Varying the Directionality of Protein Catalysts for Aldol and Retro-Aldol Reactions.

Natural aldolase enzymes and created retro-aldolase protein catalysts often catalyze both aldol and retro-aldol reactions depending on the concentrations of the reactants and the products. Here, we report that the directionality of protein catalysts can be altered by replacing one amino acid. The protein catalyst derived from a scaffold of a previously reported retro-aldolase catalyst, catalyzed aldol reactions more efficiently than the previously reported retro-aldolase catalyst. The retro-aldolase catalyst efficiently catalyzed the retro-aldol reaction but was less efficient in catalyzing the aldol reaction. The results indicate that protein catalysts with varying levels of directionality in usually reversibly catalyzed aldol and retro-aldol reactions can be generated from the same protein scaffold.

[Experimental rodent models of chronic prostatitis: effect of phosphodiesterase 5 inhibitor on chronic pelvic-pain-related behavior].

Chronic prostatitis / chronic pelvic pain syndrome (CP/CPPS) is commonly diagnosed in men younger than 50 years old. It is characterized by pelvic pain, voiding symptoms and sexual dysfunction. The considerable discomfort or pain experienced has a negative impact on the quality of life of patients and is a huge economic burden because of the high recurrence rate and the low cure rate. Appropriate animal models are essential for the development of new drugs for the treatment of CP/CPPS, and several rodent models induced by different methods and over different time frames have been established. This article reviews studies of three in vivo rodent models of prostatitis, namely, chemical-induced, autoimmune-induced and hormone-associated models reported by us and other investigators. Recent clinical investigation has suggested that tadalafil improves the International Prostatic Symptom Score and the total National Institutes of Health Chronic Prostatitis Symptom Index score of patients with benign prostatic hyperplasia with CP/CPPS, which enables us to investigate the effect of tadalafil on the pelvic-pain-related behavior and prostatic inflammation in two of these prostatitis model types, experimental autoimmune prostatitis (EAP) and hormone/castration-induced prostatitis (HCP). Both models showed the pelvic-pain-related behavior and prostatic inflammation that are characteristic of chronic prostatitis. In EAP, tadalafil suppressed both the pelvic-pain-related behavior and the prostatic inflammation. In HCP, tadalafil suppressed the pelvic-pain-related behavior. These results mimic the clinical findings. Therefore EAP and HCP are suitable for the evaluation of the potency of drugs for the treatment of CP/CPPS.

Comparison of the Effects of Tadalafil and α1-Adrenoceptor Antagonists on Spontaneous Seminal Emission and Electrical Field Stimulation-Induced Seminal Vesicle Contraction in Rats.

BACKGROUND: Although numerous reports have shown that α1-adrenoceptor (α1-AR) antagonists, which are used to treat benign prostatic hyperplasia (BPH), can cause ejaculatory disorders, few studies have investigated whether the phosphodiesterase 5 (PDE5) inhibitor tadalafil has such adverse effects. In this study, we compared the effects of tadalafil and α1-AR antagonists on seminal emission and their mechanisms of action. AIM: To evaluate in normal rats the possible effects of tadalafil on spontaneous seminal emission (SSE) and seminal contraction evoked by hypogastric nerve stimulation. METHODS: Male Sprague-Dawley rats were used. To assess SSE, plastic corsets were fitted around the thorax and upper abdomen of male Sprague-Dawley rats to prevent genital autogrooming. Rats were treated orally with tadalafil or an α1-AR antagonist (silodosin, naftopidil, or tamsulosin) for 3 days and housed in wire-bottomed cages. Ejaculatory plugs dropped on the bottoms of the cages were counted and weighed. To assess the intraluminal pressure of seminal vesicles, the hypogastric nerve of urethane-anesthetized rats was isolated and electrically stimulated. After stabilization of seminal vesicle contraction, the rats were intravenously administered test drugs. The expression of PDE5, endothelial nitric oxide synthetase (eNOS), and neuronal NOS (nNOS) in the seminal vesicle and vas deferens were measured by reverse-transcription polymerase chain reaction. MAIN OUTCOME MEASURE: The number and weight of the ejaculatory plugs produced by corset-fitted rats and the intraluminal pressure of the seminal vesicle were evaluated. RESULTS: Tadalafil did not affect the number or weight of the ejaculatory plugs of corset-fitted rats, whereas all α1-AR antagonists decreased both in a dose-dependent manner. The α1-AR antagonists, but not tadalafil, inhibited the seminal vesicle contraction evoked by electrical stimulation of the hypogastric nerve. The seminal vesicle and vas deferens expressed higher levels of PDE5 and eNOS mRNA and lower levels of nNOS mRNA relative to the urethra. CLINICAL IMPLICATIONS: Tadalafil can be a treatment option in cases where there is concern about negative effects on seminal emission. STRENGTHS AND LIMITATIONS: We demonstrated different effects of tadalafil and 3 α1-AR antagonists on rat SSE and their mechanisms of action by measuring seminal vesicle contractility in vivo. A limitation is that we used normal rats, not BPH model rats, and so our results might not apply to human BPH patients. CONCLUSION: Tadalafil did not inhibit spontaneous seminal emission or electrical field stimulation-induced seminal vesicle contraction in normal rats. The NO-cyclic guanosine monophosphate pathway is unlikely to be involved in the inhibition of seminal vesicle contraction in normal rats. Yoshinaga R, Fukui T, Yoshifuji M, et al. Comparison of the Effects of Tadalafil and α1-Adrenoceptor Antagonists on Spontaneous Seminal Emission and Electrical Field Stimulation-Induced Seminal Vesicle Contraction in Rats. J Sex Med 2019;16:680-690.

Voiding behavior and chronic pelvic pain in two types of rat nonbacterial prostatitis models: Attenuation of chronic pelvic pain by repeated administration of tadalafil.

BACKGROUND: Experimental autoimmune prostatitis (EAP) and prostatitis induced by 17ß-estradiol treatment combined with castration (hormone/castration-induced prostatitis; HCP) are the most commonly used rodent models of nonbacterial prostatitis. We studied the effect of the phosphodiesterase 5 inhibitor tadalafil on chronic pelvic pain in two such models in rats. METHODS: EAP was induced by intradermal injection of rat prostate antigen and complete Freund's adjuvant on Days 0 and 28. HCP was induced by castration followed by daily subcutaneous injection of 17ß-estradiol for 30 days. On Day 42 after antigen injection in the EAP model and Day 30 after castration in the HCP model, we investigated voiding behavior, pelvic pain (measured by applying von Frey filaments to the lower abdomen), and inflammatory changes, including changes in histopathology and IL-1ß, CCL2, and CCL3 mRNA levels. We investigated the effect of repeated administration of tadalafil on chronic pelvic pain in both models. RESULTS: In the EAP model, we observed inflammation in the ventral prostate, while in the HCP model, we observed inflammation in the lateral lobe of the prostate. Neither model showed any change in voiding behavior. As well as in the EAP model, in which chronic pelvic pain was observed, we found for the first time that HCP led to a significant increase in chronic pelvic pain. Repeated treatment with tadalafil attenuated the chronic pelvic pain in both models. CONCLUSIONS: Chronic pelvic pain was induced in both EAP and HCP models. Tadalafil significantly attenuated the chronic pelvic pain in both models.

Chronic pelvic pain and prostate inflammation in rat experimental autoimmune prostatitis: Effect of a single treatment with phosphodiesterase 5 inhibitors on chronic pelvic pain.

BACKGROUND: Experimental autoimmune prostatitis (EAP) is most often used as a nonbacterial model of chronic prostatitis/chronic pelvic pain. We investigated the development of chronic pelvic pain and inflammatory changes in rat EAP and examined the effect of a single treatment with phosphodiesterase 5 (PDE5) inhibitors on the chronic pelvic pain. METHODS: EAP was induced in rats by intradermal injection of rat prostate antigen and complete Freund's adjuvant on days 0 and 28. On day 42, after antigen injection, prostatic inflammatory changes, including the mRNA and protein levels of cytokines/chemokines, were measured and histological analysis of the prostate was performed. Pelvic pain was measured by applying von Frey filaments to the lower abdomen. To confirm that this model is appropriate for evaluating pelvic pain, we tested two drugs, celecoxib and pregabalin, which are clinically used for the treatment of prostatitis-related pain. Subsequently, we examined the effects of single treatments with three phosphodiesterase 5 inhibitors, including tadalafil, on pelvic pain in this model. RESULTS: On day 42, after antigen injection, the mRNA levels of 44 of 84 kinds of cytokines/chemokines and their receptors increased significantly in EAP rats, as did the protein levels of seven of 23 kinds of cytokines/chemokines. Histological analysis revealed inflammation characterized by neutrophils and/or mononuclear cells in the glandular and stromal tissue of the ventral prostate from rats in the EAP group. Some animals in this group showed fibrosis and hemorrhage in the stromal tissue. Pelvic pain had developed in EAP rats, which was attenuated by a single treatment with celecoxib or pregabalin, suggesting that EAP is an appropriate model for prostatitis-related pain. A single treatment with any of the three PDE5 inhibitors tested attenuated the chronic pelvic pain. CONCLUSIONS: Prostatitis leads to inflammatory changes in the prostate, which may contribute to the development and maintenance of chronic pelvic pain. PDE5 inhibitors, including tadalafil, may have the ability to block chronic pelvic pain.

Effect of tadalafil on chronic pelvic pain and prostatic inflammation in a rat model of experimental autoimmune prostatitis.

BACKGROUND: Experimental autoimmune prostatitis (EAP) shares important clinical features with clinical chronic prostatitis/chronic pelvic pain. We investigated the effect of tadalafil on pelvic pain and prostatic inflammation in a rat EAP model. METHODS: EAP was induced in rats by intradermal injection of rat prostate antigen and complete Freund's adjuvant on days 0 and 28. Rats were treated with tadalafil (2 mg/kg, p.o.; EAP-tadalafil) or vehicle (EAP-vehicle) once daily from day 0, while sham-operated animals were treated with vehicle only (Sham). Tactile allodynia was measured on days 28, 35, and 42 by applying von Frey filaments to the lower abdomen as an index of pelvic pain. On day 42, the plasma immunoglobulin G (IgG) concentration and the testosterone/estradiol ratio were measured and histopathological analysis of the prostate was performed. RESULTS: Tactile allodynia in the pelvic region was observed on days 28, 35, and 42 after EAP induction. The tactile allodynia observed on day 42 was significantly reduced by repeated treatment with tadalafil. Plasma IgG concentrations increased after EAP induction but the increase was not changed by tadalafil treatment. Prostate tissues were characterized by epithelial necrosis, infiltration of neutrophils and/or lymphocytes to acini and stroma, and fibrosis, in addition to a high stroma-to-epithelium ratio. Tadalafil treatment significantly suppressed the severity of the lesions. CONCLUSIONS: EAP rats developed pelvic pain, prostatic inflammation and increased plasma IgG concentrations. Tadalafil inhibited the chronic pelvic pain and prostatic inflammation, suggesting that its anti-inflammatory action may contribute to its blocking of pain development in the EAP model.

A comparison of vasodilation mode among selexipag (NS-304; [2-{4-[(5,6-diphenylpyrazin-2-yl)(isopropyl)amino]butoxy}-N-(methylsulfonyl)acetamide]), its active metabolite MRE-269 and various prostacyclin receptor agonists in rat, porcine and human pulmonary arteries.

Selexipag (NS-304; [2-{4-[(5,6-diphenylpyrazin-2-yl)(isopropyl)amino]butoxy}-N- (methylsulfonyl)acetamide]) is a novel, orally available non-prostanoid prostacyclin receptor (IP receptor) agonist that has recently been approved for the treatment of pulmonary arterial hypertension (PAH). We examined the effect of the active metabolite of selexipag, MRE-269, and IP receptor agonists that are currently available as PAH therapeutic drugs on the relaxation of rat, porcine and human pulmonary artery. cAMP formation in human pulmonary artery smooth muscle cells was induced by all test compounds (MRE-269, epoprostenol, iloprost, treprostinil and beraprost sodium) and suppressed by IP receptor antagonists (CAY10441 and 2-[4-(1H-indol-4-yloxymethyl)-benzyloxycarbonylamino]-3-phenyl-propionic acid). MRE-269 induced endothelium-independent vasodilation of rat extralobar pulmonary artery (EPA). In contrast, endothelial denudation or the addition of a nitric oxide synthase inhibitor markedly attenuated the vasodilation of EPA induced by epoprostenol, treprostinil and beraprost sodium but not iloprost. The vasorelaxant effects of MRE-269 on rat small intralobar pulmonary artery (SIPA) and EPA were the same, while the other IP receptor agonists induced less vasodilation in SIPA than in EPA. Furthermore, a prostaglandin E receptor 3 antagonist enhanced the vasodilation induced by all IP receptor agonists tested except MRE-269. We also investigated the relaxation induced by IP receptor agonists in pulmonary arteries from non-rodent species and found similar vasodilation modes in porcine and human as in rat preparations. These results suggest that MRE-269, in contrast to other IP receptor agonists, works as a selective IP receptor agonist, thus leading to pronounced vasorelaxation of rat, porcine and human pulmonary artery.

Effects of the phosphodiesterase 5 inhibitor Tadalafil on bladder function in a rat model of partial bladder outlet obstruction.

AIMS: To investigate the effect of the phosphodiesterase 5 (PDE5) inhibitor Tadalafil on bladder blood flow and bladder function in a rat model of partial bladder outlet obstruction (BOO). METHODS: Female 14-15-week-old Sprague-Dawley rats were divided into three groups. BOO was surgically induced in rats by placing a rubber ring around the urethra. BOO rats were administered daily oral Tadalafil (BOO-Tadalafil) or vehicle (BOO-Vehicle), while sham-operated animals were treated with vehicle (Sham). On the 14th day after surgery, micturition behavior was recorded for 24 hr by using a metabolic cage. On the 15th day after surgery, bladder blood flow and bladder weight were measured. The expression of PDE5 mRNA in the vesical and iliac arteries of intact rats was measured by reverse transcriptase-polymerase chain reaction. RESULTS: BOO led to a significant decrease in bladder blood flow and a significant increase in bladder weight. These changes were partially suppressed by Tadalafil treatment. The number of micturitions in the BOO group was significantly increased and the average micturition volume was significantly decreased, without affecting the total micturition volume. Repeated Tadalafil treatment markedly inhibited the increase in micturition frequency and the decrease in average micturition volume. PDE5 mRNA was expressed in the vesical and iliac arteries. CONCLUSION: Tadalafil suppressed the reduction in bladder blood flow caused by BOO in rats and improved urinary function. This action of Tadalafil may contribute to its amelioration of bladder function. Neurourol. Urodynam. 35:444-449, 2016. © 2015 Wiley Periodicals, Inc.

Effect of a single treatment with tadalafil on blood flow in lower urinary tract tissues in rat models of bladder overdistension/emptying and abdominal aorta clamping/release.

Impaired blood flow in lower urinary tract (LUT) tissues is a pathophysiological cause of LUT symptoms. We investigated the effects of the phosphodiesterase 5 (PDE5) inhibitor tadalafil on the sustained decrease in bladder blood flow (BBF) and time-dependent changes in BBF and prostate blood flow (PBF) resulting from ischemia/reperfusion in two rat models. In a rat model of bladder overdistension/emptying (O/E), the bladder was overdistended by saline infusion and emptied after 2h. Tadalafil was administered intraduodenally immediately after emptying. In a rat model of clamping/release (C/R), the abdominal aorta was clamped for 2h after a single oral dose of tadalafil and then the clamp was released. BBF in O/E and C/R rats and PBF in C/R rats were measured by laser Doppler flow imaging. BBF decreased on overdistension and partially recovered after emptying. A progressive decrease in BBF was observed after O/E, and this was prevented by tadalafil treatment. Both BBF and PBF decreased during clamping of the abdominal aorta and partially recovered after clamp removal. Oral pretreatment with tadalafil partially or completely prevented the decreases in BBF and PBF not only after clamp removal but also during clamping. PDE5 mRNA was highly expressed in the bladder and the supporting vasculature. Tadalafil inhibited the O/E-induced decrease in BBF and the C/R-induced time-dependent decreases in BBF and PBF. PDE5 inhibition by tadalafil may improve both BBF and PBF.

The analgesic effect of tramadol in animal models of neuropathic pain and fibromyalgia.

(±)-Tramadol hydrochloride (tramadol) is a widely used analgesic for the treatment of cancer pain and chronic pain. Although many animal studies have shown antinociceptive effects of tramadol in both acute and chronic pain, little is known about the effect of tramadol in putative animal models of fibromyalgia. In this study, we compared the antiallodynic effects of oral administration of tramadol in two kinds of rat chronic pain models, neuropathic pain induced by partial sciatic nerve ligation (PSL) and reserpine-induced myalgia (RIM). In PSL rats, the threshold for responses induced by tactile stimulation with von Frey filaments was significantly decreased seven days after the operation, suggesting that the operation induced tactile allodynia. Orally administered tramadol showed a potent and dose-dependent antiallodynic effect on PSL-induced allodynia. In RIM rats, the threshold was significantly decreased five days after reserpine treatment. Orally administered tramadol also attenuated reserpine-induced tactile allodynia. To explore the mechanism of the antiallodynic effect of tramadol in RIM rats, we investigated the effect of the opioid antagonist naloxone on the tramadol-induced analgesic effect in these rats. The effect of tramadol was partially antagonized by naloxone, suggesting that the opioid receptor is involved at least in part in the antiallodynic effect of tramadol in RIM rats. These data indicate that orally administered tramadol produced improvement in both PSL rats and RIM rats at similar doses and provide evidence that the opioid system is partly involved in the analgesic effect of tramadol in RIM rats.

Acamprosate {monocalcium bis(3-acetamidopropane-1-sulfonate)} reduces ethanol-drinking behavior in rats and glutamate-induced toxicity in ethanol-exposed primary rat cortical neuronal cultures.

Acamprosate, the calcium salt of bis(3-acetamidopropane-1-sulfonate), contributes to the maintenance of abstinence in alcohol-dependent patients, but its mechanism of action in the central nervous system is unclear. Here, we report the effect of acamprosate on ethanol-drinking behavior in standard laboratory Wistar rats, including voluntary ethanol consumption and the ethanol-deprivation effect. After forced ethanol consumption arranged by the provision of only one drinking bottle containing 10% ethanol, the rats were given a choice between two drinking bottles, one containing water and the other containing 10% ethanol. In rats selected for high ethanol preference, repeated oral administration of acamprosate diminished voluntary ethanol drinking. After three months of continuous access to two bottles, rats were deprived of ethanol for three days and then presented with two bottles again. After ethanol deprivation, ethanol preference was increased, and the increase was largely abolished by acamprosate. After exposure of primary neuronal cultures of rat cerebral cortex to ethanol for four days, neurotoxicity, as measured by the extracellular leakage of lactate dehydrogenase (LDH), was induced by incubation with glutamate for 1h followed by incubation in the absence of ethanol for 24h. The N-methyl-D-aspartate receptor blocker 5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine, the metabotropic glutamate receptor subtype 5 antagonist 6-methyl-2-(phenylethynyl)pyridine and the voltage-gated calcium-channel blocker nifedipine all inhibited glutamate-induced LDH leakage from ethanol-exposed neurons. Acamprosate inhibited the glutamate-induced LDH leakage from ethanol-exposed neurons more strongly than that from intact neurons. In conclusion, acamprosate showed effective reduction of drinking behavior in rats and protected ethanol-exposed neurons by multiple blocking of glutamate signaling.

Acamprosate suppresses ethanol-induced place preference in mice with ethanol physical dependence.

The present study investigated the effect of acamprosate on ethanol (EtOH)-induced place preference in mice with EtOH physical dependence. The expression of EtOH (2 g/kg, intraperitoneally)-induced place preference in mice without EtOH treatment before the experiment was dose-dependently suppressed by acamprosate. The levels of protein kinase A (PKA) and phospho-cAMP response element binding protein (p-CREB) in the limbic forebrain after EtOH-conditioning in naïve mice was unchanged. Furthermore, mice on the 4th day of withdrawal from continuous EtOH vapor inhalation for 9 days showed transient and significant enhancement of EtOH (1 g/kg, intraperitoneally)-induced place preference, which was significantly suppressed by acamprosate (300 mg/kg, oral administration; p.o., once a day) administered daily for 3 days after withdrawal from EtOH inhalation and during EtOH-conditioning. PKA and p-CREB proteins in the limbic forebrain of EtOH-conditioned mice on 4th day of withdrawal from continuous EtOH inhalation for 9 days significantly increased, which were completely abolished by acamprosate. These findings suggest that the signal transduction pathway via the PKA-p-CREB pathway in the limbic forebrain may be functionally related to the development of sensitization of EtOH-induced place preference and provide a possible molecular basis for the pharmacological effect of acamprosate to prevent or reduce the relapse of alcohol dependence.

Bladder function in 17ß-estradiol-induced nonbacterial prostatitis model in Wistar rat.

OBJECTIVES: To investigate bladder function in a model of nonbacterial prostatitis (NBP) induced in castrated rats by 17ß-estradiol injection. METHODS: Ten-month-old male Wistar rats were divided into two groups, sham and NBP (both N = 8). NBP was induced by castration followed by daily subcutaneous injection of 17ß-estradiol for 30 days. On the 31st day after surgery, we investigated (1) voiding behavior, (2) bladder blood flow (BBF), (3) prostate and bladder weight, and proinflammatory cytokines (TNF-α and CXCL1) levels and (4) bladder contractile responses to electrical field stimulation (EFS), carbachol and KCl. RESULTS: (1) Voiding behavior (average micturition volume, total urine volume and number of micturitions) and (2) BBF were not significantly different between the sham and NBP groups. (3) NBP led to a significant decrease in prostatic weight and increase in proinflammatory cytokine levels in the prostate, but NBP did not cause a significant change in bladder weight or proinflammatory cytokine levels in the bladder. (4) Bladder contractile forces in response to EFS, carbachol and KCl were not significantly affected by NBP. CONCLUSIONS: In this rat model, NBP did not cause a significant change in the level of proinflammatory cytokines in the bladder and affect bladder function.

Effect of the phytotherapeutic agent eviprostat on the bladder in a rat model of bladder overdistension/emptying.

AIMS: Ischemia-reperfusion injury is an important factor in the development of lower urinary tract symptoms (LUTS) that is partly mediated by the generation of free radicals. We investigate the effect of the phytotherapeutic agent Eviprostat, a treatment for benign prostatic hyperplasia (BPH) that has antioxidant and anti-inflammatory activity, on urinary bladder blood flow (BBF), and function in a rat model of bladder overdistension and emptying (OE). METHODS: For 8 days before surgery, OE rats received daily oral Eviprostat (36 mg/kg/day) or vehicle, while sham-operated animals received vehicle. The bladder was distended by infusion of saline over a period of 2 hr (overdistension) and then emptied. After 24 hr, BBF was measured with a laser speckle blood flow imager. The oxidative-stress marker malondialdehyde (MDA), proinflammatory cytokines, and myeloperoxidase were determined in the isolated bladder, and histological analysis was performed. Functional contractile responses of bladder strips to electrical field stimulation, carbachol, and KCl were measured. RESULTS: Twenty-four hours after bladder OE, a significant decrease in BBF and significant increases in bladder weight, malondialdehyde, proinflammatory cytokines, and myeloperoxidase were observed. Eviprostat almost completely prevented these changes. Histological analysis of the bladder of OE rats showed hemorrhage, accumulation of leukocytes, desquamation of epithelium, and edema, and Eviprostat suppressed these changes. The reduction in functional contractile forces in the bladder of OE rats was also prevented by Eviprostat. CONCLUSION: Eviprostat-mediated suppression of increased bladder oxidative stress and inflammation caused by bladder OE may contribute to the improvement of BBF and bladder function by Eviprostat.

Tramadol enhances urethral continence function through µ-opioid receptors in rats.

AIMS: (±)-Tramadol hydrochloride (tramadol) is a widely used analgesic that stimulates the µ-opioid receptor and inhibits the reuptake of serotonin and noradrenalin. Although tramadol is also known to inhibit the micturition reflex in rats, its effects on urethral continence function have not been reported. We therefore examined whether intravenous tramadol (1, 3, and 10 mg/kg) affects intraurethral pressure, bladder leak point pressure, and leak volume in urethane-anesthetized female rats. METHODS: (1) The intraurethral pressure was recorded with a microtip pressure transducer placed at the maximum pressure zone of the intrinsic urethral sphincter. (2) Gentle pressure was directly applied to the saline-filled bladder with a cotton bud until leakage occurred, and the bladder pressure at the moment of leakage was taken as the bladder leak point pressure. (3) The leak volume was measured as the amount of fluid leakage from the urethral orifice after electrical stimulation of abdominal muscles. RESULTS: Tramadol significantly increased the intraurethral pressure. Both tramadol and morphine increased the bladder leak point pressure and decreased the leak volume. These changes were reversed by subcutaneous pretreatment with naloxone. CONCLUSIONS: Tramadol improved urethral function and inhibited urinary incontinence through µ-opioid receptors.

Blockade of voltage-gated calcium channel Cav1.2 and α1-adrenoceptors increases vertebral artery blood flow induced by the antivertigo agent difenidol.

Difenidol (1,1-diphenyl-4-piperidino-1-butanol hydrochloride) is an effective drug for the treatment of vertigo and dizziness. This drug is known to improve the blood flow in vertebral arteries, though the precise mechanism underlying this action remains unclear. In the present study, we investigated the effect of difenidol on voltage-gated calcium channel Ca(v)1.2 and α(1)-adrenoceptor subtypes that regulate the intracellular calcium concentration ([Ca(2+)](i)), as well as their possible involvement in the action of difenidol on vertebral artery relaxation and blood flow in dogs. In vitro binding assays demonstrated that difenidol at micromolar concentrations bound to the α(1A)-, α(1B)- and α(1D)-adrenoceptor subtypes. Difenidol inhibited the phenylephrine-induced increase in [Ca(2+)](i) in Chinese hamster ovary cells expressing human α(1A)-, α(1B)- or α(1D)-adrenoceptor subtypes with similar IC(50) values in the low micromolar range. In an electrophysiological assay, difenidol inhibited L-type calcium channel (Ca(v)1.2 subunit). In dogs, i.v. difenidol preferentially enhanced vertebral over femoral arterial blood flow. Phenylephrine and potassium induced contraction of dog vertebral arterial rings, and difenidol inhibited this action. Inhibition of phenylephrine-induced contraction by difenidol was mimicked by the α(1)-adrenoceptor antagonist phentolamine, the α(1A)-adrenoceptor antagonist RS 17,053 (N-[2-(2-cyclopropylmethoxyphenoxy)ethyl]-5-chloro-α,α-dimethyl-1H-indole-3-ethanamine hydrochloride) and the α(1D)-adrenoceptor antagonist BMY 7378 (8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4,5]decane-7,9-dione dihydrochloride). In addition, the L-type calcium channel blocker nifedipine, like difenidol, attenuated the potassium-induced contraction. These findings suggest that the difenidol-induced increase in vertebral arterial blood flow may be due to vascular relaxation mediated by mixed blocking actions at α(1)-adrenoceptors and voltage-gated calcium channel Ca(v)1.2.

Suppression of bladder overactivity and oxidative stress by the phytotherapeutic agent, Eviprostat, in a rat model of atherosclerosis-induced chronic bladder ischemia.

OBJECTIVES: To clarify the mechanism by which chronic bladder ischemia causes bladder functional changes, and to investigate the involvement of oxidative stress and pro-inflammatory cytokines, and the effects of the phytotherapeutic drug, Eviprostat, on these biochemical marker levels and bladder function. METHODS: Male Sprague-Dawley rats aged 15 weeks were divided into three groups. Arterial injury was experimentally induced by balloon endothelial injury of the iliac arteries, and a 2% cholesterol diet was given for 8 weeks. Rats in the arterial-injury group were given daily oral vehicle or Eviprostat, whereas sham-operated animals on a regular diet (0.09% cholesterol) were given vehicle for the last 2 weeks. Eight weeks after surgery, the levels of bladder pro-inflammatory cytokines, as well as bladder and urinary oxidative-stress markers, were determined. Cystometrograms were carried out without anesthesia or restraint. RESULTS: Bladder and urinary oxidative-stress markers, and bladder pro-inflammatory cytokine levels were significantly increased in the arterial-injury group, and Eviprostat markedly suppressed these increase. The cystometrograms showed that arterial injury decreased the intermicturition interval without affecting the micturition pressure. This decrease was reversed by Eviprostat treatment. CONCLUSIONS: Oxidative stress and pro-inflammatory cytokines might be involved in the development of overactive bladder by atherosclerosis-induced chronic bladder ischemia. Eviprostat might provide an attractive treatment option for individuals with bladder dysfunction due to chronic bladder ischemia because of its anti-oxidant and anti-inflammatory properties.

Effect of tramadol on pain-related behaviors and bladder overactivity in rodent cystitis models.

Tramadol is a widely used analgesic that stimulates the µ opioid receptor and inhibits serotonin and noradrenalin reuptake. There have been studies on the analgesic effects of tramadol based on the tail-flick test, the formalin test, and the induction of allodynia by sciatic-nerve ligation. However, the effects of tramadol on behaviors related to bladder pain and bladder overactivity induced by cystitis have not been reported. To investigate the usefulness of tramadol for patients with cystitis, we investigated these effects of tramadol in rodent cystitis models. Intraperitoneal injection of cyclophosphamide caused bladder-specific inflammation and increases in pain-related behaviors, the number of voids and bladder weight in mice. Tramadol suppressed the cyclophosphamide-induced pain-related behaviors but did not affect the number of voids or the bladder weight. During continuous-infusion cystometrograms in anesthetized rats, cyclophosphamide shortened the intercontraction interval, indicating bladder overactivity. Tramadol significantly prolonged the intercontraction interval, and the effect was partially blocked by the opioid antagonist naloxone. This finding indicates that µ opioid receptors may be involved in the action of tramadol. In conclusion, tramadol ameliorated cyclophosphamide-induced bladder-pain-related behaviors and bladder overactivity in rodents. These findings suggest that tramadol might be a treatment option for cystitis-induced bladder pain and bladder overactivity.

Increased bladder activity is associated with elevated oxidative stress markers and proinflammatory cytokines in a rat model of atherosclerosis-induced chronic bladder ischemia.

AIMS: To further characterize, in a rat model, the effects of atherosclerosis-induced chronic bladder ischemia on bladder function and associated changes in oxidative stress markers and proinflammatory cytokines. METHODS: Adult Sprague-Dawley male rats were divided into three groups (arterial endothelial injury: AI, sham, naïve). The AI group (n = 14) underwent endothelial injury of the iliac arteries and received a 2% cholesterol diet. The sham group (n = 12) underwent sham operation and received a 2% cholesterol diet. The naïve group (n = 12) received a regular diet. After 8 weeks, cystometrograms (CMG) without anesthesia or restraint were performed. In bladders from each group, oxidative stress markers (8-hydroxy-2'-deoxyguanosine: 8-OHdG; malondialdehyde: MDA) and proinflammatory cytokines (IL-8 like cytokine CXCL1/CINC-1, TNF-α, IL-6) were quantified. Histological examination of the iliac arteries was also performed. RESULTS: At 8 weeks, the body and bladder wet weights were not significant different among the three groups. The micturition interval in the AI group decreased significantly compared with those in the other two groups, but maximum pressure during micturition did not change. The iliac arteries in the AI group revealed thickening of intima as well as diffuse media fibrosis at the sites of balloon injury. The levels of oxidative stress markers and proinflammatory cytokines were significantly higher in the AI than in the other groups. CONCLUSION: Oxidative stress and inflammation may be key factors in the development of bladder overactivity in atherosclerosis-induced chronic bladder ischemia.