Role of Extracellular Calcium and Calcium Sensitization in the Anti-Contractile Effect of Perivascular Adipose Tissue in Pregnant Rat Aorta.

Previous studies have shown that the anti-contractile effect of the perivascular adipose tissue (PVAT) is attenuated in pregnancy. In the present investigation, we have examined the possibility that this loss of anti-contractile effect could be due to changes in calcium mobilization. PVAT exerted anti-contractile effect against 5-hydroxytryptamine (5-HT)-induced contractions of aorta segments from pregnant and non-pregnant rats and this anti-contractile effect was attenuated in segments from pregnant rats. Nifedipine (10-6 mol/L), an inhibitor of L-type dihydropyridine calcium channels, significantly reduced 5-HT-induced contraction of aorta segments from non-pregnant and pregnant rats with and without PVAT. The inhibitory effect of nifedipine against 5-HT-induced contractions was attenuated in PVAT-free aorta segments from pregnant rats. However, while PVAT reduced the effectiveness of nifedipine in aorta segments from non-pregnant rats, it partially restored the inhibitory effect of nifedipine in aorta segments from pregnant rats. Inhibitors of calcium sensitization, Y-27632 (10-6 mol/L) and GF 109203X (10-6 mol/L), significantly reduced 5-HT-induced contractions of PVAT-free aorta segments from non-pregnant and pregnant rats. Both inhibitors, however, were less effective in aorta segments from pregnant rats. The presence of PVAT reduced the effectiveness of Y-27632 and GF 109203X in aorta segments from pregnant and non-pregnant rats. Protein expression of Rho-associated protein kinase (ROCK) I and II was detected in aorta segments and PVAT from pregnant and non-pregnant rats. There was a reduction in the expression of both isoforms in aorta segments but not PVAT from pregnant rats. In addition, there was no significant difference in the expression of ROCK-I and ROCK-II in PVAT from pregnant and non-pregnant rats. We concluded that the loss of anti-contractile effect of PVAT in aorta segments from pregnant rats could be due to increased influx of extracellular calcium through nifedipine-sensitive dihydropyridine channels.

Antihyperalgesic Activities of Endocannabinoids in a Mouse Model of Antiretroviral-Induced Neuropathic Pain.

Background: Nucleoside reverse transcriptase inhibitors (NRTIs) are the cornerstone of the antiretroviral therapy for human immunodeficiency virus/acquired immune deficiency syndrome (HIV/AIDS). However, their use is sometimes limited by the development of a painful sensory neuropathy, which does not respond well to drugs. Smoked cannabis has been reported in clinical trials to have efficacy in relieving painful HIV-associated sensory neuropathy. Objectives: The aim of this study was to evaluate whether the expression of endocannabinoid system molecules is altered during NRTI-induced painful neuropathy, and also whether endocannabinoids can attenuate NRTI-induced painful neuropathy. Methods: BALB/c mice were treated with 25 mg/kg of 2',3'-dideoxycytidine (ddC, zalcitabine), a NRTI, to induce thermal hyperalgesia. The expression of endocannabinoid system molecules was evaluated by real time polymerase chain reaction in the brain, spinal cord and paw skin at 6 days post ddC administration, a time point when mice had developed thermal hyperalgesia. The effects of the endocannabinoids, N-arachidonoyl ethanolamine (AEA) and 2-arachidonoyl glycerol (2-AG), the cannabinoid type 1 (CB1) receptor antagonist AM 251, CB2 receptor antagonist AM 630, and G protein-coupled receptor 55 (GPR55) antagonists ML193 and CID 16020046 on ddC-induced thermal hyperalgesia were evaluated using the hot plate test. Results: ddC treatment resulted in thermal hyperalgesia and increased transcripts of the synthesizing enzyme Plcß1 and decreased Daglß in the paw skins, but not Napepld, and Daglα compared to vehicle treatment. Transcripts of the inactivating enzymes Faah and Mgll were downregulated in the brain and/or paw skin but not in the spinal cord of ddC-treated mice. Both AEA and 2-AG had antihyperalgesic effects in mice with ddC-induced thermal hyperalgesia, but had no effect in ddC-naïve mice. The antihyperalgesic activity of AEA was antagonized by AM251 and AM630, whereas the activity of 2-AG was antagonized by AM251, ML193 and CID 16020046, but not by AM630. Conclusion: These data show that ddC induces thermal hyperalgesia, which is associated with dysregulation of the mRNA expression of some endocannabinoid system molecules. The endocannabinoids AEA and 2-AG have antihyperalgesic activity, which is dependent on cannabinoid receptor and GPR55 activation. Thus, agonists of cannabinoid receptors and GPR55 could be useful therapeutic agents for the management of NRTI-induced painful sensory neuropathy.

Loss of Anticontractile Effect of Perivascular Adipose Tissue on Pregnant Rats: A Potential Role of Tumor Necrosis Factor-α.

The present investigation examined the effect of pregnancy on the anticontractile effect of perivascular adipose tissue (PVAT) on the rat. Ring segments of the aorta, with and without PVAT, were set up in organ baths for isometric tension recording. In both groups, concentration-response curves to 5-hydroxytryptamine (5-HT) were displaced to the right with a reduction of the maximum response in aorta segments with PVAT. The anticontractile effect of PVAT was attenuated on segments from pregnant rats. 4-Aminopyridine (4-AP), an inhibitor of voltage-gated potassium (Kv) channels, enhanced 5-HT-induced contractions of aorta segments from pregnant and nonpregnant rats only when PVAT was attached. There was no difference in the effect of 4-aminopyridine on 5-HT-induced contractions of aorta segments with PVAT from pregnant and nonpregnant rats. There was also no significant difference in the expression of Kv7.4 channels in aorta segments (with PVAT) between pregnant and nonpregnant rats. Tumor necrosis factor-α (TNF-α) was detected in PVAT from pregnant and nonpregnant rats. The level of TNF-α was significantly greater in PVAT from pregnant rats. Treatment of pregnant rats with pentoxyphyline significantly reduced the level of TNF-α in the PVAT and restored the anticontractile effect of PVAT on aorta segments from pregnant rats. Finally, TNF-α (10 ng/mL) potentiated 5-HT-induced contractions of PVAT-containing pregnant rat aorta. These results would suggest that the loss of anticontractile effect of PVAT in pregnant rat aorta could be due to enhanced production of TNF-α in the PVAT in these rats.

Molecular basis for the effects of zinc deficiency on spermatogenesis: An experimental study in the Sprague-dawley rat model.

INTRODUCTION: The objective of this study is to investigate the molecular mechanisms underlying the effects of zinc deficiency on spermatogenesis in the Sprague-Dawley (SD) rat. MATERIALS AND METHODS: Three groups of eight adult male SD rats were maintained for 4 weeks on a normal diet as control, zinc deficient diet and zinc deficient diet with zinc supplementation of 28 mg zinc/kg body weight respectively. Using standard techniques, the following parameters were compared between the three groups of experimental animals at the end of 4 weeks: (a) Serum zinc, magnesium (Mg), copper (Cu), selenium (Se) and cadmium (Cd), (b) serum sex hormones, malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GPX), (c) interleukin-4 (IL-4), tumor necrosis factor-alpha (TNF-α), Bcl-2, Bax and caspase-3 expression in the testes, (d) assessment of apoptosis of testicular cells using electron microscopy and (e) testicular volume and histology using the orchidometer and Johnsen score, respectively. RESULTS: The zinc deficient group showed a reduction of testicular volume, serum concentrations of Zn, Cu, Se, Mg, SOD, GPX, IL-4, Bcl-2 and testosterone (P < 0.05), as well as increased levels of serum Cd, MDA and tissue TNF-α, Bax, caspase-3 and apoptosis of the germ cells (P < 0.05) compared with control and zinc supplementation groups. CONCLUSION: Zinc deficiency is associated with impaired spermatogenesis because of reduced testosterone production, increased oxidative stress and apoptosis. These findings suggest that zinc has a role in male reproduction.

Perivascular adipose tissue, vascular reactivity and hypertension.

Most blood vessels are surrounded by a variable amount of adventitial adipose tissue, perivascular adipose tissue (PVAT), which was originally thought to provide mechanical support for the vessel. It is now known that PVAT secretes a number of bioactive substances including vascular endothelial growth factor, tumor necrosis factor-alpha (TNF-α), leptin, adiponectin, insulin-like growth factor, interleukin-6, plasminogen activator substance, resistin and angiotensinogen. Several studies have shown that PVAT significantly modulated vascular smooth muscle contractions induced by a variety of agonists and electrical stimulation by releasing adipocyte-derived relaxing (ADRF) and contracting factors. The identity of ADRF is not yet known. However, several vasodilators have been suggested including adiponectin, angiotensin 1-7, hydrogen sulfide and methyl palmitate. The anticontractile effect of PVAT is mediated through the activation of potassium channels since it is abrogated by inhibiting potassium channels. Hypertension is characterized by a reduction in the size and amount of PVAT and this is associated with the attenuated anticontractile effect of PVAT in hypertension. However, since a reduction in size and amount of PVAT and the attenuated anticontractile effect of PVAT were already evident in prehypertensive rats with no evidence of impaired release of ADRF, there is the possibility that the anticontractile effect of PVAT was not directly related to an altered function of the adipocytes per se. Hypertension is characterized by low-grade inflammation and infiltration of macrophages. One of the adipokines secreted by macrophages is TNF-α. It has been shown that exogenously administered TNF-α enhanced agonist-induced contraction of a variety of vascular smooth muscle preparations and reduced endothelium-dependent relaxation. Other procontractile factors released by the PVAT include angiotensin II and superoxide. It is therefore possible that the loss could be due to an increased amount of these proinflammatory and procontractile factors. More studies are definitely required to confirm this.

Colonic inflammation increases the contribution of muscarinic M2 receptors to carbachol-induced contraction of the rat colon.

OBJECTIVES: Carbachol-induced contraction of the rat colon is impaired in rats with trinitrobenzene sulfonic acid (TNBS)-induced colitis. The main objective of this study was to examine the effect of colitis on the expression and function of muscarinic (M) receptor subtypes in the rat colon. MATERIALS AND METHODS: Rats (n = 80) were treated with TNBS and used 5 days later for measurement of contractility, myeloperoxidase activity, histology and expression of muscarinic receptor isoforms using Western blot analysis. RESULTS: Carbachol produced concentration-dependent contractions of colonic segments from control (n = 40) and TNBS-treated (n = 40) rats with no significant difference in potency. However, the maximum response to carbachol was significantly reduced in colon segments of TNBS-treated rats. The selective muscarinic receptor antagonists 4-diphenylacetoxy-N-methyl piperidine (4-DAMP, M(3)), pirenzepine (M(1)) and methoctramine (M(2)) antagonized carbachol-induced contraction in control (9.1 ± 0.1, 6.7 ± 0.3 and 6.0 ± 0.1, respectively) and TNBS-treated rats (9.2 ± 0.2, 6.9 ± 0.2, 6.7 ± 0.2). The -logK(B) values in control rats are consistent with an action of carbachol on muscarinic M(3) receptors. There was no significant difference in -logK(B) values for 4-DAMP and pirenzepine in control and TNBS-treated rats, but methoctramine was fivefold more potent in TNBS-treated rats, possibly indicating an increased contribution of muscarinic M(2) receptors to carbachol-induced contraction in the inflamed colon. The expression of M(2) receptors was also significantly increased in colon segments from TNBS-treated rats, confirming the increased role of muscarinic M(2) receptors in the inflamed colon. CONCLUSIONS: The data show that while only M(3) receptors appeared to mediate carbachol-induced contraction in control segments, expression of both M(2) and M(3) receptors was increased in the inflamed rat colon.

Hyperthyroidism enhances 5-HT-induced contraction of the rat pulmonary artery: role of calcium-activated chloride channel activation.

Experimentally-induced hyperthyroidism in rodents is associated with signs and symptoms of pulmonary hypertension. The main objective of the present study was to investigate the effect of thyroxine-induced pulmonary hypertension on the contractile response of the pulmonary artery to 5-HT and the possible underlying signaling pathway. 5-HT concentration-dependently contracted artery segments from control and thyroxine-treated rats with pD(2) values of 5.04 ± 0.19 and 5.34 ± 0.14, respectively. The maximum response was significantly greater in artery segments from thyroxine-treated rats. Neither BW 723C86 (5-HT(2B)-receptor agonist) nor CP 93129 (5-HT(1B)-receptor agonist) contracted ring segments of the pulmonary artery from control and thyroxine-treated rats at concentrations up to 10(-4)M. There was no significant difference in the level of expression of 5-HT(2A)-receptor protein between the two groups. Ketanserin (3 × 10(-8)M) produced a rightward shift of the concentration-response curve to 5-HT in both groups with equal potency (-logK(B) values were 8.1 ± 0.2 and 7.9 ± 0.1 in control and thyroxine-treated rats, respectively). Nifedipine (10(-6)M) inhibited 5-HT-induced contractions in artery segments from control and thyroxine-treated rats and was more effective against 5-HT-induced contraction in artery segments for thyroxine-treated rats. The calcium-activated chloride channel blocker, niflumic acid (10(-4)M) also inhibited 5-HT-induced contractions in artery segments from control and thyroxine-treated rats and was more effective against 5-HT-induced contraction in artery segments for thyroxine-treated rats. It was concluded that hyperthyroidism enhanced 5-HT-induced contractions of the rat pulmonary artery by a mechanism involving increased activity of calcium-activated chloride channels.

Lithium protects against toxic effects of cadmium in the rat testes.

PURPOSE: To investigate the protective effect of Lithium against the toxic effect of Cadmium in the rat testes. METHODS: Twenty four adult male Sprague-Dawley rats were treated with four different regimens: Cadmium only, Cadmium and lithium, lithium only and controls. Rats were sacrificed after 6 weeks and testicular levels of pro-inflammatory cytokine (IL-4), anti-inflammatory cytokine (TNF-α), Pro-apoptotic protein (Bax) and anti-apoptotic protein (Bcl-2) were measured by ELISA while serum levels of FSH, LH, Prolactin and Testosterone were measured using the Vidas parametric system. Antioxidant status (MDA, SOD) was also assessed in serum. Histopathological changes of testes were examined using light and electron microscopy. Immunohistochemical staining for Bax, Bcl-2 and Caspase 3 were performed. RESULTS: Treatment with lithium was associated with significant reduction in the toxic effects of Cadmium as shown by reduced testicular levels of TNF-α, serum levels of Malondialdehyde and testicular level of Bax, and increased levels of IL-4, Zn-Cu SOD, Bcl-2 and Testosterone. Testicular histopathology showed that Cadmium produced an extensive germ cells apoptosis and the addition of lithium in Cadmium-treated rats significantly reduced cadmium-induced testicular damage. CONCLUSION(S): Lithium has a protective effect against cadmium-induced testicular apoptosis in the rat.

Curcumin reverses attenuated carbachol-induced contraction of the colon in a rat model of colitis.

BACKGROUND: Curcumin ameliorates colitis whether it reverses colitis-induced reduction in colonic contractility remains to be investigated. OBJECTIVES: To investigate the effect of curcumin on colitis-induced reduction of carbachol-induced contraction in colon segments from rats treated with trinitrobenzenesulphonic acid. METHODS: Colitis was induced in rats by intra rectal administration of trinitrobenzenesulphonic acid and followed for 5 days. A group of animals which received trinitobenzene sulphonic acids was treated with curcumin (100 mg/Kg and 200 mg/kg body weight) 2 hrs prior to induction of colitis. The controls received phosphate buffered saline in a similar fashion. Markers of inflammation and contractility of colon were assayed using standard procedures. RESULTS: Induction of colitis was associated with increased myeloperoxidase activity and malondialdehyde levels, gross histological changes characterized by infiltration of inflammatory cells. All these changes were prevented by treatment with curcumin (100 mg/kg). Treatment with curcumin also reduced the histological scores from 3.34+/-0.40 to 1.75+/-0.30 confirming an anti-inflammatory effect of curcumin in this experimental model of colitis. Colonic reactivity to carbachol was decreased in colitis affecting the maximum response but not sensitivity. Treatment with curcumin had no effect on sensitivity of the colon to carbachol in any of the preparations. Curcumin however reversed the decrease in carbachol-induced contraction associated with trinitrobenzenesulphonic acid treatment. The same dose of curcumin had no effect on either the potency of or the maximum response to carbachol in control rats. Tissue expression of NF-kB was increased in colon segments from trinitrobenzenesulphonic acid -treated rats and this was inhibited in rats treated with curcumin. CONCLUSIONS: Based on these findings it is concluded that curcumin prevented the reduction in carbachol-induced contraction in trinitrobenzenesulphonic acid -treated rats by modulating NF-kB signaling pathway.

Role of Ca2+-sensitization in attenuated carbachol-induced contraction of the colon in a rat model of colitis.

Inflammatory bowel disease is associated with reduced colonic smooth muscle contractility. However the underlying mechanism responsible for the decrease in contractility is not fully understood. In this study we investigated the role of Ca(2+)-sensitization in reduced carbachol-induced contraction of colonic segments from rats treated with trinitrobenzenesulphonic acid (TNBS). Functional alterations in RhoA/Rho-kinase and protein kinase C (PKC) pathways were examined using specific antagonists, Y-27632 and GF-109203X respectively. In this study, TNBS-induced colitis was associated with a decrease in the maximum response but not sensitivity to carbachol. Permeabilized inflamed colonic segments showed greater sensitivity to Ca(2+) as compared to controls, indicating greater Ca(2+)-sensitivity of the myofilaments. In contrast, carbachol-induced increase in Ca(2+)-sensitization was reduced in these tissues suggesting that the reduced carbachol-induced contraction could be due to decreased Ca(2+)-sensitization. Y-27632, a Rho-kinase inhibitor, induced significantly greater relaxation in colon strips from TNBS-treated rats indicating higher basal tone in these tissues. This is consistent with increased expression of Rho-kinase in the inflamed colon. Y-27632 concentration-dependently inhibited carbachol-induced contractions in control and TNBS-treated rats. However its effect was not significantly different between the two groups. GF-109203X, a PKC antagonist, produced concentration-dependent reduction in carbachol-induced contractions in control and TNBS-treated rats. GF-109203X was less effective in reducing carbachol-induced contractions of colonic segments from TNBS-treated rats suggesting a defect in PKC activation. Western blotting analysis showed reduced expression of total PKC in inflamed colonic smooth muscle. Carbachol-induced phosphorylation of CPI-17 was also reduced in colonic segments from TNBS-treated rats. These findings suggest that Ca(2+)-sensitization in rat colon involves both the PKC and the Rho-kinase pathways and that the reduced carbachol-induced contraction in colitis was due to inflammation-induced changes in Ca(2+)-sensitization involving a defect in the PKC pathway.

Mechanism of reduced colonic contractility in experimental colitis: role of sarcoplasmic reticulum pump isoform-2.

Inflammatory bowel diseases (IBD) such as Crohn's disease and ulcerative colitis are inflammatory disorders associated with decreased colonic contractility. Here we show that, in experimental colitis in rat induced by trinitrobenzenesulfonic acid, there is a decrease in contraction in response to carbamoylcholine and the sarco/endoplasmic reticulum Ca(+2) (SERCA) pump inhibitor thapsigargin. However, the decrease in contractility may occur due to decrease in the SERCA pump levels or their inactivation. Therefore, we examined the protein and mRNA levels for SERCA2 isoform, which is predominant isoform in colonic smooth muscle. There was a decrease in the levels of SERCA2 protein and mRNA levels in inflamed colonic muscle. These findings suggest that decreased SERCA pump levels is responsible for a decrease in the Ca(+2) stores in the sarco/endoplasmic reticulum that causes a decrease in the contractility in colonic smooth muscle leading to poor bowel movements.

In vivo and in vitro assessment of human saphenous vein wall changes.

PURPOSE: To investigate if noradrenaline (NA) and 5-hydroxyptamine (5-HT) drugs induce responses of isolated control and varicose veins are altered by removal of the endothelium. SUBJECTS #ENTITYSTARTX00026; METHODS: Specimens of the great saphenous vein (GSV) were obtained from 12 subjects with primary varicose veins and 12 subjects from donor vessels at cardiac surgery. A total of 10 normal healthy volunteers were selected for comparison. The diameter changes of GSV during the resting phase, at the end of 5 minutes occlusion, and then every 30 seconds post deflation for five minutes were measured using B-mode ultrasound. Post-surgery the vein sample was collected in a tube of Krebs-Henseleit solution. RESULTS: The repeated measure ANOVA test for the diameter, percent, and difference changes of GSV diameter from maximum diameter at different time intervals showed significance difference within and between all groups. NA and 5-HT produced concentration-dependent contractions of control and varicose saphenous vein segments. There was no significant difference in the potency of NA and for 5-HT, but the maximum response, normalized for tissue weight, was less in varicose vein segments. Removal of the endothelium had no effect on the potency of NA or 5-HT but significantly (p<0.05) reduced the maximum response to NA and 5-HT in varicose vein segments but not to 5-HT in control veins. CONCLUSION: The venous endothelial damage may cause vascular smooth muscle contractions dysfunction that favours dilatation and secondary valvular insufficiency.

Pregnancy-induced modulation of calcium mobilization and down-regulation of Rho-kinase expression contribute to attenuated vasopressin-induced contraction of the rat aorta.

Normal pregnancy is characterized by attenuated vascular reactivity to a variety of contractile agonists and this, in part, has been attributed to increased circulating vasodilators and/or impaired Ca(2+)-influx through L-type Ca(2+)-channels. Our hypothesis in this study was that reduced Ca(2+)-dependent (influx) and Ca(2+)-independent (involving the RhoA/Rho-kinase pathway) mechanisms contributed to attenuated vasopressin-induced contraction of the pregnant rat aorta. AVP (10(-10) -3 x 10(-7) M) induced concentration-dependent contraction of aortic ring segments from nonpregnant and pregnant rats with no significant change in pD(2) values (8.53+/-0.11 and 8.33+/-0.18 in nonpregnant and pregnant rats, respectively). The maximum response was however significantly reduced in aorta segments from pregnant rats. Nifedipine (10(-6) M) significantly inhibited AVP-induced contraction in artery segments from nonpregnant but not pregnant rats indicating a reduced role for Ca(2+)-influx through L-type Ca(2+)-channels in AVP-induced contractions of the pregnant rat aorta. Western blot analysis revealed the expression of ROCK-1 and ROCK-II isoforms in aorta segments from both groups. There was a significant reduction in the expression of ROCK-1 and ROCK-II isoforms in aortic tissues from pregnant rats. This is consistent with the reduced potency of Y-27632 in inhibiting AVP (10(-7) M) induced contraction in aorta segments from pregnant rats. It was concluded that pregnancy-induced attenuated vascular response to AVP was due to decreased Ca(2+)-influx through L-type Ca(2+)-channels and decreased sensitization of the contractile myofilaments to Ca(2+).

Antihypertensive and vasodilator effects of methanolic and aqueous extracts of Tribulus terrestris in rats.

The effects of methanolic and aqueous extracts of Tribulus terrestris on rat blood pressure (BP) and the perfused mesenteric vascular bed were investigated. The extracts dose-dependently reduced BP in spontaneously hypertensive rats (SHRs) with the aqueous fraction being more potent than the methanolic fraction at all doses tested. In vitro, the methanolic but not aqueous extract produced a dose-dependent increase in perfusion pressure of the mesenteric vascular bed. When perfusion pressure was raised with phenylephrine (10(-5) M), the aqueous extract produced a dose-dependent reduction in perfusion pressure at all doses. A low dose of the methanolic extract produced a vasoconstrictor effect while higher doses produced dose-dependent reduction in perfusion pressure. L-NAME (10(-4) M) significantly reduced but did not abolish vasodilation induced by the extracts. Vasodilator responses to aqueous and methanolic fractions were significantly reduced in preparations where perfusion pressure was raised with KCl (60 mM). A combination of KCl and L-NAME abolished the vasodilator responses induced by the extracts. It was concluded that methanolic and aqueous extracts of Tribulus terrestris possess significant antihypertensive activity in spontaneously hypertensive rats. The antihypertensive effects appeared to result from a direct arterial smooth muscle relaxation possibly involving nitric oxide release and membrane hyperpolarization.

Increased expression and activity of heme oxygenase-2 in pregnant rat aorta is not involved in attenuated vasopressin-induced contraction.

Pregnancy is associated with attenuated vascular reactivity to a variety of contractile agonists. Heme oxygenases are expressed in the placenta, and it has been suggested that the heme oxygenase/carbon monoxide (HO/CO) pathway plays a significant role in regulating blood flow through the feto-placental unit. In this study we investigated the possible involvement of heme oxygenases in the reduced vascular reactivity associated with pregnancy. Arginine vasopressin (AVP) (10(-10)-3x10(-7) M) induced concentration-dependent contraction of aortic ring segments from non-pregnant and pregnant (16-19 days) rats. Pregnancy did not alter the sensitivity to AVP (pD2=8.5+/-0.1 and pD2=8.4+/-0.2 in non-pregnant and pregnant rats, respectively) but significantly reduced the maximum response (107.9+/-12.7% and 38.6+/-7.4%, respectively, relative to noradrenaline-induced contraction). Western blot analysis revealed the expression of HO-2 but not HO-1 isoform in both groups. There was a significant increase in the expression and activity of HO-2 protein in aortic tissues from pregnant rats compared with those from age-matched non-pregnant rats. In the presence of L-NAME to inhibit nitric oxide (NO) synthesis, tin protoporphyrin IX (SnPP-IX, 10(-5) M), an inhibitor of heme oxygenase, did not significantly affect AVP-induced contraction in aorta segments from pregnant and non-pregnant rats. It was concluded that, though pregnancy increased the expression and activity of HO-2 in the aorta, HO-2 was not involved in the attenuated response to AVP.

Attenuated noradrenaline-induced contraction of pulmonary arteries from rats treated with monocrotaline: role of rho kinase.

Noradrenaline-induced pulmonary artery contraction was reduced in monocrotaline-treated rats. The possibility that this could be due to alterations in the rho kinase pathway was examined in this study. A combination of nifedipine (10(-6) M) and thapsigargin (10(-6) M) attenuated noradrenaline-induced contraction significantly more in artery segments from monocrotaline-treated rats than in artery segments from control rats indicating a reduced role for calcium sensitization in artery segments from monocrotaline-treated rats. In artery segments permeabilized with ionomycin, CaCl(2) (1.25 mmol/l) produced significantly greater contraction in monocrotaline treated rats compared with control rats. Addition of noradrenaline (10(-5) M) to the bath produced further contractions in both groups. However, noradrenaline-induced contraction was less in monocrotaline-treated rats compared with controls. Y-27632 concentration dependently relaxed ring segments of pulmonary artery pre-contracted with noradrenaline (10(-5)M). The pIC(50) values were 6.46+/- 0.09 (n=5) 5.81+/- 0.06 (n=5) in control and pulmonary hypertensive rings, respectively. The maximum relaxation to Y-27632 was significantly higher in monocrotaline-treated rats. ROCK II was the predominant isoform of rho kinase expressed in the pulmonary artery. The level of expression was increased in rats treated with monocrotaline. These results would suggest that while basal rho kinase activity was elevated in monocrotaline-induced pulmonary hypertension, noradrenaline-induced contraction was attenuated, suggesting poor coupling of the receptor activation to rho kinase activation.

Amelioration of experimental colitis by Na-H exchanger-1 inhibitor amiloride is associated with reversal of IL-1ss and ERK mitogen-activated protein kinase.

OBJECTIVE: Na-H exchanger-1 (NHE-1) is induced in experimental colitis. It has not yet been established whether its inhibition ameliorates colitis. The effects of amiloride, an inhibitor of NHE-1, on colitis were examined in this study. Levels of mitogen-activated protein (MAP) kinases ERK, p38 and interleukin 1ss which participate in intestinal inflammation were also examined in the colonic smooth muscle of rats with colitis. MATERIAL AND METHODS: Colitis was induced in Sprague-Dawley male rats by intrarectal administration of trinitrobenzenesulphonic acid (TNBS) and treated daily with amiloride (3, 5, and 10 mg/kg b.w. (body-weight), orally) starting 1 h before induction of colitis. The animals were sacrificed on day 5 post-TNBS. Controls received phosphate buffered saline in a similar manner. RESULTS: The highest dose of amiloride (10 mg/kg) was lethal. The lowest dose (3 mg/kg) was tolerated and was used in this study. Amiloride significantly reversed the colitis-reduced contractility and induction of MPO activity, NHE-1, IL-1ss and ERK, but not of p38 in inflamed colonic smooth muscle. Splenomegaly, increased colonic mass and decreased sodium pump activity were significantly reversed by amiloride treatment. There was no recovery of b.w. loss in the treated colitic animals. Urine output was increased, whereas food and water intake remained unchanged following amiloride treatment. CONCLUSIONS: These findings suggest that the beneficial effects of NHE-1 inhibition in experimental colitis are mediated through IL-1ss and ERK MAP kinase.

Cyclo-oxygenase-2, endothelium and aortic reactivity during deoxycorticosterone acetate salt-induced hypertension.

OBJECTIVE: To test the hypothesis that the enhanced vascular responsiveness to norepinephrine that occurs during deoxycorticosterone acetate (DOCA)-salt induced hypertension is causally related to increased expression of cyclo-oxygenase (COX)-2 and oxidative stress, which diminishes the vasomodulatory influence of endothelium-derived nitric oxide. METHODS: Four groups of age-matched, male Sprague-Dawley rats were studied: Sham (normotensive); DOCA-salt (hypertensive); DOCA-salt treated with manganese(III) tetra(4-benzoic acid) porphyrin chloride [MnTBAP, an antioxidant; 15 mg/kg intraperitoneally (i.p.) for 21 days]; DOCA-salt treated with {N-[2-(cyclohexyloxy)-4-nitrophenyl]-methane sulfonamide} (NS-398, a COX-2 selective blocker; 5 mg/kg i.p. for 7 days). Contraction and relaxation were measured with FT03 force transducers coupled to a Grass polygraph in aortic rings bathed with physiologic salt solution (37 degrees C) and bubbled with a 5%CO2/95%O2 gas mixture. Aortic sensitivities (pD2 values) to norepinephrine and serum isoprostanes (8-iso-prostaglandin F2alpha, a marker of oxidative stress) were measured for each experimental paradigm. RESULTS: NS-398 significantly reduced maximal contractions in response to norepinephrine in aortic rings from Sham (44 +/- 3%) and DOCA-salt (96 +/- 2%) group rats. Expression of COX-2 protein increased significantly in vessels from DOCA-salt rats compared with those from Sham group rats. Treatment of DOCA-salt rats with either MnTBAP or NS-398 alleviated hypertension, normalized aortic pD2 values for norepinephrine and restored serum 8-isoprostane concentrations towards those observed in Sham group rats. CONCLUSIONS: COX-2 expression increases during DOCA-salt hypertension, and mediates production of factors that enhance rat aortic contractility in response to norepinephrine. Our data also suggest a role for increased oxidative stress, which is at least in part dependent on enhanced COX-2 expression, in the mechanism(s) of enhanced aortic contractility in response to norepinephrine during DOCA-salt hypertension.

Evidence for the presence of beta-3-adrenoceptors mediating relaxation in the human oviduct.

Beta1- and beta2-adrenoceptors mediate relaxation in the oviductal smooth muscle. This study examines the existence and function of beta3-adrenoceptors in the human oviduct. Ring segments of the oviduct were set up for isometric tension recording. The effect of isoprenaline and BRL 37344 on smooth muscle tone was examined. The expression of beta3-adrenoceptors in the oviduct was also examined. Isoprenaline and BRL 37344 concentration-dependently relaxed circular muscles of the oviduct. BRL 37344 was less potent than isoprenaline and was a partial agonist. Propranolol shifted isoprenaline but not BRL 37344 concentration-response curve to the right without reducing the maximum response. Cyanopindolol (1 micromol/l), a beta3-adrenoceptor antagonist, shifted the isoprenaline concentration response curve to the right. The -log K(B) value of 7.8 indicates activation of beta3-adrenoceptors by isoprenaline. mRNA for beta3-adrenoceptors was expressed in the oviduct. These results suggest that beta3-adrenoceptors, mediating relaxation, are expressed in the human oviduct.

Interaction of BKCa channel modulators with adrenergic agonists in the rat aorta is influenced by receptor reserve.

Our main objective was to study the interaction of BKCa channel modulators with adrenergic agonists UK 14304 and noradrenaline (NA), acting on alpha1-adrenoceptors, in the rat aorta and how this is affected by receptor reserve. NA and UK 14304 evoked concentration-dependent contractions of the rat aorta. UK 14304 was a partial agonist relative to NA in this preparation. The BK(Ca) channel blocker tetraethylammonium (TEA, 1 mM) and opener NS 1619 (3 x 10(-5) M) modulated NA- and UK 14304-induced contractions, and were more effective on UK 14304-induced contractions. TEA (1 mM) increased the maximum response to NA and UK 14304 by about 13% and 300%, respectively, while NS 1619 (3 x 10(-5) M) reduced the maximum response to UK 14304 by about 81% compared to 31% for noradrenaline. The effect of TEA on the noradrenaline concentration-response curve was increased after treatment of the aorta with phenoxybenzamine (PBZ), an irreversible alpha1-adrenoceptor antagonist, to reduce receptor reserve. We concluded that the interaction of BKCa channel modulators with alpha1-adrenergic agonists in the rat aorta was influenced by receptor reserve.