Concentration-dependent Contractile Responses Research Articles

Effects of lysophosphatidic acid on sling and clasp fibers of the human lower esophageal sphincter

Background This study aims to explore the role of lysophosphatidic acid receptors in the regulation mechanisms of contraction and relaxation of human lower esophageal sphincter. Methods Between July 2015 and March 2016, muscle strips were collected from a total of 30 patients (19 males, 11 females; mean age: 62±9.9 years; range, 52 to 68 years) who underwent an esophagectomy for mid-third esophageal carcinomas. The specimens were maintained in oxygenated Krebs solution. Muscle tension measurement technique in vitro was used to examine the effects of non-selective lysophosphatidic acid receptors agonists and antagonists, as well as selective lysophosphatidic acid receptors agonists on the clasp and sling fibers of human lower esophageal sphincter. Results The non-selective dopamine receptor agonist lysophosphatidic acid induced the contraction of the clasp and sling fibers of the human lower esophageal sphincter. The response induced by nonselective lysophosphatidic acid receptor agonist was inhibited completely by non-selective lysophosphatidic acid receptor antagonist. The selective lysophosphatidic acid 1 and 2 receptor agonist and the selective lysophosphatidic acid 3 receptor agonist induced a concentration-dependent contractile response of the clasp and sling fibers of the human lower esophageal sphincter. There was no significant difference in contraction rates between the clasp and sling fibers (p>0.05). Conclusion This study indicates that lysophosphatidic acid regulates the lower esophageal sphincter is through its receptor; the lysophosphatidic acid receptors may be involved in the contractile response of the human lower esophageal sphincter.

Участие Р2-рецепторов в сократительной активности мочевого пузыря пациентов с доброкачественной гиперплазией предстательной железы

AIM to study the role of P2 receptors in impaired bladder contractility in patients with lower urinary tract obstruction. MATERIALS AND METHODS in pharmacological studies, tissue samples from the bladder wall of 30 patients were used, obtained during planned surgical interventions for benign prostatic hyperplasia (transvesical simple prostatectomy without placement of cystostomy tube). Based on these tissue, isolated smooth muscle specimens were prepared. Their mechanical activity and the efficiency of ligands of purine P2 and other receptors were evaluated. With this aim, the following P2-receptor agonists were used: adenosine triphosphoric acid (ATP), adenosine diphosphoric acid (ADP), uridine-5'-triphosphoric acid (UTP), alpha, beta-methylene-ATP, 2-methylthio-ADP, as well as antagonists of P2-disulfonate receptors acid (PPADS), suramin, NF023, MRS2500. In addition, the efficiency of ligands of other receptors, including carbacholine, epinephrine, histamine, serotonin, atropine was evaluated. RESULTS the most effective agonist was alpha-beta-methylene-ATP, while ATP and 2-methylthio-ADP were significantly less active. In our experiments, ADP and UTP did not show an effect on human bladder. The influence of P2 receptor agonists was inhibited by P2 receptor antagonists PPADS and suramin, as well as MRS2500, although to a lesser extent. Carbacholine caused a strong concentration-dependent contractile response of the bladder, which was inhibited by atropine. Histamine resulted in mild bladder contractions only at high concentrations. Epinephrine and serotonin did not cause significant changes in the contractile activity of the bladder. CONCLUSION The main subtype of P2 receptors involved in the contractile activity of the human bladder is P2X1 receptors. P2Y1 receptors also have some influence on the contraction, while other subtypes of P2 receptors are not detected by pharmacological methods.

Potential vascular α1-adrenoceptor blocking properties of metformin in rat aorta and tail artery

Metformin is a widely used drug for the treatment of type 2 Diabetes Mellitus. Several studies have also suggested that metformin decreases blood pressure; although an interaction with α-adrenoceptors has been proposed, this mechanism needs to be further investigated. Since α1-adrenoceptors play a significant role to regulate vascular tone, this study has analysed the potential ability of metformin to block α1-adrenoceptors in rat aorta and tail artery. For this purpose, the contractile responses induced by noradrenaline, methoxamine, and phenylephrine were determined in the absence or presence of metformin in rat aorta and tail artery rings. In both arteries, noradrenaline, methoxamine, and phenylephrine produced concentration-dependent contractile responses. Interestingly, the contractile responses to noradrenaline, methoxamine, and phenylephrine were significantly and differentially blocked by metformin (1, 3.1 and/or 10 mM) but not by vehicle. These results suggest that metformin is capable to block α1-adrenoceptors and may explain, at least in part, the anti-hypertensive effect observed in several clinical trials.

Pharmacology of Serotonin Receptors Causing Contraction of Isolated Bovine Posterior Ciliary Arteries: Role in Ocular Blood Flow.

To determine the serotonergic (5HT) receptor subtype mediating the contraction of bovine posterior ciliary arteries (BPCAs) in vitro. Longitudinal isometric tension was measured in BPCA strips (4-5 mm) mounted in 25 mL organ baths containing oxygenated Krebs solution at 37°C. Cumulative contractile concentration-response (C-R) curves were generated for various 5-HT agonists to assess their potencies and maximal degrees of contraction. Multiple agonist C-R curves were also constructed in the presence and absence of receptor-selective antagonists to determine antagonist potencies using Schild plots. Selective and nonselective agonists for 5-HT receptors elicited concentration-dependent contractile responses in BPCAs with the following rank order of potency: MK-212 > BW723C86 > α-methyl-5-HT >5-methoxy-α-5-methyl-5-HT >> R-DO1 > >5-HT >> cabergoline >> 5-methoxy-dimethyl-tryptamine >> 2-methyl-5-HT >> tryptamine. Interestingly, both 8-OH-DPAT (5HT1A agonist) and quipazine (5HT3 agonist) did not elicit contractions in BPCAs. The contractions produced by BW723C86 (5-HT2B agonist) were antagonized by 5-HT receptor blockers, RS-127445 (5-HT2B antagonist), and M-100907 (5-HT2A antagonist), yielding antagonist pA2 values of 7.5 ± 0.12 (n = 4) and 6.2 ± 0.17 (n = 4), respectively. Furthermore, contractions elicited by MK-212 (5-HT2C agonist) was blocked by RS-102221 (5-HT2C antagonist), although noncompetitively. On the basis of the pharmacological profile of selective agonists and antagonists, we conclude that serotonin-induced contractions of the BPCA are mediated primarily by a combination of 5HT2C and/or 5HT2B receptors. It appears that 5-HT1A and 5-HT3 receptors are not involved in the contractile action of BPCAs to serotonin.

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.

Activation of angiotensin II type 1 receptors and contractile activity in human sigmoid colon in vitro.

To analyse the effects of angiotensin II (Ang II) on the contractility of human sigmoid colon, and to characterize the subtype(s) of receptor(s) involved and the related action mechanism. The contractility of sigmoid colon circular muscle strips was recorded isometrically. RT-PCR and immunohistochemistry were used to reveal the eventual existence of a local renin-angiotensin system (RAS) and the distribution of Ang II receptors. Transcripts encoding for the Ang II type 1 (AT1 ) and the Ang II type 2 (AT2 ) receptor subtypes and for the angiotensin-converting enzyme in the whole-thickness muscular wall were observed. Ang II caused a concentration-dependent contractile response, which is antagonized by losartan, AT1 receptor antagonist, but not by PD123319, AT2 receptor antagonist. The joint application of losartan and PD123319 did not produce any additive effect. The contractile response to Ang II was partially reduced by tetrodotoxin, Na(+) voltage-gated neural channel blocker, and to some extent by SR48968, tachykinin NK2 receptor antagonist. However, hexamethonium, nicotinic receptor antagonist, atropine, cholinergic muscarinic receptor antagonist and SR140333, tachykinin NK1 receptor antagonist, were ineffective. Immunohistochemical analysis showed that AT1 receptors were expressed on the smooth muscle layers and myenteric plexus. Ang II positively modulates the spontaneous contractile activity of human sigmoid colon via activation of post-junctional and pre-junctional AT1 receptors, the latter located on the enteric nerves that modulate the release of tachykinins. The presence of the components of RAS in the human colon suggests that Ang II can be also locally generated to control colonic motility.

Toll-like receptors 2 and 4 modulate the contractile response induced by serotonin in mouse ileum: analysis of the serotonin receptors involved.

Microbiota through toll-like receptors (TLR) may regulate gastrointestinal motility by activating neuroendocrine mechanisms. We evaluated the influence of TLR2 and TLR4 in the spontaneous contractions and serotonin (5-HT)-induced motor response in mouse ileum, and the 5-HT receptors involved. Muscle contractility studies to evaluate the spontaneous intestinal motility and the response to 5-HT were performed in the ileum from wild type (WT), TLR2(-/-), TLR4(-/-), and TLR2/4 double knockout (DKO) mice. 5-HT receptor expression was determined by real-time PCR. The amplitude of spontaneous contractions in ileum was higher in TLR2(-/-), TLR4(-/-), and TLR2/4 DKO mice with respect to WT. 5-HT evoked concentration-dependent contractile responses in the ileum from TLR2(-/-) and TLR4(-/-) mice similar to WT. However, in ileum from TLR2/4 DKO, 5-HT did not induce any contractile response. Expression of 5-HT2A, 5-HT2B, 5-HT2C, and 5-HT3 receptors resulted increased in ileum from TLR4(-/-) and TLR2/4 DKO. Expression of the 5-HT4 receptor was diminished in TLR2(-/-) and TLR2/4 DKO. High levels of 5-HT7 receptor expression were found in TLR2/4 DKO but not in TLR2(-/-) or TLR4(-/-). In WT and TLR4(-/-), 5-HT2, 5-HT3, 5-HT4, and 5-HT7 receptor antagonists reduced the contractile response evoked by 5-HT. In TLR2(-/-) mice, 5-HT4 antagonist did not reduce the 5-HT response. In TLR2/4 DKO mice, only 5-HT4 and 5-HT7 receptor antagonists reduced the relaxing response induced by 5-HT. TLR2 and TLR4 signaling may modulate the spontaneous contractions and the serotonin contractile response by acting on 5-HT2, 5-HT3, 5-HT4, and 5-HT7 receptors.

Two cholecystokinin receptor subtypes are identified in goldfish, being the CCKAR involved in the regulation of intestinal motility

Cholecystokinin (CCK) plays a key role in the digestive physiology of vertebrates. However, very little is known about the role of CCK on intestinal functions in fish. The present study identifies two CCK receptor subtypes in a stomachless teleost, the goldfish (Carassius auratus), and investigates by using an in vitro system their involvement mediating the effects of the sulfated octapeptide of CCK (CCK-8S) on the motility of isolated proximal intestine. Partial-length mRNAs encoding two CCK receptor isoforms (CCKAR and CCKBR.I) were sequenced and the structural analysis showed that both receptors belong to the G-protein coupled receptor superfamily. Both goldfish CCK receptor sequences were more closely related to zebrafish sequences, sharing the lowest similarities with cavefish and tilapia. The highest expression of goldfish CCKAR was observed along the whole intestine whereas the CCKBR gen was predominantly expressed in the hypothalamus, vagal lobe and posterior intestine. Application of CCK-8S to the organ bath evoked a concentration-dependent contractile response in intestine strips. The contractions were not blocked by either tetrodotoxin or atropine, suggesting that CCK-8S acts on the gut smooth muscle directly. Preincubations of intestine strips with devazepide and L365,260 (CCKAR and CCKBR receptor selective antagonists) showed that the CCK-8S-induced contraction could be partially mediated by the CCKAR receptor subtype, which is also the most abundant CCK receptor found in gastrointestinal tissues. In conclusion, two CCK receptors with a differential distribution pattern has been identified in goldfish, and the CCKAR subtype is mainly involved in the regulation of intestinal motility by the CCK-8S.

Pharmacodynamic Study of Interaction of Aqueous Leaf Extract of Psidium Guajava Linn. (Myrtaceae) with Receptor Systems Using Isolated Tissue Preparations.

The present study investigates the interaction of aqueous leaf extract of Psidium guajava with muscarinic, serotonergic and adrenergic receptor system using isolated rat ileum, gastric fundus and trachea, respectively. The concentration-dependent contractile response of aqueous leaf extract of Psidium guajava was parallel and rightward of standard agonists, ACh and 5-HT indicating agonistic activity on muscarinic and serotonergic receptor systems. The inhibition of aqueous leaf extract of Psidium guajava mediated contractions in presence of atropine (10(-7) M) and ketanserin (10(-6) M) confirmed the activity. Relaxant effect of PG (0.2 mg/ml) on carbachol induced pre-contracted rat tracheal chain indicated its agonistic action on adrenergic receptor system. Inhibition (P<0.05) of the action in the presence of propranolol (1 ng/ml) confirmed the activity. It may be concluded that PG possesses agonistic action on muscarinic, serotonergic and adrenergic receptor systems.

The Role of Tyrosine Kinase in Ca^(2+)-Independent Contraction of the Ropivacaine on Rat Aortic Smooth Muscle

The tyrosine kinase signaling pathway plays an important role in the mediation of Ca²⁺ independent mechanisms of smooth muscle contraction. Several components of this pathway, including protein kinase C (PKC), p44/42 mitogen-activated protein kinase (p44/42 MAPK) and Rho-kinase are involved in Ca²⁺ independent mechanisms. Whether the tyrosine kinase pathway mediates vasoconstriction induced by the anesthetic ropivacaine remains unclear. The present study was designed to examine the role of tyrosine kinase in ropivacaine-induced, Ca²⁺-independent contraction of rat aortic smooth muscle. The effects of tyrosine kinase inhibitor on ropivacaine-induced contractile response were observed by isometric force measurement. The protein tyrosine phosphorylation, PKC, p44/42 MAPK, and membrane translocation of Rho-kinase were examined by Western blotting. Ropivacaine induced a concentration-dependent contractile response, and showed a number of effects on protein tyrosine phosphorylation. In this study, phosphorylation levels were shown to increase at lower concentrations of ropivacaine, but the levels decline at higher concentrations in rat aortic rings attenuated by the tyrosine kinase inhibitor genistein in a concentration-dependent fashion. Ropivacaine-induced phosphorylation of PKC and p44/42 MAPK and Rho-kinase membrane translocation were also significantly attenuated by genistein in similar decreasing manner as the PKC inhibitor bisindolylmaleimide I (Bis I) and the Rho-kinase inhibitor, Y27632, but to a lesser degree than that by the p44/42 MAPK inhibitor, PD 098059. Our results showed that the ropivacaine-induced, Ca²⁺ independent-mediated contraction of rat aortic smooth muscle is, in part, regulated by tyrosine kinase-catalyzed protein tyrosine phosphorylation.

Pharmacological Studies of Tentacle Extract from the Jellyfish Cyanea capillata in Isolated Rat Aorta

Our previous studies demonstrated that tentacle extract (TE) from the jellyfish, Cyanea capillata, could cause a dose-dependent increase of systolic blood pressure, which seemed to be the result of direct constriction of vascular smooth muscle (VSM). The aim of this study is to investigate whether TE could induce vasoconstriction in vitro and to explore its potential mechanism. Using isolated aorta rings, a direct contractile response of TE was verified, which showed that TE could induce concentration-dependent contractile responses in both endothelium-intact and -denuded aortas. Interestingly, the amplitude of contraction in the endothelium-denuded aorta was much stronger than that in the endothelium-intact one, implying that TE might also bring a weak functional relaxation in addition to vasoconstriction. Further drug intervention experiments indicated that the functional vasodilation might be mediated by nitric oxide, and that TE-induced vasoconstriction could be attributed to calcium influx via voltage-operated calcium channels (VOCCs) from the extracellular space, as well as sarcoplasmic reticulum (SR) Ca2+ release via the inositol 1,4,5-trisphosphate receptor (IP3R), leading to an increase in [Ca2+]c, instead of activation of the PLC/DAG/PKC pathway or the sympathetic nerve system.

Prenatal Intestinal Obstruction Affects the Myenteric Plexus and Causes Functional Bowel Impairment in Fetal Rat Experimental Model of Intestinal Atresia

BackgroundIntestinal atresia is a rare congenital disorder with an incidence of 3/10 000 birth. About one-third of patients have severe intestinal dysfunction after surgical repair. We examined whether prenatal gastrointestinal obstruction might effect on the myenteric plexus and account for subsequent functional disorders.Methodology/Principal FindingsWe studied a rat model of surgically induced antenatal atresia, comparing intestinal samples from both sides of the obstruction and with healthy rat pups controls. Whole-mount preparations of the myenteric plexus were stained for choline acetyltransferase (ChAT) and nitric oxide synthase (nNOS). Quantitative reverse transcription PCR was used to analyze mRNAs for inflammatory markers. Functional motility and permeability analyses were performed in vitro. Phenotypic studies were also performed in 8 newborns with intestinal atresia. In the experimental model, the proportion of nNOS-immunoreactive neurons was similar in proximal and distal segments (6.7±4.6% vs 5.6±4.2%, p = 0.25), but proximal segments contained a higher proportion of ChAT-immunoreactive neurons (13.2±6.2% vs 7.5±4.3%, p = 0.005). Phenotypic changes were associated with a 100-fold lower concentration-dependent contractile response to carbachol and a 1.6-fold higher EFS-induced contractile response in proximal compared to distal segments. Transcellular (p = 0.002) but not paracellular permeability was increased. Comparison with controls showed that modifications involved not only proximal but also distal segments. Phenotypic studies in human atresia confirmed the changes in ChAT expression.ConclusionExperimental atresia in fetal rat induces differential myenteric plexus phenotypical as well as functional changes (motility and permeability) between the two sides of the obstruction. Delineating these changes might help to identify markers predictive of motility dysfunction and to define guidelines for post-surgical care.

Effects induced by inhibitors of the phosphatidylinositol 3‐kinase/Akt and nitric oxide synthase/guanylyl cyclase pathways on the isometric contraction in rat aorta: a comparative study

This work was aimed to determine whether isometric contraction in Wistar rat aorta is related to the phosphatidylinositol 3-kinase (PI3K)/Akt-dependent activation of endothelial nitric oxide synthase (eNOS). Basically, we hypothesized that additional increases in active tone occur after the pharmacological inhibition of a transduction pathway involved in NO synthesis or action. In intact aortic rings contracted with phenylephrine or high K(+), the cumulative administration of the PI3K inhibitor, LY294002, elicited significant decreases--but not supplementary increases--in tone. In endothelium-intact tissues, on the other hand, the Akt1/2 kinase inhibitor did not alter phenylephrine- and K(+)-induced isometric contractions. The PI3K inhibitor wortmannin (1 × 10(-7) m) produced a significant supplementary contraction only in endothelium-intact aortic rings precontracted with phenylephrine. Higher concentrations of this inhibitor produced relaxations of phenylephrine and high K(+)-constricted endothelium-intact and endothelium-denuded aortic rings. LY294002 and wortmannin did not cause any potentiating effect on phenylephrine- and angiotensin II-induced concentration-dependent contractile responses in endothelium-intact tissues. In intact aortic rings contracted with phenylephrine or high K(+), the addition of the NOS inhibitor, L-NAME, or the guanylyl cyclase inhibitor, ODQ, further augmented tone in a concentration-dependent manner, and these supplementary contractions were significantly reduced by endothelium removal. Taken together, our data suggest that the PI3K/Akt pathway is not counteracting aortic isometric contractions by activation of the eNOS. It appears, on the other hand, that the smooth muscle PI3K can stimulate contraction without activation of the protein kinase Akt in response to GPCR agonists and high K(+).

The Sources of Ca2+ for Muscarinic Receptor-induced Contraction in the Rat Ileum

The contractile responses obtained by activation of different muscarinic receptor subtypes in the longitudinal muscle of the rat ileum and especially the responses of this muscle to acetylcholine in a Ca(2+)-free medium have been investigated. In Ca(2+)-containing solution, acetylcholine elicited similar concentration-dependent contractile responses in the duodenum, jejunum and ileum strips of the rat intestine. The response to a maximal concentration of the agonist (1 microM) consisted of a rapid phasic response followed by a slower tonic one. Nifedipine completely relaxes or inhibits the sustained response and only partially diminishes the phasic one, which suggests that the phasic contraction depends on the release of internal Ca2+ entry from the extracellular space through voltage-dependent Ca2+ channels, but that the tonic contraction only depends on the influx of the external ion. In Ca(2+)-free medium, acetylcholine (1 microM) induced phasic contractions that depend on the release of this ion from internal stores. Participation of different subtypes of receptors (M1, M2 and M3) in these responses depends on the inhibitory action shown by methoctramine, 4-DAMP and atropine but not by pirenzepine in two different experimental models.

Contribution of Cyclooxygenase-Dependent Mechanisms to Contractile Responses to Donepezil in the Rat Urinary Bladder

Donepezil, an inhibitor of acetylcholinesterase, is used to improve cholinergic neurotransmission and cognitive function in Alzheimer’s disease. In the present study, contractile effects of donepezil on the rat urinary bladder were examined and compared with those of the nonselective cholinesterase inhibitor neostigmine. Both donepezil and neostigmine produced concentration-dependent contractile responses of the isolated rat urinary bladder strips. The neostigmine-induced contractions were abolished by atropine. However, donepezil produced contractions of urinary bladders partly through atropine-insensitive mechanisms. The atropine-resistant component of donepezil-induced contraction was significantly reduced by the cyclooxygenase inhibitor indomethacin. These results suggest that cyclooxygenase-derived prostanoids contribute, at least in part, to contractile effects of donepezil in the rat urinary bladder.

In vitro effects of &lt;i&gt;Spigelia anthelmia&lt;/i&gt; extracts on experimentally isolated guinea pig ileum

This experiment was undertaken to determine the in vitro effects of aqueous extracts of Spigelia anthelmia L (a local anthelmintic plant) on experimentally isolated guinea pig ileum, with a view to establishing its likely anthelmintic effect on nematodes. The extract induced concentrationdependent contractile responses similar to that produced by acetylcholine on isolated guinea pig ileum. The contractions produced by the extract were antagonised by atropine but not by mepyramine. It was concluded that the anthelmintic mechanism of action of Spigelia anthelmia is most probably due to paralysis of the worms.

Reduction of Urinary Bladder Activity in Transgenic Sickle Cell Disease Mice.

Abstract 2580Poster Board II-557Genitourinary infection and urinary retention have been related to impaired urinary concentrating ability and altered urinary volume in sickle cell disease (SCD) patients. This condition may be associated with changes in the detrusor smooth muscle (DSM) reactivity. Berkeley transgenic SCD mice display several clinical manifestations similar to those seen in humans, but no studies assessing the alterations of DSM reactivity in SCD mice have been performed. DSM contractions are a consequence of the activation of muscarinic receptors that promote stimulation of phospholipase C, with increased formation of inositol trisphosphate and diacylglycerol to release calcium (Ca2+) from intracellular stores, leading to bladder emptying. The Ca2+-independent RhoA/Rho-kinase pathway is also involved in the regulation of bladder smooth muscle tone by alteration of the sensitivity of contractile proteins for Ca2+. Changes in the contraction mechanisms of DSM may contribute to bladder dysfunction. The aim of this study was to evaluate the contractile mechanisms of isolated DSM of transgenic SCD mouse. SCD (SS) and C57BL/6 (CTL) mice tissues were removed and mounted in 10ml organ baths containing Krebs solution at 37°C and continuously bubbled with a mixture of 95%O2:5%CO2 (pH 7.4). Tissues were stretched to a resting tension of 10 mN and allowed to equilibrate for 60 minutes. Changes in isometric force were recorded using a PowerLab400 Data Acquisition System. Cumulative concentration-response curves to muscarinic agonist carbachol (CCh; 0.01–100 μM) were obtained in the absence and presence of the Rho-kinase inhibitor Y-27632 (1–10 μM). Cumulative concentration-response curves were constructed to hyperpolarizing solution, potassium chloride (KCl; 1–300 mM) and calcium chloride (CaCl2; 0.01–100 mM). Frequency-response curves for electrical field stimulation (EFS; 1–32 Hz) were obtained in strips in the absence and presence of non-selective muscarinic antagonist atropine (1 μM) or purinergic receptor antagonist suramin (100 μM). Cumulative addition of the CCh produced concentration-dependent contractile responses in DSM, although the potency (pEC50) did not change in SS mice (5.80±0.02) compared with CTL mice (5.69±0.05). In contrast, the maximal response (Emax) was significantly reduced in SS mice (4.20±0.42mN) compared with CTL mice (9.96±1.77mN). In addition, the pEC50 and Emax elicited by CCh were reduced in the presence of Y-27632; however, no change was observed among all group studies. Cumulative addition of the KCl produced concentration-dependent contraction in DSM, and both pEC50 (0.98±0.08) and Emax (7.63±1.03mN) were significantly reduced in SS mice, compared with CTL mice (1.24±0.07; 11.24±0.84mN, respectively). CaCl2 produced concentration-dependent contractile responses, the pEC50 did not change in SS mice (2.00±0.06); however, the Emax was significantly reduced (10.1±0.25mN) when compared to CTL mice (1.92±0.05; 17.8±2.3mN). EFS-induced contractions in DSM of SS mice were decreased at significantly higher frequencies than compared to CTL mice. In addition, EFS-induced contractions in DSM were significantly decreased in the presence of atropine and suramin in both SS mice and CTL mice, but no change was observed among all group studies. Our study shows that SS mice exhibit impaired contractile responses to muscarinic agonist, KCl, CaCl2 and EFS, with no involvement of the Rho-kinase signaling pathway. This bladder dysfunction may contribute to the increased risk of urinary tract infections observed in SCD patients. Further studies are necessary to better understand the relationship between DSM reactivity alterations and genitourinary manifestations in SCD. Disclosures:No relevant conflicts of interest to declare.

Losartan counteracts the hyper-reactivity to angiotensin II and ROCK1 over-activation in aortas isolated from streptozotocin-injected diabetic rats

BackgroundIn streptozotocin-injected rats (STZ-rats), we previously demonstrated a role for angiotensin II (AT-II) in cardiac remodelling and insulin resistance partially counteracted by in vivo treatment with losartan, an AT-II receptor antagonist.We now aimed to investigate the effect of treating diabetic STZ-rats with losartan on diabetes vascular response to vasoconstrictors.MethodsMale Wistar rats were randomly divided in four groups, two of them were assigned to receive losartan in the drinking water (20 mg/kg/day) until the experiment ending (3 weeks afterward). After 1 week, two groups, one of which receiving losartan, were injected in the tail vein with citrate buffer (normoglycemic, N and normoglycemic, losartan-treated, NL). The remaining received a single injection of streptozotocin (50 mg/kg in citrate i.v.) thus becoming diabetic (D) and diabetic losartan-treated (DL). Plasma glycaemia and blood pressure were measured in all animals before the sacrifice (15 days after diabetes induction).In aortic strips isolated from N, NL, D and DL rats we evaluated i) the isometric concentration-dependent contractile response to phenylephrine (Phe) and to AT-II; ii) the RhoA-kinase (ROCK1) activity and expression by enzyme-immunoassay and Western blot respectively.Key resultsThe concentration-dependent contractile effect of Phe was similar in aortas from all groups, whereas at all concentrations tested, AT-II contraction efficacy was 2 and half and 1 and half times higher in D and DL respectively in comparison with N and NL. AT-II contracture was similarly reduced in all groups by AT-II receptor antagonists, irbesartan or irbesartan plus PD123319. HA-1077 (10 μM), an inhibitor of ROCK1 activity, reduced AT-II efficacy (Δmg/mg tissue w.w.) by -3.5 ± 1.0, -4.6 ± 1.9, -22.1 ± 2.2 and -11.4 ± 1.3 in N, NL, D and DL respectively). ROCK1 activity and expression were higher in D than in N/NL and DL aortas.Conclusion and implicationsAortas isolated from STZ-rats present hyper-contracture to AT-II mainly dependent on the up-regulation of ROCK1 expression/activity. In vivo losartan treatment partially corrects AT-II hyper-contracture, limiting the increase in ROCK1 expression/activity. These data offer a new molecular mechanism supporting the rationale for using losartan in the prevention of diabetic vascular complications.

Stereoselectivity of satropane, a novel tropane analog, on iris muscarinic receptor activation and intraocular hypotension

To study the stereoselectivity of satropane (3-paramethylbenzene sulfonyloxy-6-acetoxy tropane), a novel tropane analog, on iris muscarinic receptor activation and intraocular hypotension. The assays for radioligand-receptor binding, the contractile responses of isolated iris muscle, the miosis response, and the intraocular hypotension of the enantiomers of satropane were investigated. In the binding analysis, S(-)satropane (lesatropane) completely competed against the [3H]quinuclydinyl benzilate-labeled ligand at muscarinic receptors in the iris muscle, whereas R(+)satropane failed to completely compete. In an isolated iris contractile assay, R,S(+/-)satropane and S(-)satropane produced a concentration-dependent contractile response with similar efficacy and potency to that of carbachol. R(+)satropane did not induce any contractile response. In the pupil diameter measurement assay in vivo, S(-)satropane induced miosis much more effectively than pilocarpine, while R(+)satropane failed to produce any miosis. In the water loading-induced and methylcellulose-induced ocular hypertensive models, S(-)satropane, but not R(+)satropane, significantly suppressed intraocular pressure at a much lower concentration than pilocarpine. The agonistic and hypotensive properties of satropane on rabbit eyes are stereoselective, with the S(-)isomer being its active form.

Regional differences in the functional and biochemical properties of endothelin receptor subtypes in the rabbit prostatic urethra

To examine the regional differences in the functional (pharmacological) and biochemical properties of endothelin (ET) receptors in the rabbit prostatic urethra. The properties of ET receptors in 6-month-old male rabbit prostatic urethras were examined using isolated muscle-bath and radioligand receptor-binding techniques. Using plasma membrane suspensions, saturation and inhibition experiments with [(125)I]ET-1 and unlabelled agonists and antagonists (ET(A)-selective antagonist BQ123, and ET(B)-selective agonist sarafotoxin 6c, STX6c) were done to determine the ET receptor densities and their subtype specificities in the different regions of the urethra. The ETs (ET-1 and ET-3) produced significant concentration-dependent contractile responses in the smooth muscle strips from the different regions of the urethra. Although the maximum contractile responses induced by ET-1 were similar in the different regions, the maximum contractile responses induced by ET-3 were greater in the distal region than in the proximal or middle regions, suggesting that the contractile response to ET-1 is more potent than that to ET-3 in all regions, and that there are region-specific differences in the responses to ET-3 but not ET-1. Moreover, the ET-3-induced contractile response was suppressed by BQ788 (a selective antagonist of the ET(B) receptor) suggesting that the ET(B) receptor subtype contributes to the contractile responses mediated by ET-3. The ET receptors were expressed in higher concentrations in the distal than in the proximal or middle regions. BQ123 and STX6c inhibited [(125)I]ET-1 binding in all regions with high and low affinity constants, indicating the presence of both ET(A) and ET(B) receptor subtypes. The proportions of high-affinity binding sites for BQ123, representing ET(A) receptors, were approximately 68%, 63% and 42% in the proximal, middle and distal regions, respectively. By contrast, the proportions of high-affinity binding sites for STX6c, representing ET(B) receptors, were approximately 27%, 35% and 52% in the proximal, middle, and distal regions, respectively. These data indicate the presence of regional differences in the densities and subtype specificities of ET receptor subtypes, and the existence of regional differences in the rabbit prostatic urethra. The results suggest regional differences in ET(B) receptor subtypes that mediate contractile responses to ET-3, reflecting differences in the densities and specificities of the ET receptor subtypes in the rabbit prostatic urethra.