calcimycin and Body-Weight

In response to changes in wall shear stress (WSS) the vascular endothelium releases several factors, among others nitric oxide. On the basis of studies of endothelial cells in culture, suggesting that platelet endothelial cell adhesion molecule-1 (PECAM-1) is specifically involved in sensing and coupling high temporal gradients of fluid shear stress with activation of eNOS, we hypothesized that dilations of isolated skeletal muscle arterioles from PECAM-1 knockout mice (PECAM-KO) will be reduced to rapid increases in WSS elicited by increases in perfusate flow.. Small and large step increases in flow resulted in substantial dilations in arterioles of WT mice (45+/-4%), but they were markedly reduced in arterioles of PECAM-KO mice (22+/-5%). The initial slope of dilations, when WSS increased rapidly, was greater in vessels of WT than those of PECAM-KO mice (slopes: 0.378 and 0.094, respectively), whereas the second phase of dilations, when flow/shear stress was steady, was similar in the 2 groups (slopes: 0.085 and 0.094, respectively). Inhibition of eNOS significantly reduced the initial phase of dilations in arterioles from WT, but not from those of PECAM-KO mice. The calcium ionophore A23187 elicited similar NO-mediated dilation in both WT and PECAM-KO mice.. In isolated arterioles of PECAM-KO mice activation of eNOS and consequent dilation by agonists is maintained, but the dilation to high temporal gradients of wall shear stress elicited by increases in perfusate flow is reduced. Thus, we propose that PECAM-1 plays an important role in the ability of the endothelium to sense and couple high temporal gradients of wall shear stress to NO-mediated arteriolar dilation during sudden changes in blood flow in vivo.

Topics: Animals; Arterioles; Body Weight; Calcimycin; Calcium; Caveolin 1; Endothelium, Vascular; Ionophores; Male; Mice; Mice, Knockout; Muscle, Skeletal; Muscle, Smooth, Vascular; Nitric Oxide; Nitric Oxide Synthase Type III; Platelet Endothelial Cell Adhesion Molecule-1; Regional Blood Flow; Stress, Mechanical; Vasodilation

Both diabetes mellitus (DM) and elevated plasma copper concentrations are risk factors for cardiovascular disease (CVD). DM is associated with impaired endothelial nitric oxide (NO) and with excess superoxide (O2*-) formation. Copper is also elevated in DM and is also associated with the generation of O2*-. To explore possible interactions between DM and copper, the effect of exogenous copper (CuCl2) on endothelium-dependent relaxation and cyclic guanosine monophosphate (GMP) formation was investigated in aortae from diabetic rabbits. Rabbits were rendered diabetic by intravenous injection of alloxan. Six months after induction of DM, the aortae were excised, cut into rings, and mounted in an organ bath for isometric measurement of acetylcholine (Ach)-evoked relaxation in rings precontracted with phenylephrine (PE). In parallel studies, cyclic (c)GMP formation by aortic rings following stimulation with Ach, calcium ionophore A23187 (A23187) and sodium nitroprusside (SNP) was assessed using radioimmunoassay. The effect of copper on these parameters was then studied using the same methods. Ach-evoked relaxation and Ach- and A23187-evoked cGMP formation were significantly impaired in aortae from diabetic rabbits compared to controls, effects that were reversed with superoxide dismutase (SOD) and catalase (CAT). In contrast, there were no significant differences in SNP-stimulated relaxation or cGMP formation in aortae from diabetic rabbits compared to controls. Copper (1 to 10 micromol/L) promoted a further significant inhibition of Ach-stimulated relaxation in aortae from diabetic but not control rabbits. This reduction by copper was again reversed by SOD and CAT. We conclude that copper augments the reduction of NO bioavailability, which is already impaired in aortae from diabetic rabbits due to excess production of O2*- and H2O2. These results indicate that patients with DM may be susceptible to copper-mediated vasculopathy at much lower concentrations than those that promote vasculopathy in nondiabetic patients.

Topics: Acetylcholine; Animals; Aorta, Thoracic; Blood Glucose; Body Weight; Calcimycin; Catalase; Cholesterol; Copper; Cyclic GMP; Diabetes Mellitus, Experimental; Endothelium, Vascular; Free Radical Scavengers; In Vitro Techniques; Male; Muscle Relaxation; Muscle, Smooth, Vascular; Nitric Oxide; Nitroprusside; Rabbits; Reactive Oxygen Species; Superoxide Dismutase; Vasodilator Agents

In this study, we have investigated the vasodilator response to acetylcholine under diabetic conditions in isolated renal arteries of Wistar rats. The effect of nitric oxide synthase (NOS) inhibition on acetylcholine-induced vasodilator response was investigated. We have also examined the effects of two endothelium-dependent agonists which induce receptor-dependent and receptor-independent vasodilator responses. Acetylcholine (10(-10) to 10(-4)M) produced a cumulative concentration-response curve in the renal arteries of both control and diabetic rats. The EC(50) values and maximal responses to acetylcholine were reduced relative to diabetic conditions. The vasodilator response to sodium nitroprusside (SNP) (10(-10) to 10(-5)M) was also investigated. SNP produced a cumulative concentration-dependent vasodilator response, which was not affected under diabetic conditions. To confirm the nitric oxide component of acetylcholine-induced vasodilator response, L-nitro-methyl arginine ester (L-NAME) (10(-4)M) was added to the Krebs' solution. The maximal vasodilator response to acetylcholine was reduced in the presence of L-NAME (10(-4)M) in both control and diabetic renal preparations, with greater attenuation in the diabetic conditions. In order to examine the possible contribution of receptor dysfunction in diabetes, the vasodilator response to ADP (receptor-dependent agonist) and the calcium ionophore A23187 (receptor-independent agonist) were investigated. ADP (10(-10) to 10(-5)M) produced a concentration-dependent vasodilator response in preparations from both control and diabetic rats. The maximal vasodilator response to ADP was significantly reduced in the renal arteries from diabetic animals. However, A23187 (10(-10) to 10(-5)M); the receptor-independent agonist, produced a concentration-dependent vasodilator response in both control and diabetic rat preparations. There was no significant change in the EC(50) values or maximal vasodilator responses to A23187 under diabetic conditions. In conclusion, our results indicated that acetylcholine-induced vasodilatation in the isolated renal arteries of streptozotocin (STZ)-induced diabetic rats was attenuated under diabetic conditions. The reduction in acetylcholine-induced vasodilatation may be attributed to acetylcholine receptor dysfunction. This is based on the results from this study in which the vasodilator response to the receptor-independent agonist A23187 were maintained, while that of the receptor-dep

Topics: Acetylcholine; Adenosine Diphosphate; Animals; Body Weight; Calcimycin; Diabetes Mellitus, Experimental; Disease Models, Animal; Dose-Response Relationship, Drug; Endothelium-Dependent Relaxing Factors; Enzyme Inhibitors; Female; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Nitroprusside; Rats; Rats, Wistar; Renal Artery; Vasodilation; Vasodilator Agents

The present study evaluated the effect of dietary vitamin E supplementation (1,000 mg/kg chow) on the alterations in vascular reactivity of streptozotocin-diabetic aorta of Wistar rats. After 12 weeks of treatment, thoracic aortic rings of rats were mounted in organ baths and contractile responses to phenylephrine and 5-hydroxytryptamine and relaxant responses to acetylcholine, calcium ionophore and sodium nitroprusside were assessed. Plasma vitamin E concentration as measured by HPLC was markedly decreased in diabetic rats and increased with dietary vitamin E supplementation. Induction of diabetes significantly impaired endothelium-dependent relaxations to acetylcholine and calcium ionophore in aortic rings, but did not change endothelium-independent relaxation to sodium nitroprusside. Vitamin E significantly improved the impaired endothelium-dependent relaxations, further it decreased the enhanced contractile response to phenylephrine and 5-hydroxytryptamine in diabetic rings. The mechanical denudation of endothelium or the chemical inhibition of endothelium-dependent relaxation with N(omega)-nitro-L-arginine methyl ester (100 micromol/l) significantly increased phenylephrine contractility in control rings and the rings of diabetic rats treated with vitamin E; such a difference was not observed in diabetic rats fed with normal diet. Liver and lung malondialdehyde concentrations, as an index of lipid peroxidation, were increased in diabetic rats and significantly decreased with vitamin E supplementation. It is concluded that dietary supplementation of vitamin E improved endothelial dysfunction in insulin-dependent model of uncontrolled diabetes, probably decreasing membranal lipid peroxidation.

Topics: Acetylcholine; Animals; Aorta, Thoracic; Blood Glucose; Body Weight; Calcimycin; Diabetes Mellitus, Experimental; Dietary Supplements; Dose-Response Relationship, Drug; In Vitro Techniques; Liver; Lung; Male; Malondialdehyde; Nitroprusside; Phenylephrine; Potassium Chloride; Rats; Serotonin; Vasoconstriction; Vasoconstrictor Agents; Vasodilation; Vasodilator Agents; Vitamin E

To investigate the effect of a chronic treatment with melatonin on arterial pressure and a possible improvement of the vascular muscarinic and NO synthase (NOS) pathways in spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats.. Mean arterial pressure (MAP), systolic (SBP), diastolic blood pressure (DBP), and heart rate (HR) were evaluated in conscious rats treated with 30 mg/kg per day of melatonin during 4 weeks. Changes in MAP were evaluated following an intravenous injection of the NOS inhibitor N-omega-nitro-L-arginine methyl ester (L-NAME). Relaxant effects of acetylcholine (Ach), sodium nitroprusside (SNP), and the calcium ionophore A23187 were examined on mesenteric beds and aortic rings with or without treatment with melatonin.. Melatonin produced a significant reduction of MAP, SBP, DBP and HR in SHR (P < 0.05). L-NAME increased the MAP of melatonin-treated SHR by the same magnitude as that of WKY rats which was significantly higher than that of non-treated SHR (P< 0.05). Melatonin treatment improved the maximal relaxation of mesenteric arteries to A23187 in SHR (P < 0.001) to the WKY level and caused a slight increment in Ach- and A23187-induced vasodilations in aorta from SHR and WKY rats (P < 0.05).. The present study showed that melatonin exerted a bradycardic and an antihypertensive action in SHR. The enhancement by melatonin of the endothelium-dependent vasodilation (Ach and/or A23187) in mesenteric artery and aorta from SHR and WKY rats and the higher increase in MAP following L-NAME treatment in melatonin-treated SHR suggest the contribution of an improved vascular NOS pathway activity in the hypotensive effect of melatonin.

Topics: Acetylcholine; Animals; Antihypertensive Agents; Aorta; Blood Pressure; Body Weight; Calcimycin; Enzyme Inhibitors; Hypertension; Ionophores; Male; Melatonin; Mesenteric Arteries; NG-Nitroarginine Methyl Ester; Nitroprusside; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Time Factors; Vasodilation; Vasodilator Agents; Vasomotor System

Sildenafil, a type V phosphodiesterase inhibitor, enhances smooth muscle relaxation in normal human and rabbit corpus cavernosum. We investigated the in vitro effects of sildenafil on non-adrenergic, non-cholinergic and nitric oxide (NO)-mediated cavernosal smooth muscle relaxation in diabetic rabbits, since alterations in this pathway are recognised in diabetic erectile dysfunction. Diabetes mellitus was induced in male New Zealand White rabbits with alloxan. Cavernosal strips from age-matched control, 3- and 6-month diabetic animals were mounted in organ baths. Relaxation responses to electrical field stimulation (1-20 Hz) or sodium nitroprusside (10(-8)-10(-4) M) were assessed in the absence and presence of sildenafil (10(-8) and 10(-7) M). The effect of sildenafil on cGMP formation by the corpus cavernosum was also assessed following stimulation with sodium nitroprusside, A23187 and acetylcholine. Sodium nitroprusside-stimulated relaxations were significantly (P<0.03) impaired in the corpus cavernosum from both diabetic groups, (IC(50)=4.6 x 10(-6) M following 3 months of diabetes mellitus and 4.0 x 10(-6) M following 6 months of diabetes mellitus; compared to 7.5 x 10(-7) M for pooled age-matched controls). Sildenafil (10(-7) M) significantly enhanced sodium nitroprusside-stimulated relaxation in control (P<0.05) and diabetic groups (P<0.03). Electrical field stimulation-mediated relaxations of the corpus cavernosum were significantly impaired after 6-month diabetes mellitus and enhanced by sildenafil (10(-8) M). cGMP formation by the diabetic corpus cavernosum was impaired significantly, but restored towards normal by sildenafil. We suggest that the impairment of NO-mediated relaxation of the corpus cavernosum reflect, at least in part, a defect in guanylyl cyclase activity. These findings support the use of sildenafil as an effective, orally administered, treatment for diabetic erectile dysfunction.

Topics: Acetylcholine; Animals; Biomarkers; Body Weight; Calcimycin; Cyclic GMP; Diabetes Mellitus, Experimental; Dose-Response Relationship, Drug; Electric Stimulation; In Vitro Techniques; Male; Muscle Relaxation; Muscle, Smooth; Nitric Oxide Donors; Nitroprusside; Penis; Phosphodiesterase Inhibitors; Piperazines; Purines; Rabbits; Sildenafil Citrate; Sulfones; Time Factors; Vasodilator Agents

Exposure to CS2, an organic solvent, is associated with an increased rate of abnormal labor or dysmenorrhea. Contraction of quiescent uteri during pregnancy can cause preterm labor. We wish to know the effects of in vivo and in vitro exposures to CS2 on uterine contractions of mid-gestation rats. After 10-d exposure to 300 or 600 mg/kg CS2, uteri of pregnant rats were measured for contractile responses to various stimuli, such as KCl, oxytocin, carbachol or A23187, a calcium ionophore, using standard muscle bath apparatus. CS2 treatment significantly increased the contractile response to KCl, carbachol, and A23187. The increase to A23187 was the greatest. In contrast, in vitro exposure to CS2 immediately suppressed carbachol-induced contraction but did not affect spontaneous and KCl-induced contractions. Results showed the pregnant uterus of the rat is susceptible to CS2. The influence of in vivo exposure to CS2 on uterine contraction was opposite to that in vitro. The increased response of CS2-treated uteri to A23187 suggests that in vivo exposure to CS2 may sensitize contraction machinery to calcium through indirect pathways.

Topics: Animals; Body Weight; Calcimycin; Carbachol; Carbon Disulfide; Female; Gap Junctions; Potassium Chloride; Pregnancy; Pregnancy, Animal; Rats; Rats, Sprague-Dawley; Uterine Contraction

We evaluated the role of SH-groups in improvement of endothelial dysfunction with ACE-inhibitors in experimental heart failure. To this end, we compared the vasoprotective effect of chronic treatment with zofenopril (plus SH-group) versus lisinopril (no SH-group), or N-acetylcysteine (only SH-group) in myocardial infarcted (MI) heart failure rats. After 11 weeks of treatment, aortas were obtained and studied as ring preparations for endothelium-dependent and -independent dilatation in continuous presence of indomethacin to avoid interference of vasoactive prostanoids, and the selective presence of the NOS-inhibitor L-NMMA to determine NO-contribution. Total dilatation after receptor-dependent stimulation with acetylcholine (ACh) was attenuated (-49%, P<0.05) in untreated MI (n=11), compared to control rats with no-MI (n=8). This was in part due to impaired NO-contribution in MI (-50%, P<0.05 versus no-MI). At the same time the capacity for generation of biologically active NO after receptor-independent stimulation with A23187 remained intact. Chronic treatment with n-acetylcysteine (n=8) selectively restored NO-contribution in total dilatation to ACh. In contrast, both ACE-inhibitors fully normalized total dilatation to ACh, including the part mediated by NO (no significant differences between zofenopril (n=10) and lisinopril (n=8)). Zofenopril, but not lisinopril, additionally potentiated the effect of endogenous NO after A23187-induced release from the endothelium (+100%) as well as that of exogenous NO provided by nitroglycerin (+22%) and sodium nitrite (+36%) (for all P<0.05 versus no-MI). We conclude that ACE-inhibition with a SH-group has a potential advantage in improvement of endothelial dysfunction through increased activity of NO after release from the endothelium into the vessel wall. Furthermore, this is the first study demonstrating the selective normalizing effect of N-actylcysteine on NO-contribution to ACh-induced dilatation in experimental heart failure.

Topics: Acetylcholine; Acetylcysteine; Angiotensin-Converting Enzyme Inhibitors; Animals; Aorta, Thoracic; Blood Pressure; Body Weight; Calcimycin; Captopril; Endothelium, Vascular; Heart Diseases; In Vitro Techniques; Lisinopril; Male; Myocardial Infarction; Nitrates; Nitrites; Nitroglycerin; omega-N-Methylarginine; Rats; Rats, Wistar; Sodium Nitrite; Sulfhydryl Compounds; Vasodilation; Vasodilator Agents

Vascular aging is mainly characterized by endothelial dysfunction. We found decreased free nitric oxide (NO) levels in aged rat aortas, in conjunction with a sevenfold higher expression and activity of endothelial NO synthase (eNOS). This is shown to be a consequence of age-associated enhanced superoxide (.O(2)(-)) production with concomitant quenching of NO by the formation of peroxynitrite leading to nitrotyrosilation of mitochondrial manganese superoxide dismutase (MnSOD), a molecular footprint of increased peroxynitrite levels, which also increased with age. Thus, vascular aging appears to be initiated by augmented.O(2)(-) release, trapping of vasorelaxant NO, and subsequent peroxynitrite formation, followed by the nitration and inhibition of MnSOD. Increased eNOS expression and activity is a compensatory, but eventually futile, mechanism to counter regulate the loss of NO. The ultrastructural distribution of 3-nitrotyrosyl suggests that mitochondrial dysfunction plays a major role in the vascular aging process.

Topics: Acetylcholine; Aging; Animals; Aorta; Body Weight; Calcimycin; Cellular Senescence; Endothelium, Vascular; Enzyme Induction; Hemodynamics; Male; Microscopy, Immunoelectron; Mitochondria; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitroprusside; Oxidative Stress; Rats; Rats, Inbred Strains; Superoxide Dismutase; Superoxides; Tyrosine; Vasodilation

The aim of this study was to measure the changes in lipid metabolism which occur during smoltification and seawater transfer in Atlantic salmon (Salmo salar). Duplicate groups of Atlantic salmon parr were fed diets containing either fish oil (FO) or a blend of linseed and rapeseed oils, vegetable oil (VO), from October (week 0) to seawater transfer in May (week 26). From May to August (weeks 26-43), all fish were fed a fish oil-containing diet. Fatty acyl desaturation and elongation activity were followed in isolated hepatocytes incubated with radioactive 18:3n-3 and 18:2n-6. Metabolism of 18:3n-3 was consistently around 5-fold greater than metabolism of 18:2n-6, and total metabolism of both substrate polyunsaturated fatty acids (PUFA) was increased in fish fed both VO and FO up to seawater transfer after which desaturation activities were reduced. Desaturation activities with both 18:3n-3 and 18:2n-6 were significantly greater in fish fed VO, compared to fish fed FO, at 22 and 26 wk. Arachidonic acid (20:4n-6; AA) in liver polar lipids (PL) of fish fed VO increased consistently from weeks 0-22 but varied after seawater transfer. In fish fed FO, AA in liver PL remained constant up to week 17 before increasing at seawater transfer and leveling off thereafter. Eicosapentaenoic acid (20:5n-3; EPA) in liver PL of fish fed VO decreased significantly from week 0-22 before rising at seawater transfer and increasing rapidly posttransfer. EPA in liver PL of fish fed FO showed a similar trend except EPA was always greater in the freshwater phase compared to fish fed VO. Docosahexaenoic acid (DHA) levels in liver PL of fish fed VO remained constant in the seawater phase before increasing following seawater transfer. In fish fed FO, DHA in liver PL increased from weeks 0-17 reducing and leveling off postseawater transfer. The levels of PGF(2 alpha) and PGF(3 alpha) were measured in isolated gill cells stimulated with calcium ionophore A23187. PGF(2 alpha) production in fish fed VO increased significantly between 0-7 wk before decreasing toward seawater transfer. After transfer, PGF(2 alpha), production increased to a peak at 35 wk. PGF(2 alpha) production in fish fed FO was not significantly altered during the trial period. The changes in PGF(3 alpha) production were broadly similar to those occurring with PGF(2 alpha), but the latter was always in excess of the former (2- to 4-fold). Plasma chloride concentrations in fish subjected to seawater challenge at 20 wk wer

Topics: Animals; Arachidonic Acid; Atlantic Ocean; Body Weight; Calcimycin; Cells, Cultured; Chlorides; Dietary Fats; Docosahexaenoic Acids; Eicosapentaenoic Acid; Fatty Acids, Unsaturated; Fish Oils; Fresh Water; Gills; Liver; Plant Oils; Prostaglandins F; Salmon; Seawater

Oxygen-derived free radicals are believed to be involved in diabetes-induced vascular complications. The role of oxygen radicals in endothelial dysfunction in diabetes is not known with certainty. In this study we tested whether inhibition of lipid peroxidation using the potent inhibitor U74389F, a 21-aminosteroid also known as lazaroid, could prevent endothelial dysfunction in diabetes. Lewis strain rats were made diabetic by intravenous injection of streptozotocin. A subgroup of diabetic animals received daily oral doses of 10 mg/kg U74389F at 72 hours post streptozotocin and throughout the 8-week duration of diabetes. Thoracic aortas were isolated and suspended in isolated tissue baths and contracted with norepinephrine. Relaxation due to the endothelium-dependent vasodilator, acetylcholine, was impaired in diabetic aorta while relaxation due to A23187 and nitroglycerin was unaltered. Chronic treatment of diabetic animals with U74389F normalized the increase in plasma lipid peroxides as assessed by thiobarbituric acid-reactive substances but did not alter serum insulin levels, blood glucose concentration, nor total glycosylated hemoglobin. Increases in aortic catalase activity resulting from diabetes was not altered by U74389F. Despite reductions in lipid peroxides, U74389F did not prevent the diabetes-induced impairment in endothelium-dependent relaxation caused by acetylcholine. These data suggest that other pathways that are antecedent to lipid peroxidation may be responsible for endothelial dysfunction in diabetes.

Topics: Acetylcholine; Animals; Antioxidants; Aorta, Thoracic; Blood Glucose; Body Weight; Calcimycin; Catalase; Diabetes Mellitus, Experimental; Endothelium, Vascular; Glycated Hemoglobin; Insulin; Lipid Peroxides; Male; Muscle Relaxation; Muscle, Smooth, Vascular; Nitroglycerin; Pregnatrienes; Rats; Rats, Inbred Lew; Streptozocin

The endothelium plays a critical role in maintaining vascular tone by releasing vasoconstrictor and vasodilator substances. Endothelium-derived nitric oxide is a vasodilator that can be rapidly inactivated by superoxide (reaction rate constant, K = 3.6 x 10(9) L/mol per second). The measurement of nitric oxide concentration in biological systems is a challenging analytic problem because nitric oxide is also rapidly inactivated by Fe(II), Fe(III), and O2, all of which are found in great abundance in biological systems. To date, no currently used instrumental technique has been suitable for direct in situ measurement of NO in isolated resistance arteries. We designed the present study to perform for the first time direct in situ measurements of NO in rat mesenteric resistance arteries and to delineate the effects of hypertension on the release of NO and/or its interaction with superoxide. We describe here an adaptation of the recently published design of a porphyrinic sensor for direct in vitro measurement of NO in a single cell. The most significant advantage of this modified porphyrinic microsensor is that its small size makes it ideal for NO measurement in resistance arteries with an internal diameter of 200 microns or less. Small segments of the third-order branch of the mesenteric artery were isolated from normotensive Wistar-Kyoto rats and stroke-prone spontaneously hypertensive rats and placed in an organ chamber filled with Hanks' balanced salt solution buffer (2 mL, 37 degrees C). The tip of the porphyrinic microsensor was inserted into the lumen of an isolated vascular ring, and NO release was monitored in situ after maximal stimulation of NO synthase with the receptor-independent agonist calcium ionophore A23187 (10 mumol/L). Maximal surface concentration of NO measured after A23187 administration was significantly smaller in 15-week-old hypertensive rats (0.28 +/- 0.03 mumol/L, n = 10) than in age-matched normotensive rats (0.38 +/- 0.03 mumol/L, n = 10, P < .03). However, in the presence of the superoxide scavenger superoxide dismutase (100 U/mL), the peak NO level from the hypertensive rats was 0.37 +/- 0.04 mumol/L (n = 10), which was comparable to that observed for the normotensive rats in the absence and presence of superoxide dismutase. In summary, our results demonstrate that in rat mesenteric resistance arteries hypertension is associated with increased NO decomposition by superoxide, whereas NO release remains unaffected. This may be im

Topics: Animals; Body Weight; Calcimycin; Cerebrovascular Disorders; Genetic Predisposition to Disease; Hypertension; Male; Mesenteric Arteries; Nitric Oxide; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Superoxide Dismutase; Superoxides; Vascular Resistance

Defective endothelium-dependent relaxation in diabetic blood vessels may be regulated at the site of synthesis. We tested the hypothesis that acute administration of L-arginine (L-Arg) as substrate for endothelium-derived relaxing factor (EDRF) would normalize defective relaxation to acetylcholine (ACh) in streptozotocin-induced diabetic rat aortic rings. Plasma concentrations of basic amino acids (e.g., arginine, lysine, and histidine) were significantly reduced by diabetes, but variable results (increased, decreased, or no change) were observed in plasma concentrations of neutral amino acids. Endothelium-dependent relaxation to ACh (but not calcium ionophore A23187) was impaired in diabetic rings. Relaxation to nitroglycerin (NTG) was not altered. Pretreatment with L-nitroarginine methyl ester (L-NAME), a nitric oxide (NO) synthase inhibitor, blocked the relaxation to ACh and A23187 but not relaxation to NTG in both control and diabetic rings. Pretreatment with 3 mM L-Arg (but not D-Arg) potentiated the relaxation to ACh in diabetic rings. L-Arg had no effect on ACh-induced relaxation in control rings or on relaxation to NTG in control or diabetic rings. A mechanism for impaired endothelium-dependent relaxation to ACh in diabetic aorta may arise from a defect in utilization of L-Arg by NO synthase for production of EDRF/NO.

Topics: Acetylcholine; Amino Acids; Animals; Arginine; Body Weight; Calcimycin; Diabetes Mellitus, Experimental; Endothelium, Vascular; Male; Muscle Relaxation; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitroglycerin; Rats; Rats, Sprague-Dawley

The pancreas has been reported as a possible target for FK506 toxicity. This study was conducted to examine the effects of FK506 on the structure and function of pancreatic acinar cells.. Male Sprague-Dawley rats received an intramuscular injection of saline or FK506, and pancreatic acini were isolated on the day of sacrifice.. FK506 caused a time-dependent suppression in amylase secretory response to cholecystokinin or carbachol at days 3-14, and increases in amylase and trypsinogen content at days 7-14. The properties of cholecystokinin and scopolamine binding sites in acini were not altered by FK506. Amylase release by A23187 and secretin were decreased by FK506, but those by phorbol ester 12-O-tetradecanoylphorbol-13-acetate, forskolin, 5'-cyclic adenosine monophosphate and vasoactive intestinal peptide were not changed. Increases in cytosolic free calcium concentration induced by cholecystokinin were not changed by FK506. Histologically, a significant increase in cytoplasmic zymogen granules was observed in pancreas from FK506-treated rats.. These data suggest that FK506 induced changes in function and metabolism in pancreatic acinar cells, and these changes might be caused by altering postreceptor loci in stimulus-secretion coupling.

Topics: Amylases; Analysis of Variance; Animals; Atropine; Body Weight; Calcimycin; Calcium; Carbachol; Cholecystokinin; Colforsin; Cytosol; Dose-Response Relationship, Drug; Feeding Behavior; Kinetics; Male; N-Methylscopolamine; Pancreas; Rats; Rats, Sprague-Dawley; Receptors, Cholecystokinin; Reference Values; Scopolamine; Scopolamine Derivatives; Secretin; Sincalide; Tacrolimus; Tetradecanoylphorbol Acetate; Time Factors; Trypsinogen; Vasoactive Intestinal Peptide

Oxidized low-density lipoproteins (LDL), which may play an important role in atherogenesis, inhibit endothelium-dependent relaxations of normal arteries in vitro. The effects of probucol, an inhibitor of LDL oxidation, on endothelium-dependent relaxations in thoracic aorta of rabbits that received a cholesterol-rich (0.5%) or standard diet for 10 weeks were determined. In some rabbits in each group, the diet was supplemented with probucol (1%) for the last 6 weeks. The cholesterol-rich diet markedly increased plasma cholesterol and resulted in increased plasma lipid peroxides. Probucol prevented the increase in lipid peroxides, but had no effect on plasma cholesterol. Rings of aorta were mounted in organ chambers for measurement of isometric tension and contracted with phenylephrine. Endothelium-dependent relaxations to acetylcholine and A23187 were significantly impaired in aortic rings from cholesterol-fed rabbits. Aortic rings from rabbits fed cholesterol and treated with probucol relaxed normally to both vasodilators. Relaxations to acetylcholine and A23187 were not significantly changed in rings from rabbits that received probucol-supplemented standard diet. Endothelium-independent relaxations to sodium nitroprusside (SNP) were not influenced by the cholesterol diet or probucol. Thus, probucol preserves endothelium-dependent relaxations of hypercholesterolemic rabbit aorta without reducing plasma cholesterol. As demonstrated by the reduction in plasma lipid peroxides, the effect of probucol may be related to its antioxidant properties and may imply that oxidized lipids have a role in endothelial cell dysfunction of atherosclerotic arteries in vivo.

Topics: Acetylcholine; Animals; Aorta, Thoracic; Body Weight; Calcimycin; Cholesterol; Coronary Artery Disease; Diet, Atherogenic; Endothelium, Vascular; In Vitro Techniques; Lipid Peroxidation; Male; Microscopy, Electron, Scanning; Muscle Relaxation; Muscle, Smooth, Vascular; Nitroprusside; Phenylephrine; Probucol; Rabbits; Thiobarbituric Acid Reactive Substances

To study the influence of membrane fatty acid composition on the formation of prostanoids and hydroxy fatty acids by rat peritoneal mast cells (MC), animals were fed three different types of fatty acids: mackerel oil (MO), abundant in n-3 fatty acids; sunflower seed oil (SO), rich in linoleic acid; and hydrogenated coconut oil (HCO), mainly containing saturated fatty acids. The presence of n-3 fatty acids in the diet resulted in the incorporation of 20:5(n-3), 22:5(n-3) and 22:6(n-3) in MC phospholipids. A decrease of arachidonic acid, 20:4(n-6), was observed in MC-phospholipids of the MO-fed animals. Furthermore, increasing the relative amounts of 18:2(n-6) in the diet (SO group) led to an increased incorporation of linoleic acid, 18:2(n-6) in MC phospholipids when compared to both other dietary groups. The changes in MC phospholipid fatty acid composition were (partly) reflected in the formation of prostanoids and hydroxy fatty acids upon stimulation with the calcium ionophore A23187. The decrease in arachidonic acid content in MC phospholipids of MO-fed rats resulted in a decreased formation of PGD2 when compared to both other groups. Also, the increased amounts of 18:2(n-6) in MC phospholipids of SO-fed rats resulted in an increased formation of 9- and 13-HODE upon stimulation. The results show that modifications in the fatty acid composition of the diet influences MC membrane fatty acid composition which ultimately results in changes in prostanoid and hydroxy fatty acid synthesis by MC upon stimulation with the calcium ionophore A23187.

Topics: Animals; Arachidonic Acid; Body Weight; Calcimycin; Cell Membrane; Coconut Oil; Dietary Fats; Fatty Acids; Fish Oils; Linoleic Acid; Linoleic Acids; Male; Mast Cells; Peritoneal Cavity; Phospholipids; Plant Oils; Prostaglandins; Rats; Rats, Wistar; Sunflower Oil

1. The effect of experimental diabetes mellitus (DM; hyperglycaemic, non-ketototic; 2 months duration) in the rat on receptor-linked prostacyclin (PGI2) synthesis (measured as 6-oxo-PGF1 alpha by radioimmunoassay) was studied in the aorta and urinary bladder using adrenaline, angiotensin II (AII) and acetylcholine (ACh). Signal transduction systems were studied via stimulation of PGI2 synthesis with phorbol ester dibutyrate (PDBU; a protein kinase C activator [PKC]), Ca2+ ionophore A23187 (A23187) and thapsigargin (both elevate intracellular Ca2+, activating phospholipase A2 [PLA2]) and arachidonate (AA; substrate for PGI2 synthesis). 2. In response to adrenaline, AII and phorbol ester, aortic PGI2 release was markedly reduced (all > 75%) in diabetic rats compared to controls. EC50s of the dose-response curves for adrenaline, AII and PDBU were also markedly increased in aortae from DM rats compared to controls. Although there was decreased output of PGI2 in response to A23187 by aortae from diabetic rats compared to controls, there was no difference in the EC50s (mean +/- s.e. mean: diabetic, 2.7 +/- 0.2 x 10(-6) M; controls 2 +/- 0.18 x 10(-6) M). There were no differences in PGI2 release (or in the EC50s) in response to thapsigargin or AA between aortae from diabetic and control rats. 3. In the urinary bladder, there was a marked increase in PGI2 output in response to ACh and a marked decrease in EC50s for the ACh-PGI2 dose-response curves in diabetic rats (EC50 = 5.8 +/- 0.32 x 10(-7) M) compared to controls (EC50 = 2.2 +/- 0.15 x 10(-6) M). Although there was an increase in PGI2 output in the urinary bladders from diabetic rats in response to A23187, there were no differences in the EC50s (control, 1.8 +/- 0.2 x 10-6 M; diabetic, 1.1 +/- 0.15 X 10-6 M). In the urinary bladders, there were no differences in PGI2 output (or the EC50s) in response to PDBU, thapsigargin or AA between diabetic or control rats.4. These data indicate that: (i) reduced PGI2 synthesis coupled to adrenoceptors and AII receptors in the aortae of diabetic rats may be due to diminished PKC activity and not to changes in receptor density and/or affinity, Ca2+ stores, PLA2, cyclo-oxygenase or PGI2 synthase; (ii) the diametrically opposite effect of DM on ACh-stimulated PGI2 synthesis is not due to an increase in PKC activity, but possibly to an increase in muscarine receptor number and/or affinity; (iii) changes in receptor-linked PGI2 synthesis are not ubiquitous in experimental DM

Topics: Angiotensin II; Animals; Aorta, Thoracic; Arachidonic Acid; Body Weight; Calcimycin; Calcium-Transporting ATPases; Diabetes Mellitus, Experimental; Epoprostenol; In Vitro Techniques; Male; Muscle, Smooth; Muscle, Smooth, Vascular; Phorbol 12,13-Dibutyrate; Rats; Rats, Sprague-Dawley; Receptors, Adrenergic; Receptors, Muscarinic; Terpenes; Thapsigargin; Urinary Bladder

The pathophysiological role of platelet activating factor (PAF) in smoking-induced disorders was examined in rats exposed daily to smoke for 10, 18 and 26 weeks. The concentration of PAF in bronchoalveolar lavage fluid and the activities of PAF biosynthetic and catabolic enzymes in alveolar macrophages and in plasma were determined. The concentration of PAF in lavage fluid of the smoke-exposed group was significantly lower than that in the sham group for each duration of smoke exposure. The PAF biosynthetic enzyme, acetyl transferase, activity in alveolar macrophages of smoked group was less than that in the sham group although the difference was not statistically significant. PAF catabolic enzyme, acetyl hydrolase, activities in alveolar macrophages and in plasma were all significantly higher in every smoked group than in the sham group. These data indicate that cigarette smoking alters PAF metabolism in the respiratory tract and in plasma and such an alteration may contribute, at least in part, to smoking induced cardiopulmonary disorders.

Topics: 1-Alkyl-2-acetylglycerophosphocholine Esterase; Acetyltransferases; Animals; Blood Platelets; Body Weight; Bronchoalveolar Lavage Fluid; Calcimycin; Macrophages, Alveolar; Male; Phospholipases A; Platelet Activating Factor; Rats; Rats, Sprague-Dawley; Tobacco Smoke Pollution

The influence of 12-O-Tetradecanoylphorbol-13-acetate, an activator of proteinkinase C and A 23187, a calcium ionophore increasing cytosolic free calcium concentration on zinc metabolism was investigated in a study with 24 eight-week old rats. Twenty-four hours before killing, the rats (235 g body weight, 8 per group) were either injected intraperitoneally with TPA (1.6 x 10(-7) mol/kg body weight) or A 23187 (1.6 x 10(-6) mol/kg body weight). Control rats received the solvent dimethylsulfoxide. The application of TPA and A 23187 provoked a marked decline in feed intake accompanied by a reduction in body weight and liver mass. Serum concentrations of zinc were reduced significantly after A 23187 injections. TPA and A 23187 increased liver zinc levels by 20 and 30% respectively, if based on fresh and dry weight. The injections, however, did not alter total liver zinc. Liver metallothionein (MT) concentration was elevated 2.4-fold after TPA administration. The increase in response to A 23187 was only 1.5-fold and not significant. Mucosa MT levels were not altered. Serum activity of alkaline phosphatase was significantly reduced (TPA: -23%, A 23187: -31%). There was no change in serum glucose after injections. However, serum creatinine and urea were increased in response to A 23187. In conclusion, TPA and A 23187 had an effect on zinc metabolism of the rat, most marked in the case of MT induction in the liver. There is evidence that the reduced feed intake caused by TPA and A 23187 resulted in effects indistinguishable from those caused by fasting. Further experiments are needed to clarify whether proteinkinase C and cytosolic free calcium are directly involved in the regulation of zinc metabolism.

Topics: Alkaline Phosphatase; Animals; Blood Glucose; Body Weight; Calcimycin; Creatinine; Eating; Liver; Male; Metallothionein; Organ Size; Rats; Rats, Inbred Strains; Tetradecanoylphorbol Acetate; Urea; Zinc

A single i.v. injection of daunomycin (10 mg/kg) into rats produced severe proteinuria and hypercholesterolemia without atherosclerosis on the 20th and 40th days after the treatment. However, these changes were not observed on the 5th day. No change in systolic blood pressure was seen through the 40-day experimental period. Relaxation to acetylcholine, A23187 and nitroprusside was examined in aortic rings precontracted with phenylephrine (3 x 10(-6) M). Acetylcholine-induced relaxation was significantly attenuated in the nephrotic rats on the 20th and 40th days, in comparison to the control animals. In aortic rings taken from control and nephrotic rats on the 40th day, removal of the endothelium or treatment with methylene blue (10(-5) M) completely abolished the relaxation induced by acetylcholine (10(-5) M). In addition, acetylcholine (10(-5) M) induced a transient increase in the aortic cyclic GMP and this increase was completely abolished by removal of the endothelium. In the preparations of nephrotic rats on the 20th and 40th days, the cyclic GMP levels stimulated by acetylcholine (10(-5) M) were decreased to about 50% in comparison to their respective control. A23187 also evoked diminished relaxation in nephrotic rats on the 20th and 40th days. However, on the 40th day after the treatment, the effects of nitroprusside in relaxing the aorta and in elevating the cyclic GMP level in the aorta were not altered by nephrosis. In addition, the nitroprusside-induced relaxation and cyclic GMP accumulation were not affected by removal of the endothelium. These results indicate that endothelium-dependent relaxation is attenuated with the development of nephrosis.

Topics: Acetylcholine; Animals; Aorta, Thoracic; Blood Pressure; Body Weight; Calcimycin; Daunorubicin; Endothelium; Injections, Intravenous; Male; Microscopy, Electron, Scanning; Muscle Relaxation; Muscle, Smooth, Vascular; Nephrosis; Nitric Oxide; Nitroprusside; Rats; Rats, Inbred Strains

Mechanisms of accelerated proteolysis were compared in denervated and unweighted (by tail-cast suspension) soleus muscles. In vitro and in vivo proteolysis were more rapid and lysosomal latency was lower in denervated than in unweighted muscle. In vitro, lysosomotropic agents (eg, chloroquine, methylamine) did not lessen the increase in proteolysis caused by unweighting, but abolished the difference in proteolysis between denervated and unweighted muscle. Leucine methylester, an indicator of lysosome fragility, lowered latency more in denervated than in unweighted muscle. 3-Methyladenine, which inhibits phagosome formation, increased latency similarly in all muscles tested. Mersalyl, a thiol protease inhibitor, and 8-(diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8), which antagonizes sarcoplasmic reticulum release of Ca2+, reduced accelerated proteolysis caused by unweighting without diminishing the faster proteolysis due to denervation. Calcium ionophore (A23187) increased proteolysis more so in unweighted than control muscles whether or not Ca2+ was present. Different mechanisms of accelerated proteolysis were studied further by treating muscles in vivo for 24 hours with chloroquine or mersalyl. Chloroquine diminished atrophy of the denervated but not the unweighted muscle, whereas mersalyl prevented atrophy of the unweighted but not of the denervated muscle, both by inhibiting in vivo proteolysis. These results suggest that (1) atrophy of denervated, but not of unweighted, soleus muscle involves increased lysosomal proteolysis, possibly caused by greater permeability of the lysosome, and (2) cytosolic proteolysis is important in unweighting atrophy, involving some role of Ca2(+)-dependent proteolysis and/or thiol proteases.

Topics: Animals; Body Weight; Calcimycin; Calcium; Chloroquine; Female; Gallic Acid; Lysosomes; Mersalyl; Methylamines; Muscle Denervation; Muscle Proteins; Muscles; Muscular Atrophy; Peptide Hydrolases; Protease Inhibitors; Rats; Rats, Inbred Strains; Sarcoplasmic Reticulum

Calimycin (A23187), a divalent cationic ionophore which increases intraneuronal concentrations of calcium, has been shown to produce neurofunctional changes associated with depression of brain excitability. The present report describes further characterization of the neurofunctional effects of acute parenteral exposure to A23187. Adult male rats were tested for the effects of A23187 on operant performance and general motor activity. In order to gain some insight into the neuronal systems which may be affected by A23187, brain levels of norepinephrine (NE), dopamine, serotonin, and their metabolites were determined. In addition, provocative pharmacologic challenges were employed to assess the interaction between A23187 and the centrally active drugs amphetamine (AMPH), scopolamine (SCOP), or chlorpromazine (CPZ). A23187 was found to effectively suppress fixed ratio-15 operant performance (p less than 0.001) at a dose of 0.5 mg/kg. This effect was temporary in nature inasmuch as performance was normalized by the day after dosing. This same dose of A23187, which produces a selective hypomotility, was found to have no apparent latent effects on the processes controlling circadian motor activity. Neurochemically, A23187 at 1 mg/kg induced a 64% increase (p less than 0.01) in the NE metabolite 3-methoxy-4-hydroxyphenylglycol, although levels of NE were unaffected. In the pharmacological challenge studies, AMPH and SCOP both attenuated and CPZ enhanced the acute hypomotility induced by 0.5 mg A23187/kg. The data show that low doses of A23187 induce a specific pattern of neurobehavioral dysfunction which may involve some interaction between the central catecholaminergic and cholinergic systems.

Topics: Animals; Body Weight; Brain Chemistry; Calcimycin; Catecholamines; Conditioning, Operant; Male; Motor Activity; Neurons; Rats; Rats, Inbred Strains; Reinforcement Schedule

The effect of in vivo exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on glucocorticoid- and calcium ionophore-induced DNA fragmentation in rat thymocytes was investigated. TCDD dose-dependently abolished DNA fragmentation in response to both agents after 7 days of exposure. Analysis of the time dependence of the effect revealed that after 1 or 2 days TCDD potentiated DNA fragmentation in untreated and glucocorticoid-treated thymocyte suspensions relative to controls. The DNA fragmentation in untreated thymocyte suspensions from TCDD-treated rats was completely prevented by inhibitors that block glucocorticoid-induced thymocyte suicide. Our results suggest that TCDD-induced thymic atrophy is due to Ca2+-dependent endonuclease activation.

Topics: Animals; Atrophy; Body Weight; Calcimycin; Calcium; Cell Survival; Dioxins; DNA; Dose-Response Relationship, Drug; Drug Synergism; Endonucleases; Glucocorticoids; Kinetics; Male; Methylprednisolone; Organ Size; Polychlorinated Dibenzodioxins; Rats; Rats, Inbred Strains; Thymus Gland

The effect of obstructive jaundice on pancreatic amylase secretion was studied in isolated pancreatic acini prepared from bile duct ligated rats (7 days postoperatively), sham operated rats being used as control. Obstructive jaundice caused increase in pancreatic wet weight, pancreatic protein content and pancreatic amylase content by 27.9%, 40.1% and 33.2%, respectively. In acini prepared from obstructive jaundice group, compared with acini from sham operation group, responsiveness to cholecystokinin (CCK) and carbachol was decreased when amylase release was expressed as the percentage of total amylase activity initially present in acini. However, sensitivity to both secretagogues was unchanged when expressed as the percentage of maximally stimulated amylase release. The dose-response curves to Ca2+ ionophore for amylase release were similarly shaped in both groups. These results suggested that a pancreatico-trophic effect, compared with altered responsiveness of pancreatic acini, should play a major role in hypersecretion in obstructive jaundice.

Topics: Amylases; Animals; Bilirubin; Body Weight; Calcimycin; Carbachol; Cholestasis; Male; Organ Size; Pancreas; Pancreatic Juice; Rats; Secretory Rate; Sincalide

Rats were given subcutaneous injections of synthetic cholecystokinin octapeptide (CCK8, 5 micrograms/kg) in a depot carrier twice daily for 7-14 days. The pancreatic wet weight increased by 20.6 and 30.9% in the rats treated with CCK8 for 7 and 14 days, respectively. The increase in pancreatic weight was associated with an increase in the amount of protein per DNA, indicating hypertrophy of the acinar cells, and with an increase in the total amount of pancreatic DNA. Moreover, CCK administration also increased the amylase content per DNA. In acini prepared from CCK8-treated rats, responsiveness to CCK8 was increased when amylase release was expressed relative to DNA but was decreased when calculated as the percentage of the initial content in the acini. The dose-response curves for CCK8 were similarly shaped in both CCK8-treated and control rats, but they were shifted 3- to 10-fold toward higher concentrations of CCK8 after 7 and 14 days of CCK8 treatment. There were no major changes in the affinity and capacity of CCK receptors determined by studying the binding of radioiodinated CCK, suggesting that alterations in pancreatic amylase release were due to changes at a postreceptor loci. In support of this hypothesis, the secretory response to carbachol, known to act on a different receptor but by a common intracellular mechanism, was altered in a manner identical to the response to CCK8. Thus chronic stimulation with CCK sufficient to induce pancreatic hypertrophy does not greatly alter CCK receptors and induces only moderate postreceptor desensitization.

Topics: Amylases; Animals; Appetite Depressants; Body Weight; Calcimycin; Carbachol; Cholecystokinin; Kinetics; Male; Organ Size; Pancreas; Peptide Fragments; Rats; Rats, Inbred Strains; Sincalide

Protein content, enzymatic activites and Ca2+ accumulation capacities were studied in plasma membrane fractions isolated from mesenteric arteries of rats made hypertensive by renal artery stenosis with and without contralateral nephrectomy, i.e., one-kidney, one clip (1-KHR) and two-kidney, one clip (2-KHR) hypertension, respectively. Both types of renovascular hypertension showed similar vascular plasma membrane abnormalities which included increased total protein contents, enhanced alkaline phosphatase activities and reduced ATP-dependent Ca2+ accumulation compared to control values. The altered alkaline phosphatase activity and ATP-dependent Ca2+ accumulation appeared to be associated with blood pressure elevation in both types of hypertension and may be related to the elevation of blood pressure insensitive to captopril (SQ 14,225) in 1-KHR and 2-KHR. These results are consistent with the current concept of biochemical abnormalities of arterial smooth muscle in the development ostem.

Topics: Alkaline Phosphatase; Animals; Blood Pressure; Body Weight; Calcimycin; Calcium; Captopril; Cell Membrane; Hypertension, Renal; Male; Mesenteric Arteries; Nucleotidases; Rats; Renal Artery Obstruction