Cligosiban, A Novel Brain-Penetrant, Selective Oxytocin Receptor Antagonist, Inhibits Ejaculatory Physiology in Rodents.

INTRODUCTION: Few treatments are available for men with premature ejaculation (PE); oxytocin (OT) receptor antagonism in the central nervous system (CNS) is a potential new approach. AIM: To determine if cligosiban selectively inhibits human OT receptors, penetrates the CNS, shows pharmacology in the CNS, and effects ejaculatory physiology in pre-clinical systems. METHODS: Experiments complied with United Kingdom legislation and were subject to local ethical review. In vitro potency and selectivity of cligosiban was assessed using recombinant and native OT receptor systems including both neuronal and non-neuronal cell types. Selectivity was determined over neighboring V1A, V1B, and V2 vasopressin receptors using a combination of recombinant and native vasopressin receptor assay systems. To determine an effect on central OT receptors and on ejaculation, cligosiban was evaluated in 2 anesthetized rat models-the electromyography model of ejaculatory physiology and a model of OT-mediated CNS neuronal firing. The CNS penetration of cligosiban was also determined by measuring cerebrospinal fluid and plasma drug concentrations following an intravenous (IV) infusion in rats. MAIN OUTCOME MEASURE: These were functional measures of pharmacology in vitro, in cell lines and tissues, and in vivo in rats. RESULTS: Cligosiban is a potent OT receptor antagonist, with a base dissociation constant of 5.7 nmol/L against native human uterine smooth muscle cell OT receptors. Cligosiban displays similar antagonistic potency against human recombinant and rat native OT receptors, including neuronal OT receptors. Cligosiban demonstrates >100-fold selectivity over human V1A, V1B, and V2 vasopressin receptors. In the electromyography model, cligosiban (0.9 mg/kg, IV bolus) reduced the bulbospongiosum burst pattern and contraction amplitude associated with ejaculation. In the anesthetized CNS neuronal firing model, the same dosing regimen of cligosiban (0.9 mg/kg IV bolus) modulated the OT-mediated response in the nucleus tractus solitarius. After systemic dosing to rats, cligosiban showed good CNS penetration. CLINICAL IMPLICATIONS: As the first highly selective and centrally penetrant OT receptor antagonist, cligosiban represents a promising compound to test the clinical hypothesis that antagonism of central OT receptors may be of therapeutic benefit in the treatment of PE. STRENGTH & LIMITATIONS: The pharmacology and selectivity of cligosiban is determined using functional assays in recombinant cell lines, native cell lines, and tissue. Functional outcomes in in vivo systems are linked to CNS measures of pharmacology. The translation of the animal models of ejaculation to PE in man is unproven. CONCLUSION: Cligosiban, a potent, selective OT receptor antagonist, demonstrated CNS penetration and pharmacology and, using the same dosing regimen, inhibited apomorphine-induced ejaculation in rats. Cligosiban is a promising compound to test the clinical hypothesis that antagonism of central OT receptors may be of therapeutic benefit in the treatment of PE. Wayman C, Russell R, Tang K, et al. Cligosiban, A Novel Brain Penetrant Selective Oxytocin Receptor Antagonist, Inhibits Ejaculatory Physiology in Rodents. J Sex Med 2018;15:1698-1706.

Effects of multikinase inhibitors on pressure overload-induced right ventricular remodeling.

BACKGROUND: Little is known about the effects of current PAH therapies and receptor tyrosine kinase inhibitors on heart remodeling. We sought to investigate the effects of the multikinase inhibitors sunitinib (PDGFR-, VEGFR- and KIT-inhibitor) and sorafenib (raf1/b-, VEGFR-, PDGFR-inhibitor) on pressure overload induced right ventricular (RV) remodeling. METHODS: We investigated the effects of the kinase inhibitors on hemodynamics and remodeling in rats subjected either to monocrotaline (MCT)-induced PH or to surgical pulmonary artery banding (PAB). MCT rats were treated from days 21 to 35 with either vehicle, sunitinib (1mg/kg, 5mg/kg and 10mg/kg/day) or sorafenib (10mg/kg/day). PAB rats were treated with vehicle, sunitinib (10mg/kg/day) or sorafenib (10mg/kg/day) from days 7 to 21. RV function and remodeling were determined using echocardiography, invasive hemodynamic measurement and histomorphometry. RESULTS: Treatment with both sorafenib and sunitinib decreased right ventricular systolic pressure, pulmonary vascular remodeling, RV hypertrophy and fibrosis in MCT rats. This was associated with an improvement of RV function. Importantly, after PAB, both compounds reversed RV chamber and cellular hypertrophy, reduced RV interstitial and perivascular fibrosis, and improved RV function. CONCLUSION: We demonstrated that sunitinib and sorafenib reversed RV remodeling and significantly improved RV function measured via a range of invasive and non-invasive cardiopulmonary endpoints in experimental models of RV hypertrophy.

Lack of abuse potential in a highly selective dopamine D3 agonist, PF-592,379, in drug self-administration and drug discrimination in rats.

Dopamine D3-preferring agonists are commonly used to treat Parkinson's disease and restless leg syndrome; however, laboratory animal studies suggest that they may possess a moderate abuse potential. These studies aimed to compare the highly selective, full D3 agonist PF-592,379 to the less selective D3 agonist 7-OH-DPAT, and the indirect dopamine agonist cocaine in drug self-administration and discrimination assays. Although rats readily acquired high rates of fixed ratio (FR)1 responding for cocaine, experimentally naive rats failed to acquire responding when 7-OH-DPAT or PF-592,379 was made available during an 18-session acquisition period. Cocaine also maintained dose-dependent levels of responding when available under a FR5 or a progressive ratio (PR) schedule of reinforcement. Although 7-OH-DPAT maintained modest levels of responding when substituted under a FR5, it failed to maintain significant levels of PR responding. PF-592,379 maintained saline-like rates of responding when substituted under FR5 or PR schedules of reinforcement. Similar behavioral profiles were observed in cocaine discrimination assays, with 7-OH-DPAT partially substituting for cocaine, and PF-592,379 producing saline-like effects over a wide range of doses. Together, the results of these studies predict that highly selective D3 agonists, such as PF-592,379, will have low abuse potential in humans.

Neuroanatomical distribution of the melanocortin-4 receptors in male and female rodent brain.

The melanocortin-4 receptor (MC4-R) plays a critical role in several physiological functions, from food intake, energy homeostasis, neuroendocrine and cardiovascular function, to sexual responses. The brain regions and the central neuronal pathways mediating the different actions of MC4-R remain largely unknown. We aimed to use immunocytochemistry using a specific antibody against rat MC4-R, to establish the detailed neuroanatomical distribution of MC4-R in brain slices of male and estrous female rats. We demonstrated that MC4-R-positive neurons were widely distributed in several brain regions including the cortex, thalamus, hypothalamus, and brainstem. In both male and female brains, MC4-R-positive cells were especially abundant in the hypothalamus, including the paraventricular hypothalamic nucleus, lateral septal nucleus, arcuate nucleus, supraoptic nucleus, medial preoptic area and lateral hypothalamic area. A moderate number of MC4-R-positive neurons were found in the piriform cortex, bed nucleus of the stria terminalis, medial and basolateral nuclei of amygdala, periaqueductal gray, red nucleus and raphe nucleus. A dimorphic sexual difference in the number of MC4-R-positive neurons was observed in some brain regions. In the medial preoptic area and arcuate nucleus, MC4-R-positive neurons were significantly more abundant in female than in males, whereas in the lateral hypothalamus the opposite proportion was observed. This is the first time the neuroanatomical distribution, and sex differences, of brain MC4-R localisation have been described. The distribution of MC4-R is consistent with the proposed roles of MC4-R-positive neurons and provides further information about the circuitry controlling food intake, energy balance and sexual responses in both males and females.

Neuroanatomical evidence for a role of central melanocortin-4 receptors and oxytocin in the efferent control of the rodent clitoris and vagina.

INTRODUCTION: The clitoris and the vagina are the main peripheral anatomical structures involved in physiological changes related to sexual arousal and orgasm. Their efferent control and, more particularly, the neurochemical phenotype of these descending neuronal pathways remain largely uncharacterized. AIM: To examine if brain neurons involved in the efferent control of the clitoris and the vagina possess melanocortin-4 receptor (MC4-R) and/or contain oxytocin (OT). METHODS: Neurons involved in the efferent control of the vagina and clitoris were identified following visualization of pseudorabies virus (PRV) retrograde tracing. PRV was injected into the vagina and clitoris in adult rats in estrous. On the fifth day postinjection, animals were humanely sacrificed, and brains were removed and sectioned, and processed for PRV visualization. The neurochemical phenotype of PRV-positive neurons was identified using double or triple immunocytochemical labeling against PRV, MC4-R, and OT. Double and triple labeling were quantified using confocal laser scanning microscopy. MAIN OUTCOME MEASURE: Neuroanatomical brain distribution, number and percentage of double-labeled PRV/MC4-R and PRV-/OT-positive neurons, and triple PRV-/MC4-R-/OT-labeled neurons. RESULTS: The majority of PRV immunopositive neurons which also expressed immunoreactivity for MC4-R were located in the paraventricular and arcuate nuclei of the hypothalamus. The majority of PRV positive neurons which were immunoreactive (IR) for OT were located in the paraventricular nucleus (PVN), medial preoptic area (MPOA), and lateral hypothalamus. PRV positive neurons were more likely to be IR for MC4-R than for OT. Scattered triple-labeled PRV/MC4-R/OT neurons were detected in the MPOA and the PVN. CONCLUSION: These data strongly suggest that MC4-R and, to a less extent, OT are involved in the efferent neuronal control of the clitoris and vagina, and consequently facilitate our understanding of how the melanocortinergic pathway regulates female sexual function.

Discovery of a selective small-molecule melanocortin-4 receptor agonist with efficacy in a pilot study of sexual dysfunction in humans.

The relevance of the melanocortin system to sexual activity is well established, and nonselective peptide agonists of the melanocortin receptors have shown evidence of efficacy in human sexual dysfunction. The role of the MC4 receptor subtype has received particular scrutiny, but the sufficiency of its selective activation in potentiating sexual response has remained uncertain owing to conflicting data from studies in preclinical species. We describe here the discovery of a novel series of small-molecule MC4 receptor agonists derived from library hit 2. The addition of methyl substituents at C3 and C5 of the 4-phenylpiperidin-4-ol ring was found to be markedly potency-enhancing, enabling the combination of low nanomolar potencies with full rule-of-five compliance. In general, the series shows only micromolar activity at other melanocortin receptors. Our preferred compound 40a provided significant systemic exposure in humans on both sublingual and oral administration and was safe and well tolerated up to the maximum tested dose. In a pilot clinical study of male erectile dysfunction, the highest dose of 40a tested (200 mg) provided a similar level of efficacy to sildenafil.

Neuronal activity in the hypothalamic paraventricular nucleus varies across the estrous cycle in anesthetized female rats: effects of dopamine receptor agonism.

PURPOSE: The central nervous system plays a pivotal role in sexual behavior. The role of the paraventricular nucleus (PVN) of the hypothalamus in female sexual behavior is poorly characterized. In males, there is a correlation between PVN neuron activity and erectile activity, and in mediating dopamine receptor agonist-induced sexual arousal. MATERIAL AND METHODS: To understand the role of the PVN in female sexual function, baseline PVN neuronal activity and responses to dopamine receptor agonism were assessed in anesthetized rats. Single unit recordings were used to assess the firing properties of individual PVN neurons; and local field potentials quantified PVN network activity (combined activity of large numbers of PVN neurons). Baseline and apomorphine-stimulated activity was measured across the estrous cycle. RESULTS: Baseline firing rates of single units were found to differ across the stages of the estrous cycle with metestrus showing the highest firing rate (3.7 vs. 0.9 Hz in diestrus). Apomorphine administration caused significant increases in firing rate in 29% of neurons, and significant decreases in 71%. Basal local field potentials also varied between estrous stages and in response to apomorphine; significant differences in the total power of alpha1 and beta1 bands were observed in both cases. CONCLUSIONS: This study shows that PVN neuronal activity varies with hormonal state, and these neurons are differentially affected by apomorphine, suggesting two different populations. These data are indicative of a critical role for the PVN in female sexual function. It is important that the hormonal state should be considered when investigating sexual physiology and the effect of pharmacological agents.

II. Slow oscillations in vaginal blood flow: regulation of vaginal blood flow patterns in rat by central and autonomic mechanisms.

INTRODUCTION: A new method for assessing female sexual arousal through changes in slow oscillatory patterns in vaginal blood flow was first described in the previous manuscript [1]. This method was translational and discriminated between normal healthy volunteers and women with female sexual arousal disorder. AIM: These studies addressed the influence of autonomic and central nervous systems on slow vaginal blood flow oscillations in rats. METHODS: Vaginal blood flow oscillations were measured in urethane-anesthetized rodents using laser Doppler flowmetry. Acquired data were filtered for frequency analysis range of 0.013-2.5 Hz. MAIN OUTCOME MEASURES: Data were assessed for changes in a high frequency range (HF = 0.6-2.5 Hz), and low frequency range (LF = 0.013-0.6 Hz). RESULTS: The basal HF oscillatory component of vaginal blood flow was primarily vagally mediated, although could be modulated pharmacologically with p-chloroamphetamine in the absence of vagal innervation. The LF component could be modulated by antagonists of noradrenergic receptors but did not appear to be dependent upon tonic activation of sympathetic circuitry. The non-selective dopamine receptor agonist apomorphine induced changes in vaginal blood flow oscillations consistent with sexual arousal during metestrus in the presence of the peripheral antagonist domperidone but not in the presence of the centrally acting antagonist haloperidol. Electrical stimulation of the paraventricular nucleus (PVN) of the hypothalamus induced an anti-arousal response in vaginal blood flow oscillations. These data demonstrated that manipulation of the central nervous system alone (via centrally acting apomorphine or electrical stimulation of the PVN) could produce either a pro-arousal or an anti-arousal response in vaginal blood flow oscillations. Alterations in the LF/HF ratio measured from vaginal laser Doppler flowmetry were independently regulated from vasculature in the trunk, the tongue, and electrocardiogram-derived heart rate variability, and were independent of overall vasocongestion of the vagina as measured by mean blood flow. CONCLUSIONS: These data indicated that slow oscillations in vaginal blood flow from rodents may be utilized as an animal model of female sexual arousal. Changes in these oscillations are driven by the central nervous system and modulated by the autonomic nervous system.

I. Slow oscillations in vaginal blood flow: alterations during sexual arousal in rodents and humans.

PURPOSE: This study investigated slow oscillatory rhythms in vaginal blood flow as a physiological marker of female sexual arousal in rodents, human healthy volunteers, and women with female sexual arousal disorder (FSAD). MATERIALS AND METHODS: Vaginal blood flow was measured in urethane-anesthetized rodents using laser Doppler flowmetry, while in humans, vaginal photoplethysmography was used. Acquired data were filtered for frequency analysis in the range of 0.013-2.5 Hz in rodents and 0.01-0.5 Hz in humans. Rodents were assessed for changes in a high frequency range (HF = 0.6-2.5 Hz), and a low frequency range (LF = 0.013-0.6 Hz). Human data were assessed for total spectral power in the entire frequency range. RESULTS: During naturally induced arousal (exposure to male), oscillatory rhythms in vaginal blood flow from rodents demonstrated an increase in the ratio of LF oscillations to HF oscillations (LF/HF ratio). Drugs known to induce sexual arousal (apomorphine and melanotan II) were tested in anesthetized rodents. Both compounds induced an increase in the LF/HF ratio. In humans, visual sexual stimulation induced an increase in the total power of slow oscillatory activity in vaginal blood flow in healthy human volunteers. No such increase was observed in women with FSAD. CONCLUSIONS: This study demonstrated that slow oscillations in vaginal blood flow are correlated with subjective physiological arousal and display diminished responsiveness in women with FSAD. Slow oscillations in vaginal blood flow are entirely independent of vaginal vasocongestion as women with FSAD demonstrated a normal vasocongestion response to visual sexual stimulation. In conditions where rodents would be expected to be sexually aroused, slow oscillations in vaginal blood flow showed a shift from HFs to LFs. This technique will greatly enhance the investigation of female sexual function both clinically and preclinically.

Electrophysiological actions of the dopamine agonist apomorphine in the paraventricular nucleus during penile erection.

The ability to achieve and maintain penile erection is necessary for successful copulation. Studies have demonstrated that dopamine receptor stimulation in the paraventricular nucleus (PVN) of the hypothalamus induces penile erection in rodents, and the dopamine agonist apomorphine has been used to treat erectile dysfunction. The aim of this study was to determine the electrophysiological characteristics of PVN neuronal firing activity in anaesthetised rodents during apomorphine-induced erection. Our findings can be placed in two categories; those effects that occur immediately upon apomorphine administration and continue for up to several minutes prior to penile erection, deemed 'pre-erectile', and those effects that were only observed during penile erection and seminal emission. In the pre-erectile period, apomorphine acts on two different populations of PVN neurons to increase or decrease firing rates and increases alpha1 frequency band power in local field potentials. Decreased delta and increased theta frequency power in PVN local field potentials occur only during penile erection and seminal emission. These studies provide further understanding of the coordinated neuronal activity that occurs in the PVN during apomorphine-induced penile erection.

Neuropeptide modulation of a lumbar spinal reflex: potential implications for female sexual function.

INTRODUCTION: Neuropeptides are known to modulate female receptivity. However, even though receptivity is a spinal reflex, the role of neuropeptides within the spinal cord remains to be elucidated. AIM: The aims were to (i) investigate neuropeptides in the lumbosacral region; and (ii) determine how neuropeptides modulate glutamate release from stretch Ia fibers, touch sensation Abeta fibers and Adelta/C pain fibers. MAIN OUTCOME MEASURES: Neuropeptide modulation of the lumbosacral dorsal-root ventral-root reflex in vitro. METHODS: Spinal cords were removed from Sprague-Dawley rats in compliance with UK Home Office guidelines. Hemisected cords were superfused with aCSF and the dorsal root (L4-S1) was stimulated to evoke glutamate release. A biphasic reflex response was evoked from the opposite ventral root consisting of a monosynaptic (Ia fibers) and polysynaptic (Abeta, Adelta/C fibers) component. RESULTS: The micro opioid receptor (MOR) agonist DAMGO inhibited the monosynaptic (EC(50) 0.02 +/- 0.02 nM) and polysynaptic area (EC(50) 125 +/- 167 nM) but not polysynaptic amplitude. Oxytocin and corticotrophin releasing factor (CRF) inhibited the monosynaptic amplitude (EC(50), 1.4 +/- 1.0 nM and EC(50) 4.3 +/- 3.5 nM, respectively), polysynaptic amplitude (EC(50) 18.2 +/- 28.0 nM and EC(50), 9.5 +/- 13.3 nM, respectively), and area (EC(50) 11.6 +/- 13.0 nM and EC(50), 2.8 +/- 3.3 nM, respectively); effects that were abolished by oxytocin and CRF(1) antagonists, L-368899 and 8w. Melanocortin agonists solely inhibited the monosynaptic component, which were blocked by the MC(3/4) receptor antagonist SHU9119. CONCLUSION: These data suggest endogenous neuropeptides are released within the lumbosacral spinal cord. Melanocortin agonists, oxytocin, CRF, and DAMGO via MC(4), oxytocin, CRF(1), and MOR inhibit glutamate release but with differing effects on afferent fiber subtypes. Melanocortins, oxytocin, CRF, and DAMGO have the ability to modulate orgasm whereas oxytocin, CRF and DAMGO can increase pain threshold. Oxytocin and CRF may dampen touch sensation.

Combination of doxazosin and sildenafil exerts an additive relaxing effect compared with each compound alone on human cavernosal and prostatic tissue.

INTRODUCTION: Phosphodiesterase 5 inhibitors (PDE5) such as sildenafil are first-line treatment for erectile dysfunction (ED). Alpha1 (alpha1)-adrenoceptor antagonists such as doxazosin are indicated for the treatment of patients with lower urinary tract symptoms (LUTS)/benign prostatic hyperplasia (BPH). ED and LUTS/BPH are conditions that are often associated. Accordingly, alpha1-adrenoceptor antagonists and PDE5 inhibitors will be often prescribed in real life setting together. AIM: To evaluate the effects of the combination of sildenafil and doxazosin on human cavernosal and prostatic tissue. METHODS: Prostatic and erectile tissues were obtained from nine to 12 patients, respectively. Patients underwent cystoprostatectomy for infiltrating bladder cancer or penile surgery for penile implant, congenital curvature or Peyronie's disease. MAIN OUTCOME MEASURES: In organ baths, prostatic and cavernosal strips were submitted to either concentration-response curves (CRC) to phenylephrine (Phe) or norepinephrine (NE), respectively, in presence of vehicle, sildenafil (10(-6) M, 10(-5) M), doxazosin (10(-8) M, 3.10(-8) M, or 10(-7) M), or a combination of both. Continuous electrical field stimulation (EFS; 32 Hz, 5 ms, 5 seconds, 300 mA) was performed on prostatic strips which were incubated with sildenafil 10(-6) M or vehicle before the successive addition of doxazosin (10(-7) M, 10(-6) M) or vehicle. Cavernosal strips were pre-incubated with doxazosin (10(-9) M, 10(-8) M) or vehicle, then CRC to sildenafil were constructed on NE (3.10(-6) M) precontracted cavernosal strips. RESULTS: Combination of sildenafil and doxazosin exerted a greater relaxing effect on CRC to Phe or NE compared with each compound alone in both tissues. Sildenafil significantly enhanced the relaxing effect of doxazosin on EFS-induced contractions in prostatic strips. Doxazosin significantly increased the ability of sildenafil to inhibit NE-induced contractions in cavernosal strips. CONCLUSIONS: Sildenafil and doxazosin reduced adrenergic tone of prostatic and cavernosal smooth muscle and their combination provided a significant benefit when targeting relaxation of both tissues. These experiments provide support for further clinical evaluation of the sildenafil and doxazosin combination in ED patients with LUTS/BPH.

Impact of a long-term sildenafil treatment on pressor response in conscious rats with insulin resistance and hypertriglyceridemia.

BACKGROUND: Insulin resistance constitutes a risk factor for endothelial dysfunction and subsequent cardiovascular diseases including hypertension. Daily treatment with phosphodiesterase type 5 (PDE5) inhibitors has beneficial effects on endothelial function in men with increased cardiovascular risk. Endothelium-dependent vasomotor function is ultimately linked to blood pressure (BP) regulation. We postulated that sildenafil would ameliorate BP and biological markers of endothelial function in fructose-fed rats (FFRs). METHODS: Wistar rats were fed a standard chow or a 60% fructose-enriched diet containing 12% fat for 8 weeks (FFR). From week 6 through 8, sildenafil (twice a day subcutaneously, 20 mg/kg) was administered followed by a 1-week washout period. At the end of the washout period, BP was recorded using radiotelemetry following cumulative infusion of norepinephrine (from 50 to 400 ng/kg/min). RESULTS: FFR displayed both an impaired glucose tolerance and elevated triglyceridemia. The latter was corrected by sildenafil treatment. Resting BP was similar in all rats, whereas pressor responses were significantly enhanced in FFR (maximal increase in mean BP to norepinephrine: 25.6 +/- 3.8 vs. 40.8 +/- 4.0 mm Hg, P < 0.05) and normalized by sildenafil treatment (24.9 +/- 5.3 mm Hg, not significant vs. control). Urinary levels of 8-isoprostanes and thromboxane B(2) were increased in FFR and corrected by sildenafil treatment. CONCLUSION: Thus, chronic treatment with sildenafil normalized BP regulation in an experimental model of insulin resistance and hypertriglyceridemia while restoring normal excretion of urinary biological markers of oxidative stress and cyclooxygenase-derived vasoconstrictors. The modulation of ROS and cyclooxygenase-derived vasoconstrictors generation by a chronic treatment with sildenafil may represent an added benefit beyond PDE5 inhibition.

Daily treatment with sildenafil reverses endothelial dysfunction and oxidative stress in an animal model of insulin resistance.

OBJECTIVES: Patients with insulin resistance exhibit endothelial dysfunction with decreased nitric oxide (NO) production and increased oxidative stress. We postulated that daily sildenafil improved endothelial function in fructose-fed rats. METHODS AND RESULTS: Wistar rats were fed a standard or fructose-enriched diet (FFR) for 9 wk. From weeks 6-8, sildenafil was administered twice daily (sc, 20 m g/kg), followed by a 1-wk washout. Concentration-response curves (CRCs) to endothelium-dependent (acetylcholine [Ach] and A23187) and -independent (sodium nitroprusside [SNP]) relaxing agents were performed on isolated precontracted aortas and superior mesenteric arteries (SMAs). Vascular cyclic guanosine monophosphate (cGMP) content, urinary excretion of nitrates/nitrites (NOx) and 8-isoprostanes (IPT), and plasma levels of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) were evaluated. Relaxations to ACh were significantly reduced in aortas and SMAs of FFR. Sildenafil restored ACh-induced relaxations in aortas and provoked a significant leftward shift of the CRC to ACh in SMAs, whereas it did not modify the enhanced relaxations to SNP in FFR. IL-6, TNF-alpha, vascular cGMP, and urinary NOx levels were not modified by the fructose or sildenafil treatment. Urinary IPT levels were significantly elevated in FFR and normalized by sildenafil. CONCLUSIONS: Endothelial dysfunction and oxidative stress associated with insulin resistance can be reversed by daily sildenafil, even 1 wk after treatment cessation.

Chronic sildenafil improves erectile function and endothelium-dependent cavernosal relaxations in rats: lack of tachyphylaxis.

OBJECTIVES: Sildenafil is a widely-prescribed effective on-demand treatment of erectile dysfunction (ED). Chronic treatment with sildenafil could help patients with ED. METHODS: The effects of an 8-week long treatment with sildenafil (60 mg/kg/d sc) in male Sprague Dawley rats were evaluated on electrically-elicited erectile responses in vivo before and after an acute injection of sildenafil (0.3mg/kg iv). In addition, endothelium-dependent and -independent relaxations of strips of corpus cavernosum in vitro were examined. All experiments were performed 36 hours after the last injection of sildenafil. RESULTS: Endothelium-dependent relaxations of cavernosal strips to acetylcholine were enhanced after chronic treatment with sildenafil while relaxations to A23187 or sodium nitroprusside were unchanged. Frequency-dependent erectile responses elicited by cavernous nerve stimulation were significantly improved. Moreover, the erectile responses to acute sildenafil were greater in chronically-treated rats with sildenafil. CONCLUSIONS: This is the first report providing experimental support for chronic dosing with sildenafil which could be of use for patients that are poor responders to on-demand treatment. Chronic sildenafil may regulate the transduction pathway leading to the activation of eNOS but has no effect on NO bioavailability or on the cGMP pathway, thereby eliminating a possible concern for tachyphylaxis.

Receptor-independent activation of Rho-kinase-mediated calcium sensitisation in smooth muscle.

1. The aim of this work was to determine whether Rho-kinase-mediated calcium sensitisation contributes to contractions of the mouse anococcygeus smooth muscle and, if so, whether the process was activated by receptor-dependent or receptor-independent mechanisms. 2. The Rho-kinase inhibitor Y27632 produced concentration-dependent decreases in tone raised by either the muscarinic receptor agonist carbachol (CCh), or the sarco-endoplasmic reticulum calcium ATPase inhibitor thapsigargin (Tg) (EC(50) values against CCh and Tg of 8.4+/-3.3 (n=6) and 6.1+/-2.1 (n=7) micro M, respectively). Pretreatment of tissues with Y27632 also inhibited contractions produced by 65 mM external potassium (69+/-7% (n=4) inhibition using 10 micro M Y27632). Y27632 had no effect on contractions produced by the inhibitor of smooth muscle myosin light-chain phosphatase, calyculin-A. 3. In beta-escin-permeabilised preparations, both CCh and Tg produced significant increases in tone over-and-above that produced by a combination of calcium (1 micro M) and GTP (100 micro M). These responses to CCh and Tg were inhibited by Y27632 (10 micro M). 4. Western blot analysis of fractionated tissue samples probed for RhoA immunoreactivity, indicated that both CCh and Tg were able to induce translocation of RhoA from the cytosol to the membrane. 5. These findings suggest that Rho-kinase-mediated calcium sensitisation is activated by both receptor-dependent and receptor-independent mechanisms in the mouse anococcygeus.