The nonadrenergic noncholinergic relaxation of anococcygeus muscles of bile duct-ligated rats

Previous studies have shown the naloxone-induced withdrawal syndrome and the development of tolerance in the tissues of cholestatic animals. Increased neuronal nitric oxide synthase (nNOS) expression is reported to exist in morphine-tolerant animals. This, together with evidence for nitric oxide (NO) overproduction in cholestasis, suggested the possibility of an alteration of nonadrenergic noncholinergic (NANC) relaxation of anococcygeus muscles of cholestatic rats. To study this, we used three main groups of animals: unoperated, sham-operated and bile duct-ligated. Electrical field stimulation, in the presence of atropine and guanethidine, caused NANC relaxation in the anococcygeus muscle which was enhanced in bile duct-ligated animals. N(G)-nitro-L-arginine methyl ester (L-NAME), a NOS blocker, caused a dose-dependent inhibition of the NANC relaxation. The IC(50)'s of L-NAME in 7-day (7.30+/-0.87 microM), 14-day (6.98+/-0.70 microM) and 21-day (8.25+/-1.40 microM) bile duct-ligated groups were significantly different from those of unoperated (1.69+/-0.30 microM) and sham-operated groups (1.90+/-0.27 microM). L-NAME (100 microM) completely inhibited the NANC relaxation response, suggesting that NANC relaxation in the rat anococcygeus muscle is mediated mainly via NO. The contraction response of the intact muscle to phenylephrine, an alpha(1)-adrenoceptor agonist, and the relaxation response of the phenylephrine-contracted muscle to sodium nitroprusside, an NO donor, were not different in unoperated, sham-operated and 7-day bile duct-ligated groups. These results showed that the smooth muscle component of NANC relaxation is not altered in anococcygeus muscles of bile duct-ligated rats. It can thus be concluded that the NANC relaxation in the anococcygeus of cholestatic rats is more resistant to a NOS blocker, providing evidence for increased nitrergic neurotransmission in the anococcygeus muscles of cholestatic rats.     

Mesenteric vascular bed responsiveness in bile duct-ligated rats: roles of opioid and nitric oxide systems.

Changes in vascular responsiveness are proposed as the basis for some of the cardiovascular complications in cholestasis. Cholestasis is also associated with accumulation of endogenous opioid peptides and evidence of overproduction of nitric oxide (NO). The possible role of NO or opioid system in cholestasis-induced mesenteric vascular bed responsiveness was investigated. Bile duct-ligated and sham-operated rats were treated for 6 days with either normal saline, naltrexone, an opioid antagonist (20 mg/kg/day) or L-NAME (N(omega)-nitro-L-arginine methyl ester), a nitric oxide synthase inhibitor (3 mg/kg/day). After 7 days, the superior mesenteric artery was cannulated and the mesenteric vascular bed was perfused according to the McGregor method. Baseline perfusion pressure of the mesenteric vascular bed was decreased in bile duct-ligated compared to sham-operated animals. ED(50) of phenylephrine-induced vasoconstriction was increased, but vasoconstriction R(max) was not different in the vascular bed of bile duct-ligated rats and of sham-operated ones. Acetylcholine-induced vasorelaxation was impaired in bile duct-ligated rats (increased ED(50) and decreased vasorelaxation R(max)). Sodium nitroprusside-induced vasorelaxation was not different between bile duct-ligated and sham-operated rats, implying that the smooth muscle components of vasorelaxation were intact. Chronic treatment with L-NAME partially restored both the acetylcholine-induced vasorelaxation and phenylephrine-induced vasoconstriction response in bile duct-ligated rats. Naltrexone treatment also partially restored the acetylcholine-induced vasorelaxation and phenylephrine-induced vasoconstriction in bile duct-ligated rats. There is impaired acetylcholine-induced vasorelaxation in cholestatic rats, probably due to a defect in endothelial function. This study also provided evidence for the involvement of increased opioidergic tone and NO overproduction in cholestasis-induced vascular hyporesponsiveness.     

Time-dependent reduction of acetylcholine-induced relaxation in corpus cavernosum of cholestatic rats: role of nitric oxide and cyclooxygenase pathway

The endothelium-dependent relaxation of corpus cavernosum smooth muscle and the roles of nitric oxide (NO) and arachidonic acid products of cyclooxygenase were investigated in non-operated, SHAM-operated, and bile duct-ligated rats. We further investigated the time-dependent alterations of corpus cavernosum relaxation in 2-, 7-, and 14-day bile duct-ligated animals. Acetylcholine produced concentration-dependent relaxation in phenylephrine-precontracted strips of corpus cavernosum. A significant reduction in the acetylcholine-induced relaxation was observed 2 days after bile duct ligation, and a greater reduction was observed on subsequent days. Incubation with 20 microM indomethacin reduced the acetylcholine-induced relaxation of the corpus cavernosum of unoperated rats while it had no effect in the corpus cavernosum of bile duct-ligated rats. Chronic treatment with Nomega-Nitro-L-Arginine Methyl Ester (L-NAME, 3 mg/kg/day, intraperitoneally) reduced the relaxation responses in the unoperated group while it had no effect in the bile duct-ligated group. These results show that acetylcholine-induced corporal relaxation is impaired in cholestatic rats, and this may be related to deficient nitric oxide production by the endothelium. The involvement of prostaglandins in this impairment seems unlikely.     

The role of nitric oxide in bradycardia of rats with obstructive cholestasis

Nitric oxide (NO) has an important role in controlling heart rate and contributes to the cholinergic antagonism of the positive chronotropic response to adrenergic stimulation. Based on evidence of NO overproduction in cholestasis and also on the existence of bradycardia in cholestatic subjects, this study aimed to evaluate the chronotropic effect of epinephrine in isolated atria of cholestatic rats and determine whether alterations in epinephrine-induced chronotropic responses of cholestatic rats are corrected after systemic inhibition of NO synthase (NOS) with N(G)-nitro-L-arginine (L-NNA). Male Sprague-Dawley rats were used. Cholestasis was induced by surgical ligation of the bile duct under general anesthesia and sham-operated animals were considered as control. The animals were divided into three groups, which received either L-arginine (200 mg/kg/day), L-NNA (10 mg/kg/day) or saline. One week after the operation, a lead II ECG was recorded from the animals, then spontaneously beating atria were isolated and chronotropic responses to epinephrine were evaluated in a standard oxygenated organ bath. The results showed that plasma gamma-glutamyl transpeptidase and alanine aminotransferase activity was increased by bile-duct ligation, and that L-aginine treatment partially, but significantly, prevented the elevation of these markers of liver damage. The results showed that heart rate of cholestatic animals was significantly less than that of sham-operated control rats in vivo and this bradycardia was corrected with daily administration of L-NNA. The basal spontaneous beating rate of atria in cholestatic animals was not significantly different from that of sham-operated rats in vitro. Meanwhile, cholestasis induced a significant decrease in chronotropic effect of epinephrine. These effects were corrected by daily administration of L-NNA. Surprisingly L-arginine was as effective as L-NNA and increased the chronotropic effect of epinephrine in cholestatic rats but not in sham-operated animals. Systemic NOS inhibition corrected the decreased chronotropic response to adrenergic stimulation in cholestatic rats, and suggests an important role for NO in the pathophysiology of heart rate complications in cholestatic subjects. The opposite effect of chronic L-arginine administration in cholestasis and in control rats could be explained theoretically by an amelioration of cholestasis-induced liver damage by chronic L-arginine administration in bile duct-ligated rats.     

Potentiation of anandamide effects in mesenteric beds isolated from bile duct-ligated rats: role of nitric oxide

Changes in vascular responsiveness are proposed as the basis for some of the cardiovascular complications in cholestasis. Cholestasis is also associated with accumulation of endogenous opioid peptides and evidence of nitric oxide (NO) overproduction. On the other hand, it is well known that anandamide, an endogenous cannabinoid ligand, causes hypotension and a decrease in systemic vascular resistance. In the present study, the possible role of the cannabinoid system in cholestasis-induced mesenteric vascular bed responsiveness was investigated. Mesenteric arteries of bile duct-ligated and sham-operated rats receiving daily administrations of saline were used for evaluating phenylephrine or anandamide dose-response, acute effects of N(G)-nitro-L-arginine methyl ester (L-NAME, 100 microM), a non-selective inhibitor of NO synthase (NOS), or naltrexone, an opioid receptors antagonist (1 microM). The other groups of bile duct-ligated and sham-operated rats received daily intraperitoneal administration of L-NAME (20 mg/kg/day), aminoguanidine, a selective inducible NOS (iNOS) inhibitor (150 mg/kg/day) or naltrexone (10 mg/kg/day). After 7 days, the superior mesenteric artery was cannulated and the mesenteric vascular bed was perfused according to the McGregor method. Anandamide-induced relaxation was significantly potentiated in mesenteric vascular beds of bile duct-ligated rats. Chronic treatment of bile duct-ligated animals with L-NAME and aminoguanidine blocked this hyperresponsiveness while the hyperresponsiveness was potentiated at large doses of anandamide on chronic treatment of these animals with naltrexone. Although acute L-NAME treatment of mesenteric beds completely blocked the anandamide-induced vasorelaxation in sham-operated rats, this vasorelaxation still was present in bile duct-ligated animals. Anandamide-induced vasorelaxation remained unaffected after acute naltrexone treatment of mesenteric beds in both bile duct-ligated and sham-operated rats. Our results indicate that (1) there is enhanced anandamide-induced vasorelaxation in cholestatic rats, probably due to a defect in cannabinoid or vanilloid receptors and (2) NO overproduction may be involved in cholestasis-induced vascular hyperresponsiveness.     

Modulation of cholestasis-induced antinociception by CCK receptor agonists and antagonists.

Acute cholestasis is associated with increased activity of the endogenous opioid system. Agonists and antagonists of cholecystokinin (CCK) receptors are known to modulate opioid-induced antinociception. In the present study, the effect of the CCK receptor agonist caerulein and the antagonist proglumide on antinociception induced during acute cholestasis was investigated in rats using the tail-flick test. A significant increase in nociception threshold was observed in bile duct ligated (BDL) rats compared to sham-operated controls that was maximum on day 7 after the operation and decreased thereafter. Proglumide (40 mg/kg, i.p.) did not affect nociception in unoperated and sham-operated animals, but exerted a significant potentiation of antinociception in cholestatic rats in a way similar to its potentiation effect on unoperated morphine-treated (2 mg/kg, s.c.) animals. Caerulein (0.005, 0.001, 0.01 and 0.02 mg/kg, s.c.), which did not change nociception per se or in sham-operated animals, also significantly potentiated the antinociception in BDL rats as well as in morphine-treated unoperated controls. Caerulein-induced potentiation of antinociception in BDL animals was completely reversed by proglumide pretreatment. Our findings show that, in cholestatic animals, modulation of nociception by the CCK system is different from normal subjects and resembles the state observable in morphine-administered subjects.     

Role of nitric oxide in hypodipsia of rats with obstructive cholestasis

Cholestasis is associated with the overproduction of nitric oxide (NO), and NO acts as an inhibitory mechanism when thirst is stimulated by water deprivation or by angiotensin II. Due to the presence of hypodipsia in the cholestatic condition, we have compared the rate of water intake between bile duct-ligated (cholestatic) and sham-operated rats. We have evaluated the effect of NO synthesis inhibition by N(G)-nitro-L-arginine (L-NNA, 10 mg kg(-1)/day) on the rate of water intake in cholestatic rats. The results showed that plasma alkaline phosphatase activity (a marker of liver damage) increased after bile-duct ligation, and that its elevation was partially (but significantly) prevented by treatment with L-arginine. A two-week bile-duct obstruction induced a significant decrease in the rate of water intake compared with sham-operated animals (35.87 +/- 1.45 vs 42.37 +/- 1.99 mL/day, P < 0.05). This effect was corrected by the daily administration of L-NNA. Surprisingly, L-arginine (200 mg kg(-1)/day) showed similar activity as L-NNA in cholestatic rats and increased water intake, but not in control animals. Systemic NO synthesis inhibition corrected the decrease in water intake observed in cholestatic rats. This suggests an important role for NO in the pathophysiology of hypodipsia in cholestatic subjects. The effect of chronic L-arginine administration observed in cholestatic rats but not seen in the control rats could be explained theoretically by the amelioration of cholestasis-induced liver damage by chronic L-arginine administration in bile duct-ligated rats.     

Effects of subacute lead acetate administration on nitric oxide and cyclooxygenase pathways in rat isolated aortic ring.

Low level exposure to lead increases blood pressure in human and rats. In this study, we investigated the contribution of the nitric oxide (NO) and cyclooxygenase pathways of aortic rings of 28-day lead-treated and control rats, to the responsiveness to phenylephrine and acetylcholine. There were no differences in phenylephrine contractions between the two groups. N(omega)-nitro-L-Arginine-methyl ester (L-NAME), a NO synthase inhibitor, caused attenuation in contraction response to phenylephrine in the aortic rings of the lead-treated rats, while endothelium-denudation caused attenuation in those of controls. This may be due to either endothelium-derived vasoconstrictor(s) (such as reactive oxygen species or endothelins) or a source of NO in smooth muscle cells. There is a left-shift in acetylcholine relaxation response. Indomethacin incubation caused a left-shift in relaxation response to acetylcholine in controls but without any effect on lead-treated ones. Indomethacin incubation caused attenuation in contraction to phenylephrine in both groups. The relaxation response to sodium nitroprusside is not different between the two groups, suggesting that smooth muscle relaxation component is intact. However, the relaxation response to glyceryl trinitrate is impaired in aortic rings of lead-treated rats. It can be concluded that NO and cyclooxygenase pathways are altered in aortic rings of lead-treated rats, with possible involvement of endothelium-derived vasoconstrictors.     

Contribution of endogenous opioids and nitric oxide to papillary muscle contractile impairment in cholestatic rats

Attenuated responsiveness to adrenoceptor stimulation has been proposed as an important factor underlying cardiovascular complications of cholestasis. We examined isolated papillary muscle responsiveness to alpha (phenylephrine) and beta-adrenoceptor (isoproterenol) agonists in 7-day bile duct-ligated rats. We investigated the role of nitric oxide (NO) and endogenous opioids in papillary muscle hyporesponsiveness to isoproterenol stimulation. In order to evaluate the effect of NO and endogenous opioids, animals were treated with chronic subcutaneous injections of N(omega)-nitro-L-arginine methyl ester (L-NAME, 10 mg/kg/day) or naltrexone (20 mg/kg/day), or isolated papillary muscles were exposed acutely to the same drugs (10(-4) and 10(-6) M, respectively) in an organ bath. The basal contractile force of papillary muscle, +dT/dtmax and -dT/dtmax, was significantly decreased in bile duct-ligated rats compared to sham-operated ones (P<0.05, for each value). The concentration-response curve for phenylephrine and isoproterenol demonstrated a reduced maximum effect in bile duct-ligated rats compared to the sham-operated group (P<0.01 and 0.05, respectively). Basal contractile abnormalities of bile duct-ligated rats were corrected by L-NAME or naltrexone treatment, either acute or chronic. While chronic L-NAME treatment resulted in a left-ward shift (P<0.05), it had no effect on the maximum effect in bile duct-ligated rats. Acute L-NAME treatment did not influence isoproterenol responsiveness. Acute and chronic naltrexone treatment resulted in partial and complete correction of the hyporesponsiveness of bile duct-ligated rats, respectively (P<0.05). This investigation demonstrates that the papillary muscles of 7-day bile duct ligated-rats have an impaired basal contractility and hyporesponsiveness to both alpha and beta-adrenoceptor stimulation. It also provides evidence for the involvement of increased opioidergic tone and NO overproduction in cholestasis-induced cardiac impairment.     

Role of opioid and nitric oxide systems in the nonadrenergic noncholinergic-mediated relaxation of corpus cavernosum in bile duct-ligated rats

Changes in nonadrenergic noncholinergic (NANC)-mediated relaxation of the anococcygeus muscle have been demonstrated in cholestasis. Cholestasis is also associated with accumulation of endogenous opioid peptides and nitric oxide (NO) overproduction. This study was therefore undertaken to investigate the effect of cholestasis on the NANC-mediated relaxation of corpus cavernosum in bile duct-ligated rats and to examine the possible roles of the opioid system and nitric oxide in the cholestasis-associated alterations of corpus relaxation. Bile duct-ligated and sham-operated rats were treated for 2 weeks with either normal saline, N (omega)-nitro L-arginine methylester (L-NAME) (3 mg/kg/day, i.p.) or naltrexone (20 mg/kg/day, i.p.). On the 14th day, the strips of corpus cavernosum were mounted under tension in a standard oxygenated organ bath with guanethidine sulfate (5 microM) and atropine sulfate (1 microM) (to produce adrenergic and cholinergic blockade). The strips were precontracted with phenylephrine hydrochloride (7.5 microM) and electrical field stimulation was applied at different frequencies to obtain NANC-mediated frequency-dependent relaxant responses. The results showed that the amplitudes of relaxation responses at each frequency in bile duct-ligated rats were greater than the responses of sham-operated animals. This increase in relaxation responses in bile duct-ligated rats was inhibited by chronic L-NAME administration for 2 weeks so it seemed that it might be due to the nitric oxide overproduction in cholestatic states. Chronic administration of naltrexone for 2 weeks to bile duct-ligated rats had the same inhibitory effect on the relaxation responses. Our results demonstrated that in cholestasis, there was an increase in NANC-mediated relaxation of corpus cavernosum and both opioid and nitric oxide systems were involved in this increase.     

The calpain inhibitor, A-705253, corrects penile nitrergic nerve dysfunction in diabetic mice

Despite the well-known involvement of the peripheral sympathetic abnormalities in the development of cardiovascular complications of cholestasis, the role of the central sympathetic system is still elusive. The goal of this study was to evaluate the effects of central sympathetic tone reduction, through clonidine administration, on hemodynamic parameters of 7-day bile duct-ligated rats. The contributions of nitric oxide and endogenous opioids were also examined by acute intravenous (10 min before clonidine) or chronic daily subcutaneous administrations of N(omega)-nitro-L-arginine methyl ester (L-NAME, 3 mg/kg) or naltrexone (20 mg/kg). Seven days after bile duct ligation or sham operation, animals were anesthetized with sodium pentobarbital. After hemodynamic stabilization, clonidine (10 microg/kg) was injected intravenously, which elicited an initial hypertension (the peripheral effect) followed by persistent hypotension and bradycardia (the central effects). Cholestatic rats demonstrated significant basal bradycardia (P<0.001) and hypotension (P<0.05), which were corrected by chronic naltrexone but not L-NAME treatment. While the peripheral effect of clonidine was blunted, the central effects were exaggerated in cholestatic rats (P<0.01). Acute L-NAME treatment accentuated the hypertensive phase in sham-operated and cholestatic rats (P<0.05). However, the difference between the two groups was preserved (P<0.01). This treatment attenuated the central effects in both sham-operated and cholestatic rats to the same level (P<0.001). Chronic L-NAME treatment resulted in exaggeration of the peripheral response in cholestatic and central responses in sham-operated rats (P<0.05), and abolished the difference between the groups. Naltrexone treatment had no significant effect on either the central or the peripheral responses to clonidine. This study shows that both central and peripheral hemodynamic responses to clonidine are altered in cholestasis. It also provides evidence that nitric oxide contributes to the development of these abnormalities.     

Altered vascular reactivity in mice made hypertensive by nitric oxide synthase inhibition

This study tested the hypothesis that nitric oxide (NO) synthase inhibition in mice would result in hypertension characterized by increased agonist-induced vasoconstrictor responsiveness and attenuated endothelium-dependent vasodilation. Administration of N-nitro-L-arginine (L-NNA), an NO synthase inhibitor (1 g/L, 4 weeks), via drinking water to mice resulted in significant elevations in blood pressure. Phenylephrine-induced contraction was significantly increased in aortic rings from L-NNA-treated mice compared with rings from control mice. Aortic rings from control mice showed a concentration-dependent relaxation to acetylcholine whereas those obtained from L-NNA-treated mice showed a biphasic response, contracting at lower concentrations while relaxing at higher concentrations. Aortic rings from L-NNA-treated mice had decreased relaxation to acetylcholine and increased sensitivity to sodium nitroprusside compared with control rings. The relaxation induced by an NO-independent soluble guanylyl cyclase activator was not different between groups. In aortic rings from control and L-NNA-treated mice pre-contracted with phenylephrine, the administration of L-NNA to the organ bath caused additional and sustained contraction. When compared with the contraction induced by phenylephrine, L-NNA-induced contraction in aorta from control mice was significantly higher than that in aorta from L-NNA-treated mice. We conclude that mice treated with L-NNA develop hypertension and that a reduction in NO availability is responsible for the changes observed in vascular reactivity.     

N(G)-nitro-L-arginine methylester is protective against ethanol-induced gastric damage in cholestatic rats.

In this study the effect of nitric oxide (NO) synthesis inhibition on ethanol-induced gastric damage was evaluated in bile duct-ligated, sham-operated and unoperated rats. The animals were injected intraperitoneally with saline, L-arginine (200 mg/kg) or N(G)-nitro-L-arginine methylester (L-NAME) in doses of 5, 15 and 30 mg/kg, 30 min before ethanol administration. The animals were killed 1 h after ethanol administration and their stomachs were removed for measurement of gastric mucosal damage. The results showed that L-NAME significantly enhanced the development of gastric mucosal lesions in sham-operated and unoperated rats, while in bile duct-ligated animals, L-NAME decreased and L-arginine enhanced the potentiation of ethanol-induced gastric mucosal damage. The plasma level of nitrite and nitrate was also measured and was significantly higher in bile duct-ligated rats than in control groups. The results suggest that inhibition of NO synthase with L-NAME has different effects on ethanol-induced gastric damage in cholestatic groups and in normal rats and that these effects can be explained by overproduction of NO in bile duct-ligated animals.     

Chronic 17β-estradiol augments relaxant role of basal nitric oxide in blood vessels from rats with heart failure

The effects of chronic 17β-estradiol on endothelium-dependent relaxation to acetylcholine (ACh) and contraction to N ^G-nitro-l-arginine methyl ester (l-NAME), and endothelium-independent relaxation to sodium nitroprusside (SNP) were examined on blood vessels from rats with chronic heart failure (CHF). Two groups of ovariectomized female (50–60 days) rats were implanted with pellets containing 17β-estradiol (25 μg/day) or vehicle, and given ligation of the left main coronary artery 1 week later. Another group of ovariectomized rats was implanted with vehicle pellets, and sham-operated. After 7 weeks, thoracic aortic rings, pulmonary artery rings, and portal vein strips were prepared for in vitro studies. Relative to sham-operated rats treated with the vehicle, vessels from vehicle-treated, coronary-ligated rats had similar relaxation to ACh and SNP but reduced response to l-NAME that was significant (P<0.05) for the aorta and portal vein but not pulmonary artery. Treatment of ligated rats with 17β-estradiol augmented responses to l-NAME in the aorta, pulmonary artery and portal vein to values above those in sham-operated rat. 17β-Estradiol did not affect relaxation of any vessels to SNP and increased maximum relaxation to ACh only in the portal vein. Hence, 17β-estradiol enhances the relaxant role of basal nitric oxide in CHF. Received: 17 June 1998 / Accepted: 21 September 1998     

N-nitro- -arginine methylester is protective against ethanol-induced gastric damage in cholestatic rats

In this study the effect of nitric oxide (NO) synthesis inhibition on ethanol-induced gastric damage was evaluated in bile duct-ligated, sham-operated and unoperated rats. The animals were injected intraperitoneally with saline, -arginine (200 mg/kg) or N^G-nitro- -arginine methylester ( -NAME) in doses of 5, 15 and 30 mg/kg, 30 min before ethanol administration. The animals were killed 1 h after ethanol administration and their stomachs were removed for measurement of gastric mucosal damage. The results showed that -NAME significantly enhanced the development of gastric mucosal lesions in sham-operated and unoperated rats, while in bile duct-ligated animals, -NAME decreased and -arginine enhanced the potentiation of ethanol-induced gastric mucosal damage. The plasma level of nitrite and nitrate was also measured and was significantly higher in bile duct-ligated rats than in control groups. The results suggest that inhibition of NO synthase with -NAME has different effects on ethanol-induced gastric damage in cholestatic groups and in normal rats and that these effects can be explained by overproduction of NO in bile duct-ligated animals.     

Effect of clofibrate on vascular reactivity in a model of high blood pressure secondary to aortic coarctation

The aims of this study were to identify the effect of clofibrate administration in the development of high blood pressure secondary to aortic coarctation (AoCo) and to assess its effect on vascular reactivity. Three experimental groups of rats were used: sham-operated, aortic coarctated vehicle-treated (AoCo-V), and aortic coarctated clofibrate-treated (AoCo-C100). The rats were treated for seven days. Blood pressure was measured, and the vascular response to angiotensin II (AngII), norepinephrine (NE), and acetylcholine (ACh) were evaluated in aortic rings. The activity and expression of endothelial nitric oxide synthase (eNOS) was also evaluated. The major findings of this study include the following: AoCo induced a rise in blood pressure, and this effect was attenuated by clofibrate. The vascular response to AngII was higher in aortic rings from the AoCo-V group compared to the Sham-V or AoCo-C100 groups. ACh-elicited vasorelaxation was lower in the arteries of AoCo-V rats than Sham-V or AoCo-C100, while it was comparable between the Sham-V and AoCo-C100 groups. In every case, vasorelaxation was dependent on NO. However, the ACh-induced release of NO as well as NOS activity and expression were reduced in the arteries of AoCo-V rats. Clofibrate maintained normal NOS activity and increased eNOS expression. In conclusion, clofibrate administration attenuated the AoCo-induced rise in blood pressure by a mechanism that involves the participation of the NO system at both the NO synthesis and the eNOS protein expression levels. These events improved endothelial function, preserved normal vascular responses to both vasorelaxants and vasoconstrictors, and led to better blood pressure control.     

Different contribution of endothelial nitric oxide in the relaxation of human coronary arteries of ischemic and dilated cardiomyopathic hearts

Coronary artery disease and congestive heart failure (CHF) have been associated with a reduction in nitric oxide (NO) release or bioavailability from the vascular endothelium. The objectives of this study were to compare the role of NO in human coronary vessels isolated from nonischemic dilated (DCM) (n = 10) and ischemic (ICM) (n = 12) cardiomyopathic hearts. Segments were mounted on a wire myograph to record changes in isometric tension. All experiments were performed in the presence of indomethacin (10 microM). Contractions induced by angiotensin II (0.1 microM) or a depolarizing physiologic solution containing 40 mM KCl, were of similar amplitude in DCM and ICM. In vessels precontracted with angiotensin II, acetylcholine (1 microM) caused an endothelium-dependent relaxation of rings from DCM but a paradoxical contraction of rings from ICM; NO synthase inhibition with Nomega-nitro-L-arginine (L-NNA, 100 microM) did not affect acetylcholine-induced relaxation or contraction of DCM or ICM vessels, respectively. By contrast, substance P (0.1 microM) induced an endothelium-dependent relaxation in both groups of vessels; this relaxation was prevented (p < 0.05) by L-NNA in vessels from ICM hearts but only reduced (p < 0.05) by L-NNA in vessels from DCM hearts. In depolarized conditions, acetylcholine contracted (p < 0.05) whereas substance P induced a complete relaxation (p < 0.05) of vessels from both groups: substance P-induced relaxation was abolished (p < 0.05) by L-NNA. Our data suggest that in the presence of indomethacin, NO does not contribute to acetylcholine-induced relaxation of human epicardial coronary arteries isolated from DCM hearts. Furthermore, whereas NO and a secondary endothelium-derived relaxing factor sensitive to high K+ contribute to substance P-induced relaxation of rings from DCM hearts, only NO is involved in ICM hearts.     

Relaxation of rat aorta by adenosine in diabetes with and without hypertension: role of endothelium

Effects of diabetes on the responses of aortic rings of normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rat to adenosine analogues were examined. Streptozotocin-induced diabetes caused an increase in blood glucose and plasma levels of cholesterol and triglycerides in normotensive (diabetic-WKY) as well as hypertensive (diabetic-SHR) rats. In diabetic-SHR group, the body weight was significantly low (50%) as compared to SHR (non-diabetic). Diabetic-SHR group showed the largest heart weight-to-body weight ratio indicating cardiac enlargement. The relaxation responses to adenosine analogues were obtained in endothelium-intact and -denuded aortic rings precontracted with phenylephrine. The IC(50) values of adenosine analogues were lower in endothelium-intact aortic rings of WKY as compared to diabetic-WKY and -SHR. Aortic rings from diabetic-SHR showed the greatest attenuation in adenosine analogue-mediated relaxation. Removal of endothelium from the aortic rings inhibited the relaxant response of adenosine analogues and abolished the differences among the groups. Nitric oxide (NO) synthase inhibitor L-monomethylarginine (L-NMMA) caused a significant rightward shift in the concentration-response curves in WKY and diabetic-WKY groups, only a small shift in SHR and no change in diabetic-SHR group indicating that it is primarily the inhibition of NO release which is responsible for attenuation of adenosine receptor responses in SHR and diabetic-WKY and there was absence of NO release in diabetic-SHR. Forskolin and sodium nitroprusside equally relaxed the aortic rings in all the groups. This suggested that there was no abnormality in the relaxant property of vascular smooth muscle due to hypertension and/or diabetes. Therefore, it is concluded that streptozotocin-induced diabetes in SHR aggravates the severity of vascular endothelial dysfunction which led to impairment in adenosine receptor-mediated vascular responses.     

Quinapril treatment restores the vasodilator action of insulin in fructose-hypertensive rats

1. Angiotensin-converting enzyme (ACE) inhibitors have been shown to improve insulin-resistance both experimentally and clinically. We therefore investigated the effects of quinapril, which has high tissue specificity for ACE, regarding the contribution of insulin to vascular contractions, as well as insulin sensitivity in a dietary rat model of insulin resistance. 2. Male Sprague-Dawley rats were divided into three groups: (i) rats fed normal chow (normal diet group); (ii) rats fed fructose-rich chow containing 40% fructose and 7% lard (fructose diet group); and (iii) rats fed fructose-rich chow plus quinapril (10 mg/kg per day; quinapril-treated group). 3. After 2 weeks, we evaluated systolic blood pressure, insulin sensitivity as assessed by steady state plasma glucose (SSPG) levels, response of aortic rings to phenylephrine (10-9 to 10-6 mol/L) in the presence or absence of insulin and the response of aortic rings to acetylcholine. 4. Feeding rats fructose-rich chow resulted in an elevation of blood pressure (P < 0.01) and SSPG levels (P < 0.01). Quinapril treatment significantly prevented increases in both blood pressure and SSPG, with a return to the levels seen in the normal diet group. 5. In the absence of insulin, the maximal contractile response to phenylephrine did not differ between the three groups. However, in the presence of insulin (100 mU/mL), the contractile response to phenylephrine (10-6 mol/L) was reduced by 22.8 +/- 1.2% in the normal diet group, although no insulin effects were observed in the fructose diet group (P < 0.01). Quinapril restored the inhibitory effect of insulin on phenylephrine-induced contractions. 6. In addition, the reduction in relaxation induced by acetylcholine in the fructose diet group was significantly reversed by quinapril treatment. 7. It is concluded that the fructose diet impairs the vasodilator effects of insulin as well as acetylcholine-induced relaxation in rat thoracic aortas. Quinapril prevented deterioration in the responses of the aortic rings, suggesting that ACE inhibitors may be useful for treating vascular insulin resistance.     

Pharmacokinetics of Intragastrically Administered Digoxin in Rabbits with Experimental Bile Duct Obstruction

A change in the functioning of the liver as a result of experimental cholestasis could result in a change in the biotransformation of drugs. The aim of this study was to evaluate the effect of extrahepatic cholestasis on the pharmacokinetics of digoxin. The investigation was performed on male rabbits randomly divided into two groups: sham-operated and animals with bile-duct ligation. Digoxin (0.02 mg kg^?1) was administered intragastrically as a single dose. Biomedical and anatomo-pathological tests and pharmacokinetic assays were performed before the operation and on the 6th day after surgery. A significant increase in area under the serum concentration-time curve and in mean residence time, a decrease in total body clearance, a reduction in the volume of distribution and increases in maximum concentration and the time to reach maximum concentration were observed in animals with the bile-duct ligation. These results suggest reduced elimination of digoxin in animals with obstructive cholestasis.