Concentration-dependent Contractions Research Articles

Residual NO modulates contractile responses and membrane potential in isolated rat mesenteric arteries

Shear stress or vasocontriction causes endothelial nitric oxide (NO) release resulting in the regulation of vascular smooth muscle tone in small resistance arteries. Generation of NO is inhibited by nitric oxide synthase (NOS) inhibitors. In this study, we investigated the effect of residual NO, released even in the presence of NOS inhibitors, on the membrane depolarization and phenylephrine-induced contractions of smooth muscle. For this purpose, we used hydroxocobalamin (HC), an NO scavenger, in the presence of NOS inhibitiors, Nω-nitro- L-arginine (L-NA) or Nω-nitro-L-arginine methyl ester (L-NAME) in mesenteric arteries isolated from rats. Phenylephrine (0,01–10 μM), an α1-adrenoceptor agonist, led to depolarisation and concentration-dependent contraction in mesenteric arteries. The depolarisation and contractile responses were augmented by L-NA or L-NAME. Hydroxocobalamine (HC) or carboxy-PTIO (c-PTIO) also caused to further increase the membrane depolarization and contractions induced by phenylephrine in the presence of NOS inhibitors. Chemical removal of endothelium by saponin, tyrosin kinase inhibitor erbstatin A, but not calmodulin inhibitor calmidazolium inhibited the additional membrane depolarisation and contractile responses induced by L-NA or L-NAME and L-NA or L-NAME plus HC. These findings show that residual NO modulates the contractile responses in isolated rat mesenteric arteries by exerting a tonic inhibitor effect on the depolarization and vasoconstriction induced by phenylephrine.

Attenuation of the anti-contractile effect of cooling in the rat aorta by perivascular adipose tissue.

In addition to providing mechanical support for blood vessels, the perivascular adipose tissue (PVAT) secretes a number of vasoactive substances and exerts an anticontractile effect. The main objective of this study was to find out whether the anticontractile effect of cooling in the rat aorta is affected by PVAT. Our hypothesis was that PVAT would enhance the anticontractile effect of cooling in the rat aorta. Aorta segments, with or without PVAT, were used in this investigation. Cumulative concentration-response curves were established for phenylephrine at 37°C or 24°C. Phenylephrine (10-9 M - 10-5 M) induced concentration-dependent contractions of aorta segments with or without PVAT at 37°C. The maximum response, but not pD2 value, was reduced in aorta segments with PVAT. Cooling the tissues to 24 °C resulted in a significant reduction in the maximum response in aorta segments without PVAT with no change in pD2 values. However, the anticontractile effect of cooling was attenuated in the presence of PVAT with no significant (p > 0.05) change in either the maximum response or pD2 value. L-NAME potentiated PE-induced contractions and this was greater in aorta segments without PVAT at both temperatures. The expression of eNOS protein and basal tissue level of nitric oxide (NO) were greater in aorta segments with PVAT at both temperatures. However, PE significantly increased tissue levels of NO only in aorta segments without PVAT. We concluded that PVAT-induced loss of anticontractile effect of cooling against PE-induced contractions could be due to impaired generation of NO in aorta segments with PVAT.

Atypical sympathomimetic drug lerimazoline mediates contractile effects in rat aorta predominantly by 5-HT2A receptors.

Lerimazoline is a sympathomimetic drug that belongs to the imidazoline class of compounds, and is used as a nasal decongestant. Studies on lerimazoline are rare, and its pharmacological profile is not completely understood. Here, we analyzed the affinity of lerimazoline for dopamine receptor D2, serotonin 5-HT1A and 5-HT2A receptors and α1-adrenoceptor, and investigated lerimazoline contractile effects in isolated rat thoracic aorta. We also determined the effect of several antagonists on the contractile response to lerimazoline, including prazosin (α1-adrenoceptor antagonist), RX 821002 and rauwolscine (α2-adrenoceptor antagonists), JP 1302 (α2C-adrenoceptor antagonist), methiothepin (non-selective 5-HT receptor antagonist), SB 224289 (5-HT1B receptor antagonist), BRL 15572 (5-HT1D receptor antagonist), and ketanserin (5-HT2A receptor antagonist). Lerimazoline displayed high affinity for the 5-HT1A receptor (Ki = 162.5 nM), similar to the previously reported affinity for the 5-HT1D receptor. Binding affinity estimates (Ki) for α1, 5-HT2A, and D2 receptors were 6656, 4202 and 3437.5 nM, respectively (the literature reported Ki for 5-HT1B receptor is 3480 nM). Lerimazoline caused concentration-dependent contractions in 70% of preparations, varying in the range between 40% and 55% of the maximal contraction elicited by phenylephrine. While prazosin reduced the maximum contractile response to lerimazoline, rauwolscine showed a non-significant trend in reduction of the response. Both ketanserin (10 nM and 1 µM) and methiothepin strongly suppressed the maximum response to lerimazoline. Overall, our results suggest that 5-HT2A and, less distinctly, α1-adrenergic receptors are involved in the lerimazoline-induced contractions, which makes lerimazoline an "atypical" decongestant.

A critical role for cystathionine-β-synthase in hydrogen sulfide-mediated hypoxic relaxation of the coronary artery

Hypoxia-induced coronary artery vasodilatation protects the heart by increasing blood flow under ischemic conditions, however its mechanism is not fully elucidated. Hydrogen sulfide (H2S) is reported to be an oxygen sensor/transducer in the vasculature. The present study aimed to identify and characterise the role of H2S in the hypoxic response of the coronary artery, and to define the H2S synthetic enzymes involved. Immunoblotting and immunohistochemistry showed expression of all three H2S-producing enzymes, cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (MPST), in porcine coronary artery. Artery segments were mounted for isometric tension recording; hypoxia caused a transient endothelium-dependent contraction followed by prolonged endothelium-independent relaxation. The CBS inhibitor amino-oxyacetate (AOAA) reduced both phases of the hypoxic response. The CSE inhibitor dl-propargylglycine (PPG) and aspartate (limits MPST) had no effect alone, but when applied together with AOAA the hypoxic relaxation response was further reduced. Exogenous H2S (Na2S and NaHS) produced concentration-dependent contraction followed by prolonged relaxation. Responses to both hypoxia and exogenous H2S were dependent on the endothelium, NO, cGMP, K+ channels and Cl−/HCO3– exchange. H2S production in coronary arteries was blocked by CBS inhibition (AOAA), but not by CSE inhibition (PPG). These data show that H2S is an endogenous mediator of the hypoxic response in coronary arteries. Of the three H2S-producing enzymes, CBS, expressed in the vascular smooth muscle, appears to be the most important for H2S generated during hypoxic relaxation of the coronary artery. A contribution from other H2S-producing enzymes only becomes apparent when CBS activity is inhibited.

Hypercholesterolemia impairs oxytocin-induced uterine contractility in late pregnant mouse.

High cholesterol is known to negatively affect uterine contractility in ex vivo conditions. The aim of the present study was to reveal the effect of in vivo hypercholesterolemia on spontaneous and oxytocin-induced uterine contractility in late pregnant mouse uterus. Female Swiss albino mice were fed with high cholesterol (HC) diet (0.5% sodium cholate, 1.25% cholesterol and 15% fat) for 6 weeks and then throughout the gestation period after mating. On day 19 of gestation, serum cholesterol level was increased more than 3-fold while triglycerides level was reduced in HC diet-fed animals as compared to control animals fed with a standard diet. In tension experiments, neither the mean integral tension of spontaneous contractility nor the response to CaCl2 in high K+-depolarized tissues was altered, but the oxytocin-induced concentration-dependent contractile response in uterine strips was attenuated in hypercholesterolemic mice as compared to control. Similarly, hypercholesterolemia dampened concentration-dependent uterine contractions elicited by a GNAQ protein activator, Pasteurella multocida toxin. However, it had no effect on endogenous oxytocin level either in plasma or in uterine tissue. It also did not affect the prostaglandin release in oxytocin-stimulated tissues. Western blot data showed a significant increase in caveolin-1 and GRK6 proteins but decline in oxytocin receptor, GNAQ and RHOA protein expressions in hypercholesterolemic mouse uterus. The results of the present study suggest that hypercholesterolemia may attenuate the uterotonic action of oxytocin in late pregnancy by causing downregulation of oxytocin receptors and suppressing the signaling efficacy through GNAQ and RHOA proteins.

Inhibition of agonist-induced smooth muscle contraction by picotamide in the male human lower urinary tract outflow region

Male lower urinary tract symptoms (LUTS) due to bladder outlet obstruction are characterized by abnormal smooth muscle contractions in the lower urinary tract. Alpha1-adrenoceptor antagonists may induce smooth muscle relaxation in the outflow region and represent the current gold standard of medical treatment. However, results may be unsatisfactory or inadequate. Apart from α1-adrenoceptor agonists, smooth muscle contraction in the outflow region may be induced by thromboxane A2 (TXA2), endothelins, or muscarinic receptor agonists. Here, we studied effects of the thromboxane A2 receptor (TP receptor) antagonist picotamide on contraction in the human male bladder trigone and prostate. Carbachol, the α1-adrenoceptor agonist phenylephrine, the thromboxane A2 analog U46619, and electric field stimulation (EFS) induced concentration- or frequency-dependent contractions of trigone tissues in an organ bath. Picotamide (300µM) inhibited carbachol-, phenylephrine-, U46619-, and EFS-induced contractions. Endothelins 1-3 induced concentration-dependent contractions of prostate tissues, which were inhibited by picotamide. Analyses using real time polymerase chain reaction and antibodies suggested expression of thromboxane A2 receptors and synthase in trigone smooth muscle cells. Thromboxane B2 (the stable metabolite of thromboxane A2) was detectable by enzyme immune assay in trigone samples, with most values ranging between 50 and 150pg/mg trigone protein. Picotamide inhibits contractions induced by different stimuli in the human lower urinary tract, including cholinergic, adrenergic, thromboxane A2- and endothelin-induced, and neurogenic contractions in different locations of the outflow region. This distinguishes picotamide from current medical treatments for LUTS, and suggests that picotamide may induce urodynamic effects in vivo.

Impaired Aortic Contractility to Uridine Adenosine Tetraphosphate in Angiotensin II-Induced Hypertensive Mice: Receptor Desensitization?

We previously showed that uridine adenosine tetraphosphate (Up4A)-mediated aortic contraction is partly mediated through purinergic P2X1 receptors (P2X1R). It has been reported that the plasma level of Up4A is elevated in hypertensive patients, implying a potential role for Up4A-P2X1R signaling in hypertension. This study investigated the vasoactive effect of Up4A in aortas isolated from angiotensin (Ang) II-infused (21 days) hypertensive mice. Blood pressure was measured by tail cuff plethysmography. Aortas were isolated for isometric tension measurements, and protein expression was analyzed by western blot. Mean and systolic arterial pressures were elevated by ~50% in Ang II-infused mice. Protein levels of both AT1R and P2X1R were upregulated in Ang II-infused aortas. Surprisingly, Up4A (10-9-10-5 M)-induced concentration-dependent contraction was significantly impaired in Ang II-infused mice. Studies in control mice revealed that both P2X1R (MRS2159) and AT1R (losartan) antagonists significantly attenuated Up4A-induced aortic contraction. In addition, desensitization of AT1R by prior Ang II (100 nM) exposure had no effect on Up4A-induced aortic contraction. However, subsequent serial exposure responses to Up4A-induced aortic contraction were markedly reduced, suggesting a desensitization of purinergic receptors. This desensitization was further confirmed in control mice by prior exposure of aortas to the P2X1R desensitizer α, β-methylene ATP (10 μM). Despite upregulation of AT1R and P2X1R in hypertension, Up4A-mediated aortic contraction was impaired in Ang II-infused mice, likely through the desensitization of P2X1R but not AT1R. This implies that vascular P2X1R activity, rather than plasma Up4A level, may determine the role of Up4A in hypertension.

Moderate hypothermia attenuates α1-adrenoceptor-mediated contraction in human varicose spermatic vein: The role of nitric oxide(Short communication).

The effects of moderate hypothermia (28°C) on the response of human varicose spermatic vein to α1-adrenoceptor agonist phenylephrine and the role of endothelial nitric oxide (NO) in these effects were studied. Concentration-response curves for phenylephrine (10-9 to 3 × 10-4 M) were recorded in rings with and without endothelium at 37 and 28°C. To further analyze the role of NO, in the response to phenylephrine during hypothermia, the effects of this agonist in the presence of NG-nitro-L-arginine methyl ester (10-4 M) were also determined. Under every condition tested, phenylephrine produced a marked, concentration-dependent contraction. Sensitivity of intact veins to the agonist was consistently lower at 28°C than at 37°C. There was no significant difference in phenylephrine response at 28 and 37°C in vessels without endothelium but at 28°C veins without endothelium showed a higher sensitivity than intact veins to phenylephrine. The sensitivity of veins with and without endothelium to nitroprusside (10-9 to 3 × 10-3 M) was significantly decreased during hypothermia, and endothelium removal did not affect the relaxation to this nitrovasodilator. These results suggest that moderate hypothermia decreases the sensitivity of human varicose spermatic vein to phenylephrine probably by increasing the availability of endothelial NO.

Chemerin-induced arterial contraction is Gi- and calcium-dependent

Chemerin is an adipokine associated with increased blood pressure, and may link obesity with hypertension. We tested the hypothesis that chemerin-induced contraction of the vasculature occurs via calcium flux in smooth muscle cells. Isometric contraction of rat aortic rings was performed in parallel with calcium kinetics of rat aortic smooth muscle cells to assess the possible signaling pathway. Chemerin-9 (nonapeptide of the chemerin S157 isoform) caused a concentration-dependent contraction of isolated aorta (EC50 100nM) and elicited a concentration-dependent intracellular calcium response (EC50 10nM). Pertussis toxin (Gi inhibitor), verapamil (L-type Ca2+ channel inhibitor), PP1 (Src inhibitor), and Y27632 (Rho kinase inhibitor) reduced both calcium influx and isometric contraction to chemerin-9 but PD098059 (Erk MAPK inhibitor) and U73122 (PLC inhibitor) had little to no effect on either measure of chemerin signaling. Although our primary aim was to examine chemerin signaling, we also highlight differences in the mechanisms of chemerin-9 and recombinant chemerin S157. These data support a chemerin-induced contractile mechanism in vascular smooth muscle that functions through Gi proteins to activate L-type Ca2+ channels, Src, and Rho kinase. There is mounting evidence linking chemerin to hypertension and this mechanism brings us closer to targeting chemerin as a form of therapy.

Abstract P104: Role of Adenosine Receptors in Angiotensin II Dependent Hypertension in Mice

Our recent finding showed that acute angiotensin (ANG) II stimulation enhanced A 1 adenosine receptor (AR) dependent cytochrome P450 (CYP) 4A mediated contraction in mouse mesenteric arteries (MA). In addition, A 2B AR dependent and partly K ATP channel mediated relaxation was also reduced with ANGII stimulation. These data suggest a possible interaction between ANG II and ARs at vascular level which may play a role in ANG II dependent hypertension. We developed ANG II (1000 ng/kg/min, 21 days) infused hypertension mouse model and tail cuff was used to measure blood pressure. Aorta as a conduit and MA (second order) as a resistance artery were chosen for muscle tension studies. Protein expressions in aortas and MAs were analyzed by western blot. Infusion of ANG II increased systolic arterial blood pressure (SAP) in mice. Particularly, in ANG II infused mice, SAP (in mmHg, mean ± SD) at day 0 was 101.6 ± 6.675 which increased to 156.7 ± 15.68 on day 21 (p<0.05) while in saline control mice, SAP was 100.8 ± 6.753 (day 0) and changed to 105.4 ± 7.620 (day 21). Higher expression of A 1 AR (~155% over 100%) and CYP4A (~50%, ~0.6 over 0.3, ratio to actin) was present in MAs in hypertensive compared to control mice. Expression of A 1 AR and CYP4A was comparable in aortas of hypertensive and control mice. CCPA (A 1 AR agonist) -induced concentration dependent contraction (% normalize to phenylephrine contraction) was significantly reduced (~15% from 40% at 100 nM) in aorta of hypertensive mice compared to control. NECA (non-selective AR agonist) produced a significantly higher (~ -15% from 0% at 10 μM) relaxation in aorta of hypertensive mice. In MAs, CCPA-induced contraction was reduced in hypertensive mice. NECA induced relaxation was comparable in MAs. Pinacidil (K ATP channel opener) induced relaxation was significantly lower (~0% from -25% at 100 nM) in MAs of hypertensive mice. In conclusion, our data reveal important role for A 1 AR dependent signaling in ANG II induced hypertension. Higher A1AR and CYP4A may reduce K ATP channel dependent relaxation of MAs aiding vascular contraction and blood pressure. Further studies involving ARs pharmacological inhibitors and genetic models are required to fully understand the relationship between ANG II and ARs in the development of hypertension.

Abstract P182: The Novel Perivascular Adipose Tissue Adipokine, Chemerin, Signals Through G i - and Calcium-Dependent Mechanisms

Chemerin is an adipokine associated with inflammation, increased blood pressure, and may be a link between the pathologies of obesity and hypertension. We tested the hypothesis that chemerin-induced contraction of the vasculature occurs via the chemerin receptor and calcium flux in smooth muscle cells. Known mediators of the amplified arterial responsiveness seen in hypertension (L-type Ca 2+ channels, Src, and Rho kinase) were interrogated by isometric contraction of rat aortic rings in parallel with calcium kinetics of rat aortic smooth muscle cells. Western blots were also used to observe phosphorylation of Erk/MAPK. Chemerin-9 (nonapeptide of the chemerin S 157 isoform) caused a concentration-dependent contraction of isolated aorta (EC 50 100 nM) and elicited a concentration-dependent intracellular calcium response (EC 50 10 nM). Both calcium influx and isometric contraction, respectively, were reduced (units of “% of vehicle response”) by Pertussis toxin (G i inhibitor; 0±3% and 23±9%), verapamil (L-type Ca 2+ channel inhibitor; 38±20% and 23±4%), PP1 (Src inhibitor; 43±23% and 15±4%), and Y27632 (Rho Kinase inhibitor; 58±23% and 22±4%) but U73122 (PLC inhibitor) had little to no effect (71±31% and 71±12%). PD098059 (Erk/MAPK inhibitor) did not inhibit chemerin-9 induced contraction (117±19%) and phosphorylation did not change after chemerin-9 stimulation [1.12±0.14 (44 kDa) and 1.11±0.29 (42 kDa) fold-increase with chemerin-9 contraction compared to vehicle, p>0.05]. The chemerin receptor-selective antagonist CCX832 inhibited chemerin-9-induced calcium flux and aortic contraction and calcium flux (0.1±10.3% and 10±7%). These data support a chemerin-induced contractile mechanism in vascular smooth muscle that functions through the G i -linked chemerin receptor to activate L-type Ca 2+ channels, Src, and Rho kinase. There is mounting evidence linking chemerin to hypertension and this mechanism brings us one step closer to targeting chemerin as a unique form of therapy.

Novel pentapeptide, PALAL, derived from a bony fish elicits contraction of the muscle in starfish Patiria pectinifera.

A bioactive peptide mimicking peptide-signaling molecules has been isolated from the skin extract of fish Channa argus which caused contraction of the apical muscle of a starfish Patiria pectinifera, a deuterostomian invertebrate. The primary structure of the isolated pentapeptide comprises amino acid sequence of H-Pro-Ala-Leu-Ala-Leu-OH (PALAL) with a molecular mass of 483.7 Da. Pharmacological activity of PALAL, dosage ranging from 10-9 to 10-5 M, revealed concentration-dependent contraction of the apical muscles of P. pectinifera and Asterias amurensis. However, PALAL was not active on the intestinal smooth muscle of the goldfish Carassius auratus and has presumably other physiological roles in fish skin. Investigation of structure-activity relationship using truncated and substituted analogs of PALAL demonstrated that H-Ala-Leu-Ala-Leu-OH was necessary and should be sufficient to constrict apical muscle of P. pectinifera. Furthermore, the second alanine residue was required to display the activity, and the fifth leucine residue was responsible for its potency. Comparison with PALAL's primary structure with those of other known bioactive peptides from fish and starfish revealed that PALAL does not have any significant homology. Consequently, PALAL is a bioactive peptide that elicits a muscle contraction in starfish, and the isolation of PALAL may lead to develop other bioactive peptides sharing its similar sequence and/or activity. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Inhibition of Adrenergic and Non-Adrenergic Smooth Muscle Contraction in the Human Prostate by the Phosphodiesterase 10-Selective Inhibitor TC-E 5005.

The phosphodiesterase (PDE) 5 inhibitor tadalafil is available for treatment of male lower urinary tract symptoms (LUTS), while the role of other PDE isoforms for prostate smooth muscle tone is still unknown. Here, we examined effects of the PDE10-selective inhibitor TC-E 5005 on smooth muscle contraction in human prostate tissue. Prostate samples were obtained from patients undergoing radical prostatectomy. Expression of PDE10 was addressed by RT-PCR, Western blot, and fluorescence staining with different markers. Effects of TC-E 5005 and tadalafil on contraction, and relaxation of prostate strips were studied via organ bath. PDE10A was detectable by RT-PCR, Western blot, and fluorescence staining in prostate tissues. Colocalization with markers suggested expression of PDE10A in smooth muscle cells and catecholaminergic nerves. Norepinephrine, the α1 -adrenergic agonist phenylephrine, the thromboxane A2 analogue U46619, and endothelins 1-3 induced concentration-dependent contractions of prostate strips, while electric field stimulation (EFS) induced frequence-dependent contractions. Application of TC-E 5005 (500 nM) caused significant inhibition of norepinephrine-, phenylephrine-, and endothelin-3-induced contractions. Inhibition of EFS-induced contractions by TC-E 5005 ranged around 50%, resembling inhibition of EFS-induced contractions by tadalafil (10 μM). The prostacyclin analog treprostinil and the nitric oxide donor DEA NONOate induced relaxations of precontracted prostate strips, which were significantly amplified by TCE 5005. The PDE10-selective inhibitor TC-E 5005 inhibits adrenergic and neurogenic smooth muscle contractions in the human prostate. TC-E 5005 inhibits neurogenic contractions with similar efficacy than tadalafil, so that urodynamic effects in vivo appear possible. Prostate 76:1364-1374, 2016. © 2016 Wiley Periodicals, Inc.

Pancreatic polypeptide stimulates mouse gastric motor activity through peripheral neural mechanisms.

Pancreatic polypeptide (PP) is supposed to be one of the major endogenous agonists of the neuropeptide Y4 receptor. Pancreatic polypeptide can influence gastrointestinal motility, acting mainly through vagal mechanisms, but whether PP acts directly on the stomach has not been explored yet. The aims of this study were to investigate the effects of PP on mouse gastric emptying, on spontaneous tone of whole stomach in vitro and to examine the mechanism of action. Gastric emptying was measured by red phenol method after i.p. PP administration (1-3nmol per mouse). Responses induced by PP (1-300mmolL-1 ) on gastric endoluminal pressure were analyzed in vitro in the presence of different drugs. Gastric genic expression of Y4 receptor was verified by RT-PCR. Pancreatic polypeptide dose-dependently increased non-nutrient liquid gastric emptying rate. In vitro, PP produced a concentration-dependent contraction that was abolished by tetrodotoxin, a neural blocker of Na+ voltage-dependent channels. The contractile response was significantly reduced by atropine, a muscarinic receptor antagonist, and by SR48968, an NK2 receptor antagonist, while it was potentiated by neostigmine, an inhibitor of acetylcholinesterase. The joint application of atropine and SR48968 fully abolished PP contractile effect. Reverse transcriptase-polymerase chain reaction analysis revealed the presence of Y4 receptor mRNA in mouse stomach with a greater expression in antrum than in fundus. The present findings demonstrate that exogenous PP stimulates mouse gastric motor activity, by acting directly on the stomach. This effect appears due to the activation of enteric excitatory neurons releasing acetylcholine and tachykinins.

Application of Outlier Robust Nonlinear Mixed Effect Estimation in Examining the Effect of Phenylephrine in Rat Corpus Cavernosum

Ignoring two main characteristics of the concentration-response data, correlation between observations and presence of outliers, may lead to misleading results. Therefore the special method should be considered. In this paper in to examine the effect of phenylephrine in rat Corpus cavernosum, outlier robust nonlinear mixed estimation is used. in this study, eight different doses of phenylephrine in eight experimental groups were used. Each group consisted of eight rats. The concentration–response curves to phenylephrine (0.1µM to 300µM) were obtained by the cumulative addition of phenylephrine to the chamber. Because of the existence of an outlier to achieve robust estimations, M-estimation method and Huber function as a dispersion function were used. Cumulative administration of phenylephrine (0.1µM - 300µM) caused concentration-dependent contractions in strips of rat corpus cavernosum (-Log EC50 was 5 ± 0.31, 95% CI= 5.92 to 4.21). The contraction of corpus cavernosum started in the concentration of 0.3 μM and then gradually increased in a dose-dependent manner till it reached a plateau in 100 μM. To consider the clustering feature of concentration-response data, the 4pl regression with a random term has been used. To estimate parameters, because of existence of an outlier in dataset, the robust procedure has been applied. The contraction of corpus cavernosum started in the concentration of 0.3 μM and then gradually increased in a dose-dependent manner till it reached a plateau in 100 μM.

Impaired Excitatory Neurotransmission in the Urinary Bladder from the Obese Zucker Rat: Role of Cannabinoid Receptors.

Metabolic syndrome (MS) is a known risk factor for lower urinary tract symptoms. This study investigates whether functional and expression changes of cannabinoid CB1 and CB2 receptors are involved in the bladder dysfunction in an obese rat model with insulin resistance. Bladder samples from obese Zucker rat (OZR) and their respective controls lean Zucker rat (LZR) were processed for immunohistochemistry and western blot for studying the cannabinoid receptors expression. Detrusor smooth muscle (DSM) strips from LZR and OZR were also mounted in myographs for isometric force recordings. Neuronal and smooth muscle CB1 and CB2 receptor expression and the nerve fiber density was diminished in the OZR bladder. Electrical field stimulation (EFS) and acetylcholine (ACh) induced frequency- and concentration-dependent contractions of LZR and OZR DSM. ACh contractile responses were similar in LZR and OZR. EFS-elicited contractions, however, were reduced in OZR bladder. Cannabinoid receptor agonists and antagonists failed to modify the DSM basal tension in LZR and OZR In LZR bladder, EFS responses were inhibited by ACEA and SER-601, CB1 and CB2 receptor agonists, respectively, these effects being reversed by ACEA plus the CB1 antagonist, AM-251 or SER-601 plus the CB2 antagonist, AM-630. In OZR bladder, the inhibitory action of ACEA on nerve-evoked contractions was diminished, whereas that SER-601 did not change EFS responses. These results suggest that a diminished function and expression of neuronal cannabinoid CB1 and CB2 receptors, as well as a lower nerve fiber density is involved in the impaired excitatory neurotransmission of the urinary bladder from the OZR.

MP68-08 INHIBITION OF NEUROGENIC, CHOLINERGIC, AND ADRENERGIC CONTRACTION OF HUMAN BLADDER SMOOTH MUSCLE BY THE THROMBOXANE RECEPTOR ANTAGONIST, PICOTAMIDE

You have accessJournal of UrologyUrodynamics/Lower Urinary Tract Dysfunction/Female Pelvic Medicine: Basic Research & Pathophysiology II1 Apr 2016MP68-08 INHIBITION OF NEUROGENIC, CHOLINERGIC, AND ADRENERGIC CONTRACTION OF HUMAN BLADDER SMOOTH MUSCLE BY THE THROMBOXANE RECEPTOR ANTAGONIST, PICOTAMIDE Martin Hennenberg, Yiming Wang, Frank Strittmatter, Anna Ciotkowska, Beata Rutz, Christian Stief, and Christian Gratzke Martin HennenbergMartin Hennenberg More articles by this author , Yiming WangYiming Wang More articles by this author , Frank StrittmatterFrank Strittmatter More articles by this author , Anna CiotkowskaAnna Ciotkowska More articles by this author , Beata RutzBeata Rutz More articles by this author , Christian StiefChristian Stief More articles by this author , and Christian GratzkeChristian Gratzke More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.1345AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Male lower urinary tract symptoms include storage symptoms, which are mostly caused by an overactive bladder (OAB) due to spontaneous detrusor contractions. Anticholinergic treatment represents the goldstandard of medical therapy, but discontinuation rates are high, due to disappointing efficacy and side effects. In addition to muscarinic receptors, detrusor contraction may be induced by thromboxane A2 (TXA2) and α1-adrenoceptors. Here, we studied effects of the TXA2 receptor (TXA2R) antagonist picotamide on detrusor contraction in the male human bladder. METHODS Tissues were obtained from trigones of male patients (n=55) undergoing radical cystectomy. Contractility was studied in an organ bath. Expression of the TXA2 system was examined by RT-PCR, Western blot, immunofluorescence staining, and enzyme immunoassay (EIA). RESULTS Carbachol, the α1-selective adrenoceptor agonist phenylephrine, and the TXA2 analogue U46619 induced concentration-dependent contractions of trigone tissues. Electric field stimulation (EFS) induced frequency-dependent contractions. Picotamide (300 µM) inhibited carbachol-, phenylephrine-, U46619-, and EFS-induced contractions. Inhibition was significant at 0.1-3 µM carbachol, 1-3 µM phenylephrine, 10-30 µM U46619, and at EFS with 32 Hz. TXA2R and TXA2 synthase (TXS) were detectable by RT-PCR and Western blot analysis. Immunoreactivity for TXA2R and TXS colocalized with calponin, indicating expression in smooth muscle. TXB2 was detectable by EIA in each sample, with most values ranging between 50-150 pg/mg trigone protein. CONCLUSIONS Picotamide inhibits different contractile systems in the human detrusor at once, including cholinergic, adrenergic, TXA2-induced, and neurogenic contractions. This distinguishes picotamide from available medications for OAB treatment. Because picotamide is well tolerated even under long-term administration, clinical studies and urodynamic effects appear possible. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e891-e892 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Martin Hennenberg More articles by this author Yiming Wang More articles by this author Frank Strittmatter More articles by this author Anna Ciotkowska More articles by this author Beata Rutz More articles by this author Christian Stief More articles by this author Christian Gratzke More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

MP44-15 SPONTANEOUS MYOGENIC CONTRACTILITY IN THE HUMAN PROSTATE GLAND: IMPLICATIONS FOR THE TREATMENT OF LUTS ASSOCIATED WITH BPH.

INTRODUCTION AND OBJECTIVES: The phosphodiesterase (PDE) 5 inhibitor tadalafil is available for treatment of male lower urinary tract symptoms, while the role of other PDE isoforms for prostate smooth muscle tone is still unknown. Here, we examined effects of the PDE7-selective inhibitor BRL 50481, and the PDE10-selective inhibitor TC-E 5005 on smooth muscle contraction in the hyperplastic human prostate. METHODS: Prostate samples were obtained from patients (n1⁄495) undergoing radical prostatectomy. Expression of PDE isoforms was addressed by RT-PCR, Western blot, and immunofluorescence. Effects of TC-E 5005, BRL 50481, and tadalafil on contractility of prostate strips were studied in an organ bath. RESULTS: PDE7A1/2 and PDE10A were detectable by RTPCR, Western blot, and fluorescence staining. Colocalization with calponin and tyrosine hydroxylase suggested expression of PDE7A1/2 and PDE10A in smooth muscle cells and catecholaminergic nerves. Noradrenaline, the a1-adrenergic agonist phenylephrine, the thromboxane A2 analogue U46619, and endothelins 1-3 induced concentration-dependent contractions of prostate strips, while electric field stimulation (EFS) induced frequence-dependent contractions. Application of TC-E 5005 (500 nM) caused significant inhibition of noradrenaline-, phenylephrine-, and endothelin-3-induced contractions. Similarly, TC-E 5005 caused significant inhibiton of EFS-induced contractions. Inhibition of EFS-induced contraction by TC-E 5005 amounted to approximately 50 %, resembling inhibition of EFS-induced contraction by tadalafil (10 mM) (also around 50 %) (see figure). EFSand norepinephrine-induced tension were similar after application of tadalafil, and after combined application of tadalafil and TC-E 5005. BRL 50481 was without effect on noradrenaline-induced contraction. CONCLUSIONS: The PDE10-selective inhibitor TC-E 5005 inhibits neurogenic, adrenergic, and endothelin-3-induced smooth muscle contractions in the hyperplastic human prostate. TC-E 5005 inhibits neurogenic contractions with an efficacy similar to tadalafil. Urodynamic effects in vivo appear possible.

MP44-14 INHIBITION OF ADRENERGIC AND NON-ADRENERGIC SMOOTH MUSCLE CONTRACTION IN THE HYPERPLASTIC HUMAN PROSTATE BY THE PHOSPHODIESTERASE 10-SELECTIVE INHIBITOR TC-E 5005

You have accessJournal of UrologyBenign Prostatic Hyperplasia: Basic Research & Pathophysiology1 Apr 2016MP44-14 INHIBITION OF ADRENERGIC AND NON-ADRENERGIC SMOOTH MUSCLE CONTRACTION IN THE HYPERPLASTIC HUMAN PROSTATE BY THE PHOSPHODIESTERASE 10-SELECTIVE INHIBITOR TC-E 5005 Martin Hennenberg, Melanie Schott, Patrick Keller, Alexander Tamalunas, Anna Ciotkowska, Beata Rutz, Raphaela Waidelich, Frank Strittmatter, Christian Stief, and Christian Gratzke Martin HennenbergMartin Hennenberg More articles by this author , Melanie SchottMelanie Schott More articles by this author , Patrick KellerPatrick Keller More articles by this author , Alexander TamalunasAlexander Tamalunas More articles by this author , Anna CiotkowskaAnna Ciotkowska More articles by this author , Beata RutzBeata Rutz More articles by this author , Raphaela WaidelichRaphaela Waidelich More articles by this author , Frank StrittmatterFrank Strittmatter More articles by this author , Christian StiefChristian Stief More articles by this author , and Christian GratzkeChristian Gratzke More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.270AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The phosphodiesterase (PDE) 5 inhibitor tadalafil is available for treatment of male lower urinary tract symptoms, while the role of other PDE isoforms for prostate smooth muscle tone is still unknown. Here, we examined effects of the PDE7-selective inhibitor BRL 50481, and the PDE10-selective inhibitor TC-E 5005 on smooth muscle contraction in the hyperplastic human prostate. METHODS Prostate samples were obtained from patients (n=95) undergoing radical prostatectomy. Expression of PDE isoforms was addressed by RT-PCR, Western blot, and immunofluorescence. Effects of TC-E 5005, BRL 50481, and tadalafil on contractility of prostate strips were studied in an organ bath. RESULTS PDE7A1/2 and PDE10A were detectable by RT-PCR, Western blot, and fluorescence staining. Colocalization with calponin and tyrosine hydroxylase suggested expression of PDE7A1/2 and PDE10A in smooth muscle cells and catecholaminergic nerves. Noradrenaline, the α1-adrenergic agonist phenylephrine, the thromboxane A2 analogue U46619, and endothelins 1-3 induced concentration-dependent contractions of prostate strips, while electric field stimulation (EFS) induced frequence-dependent contractions. Application of TC-E 5005 (500 nM) caused significant inhibition of noradrenaline-, phenylephrine-, and endothelin-3-induced contractions. Similarly, TC-E 5005 caused significant inhibiton of EFS-induced contractions. Inhibition of EFS-induced contraction by TC-E 5005 amounted to approximately 50 %, resembling inhibition of EFS-induced contraction by tadalafil (10 µM) (also around 50 %) (see figure). EFS- and norepinephrine-induced tension were similar after application of tadalafil, and after combined application of tadalafil and TC-E 5005. BRL 50481 was without effect on noradrenaline-induced contraction. CONCLUSIONS The PDE10-selective inhibitor TC-E 5005 inhibits neurogenic, adrenergic, and endothelin-3-induced smooth muscle contractions in the hyperplastic human prostate. TC-E 5005 inhibits neurogenic contractions with an efficacy similar to tadalafil. Urodynamic effects in vivo appear possible. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e604 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Martin Hennenberg More articles by this author Melanie Schott More articles by this author Patrick Keller More articles by this author Alexander Tamalunas More articles by this author Anna Ciotkowska More articles by this author Beata Rutz More articles by this author Raphaela Waidelich More articles by this author Frank Strittmatter More articles by this author Christian Stief More articles by this author Christian Gratzke More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

The effect of urapidil, an alpha-1 adrenoceptor antagonist and a 5-HT1A agonist, on the vascular tone of the porcine coronary and pulmonary arteries, the rat aorta and the human pulmonary artery

Urapidil (Eupressyl®) an antihypertensive drug acting as an α1 antagonist and a 5-HT1A agonist, may be of special interest in the treatment of hypertension associated with preeclamptic toxaemia and hypoxia-induced pulmonary arterial vasoconstriction. However, the effect of urapidil on vascular tone has been poorly investigated. Vascular reactivity was evaluated using pulmonary and coronary arteries from 36 pigs, aortae from 22 rats and 9 human pulmonary artery samples suspended in organ chambers. Concentration-relaxation curves either to urapidil, 5-HT, or the 5-HT1A receptor agonist 8-OH-DPAT were constructed after pre-contraction of rings. Pig pulmonary and coronary artery rings were contracted with U46619, a thromboxane mimetic, rat aortic rings with either endothelin-1 or phenylephrine, and human pulmonary artery rings with U46619 or phenylephrine. Urapidil markedly inhibited phenylephrine-induced contractions in rat aortic rings with and without endothelium with a more pronounced effect observed in rings without endothelium. Both 5-HT and 8-OH-DPAT failed to induce relaxation in rat aortic rings with an intact endothelium. 5-HT, but not urapidil and 8-OH-DPAT, induced a concentration-dependent relaxation in the porcine coronary and pulmonary artery rings with an intact endothelium (P<0.05). 5-HT and phenylephrine but not urapidil caused concentration-dependent contractions in human pulmonary artery rings. The present findings, while confirming that urapidil is a potent inhibitor of α1-adrenoceptor-induced contraction, do not support the role of 5-HT1A receptor activation in the control of the vascular tone of the different types of arteries tested in response to urapidil. In addition, they indicate that urapidil seems to preferentially target arteries with endothelial dysfunction.