Angiotensin II increases corpus cavernosal contractility and oxidative stress in partial bladder outlet obstructed rabbits: relevance to erectile dysfunction.

INTRODUCTION: We investigated the effect angiotensin II (Ang II), a corpus cavernosal smooth muscle (CCSM) constrictor peptide, has on tissue taken from rabbits following chronic partial bladder outlet obstruction (PBOO), as this model is characterized by an increase in corpus cavernosal collagen deposition and a marked reduction and impaired relaxation of CCSM cells. AIM: To determine the interaction between Ang II and nitric oxide (NO) and the development of oxidative stress (OS) in a rabbit model of chronic PBOO. METHODS: Corpus cavernosal tissue was obtained from 12 sham-operated and 20 PBOO rabbits. Organ bath studies determined Ang II/NO interaction on CCSM function using losartan (AT1 receptor antagonist), sodium nitroprusside (SNP, NO donor), electrical field stimulation (EFS), and vardenafil (phosphodiesterase type 5 inhibitor). The role of OS in the Ang II response was also determined using diphenylene iodonium chloride (DPI), the nicotinamide adenine dinucleotide phosphate oxidase inhibitor, which inhibits superoxide production and superoxide dismutase (SOD, the enzyme that accelerates the breakdown of superoxide). MAIN OUTCOME MEASURE: Action of Ang II and AT1 receptor antagonist, as well as SOD and DPI on CCSM function. RESULTS: Ang II caused a dose-dependent contraction of CCSM strips that was enhanced in PBOO rabbits and inhibited by losartan, DPI, and SOD. CCSM relaxation induced by SNP/EFS was impaired in this model and improved by vardenafil and losartan. CONCLUSIONS: These findings imply that the increased Ang II contractile response is a pathological consequence of PBOO and that AT1 receptor inhibition may be a therapeutic approach to treat ED associated with PBOO.

Effect of angiotensin II and its receptor antagonists on human corpus cavernous contractility and oxidative stress: modulation of nitric oxide mediated relaxation.

PURPOSE: To our knowledge the interaction between angiotensin II and nitric oxide in the control of human corpus cavernous function has not been assessed previously. We determined the presence and role of angiotensin II and its receptors in human penile function. MATERIALS AND METHODS: Corpus cavernous tissue was obtained from 35 patients undergoing gender reassignment surgery. Immunohistochemical analysis was done to determine angiotensin II peptide tissue distribution. Organ bath studies were done to determine the angiotensin II/nitric oxide interaction on corpus cavernous smooth muscle function. The role of oxidative stress in the angiotensin II response was also examined using the nicotinamide adenine dinucleotide phosphate oxidase inhibitor apocynin. RESULTS: Angiotensin II was distributed in arteriolar endothelium, endothelium lining sinusoids and smooth muscle cells, and caused dose dependent contraction of human corpus cavernous smooth muscle strips that was inhibited by the angiotensin type 1 receptor antagonist losartan. Relaxation of corpus cavernous smooth muscle induced by the nitric oxide donor sodium nitroprusside or electrical field stimulation was potentiated by losartan. Apocynin decreased angiotensin II induced corpus cavernous contraction. CONCLUSIONS: Angiotensin II and nitric oxide interact to modulate human cavernous function since losartan potentiated sodium nitroprusside and electrical field stimulation mediated corpus cavernous smooth muscle relaxation. The angiotensin II response involves the production of superoxide and the development of oxidative stress. These findings support the role of angiotensin II in the regulation of human penile smooth muscle tone and suggest that angiotensin type 1 receptor inhibition may be a therapeutic approach to erectile dysfunction.

A functional study of purinergic signalling in the normal and pathological rabbit corpus cavernosum.

OBJECTIVE: To examine rabbit cavernosal smooth muscle (CSM) relaxation to ATP, ADP and UTP in normal rabbits and in models of conditions that predispose to erectile dysfunction (ED), diabetes mellitus (DM; induced for 6 months) and bladder outlet obstruction (BOO, 6 weeks after surgery). MATERIALS AND METHODS: Concentration-response curves (CRCs) were constructed to ATP, ADP and UTP on CSM from control rabbits in the absence and presence of antagonists. In addition, CRCs were constructed to ATP in CSM from rabbits with DM and BOO. RESULTS: ATP and UTP caused equipotent, dose-dependent relaxations of pre-contracted normal rabbit CSM; ADP was more potent. Relaxation was inhibited by Reactive Blue 2, but not by suramin, 8-p-sulfophenyltheophylline or L-N(G)-nitroarginine methyl ester. In rabbits with DM and those with partial BOO, ATP-mediated CSM relaxation was less than in control rabbits. Pharmacological profiling suggests that purine-induced CSM relaxation might be mediated by P2Y(1) and P2Y(4) receptors in the rabbit. CONCLUSIONS: In healthy rabbits, ATP released from nerves appears to produce relaxation of CSM via P2Y(4) receptors on smooth muscle, while ADP, acting on P2Y(1) receptors on endothelial cells, produces relaxation via nitric oxide. Alterations in CSM purinergic signalling might be implicated in the pathophysiology of ED associated with DM and BOO. Characterization of purinergic signalling in CSM might highlight new therapeutic targets for treating ED.

Gene therapy and erectile dysfunction: the current status.

Current available treatment options for erectile dysfunction (ED) are effective but not without failure and/or side effects. Although the development of phosphodiesterase type 5 (PDE5) inhibitors (i.e. sildenafil, tadalafil and vardenafil) has revolutionized the treatment of ED, these oral medications require on-demand access and are not as effective in treating ED related to diabetic, post-prostatectomy and severe veno-occlusive disease states. Improvement in the treatment of ED is dependent on understanding the regulation of human corporal smooth muscle tone and on the identification of relevant molecular targets. Future ED therapies might consider the application of molecular technologies such as gene therapy. As a potential therapeutic tool, gene therapy might provide an effective and specific means for altering intracavernous pressure "on demand" without affecting resting penile function. However, the safety of gene therapy remains a major hurdle to overcome before being accepted as a mainstream treatment for ED. Gene therapy aims to cure the underlying conditions in ED, including fibrosis. Furthermore, gene therapy might help prolong the efficacy of the PDE5 inhibitors by improving penile nitric oxide bioactivity. It is feasible to apply gene therapy to the penis because of its location and accessibility, low penile circulatory flow in the flaccid state and the presence of endothelial lined (lacunar) spaces. This review provides a brief insight of the current role of gene therapy in the management of ED.

The effect of vardenafil (a PDE type 5 inhibitor) on renal function in the diabetic rabbit: a pilot study.

BACKGROUND: Diabetic nephropathy is a common cause of impaired renal function. We investigated the effect vardenafil, a phosphodiesterase type 5 (PDE-5) inhibitor, has on renal function in the diabetic rabbit. MATERIALS AND METHODS: Blood was taken at 4 and 6 months from control and alloxan-induced diabetic animals (n=8, in each group) and biochemical variables pertaining to renal function determined. A 7-month sample was also analysed after giving control and diabetic animals (n=4 in each group) either vardenafil (3 mg/kg) or vehicle to drink for 4 weeks. Spot urine total protein/ creatinine ratio (TP/C) was determined at 4 and 6 months. At 7 months a 24 h-urine sample was collected to measure TP/C and creatinine clearance (CrCl). RESULTS: There was a significant increase in serum creatinine concentration after 6 months diabetes, which was significantly reduced by vardenafil. TP/C from diabetic rabbit spot urine samples at 6 months were significantly elevated compared to control animals, indicating the presence of proteinuria. Vardenafil treatment caused a normalisation of TP/C. Diabetic animals receiving vardenafil showed a significant improvement in CrCl when compared with diabetic animals given vehicle. CONCLUSION: These findings highlight a potential role for vardenafil in the treatment of diabetic nephropathy.

Serotonin induces a biphasic response in rabbit cavernosal smooth muscle: relevance to the erectile process.

INTRODUCTION: Serotonin (5-hydroxytryptamine; 5-HT) can cause contraction in cavernosal smooth muscle. We further evaluated this effect of 5-HT. METHODS: Organ bath studies were used. RESULTS: 5-HT induced a sustained contraction occasionally accompanied by a transient relaxation (in 30% of rabbit cavernosal tissues) that preceded the contraction. Ondansetron and Y-25130 (both 5-HT3 receptor antagonists) but not SB-269970 (a 5-HT7 receptor antagonist) significantly inhibited or abolished this transient relaxation. Doxazosin (dox, an alpha1-receptor antagonist) and ketanserin (ketan, a 5-HT2A receptor antagonist) significantly inhibited or abolished the sustained contraction. The effects of dox on 5-HT-mediated contraction were concentration-dependent. CONCLUSIONS: Our findings further confirm that the peripheral serotonergic pathway may play a part in the erectile process via 5-HT2A receptor-mediated contractile and 5-HT3 receptor-mediated relaxant activities. Our results also support the findings of human studies, which suggest that both ketan and dox may exert beneficial effects on the erectile process.

The management of phosphodiesterase-5 (PDE5) inhibitor failure.

The oral phosphodiesterase type 5 (PDE5) inhibitors have made a valuable contribution to the treatment of erectile dysfunction (ED). PDE5 inhibitors enhance cavernosal smooth muscle relaxation, vasodilatation and penile erection. However, PDE5 inhibitors are not always effective. Decreased efficacy, cost, incorrect administration, lack of sexual stimulation, vascular risk factors associated with ED and vascular or neurogenic diseases are causes of PDE5 inhibitor failure. Tachyphylaxis may also occur. This is defined as reduced tissue responsiveness to a drug in the presence of a constant concentration of this drug. Treatment failure may cause considerable distress. If dose titration, more attempts and continuous dosing of PDE5 inhibitors (taken on a daily basis) fail to resolve the initial PDE5 inhibitor failure, clinicians need to consider alternative treatments. These include sublingual apomorphine, intracavernosal/intraurethral pharmacotherapy, vacuum devices, the insertion of a prosthesis and penile vascular surgery. Combination therapy like prostaglandin E(1) (PGE(1)) with doxazosin (dox; an alpha-1-blocker) or ketanserin (ketan; a 5-HT(2) antagonist) as well as other pro-erection agents, like Endothelin-1 antagonists, angiotensin II antagonists (valsartan/losartan), adrenomedullin, Rho kinase inhibitors and nitric oxide (NO) donors may be beneficial in the treatment of ED. However, these combination therapies need to be validated. Adding an androgen to a PDE5 inhibitor may help when circulatory testosterone levels are low. The early use of PDE5 inhibitors in patients with hypertension, hyperlipidaemia or diabetes with concomitant ED and treating these risk factors may improve corporeal blood flow and lead to long-term preservation of cavernosal function. Therefore, the efficacy of PDE5 inhibitors may be maintained. Targeting the risk factors of ED (similar to those for arteriosclerosis) in the early stages of the disease may prevent the development or decrease the severity of ED.

Doxazosin and serotonin (5-HT) receptor (1A, 2A, and 4) antagonists inhibit 5-HT-mediated human cavernosal contraction.

Penile erection results from the balance between relaxation and contractile mechanisms of the corpus cavernosum. Only a few studies suggest a role for endogenous contractile agents such as 5-hydroxytryptamine (5-HT). Our aim was to confirm the possible role of 5-HT in human erection. The effect of 5-HT on human cavernosal tissues, as well as those of doxazosin (shown previously to have 5-HT inhibitory action), ketanserin (5-HT (2A) receptor antagonist), NAN-190 (5-HT (1A) receptor antagonist), and SB 203186 (5-HT (4) receptor antagonist) on 5-HT-mediated effects, were assessed using the organ bath technique, including electrical field stimulation study (EFS). Results are presented as median (mg/mg = mg contraction/mg of tissue). Consistent 5-HT-mediated (10(-3) M) contractions were demonstrated (n = 18; 63 mg/mg). These contractions were inhibited with ketanserin by 90% (n = 8), NAN-190 by 68% (n = 12), and SB 203186 by 55% (n = 12). Doxazosin showed a similar 5-HT inhibitory action in a concentration-dependent manner (10(-4) M; 94% reduction; n = 8, 10(-6) M; 68.3% reduction; n = 8). Our EFS studies indicated the presence of neuronally derived 5-HT and that a majority of the nonnoradrenogenic contraction (54%) was mediated via 5-HT(2A) receptors. These findings suggest that 5-HT may play a role in the human detumescence process via 5-HT(1A), 5-HT(2A), and 5-HT(4) receptors. Neuronally released 5-HT is probably an important contractile neurotransmitter in the erectile process. Doxazosin, ketanserin, and 5-HT(1A) and 5-HT(4) receptor antagonists may be useful as part of combination therapy used to treat erectile dysfunction.

Pharmacological properties of endothelin-1 in the rabbit corpus cavernosum.

BACKGROUND: Endothelin (ET-1) may play a role in the regulation of erection but this has not been conclusively demonstrated. Augmented cavernosal smooth muscle (CSM) contraction in the rat occurs following exposure to both ET-1 and phenylephrine (PE; alpha-1 agonist). The aim of this study was to assess the effect of ET-1 and its possible role in the alpha1-adrenergic pathway during the erectile process. MATERIALS AND METHODS: Organ bath studies were performed on CSM strips of penises obtained from 12 age-matched New Zealand White rabbits. The effect of ET-1 and PE alone on CSM tone in the absence and presence of ETA (BQ123) and ETB (BQ788) antagonists was assessed. Tissue responses were measured as tension (newton, N). EC50 values are expressed as mean +/- S.E.M. RESULTS: PE (10(8) - 10(-4) M) and ET-1 (10(-10) - 10(-6) M) produced a concentration-dependent contraction in rabbit CSM strips. The EC50 values were 1.7 x 10(-7) M +/- 1.1 and 3.4 x 10(-9) M +/- 1.5, respectively. BQ123 10(-5) M significantly inhibited ET-1-mediated CSM contractions more than BQ788 10(-5) M (both ANOVA p<0.01). The EC50 were 1.3 x 10(-6) M +/- 2.6 and 2.0 x 10(-7) M +/- 2.1, respectively. Neither the ETA or ETB receptor antagonist had a significant influence on alpha1-adrenergic receptor-mediated CSM contraction. CONCLUSION: ETA receptors may play a greater role than ETB receptors in ET-1-induced rabbit CSM contraction and the detumescence process. The a1-adrenergic-dependent pathway does not involve the ETA or ETB receptors.

The role of serotonin in tumour growth (review).

Serotonin (5-hydroxytryptamine; 5HT) a monoamine neurotransmitter mediates a wide range of physiological actions in the human body. For example 5HT is implicated in psychiatric and neurological disorders and also plays a fundamental role in tumour growth, differentiation and gene expression. 5HT acts as a growth factor for several types of tumoural and non-tumoural cells. This review considers the role of 5HT and its receptors in the human body with particular reference to carcinogenesis. We conclude that 5HT causes growth proliferation and 5HT antagonists cause growth inhibition in a variety of tumour cells (e.g. prostate carcinoma, lung carcinoma and colonic carcinoma). Therefore, further studies should look into the potential use of 5HT antagonists in the treatment of cancer.

Growth inhibitory effect of doxazosin on prostate and bladder cancer cells. Is the serotonin receptor pathway involved?

UNLABELLED: Doxazosin, an alpha1-adrenoceptor antagonist, is used for the treatment of benign prostatic hyperplasia (BPH) and hypertension. Alpha-adrenoceptor antagonists also inhibit growth and induce apoptosis in malignant prostatic cells. The apoptotic activity is independent of their capacity to antagonize alpha-adrenoceptors. The effect of doxazosin on the growth of prostate and bladder cancer cell lines was assessed and whether the growth inhibitory effect of doxazosin on prostate cancer cells is serotonin (5-hydroxtryptamine; 5HT)-dependent was investigated. MATERIALS AND METHODS: PC3 (androgen-independent prostate cancer) and HT1376 (grade III transitional cell carcinoma) cells were plated. The cells were incubated with doxazosin. After 72 h, cell viability was assessed (crystal violet assay). Studies were also performed after incubating the PC3 cells with 5HT or 5HT(1B) agonists for a short duration, followed by the addition of doxazosin. Cell viability was assessed at 72 h. RESULTS: Doxazosin caused a dose-dependent inhibition of PC3 and HT1376 cell growth with a maximum inhibition of 80% (n=12, p < 0.0001) and 91% (n=12, p < 0.0001), respectively, at a concentration of 10(-4)M, at 72 h. Incubation of PC3 cells with 5HT or 5HT(1B) agonist, followed by addition of doxazosin, increased the percent of viable cells as compared to when the cells were treated with doxazosin alone. CONCLUSION: Doxazosin significantly inhibited prostate (PC3) and bladder cancer (HT1376) cell growth. Furthermore, prior incubation of PC3 cells with 5HT or 5HT(1B) agonist increased cell viability as compared to treatment with doxazosin alone. These findings may be related to the similarity between subtype 1 serotonin and adrenergic receptors. The effect of alpha1-adrenoceptor antagonists on tumour cell growth merits further investigation.

Immunocytochemical and pharmacological characterisation of P2-purinoceptor-mediated cell growth and death in PC-3 hormone refractory prostate cancer cells.

BACKGROUND: Extracellular nucleotides (e.g adenosine 5'-triphosphate, ATP) influence biological processes via purinergic receptors. We characterised the P2-purinoreceptors in human hormone refractory prostate cancer (HRPC) cells (PC-3 cells). RESULTS: 1. Immunofluorescent staining demonstrated P2X3 P2X, P2X5 P2X7 and P2Y2 receptors. 2. ATP inhibited cell growth by up to 91% over 72h. Pharmacological characterisation indicated a P2X-purinoreceptor-mediated response. 3. Comparable maximum growth inhibition was seen after either a single addition of 1mM or daily addition of 100mM ATP. ATP concentrations ([ATP]) returned to baseline levels within 24h if the initial [ATP] was < or =100 HM, while [ATP] remained high for 72h if a single concentration of 1 mM was used. 4. ATP 1 mM significantly (p<0.001) increased the proportion of cells undergoing apoptosis from 0.27% (+/- 0.04%) to 5.28% (+/- 0.77%). CONCLUSION: Threshold concentrations of ATP inhibited HRPC cell growth in vitro via the activation of P2X-purinoreceptors. The role of nucleotides in the treatment of HRPC requires further investigation.

Erectile dysfunction: a need for greater awareness.

Research has led to effective treatment regimes for erectile dysfunction (ED). Convincing evidence links vascular risk factors (hypertension, diabetes mellitus, hyperlipidaemia and smoking) with ED. This association is not surprising since the corpus cavernosum is a modified vascular tissue. This review presents a brief account of the aetiology, diagnosis and treatment of ED. There is a need to raise awareness of this condition and to make appropriate treatment available to patients.

Animal models of diabetes mellitus: relevance to vascular complications.

The prevalence of diabetes mellitus is increasing worldwide at an alarming rate due to population growth, obesity, sedentary lifestyle and aging. Consequently, diabetic microvascular complications (retinopathy and nephropathy) and macrovascular complications (coronary heart disease, peripheral arterial disease and cerebrovascular disease) are also rising. Traditional oral hypoglycaemic agents only partially prevent the development of these complications. This suggests that selective treatment options that target specific biological pathways (i.e. metabolic factors, intracellular signaling proteins and growth factors) may be a more effective strategy. Type 1 and Type 2 diabetic animal models have been produced spontaneously by selective inbreeding or by genetic modification, as well as, pharmacological induction. These models have become a safe and reliable option to test the therapeutic potential of novel drugs. They also help to understand the pathophysiology of diabetes mellitus. This review highlights the most commonly used animal models for the treatment of diabetic micro and macrovascular complications.

The role of serotonin (5-hydroxytryptamine1A and 1B) receptors in prostate cancer cell proliferation.

PURPOSE: Serotonin (5-hydroxytryptamine), a monoamine neurotransmitter released by prostate neuroendocrine cells, has a fundamental role in tumor growth, differentiation and gene expression. We investigated the effect of 5-hydroxytryptamine and 5-hydroxytryptamine antagonists on the growth of prostate cancer cells and we identified 5-hydroxytryptamine receptor expression in PC3 cells and in human hormone refractory prostate cancer tissue. MATERIALS AND METHODS: A total of 12 preparations of hormone refractory PC3 human prostate cancer cells were incubated with 5-hydroxytryptamine, or the 5-hydroxytryptamine receptor antagonists 5-hydroxytryptamine1A, 1B, 1D, 2, 3 or 4. After 72 hours cell viability was assessed using the crystal violet assay. PC3 cells treated with 5-hydroxytryptamine1A and 1B antagonists were investigated for apoptosis using flow cytometry. PC3 cells and sections of hormone refractory human prostate cancer tissue were studied by immunohistochemistry and Western blot analysis. RESULTS: In PC3 cells 5-hydroxytryptamine caused dose dependent proliferation with a maximum increase of 15% in 12 preparations at a concentration of 10(-8) M at 72 hours compared to controls (p < 0.0001). At a concentration of 10(-4) M at 72 hours the 5HT1A antagonist NAN-190 hydrobromide and the 5-hydroxytryptamine1B antagonist SB224289 HCl (Tocris Laboratories, Bristol, United Kingdom) induced a 20% and 78% inhibitory effect, respectively, on PC3 cell growth compared to that in controls (p < 0.0001). In PC3 cells 5-hydroxytryptamine1A and 1B antagonists demonstrated apoptosis after 24 and 48 hours of incubation. Immunostaining for 5-hydroxytryptamine1A and 1B receptors was seen in PC3 cells and prostate cancer tissue. Western blot analysis demonstrated 5-hydroxytryptamine1A and 1B receptor proteins with 46 and 43 kDa bands, respectively. CONCLUSIONS: In PC3 prostate cancer cells 5-hydroxytryptamine1A and to a greater extent 5-hydroxytryptamine1B antagonists significantly inhibit growth and induce apoptosis. To our knowledge growth inhibition caused by the 5-hydroxytryptamine1B antagonist SB224289 HCl is a novel finding, as is apoptosis caused by the 2 antagonists 5-hydroxytryptamine1A and 1B. This effect is most likely mediated via 5-hydroxytryptamine1A and 1B receptors. Therefore, our results imply that 5-hydroxytryptamine1A and in particular 5-hydroxytryptamine1B receptor antagonists warrant further investigations as potential anti-neoplastic agents.

The effect of serotonin and serotonin antagonists on bladder cancer cell proliferation.

OBJECTIVE: To investigate the role of serotonin (5-hydroxytryptamine, 5HT) and its antagonists in the proliferation of high-grade bladder cancer cells (HT1376), as high-grade bladder cancer has a rapid rate of progression, invasion and recurrence, and 5HT antagonists inhibit the growth of the prostate cancer cell line (PC3). MATERIALS AND METHODS: HT1376 (human grade III transitional cell carcinoma) cells were incubated with either 5HT or 5HT antagonists (5HT(1A), 5HT(1B), 5HT(1D), 5HT(2), 5HT(3) and 5HT(4)). After 72 h, cell viability was assessed using the crystal violet assay. The presence of 5HT receptor subtypes on HT1376 cells and sections of human bladder cancer tissue was determined by immunohistochemistry and Western blot analysis. RESULTS: 5HT caused a dose-dependent increase in the proliferation of HT1376 cells. The maximum increase in cell proliferation (12%; 12 samples, P < 0.001) was at 10(-8)m as compared to the control at 72 h. At 10(-4)m, 5HT(1A) antagonist (NAN-190 hydrobromide) and 5HT(1B) antagonist (SB224289 hydrochloride) had a 10% (12 samples, P < 0.001) and 93% (12, P < 0.001) inhibitory effect on HT1376 cell growth, respectively, compared to the control at 72 h. There was immunostaining for 5HT(1A) and 5HT(1B) receptors in HT1376 cells and malignant bladder tissue, confirming the presence of these two receptor subtypes. Western blot analysis showed the presence of 5HT(1A) and 5HT(1B) receptor proteins with bands of 46 kDa and 43 kDa, respectively. CONCLUSION: 5HT(1A) and to a greater extent 5HT(1B) antagonists significantly inhibit bladder cancer cell growth. This effect is probably mediated via the 5HT(1A) and 5HT(1B) receptors. These results highlight the potential use of 5HT(1A) and 5HT(1B) antagonists in the treatment of bladder cancer.

The purinergic component of human vas deferens contraction.

OBJECTIVE: To examine purinergic signaling in human vas deferens. DESIGN: To study contractile responses of the scrotal vas deferens. SETTING: Research department of a university teaching hospital. PATIENT(S): Undergoing vasectomy or orchidectomy (aged 27-88 years, n = 14). INTERVENTION(S): Vasectomy or orchidectomy. MAIN OUTCOME MEASURE(S): Strips of vas deferens were suspended in an organ bath and subjected to electrical stimulation to establish frequency-response curves. These stimulations were repeated in the presence of pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS, P2 receptor antagonist), prazosin (adrenergic alpha1 antagonist), and tetrodotoxin. Concentration-response curves were constructed to noradrenaline and the P2X agonists ATP and alpha,beta-methylene ATP (alpha,beta-meATP). The P2X receptor subtype distribution was assessed by immunohistochemistry using specific antibodies. RESULT(S): The response at 32 Hz in the presence of PPADS was reduced by 40% and in the presence of prazosin by 80%. Noradrenaline caused concentration-dependent contractions (EC50 = 11.8 microM). Contractions to ATP and alpha,beta-meATP (EC50 = 6.27 microM) suggested that the functional receptor was P2X1 and/or P2X3. However, immunohistochemistry demonstrated P2X1, but not P2X3, receptor immunoreactivity on the smooth muscle cells. CONCLUSION(S): This study demonstrated that ATP is a co-transmitter with noradrenaline in the contraction of the human vas deferens predominantly acting through the P2X1 receptor.

Smooth muscle and purinergic contraction of the human, rabbit, rat, and mouse testicular capsule.

The smooth-muscle cells of the testicular capsule (tunica albuginea) of man, rat, and mouse were examined by electron microscopy. They were characteristically flattened, elongated, branching cells and diffusely incorporated into the collagenous matrix and did not form a compact muscle layer. Contractile and synthetic smooth-muscle cell phenotypes were identified. Nerve varicosities in close apposition to smooth muscle were seen in human tissue. Contractions induced by adenosine 5'-triphosphate (ATP), alpha, beta-methylene ATP, noradrenaline (NA), acetylcholine (ACh), and electrical field stimulation (EFS) of autonomic nerves were investigated. Nerve-mediated responses of the rabbit and human tunica albuginea were recorded. The EFS-induced human responses were completely abolished by prazosin. In the rabbit, EFS-induced contractile responses were reduced by pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid by 36% and by prazosin by 77%. Both antagonists together almost completely abolished all EFS-induced contractions. The human tunica albuginea was contracted by NA, ATP, and alpha, beta-methylene ATP, but not by ACh. The rabbit and rat tunica albuginea were contracted by NA, ATP, alpha, beta-methylene ATP, and ACh. The mouse tunica albuginea was contracted by ACh, ATP, and alpha, beta-methylene ATP, but relaxed to NA. Immunohistochemical studies showed that P2X1 (also known as P2RX1) and P2X2 (also known as P2RX2) receptors were expressed on the smooth muscle of the rodent testicular capsule, expression being less pronounced in man. The testicular capsule of the rat, mouse, rabbit, and man all contain contractile smooth muscle. ATP, released as a cotransmitter from sympathetic nerves, can stimulate the contraction of rabbit smooth muscle. Human, rat, and mouse testicular smooth muscle demonstrated purinergic responsiveness, probably mediated through the P2X1 and/or P2X2 receptors.