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The Unexpected Evolution of Basic Science Studies about Cyclic Nucleotide Action into a Treatment for Erectile Dysfunction

In these Reflections, I describe my perceived role in discoveries made in the cyclic nucleotide field that culminated in the advent of PDE5 inhibitors that treat erectile dysfunction, such as Viagra, Levitra, and Cialis. The discoveries emphasize the critical role of basic science, which often evolves in unpredictable and circuitous paths, in improving human health.

Keywords: Cyclic AMP (cAMP), Cyclic GMP (cGMP), Phosphodiesterases, Protein Kinase A (PKA), Protein Kinase G (PKG)

Introduction

I grew up in two towns in western North Carolina, Franklin and Bryson City, both of which had populations of less than 3000. Most Americans refer to this region as Southern Appalachia, which is more widely known for its poverty than its scenic mountains and spectacular waterfalls. Perhaps the experiences with nature provide a good beginning to a scientific career; at least they provide inspiration for solving how nature works. I had five brothers and no sisters. My mother was a stay-at-home mom, and my father was a hospital laboratory technician and did some medical work, including house calls, although he only completed two years at Emory Medical School. I owe my father significant credit for inspiring me to pursue a scientific career later on. In high school, I succeeded in several sports, especially football, and was awarded a scholarship to attend Tennessee Technological University ( Fig. 1 ). I played in the Tangerine Bowl in 1961 and was co-captain of the team in 1962. We won the Ohio Valley Conference championship three of my four college years. After finishing the three-year pre-medicine program, I switched my major to biology and was able to play football for one more season. It was during that year that I was introduced to a scientific career through the influence of one of my biology professors, Dr. William Downs, who was a colleague of Charles Rawlinson (Rollo) Park, chairman of the Department of Physiology at Vanderbilt University.

Jackie Corbin playing football for Tennessee Tech in 1961.

A scientific career for me would have been improbable, particularly because of financial considerations, were it not for a highly significant world event. The first man-made satellite, Sputnik, was launched by the Soviet Union in 1957. Sputnik unleashed the Space Race between the United States and Soviet Union and led to the moon landings and many other achievements in space. It also ushered in a scientific renaissance within the American public and government during the Kennedy and Johnson presidential administrations. Among many new programs, fellowships were made available to prepare scientists for advanced degrees not only in space technology, but in practically all of the sciences, including the biosciences. The years that followed were fruitful for the scientific community and inspired those who had an appreciation for and a belief in the improvement of the quality of life through scientific advances. Due in no small part to Sputnik, I received a training grant for graduate level research in physiology at Vanderbilt University in 1963.

I chose Rollo Park as my Ph.D. thesis advisor at Vanderbilt ( Fig. 2 ). He is a very decent and dignified man who is exceptionally wise, patient, and scientifically competent. After his undergraduate education at Harvard University, Rollo received his medical degree at Johns Hopkins University and then did research training at Washington University in St. Louis under Carl Cori. Rollo went directly from his postdoctoral studies under Cori to chairman of the Department of Physiology at Vanderbilt, a remarkable career ascent! I remember fondly many of our scientific and political discussions, as well as our outdoor excursions together in the Tennessee hills and streams. At the time of this writing, Rollo, at 98 years of age, resides with his wife, Janey, in a Nashville suburb.

Rollo Park, chairman of the Department of Physiology at Vanderbilt University in the 1950s–1980s.

Earl Sutherland, who had discovered cAMP in the late 1950s, was a longtime friend of Rollo Park since they had both done postdoctoral studies in the Cori laboratory at the same time. Sutherland was recruited by Rollo to Vanderbilt’s Department of Physiology in 1963. This event profoundly affected my scientific career. At that time, Sutherland and his collaborators, who included Theodore (Ted) Rall, Reginald (Bill) Butcher, Joel Hardman, G. Alan (Al) Robison, Rollo Park, Grant Liddle, Howard Morgan, and John Exton among many others, had already demonstrated a role for cAMP in the regulation of a few physiological processes, particularly for epinephrine stimulation of glycogen breakdown in the liver. Following their discovery, they fully realized that cAMP was likely involved in the regulation of many physiological processes (1). This subject was the center of attention within our department and throughout the Vanderbilt University Medical Center. Discussions in the hallways, seminars, workshops, and other gatherings were invariably lively and often centered on the actions of cyclic nucleotides. I remember vividly the heated debates and shouting matches that took place during the departmental and interdepartmental seminars, at Gordon Conferences, and at national meetings, which rarely occur in the relatively polite atmosphere of scientific presentations today.

Early History of Research on Cyclic Nucleotide Action

Most of the early investigations of cyclic nucleotides were centered on the cyclic nucleotides themselves and the roles they played in body tissues (1). Some of this work was done by Sutherland at Case Western Reserve University before he arrived at Vanderbilt. Sutherland and his colleague Ted Rall discovered cAMP as a heat-stable factor produced in cell-free liver homogenates by the addition of adrenalin or glucagon. cAMP caused the conversion of glycogen phosphorylase to a phosphorylated active form when added to supernatant fractions of the homogenates. In the intact organism, elevation of the cAMP level in the liver resulted in increased glycogen breakdown to glucose during physiological stresses, such as exercise and starvation. Together with David Lipkin at Washington University in St. Louis, who had worked previously on the Manhattan atomic bomb project in Los Alamos, New Mexico, they identified the crystallized factor chemically as a novel compound, cAMP. Sutherland showed that cAMP is synthesized by the enzyme “adenyl” cyclase (adenylyl cyclase) and that it is inactivated by the hydrolysis of the high-energy cyclic phosphate ring to form 5′-AMP by a “cyclic 3,5-AMP-inactivation enzyme” activity from heart, brain, or liver extracts, which was termed a phosphodiesterase (PDE) 1 activity (1). Caffeine, a methylxanthine, was found to inhibit the PDE, which was the first demonstration of a mechanistic effect of caffeine on an enzyme. As discussed below, this was a critical milestone leading to our research that resulted in the advent of PDE5 inhibitors for treatment of erectile dysfunction.

At Vanderbilt, much of the effort of the Sutherland group emphasized the broad regulatory functions of cAMP. Some of these included adrenalin stimulation of liver glycogenolysis and gluconeogenesis, as well as of adipose tissue lipolysis. During the 1960s, numerous laboratories were demonstrating myriad roles for cAMP in mediating the effects of many hormones (1). On the basis of the intracellular/extracellular locations of the steps within the hormonal pathways and abundant regulatory roles for cAMP established at that time, Sutherland proposed the “second messenger hypothesis.” According to this concept, a signal (e.g. a hormone (first messenger) derived from one cell type or organ) interacts with the exterior of another cell to elicit generation of cAMP (second messenger). By this process, the first messenger transfers the message to the inside of a target cell, thereby altering metabolism and other physiological processes. Sutherland was awarded the Nobel Prize in Physiology or Medicine in 1971. In the week of the Nobel announcement, I recall that a member of the Nashville press interviewed him and asked for advice for young people on how to win a Nobel Prize. He said that, first of all, one must be lucky, and, second of all, one must recognize the luck when it comes!

Although cAMP was the only established second messenger during the early 1960s, Sutherland and his co-workers stated that it seemed “possible or even likely that other second messengers will be discovered” (1). This prediction turned out to be accurate for many signaling pathways, even though some exhibited slight deviations from the original theory. For example, several first messengers were found to traverse the cell membranes to generate second messengers within cells.

In 1963, T. D. Price and his co-workers at Columbia University discovered cGMP in urine as a radioactive compound following the injection of radioactive inorganic phosphate into rats (2). Subsequently, cGMP was shown to be present in all tissues tested (1). However, many attempts during the 1960s to demonstrate hormonal alteration of the level of cGMP in tissues were unsuccessful, even though rapid turnover of this nucleotide was well documented (1). “Guanyl” cyclase (guanylyl cyclase), which catalyzes the synthesis of cGMP from GTP, was discovered in 1969 (3), and by 1970, crude preparations of PDEs were shown to catalyze breakdown of cGMP as well as cAMP (4).

During the 1970s, the groups of Günter Schultz with Joel Hardman at Vanderbilt and the University of Heidelberg, Germany, and Ferid Murad at the University of Virginia showed that treatment of several smooth muscle tissues with compounds that were capable of generating nitric oxide causes elevation of cGMP levels and concomitant relaxation of those tissues (5, 6). This was the first indication of a possible physiological role for cGMP, even though it was known that cAMP elevation would also cause smooth muscle relaxation. The fact that blood vessel walls were known to be rich in smooth muscle cells suggested that cGMP could play an important role in the regulation of blood flow and pressure, but there was strong bias that cAMP signaling was more important in this process, given the proven role of cAMP in so many other biological processes. The role of cGMP in processes other than visual transduction still was not established. It was also known during the 1970s that penile tissue is rich in smooth muscle and that relaxation of this muscle plays a critical role in penile erection (7).

Cyclic Nucleotide-dependent Protein Kinases

Sutherland and his collaborators demonstrated that cAMP is the mediator of many hormonal signals, but they did not pursue, to a significant extent, the molecular mechanisms of its action or that of cGMP action on body functions. During the 1960s, the groups of Joe Larner at the University of Virginia (8) and Edwin Krebs at the University of Washington (9) did pioneering work in elucidating the biochemical mechanism of action of cAMP in inhibiting glycogen synthesis and stimulating glycogen breakdown, respectively, in skeletal muscle. They deduced that these effects are mediated by a protein kinase that is stimulated by cAMP, which Krebs named cAMP-dependent protein kinase (protein kinase A (PKA)), implying that it phosphorylates more than a single protein.

I conducted my Vanderbilt thesis project (10) on the anti-lipolytic effect of insulin in adipose tissue. Using an isolated fat cell preparation that had recently been described by Martin Rodbell at the National Institutes of Health (11), I demonstrated that insulin inhibits adrenalin-stimulated lipolysis by lowering cAMP and that this effect, or the effect of adrenalin to stimulate lipolysis, occurs within a very narrow 2–3-fold range of concentrations of cAMP. Because I found that insulin blocks the lipolytic effect of adrenalin or exogenous cAMP, but not that of exogenous dibutyryl-cAMP, which is resistant to breakdown by a PDE, I proposed that insulin acts by stimulating the activity of a PDE to lower cAMP. Some of this work was done in collaboration with Bill Butcher and Sam Sneyd. At that time, Sneyd was doing postdoctoral studies at Vanderbilt on cAMP and insulin action with my mentor, Rollo Park. Somewhat later, Sneyd returned to his native University of Otago in New Zealand and, along with his student David Loten, who had also studied at Vanderbilt as a postdoctoral fellow, showed using liver slices that insulin does indeed stimulate a particular “low K_m” PDE, later named PDE3 (12, 13). Today, we recognize PDE3 as a critical mediator of insulin action in the liver and adipose tissue during starvation, exercise, and obesity.

Having completed my doctoral thesis project at Vanderbilt, I had a strong desire to continue studies on the mechanism of action of cAMP in adipose tissue. Rollo Park wisely advised me to do postdoctoral studies with Edwin Krebs ( Fig. 3 ). Rollo thought that experience in the Krebs’ laboratory would have the added advantage of broadening my knowledge base and enhancing my career experiences because it would be more biochemical in nature and would involve learning enzyme purification procedures. Krebs agreed wholeheartedly to carry out the adipose tissue project. Despite Krebs moving to the University of California, Davis, just before my arrival, I was not deterred, although I did have some inhibitions about living outside The South for the first time. While at Davis, my wife, Ann, gave birth to our only child, Amy, in 1970.

Jackie Corbin with Edwin Krebs at a FASEB Summer Research Conference at Copper Mountain, Colorado, in the 1980s.

While working in the Krebs’ laboratory, I demonstrated that PKA mediates the ATP-dependent cAMP activation of lipolysis by hormone-sensitive lipase stimulation (13). Another member of the Krebs’ group, Tom Soderling (14), showed simultaneously that the same PKA mediates phosphorylation and inactivation of liver glycogen synthase; this had also been shown by Joe Larner earlier, as mentioned above. Taken together with the previous finding that PKA mediates cAMP-stimulated glycogenolysis, these observations provided strong evidence that PKA mediates many effects of cAMP elevation in tissues. In fact, during the 1970s, many proteins were shown to be phosphorylated and modulated by this enzyme. It seemed unconventional at the time to suggest that a single enzyme could carry out the same catalytic effect on so many diverse substrates, especially because scientists had become indoctrinated about the reasons and mechanisms for enzyme specificity, yet it proved to be the case. The dogma that PKA mediated all of the myriad effects of cAMP in mammals prevailed for many years until other cAMP mediators were discovered (15).

In collaboration with Margaret Brostrom, Charles Brostrom, and Erwin Reimann in the Krebs’ laboratory, I carried out enzyme work to establish that PKA is composed of a catalytic subunit that is inhibited by an associated regulatory subunit (16). Subsequently, I established that the inhibitory mechanism of the regulatory subunit was conferred by a competitive substrate mechanism within its structure (17). At the time, this was a novel inhibitor mechanism and has often been referred to as a pseudo-substrate mechanism. Subsequent studies established that this is a relatively common inhibitory mechanism for many other protein kinases. In the case of PKA, cAMP binding to the regulatory subunit leads to dissociation of the regulatory and catalytic subunits, thereby resulting in removal of inhibition of the catalytic subunit and followed by catalysis of the phosphorylation reaction. In those days, it was considered very unusual for an enzyme to be activated by the dissociation of its subunits.

In 1971, I returned to Vanderbilt (where I remain to this day) to join the faculty of my old Department of Physiology, which was still chaired by Rollo Park. The department is now known as the Department of Molecular Physiology and Biophysics. My first significant achievement after my arrival was to show that PKA exhibits two major isozymic forms, which I named types I and II (18). I found that type II is bound primarily to subcellular particles of heart homogenates, whereas type I is mainly soluble (19). The bound holoenzyme adheres to the particles through its regulatory subunit. When cAMP is elevated and binds to the regulatory subunit, the catalytic subunit dissociates from the regulatory subunit and from the particles. I proposed a model for cells in which the bound holoenzyme is compartmentalized, or anchored, near its substrate(s). This would be an efficient mechanism for phosphorylation and modulation of a substrate when the catalytic subunit is activated by its dissociation from the regulatory subunit following cAMP elevation. On the basis of the characteristics of its particle binding, I suggested that the regulatory subunit is anchored by binding to other particle protein(s) rather than being an intrinsic membrane protein. In subsequent years, the membrane proteins to which the regulatory subunit is anchored were identified by others as a family of A-kinase anchoring proteins (20, 21).

Tom Lincoln arrived at Vanderbilt as my first postdoctoral fellow in 1974. He grew up mainly in Decatur, Alabama, and received his Ph.D. degree from the University of Tennessee. Tom had contacted Rollo Park regarding postdoctoral study, and Rollo then directed him to my laboratory. In 1970, the group of Paul Greengard (Nobel laureate in 2000) discovered cGMP-dependent protein kinase (PKG) in lobster tail muscle (22, 23). Because very few biochemical studies on PKG had been done by that time, Tom and I felt that some significant findings concerning cGMP action could be made by carrying out extensive enzyme purification and kinetic studies on PKG much as I had already done on PKA. Greengard’s group, working with lobster tail tissue, had reported that PKG, like PKA, is composed of a cGMP-binding regulatory subunit and a catalytic subunit, which dissociated from each other upon the addition of cGMP when examined in a crude preparation. In his initial work in my laboratory, Tom identified a specific PKG activity in mammalian tissues. On the basis of earlier experience with PKA, Tom and I were biased that our purified lung PKG should contain separate regulatory and catalytic subunits that dissociate from each other during cGMP binding to a regulatory subunit; however, Tom was unable to establish this process. We reasoned that the regulatory and catalytic components of PKG, unlike those of PKA, are located on a single subunit and that cGMP activates the lung PKG by causing a conformational change rather than by causing a physical dissociation of subunits (23). We thought that the lobster tail PKG actually has a similar structure and mechanism of activation as the lung PKG, but that regulatory and catalytic components on the same protein chain of the lobster tail PKG are cleaved into separate fragments by contaminating proteases during activation of the enzyme by cGMP. Likewise, Gordon Gill at the University of California, San Diego, concluded that PKG does not contain separate regulatory and catalytic subunits (24), and from their studies of PKG from silkworm, Yasutomi Nishizuka’s group at Kobe University in Japan independently drew the same conclusion about PKG (25). Although this proved to be the correct mechanism, it turns out that PKG is more common in its activation mechanism and that PKA is more unusual in comparison with other enzymes. Most enzymes do not dissociate into free subunits during activation.

Despite the apparent differences in subunit composition and structure, both PKA and PKG proved to be remarkably similar in other respects. For example, both are inhibited by pseudo-substrate mechanisms and activated by binding of cyclic nucleotides to their regulatory regions/subunits; crude structural properties are similar; and importantly, both kinases recognize similar primary structural cues in substrate proteins to catalyze very specific serine/threonine phosphorylation. In 1977, we proposed that PKA and PKG are homologous proteins and predicted that the protein kinase family might be a much larger one (26). In 1977, only a handful of protein kinases were known; protein kinase C, calcium/calmodulin-dependent protein kinase II, and the tyrosine kinases were yet to be discovered.

Cyclic Nucleotide-binding Proteins and the Discovery of PDE5

The investigations that I carried out with my collaborators on PKA and PKG eventually led to the discovery of PDE5, the receptor for Viagra, Levitra, Cialis, and other commercial PDE5 inhibitors.

While I was doing my postdoctoral studies in the laboratory of Edwin Krebs, the regulatory subunit of PKA was being studied not only for its inhibitory activity toward the catalytic subunit of the enzyme, but also for its cAMP-binding activity. When I returned to Vanderbilt, I optimized a [ 3 H]cAMP-binding assay for the regulatory subunit (27). Using this assay, a [ 3 H]cAMP-binding stoichiometry of two cAMP molecules/regulatory subunit was established. These two binding sites were demonstrated to be quite different from each other even though both were important for enzyme activation (12). The [ 3 H]cGMP-binding assay for the regulatory component of PKG was also optimized, and I showed that this kinase also contains two binding sites/subunit, albeit both are selective for cGMP over cAMP (28).

In the mid-1970s, the dogma was widespread that PKA and PKG mediated all of the mammalian physiological effects of cAMP and cGMP, respectively. Tom Lincoln and I believed that there could be other mediators. In 1970, the group of Zubay (29) at Columbia University had reported the presence of a non-protein kinase cAMP receptor in Escherichia coli named the catabolite activator protein, but the presence of this protein or any cAMP or cGMP receptor, other than PKA and PKG, had not been demonstrated in mammals. The development of stoichiometric cyclic nucleotide-binding assays offered an opportunity to search for such proteins that specifically bind cAMP or cGMP. Believing that we were among the few scientists in the world vigorously pursuing cGMP action at that time, we decided to search for cGMP-binding proteins instead of cAMP-binding proteins. We made homogenates from several rat tissues, centrifuged them, and then subjected the supernatants to DEAE-cellulose chromatography to obtain a crude separation of the soluble proteins from the various tissues (30). The [ 3 H]cGMP-binding assay of the fractions obtained by chromatography of several tissue extracts revealed a PKG peak in its typical elution position as expected. Importantly, some of the tissue extracts, such as that from the lung, exhibited an additional peak of [ 3 H]cGMP-binding activity eluting ahead of the PKG peak. This fraction was clearly highly specific for cGMP over cAMP, was not a protein kinase, and possessed other physical and kinetic properties indicating that it was unique. Our report of these studies was the first to indicate the presence of a mammalian cAMP or cGMP receptor other than the cyclic nucleotide-dependent protein kinases. Among several kinetic features of this newly discovered protein was its stimulation of [ 3 H]cGMP binding by the PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX). Although this finding suggested that it could be a PDE, more definitive proof was needed because some thought that this activity could be due to a breakdown product of PKG or that the “stimulation” by IBMX was simply due to protection of the substrate cGMP from breakdown. Other interesting features surrounding this protein, including important medical breakthroughs, would be forthcoming ( Fig. 4 ).

Group of scientists from the Vanderbilt Department of Physiology in 1978. 1, John Exton; 2, Rollo Park; 3, Tom Lincoln; 4, Sharron Francis; 5, Jackie Corbin.

A key event in uncovering the identity, function, and eventual medical use of PDE5 inhibitors such as Viagra was the return of Sharron Francis as a faculty member in the Vanderbilt Department of Physiology in 1975. Sharron and I had experienced similar prescientific and early scientific careers during the 1940s–1960s. Sharron grew up in eastern and central Kentucky and I in the Appalachian Mountains of western North Carolina. Sharron did her undergraduate work at Western Kentucky University and I at Tennessee Tech. As undergraduates, both of us had majored in biology. Both of us had received post-Sputnik government training grants to do Ph.D. thesis research in the Vanderbilt Department of Physiology. Both of us had been exposed to the cyclic nucleotide research done at Vanderbilt during the 1960s. I had chosen Rollo Park as my thesis advisor, and Sharron had chosen Rollo’s wife, Janey. Sharron had done postdoctoral studies involving protein structure and regulatory processes in two outstanding laboratories, those of Herman Eisen at Washington University and of Earl Stadtman at the National Heart, Lung, and Blood Institute ( Fig. 5 ).

Jackie Corbin with Sharron Francis in the lab in the 1990s.

In 1977, Sharron joined Tom and me in a collaboration to determine the function of the newly discovered cGMP-binding protein. In those days, very few consensus amino acid or nucleotide sequences of functional domains for any protein family were available to guide us. Biochemists ordinarily used the approach of purifying a protein extensively and establishing if a particular functional property was retained in that protein. Because of the stimulatory effect of IBMX on the cGMP-binding activity that Tom had observed, we believed that the protein could be a PDE, but many people doubted that interpretation. Therefore, we tested whether or not the IBMX-stimulated cGMP-binding activity correlated with PDE activity in the chromatographic profiles during several steps of purification of the protein (31). Using rat lung as starting material, Sharron spent more than a year in carefully developing conventional purification steps. She found that the two activities do indeed co-chromatograph precisely. Moreover, the stimulatory effect of IBMX on [ 3 H]cGMP-binding activity, which is also obtained using other PDE inhibitors, such as papaverine, theophylline, and caffeine, is retained in the purified protein. The PDE was found to be highly specific for cGMP over cAMP as a substrate. Some predicted that the [ 3 H]cGMP-binding activity of the enzyme was conferred by its substrate site and even perhaps a modified substrate site. However, because PDE inhibitors were known to be competitive with cGMP for the substrate site of PDEs, it would be predicted that PDE inhibitors should be inhibitory, not stimulatory, toward [ 3 H]cGMP binding. Moreover, binding of [ 3 H]cGMP to the PDE substrate site did not have sufficient affinity to be detected by our [ 3 H]cGMP-binding assay. The combined results suggested that the protein possesses two different kinds of sites for cGMP action, a catalytic site for breakdown of cGMP and a binding site for regulation by cGMP. This was confirmed by the findings that heat or trypsin treatment of the purified protein destroys PDE activity but stimulates cGMP-binding activity and that certain cGMP analogs inhibit PDE activity but not cGMP-binding activity. The catalytic site and cGMP-binding site are non-homologous (32). This PDE, which would ultimately be the target for PDE5 inhibitors in treating erectile dysfunction and pulmonary hypertension, thus represented the first recognized mammalian cyclic nucleotide receptor other than PKA and PKG. We named it the cGMP binding protein-PDE, which was later changed to PDE5 in the 1990s.

Physical and Functional Characterization of the Newly Discovered cGMP-binding PDE5

Tom Lincoln finished his postdoctoral studies in my laboratory in 1979 and continued to work on PKG in smooth muscle. His laboratory at the University of South Carolina was the first to propose that cGMP lowers intracellular calcium levels in smooth muscle to induce relaxation (33). From this point, Sharron and I continued our close collaborative investigations of PDE5. We shared all of the laboratory functions about equally, including mentorships, writing grants and papers, and presenting talks. We discussed experimental results and ideas daily. In addition to inserting more enjoyment into our research experiences, this arrangement provided enormous advantages for our investigations. I highly recommend a similar organization to developing scientists. There are many examples of such successful collaborations in the scientific world ( Fig. 6 ).

Backpacking with lab members in the Smokies in the 1980s. 1, Jackie Corbin; 2, guest Roger Colbran; 3, Sharron Francis.

PDE5 potentially represented a new direction for research on cyclic nucleotide action. The function(s) of the cGMP-binding site of the enzyme were unknown. Biochemical studies could reveal interesting features not only about the regulation of the enzyme, but also about its catalytic properties and physiological roles. The literature already contained implications for important roles for cGMP in smooth muscle regulation, particularly for cardiovascular diseases such as hypertension. We were well aware of the potentiating effect of PDE inhibitors to raise the tissue levels of cAMP or cGMP by inhibiting PDEs. For example, such an effect would raise the cGMP level in arterial smooth muscle, relax the muscle, and lower blood pressure, which suggested that a cGMP PDE inhibitor could be used to treat hypertension.

Having worked out the arduous process of purifying PDE5 from rat lung, we decided to switch from rat lung to bovine lung as the tissue source to obtain larger quantities of PDE5 for molecular studies. Through much diligent work by Melissa Thomas, Linda McAllister, and Janet Colbran, the first amino acid sequences were obtained, and this led to the cloning of the cDNA of the enzyme via collaboration with Joseph Beavo’s laboratory at the University of Washington (32, 34, 35). Melissa Thomas discovered that PKG phosphorylates the dimeric enzyme at a single serine residue in its regulatory domain and that this modification stimulates the PDE catalytic activity (26). Working together with our students Mitsi Blount and Emmanuel Bessay, we uncovered several other regulatory features of the enzyme. Phosphorylation of PDE5 stimulates cGMP binding to the regulatory site (36), which stimulates the catalytic site of PDE5, as well as the phosphorylation of the enzyme (37). Finally, we found that when a substrate binds at the substrate site, it stimulates both cGMP binding at the regulatory site and phosphorylation of the enzyme. These findings together indicated that elevation of cGMP in cells causes a concerted feedback response to activate PDE5 and results in lowering of the cGMP level, a classic negative feedback effect.

Regulation of Smooth Muscle Function, Blood Pressure, and Blood Flow

Because of their critical roles in species survival, blood flow and blood pressure are highly regulated processes ( Fig. 7 ). A plethora of mechanisms exist for their control. One salient mechanism targets the smooth muscle cells in the walls of blood vessels. In general, contraction of these smooth muscles constricts arteries, decreasing downstream blood flow and increasing upstream blood pressure. Conversely, relaxation of these smooth muscles dilates arteries, increasing downstream blood flow and decreasing upstream blood pressure. The sympathetic and parasympathetic nervous systems are prominent regulators of contraction/relaxation of blood vessel smooth muscles. Common neurotransmitters for sympathetic nerves are adrenalin and noradrenalin, thereby referred to as “adrenergic.” Parasympathetic nerves commonly employ acetylcholine; thus, they are referred to as “cholinergic.” In the early 1980s, the dilation of penile cavernosal smooth muscle to produce erections was thought to be mediated by parasympathetic nerve stimulation.

Jackie Corbin clogging with Ramsey Wall at one of his Vanderbilt Medical School cardiac physiology lectures.

Up until the mid-1980s, either cAMP or cGMP elevation was known to produce smooth muscle relaxation, but the mechanism for each nucleotide was unknown, even though many workers in the field believed that PKA or PKG mediated the respective cyclic nucleotide effect. However, we found that although either cAMP or cGMP analogs caused relaxation of vascular and tracheal smooth muscles, only those analogs that activated PKG relaxed the muscles (some cAMP analogs activated PKG) (38). We could find no evidence for PKA involvement in mediating the relaxation effects. We suggested that cAMP produces its physiological relaxation effect by “cross-activation” of PKG (39). Our attention centered on the cGMP pathway, and because we had shown that smooth muscle (and lung tissue) was rich in both PKG and PDE5, we surmised that coordinated activation/inhibition of these respective proteins would lead to development of a particularly potent vasodilator or bronchodilator. On this premise, we devised the idea that perhaps a drug, such as a PDE5-resistant cGMP analog, could be developed that would serve as a dual-acting compound to activate PKG and inhibit PDE5, thus substantially elevating cGMP to produce vasodilation or bronchodilation for treating certain diseases.

Penile Erection

It was known by the early 1980s that the penis contains a paired cylindrical structure termed the corpus cavernosum, which is primarily responsible for penile erection ( Fig. 8 ) (7). This vascularized spongy structure is highly enriched in smooth muscle cells that surround blood vessels and cavities known as sinusoids. It was known that sexual arousal in men specifically induces parasympathetic nerve stimulation in penile tissue and was believed to be non-cholinergic and non-adrenergic. However, the neurotransmitter had not been identified. The nerve stimulation brings about relaxation of the penile smooth muscle cells, leading to increased blood inflow to and expansion of the sinusoids, which compresses the veins to reduce outflow of blood from the sinusoids. Together, these processes cause an ∼8-fold increase in penile blood volume, resulting in a full erection due to the swelling and rigidity of the penis.

Sexual arousal is the natural psychological stimulus that causes nerve stimulation to the penis, release of nitric oxide at the nerve ending, and increase in cGMP in smooth muscle cells of the corpus cavernosum. From this cross-section of the penis, an increase in cGMP in these smooth muscle cells (brownish network) causes relaxation of the muscle and engorgement of the sinusoids (cavities) with blood (shown in red), resulting in dilation of arterioles, expansion of sinusoids, and compression of veins. This leads to rigidity, or penile erection. PDE5 inhibitors potentiate the increase in cGMP to cause further erection.

Nitric Oxide and Its Role in Stimulating Penile Erection

Before 1990, the organic cause(s) of erectile dysfunction were largely unknown, and psychotherapy was a method of choice for treating this condition. Penile implants, vacuum constriction devices, and surgeries also were used, although understandably, most patients were not attracted to these methods. Direct penile administration of prostaglandin E₁ and other agents was sometimes effective, but was also unacceptable to most patients. Various oral treatments were in use, but they were generally ineffective.

In the 1980s, nitric oxide was discovered as an important signaling molecule. The groups of Ferid Murad at the University of Texas, Robert Furchgott at the State University of New York Health Science Center, and Louis Ignarro at UCLA received the Nobel Prize in 1998 for their work on this subject. In 1990, the Ignarro group reported that nitric oxide-induced cGMP elevation in penile smooth muscle mediates stimulation of penile erection in rabbits (40). Two sources of nitric oxide came to be recognized in this process: parasympathetic nerves, which mediate the effects of sexual arousal; and vascular endothelial cells, which mediate the effects of local penile factors. The idea was emerging that erectile dysfunction was caused primarily by insufficient nitric oxide release within the penis to adequately elevate cGMP in the vascular smooth muscle cells of this tissue (41). If so, we thought that a PDE5 inhibitor, which would elevate cGMP by preventing its breakdown, would be therapeutic for men with this condition.

The general principle of synergism (potentiation) was established in the early 1960s by Rall and Sutherland. They showed in liver preparations that inhibition of PDE-catalyzed breakdown of cAMP by a PDE inhibitor (caffeine in this case) produces a much bigger elevation in cAMP and thus a bigger downstream physiological response in the presence of a stimulator of adenylyl cyclase-catalyzed cAMP synthesis, such as epinephrine or glucagon. Synergism works for the cGMP pathway as well as for the cAMP pathway. Therefore, a PDE5 inhibitor would have its greatest effect in tissues that already have a stimulated rate of cGMP synthesis. In the case of penile erection, this stimulated rate is provided by sexual arousal. The arousal causes increased cGMP synthesis, and the PDE5 inhibitor causes decreased cGMP breakdown, which synergistically and selectively elevates the penile smooth muscle cGMP level and leads to the relaxation of the muscle and penile erection. Viagra and the other commercial PDE5 inhibitors have very little effect unless a man is sexually aroused. The synergism provides a targeting effect of a PDE5 inhibitor to cause penile erection in the absence of significant side effects because cGMP synthesis in body tissues, other than penile smooth muscle, is not activated by sexual arousal. As Ken Murray at SmithKline Beecham Pharmaceuticals (now GlaxoSmithKline) stated in a review in 1993 (42), “there could be considerable value in a therapeutic agent that has little activity in its own right, but potentiates the effects of endogenous mediators.”

After the advent of Viagra in 1998, I got numerous invitations to present lectures on treatment of erectile dysfunction. In addition to universities and international meetings, I also enjoyed giving PowerPoint lectures to rural Tennessee doctors and nurses, senior citizen groups, service organizations, and schools. One memorable experience followed a lecture I gave to a small-town middle school group. Part of my presentation included the slide depicted in Fig. 8 , which is a picture of the statue of David by Michelangelo with an inset showing the cross-section of a penis. Although this picture and its description may have been well received by the students, this was not the case for many of the teachers, parents, townspeople, and local politicians, who were in attendance. Before I arrived home after a two-hour drive from the school, the Nashville television channels presented interviews with some of those irate citizens on the evening news. The slide of Fig. 8 was used as a backdrop by one of the channels during the presentation, albeit with the penis covered by a black rectangle. The local reporters had visited the students in my laboratory, who gave them the requested slide before I returned from my trip. News of this embarrassing incident reached many of my colleagues around the world. Several Europeans were particularly amused by the implication that the statue of David was a pornographic image.

The Vanderbilt Lawsuit

In the late 1980s, Sharron and I pursued the synthesis and study of cGMP analogs to determine whether, by appending certain chemical groups, the resulting analogs might more effectively activate PKG. Through the phosphorylation of other proteins, the enzyme reduces the level of cellular calcium, resulting in relaxation of the smooth muscle cell. In 1989, we were awarded a three-year Glaxo Cardiovascular Discovery Grant to further pursue our research. During 1991, the final year of the grant, Glaxo encouraged us to emphasize the development of PDE5 inhibitors over PKG activators because cGMP analogs break down in the digestive system before reaching targeted smooth muscle cells and thus were not good candidates for orally administered drugs. PDE5 inhibitors act by competing with the enzyme substrate cGMP, so these inhibitors have some structural similarity to cGMP. On the basis of the knowledge of PDE5 gained from our studies of cGMP analogs, Sharron and I, along with Sekhar Konjeti (known as Raja), who was an excellent chemist and had joined us as a postdoctoral fellow to synthesize compounds in connection with the 1989 grant, theorized that more potent PDE5 inhibitors could be created by attaching hydrophobic groups, such as a phenyl ring, with additional electron-donating groups appended to the ring, in positions where they could bind to the same site on the PDE5 molecule as does the ribose phosphate moiety of cGMP. Moreover, because our research suggested that cGMP bound more tightly to the catalytic site on PDE5 when the ribose phosphate moiety was in the anti-position in relation to the guanine moiety, we further theorized that inhibitor potency would be enhanced by appending the phenyl ring at the 8-position on the six-member/five-member ring scaffold, as opposed to the 9-position, where the ribose phosphate moiety of cGMP joins its guanine moiety. Applying these theories to IBMX, we conceived 8-(4-OH-phenylthio)-IBMX (Scheme 1). Tests showed that 8-(4-OH-phenylthio)-IBMX was 160 times more potent than IBMX in inhibiting PDE5 and six times more potent than zaprinast, which was the most potent inhibitor of PDE5 then known.

On November 12, 1991, we sent a letter to the Vanderbilt Department of Technology Transfer explaining the potential commercial applications for our PDE5 inhibitors. Having read the Ignarro report concerning cGMP involvement in penile erection (40), we explained that the inhibitors could be useful to treat “male impotence” or “any malady that involves smooth muscle.” To our knowledge, this was the first written mention that PDE5 inhibitors could be used to treat this condition.

During a telephone conversation in late December 1991, I discussed our success using PDE5 inhibitors with the head of the Glaxo Group Research’s cardiovascular research management committee. He requested that I send him a letter summarizing our research and a proposal for a possible second grant from Glaxo to fund further research. In the letter we sent on January 3, 1992, years before inhibitors such as sildenafil (Viagra) were tested for that purpose, we pointed out that PDE5 “is present in high concentrations in vascular smooth muscle,” such as the penile corpus cavernosum, and that PDE5 inhibitors could be used to treat “male impotence” in addition to other diseases. We also described our theories for designing PDE inhibitors and the significant increases in potency that we had achieved. However, we identified our inhibitors only as IBMX analogs.

Our letter prompted the Glaxo representative to fly from London to meet Sharron, Raja, and me at Vanderbilt on February 3, 1992, to discuss a formal Glaxo grant application for us to conduct further research on cGMP analogs and PDE5 inhibitors. He told us that we should submit a research proposal with more detail describing our experimental design. We did so on February 24, 1992. The proposal repeated our proposition that newly synthesized PDE5 inhibitors, based on our scheme of appending favorable chemical groups to IBMX, could be used to treat male impotence, later referred to as erectile dysfunction. It also disclosed and outlined the chemical structure of our potent inhibitor, 8-(4-OH-phenylthio)-IBMX, which we had synthesized in November 1991.

On April 8, 1992, the head of Glaxo Group Research’s cardiovascular research management committee sent copies of our research proposal to a number of Glaxo scientists, including the lead chemist at a Glaxo facility near Paris, France, who was leading a team tasked with developing new PDE5 inhibitors. On April 23, 1992, members of that Glaxo team tested 29 compounds (including, as a control, the known PDE5 inhibitor zaprinast) for PDE5 inhibition, each of which included a six-member/five-member ring. Twenty-six of the compounds included some substitution at the 8-position of the six-member/five-member ring scaffold. Eleven of the 29 compounds tested on April 23 contained structural features found in our inhibitor, 8-(4-OH-phenylthio)-IBMX, shown here in red (Scheme 2). Prior to that time, Glaxo had never tested a single compound containing those features for PDE5 inhibition. One of those showed promise for further development (Scheme 3). The first modification Glaxo made to that compound was the replacement of the pyridine ring with a combination of a phenyl ring and an electron-donating methoxy substituent, which resulted in an even more potent inhibitor, shown here alongside 8-(4-OH-phenylthio)-IBMX (Scheme 4). Further modifications ultimately led to Glaxo’s synthesis of tadalafil, which later came to be marketed commercially as the erectile dysfunction medication Cialis.

Cialis Commercial 2015 Pool

Traveling in Tuscany – Tips and suggestions for your holidays in Tuscany. Holiday accomodation in Tuscany, Umbria and on Elba Island. – Contact us for your holiday home with swimming pool, villa with private pool, or other holiday rentals in Tuscany and for informal advice on the choice of your travel destination and your holiday accomodation in Tuscany or on Elba Island.

Pages

The much discussed Fiat 500X commercial in Tuscany

the new Fiat 500X (photo www.alvolante.it)

By now almost everyone will have seen the commercial for the new Fiat 500X, also known as the commercial with the “Viagra”. In America everybody saw the TV-spot at the Super Bowl, and now the spot has become famous in Europe too. The commercial was recorded in the picturesque town of Pitigliano in the Tuscan area of Maremma.

Pitigliano is located in the most southern province of Tuscany, Grosseto. Most of the houses are built with tuff bricks and the hill on which the town of Pitigliano is built consists of tuff rocks. The Italian brand Fiat chose this beautiful town because it perfectly combines tradition with the irony of the commercial.

The spot begins with a panoramic scene of the idyllic hillside town Pitigliano and then a view inside a typical Tuscan house of an Italian couple. The older man wants to take his last Viagra tablet, but he’s just a little too excited and accidentally the blue tablet flies out the window. It rolls along the streets and alleys of Pitigliano and along the fountain in the central square of Piazza della Repubblica . After a whole detour the blue pill eventually ends up in the petrol tank of a Fiat 500 of a young man who’s about to refuel and the Viagra starts working . the Fiat 500 grows and becomes a Fiat 500X.

Certainly a wonderful advertising spot for Fiat, but also extraordinary exposure for one of the most beautiful towns of Italy and Tuscany, Pitigliano, which belongs to the club of I Borghi più Belli d’Italia.

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For example, this is called the fourth area by the demons and the demons, and the aborigines call it the barrier of the Alfano region.

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Bang bang, Bang bang, Like a heartbeat, the dark light door quivered slightly, The black portal slowly opened, and the two Abyss Kings sitting on the Demon King s seat revealed themselves.

this is, Barr looked at the cave that was gradually taking shape, and then at the wanton flames of the droplets, which new viagra commercial 2015 took off the burning light of the entire mountain.

The dark starry sky, the deep nebula, the floating continent fragments, everything is so similar to that extreme bath hydro shower max enlarger enhancer enlargement penis pump place.

The entire junction of the Hell of Fire and the Hell of Ice and Snow was filled with a strong bloody atmosphere, and it was nasty.

Under the horrified new viagra commercial 2015 gaze of all the demons, they disappeared little by little, and even for some reason, their previous memories in the minds of the demons also disappeared little by little.

The atmosphere was extremely condensed for a while, It seems that all the auras between heaven and earth converge here.

As the impetus of this violent conquest, the Demon Emperor Yugust Tenis has become what doctor prescribes viagra a powerful figure that has attracted worldwide attention.

Diablo sneered and waved his hand, endless lightning erupted from his hand, distorting the space, following Andariel s projection, into the void.

Hum, A peculiar power spread out, and the three-element crown that rioted in an instant, completely quieted down.

Furthermore, under the feet of this holy city, there is an extraordinary thing buried, these stupid things, living here for tens of thousands of years have not discovered this secret, if it weren t for the speculation what doctor prescribes viagra of the masters of the dick proenneke temple.

viagra 25 mg price 60 Capsules Viagra can i take revatio for ed The black and gorgeous wings represent their strength, The stronger the one, the bigger and more wings they have.

Although it do male libido pills work is possible that even this imaginary god did not know it, Diablo was clear about it.

They new viagra commercial 2015 forcibly opened a plane channel in the heaven mountain, and a group of extraordinary angel army walked out from it, and blasted towards the surrounding area.

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The God King An seemed to pant slightly and said, his expressing attitude instantly made the elves dominate them to relax a little.

As the lord of heaven, Diablo could not reach the new viagra commercial 2015 dreamlike height of using the universe as a chessboard, but he new viagra commercial 2015 new viagra commercial 2015 Buying could see the mystery of this plane new viagra commercial 2015 Buying at a glance.

The escape method that just disappeared in an instant could not even be done by their New Viagra Commercial 2015 new viagra commercial 2015 angels.

Your Mightiness, Tolstoy frowned and continued, Then you will destroy it! This time, the are testosterone boosters necesary for older men female devil did not let the gods of the Sky City choose, the vast and huge projection suddenly collapsed, and a wave of shocking dark energy spread in all directions.

Puff, A mouthful of blood was spit out, containing high testosterone problems a large stock of ordinary energy, and Diablo s expression was clear.

itself? Barr frowned tightly, How could he be in crisis? It s more reliable to say that the crisis comes from itself, it is better to say that the crisis comes from the mysterious New Viagra Commercial 2015 area below the sixth floor of the abyss.

The black flame in his eyes flashed slightly again, The new viagra commercial 2015 human race and the angel race, the New Viagra Commercial 2015 seeds how to get bigger girth of hatred have been sown, and the eyes of the gods have also been successfully shifted, and they all began to be suspicious.

An offensive portal! The first layer of the abyss portal, a strong breakthrough.

That is the mark of the abyss viagra prescription cost that has never been erased, Just like destroying and destroying fear, the demons transformed by the three dark powers, they call themselves burning demons.

Tsk tusk tusk, it s been so long, new viagra commercial 2015 you top male enhancement pills are still so cold, and you have a lot of secrets, I erect xl male enhancement pills heard that in the ancient times, you were the new viagra commercial 2015 Buying celebrity beside the Destroyer Lord, this time I new viagra commercial 2015 met the one who also possessed destruction Your Majesty Barr, your situation may not be much better than mine! Samuel flashed his bright wings and smiled coldly.

Horror, horrible! The black New Viagra Commercial 2015 shadow slowly turned his body, what doctor prescribes viagra his eyes bursting with radiance.

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What? This guy? Really exist? Everyone was shocked and looked at the wall of the conference room, where the tail of the shark was hung in it.

This battle is not something that the Demon Kings can participate in, Those viagra for women online who come are New Viagra Commercial 2015 absolutely terrifying existences in the Divine King level, and the gathered how to get cialis without doctor gods, such as destroying the descendants, are still useful.

The man in front of him is not a friendly person, Diablo has 100% confidence new viagra commercial 2015 in the New Viagra Commercial 2015 belief that whether it is the past several undead natural disasters, or the sorcerer s conspiracy by the magic sword, the transformation into the lich Arandica, etc, tadalafil vardenafil absolutely It s all related to the new viagra commercial 2015 behind-the-scenes man in front of me.

With its appearance, the surrounding time and space can be clearly seen, new viagra commercial 2015 and vigrx plus harga the phenomenon of overlapping folds has occurred.

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Possess a three-point god, create a trinity demon god, a forbidden ability, fear, time and space, destruction and destruction.

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Mephisto murmured to himself, spinning his mind rapidly, recalling every detail.

Whether it is foreign gods, or all ghosts, sneaky and strange things, even new viagra commercial 2015 those so-called behind-the-scenes, ancient gods, they are all under his plan.

But it s too small, If you attack by yourself, a few hundred meters will not necessarily erase the outer brilliance.

Bang, A few sharp sword-like tentacles broke through the flame blockade, arrived in the air, and instantly fell on the flame demon phoenix body.

First, the source of fear entangled and invaded the black dragon princess, In his opinion, this was entirely because the black dragon princess had a deeper dark ability after mutation.

She had only heard about mens supplement it by other forms of viagra chance, and groupon products reviews she asked about it quietly, and guessed privately that the secret should be related to the ancient Sea-Monster clan being destroyed by the ancient Destroyer Demon but luckily survived.

This seat can feel that the king who truly belongs to us, the darkness how to use a penis extender of the universe, and the great existence that dominates this universe is coming soon.

blazing! In an instant, a powerful energy suddenly tore out, and then the naked eye could see that a weird void that was very different from the void crack appeared in the very center new viagra commercial 2015 of new viagra commercial 2015 the Chaos God Pool.

It used to be a taboo ability that only interfered slightly in the past, At this moment, it has the weird ability to change the future.

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The earth gradually cracked, huge rocks directly flying into the sky from the ground, and what doctor prescribes viagra a trace of powerful aura spread from the cracks in the ground.

Humph! Damn guy, hiding his head and revealing his tail, dare to belittle the three Demon Lords and come out to this seat.

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This guy was sinister enough, he didn t know what he was calculating, he didn t even show up when the bloody lair was besieging the demon in the west and new viagra commercial 2015 suffered heavy losses.

This thing is the trophy of this seat, I want that life in exchange! The stalwart body of the Three Demon Gods continued to new viagra commercial 2015 rise, as if even the lower body hidden in the source of chaotic energy was about to emerge.

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Master Great Sacrifice, the leaders of several organizations, asked us what happened to the sudden update of the virtual world? How should we answer? A white-robed man with a long sword on his back flew over.

This sub-element is war energy, It is different from the various elements commonly used in the universe, but it is not like the abyssal demon energy, new viagra commercial 2015 which is absolutely hostile to the universe.

Everyone who what is sildenafil citrate tablets becomes a god king is not a simple new viagra commercial 2015 existence, They are all guys who follow through on ed herbal supplements their own path.

Among ordinary civilians, it is impossible for such a perfect human to be born.

Their strength, even their closeness to Baal, is self-evident, The most important thing cialis didnt work for me is that these seven guys, but with the three most powerful commanders, Isabella, No.

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Its body is twisting, and its eyes are cold, revealing a terrifying aura, Boom.

Even the will of the abyss will not work! Ya Ya Well, hateful guy called the other end of Cang skeleton, witch definitely make this big man to come when I caught a toy.

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With the soul strength almost comparable to the legendary spirit, it is incredible that he can t detect the rhino for men abnormality.

It was the angry roar of the will of the abyss outside, and it seemed that this guy was very angry after being threatened sexual health clinic hounslow by Barr.

But it is undeniable that the blockade of the thirteenth portal has latest in penis enlargement made the abyss much weaker, and it scene de sex doesn t even have male drive max pills the power to interfere with us.

Monastery organization, other heads of state, other erectile dysfunction medication reviews city leaders, viagra alternative online All of them fell into a daze.

Come on, defeat the servant army new viagra commercial 2015 of this seat, come to the dark hell, and conquer this seat.

As if the soul was torn apart, He knew that there was some kind of power covering up everything New Viagra Commercial 2015 about original sin, even his appearance, but what new viagra commercial 2015 kind of power it was that could even cover the detection levitra tabs of the king of gods, from all the rule information of the heavens what doctor prescribes viagra and the world, no matter what kind of power it was.

His portrait is also known by the people of the God Realm, Oh, The low voice of the three demon gods emerged, This kind of voice was very peculiar, like a mixture of three voices, what doctor prescribes viagra with a hoarse and cold feeling, in the chaos and chaos, it seemed vague, but new viagra commercial 2015 it seemed very clear.

Including Avril Lavigne, now she, even if she is a legendary demon, is not in the eyes of the true blood demon king.

Bar You does viagra cause heartburn thought leisurely, touched the half scarlet key in front of him with his other hand, and after looking indifferently for a moment, he sneered.

Now after Tyrael s reminder, everyone is what doctor prescribes viagra terrified, If this is true, And at this time, Tyrael made up a knife nakedly when everyone hesitated, It stands to reason that the God Realm and the Abyss appeared in the universe almost at the same time.

However, New Viagra Commercial 2015 now he has been rejected and can only negotiate! As the new viagra commercial 2015 most domineering demon free trial sex enhancement pills master, how to raise testosterone fast just being unhappy is new viagra commercial 2015 already restraining himself.

If he doesn t take the opportunity to threaten him, he what doctor prescribes viagra just wants to leave.

Lord Angel s plan, then people like them can just go to death! The angel male enhancement surgery 2019 new viagra commercial 2015 inlaid with golden patterns said fiercely, looking very vicious.

The demons: No. The illusory gods live in the realm of the gods, We can only be tied up in a small abyss, I am not angry and feel injustice! I once sweared that one day, I will lead all the demons to destroy the universe and destroy those illusory Gods, I swear that we will let our abyssal demons truly dominate our lives! We will destroy everything that dares to how much is cialis 20mg stop us.

Destroy the legion s parliament land-Destroy the cave! The huge space-time magic circle, the beam of light that penetrated the sky completely, it was this guy who summoned it.

The man glanced at the chaotic battlefield again, and after a while, the stars disappeared in his hands.

New Viagra Commercial 2015 | Ryan Clark Music

As for who this someone is, Later, after everyone collected some experimental specimens, the Amonro food crops were burned to the ground under the massive witchcraft destruction, and then under the continued guidance of new viagra commercial 2015 the Crystal Turtle, they proceeded toward the deeper underground.

A vessel with a very high colloidal substance, and then stimulate the annihilation force inside the vessel, and then observe the most basic nature of the annihilation force through the gaps and traces left by the annihilation reaction inside the vessel through the annihilation force, and then decide the next step the study. At this time, the shadow cloud layer above the battlefield is gathered by the shadow cloud layer temporarily mobilized by many mystery prophets.

In this case, if you don t buy a non-toxic New Viagra Commercial 2015 formula through your own long-term research, you will save a lot of time in a New Viagra Commercial 2015 short period of time. Three new viagra commercial 2015 monster erection days later, Simon snapped his fingers new viagra commercial 2015 and said to the starling on his shoulder: You can play outside.

It is speculated that as long as the original ght male space fortress has not obtained the specific information of the Zhangma tree, the first different extenze pills one of the people to return to the original space New Viagra Commercial 2015 fortress to submit task information will inevitably receive a medal of honor. The advantage of killing wildly, One day later, The fight between the vitamin coffee for male enhancement army of monsters and the army of shadows has slowed New Viagra Commercial 2015 down.

These lava new viagra commercial 2015 giants vary in size, The new viagra commercial 2015 tallest elite male enhancement testosterone booster body is more than ten meters long, and the lowest is only two or three meters, which is less than the first-level biological criterion, but compared to the wizards, they are still sex shop dc much larger. high blood pressure and low libido, Quack, don t worry about York Liana, my young master will how to make your penis bigger pills recommended by doctor definitely protect you.

This is a noble milky-white skin mystery Amonro, whose right eye is closed for some reason New Viagra Commercial 2015 all the time. According to Yorklianna, these black witch apprentices are a bit unusual, new viagra commercial 2015 and they seem to have suddenly proliferated in a certain period of time.

New libido pills cvs clinically proven male enhancement products. The Liuliruohuo physique pe head touches male enhancement on the test bench Amonro had a reaction instrument on his left eye.

After quickly reflecting on it, he was pleasantly surprised: Okay! In one minute, if you successfully kill the witch hunter and we fail, we will withdraw from the witch hunt record. An afterimage was fleeting, and the monster with evil New Viagra Commercial 2015 eyes bowed its head, looking at the tail inserted into his chest in 25 pill disbelief, the long fork in his hand fell.

This female witch hunter could only grit her teeth and best male enhancer pills use her aircraft to penis enlargement surgery prices passively defend her. The starling s voice what are the best male enhancement pills was new viagra commercial 2015 instantly drowned out by the new viagra commercial 2015 opponent, and what are the common side effects of ed pills the head of the giant bird suddenly showed a complacent look.

In a world, certain existences that exceed the characteristics magnesium oil testosterone of New Viagra Commercial 2015 its own rules will cause the new viagra commercial 2015 world s original rules to rebound With a cry of despair, several sailors stumbled and fell into the sea, being submerged by huge waves.

Just keep attacking the underground lair, Since there is no group of intelligent creatures like the new viagra commercial 2015 lava giants of the shadow mystery world or Amonro at all, it is very difficult for world guardians to appear. A bright wizard camp who went to Lilith s hut turned into a terrifying and cruel dark wizard camp at Hesota Academy.

Taking a deep breath, Simon glanced at viagra on an empty stomach the flame-greedy giant who seemed a little confused behind him, and strode down the shock in his heart and walked towards the direction of the crystal turtle. The black bone armor squeezed the skin of the body, lying quietly on another ed meds online review experimental platform, with a new viagra commercial 2015 lot of pipes inserted in it.

Barely showing a smile, Simon nodded, and as Millie new viagra commercial 2015 watched, holding the Tomb New Viagra Commercial 2015 Words Stigma Wizard silverback supplements s order, turned and walked towards the World Teleportation Array of the Fortress New Viagra Commercial 2015 Wizard Tower. In the fat barriers of the underground new viagra commercial 2015 passages, this kind of creature walks in it like a fish in water.

Especially the emergence new viagra commercial 2015 of dark wizards in recent years has New Viagra Commercial 2015 exacerbated such rumors. After a moment of stunned, Peranos suddenly realized: Oh, I see, you are the life consciousness that has not been fully promoted to the official wizard, and the length of the perception time stays between the wizard apprentice and the official wizard.

New Viagra Commercial 2015 Unexplainable, unclear feeling, If he could how to improve my libido as a woman grasp the talents of deeper relative spatial boundaries and confirm the absolute boundaries of that layer of paper, Simon would be able to try to tear the relative boundaries and penetrate the emptiness.

after one new viagra commercial 2015 day, Flying for a long time in the sky twisted and refracted by the air, I finally reached the volcano New Viagra Commercial 2015 penis enhancement supplements group of permanent penis enlargement pump the mission target. This is an elemental male enhancement pills genix refining wizard wearing a blood-red metal armor, The bloody energy all over his body is unsteady.

It s also time for the Shadow Mystery new viagra commercial 2015 World to be renamed the Fire Crystal World. In the eyes prescription metabolism boosters of ordinary people, wizards are always new viagra commercial 2015 mysterious, unknown and frightening.

Simon nodded and noticed that the little boy had only been igniting magic power soon. This element-activated witchcraft ice crystal phoenix has some mysteries even stronger than its own flame-greedy giant.

Countless human bones and mechanical remains were stacked into a dense carpet, as if vaguely hearing the what pills make your penis bigger bloody roars of countless ancient wizards lingering in their ears. The blockbuster energizes the outer armor to blur, boom! The ground was bombarded with a large concussion wave alpha man extreme pill by the giant flesh and blood artillery.

At least, Simon knew that after the First Civilization War, the ancient mechanical wizards and dark wizards of the wizarding world gave birth to more advanced elemental vigrx plus for man 60 caps wizards, and surgical treatments for erectile dysfunction gradually replaced the dominant position of mechanical wizards. Amonro World? In the endless world, maybe it s just new viagra commercial 2015 a toddler, The mountain beetle under the throne of the ancestor of Amonro moved away, and suddenly, an extremely beautiful and calm thunder mirror appeared.

After a pause, Alowoz continued: If her father knew that this would be the result, maybe he New Viagra Commercial 2015 wouldn t be so persistent anymore, hey. In the eyes of these novice wizard apprentices, the imprint points are all their New Viagra Commercial 2015 harvard health sexual abuse definition own goals at this time, the black wizard, the seven-ring sacred tower, the witch hunter, etc, are too far away.

As early as the wizard academy, I stayed in the wizard, After 60 years of apprenticeship, plus the 30 aloe vera plant male enhancement years of the seven-ring sacred tower before, I am voodoo penis enlargement very well prepared for promotion to the official wizard. It s just that Simon s New.

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words have just been spoken, but they have naturally been infected with the gloomy hoarseness of the dark wizard, and even the joy in romans login it has been obscured by the cruel calm.

Among them, there are some mysterious wizards new viagra commercial 2015 who sell time, is ageless male safe to take conduct experiments and observations on behalf of witch hunters, and conduct experiments on ordering experimental equipment. However, considering Simon s identity as a dark wizard, he sex ii is a new viagra commercial 2015 first offender, He took the initiative to repent of his sins and new viagra commercial 2015 did not cause actual heavy losses to new viagra commercial 2015 the how to improve libido in females wizarding world order rules.

Simon nodded and said nothing, I have seen two wizards, thank you for your help On the ground, the already desperate apprentice wizard shouted excitedly in a most respectful manner. No, Simon rejected Millie s invitation, and then asked: I have to register for the hunt, how about you.

It won t be so coincidental! At this time, Simon knew what what can increase testosterone levels kind of viagra samples free pfizer wealth gnc penis pills it would be for the Wizarding World if he could discover a world crack. No wonder this is the case, Generally speaking, after the selected children enter the Wizarding Academy, even if they do not die by chance, it will take a hundred years for the selected children to return to their birthplace of ordinary human time.

Although such an elemental wizard cannot display the elemental decomposition ability, only has the elemental teleport ability, but it can give the self-evolution potential of its boron and testosterone own cells. According to simple experiments, the flame element below a thousand degrees can not pose any threat to this system at all.

Somewhat proud of his correct judgment, The magic power surged, viagra works and a group of black flames ignited with a boom. After establishing the coordinate line in the crystal ball, he commanded Fly to the nearest coordinate point with the spaceship.

Since you are a potential junior in the academy, and Master Peranos male enhancement vivax said that you have no intention of competing for the spot in the hunting record, then we are not in a competitive relationship. However, they have lost any resistance to capital, including, courage, Simon flew over the heads of these creatures, watching all this male enhancer underwear indifferently, without the slightest emotional fluctuations.

Goldfin fish steel libido reviews male extra enhancement pills is a strange creature in new viagra commercial 2015 the deep sea, This marine creature will jump out of the water after being exposed to danger, glide in the sky for a period of time to avoid kangaroo for men the new viagra commercial 2015 danger, and then return to the ocean again after the danger disappears. Viable path, At least, the new viagra commercial 2015 low-level new viagra commercial 2015 witch New Viagra Commercial 2015 hunting period is a viable path, It will save myself a lot of time to study advanced elemental knowledge, and it will also increase my life-saving ability during ed pills at walmart the vacuum period of elemental witchcraft strength Simon added.

Pry male enhancement gadgets the Endless World warning; the third is the seventy-ninth floor of the Black Tower. After a few hourglass flight time, a vague black shadow appeared over the sea, and Peranos turned and said: The Maglev Island has fallen, and the great Hesota Stigma wizard serexin male enhancement lives on it all year round.

The distant mountains and corpses, Xuehe River, and weird fat buildings quickly passed Simon s vision. Is this the real transition from an apprentice wizard to a formal wizard? No extensions 2 male enhancement side effects wonder Teacher Peranos said that all the apprentices who are newly promoted to official wizards are just a new viagra commercial 2015 group of apprentices with wizarding power in testosterone booster nutrition solutions a short period of time.

On the side, Dadalong Second Master Uncle Simon nodded silently, and said: In your case, I can t do anything. During New.

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Simon New Viagra Commercial 2015 s astonishing exchanges with Mina, he had new viagra commercial 2015 gradually stepped fast penis enlargement pills into the depths of the tree hole, and gradually, a fresh life essence of flowers and plants began to permeate.

Gently new viagra commercial 2015 unscrew the bottle cap, as the ready man pills review unique fragrance of Venus, the god of love, diffused from the bottle, Simon suddenly trembled! After enjoying it for nearly half an hour, Simon s closed eyes slowly opened, unbelievable. With Simon, generic name of viagra the four-meter-long mine elemental giant sword swept through the air, and this golden Amonro banged into a corpse.

Across the bustling chaotic square, Simon extenze fast acting extended release came to the small wizard whats the side effects of extenze tower in the center of the square. Nodding, Simon motioned to Guilinie and Victor to enter the room, As soon as new viagra commercial 2015 Guilinie and Victor entered the room, they saw a bright feathered starling with a red bow tie on his chest, holding a mallet, and slowly performing wizarding etiquette toward the two of them.

At this time, not to mention the encirclement and suppression new viagra commercial 2015 of the three-level mystery Amonro that broke in, but now it is one thing for a few people to say whether they can escape under the hands of a home remedy erectile dysfunction third-level Amonro and new viagra commercial 2015 a mystery prophet. For a moment, the vast space fortress fell into silence, and all the witch hunters held their breath and looked up, waiting for the stigmata wizard to come.

Now follow us, Take the trial in the hunting courtroom, Nodded, Simon stretched out both arms, Click. boom, The explosion sounded far away across the sea of gems, and large waves of waves swept across.

After a full hourglass time, after a simple test of the magic circle, Simon nodded in satisfaction under the face of truth, and disappeared from the hall in a flash. When the palm was extended, it was slightly new viagra commercial 2015 crimson, and there was a difference between the heat of the blood vessels in the palm and the red of the muscles.

He tore a small piece of the oil flower cake from Simon s plate and New Viagra Commercial 2015 ate it, and said as he ate, That guy Ungle, he s not advanced to the second level. Huh? Then what do you, Wow, quack, quack, Simon s words were only halfway through, and Mynah burst into laughter, Mynah said excitedly: This witchcraft is a great and unique witchcraft that I used to create with seven thousand years of sweat and wisdom.

boom! While Simon was thinking, suddenly, a wave can i buy extenze with hsa credit of energy erupted at the far entrance blue male supplement enhancer of the cave, and the super-oscillating sound wave swayed away. Then go to practice, Simon s passive dual evolution, During the wizard apprenticeship, Simon has already eliminated the steps of the physical experiment of new viagra commercial 2015 the mixed poison, which saves a lot of jelqing bad side effects time.

With extremely steady, deep, and calm eyes, Simon faintly smiled under the messy golden hair. Simon, this demon hunting expedition has undergone such a change, you can be safe, but the fellow Peranos has accepted another good disciple.

So many Amonro Shadow Legion with flying ability naturally shocked Simon, You must know that this is already the deep region of the power of shadows. Simon said lightly: Without my order, no one is allowed to approach this camphor tree tunnel.

Among them, it is mentioned that the soul will of creatures is New.

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divided into two types: potential will and self-will. After the initial differentiation and cultivation, Simon collected the activated flesh and blood in each jar and carefully cultivated the nutrient solution.

These wizards new viagra commercial 2015 are too powerful, evil, and terrifying, The most critical moment of the great Amonro world has arrived. Because for most demon hunting expeditions, two or even three demon New Viagra Commercial 2015 hunting New Viagra Commercial 2015 wizards are required to complete it, but now there is only one trace of the stigmata wizard.

And this spherical black blank area, with a diameter of a hundred meters, is actually a huge creature that is trying to pull the natural extenze vasodilaarot force around the body that is less than two meters in contrast to Simon sexual health dr near me s body. It will take at least ten years to return to the wizarding world, It cannot be wasted.

These were a group of half-meter-long small creatures, Except for the six leading ones with white sex hd mp4 skin, 72 hour male enhancement pill all the others were golden yellow. The young sapling said angrily: Young little witch hunter, don t you know that Eye of the World does not allow creatures above zebra maximum male enhancement the first level of the Wizarding World to approach.

With a bang, the magic beam of light broke through the clouds in the sky, and the rolling shadow clouds stirred and wandered, causing the New Viagra Commercial 2015 sky to reveal a testosterone seed booster woman red sun light, shining on the earth. It seems that the wizard completely controls the body and soul of the best memory enhancer supplements soul slave, but the new viagra commercial 2015 soul slave still has his own emotions.

However, because the number of blood evil moss is really small, Simon has only made a dozen blood evil bombs. Quack, little turtle, supplements for stamina in bed your eighth master is back, Myna picked up a fat fruit and threw it into the distance, shouting arrogantly: Go and pick it enhanced male pills up for Baye, hurry up.

It seems that it is due to the relationship best natural over counter male enhancement pills 2019 between the fat barrier layer, There is no communication between Amonro and Simon New Viagra Commercial 2015 Go in, Rumble! The moment the metal barrier of the Honma Fortress touched the Mystery Hurricane, there was where can you buy male enhancement products online a loud noise like metal friction, and after a shock, it rushed in without any hindrance.

A pair of icy eyes under the metal helmet, four long swords enzyte male enhancement free sample inserted in the new viagra commercial 2015 back, hundreds of dark red energy bars that resemble the life of strings, new viagra commercial 2015 wandering around, faintly revealing the armor and graceful body new viagra commercial 2015 inside. Guru, Guru, Bang! At this moment, the lava lake fluctuated violently, In the eyes of all the surviving wizards, a behemoth slowly rose from the bottom of the magma pool, and magma slowly flowed down from the flame-bearing karst rocks.

Sure enough, there are quite titanax male enhancement reviews a few backup methods in the original space fortress. For now, Simon intends to collect as many biological specimens as possible here, observe and speculate the theory of address modification, and then go male enhancement supplement pill manufacturers to the next destination.

But wisdom is attributed new viagra commercial 2015 to foresight, and only when you really experience it can you New Viagra Commercial 2015 experience the painful torment. The weakest new viagra commercial 2015 emotion in this kind of heart is what Peranos male enhancement cup mentioned, The wizard who has never experienced all of this is just a wizard apprentice with the power of a wizard.

From time to time, in the Ming Wizard Corps, there will be some compound witchcraft and magic circle assembly witchcraft composed of dozens of Ming wizards, and amazing energy alcohol consumption and men sexual health attacks are surging. Entering the void world male enhancement pills in stores from the chaos layer is like entering the shadow of the paper through the edge of the paper, and entering the void from the relative limit, the living being directly feels that it is on what can i take to increase my sex drive female the invisible space paper, new viagra commercial 2015 and the will penetrates The paper feels the shadow side of the paper.

There are two levels of difference in life levels, and the gap between them is almost incalculable. This is an empty area that has been completely erased by destruction energy, new viagra commercial 2015 the sexual health awareness month 2019 scars of the shadow mystery world, a ravine of tens of thousands of meters that replaces many peaks, and is printed on the ground.

gop health care plan and sexual assault health promotion sexual health I love you Simon was extremely serious and focused, looking straight into Lafite s eyes, as if to remember this moment in his mind forever.

Under the guidance of steroids sexdrive the Crystal Turtle, Simon and his party of four continued sex gel walmart to advance through the twisty and fat barrier passage. Puff, puff, While traveling, Simon saw the two low-level Amuros who had hidden their bodies because they found themselves.

In the future, even if this experimental body synthetic beast Simon is no longer needed, this unsynthesized natural elemental soul of the big blood waterfall lava dragon can still be the material for the next synthetic beast until it is artificially synthesized by Simon into a high-level penise pills elemental soul. After a pause, Zuo-eared bear continued: I have contacted a truly great wizard through the ancestor s token contract, and new viagra commercial 2015 will come here to sign male enhancement supplements uk a contract with me in the near future.

Spring comes and autumn goes, the four seasons reincarnate, Thirty years have passed. Even the Stigma Wizard could not ignore its existence, and had to stop, expelling these little new viagra commercial 2015 things to protect themselves.

The golden long tail and the milky white long tail once again pierced New Viagra Commercial 2015 the body of the elemental giant. After the body of Jiu Rong Mountain was shaken, it continued to walk forward with difficulty.

A pair of eyes that have gone through the vicissitudes of time and are no longer clear, looking at Simon, this feeling of security, this feeling of trust, this feeling of being cared for by another person, Rafi feels that his heart is about to break. Half a quarter of an hour later, Simon released his left hand grasping on the soul of the crystal turtle, and said lightly: Now, do you know.

There were few cases of neighboring each other, Now, a village with hundreds of families in close new viagra commercial 2015 proximity has completely replaced the empty ditches in memory.

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