Effect of phorbol ester on contractile response of aorta from endotoxic rats

The influence of phorbol ester on the isometric contractile response of aorta from endotoxic rats was examined. In endotoxic rat aorta, the contractile responses to KCl and phorbol 12,13-dibutyrate (PDBu) were both remarkably diminished, compared to those in control rat aorta. Preincubation with PDBu augmented the aortic contractile response to KCl in both control and endotoxic rats. This augmentative effect of PDBu was significantly more pronounced in endotoxic rats than in controls. When the contractile response to 80 mM KCl reached a plateau after PDBu pretreatment, addition of 5 mM CaCl2 (final concentration) to the organ bath completely reversed the diminished contractile response of endotoxic rat aorta to the control level. These results suggest that the hyporesponsiveness of endotoxic rat aorta to KCl may be caused by decreases in both protein kinase C mediated response and calcium sensitivity of vascular smooth muscle cells.     

Effect of melatonin on vascular reactivity in pancreatectomized rats

The present study was undertaken to assess whether the improvement of contractile performance of aortic rings by melatonin described in streptozotocin diabetic rats also occurs in another model of type I diabetes, the pancreatectomized rats. Adult male Wistar rats submitted to a subtotal pancreatectomy and exhibiting altered levels of fasting glucose and an abnormal tolerance glucose test, were used. Sham-operated laparotomized rats were employed as controls. Dose-response curves for acetylcholine-induced, endothelium-related relaxation of aortic rings (after previous exposure to phenylephrine) and for phenylephrine-induced vasoconstriction were conducted. This protocol was repeated with rings pre-incubated in a high glucose solution (44 mmol/l). Pancreatectomy decreased significantly acetylcholine-induced relaxation of aortic rings, but not phenylephrine-induced vasoconstriction, the effect being amplified by preincubation in high glucose solution. The deleterious effect of a high glucose medium was more pronounced in pancreatectomized rats. Melatonin (10(-5) M) did not modify acetylcholine-induced relaxation in normal glucose concentration but was effective to prevent the impairment of relaxation brought about by exposure to high glucose solution. The contractile response to phenylephrine of aortic rings obtained from pancreatectomized rats was not affected by melatonin. The results further support the improvement by melatonin of endothelial-mediated relaxation in blood vessels of diabetic rats.     

Low mercury concentrations cause oxidative stress and endothelial dysfunction in conductance and resistance arteries

Increased cardiovascular risk after mercury exposure has been described, but the underlying mechanisms are not well explored. We analyzed the effects of chronic exposure to low mercury concentrations on endothelium-dependent responses in aorta and mesenteric resistance arteries (MRA). Wistar rats were treated with mercury chloride (1st dose 4.6 microg/kg, subsequent dose 0.07 microg.kg(-1).day(-1) im, 30 days) or vehicle. Blood levels at the end of treatment were 7.97 +/- 0.59 ng/ml. Mercury treatment: 1) did not affect systolic blood pressure; 2) increased phenylephrine-induced vasoconstriction; 3) reduced acetylcholine-induced vasodilatation; and 4) reduced in aorta and abolished in MRA the increased phenylephrine responses induced by either endothelium removal or the nitric oxide synthase (NOS) inhibitor N(G)-nitro-l-arginine methyl ester (l-NAME, 100 microM). Superoxide dismutase (SOD, 150 U/ml) and the NADPH oxidase inhibitor apocynin (0.3 mM) decreased the phenylephrine-induced contraction in aorta more in mercury-treated rats than controls. In MRA, SOD did not affect phenylephrine responses; however, when coincubated with l-NAME, the l-NAME effect on phenylephrine response was restored in mercury-treated rats. Both apocynin and SOD restored the impaired acetylcholine-induced vasodilatation in vessels from treated rats. Endothelial NOS expression did not change in aorta but was increased in MRA from mercury-treated rats. Vascular O2(-) production, plasmatic malondialdehyde levels, and total antioxidant status increased with the mercury treatment. In conclusion, chronic exposure to low concentrations of mercury promotes endothelial dysfunction as a result of the decreased NO bioavailability induced by increases in oxidative stress. These findings offer further evidence that mercury, even at low concentrations, is an environmental risk factor for cardiovascular disease.     

Contractile effects of vanadate on aorta rings from virgin and pregnant rats

The present study was undertaken to characterize the contractile effects of vanadate on thoracic aorta rings from virgin and term-pregnant rats. Vanadate caused concentration-dependent contraction in rat aortic rings with an EC₅₀ (concentration producing 50% maximum response) of 0.10 mM. Contractions in response to vanadate were equivalent to the ones measured with 1 M phenylephrine. The effects of vanadate were not affected by indomethacin (up to 10 M), an inhibitor of prostanoid cyclooxygenase, but were blocked in a concentration-dependent manner by staurosporine (0.1–1.0 M), an inhibitor of protein kinase C. Vanadate exhibited a significant decrease of contractile responses in aorta of pregnant as compared to virgin rats. When aortic rings were bathed in presence of different concentrations of vanadate, the concentration-response curve to phenylephrine was shifted to the left, but maximum response was not affected. The potentiation of the contractions to phenylephrine by vanadate was significantly more prominent in aorta of virgin than of pregnant rats. These results suggest that the contractile effect of vanadate on rat aorta is independent of endogenous prostanoids and may be mediated by protein kinase C-dependent pathway. These results also show that the contractile response to vanadate on the rat aorta is impaired during pregnancy.     

Chronic Lead Exposure Decreases the Vascular Reactivity of Rat Aortas: The Role of Hydrogen Peroxide

We investigated whether exposure to small concentrations of lead alters blood pressure and vascular reactivity. Male Wistar rats were sorted randomly into the following two groups: control (Ct) and treatment with 100 ppm of lead (Pb), which was added to drinking water, for 30 days. Systolic blood pressure (BP) was measured weekly. Following treatment, aortic ring vascular reactivity was assessed. Tissue samples were properly stored for further biochemical investigation. The lead concentration in the blood reached approximately 8 μg/dL. Treatment increased blood pressure and decreased the contractile responses of the aortic rings to phenylephrine (1 nM–100 mM). Following N-nitro-L arginine methyl ester (L-NAME) administration, contractile responses increased in both groups but did not differ significantly between them. Lead effects on R_max were decreased compared to control subjects following superoxide dismutase (SOD) administration. Catalase, diethyldithiocarbamic acid (DETCA), and apocynin increased the vasoconstrictor response induced by phenylephrine in the aortas of lead-treated rats but did not increase the vasoconstrictor response in the aortas of untreated rats. Tetraethylammonium (TEA) potentiated the vasoconstrictor response induced by phenylephrine in aortic segments in both groups, but these effects were greater in lead-treated rats. The co-incubation of TEA and catalase abolished the vasodilatory effect noted in the lead group. The present study is the first to demonstrate that blood lead concentrations well below the values established by international legislation increased blood pressure and decreased phenylephrine-induced vascular reactivity. The latter effect was associated with oxidative stress, specifically oxidative stress induced via increases in hydrogen peroxide levels and the subsequent effects of hydrogen peroxide on potassium channels.     

Superoxide scavengers augment contractile but not energetic responses to hypoxia in rat diaphragm.

Acute exposure to severe hypoxia depresses contractile function and induces adaptations in skeletal muscle that are only partially understood. Previous studies have demonstrated that antioxidants (AOXs) given during hypoxia partially protect contractile function, but this has not been a universal finding. This study confirms that specific AOXs, known to act primarily as superoxide scavengers, protect contractile function in severe hypoxia. Furthermore, the hypothesis is tested that the mechanism of protection involves preservation of high-energy phosphates (ATP, creatine phosphate) and reductions of P(i). Rat diaphragm muscle strips were treated with AOXs and subjected to 30 min of hypoxia. Contractile function was examined by using twitch and tetanic stimulations and the degree of elevation in passive force occurring during hypoxia (contracture). High-energy phosphates were measured at the end of 30-min hypoxia exposure. Treatment with the superoxide scavengers 4,5-dihydroxy-1,3-benzenedisulfonic acid (Tiron, 10 mM) or Mn(III)tetrakis(1-methyl-4-pyridyl) porphyrin pentachloride (50 microM) suppressed contracture during hypoxia and protected maximum tetanic force. N-acetylcysteine (10 or 18 mM) had no influence on tetanic force production. Contracture during hypoxia without AOXs was also shown to be dependent on the extracellular Ca(2+) concentration. Although hypoxia resulted in only small reductions in ATP concentration, creatine phosphate concentration was decreased to approximately 10% of control. There were no consistent influences of the AOX treatments on high-energy phosphates during hypoxia. The results demonstrate that superoxide scavengers can protect contractile function and reduce contracture in hypoxia through a mechanism that does not involve preservation of high-energy phosphates.     

Endotoxin administration alters the force vs. pCa relationship of skeletal muscle fibers

Recent work indicates that endotoxemia elicits severe reductions in skeletal muscle force-generating capacity. The subcellular alterations responsible for these decrements have not, however, been fully characterized. One possibility is that the contractile proteins per se are altered in endotoxemia and another is that the mechanism by which these proteins are activated is affected. The purpose of the present study was to assess the effects of endotoxin administration on the contractile proteins by examining the maximum calcium-activated force (F(max)) and calcium sensitivity of single Triton-skinned fibers of diaphragm, soleus, and extensor digitorum longus (EDL) muscles taken from control and endotoxin-treated (8 mg/kg) rats. Fibers were mounted on a force transducer and sequentially activated by serial immersion in solutions of increasing Ca(2+) concentration (i.e., pCa 6.0 to pCa 5.0); force vs. pCa data were fit to the Hill equation. All fibers were typed at the conclusion of studies using gel electrophoresis. F(max), the calcium concentration required for half-maximal activation (Ca(50)), and the Hill coefficient were compared as a function of muscle and fiber type for the control and endotoxin-treated animals. Control group F(max) was similar for diaphragm, soleus, and EDL fibers, i.e., 112.34 +/- 2.64, 111.55 +/- 3.66, and 104.05 +/- 4.33 kPa, respectively. Endotoxin administration reduced the average F(max) for fibers from all three muscles to 80.25 +/- 2.30, 72.47 +/- 2.97, and 78.32 +/- 2.43 kPa, respectively (P < 0.001 for comparison of each to control). All fiber types in diaphragm, soleus, and EDL muscles manifested similar endotoxin-related reductions in F(max). The Ca(50) and the Hill coefficient for all fiber types and all muscles were unaffected by endotoxin administration. We speculate that these alterations in the intrinsic properties of the contractile proteins represent a major mechanism by which endotoxemia reduces muscle force-generating capacity.     

Despite minimal hemodynamic alterations endotoxemia modulates NOS and p38-MAPK phosphorylation via metalloendopeptidases

In the present study, we hypothesized that endotoxemia produces metalloendopeptidase (MEPD)-dependent generation of endothelin-1 (ET-1) and alters NOS expression correlating with p38-mitogen-activated protein kinase (MAPK) phosphorylation in thoracic aorta. Male Sprague-Dawley rats (350-400 g) were subjected to two groups randomly; sham-treated (N = 10) and lipopolysaccharide (LPS)-treated (N = 10) (E. coli LPS 2 mg/kg bolus + 2 mg/kg infusion for 30 min). The animals in each group were further subdivided into vehicle and MEPD inhibitor phosphoramidon (1 mg/kg bolus, PHOS)-treated groups. LPS produces a significant decrease in mean arterial pressure (MAP) at 2 h post endotoxemia that was blocked by PHOS. PHOS attenuated LPS-induced increase in tumor necrosis factor-alpha (TNF-alpha) concentration at 2- and 24 h post-LPS administration. LPS significantly elevated plasma concentrations of ET-1 at 2- and 24 h post endotoxemia. An upregulated preproET-1 expression following both LPS and MEPD inhibition was observed in thoracic aorta at 2 h post treatment. PHOS effectively blocked conversion of preproET-1 to ET-1 in thoracic aorta locally at 24 h post treatment in endotoxic rats. PHOS inhibited LPS-induced upregulation of inducible NOS (iNOS), downregulation of endothelial NOS (eNOS) and elevation of NO byproducts (NOx) in thoracic aorta. PHOS also blocked LPS-induced upregulated p38-MAPK phosphorylation in thoracic aorta at 24 h post endotoxemia. The data revealed that LPS induces MEPD-sensitive inflammatory response syndrome (SIRS) at 2- and 24 h post endotoxemia. We concluded that inhibition of MEPD not only decreases the levels of ET-1 but also simultaneously downregulates protein expression of iNOS and phosphorylated p38-MAPK while increasing eNOS in thoracic aorta during SIRS in endotoxemia. We suggest that MEPD-dependent ET-1 and NO mechanisms may be involved in endotoxemia-induced altered p38-MAPK phosphorylation.     

Plasma levels of nitrites, PGF1α and nitrotyrosine in LPS-treated rats: functional and histochemical implications in aorta

We investigated the effects of lipopolysaccharide (LPS) administration on plasma nitrite, nitrotyrosine and 6-keto prostaglandin F1alpha, (PGF1alpha) levels and the related resultant changes in function and histochemistry of aorta in rats. Plasma nitrite and PGF1alpha nitrotyrosine levels were analysed after 5 mg/kg intravenous LPS was administered to rats compared with those in non-treated rats. The distribution of nitrotyrosine in the aorta was studied immunohistochemically. The contractile responses of aortic rings to phenylephrine (PE) from both the LPS-treated and control rats were studied in the organ baths. There were increases in plasma nitrite, PGF1alpha, and nitrotyrosine concentrations of LPS-treated rats compared to non-treated rats. Immunoreactivity of nitrotyrosine residues were detected in the endothelial and smooth muscle cells in LPS-treated but not in control rat aorta. The contractile responses to PE of the LPS-treated rat aortic rings were significantly reduced as compared with those of control rat's. Incubation of the aortic rings from LPS-treated rats with cyclooxygenase inhibitor indomethacine or with a combination of indomethacine and nitric oxide synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) increased the contractile responses to the levels observed in control rats suggesting that both prostanoids and particularly nitric oxide (NO) are involved in the reduced contractile responses in LPS-treated rats. These results supported the view that LPS might cause an increment in both NO and PGI2 levels. This increase in the NO and PGI2 levels may be responsible from the reduction in responses of aorta to contractile agents in LPS-treated rats. Increased peroxynitrite formation in LPS-treated rats may lead to nitration of the tyrosil residues of the proteins in the aorta.     

Ouabain-induced hypertension is accompanied by increases in endothelial vasodilator factors

The involvement of nitric oxide (NO), prostaglandins, and calcium-dependent potassium channel (K(Ca)) activators on the negative modulation of phenylephrine-induced contractions was evaluated on the isolated aorta and caudal (CAU) artery obtained from rats treated with ouabain for 5 wk to induce hypertension. In ouabain-treated rats, the reactivity to phenylephrine was reduced in the endothelium-intact aorta but not the CAU segments. Endothelial modulation of phenylephrine contraction, as demonstrated by endothelium removal, NO synthase (NOS) inhibition with N(omega)-nitro-L-arginine methyl ester and aminoguanidine, as well as K(Ca) inhibition with tetraethylammonium, was more pronounced in segments from ouabain-treated animals, and here greater effects were seen in the aorta than in CAU. An increased expression of endothelial NOS and neuronal NOS was seen in the aorta after ouabain treatment. In CAU, only endothelial NOS was detected and ouabain treatment did not alter its expression. These results suggest that ouabain-induced hypertension is accompanied by increased NO release derived from endothelial NOS and neuronal NOS and increased release of an endothelial hyperpolarizing factor that presumably opens K(Ca), all of which contribute to the increased negative modulation of the phenylephrine contraction.     

Myosin light chain phosphorylation in 32P-labeled rabbit aorta stimulated by phorbol 12,13-dibutyrate and phenylephrine

The mechanism(s) of force development in vascular smooth muscle following pharmacological activation of protein kinase C by phorbol esters are not known. In this study, we examined the myosin light chain phosphorylation response following stimulation by phorbol 12,13-dibutyrate (PDB) or phenylephrine in rabbit aorta which had been incubated with 32PO4 in order to label ATP pools. Through tryptic phosphopeptide mapping of myosin light chain from intact tissue and comparison to controls using purified components, we inferred that Ca2+-dependent force stimulated by PDB was associated with small increases in serine-19 phosphorylation, consistent with a contractile mechanism involving indirect activation of myosin light chain kinase. Additional residues, consistent with the in vitro substrate specificity of protein kinase C, were also observed to be phosphorylated in response to PDB and represented proportionately a larger fraction of the total phosphorylated myosin light chain in Ca2+-depleted tissues. Stimulation by an alpha 1-adrenergic agonist (phenylephrine) resulted in phosphorylation of residues which were consistent with an activation mechanism involving myosin light chain kinase only. These results indicate that in rabbit aorta the contractile effects of PDB may be partially mediated by Ca2+-dependent activation of myosin light chain kinase. However, the data do not rule out a component of the PDB-stimulated contractile response which is independent of myosin light chain phosphorylation on the serine-19 residue. In addition, activation by a more physiological stimulus, phenylephrine, does not result in protein kinase C-mediated myosin light chain phosphorylation.     

Ruthenium Red inhibits the activation of pyruvate dehydrogenase caused by positive inotropic agents in the perfused rat heart.

The increases in the amount of active, non-phosphorylated, pyruvate dehydrogenase caused by positive inotropic agents (from a control value of about 10%, to 40% of total enzyme) in the perfused rat heart could be completely blocked by prior perfusion with 2.5 micrograms of Ruthenium Red/ml. A similar increase caused by 5 mM-pyruvate was not blocked. This concentration of Ruthenium Red caused a 25% decrease in contractile force of hearts perfused in the absence of positive inotropic agents; however, in their presence the contractile force reached the same value in the absence or presence of Ruthenium Red. Neither control nor stimulated phosphorylase a content was affected by Ruthenium Red. Verapamil (0.1 microM) also decreased control contraction (by 40%), but did not block the activation of pyruvate dehydrogenase caused by a rise in extracellular [Ca2+]. The results support the hypothesis that positive inotropic agents activate pyruvate dehydrogenase in rat heart by increasing intramitochondrial [Ca2+].     

Tissue and concentration-dependent effects of sodium selenite on muscle contraction

In this study, we demonstrated that sodium selenite with high doses (≥ 10^-3 M) were potent in inducing a contracture type effect on heart and smooth muscles. Selenite (Se), at a concentration of 10^-3 M, caused a contracture effect in heart preparations. Also, low Se concentrations did not have any significant effect. Although low concentrations of Se (≥ 10^-5 M) had a biphasic effects on acetylcholine (ACh) induced and spontaneous ileum contractions, 10^-3 M selenite enhanced once more a contracture effect similar to that of the heart preparations. Replacing Ca²⁺ concentration of the bathing solution by twofold Ca²⁺ or Ca²⁺-free did not change the effects of selenite (10^-5 M) on contractility of ileum preparations. In vascular smooth muscle, low concentration of selenite (<10^-4) had no significant effects on KC1, and phenylephrine-induced contractions and acethylcholineinduced endothelium-dependent relaxations of isolated rabbit aorta. However, the contractions induced by phenylephrine and the relaxations induced by acetylcholine in rabbit aorta were depressed significantly by high concentration of selenite (10^-3 M). The results obtained by selenite exposure from these three different types of tissue preparations first suggest that the high concentration of selenite exposure induces some alterations in the functions of muscles and endothelium in a tissue and dose-dependent manner. Second, this observed irreversible type of dysfunction of tissues induced by l0^-3 M selenite is not directly dependent on the Ca²⁺ entrance into the cytosol, but might be induced by the increase of intracellular Ca²⁺ with the disturbance of Ca²⁺ regulation.     

Alterations in vascular endothelial function in the aorta and mesenteric artery in type II diabetic rats

We used the partial protection exerted by suitable dosages of nicotinamide against the beta-cytotoxic effect of streptozotocin (STZ) to create an experimental diabetic syndrome in adult rats that appears closer to type II diabetes mellitus than other available animal models. The dosage of 230 mg/kg of nicotinamide given intraperitoneally 15 min before STZ administration (65 mg/kg i.v.) yielded animals with hyperglycemia (187.8 +/- 17.8 vs. 103.8 +/- 2.8 mg/dL in controls; P < 0.001) and preservation of plasma insulin levels. This study assessed the relationship between endothelial dysfunction and agonist-induced contractile responses in such rats. In the thoracic aorta, the acetylcholine (ACh) induced relaxation was significantly reduced and the noradrenaline (NA) induced contractile response was significantly increased in diabetic rats compared with age-matched control rats. In the superior mesenteric artery, the ACh-induced relaxation was similar in magnitude between diabetic and age-matched control rats; however, the ACh-induced endothelium-derived hyperpolarizing factor (EDHF) type relaxation was significantly weaker in diabetic rats than in the controls. The phenylephrine (PE) induced contractile response was not different between the two groups. The plasma concentration of NOx (NO2- + NO3-) was significantly lower in diabetic rats than in control rats. We conclude that vasomotor activities in conduit arteries are impaired in this type II diabetes model.     

Increased tail artery vascular responsiveness to angiotensin II in cold-treated rats

Chronic exposure of rats to cold for 1-3 weeks results in a mild form of hypertension. The renin-angiotensin system (RAS) has been implicated in this model of cold-induced hypertension. Previously we have characterized the vascular responsiveness in cold-acclimated animals, using aortic tissue, and recent studies have focused on the thermoregulatory responses of angiotensin II (AngII), utilizing the tail artery of the rat. Therefore in the current study we evaluated the vascular responsiveness of cold-treated rats to AngII in both aorta and tail artery at 2 and 4 weeks of cold exposure (5+/-2 degrees C). Systolic blood pressures were significantly elevated in cold-treated animals compared with control animals at both 2 and 4 weeks of cold exposure. At both of these time points body weights were reduced and ventricular weights were increased in cold-treated animals. After 2 weeks of cold exposure the vascular responsiveness of the aorta to AngII was significantly lower than that of controls. This vascular responsiveness to AngII was elevated and returned to control levels after 5 weeks of cold exposure. However, this pattern was not observed in the tail artery. The vascular responsiveness of tail artery rings from cold-treated rats to AngII was significantly greater than that of control animals during both 2 and 5 weeks of exposure to cold. The vascular contractile responses of both the aorta and tail artery to KCI in the cold-treated animals was not different from that of the control animals maintained at ambient room temperature, suggesting that the vascular smooth muscle contractile components were not altered by the cold exposure. Thus, the in vitro vascular reactivity to the receptor-mediated vasoconstrictor AngII was decreased in the sparsely innervated aorta and increased in the more densely innervated tail artery of the cold-treated animals when compared with controls. These results suggest that the increased responsiveness of AngII on the smooth muscle of the tail artery may play a role in adaptation to the cold and the maintenance of cold-induced hypertension.     

Epinephrine infusion induces hyporesponsiveness of vascular smooth muscle

Exposure to vasoactive drugs may lead to desensitization of vascular smooth muscle responsiveness. We have explored this phenomenon by infusing epinephrine into awake rabbits for 2h and then assessing smooth muscle contraction, both in vivo and ex vivo. Epinephrine was infused at a rate of 1 microgram X min-1 which resulted in a 15-fold increase in the plasma epinephrine concentration. The dose of phenylephrine required to cause a 25 mmHg increase in mean arterial pressure significantly increased from 109 +/- 56 micrograms prior to the infusion to 261 +/- 143 at the end of the 2h infusion (p less than 0.01). The sensitivity to phenylephrine remained decreased when reassessed 2h later. Untreated rabbits displayed no change in alpha-adrenergic responsiveness when assessed at 2 hourly intervals over the time-course of the experiment. Contraction of aortic rings removed from both epinephrine-treated and control rabbits was determined in vitro in tissue baths. The EC50 of norepinephrine-induced contraction increased from 31 +/- 6 to 210 +/- 20 nM while there was also a 30% decrease in the maximal force of contraction (EMax) in treated vessels. The EC50 only partially recovered after 4h of incubation ex vivo, while the EMax was restored to the control value. The EC50 for histamine in the aortic rings from epinephrine-treated rabbits was not different from controls although there was a 25% reduction in the EMax at 2h. We conclude that desensitization of alpha-adrenergic mediated vascular contractility develops rapidly in vivo and is only slowly reversible after removal of the agonist.     

Enhancement of endotoxin-induced vascular hyporeactivity to phenylephrine in the thoracic aortas of Mg-deficient rats ex vivo

Since endotoxin lethality is enhanced by Mg deficiency in animals, we determined whether endotoxin-induced vascular hyporeactivity to phenylephrine (PE) is enhanced in Mg-deficient rats. Normal and Mg-deficient adult male Wistar rats were injected with Escherichia coli 011: B4 lipopolysaccharide (1 or 5 mg/kg, i.p.). Six h later, rings prepared from their thoracic aortas showed severe hyporeactivity to PE. This was more pronounced in the Mg-deficient rats, and was reversed by in vitro treatment with a highly selective inducible nitric oxide (NO) synthase inhibitor, 1400 W, or a highly selective soluble guanylyl cyclase inhibitor, ODQ. However, reversal required high doses of both inhibitors in Mg-deficient rats. Endotoxemia for 6 h was associated with elevated serum interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha levels, and strong TNF receptor mRNA expression in the abdominal aortas, which were significantly greater in the Mg-deficient rats. Treatment of the thoracic aortas, isolated from control and Mg-deficient rats before endotoxic challenge, with IL-1beta or TNF-alpha for 6 h in vitro caused hyporeactivity to PE, but its severity did not differ significantly between the two groups. These results suggest that high serum IL-1beta and TNF-alpha levels, and increased TNF receptor production in the vascular tissue, contribute to vascular hyporeactivity to PE in endotoxemia, and to its enhancement in Mg-deficient rats, via NO/cGMP signaling.     

Cyclic AMP and the positive inotropic effect of norepinephrine and phenylephrine.

Time-response studies of the effects of norepinephrine and phenylephrine revealed that both agonists caused an increase in cyclic AMP levels before increases in contractile force in either the electrically stimulated left atria or spontaneously beating right atria of the rat. Norepinephrine caused a nearly sixfold increase in cyclic AMP, whereas phenylephrine produced only a 50% increase in the nucleotide. Pretreatment with reserpine did not affect the norepinephrine cyclic AMP response; however, the phenylephrine cyclic AMP response was abolished. Reserpine pretreatment did not significantly affect the contractile responses of either amine. In the presence of propranolol, norepinephrine was found to have the ability to produce an increace in contractile force in which cyclic AMP was apparently not involved. The time course of the contractile response induced by adrenergic amines was found to be remarkably influenced by the chronotropic response in spontaneously beating preparations while the cyclic AMP response was not greatly affected. This difference in the contractile response may be due to the ability of the chronotropic response to influence the flux of calcium through the cell membrane.     

Mechanisms of aortic smooth muscle hyporeactivity after prolonged hypoxia in rats.

The aim of this study was to determine whether the effects of hypoxia on aortic contractility reflect a decrease in smooth muscle activation [phosphorylation of the 20-kDa myosin regulatory light chain (LC(20))], the capacity for myofibrillar ATP hydrolysis (mATPase activity), or both. Our results indicate that, in endothelium-denuded aortic rings from rats exposed to hypoxia for 48 h (inspired O(2) concentration = 10%), contractions to phenylephrine and potassium chloride (KCl) are impaired compared with rings from normoxic rats. The proportion of phosphorylated to total LC(20) during aortic contraction induced by 10(-5) M phenylephrine was reduced after hypoxia (51.4 +/- 5.4% in normoxic control rats vs. 32.5 +/- 4.7% in hypoxic rats, P < 0.01). Aortic mATPase activity was also decreased (maximum ATPase rate = 29.6 +/- 3.4 and 20.7 +/- 3.7 nmol. min(-1). mg protein(-1) in control and hypoxic rats, respectively, P < 0.05). Neither proliferation nor dedifferentiation of aortic smooth muscle was evident in this model; immunostaining for smooth muscle expression of the proliferating cell nuclear antigen was negative and smooth muscle-specific isoforms of myosin heavy chains, h-caldesmon, and calponin were increased, not decreased, after hypoxic exposure. Decreased aortic reactivity after hypoxia is associated with both impairment of smooth muscle activation and diminished capacity of the actomyosin complex, once activated, to hydrolyze ATP. These changes cannot be attributed to smooth muscle dedifferentiation or to reduced contractile protein expression.     

The effects of EGTA on vascular smooth muscle contractility in calcium-free medium

The effect of EGTA, commonly present in Ca2+-free physiological saline solution, on the contractile responses induced by Ca2+ and phenylephrine was studied in dog mesenteric arteries and aortas of rats and rabbits. EGTA substantially enhanced the contractile responses of these vascular strips or rings to added Ca2+ after a prolonged preincubation period in the Ca2+-free medium. The maximal level of the enhanced contractile responses was independent of EGTA concentration, but the rate of the maximal responses was faster at higher EGTA concentration, presumably as a result of faster removal of intracellular Ca2+. Such a Ca2+-induced response was sensitive to the Ca2+ antagonist, nifedipine. EGTA present at low concentrations (50 and 400 microM) in Ca2+-free medium also inhibited the phenylephrine-induced contractile response more prominently for the longer preincubation periods of the vascular tissues in Ca2+-free medium. Our results suggest that EGTA, even when added at low concentrations to the vascular smooth muscle for a sufficiently long period in Ca2+-free medium, may cause destabilization of the cell membranes leading to increased permeability to subsequently added Ca2+. EGTA may also remove the superficially bound Ca2+ and subsequently reduce the intracellular Ca2+ pool via extraction of the intracellular Ca2+ at the cell membrane surfaces.