Maximal Contractile Response Research Articles

Growth hormone receptor deficiency in mice results in reduced systolic blood pressure and plasma renin, increased aortic eNOS expression, and altered cardiovascular structure and function

To study the role of the growth hormone receptor (GHR) in the development of cardiovascular structure and function, female GHR gene-disrupted or knockout (KO) and wild-type (WT) mice at age 18 wk were used. GHR KO mice had lower plasma renin levels (12 +/- 2 vs. 20 +/- 4 mGU/ml, P < 0.05) and increased aortic endothelial NO synthase (eNOS) expression (146%, P < 0.05) accompanied by a 25% reduction in systolic blood pressure (BP, 110 +/- 4 vs. 147 +/- 3 mmHg, P < 0.001) compared with WT mice. Aldosterone levels were unchanged, whereas the plasma potassium concentration was elevated by 14% (P < 0.05) in GHR KO. Relative left ventricular weight was 14% lower in GHR KO mice (P < 0.05), and cardiac dimensions as analyzed by echocardiography were similarly reduced. Myograph studies revealed a reduced maximum contractile response in the aorta to norepinephrine (NE) and K(+) (P < 0.05), and aorta media thickness was decreased in GHR KO (P < 0.05). However, contractile force was normal in mesenteric arteries, whereas sensitivity to NE was increased (P < 0.05). Maximal acetylcholine-mediated dilatation was similar in WT and GHR KO mice, whereas the aorta of GHR KO mice showed an increased sensitivity to acetylcholine (P < 0.05). In conclusion, loss of GHR leads to low BP and decreased levels of renin in plasma as well as increase in aortic eNOS expression. Furthermore, GHR deficiency causes functional and morphological changes in both heart and vasculature that are beyond the observed alterations in body size. These data suggest an important role for an intact GH/IGF-I axis in the maintenance of a normal cardiovascular system.

The Effects of Bumetanide on Human Umbilical Artery Contractions

The authors investigate the effect of bumetan ide, an inhibitor of NKCC1 and a loop diuretic, on the tone of human umbilical artery (HUA). Rings of HUA (n = 35) from vaginal deliveries were suspended for isometric tension recordings in organ baths. Cumulative concentration-response curves to serotonin, histamine, and KCl were performed in the absence (control) or in the presence of bumetanide. The relaxant effect of bumetanide was also evaluated in serotonin- and histamine-induced contractions. Bumetanide inhibited HUA tone in serotonin- and histamine-induced contractions with significant changes in the potency (pD(2)) and maximum contractile response (E(max)) values. However, only pD( 2) values for KCl-induced contraction significantly changed in the presence of bumetanide. Bumetanide caused concentration-dependent and sustained relaxations in serotonin-induced contraction; however, there was refractoriness in histamine-induced contraction. These findings raise the possibility that NKCC1 may play a role in the regulation of the umbilical artery tone.

A61603‐Induced Contractions of the Porcine Meningeal Artery Are Mediated by α1‐ and α2‐Adrenoceptors

It has recently been shown that A61603 (N-[5-(4,5-dihydro-1H-imidazol-2yl)-2-hydroxy-5,6,7,8-tetrahydro-naphthalen-1-yl]methane sulphonamide), a potent alpha(1A)-adrenoceptor agonist, decreased carotid artery conductance in anaesthetized pigs by a novel non-adrenergic mechanism. In this study, we set out to pharmacologically characterize A61603-induced contractions of the porcine isolated meningeal artery. While the maximum contractile responses of the artery were similar, A61603 (E(max): 183 +/- 23% of 100 mM KCl; pEC(50): 7.25 +/- 0.18) was more potent than noradrenaline (E(max): 156 +/- 16%; pEC(50): 5.75 +/- 0.17) or phenylephrine (E(max): 163 +/- 20%; pEC(50): 5.63 +/- 0.02). Prazosin (pA(2): 9.36 +/- 0.23) and, to a lesser extent, rauwolscine (pK(b): 6.36 +/- 0.38) and yohimbine (pK(b): 7.30 +/- 0.15) antagonised the contractions to A61603. The 5-HT(1B) (GR127935; N-[4-methoxy-3-(4-methyl-1-piperazinyl) phenyl]-2'-methyl-4'-(5-methyl-1,2,4-oxadiazol-3-yl)[1,1-biphenyl]-4-carboxamide) and 5-HT(2) (ritanserin) receptor antagonists failed to affect the responses to A61603, but methiothepin, which, in addition, has a high affinity for alpha-adrenoceptors, proved an effective antagonist. The A61603-induced responses were suppressed by the cAMP stimulator forskolin, but not by the protein kinase C inhibitor chelerythrine. Our results suggest that the contraction of porcine isolated meningeal artery by A61603 is mediated via mainly alpha(1)-(probably alpha(1A)) and, to a lesser extent, alpha(2)-adrenoceptors, involving the adenylyl cyclase, but not the diacylglycerol pathway.

Effect of chronic ethanol consumption on endothelin-1 generation and conversion of exogenous big-endothelin-1 by the rat carotid artery

The purpose of the present work was to investigate whether conversion of exogenous applied big-endothelin-1 (Big-ET-1) as well as the basal release and mRNA levels of endothelin-1 (ET-1) is altered by ethanol consumption in the rat carotid. The measurement of the contraction induced by Big-ET-1 served as an indicative of functional endothelin (ET)-converting enzyme (ECE) activity. Cumulative application of exogenous Big-ET-1 elicited a concentration-related contraction with the concentration–response curve shifted to the right when compared to ET-1. In endothelium-intact rings, phosphoramidon (1 mmol/l), a nonselective ECE/neutral endopeptidase (NEP) inhibitor, produced a rightward displacement of the concentration–response curves and reduced the maximal contractile response to Big-ET-1. However, in endothelium-denuded rings phosphoramidon reduced the maximum contraction for Big-ET-1 but did not alter the potency when compared to the curves obtained in the absence of the inhibitor. Ethanol consumption for 2, 6, or 10 weeks reduced the contractile effect elicited by Big-ET-1 in carotid rings with intact endothelium when compared to control or isocaloric rings. However, no differences on Big-ET-1–induced contraction were observed after endothelial denudation. On the other hand, ethanol consumption increased ET-1–induced contraction. Finally, chronic ethanol consumption did not alter either the mRNA levels for pre–pro-ET-1 nor the basal release of ET-1. The present findings show that chronic ethanol consumption does not alter the mRNA levels for ET-1 or its basal release in the rat carotid. Moreover, ethanol intake reduces the contraction induced by exogenously applied Big-ET-1 in carotid rings with intact endothelium, a fact that might be the result of a reduced conversion of this peptide by ECE on its mature active peptide ET-1.

The role of the urothelium in mediating bladder responses to isoprenaline

To investigate whether the responses of the pig bladder to isoprenaline (a nonselective beta-adrenoceptor agonist) are influenced by the presence of an intact urothelium and whether any influence might be attributed to the release of nitric oxide (NO), since stimulation of beta-adrenoceptors induces a direct relaxation of detrusor smooth muscle and beta-adrenoceptors are also present on the urothelium. Paired (in the presence or absence of urothelium) longitudinal strips of pig bladder dome were set up in tissue baths and the developed tension recorded. Relaxation responses to isoprenaline were examined after pre-contraction with carbachol. The inhibitory effects of isoprenaline were examined by comparing responses to carbachol in the absence and presence of isoprenaline. To examine a possible role for NO, similar experiments were performed in the presence of the NO synthase inhibitor N(G)-nitro-L-arginine (L-NNA). In the presence of the urothelium, both the potency (pEC(50)) and the maximum contractile responses to carbachol were depressed. In relaxation experiments, isoprenaline relaxed carbachol pre-contracted tissues by approximately 75%, and the potency and maximum relaxation were similar in the absence and presence of the urothelium. In the inhibition experiments, the presence of isoprenaline caused rightward parallel shifts of the concentration-response curves to carbachol, but isoprenaline did not influence the maximum contractions. In the presence of the urothelium there was a greater shift with 0.1 microm isoprenaline than in denuded tissues. Incubation with L-NNA did not affect the influence of the urothelium on responses to isoprenaline in any experimental group. The relaxation responses of the bladder to isoprenaline do not appear to involve the urothelium or NO release in vitro. However, contractile responses to carbachol were inhibited in the presence of an intact urothelium, and this might reflect the release of an inhibitory factor other than NO.

PPARγ agonists partially restores hyperglycemia induced aggravation of vascular dysfunction to angiotensin II in thoracic aorta isolated from rats with insulin resistance

Altered vascular responses to various vasopressors in animal models of insulin resistance (IR) and diabetes have been well documented. However, the precise mechanisms about vascular responses in IR with or without frank hyperglycemia (prediabetic state) are not available. Moreover, recently the role of peroxisome proliferators activated receptor-γ (PPARγ) has been linked to influence the vascular responses in hypertensive and diabetic state. Hence, the present study was conceived to determine the role of hyperglycemia on angiotensin II (Ang II) mediated vascular responses in the high fat diet (HFD) induced insulin resistance either with mild or frank hyperglycemia [induced by injection of low dose streptozotocin (STZ) to HFD fed rats (HFD + STZ)]. In addition, insulin-sensitizing agent such as rosiglitazone and pioglitazone were also studied on biochemical and vascular responses. Ang II-induced contractions were studied isometrically in thoracic aortic rings isolated from 4 weeks of normal pellet diet (NPD) fed control, HFD and HFD + STZ fed insulin resistant rats. Specific binding of Ang II receptors were carried out using radioligand ([ 3H]–Ang II) binding studies. After 4 weeks of HFD feeding, rats exhibited characteristics features of insulin resistance such as mild hyperglycemia, hyperinsulinemia, hypertriglyceridemia, hypercholesterolemia and hypertension; whereas HFD + STZ treated rats showed all above parameters along with frank hyperglycemia. Maximal contractile response ( E max) to Ang II is increased in HFD fed rats as compared to control rats. Moreover, E max values are further elevated in HFD + STZ group where the frank hyperglycemia was induced by low dose of streptozotocin. Rosiglitazone (5 mg kg −1, p.o.) and pioglitazone (10 mg kg −1, p.o.) treatment significantly lowered the plasma glucose, triglycerides, insulin and cholesterol levels in insulin resistance rats. In addition, it also restored the elevated systolic, mean arterial, diastolic blood pressure and attenuated the enhanced contractile responses to Ang II in thoracic aortic rings obtained from both HFD and HFD + STZ treated rats. Specific binding of [ 3H]–Ang II is upregulated in HFD-fed and HFD + STZ treated rats. Treatment with pioglitazone and rosiglitazone significantly decreased the AT 1R specific binding in HFD fed rats. Our results indicate the role of hyperglycemia in the elevation of Ang II induced vascular responses in thoracic aorta isolated from insulin resistant rats and PPARγ agonists can attenuate these responses.

52: Non-hypoxic ischemia: An accurate cell culture model of ischemia reperfusion injury in lung transplantation

“non-treated” control. Furthermore another control investigating the impact of the vehicle ethyl alcohol was obtained. Results: The maximal contractile response for 1mmol NA was similar between the different concentrations of RAPA and the controls. Comparing the ACh induced relaxation in relation to the NA (1mmol) induced precontraction before versus after incubation in each group, we found a concentration dependent decrease in relaxation, being significant for 1mmol and 10mmol RAPA compared to the control (in %: control: 48.0 4.4; 1mmol :30.2 2.8, p 0.01; 10mmol: 12.9 1.2, p 0.001, ethyl alcohol: 51.2 3.3 n.s.). Conclusions: Our results revealed, that contractility induced by NA revealed no direct effect of RAPA on muscular contractile function. However, relaxation of ITA induced by ACh is concentration-dependently decreased by RAPA indicating impaired endothelial function after only short-term exposure to RAPA.

Impact of freezing/thawing procedures on the post-thaw viability of cryopreserved human saphenous vein conduits

Background: Cryopreserved human blood vessels are important tools in reconstructive surgery. However, patency of frozen/thawed conduits depends largely on the freezing/thawing procedures employed. Methods: Changes in tone were recorded on rings from human saphenous vein (SV) and used to quantify the degree of cryoinjury after different periods of exposure at room temperature to the cryomedium (Krebs–Henseleit solution containing 1.8 M dimethyl sulfoxide and 0.1 M sucrose) and after different cooling speeds and thawing rates following storage at −196 °C. Results: Without freezing, exposure of SV to the cryomedium for up to 240 min did not modify contractile responses to noradrenaline (NA). Pre-freezing exposure to the cryomedium for 10–120 min attenuated significantly post-thaw maximal contractile responses to NA, endothelin-1 (ET-1) and potassium chloride (KCl) by 30–44%. Exposure for 240 min attenuated post-thaw contractile responses to all tested agents markedly by 62–67%. Optimal post-thaw contractile activity was obtained with SV frozen at about −1.2 °C/min and thawed slowly at about 15 °C/min. In these SV maximal contractile responses to NA, ET-1 and KCl amounted to 66%, 70% and 60% of that produced by unfrozen controls. Following cryostorage of veins for up to 10 years the responsiveness of vascular smooth muscle to NA was well maintained. Conclusion: Cryopreservation allows long-term banking of viable human SV with only minor loss in contractility.

Omalizumab decreases nonspecific airway hyperresponsiveness in vitro

In asthmatic patients, both symptoms and hyperresponsiveness are related to immunoglobulin E (IgE) concentration in serum. The anti-IgE monoclonal antibody omalizumab improved the control of asthma, but its effect on airway hyperresponsiveness is controversial. Passive sensitization reproduced in vitro a bronchial hyperresponsiveness, an increase in IgE bearing cells, and a mast cell degranulation. This study was designed to examine the effect of omalizumab on passive sensitization-induced hyperresponsiveness, alterations in IgE positive inflammatory cells and mast cell degranulation within the bronchial wall. Proximal (3-5 mm diameter) and distal (0.5-1.5 mm diameter) human bronchi dissected out from 10 lung specimens were incubated in normal or asthmatic serum containing various concentrations of omalizumab. Contractile responses to histamine or Dermatophagoides pteronyssinus (D. pter) were recorded using an organ bath system and expressed as percentage of maximal contractile response to acetylcholine (ACh). Immunohistochemistry was performed using monoclonal antibodies directed against IgE or tryptase. Mast cells were classified as fully granulated (type I), partly (type II) or largely degranulated (type III). The specific bronchial hyperresponsiveness to D. pter and the nonspecific bronchial hyperresponsiveness to histamine following passive sensitization were significantly inhibited by omalizumab in both distal and proximal airways. Passive sensitization-induced increase in IgE positive cells was also abolished by omalizumab in a concentration dependent manner. Mast cell degranulation which was inhibited by omalizumab was positively correlated with the contractile response to D. pter. Omalizumab blocks specific and nonspecific bronchial hyperresponsiveness. Anti-IgE also decreases IgE bearing cell number and mast cell degranulation.

Intraindividual comparison of human radial and internal thoracic arteries in vitro and the effect of preoperative calcium blocker therapy

The patency rate of radial artery (RA) conduits is considerably lower than that of internal thoracic artery (ITA) grafts and the evidence suggests that this is due to a clinically suspected higher incidence of vasospasm. The aim of this study was, therefore, to compare intraindividually the pharmacological reactivity of RA with that of ITA. Both RA and ITA were taken from the same patients and investigated in parallel. Changes in tone were monitored isometrically on ring preparations from both arteries in organ baths with modified Krebs-Henseleit solution containing 1.25 mm calcium chloride at 1 g passive preload. In intraindividual comparisons maximal receptor-mediated contractile responses to noradrenaline and endothelin-1 and endothelium-dependent acetylcholine-induced relaxant responses revealed no differences between both arteries. By contrast, depolarization-induced contractions to potassium chloride (KCl) appeared to be significantly higher in RA than in ITA. Further analysis, however, revealed that this difference was due to preoperative calcium entry blocker (Ca(2+)eB) therapy. Compared with control tissues, maximal responses to KCl were significantly attenuated in the ITA but unchanged in RA when arteries were obtained from patients with preoperative Ca(2+)eB therapy. The present results suggested that functional responses to pharmacological stimuli of both RA and ITA were quite similar. Preoperative Ca(2+)eB therapy, however, attenuated markedly responses to KCl of the ITA leaving those of RA unchanged. These results, demonstrating a lower sensitivity to Ca(2+)eB of RA, therefore suggested that in addition to Ca(2+)eB other classes of drug may be more effective at reducing the propensity of RA conduits to vasospasm.

Effects of adenosine receptor antagonists on amitriptyline-induced vasodilation in rat isolated aorta

Background. Although we have previously demonstrated the beneficial effects of adenosine receptor antagonists in preventing cardiovascular toxicity of amitriptyline in rats, it is not clear whether adenosine receptors in heart or in vasculature are dominant. The aim of the current study was to investigate the role of adenosine A2a receptors on amitriptyline-induced vasodilation in rat isolated aorta. Methods. After determining EC80 of noradrenalin (NA) (the concentration of noradrenalin that produces 80% of maximal contractile response) as 10−5M, the IC50 value of amitriptyline was measured in rat isolated aorta (the drug concentration causing a half- maximal inhibition of contractile responses to NA); IC50 of amitriptyline was then compared in the presence of the DPCPX (a selective adenosine A1 antagonist), CSC (a selective A2a antagonist) or DMSO (a solvent for adenosine antagonists). Statistical analysis was done using the Student t test. Results. Amitriptyline-inhibited 49.9 ± 3.7 % contractile response to NA on aorta segments at 1.8 × 10−5M (IC50). While DPCPX increased amitriptyline-induced inhibition on contractile response to NA dose dependently, CSC decreased the contractile response to NA only at 10−5M. DMSO did not change amitriptyline-induced IC50. Conclusion. Adenosine A2a receptor stimulation seems to be responsible partly for amitriptyline-induced vasodilation and hypotension since the adenosine A1 antagonist, DPCPX, increased amitriptyline-induced vasodilation in rat isolated aorta.

DO ANTIBIOTICS CONTRIBUTE TO POSTOPERATIVE ILEUS? CONTRACTILE RESPONSES OF ILEUM SMOOTH MUSCLE IN GUINEA PIGS TO LONG‐TERM PARENTERAL CEFTRIAXONE AND AMPICILLIN

Antibiotics may impair small bowel smooth muscle contractility and contribute to postoperative ileus. The aim of this study was to compare the contractile responses of ileum smooth muscle to different agonists in guinea pigs treated with ceftriaxone (Rocephin; F. Hoffman-La Roche, Kaiseraugst, Switzerland) or ampicillin (Ampisina; Mustafa Nevzat Ilaç Sanayii AS, Istanbul, Turkey). Twenty-four adult guinea pigs were randomly divided into three groups. Whereas eight of these received ceftriaxone sodium (100 mg/kg per day, i.m.) for 10 days, another eight guinea pigs received ampicillin (50 mg/kg per day, i.m.) for 10 days and the remaining eight served as the control group receiving 1 mL distilled water during 10 days as placebo. By the end of 10 days, the animals were killed and their ilea were excised. Ileum segments were placed in an organ bath; concentration-response relationship for carbachol and histamine were obtained by adding the reagent cumulatively to the bath. pD(2) values being the same, maximum contractile responses (E(max)) to carbachol and histamine were significantly reduced in the ceftriaxone sodium group compared with the control group. No significant differences in E(max) and pD(2) values to carbachol and histamine were observed between the ampicillin group and the control group. These data indicate that whereas ceftriaxone may impair small bowel smooth muscle contractility, ampicillin does not. There are implications for the long-term use of parenteral antibiotics in the postoperative period.

Intestinal edema decreases intestinal contractile activity via decreased myosin light chain phosphorylation

The purpose of this study was to investigate the effects of interstitial edema on intestinal contractile activity. Randomized animal study. University laboratory. Male Sprague-Dawley rats. Intestinal edema was induced in rats by a combination of fluid infusion and mesenteric venous hypertension. Rats were divided into four groups: CONTROL, sham; RESUS, saline infusion only; RESUS+VH, saline infusion and venous hypertension; and VH, venous hypertension only. Edema development, basal contractile activity, maximum agonist-induced contractile response (measured as total force generation during the first 2 mins after carbachol treatment), and myosin light chain phosphorylation were measured in the distal small intestine. The amount of interstitial fluid, indicated by the wet-to-dry ratio, increased significantly in both the RESUS and RESUS+VH groups as early as 30 mins after surgery compared with the CONTROL group. Whereas the tissue fluid remained significantly elevated in the RESUS+VH group up to 6 hrs after surgery, the RESUS group wet-to-dry ratios returned to CONTROL group levels by 2 hrs after surgery. Basal contractile activity was significantly less in the RESUS+VH group compared with either the RESUS group or the CONTROL group 6 hrs after surgery. Maximum contractile response decreased significantly in the RESUS+VH group compared with the RESUS group. Basal contractile activity and maximum contractile response did not change significantly in the VH group compared with the CONTROL group. The phosphorylated fraction of myosin light chain was significantly lower in the RESUS+VH group compared with the CONTROL group at 0.5, 2, and 6 hrs after surgery. We conclude that edema decreases myosin light chain phosphorylation, leading to decreased intestinal contractile activity.

Enhancement of rat bladder contraction by artificial sweeteners via increased extracellular Ca2+ influx

Consumption of carbonated soft drinks has been shown to be independently associated with the development of overactive bladder symptoms (OR 1.62, 95% CI 1.18, 2.22) [Dallosso, H.M., McGrother, C.W., Matthews, R.J., Donaldson, M.M.K., 2003. The association of diet and other lifestyle factors with overactive bladder and stress incontinence: a longitudinal study in women. BJU Int. 92, 69-77]. We evaluated the effects of three artificial sweeteners, acesulfame K, aspartame and sodium saccharin, on the contractile response of isolated rat detrusor muscle strips. Strips of detrusor muscle were placed in an organ bath and stimulated with electrical field stimulation (EFS) in the absence and presence of atropine, and with alpha,beta methylene ATP, potassium, calcium and carbachol. Sweeteners 10(-7) M to 10(-2) M enhanced the contractile response to 10 Hz EFS compared to control (p<0.01). The atropine-resistant response to EFS was marginally increased by acesulfame K 10(-6) M, aspartame 10(-7) M and sodium saccharin 10(-7) M. Acesulfame K 10(-6) M increased the maximum contractile response to alpha,beta methylene ATP by 35% (+/-9.6%) (p<0.05) and to KCl by 12% (+/-3.1%) (p<0.01). Sodium saccharin also increased the response to KCl by 37% (+/-15.2%) (p<0.05). These sweeteners shifted the calcium concentration-response curves to the left. Acesulfame K 10(-6) M increased the log EC(50) from -2.79 (+/-0.037) to -3.03 (+/-0.048, p<0.01) and sodium saccharin 10(-7) M from -2.74 (+/-0.03) to 2.86 (+/-0.031, p<0.05). The sweeteners had no significant effect on the contractile response to carbachol but they did increase the amplitude of spontaneous bladder contractions. These results suggest that low concentrations of artificial sweeteners enhanced detrusor muscle contraction via modulation of L-type Ca(+2) channels.

Essential oil of Pterodon polygalaeflorus inhibits electromechanical coupling on rat isolated trachea

The present work studied the effects of the essential oil of Pterodon polygalaeflorus (EOPP), a plant used to treat bronchitis and amigdalytis, on rat airway smooth muscle in vitro. In Ca 2+-containing medium, EOPP (100–1300 μg/ml) inhibited preferentially high KCl- than 5-HT-induced muscle contractions in a concentration-dependent fashion, but did affect neither basal muscle tension nor ACh-induced contractions. In preparations maintained in either 60 mM K + or 10 μM ACh in Ca 2+-free medium, EOPP (100, 600 and 1300 μg/ml) inhibited maximum contractile response induced by cumulative Ca 2+ addition (0.1–20 mM). Verapamil (10, 30 and 100 μg/ml), a Ca 2+ channel blocker, also inhibited Ca 2+-induced concentration–effect curve in presence of ACh in Ca 2+-free medium, whilst it was ineffective to decrease cholinergic contractions in Ca 2+-containing medium. In presence of 150 mM K + in Ca 2+-containing medium, EOPP (1300 μg/ml) did not reversed ACh-induced contractions. In contrast, under similar conditions, EOPP almost fully relaxed cholinergic contractions of tracheal smooth muscle in Ba 2+-containing medium. In medium containing 10 mM tetraethylammonium and 2 mM Ba 2+ instead of Ca 2+, both EOPP (1300 μg/ml) and verapamil (∼5 μg/ml) significantly decreased ACh-induced contractions. Thus, in rat isolated trachea, EOPP induces inhibitor effects on contractions preferentially triggered by an electromechanical coupling mode.

Ischemia/Reperfusion-Induced Low Reactivity of the Rat Superior Mesenteric Vascular Bed is Associated With Expression of Nitric Oxide Synthases

Our objective was to investigate the mRNA and protein expressions of eNOS and iNOS in the mesenteric vascular bed after ischemia and reperfusion of the rat superior mesenteric artery (SMA) and the role of nitric oxide (NO) in the response of the vascular bed to vasoconstrictors following reperfusion of the SMA. Methods Real-time polymerase chain reaction and immunohistochemistry were used to monitor the mRNA and protein expression of eNOS and iNOS after I/R challenge to the rat SMA. Ischemia was induced by clamping the SMA for 40 minutes, after which the flow was restored and the vessels were reperfused for 300 minutes. Blood samples were collected for assays of lactic dehydrogenase, tumor necrosis factor (TNF), hydroxyl radical, and NO. After ischemia/reperfusion, the vascular beds were separated for analysis of the expression of eNOS and iNOS. The SMA with its associated intestinal tissue was isolated and perfused in vitro with Tyrode’s solution ( N = 8) then challenged with phenylephrine. Results Reperfusion of the SMA induced an increase in blood concentrations of lactic dehydrogenase ( P < .001; N = 8), hydroxyl radical ( P < .05), TNF ( P < .001), and NO ( P < .05). ENOS and iNOS mRNA expression increased 1.3 ± 0.1-fold and 19.6 ± 3.5-fold, respectively when compared to the sham-operated group. Protein expression increased 1.9 ± 0.4-fold and 12.6 ± 3.1-fold, respectively, after reperfusion (N = 3) when compared with sham-treated rats. In vitro challenge showed that administration of phenylephrine (10 −8 ∼ 10 −4 nmol) produced vasoconstriction in a dose-related manner. Maximum contractile responses to phenylephrine were attenuated in reperfused SMA. Addition of the NOS inhibitor N G-nitro-L-arginine (L-NNA, 10 −4 M) resulted in full recovery of the response to phenylephrine. Conclusions Ischemia/reperfusion of the SMA results in a decrease in vascular reactivity of the mesenteric vessels that is dependent on NOS expression by the intestinal vascular bed.

Effects of endothelin receptor blockade on hypervasoreactivity in streptozotocin-diabetic rats: vessel-specific involvement of thromboxane A2

Increased vasoconstrictor response to norepinephrine (NE) and endothelin (ET)-1 in arteries from diabetic animals is ameliorated by chronic endothelin receptor blockade with bosentan and was absent in endothelium-denuded arteries, suggesting the involvement of ET-1 and an endothelium-derived contracting factor such as thromboxane A2 (TxA2). To examine this possibility, we determined the effects of acute blockade of ET receptors or inhibition of TxA2 synthesis on the vascular function of superior mesenteric arteries (SMA) and renal arteries (RA) isolated from nondiabetic and 11-week streptozotocin (STZ) diabetic rats chronically treated with either bosentan or vehicle. Both in vitro incubation with bosentan and a selective ETA receptor blocker, BQ123, eradicated the increase in NE contractile responses in diabetic SMA. Additionally, in vitro incubation with the thromboxane synthase inhibitor, dazmegrel, abrogated the exaggerated NE and ET-1 contractile responses in diabetic SMA. Conversely, in RA, no significant acute effect of bosentan, BQ123, nor dazmegrel on vascular responses to NE was observed. Dazmegrel incubation attenuated the maximum contractile responses to ET-1 in diabetic RA; however, these responses in diabetic RA remained significantly greater than those of other groups. Diabetic RA but not SMA exhibited an enhanced contractile response to the TxA2 analogue U46619, which was corrected by chronic bosentan treatment. Immunohistochemical analyses in diabetic SMA revealed an increase in ETA receptor level that was normalized by chronic bosentan treatment. These data indicate that an interaction between ET-1 and TxA2 may be involved in mediating the exaggerated vasoconstrictor responses in diabetic arteries. Furthermore, the underlying mechanisms appear to be vessel specific.

A Rho-kinase inhibitor, Y-27632, reduces cholinergic contraction but not neurotransmitter release

This study examined the effects of the selective Rho-kinase inhibitor Y-27632 [(+)-( R)- trans-4-(1-aminoethyl)-(4-pyridyl)cyclohexanecarboxamide dihydrochloride]) on cholinergic nerve-mediated contraction and neurotransmitter release in murine and guinea-pig isolated tracheal preparations. In tracheal preparations obtained from both species, Y-27632 shifted carbachol concentration–effect curves to the right and reduced the maximal contractile response. Repeated electrical field stimulation (EFS) evoked transient, consistent and reproducible contractions in murine and guinea-pig tracheal preparations. Y-27632 inhibited these cholinergic nerve-mediated contractions in a concentration-dependent manner. EFS (0.1–30 Hz) elicited frequency-dependent cholinergic nerve-mediated contractile responses. In murine tracheal preparations, Y-27632 (3 μM and 10 μM) shifted frequency–response curves to EFS to the right by 5.5 and 13.0 fold respectively and markedly reduced the maximal contractile response. In murine and guinea-pig tracheal preparations loaded with [ 3H]-choline, Y-27632 (10 μM) significantly increased the EFS-induced outflow of radioactivity from airway cholinergic nerves by 27% and 54% respectively. Thus, Y-27632 inhibited both carbachol-induced and cholinergic nerve-mediated contractile responses. Conversely, Y-27632 increased neurotransmitter release from airway cholinergic nerves. However, since antagonism of acetylcholine-induced contraction by Y-27632 overwhelmed the increased neurotransmitter release, the overall effect of this Rho-kinase inhibitor was to inhibit cholinergic nerve-mediated contraction.

Age Related Changes in the Functional, Biochemical and Molecular Properties of α 1-Adrenoceptors in the Rat Genitourinary Tract

Because age related changes occur in the properties of alpha(1)-adrenoceptor in several mammalian tissues and alpha(1)-adrenoceptor antagonists are extensively used to treat lower urinary tract symptoms secondary to benign prostatic hyperplasia, we investigated age related changes in the functional, biochemical and molecular properties of alpha(1)-adrenoceptor in the rat genitourinary tract. The characteristics of alpha(1)-adrenoceptor in the ventral and dorsolateral prostate, and bladder base and dome of 3 and 22-month-old rats were determined using an isolated muscle bath, radioligand receptor binding and real-time reverse transcriptase-polymerase chain reaction techniques. Old rats had significantly higher body weight, lower testosterone, a smaller ventral prostate and a larger bladder dome than young rats. Although there was no significant age dependent difference in the properties of alpha(1)-adrenoceptor in the bladder base and dome, total alpha(1)-adrenoceptor density, mRNA expression of all 3 alpha(1)-adrenoceptor subtypes (alpha(1A), alpha(1B) and alpha(1D)) and the maximum contractile responses to phenylephrine were significantly lower in the ventral and dorsolateral prostate of 22 vs 3-month-old rats. Age related differences in the molecular, biochemical and functional properties of alpha(1)-adrenoceptors in the rat genitourinary tract may indicate potential differences in the response to alpha(1)-adrenoceptor antagonists with aging, ie a decrease in the therapeutic response in old vs young rats in the response to alpha(1)-adrenoceptor antagonists when used to treat lower urinary tract symptoms secondary to benign prostatic hyperplasia.

Plasticity of rat small intestine after removal of a chronic mechanical obstruction

Chronic intestinal obstruction is associated with morphological changes and functional disorders clinically reported and experimentally documented in laboratory animals. In contrast, little is known about the properties of the hypertrophied intestine after removal of the obstruction. In the present study, we removed the ileal obstruction previously applied to the ileum of rats and, after 1 or 2 weeks, studied in vitro the motor responses of de-obstructed segments of intestine to pharmacological or electrical field stimulation (EFS). By 2 weeks after de-obstruction, maximal contractile responses to receptor (acetylcholine) and non-receptor (K(+)) mediated stimuli were comparable in operated and control tissues; furthermore, the loss of sensitivity to nitric oxide (NO) unmasked in obstructed tissues was, after de-obstruction, replaced by supersensitivity to exogenous NO and vasoactive intestinal polypeptide, probably acting through cyclic nucleotide-independent pathways. Despite the complete recovery of smooth muscle responses, neurogenic contractions remained impaired in de-obstructed tissue; however, the equal contribution of cholinergic/peptidergic components to EFS responses could represent a sign of gradual but delayed recovery of enteric neurotransmission.