Corporal Smooth Muscle Research Articles

RECONSTITUTION OF HUMAN CORPORAL SMOOTH MUSCLE AND ENDOTHELIAL CELLS IN VIVO

Purpose The availability of autologous erectile tissue composed of corporal smooth muscle and endothelial cells would be beneficial in patients undergoing penile reconstruction. We previously showed that cultured cavernous cells seeded on polymer scaffolds form corporal muscle when implanted in vivo. However, to reconstruct corporal tissue endothelial and corporal muscle cells are necessary. In this study we investigated the possibility of developing tissue composed of corporal cells in vivo by combining smooth muscle and endothelial cells. Materials and Methods Human corporal smooth muscle and endothelial cells were seeded on biodegradable polyglycolic acid polymer scaffolds at concentrations of 20 x 10 6 and 10 x 10 6 cells per cm. 3, respectively. A total of 60 polymer scaffolds seeded with cells and 20 control polymers without cells were implanted in the subcutaneous space of 20 athymic mice. Mice were sacrificed 1, 3, 5, 7, 14, 21, 28 and 42 days, respectively, after implantation. Immunocytochemical and histochemical analyses were performed with antifactor VIII, antipancytokeratins and anti-alpha actin antibodies. Results Histologically the retrieved polymers seeded with corporal smooth muscle and endothelial cells showed the formation of multilayered smooth muscle strips adjacent to endothelial cells 7 days after implantation. Increased organization of the smooth muscle tissue and accumulation of endothelium lining the luminal structures were evident by 14 days. A well organized tissue construct was noted 28 and 42 days after implantation. There was no evidence of tissue formation in controls. Immunocytochemical analysis using antifactor VIII to identify native vasculature only and antipancytokeratins to identify ECV 304 endothelial cells only distinguished the origin of the vascular structures in each construct. Anti-alpha-actin confirmed the smooth muscle phenotype. Conclusions Human corporal smooth muscle and endothelial cells seeded on biodegradable polymer scaffolds formed vascularized corpus cavernosum muscle when implanted in vivo. To our knowledge this is the first demonstration in tissue engineering in which capillary formation was facilitated by the addition of endothelial cells in composite tissue in vivo.

TRANSFORMING GROWTH FACTOR-beta 1 (TGF-beta 1) IS SUFFICIENT TO INDUCE FIBROSIS OF RABBIT CORPUS CAVERNOSUM IN VIVO

Purpose The pleotropic cytokine TGF-beta 1 which induces connective tissue synthesis, and inhibits the growth of smooth muscle cells, has been implicated in corpus cavernosum fibrosis. The objective of this study was to determine the dose and time dependence of TGF-beta 1 as an active agent in penile corporal fibrosis in an animal model. Materials and Methods A time release method of delivery was developed using sodium alginate microspheres containing recombinant human (rh) TGF-beta ( 1). New Zealand White rabbits were injected intracorporally with a single alginate microsphere either with or without rh-TGF-beta 1. Dosage was varied from 325 to 1500 ng./bead. Animals were sacrificed at either three or five days post injection and the penises removed en bloc, examined, and processed for quantitative histomorphometric analysis, staining the sections with Masson's trichrome. Results Alginate microspheres containing [( 125) I]-rh-TGF-beta 1 showed slow-release kinetics (t 1/2 = 10.5 hours). Histomorphometric analysis of 60 sets of high powered fields/treatment/animal showed dose dependent decreases in percentage of corporal smooth muscle with TGF-beta 1 treatment (750 to 1500 ng./bead). Placebo (alginate microspheres alone) had trabecular smooth muscle content comparable to values previously reported for untreated rabbit corpus cavernosum. Conclusions This study confirms that TGF-beta 1 induces fibrosis in situ by altering connective tissue synthesis and hence the structure of the corpus cavernosum. Injection of rh-TGF-beta 1 impregnated alginate microspheres into the corpus cavernosum resulted in dose-dependent decreases in percentage of corporal smooth muscle.

Enhanced Contractility of Rabbit Corpus Cavernosum Smooth Muscle by Oxidized Low Density Lipoproteins

We have investigated the effect of oxidatively-modified low density lipoproteins (ox-LDL) on the contractility of rabbit trabecular smooth muscle. Low density lipoproteins (LDL) were isolated from fresh human plasma pooled from multiple donors and oxidized by exposure to copper. Corpus cavernosum strips from New Zealand White rabbits were studied in organ chambers for isometric tension measurement. Corporeal strips in which moderate tone was induced by phenylephrine, contracted when exposed to ox-LDL, but not when exposed to either native LDL (nLDL) or LDL protected from oxidation by butylated hydroxytoleune (BHT-LDL). Removal of the endothelium, or treatment of the corporeal strips with N(omega)-nitro-L-arginine (nitric oxide synthase inhibitor), methylene blue of LY83583 (guanylate cyclase inhibitors/superoxide producing agents), did not prevent ox-LDL-induced contraction. ox-LDL, dose-dependently, enhanced the contractile response of corporeal strips to low and moderate concentrations by phenylephrine. nLDL had no significant effect on phenylephrine-induced contraction of corporeal strips. ox-LDL, nLDL or BHT-LDL had no effect on relaxation induced by the endothelium-dependent dilator, acetylcholine, or the nitric oxide donor, nitroprusside. In conclusion, this present study demonstrates significant pro-contractile effects of ox-LDL on corporeal smooth muscle, this effect is independent of the endothelium or the nitric oxide/cGMP pathway. The pro-contractile effect of ox-LDL may interfere with penile smooth muscle relaxation, necessary for the initiation and maintenance of penile erection.

Prostaglandin E1 activates the large-conductance KCa channel in human corporal smooth muscle cells.

The large conductance calcium-sensitive potassium channel (KCa or maxi-K) is an important modulator of human corporal smooth muscle tone, and therefore, erectile capacity. The goal of this investigation was to evaluate the actions of prostaglandin E1 (PGE1), the most widely used and effective drug for the treatment of impotence, on the activity of the KCa channel, a prominent K+ current present in human corporal smooth muscle. Whole-cell patch clamp studies conducted on short-term cultured and enzymatically dissociated human corporal smooth muscle cells, revealed mean resting potentials of -50.8 +/- 2.1 mV (n = 8) and -34 +/- 4 mV (n = 8), respectively. In the attached-patch configuration, the corresponding single-channel slope conductance values for the KCa channel subtype were 173 +/- 4 pS (n = 8) in cultured cells, and 190 +/- 13 pS (n = 3) in freshly isolated myocytes. Furthermore, voltage clamp experiments revealed that relative to control values, the application of PGE1 to cultured cells (3.3 or 33 microM) elicited an apparent increase in both the open probability (Po; ranging from 1.2-23 fold), and the mean open time (5-6 fold) of the KCa channel at membrane potentials of +90 mV and +110 mV. PGE1-induced alterations in KCa channel activity were also observed in freshly isolated corporal myocytes. In the whole cell-recording mode, statistically significant, Charybdotoxin-sensitive (100 nM) 2-3 fold increases in the outward K+ currents were observed in both cultured and freshly isolated corporal myocytes. The presence of a PKA inhibitor (fragment 6-22 amide; 10 microM) in the pipette tip was also associated with a nearly complete ablation of the observed PGE1-induced whole cell K+ currents. Taken together, these data confirm and extend our previous observations, and indicate that PGE1-induced relaxation of human corporal smooth muscle is related, at least in part, to activation of the KCa channel subtype resulting in cellular hyperpolarization.

Characterization of ATP-sensitive potassium channels in human corporal smooth muscle cells.

Potassium (K) channels play a significant role in modulating human corporal smooth muscle tone, and thus, erectile capacity. Recent pharmacological studies indicate that the metabolically-regulated K channel (KATP) may be an important modulator of human penile erection with significant therapeutic potential. The goal of these initial studies, therefore, was to utilize patch clamp techniques to characterize the putative KATP subtype(s) present in cultured and freshly isolated human corporal smooth muscle cells. In the cell-attached patch mode, two distinct unitary K+ currents were identified whose respective conductance values were similar in cultured and freshly isolated smooth muscle cells. In cultured myocytes, the measured conductance values in symmetric KCl (140 mM) solutions were 59.1 +/- 2.7 pS and 18.4 +/- 2.1 pS (n = 5 cells). Under identical experimental conditions in freshly isolated myocytes, corresponding conductance values were 59.2 +/- 3.7 pS and 18.5 +/- 2.4 pS, respectively (n = 4 cells). I-V curves constructed during step depolarization (-60 to +80 mV), revealed a linear I-V relationship for both unitary conductances. Single channel records documented that both conductances were reversibly inhibited by the application of ATP (1-3 mM) to the bath solution in the inside-out attached patch configuration. The unitary activity of both K channel subtypes was significantly increased by the application of pinacidil (10 microM) and levcromakalim (10 microM). Whole cell patch recordings documented a glibenclamide-sensitive, pinacidil- and levcromakalim-induced increase in the whole cell outward K+ current during step depolarization (-70 mV to +130 mV) of 105 +/- 37%, 139 +/- 42%, respectively. These data confirm and extend our previous observations, and provide the first evidence for the presence of KATP channel subtypes in human corporal smooth muscle cells.

5-HT4 receptors in isolated human corpus cavernosum?

The novel serotonin subtype-4 (5-HT4) receptor agonist, SC53116 (SC), produced a limited relaxation of noradrenaline (NA) pre-contracted human corpus cavernosum (CC) smooth muscle in vitro. This effect was not significantly attenuated by the 5-HT4 antagonist SDZ250557 (SDZ). In the presence of (+/-) pindolol (1 microM) and methysergide (1 microM), employed to mask 5-HT1 and beta-adrenergic, and 5-HT2 receptors respectively, SC failed to relax NA pre-contracted CC strips to a greater extent than saline. Functional cAMP dependent relaxation pathways were demonstrated by a significant reduction in NA induced tone by prostaglandin E1 (PGE1) and isopropylnoradrenaline (IPNA), the action of the latter compound was effectively eliminated in the presence of (+/-) pindolol. Relaxation of NA induced tone caused by the nitric oxide donor nitro-glycerine (NTG) was significant and similar in the absence and presence of the 5-HT and beta-adrenergic antagonists. The results of this present study indicate that human corporal smooth muscle does not contain 5-HT4 receptors and that, although compounds like SC act to relax non-vascular smooth muscle via cAMP dependent mechanisms, 5-HT4 receptor agonists may be expected to be of limited utility in triggering cAMP dependent relaxation responses in human CC.

Correlation of calcium-activated ATPase activity, lipid peroxidation, and the contractile response of rabbit corporal smooth muscle treated with in vitro ischemia

Oxygen and glucose are critical to support the survival and integrity of all smooth muscles. Hypoxia alone has been demonstrated to suppress the contractile response of corporal smooth muscle, and one might expect simultaneous deprivation of oxygen and glucose (in vitro model of ischemia) to exert more serious damage to corporal smooth muscle contraction. The effect of in vitro ischemia on the pharmacological responses of isolated rabbit corporal smooth muscle was correlated with the level of tissue lipid peroxidation. The effects of in vitro ischemia were as follows: (1) In vitro ischemia resulted in an 85% reduction in the contractile response to phenylephrine; (2) more than a 50% reduction in the activity of thapsigargin-sensitive calcium-activated ATPase activity of the microsomes (sarcoplasmic reticulum [SR]); (3) more than a fourfold increase in the tissue concentration of thiobarbituric acid reactive substances (TBARS) (level of lipid peroxidation). In conclusion, stimulation of lipid peroxidation in part may be responsible for the decrease in thapsigargin-sensitive calcium-activated ATPase activity of the SR (SERCA), and the correlated decrease in the contractile response to phenylephrine in response to ischemia.

Enhanced contractility of rabbit corpus cavernosum smooth muscle by oxidized low density lipoproteins.

We have investigated the effect of oxidatively-modified low density lipoproteins (ox-LDL) on the contractility of rabbit trabecular smooth muscle. Low density lipoproteins (LDL) were isolated from fresh human plasma pooled from multiple donors and oxidized by exposure to copper. Corpus cavernosum strips from New Zealand White rabbits were studied in organ chambers for isometric tension measurement. Corporeal strips in which moderate tone was induced by phenylephrine, contracted when exposed to ox-LDL, but not when exposed to either native LDL (nLDL) or LDL protected from oxidation by butylated hydroxytoleune (BHT-LDL). Removal of the endothelium, or treatment of the corporeal strips with N(omega)-nitro-L-arginine (nitric oxide synthase inhibitor), methylene blue of LY83583 (guanylate cyclase inhibitors/superoxide producing agents), did not prevent ox-LDL-induced contraction. ox-LDL, dose-dependently, enhanced the contractile response of corporeal strips to low and moderate concentrations by phenylephrine. nLDL had no significant effect on phenylephrine-induced contraction of corporeal strips. ox-LDL, nLDL or BHT-LDL had no effect on relaxation induced by the endothelium-dependent dilator, acetylcholine, or the nitric oxide donor, nitroprusside. In conclusion, this present study demonstrates significant pro-contractile effects of ox-LDL on corporeal smooth muscle, this effect is independent of the endothelium or the nitric oxide/cGMP pathway. The pro-contractile effect of ox-LDL may interfere with penile smooth muscle relaxation, necessary for the initiation and maintenance of penile erection.

THE EPIDEMIOLOGY AND PATHOPHYSIOLOGY OF ERECTILE DYSFUNCTION

Published studies on the epidemiology of erectile dysfunction and the physiology/ pathophysiology of erectile function are reviewed. A literature search of more than 400 studies of the epidemiology and pathophysiology of impotence and erectile dysfunction published during the last 3 decades was conducted and the most pertinent articles are discussed. It has been estimated that the prevalence of erectile dysfunction of all degrees is 52% in men 40 to 70 years old, with higher rates in those older than 70 years. Erectile dysfunction has a significant negative impact on quality of life. Risk factors for erectile dysfunction include aging, chronic illnesses, various medications and cigarette smoking. A nitric oxide/cyclic guanosine monophosphate mechanism has an important role in mediating the corporal smooth muscle relaxation necessary for erectile function. Other mechanisms involving neuropeptides, gap junctions and ion channels also may modulate corporal smooth muscle tone. Erectile dysfunction can be due to vasculogenic, neurogenic, hormonal and/or psychogenic factors as well as alterations in the nitric oxide/cyclic guanosine monophosphate pathway or other regulatory mechanisms, resulting in an imbalance in corporal smooth muscle contraction and relaxation. Erectile dysfunction is a common condition associated with aging, chronic illnesses and various modifiable risk factors. Normal penile erection is a hemodynamic process that is dependent on corporal smooth muscle relaxation mediated by parasympathetic neurotransmission, nitric oxide, and possibly other regulatory factors and electrophysiological events. As more knowledge is gained of the physiology and regulatory factors that mediate normal erectile function, the mechanisms involved in the pathophysiology of erectile dysfunction should be further elucidated.

Sildenafil: a new oral therapy for erectile dysfunction.

Sildenafil citrate (Viagra) is a relatively selective 5-phosphodiesterase (PDE) inhibitor. It is the first oral medication approved for the treatment of erectile dysfunction (ED). The neuronal release of NO which binds to the heme-containing region of guanylate cyclase increases levels of cGMP. This leads to a cascade of reaction which results in corporal smooth muscle relaxation and penile erection. Sildenafil causes an erection by inhibiting PDE5, which in turn causes an increase in the intracellular levels of cGMP. Sildenafil is well absorbed after a single oral administration with a t(1/2) of approximately 4 h. The mode of onset varies from 0.5-4 h. The drug has been used in millions of men since first approved by the U.S. FDA 1 year ago and has revolutionized the approach to, and therapy of, erectile dysfunction.

ENDOTHELIN-1 AS A PUTATIVE MODULATOR OF GENE EXPRESSION AND CELLULAR PHYSIOLOGY IN CULTURED HUMAN CORPORAL SMOOTH MUSCLE CELLS

Purpose Increases in cytosolic calcium levels trigger smooth muscle contraction while nuclear calcium increases are thought to regulate gene expression. Endothelin-1 (ET-1) affects both. The goal of these studies was to further investigate the importance of ET-1 to corporal physiology by examining its actions on proliferation and immediate early gene (IEG) expression in cultured human corporal smooth muscle cells. Materials & Methods Early passage (1-3) smooth muscle cells were grown in culture and exposed to either phenylephrine (PE) or ET-1 in the absence and presence of serum, the ET A or ET B selective antagonist BQ123 or IRL1038, or the L-type Ca 2+ channel blocker, verapamil. Cell proliferation was assessed with a hemocytometer. The effects of ET-1 on c-myc and c-fos were evaluated using Northern blot analysis. Parametric or nonparametric statistics were used as appropriate. Results Addition of ET-1 (100 nM) to serum-starved cultured corporal smooth muscle cells was associated with a nearly 2-fold increase in cell number, as well as 2 to 6-fold increases in c-myc and c-fos levels. Cellular proliferation was inhibited by ET A- or ET B-receptor subtype blockade with BQ123 (1 micro M) or IRL1038 (1 micro M), respectively, or blockade of Ca 2+ channels with verapamil (10 micro M). PE (3 micro M) had no detectable effect on smooth muscle proliferation. Conclusions Cell proliferation was mediated by activation of the ET A and ET B receptor subtypes, dependent on transmembrane Ca 2+ flux, and correlated with significant increases in c-myc and c-fos mRNA levels. These studies extend previous observations to indicate the potential pleotropic actions of this peptide in the regulation of human corporal smooth muscle physiology in vivo.

The relaxation effects of ginseng saponin in rabbit corporal smooth muscle: is it a nitric oxide donor?

To determine whether crude extracts of ginseng saponin (GCS), containing the active ingredients from Panax ginseng and used as an aphrodisiac in oriental countries, relax corpus cavernosal smooth muscle in the rabbit. Corpus cavernosal strips were prepared from rabbit penises. Isometric tension changes, recorded with a pressure transducer, in response to various drugs and electrical stimulation were assessed in an organ chamber, after active muscle tone had been induced by 10 micromol/L phenylephrine. GCS (0.2-8.0 mg) relaxed the smooth muscle of rabbit corpus cavernosum (SMRCC) pre-contracted with phenylephrine in a dose-dependent manner. GCS at 0.75 mg significantly enhanced the relaxation of SMRCC induced by electrical field stimulation. The relaxation induced by 0.2-8.0 mg GCS was significantly attenuated by atropine (1 micromol/L), methylene blue (100 micromol/L) and N-omega-nitro-L-arginine methyl ester (L-NAME, 10 micromol/L). However, there was no significant difference in the attenuation of GCS-induced relaxation of SMRCC by adding vasoactive intestinal peptide antagonists or indomethacin. In addition, the decreasing rate of GCS-induced relaxation of SMRCC by methylene blue and L-NAME was greater than that by atropine. L-arginine (10 mmol/L) reversed the inhibitory effect induced by L-NAME (1 mmol/L) on the attenuation of GCS-induced relaxation. These data suggest that GCS, as a nitric oxide donor, induces the relaxation of SMRCC through the L-arginine/nitric oxide pathway. For the clinical application of ginseng saponin, further studies are required to clarify the active subfraction(s) of GCS.

Electrical activity from rat corpora is affected by pharmacological but not mechanical intracorporal manipulation.

Smooth muscle electromyographic (EMG) recording from the corpora cavernosa is a potentially useful clinical tool for the diagnosis of impotence. Controversy still exists regarding the nature of this signal, which could be resolved using animal models. The signal recorded from denuded rat corporal surface was Fourier transformed, and power at 0.01-1 Hz was calculated. Measurements were taken in the flaccid state and during erection induced by intracorporal injection of smooth muscle relaxants [the phosphodiesterase inhibitors papaverin and isobutylmethylxanthine (IBMX)]. Recorded activity decreased significantly during the pharmacologically evoked erection: the mean square root power of 250.7+/-137.1 microV decreased to 49.8+/-14.1 microV (P<0.0001) after papaverin, and IBMX caused it to decrease from 222.8+/-132.0 microV to 58.1+/-40.8 microV (P<0.0001) (mean +/- SD). Intracorporal injection of adrenaline partially reversed the response, increasing activity to 135.3+/-72.3 microV (P=0.01) and 137.5+/-122.6 microV (P=0.015), respectively, in parallel with detumescence. In order to rule out a mechanical artefactual explanation for these results, recording was also performed before and during artificial erection induced by the intracorporal infusion of saline. This manipulation did not cause any change in corporal activity, despite corporal engorgement and increased pressure. In conclusion, the signal indeed reflects changes in the tone of corporal smooth muscle, and therefore should be regarded as a genuine EMG recording.

The Pharmacologic Management of Erectile Dysfunction: Sildenafil Citrate (Viagra)

Sildenafil citrate (Viagra) is the first effective, orally administered agent for the management of erectile dysfunction (ED). It is a potent and selective inhibitor of phosphodiesterase type 5 (PDE5), the predominant isoform found in human corporal smooth muscle. Sexual stimulation releases, in the penis, the neurotransmitter nitric oxide, which in turn increases cyclic GMP (cGMP) within the smooth muscle cell. Cyclic GMP produces penile smooth muscle relaxation, which increases blood inflow and entrapment within the penis. The inhibition of PDE5 by sildenafil increases the duration and activity of cGMP and thus amplifies the effect of sexual stimulation. Sildenafil has demonstrated significant efficacy (57-83%) regardless of the etiology (organic, psychogenic, mixed), age, or baseline severity of the ED. The adverse events related to sildenafil include headaches, flushing, dyspepsia, and nasal stuffiness. In over 50 million patient administrations in the field since FDA approval, sildenafil has maintain...

Molecular studies of human connexin 43 (Cx43) expression in isolated corporal tissue strips and cultured corporal smooth muscle cells

Intercellular communication plays an important role in erectile function. The goal of this study, therefore, was two-fold. Firstly, to determine if cultured corporal smooth muscle cells provide a valid model system for evaluating the role of junctional communication to erectile physiology, and secondly, to explore the possibility that there may be age-related alterations in Cx43 mRNA expression. Human corpus cavernosum tissue was obtained from 31 patients with a mean age of 58 (range 27-89), while cell cultures were developed from 21 distinct patients with a mean age of 57 (range 26-59). Northern blots revealed that mRNA for Cx43 was expressed at detectable levels in all samples examined. It migrated as a transcript with an apparent size of 3.1 Kb. Western blots revealed the presence of multiple bands of Cx43 protein in both tissues and cells. However, Cx43 protein in tissue predominantly migrated as a 45 kDa band, while the Cx43 from cultured cells predominantly migrated as 41 kDa band. Cx43 mRNA expression was similarly heterogeneous in both frozen tissues and cultured cells. An approximately 3-5-fold increase in Cx43 mRNA levels was observed in cultured cells relative to frozen tissue, but the expression of Cx43 mRNA was not further altered upon passaging (p1-5). When Cx43 mRNA levels were normalized, and expressed as a ratio of the Cx43/beta-tubulin mRNA, there was a significant negative correlation between patient age and Cx43 levels on frozen tissues, but not on cultured cells. We conclude that: (1) There is similar heterogeneity/variability in Cx43 mRNA levels in frozen tissues and cultured cells derived from human corpus cavernosum. (2) That the expression of Cx43 mRNA in cultured cells is sufficiently stable, and similar to, expression levels in tissue as to provide a valid and physiologically relevant model system for further studying the role(s) of Cx43 in the regulation of penile erection. (3) There is a statistically significant, albeit modest, negative correlation between the Cx43/beta-tubulin ratio and patient age in frozen corporal tissue strips, but not on cultured corporal smooth muscle cells. Such observations provide further evidence for the plasticity of intercellular communication in the erectile process. Moreover, the similarities in the apparent regulation of Cx43 mRNA levels and that of the putative 'housekeeping' gene beta-tubulin, may suggest that Cx43 is constitutively synthesized in this tissue.

The Control of Corporal Smooth Muscle Tone, the Coordination of Penile Erection, and the Etiology of Erectile Dysfunction: The Devil is in the Details

Penile erection is a complex neurovascular event with hemodynamic features analogous to those observed in vascular tissues throughout the vascular tree. A spate of recent basic research activity into the mechanistic basis for the control of penile erection and the etiology of erectile dysfunction has highlighted the central role of the corporal smooth muscle cell in this process. It is now clear that penile erection is highly dependent not only on the integrity of the corporal smooth muscle cells that comprise the vast majority of the corporal parenchyma, but also on their ability to respond to stimuli for both contraction and relaxation in a coordinated and syncytial fashion. As such, further elaboration of the precise mechanistic details concerning the physiology and pathophysiology of the control of corporal smooth muscle tone represents the greatest remaining barrier to the improved understanding, diagnosis, and treatment of erectile dysfunction. It is anticipated that multidisciplinary basic research...

Cyclic AMP modulates TGF-beta 1-induced fibrillar collagen synthesis in cultured human corpus cavernosum smooth muscle cells.

The mechanisms by which PGE1 suppresses transforming growth factor beta 1 (TGF-beta 1) induced fibrillar collagen synthesis in human corpus cavernosum smooth muscle cells (HCC SMC) remain undefined. Since PGE1 induces cyclic AMP (cAMP) synthesis in HCC SMC, the aim of this research is to investigate the role of cAMP in the regulation of connective tissue biosynthesis in human corporal smooth muscle cells in culture. HCC SMC were incubated for 24 h in media containing [3H] proline with and without TGF-beta 1 in the presence or absence of agents which modulate cAMP or cGMP synthesis, or hydrolysis. Fibrillar collagen synthesis was determined by [3H]-proline incorporation into pepsin-resistant, trichloroacetic acid and precipitable protein. TGF-beta 1-induced collagen synthesis in HCC SMC was inhibited by receptor-mediated (PGE1) and non-receptor-mediated (forskolin) increases in cAMP synthesis, as well as the cell-permeable analog, dibutyryl cAMP. Sodium nitroprusside, a nitric oxide donor, and the cell-permeable analog, dibutyryl cGMP had no effect on TGF-beta 1-induced collagen synthesis. Cyclic AMP synthesis in HCC SMC inhibits TGF-beta 1-induced collagen synthesis. Agents which increase cAMP through specific G-protein-coupled receptors, direct stimulation of adenylate cyclase, or inhibition of phosphodiesterase activity could have beneficial effects as modulators of corpus cavernosum connective tissue biosynthesis.

Intracorporal injection of hSlo cDNA in rats produces physiologically relevant alterations in penile function.

The Ca2+-sensitive K+ channel (maxi-K+) is an important modulator of corporal smooth muscle tone. The goal of these studies was twofold: 1) to determine the feasibility of transfecting corporal smooth muscle cells in vivo with the hSlo cDNA, which encodes for the human smooth muscle maxi-K+ channel, and 2) to determine whether transfection of the maxi-K+ channel would affect the physiological response to cavernous nerve stimulation in a rat model in vivo. Intracorporal microinjection of pCMVbeta/Lac Z DNA in 10-wk-old rats resulted in significant incorporation and expression of beta-galactosidase activity in 10 of 12 injected animals for up to 75 days postinjection. Moreover, electrical stimulation of the cavernous nerve revealed that, relative to the responses obtained in age-matched control animals (N = 12), intracavernous injection of naked pcDNA/hSlo DNA was associated with a statistically significant elevation in the mean amplitude of the intracavernous pressure response at all levels of current stimulation (range 0.5-10 mA) at both 1 mo (N = 5) and 2 mo (N = 8) postinjection. Furthermore, qualitatively similar observations were made at 3 mo (N = 2) and 4 mo (N = 2) postinjection. These data indicate that naked hSlo DNA is quite easily incorporated into corporal smooth muscle and, furthermore, that expression is sustained for at least 2 mo in corporal smooth muscle cells in vivo. Finally, after expression, hSlo is capable of measurably altering nerve-stimulated penile erection. Taken together, these data provide compelling evidence for the potential utility of gene therapy in the treatment of erectile dysfunction.

A POSSIBLE MECHANISM FOR ALTERATION OF HUMAN ERECTILE FUNCTION BY DIGOXIN: INHIBITION OF CORPUS CAVERNOSUM SODIUM/POTASSIUM ADENOSINE TRIPHOSPHATASE ACTIVITY

Purpose Digoxin use has long been recognized to affect adversely male sexual function but the underlying mechanism is poorly understood. Digoxin is a known inhibitor of sodium/potassium adenosine triphosphatase (sodium pump), a plasma membrane enzyme that has a role in the regulation of smooth muscle tone. We investigated the effects of digoxin on human corpus cavernosum smooth muscle contractility and overall erectile function. Materials and Methods In human corporeal smooth muscle strips the in vitro effects of digoxin were assessed on sodium pump activity as measured by digoxin inhibitable uptake of 86 rubidium, basal tone and endothelium dependent, neurogenic and nitric oxide donor induced relaxation. An in vivo prospective double-blind, placebo controlled, crossover, 4-period investigation was performed in 6 healthy male volunteers. The effects of digoxin on serum hormones, erectile function questionnaire, visual sexual stimulation and nocturnal penile tumescence were recorded. Results In vitro digoxin caused concentration dependent inhibition of ( 86) rubidium uptake (half maximum effect at 0.01 micro M.) and contraction of corporeal smooth muscle (half maximum effect at 0.8 micro M.). Therapeutic concentrations of digoxin (2 nM.) also inhibited relaxation induced by acetylcholine and electrical field stimulation, which release nitric oxide from corpus cavernosum endothelial cells and nonadrenergic noncholinergic nerves, respectively. In vivo digoxin diminished penile rigidity during visual sexual stimulation and nocturnal penile tumescence testing compared to placebo without influencing libido or serum testosterone, estrogen or luteinizing hormone levels. Conclusions Digoxin associated alteration of human erectile function may be explained, in part, by inhibition of corporeal smooth muscle sodium pump activity, which promotes contraction and impedes nitric oxide induced relaxation. Such findings suggest therapeutic use of digoxin for treatment of recurrent priapism states.

Digital Analysis of the Pharmacological Effects of in vitro Ischemia on Rabbit Corpus cavernosum

Purpose: Regulation of corporal smooth muscle tone is essential for the initiation of penile erection. In recent years, in vitro isometric tension studies using isolated corpus cavernosal tissue have been used extensively to investigate the mechanisms regulating corporal smooth muscle tone and tension. In the present study, we utilized digital analysis of contractile data generated from investigation of contractile and relaxation responses of isolated rabbit corpus cavenosum to various forms of stimulation. Digital analysis of the contractile and relaxation data allows quantitation of both maximal and mean rates of tension change, and time elapsed to maximal response. Rates of tension changes may provide additional important information regarding cellular events that mediate corporal tone and tension changes. Methods: Sexually mature male New Zealand White rabbits were used. Each corpus cavernosum was dissected sharply from the removed penis, then two longitudinal strips were prepared for isometric tension studies and placed in individual baths. Tension was monitored continually using an 8-channel Grass Polygraph. The Grass PolyVIEW system simultaneously converted analog signals to digital information and stored data using a 486 PC computer. Each corporal strip was prestimulated with 300 µmol/l phenylephrine to produce a maximal contraction, then field stimulation (FS), carbachol and nitroprusside were applied consecutively to determine relaxation effects. This procedure was repeated after strips were deprived of glucose and oxygen (in vitro ischemia) for 1 h. The following parameters were quantitated for all responses: maximal tension change; maximal and mean rates of tension change, and time to maximal response. Results: Effects of 1-hour in vitro ischemia on rabbit corporal tissue were as follows: (1) an 85% decrease in contractile response to phenylephrine, and (2) a marked increase in rate of contractile response to phenylephrine. Phenylephrine precontracted strips exhibited: (3) no change in relaxant response to FS; (4) increased relaxant responses to carbachol and nitroprusside; (5) no change in rates of relaxation in response to FS; (6) increased rates of relaxation in response to carbachol and nitroprusside, (7) no change in elapsed time to maximal relaxation in response to FS, and (8) decreased elapsed time to maximal relaxation in response to carbachol and nitroprusside. Conclusion: Digital analysis of the data generated facilitates recording, reviewing and analyzing of in vitro isometric tension studies on rabbit corpus cavernosum. Digital analysis allows quantification of additional parameters that have important implications for determination of mechanisms by which specific pathological processes occur.