Integrins inhibit angiotensin II-induced contraction in rat aortic rings

Many extracellular matrix proteins contain the tripeptide sequence arginine-glycine-aspartate (RGD). This RGD motif is recognized by integrins, a family of adhesion receptors present on vascular smooth muscle cells. In the present study, we examined the ability of different RGD-containing peptides to affect the contraction of rat aortic rings in response to different agonists. We found that the peptide RGDS inhibited angiotensin-induced contraction in a dose dependent manner. In contrast, the peptides RGDW and RGES had no effect on angiotensin-induced contractility. We show that function-blocking antibodies to the integrins alphavbeta3 and alpha5beta1 also inhibit angiotensin-induced contraction. These effects were observed in the absence of an intact endothelium. In contrast, neither an antibody directed against the beta1 subunit nor the peptide RGDS had an effect on phenylephrine or 5-hydroxytryptamine-induced contraction. These data suggest that interactions of vascular smooth muscle with components of the surrounding extracellular matrix may influence the response of smooth muscle to agonists.     

Vascular hyporesponsiveness in aortic rings from cirrhotic rats: role of nitric oxide and endothelium

1. The role of nitric oxide as mediator of the vascular alterations present in different models of experimental liver cirrhosis is controversial. In the present study, we evaluated the role of nitric oxide and that of the endothelium in the response to phenylephrine and acetylcholine of isolated aortic rings from chronic bile duct-ligated (29 days) rats and their corresponding controls. Experiments were performed in rings with or without endothelium, in rings pretreated with N-omega-nitro-L-arginine methyl ester (10(-4) mol/l) to inhibit nitric oxide synthesis and in rings pretreated with aminoguanidine (10(-4) mol/l) to inhibit inducible nitric oxide synthesis. 2. Under basal conditions, the maximum absolute tension developed in response to cumulative addition of phenylephrine was significantly decreased in rings from bile duct-ligated animals (1.62 +/- 0.06 g) compared with the control rings (2.15 +/- 0.099). This hyporesponsiveness to phenylephrine of rings from bile duct-ligated animals was corrected after treatment with N-omega-nitro-L-arginine methyl ester and reduced, but not completely eliminated, in rings without endothelium. In contrast, aminoguanidine did not modify the lower response to phenylephrine rings from bile duct-ligated animals. ED50 values were not different between groups under any experimental conditions. 3. The endothelium-dependent vasodilatation to acetylcholine in phenylephrine-constricted rings was similar in both groups of animals, control and bile duct ligated, under all experimental conditions. N-omega-nitro-L-arginine methyl ester pretreatment and removal of the endothelium completely abolished the response to acetylcholine in cirrhotic and control rings. 4. These results demonstrate that in aortic rings from cirrhotic, bile duct-ligated rats, increased production of nitric oxide, mainly of endothelial origin, is responsible for the lower contractile response to phenylephrine. Our data, however, do not support the involvement of the inducible nitric oxide synthase isoform in this alteration. In contrast, endothelial vasodilatory response to acetylcholine is not altered in this model of cirrhosis, which indicates that not all mechanisms of nitric oxide release are abnormal.     

Denaturing behavior of glutathione reductase from cyanobacterium Spirulina maxima in guanidine hydrochloride

The influence of guanidine hydrochloride (Gdn-HCl) on glutathione reductase from Spirulina maxima has been studied by measuring the changes in enzymatic activity, protein fluorescence, circular dichroism, thiol groups accessibility, and gel filtration chromatography. It was found that the denaturation process involves several intermediate states. At low, Gdn-HCl concentrations (Cm = 0.4 M), reductase activity was fully lost. However, below 3 M Gdn-HCl, this inhibition was freely reversible upon removal of the denaturing agent. Gel filtration experiments revealed that this reversible inhibition was not due to dissociation of the tetrameric enzyme. Structural studies strongly suggest that the conformation of this intermediate state is similar to that of native enzyme. A model in which a local region of the polypeptide chain assumes an extended conformation (D. T. Haynie, and E. Freire, Proteins 16,115-140) is proposed for the reversibly inactivated enzyme. Between 3 and 4 M Gdn-HCl (Cm = 3.5), the enzyme activity was irreversibly lost, this inhibition being concomitant with the loss of ellipticity, changes in both wavelength and intensity at the maximum of fluorescence emission, and dissociation of the enzyme into unfolded monomers; these results reveal that gross changes in the protein conformation occur under these conditions. At 4 M Gdn-HCl an equilibrium exists between the denatured forms of dimer and monomer, which is completely shifted toward the unfolded monomers at 5 M Gdn-HCl. Irreversibility in the Gdn-HCl-induced denaturation of S. maxima glutathione reductase was not due to aggregation of the unfolded enzyme.     

Norepinephrine induces expression of c-fos mRNA through the alpha-adrenoceptor in rat aortic rings

We examined whether norepinephrine at pharmacologically relevant doses induces increased expression of c-fos mRNA in rat aortic rings. c-fos mRNA was expressed at norepinephrine concentrations known to cause minimum and maximum contraction of rat aorta in vitro. At the concentration known to cause maximum contraction, norepinephrine produced a marked and sustained increase of c-fos mRNA expression. Induction of c-fos was blocked completely by the alpha 1-adrenergic antagonist prazosin, partially by the alpha 2-adrenergic antagonist yohimbine, and not at all by the beta-adrenergic antagonist propranolol. A prazosin inhibition curve showed that 1 nmol/L prazosin inhibited 10 micromol/L norepinephrine induced c-fos expression by 40%. At the pharmacologic dose known to cause maximum contraction, norepinephrine induces c-fos mRNA expression through the alpha-adrenoceptor in rat aortic rings.     

Expression of platelet-derived growth factor ligands and receptors by rat aortic endothelium in vivo

In situ hybridization and immunostaining were used to study the time course of expression for platelet-derived growth factor (PDGF) ligands and receptors in endothelium of the rat aorta after injury. The PDGF-A and -B chains were expressed in endothelial cells at the wound edge within 4 h after injury, but no expression was detectable in uninjured endothelium. PDGF alpha-receptor was expressed in a pattern similar to the PDGF-A chain, while expression of PDGF beta-receptor was not detected at any time. Expression of the PDGF-B chain remained elevated in endothelial cells at the leading edge even at later measurements when these cells had stopped replicating. Smooth muscle cells (SMCs), which are absent from the intima of the normal aorta and are known to express PDGF beta-receptors, were predominantly found to migrate into the intima near the endothelial leading edge where PDGF-B was expressed. These data suggest a paracrine role for endothelial PDGF in SMC migration.     

Effects of vitamin E deficiency on vasomotor activity and ultrastructural organisation of rat thoracic aorta

1. The effects of vitamin E deficiency were evaluated in aortic rings isolated from rats maintained on a diet deficient in vitamin E. 2. Endothelium-dependent vasodilator responses to acetylcholine (ACh) and calcium ionophore, A23187, were reduced in preparations from treated animals, compared to the age-matched controls. The maximal vasodilation to ACh was 66.4 +/- 9 (n = 4) and 38.8 +/- 7 (n = 4) % in control and 10 month-treated preparations, respectively. 3. The endothelium-independent vasodilator responses to sodium nitroprusside as well as the concentration-dependent contractile responses to noradrenaline, did not differ between treated and control preparations. 4. Electron microscopic examination of vascular segments and revealed that, following vitamin E deficiency, normal tissue organisation was disrupted, the endothelial monolayer either not being in contact with the underlying tissue or being absent in most of the areas analysed. 5. It is concluded that during vitamin E deficiency both morphological disruption and functional impairment of endothelium occur without observable modification of muscle cell function and morphology.     

Endothelin-induced prostacyclin production in rat aortic rings is mediated by protein kinase C

Endothelin (ET) is a vasoconstrictor peptide released from endothelial cells that is known to cause prostaglandin release. The mechanism remains unclear. To determine whether the protein kinase C (PKC) signaling pathway is stimulated by endothelin, we pretreated rat aortic rings with either PKC activator or inhibitors and measured the release of prostacyclin (PGI2) by radioimmunoassay. ET (10(-9) M) produced a 10-fold increase in PGI2 release. Pretreatment with 10(-9) M of three different PKC inhibitors, 1-(5-isoquinolinesulfonyl)piperazine(CL), staurosporine, and 1-(5-isoquinolinesulfonyltmethyl)piperazine (H7), blocked ET-induced PGI2 release. ET-induced PGI2 release was also blocked by pretreatment with inhibitors of either phospholipase A2 7,7-dimethyleicosadienoic acid or trifluoromethyl ketone analogue) (10(-9) M) or cyclooxygenase (indomethacin) (10(-9) M). We conclude that ET activates PKC, which activates phospholipase A2, which liberates arachidonic acid, which increases PGI2 production and release.     

Effects of dietary vegetable oils on behavior and drug responses in mice

Previously, we noted significant differences in the behavioral patterns of mice fed safflower oil with a very low alpha-linolenate/linoleate ratio and perilla oil with a high alpha-linolenate/linoleate ratio from mothers to offsprings. In this report, we compared the behavior and drug responses in mice fed diets containing six different vegetable oils-corn, rapeseed, soybean, safflower, perilla and a mixture of perilla and safflower oils- for a relatively short period: 8 months after weaning. Soybean oil is a component of most conventional diets and was used as a control. The alpha-linolenate/linoleate ratios of the oils appeared to affect the locomotor activities in a wheel cage: the activity decreased in the order of safflower, the mixture (1:1) and the perilla oil groups. However, the rapeseed oil group exhibited much higher locomotor activity than that expected from the alpha-linolenate/linoleate ratio. Additionally, the rapeseed oil group exhibited unusual behavior patterns, including higher ambulation and rearing activities, faster acquisition of the water maze task and slower habituation behavior as compared with the control group. Susceptibility to pentobarbital anesthesia tended to be higher in the rapeseed oil group. The differences in the alpha-linolenate/linoleate ratios of these oils alone do not account for the observed differences in the behavioral patterns among the six dietary groups. Although we cannot exclude the possibility that the observed behavioral anomaly is due to the unique fatty acid composition of rapeseed oil, we speculate that a factor(s) other than fatty acids in rapeseed oil affected nervous system functions.     

Effects of changes in pH and CO2 on pulmonary arterial wall tension are not endothelium dependent

We examined the changes in isolated pulmonary artery (PA) wall tension on switching from control conditions (pH 7.38 +/- 0.01, PCO2 32.9 +/- 0.4 Torr) to isohydric hypercapnia (pH change 0.00 +/- 0.01, PCO2 change 24.9 +/- 1.1 Torr) or normocapnic acidosis (pH change -0.28 +/- 0.01, PCO2 change -0.3 +/- 0.04 Torr) and the role of the endothelium in these responses. In rat PA, submaximally contracted with phenylephrine, isohydric hypercapnia did not cause a significant change in mean (+/- SE) tension [3.0 +/- 1.8% maximal phenylephrine-induced tension (Po)]. Endothelial removal did not alter this response. In aortic preparations, isohydric hypercapnia caused significant (P < 0.01) relaxation (-27.4 +/- 3.2% Po), which was largely endothelium dependent. Normocapnic acidosis caused relaxation of PA (-20.2 +/- 2.6% Po), which was less (P < 0.01) than that observed in aortic preparations (-35.7 +/- 3.4% Po). Endothelial removal left the pulmonary response unchanged while increasing (P < 0.01) the aortic relaxation (-53.1 +/- 4.4% Po). These data show that isohydric hypercapnia does not alter PA tone. Reduction of PA tone in normocapnic acidosis is endothelium independent and substantially less than that of systemic vessels.     

Effects of dietary biotin on enhanced sucrose intake and enhanced gustatory nerve responses to sucrose seen in diabetic OLETF rat

Vicilin (7S storage proteins) isolated from different legume seeds were shown to inhibit yeast growth and glucose stimulated acidification of the medium by yeast cells. The degree of growth inhibition varied with the origin of vicilins. It was more than 90% for vicilins from cowpea (Vigna unguiculata, cultivar pitiuba) and equal to 65% for vicilins from Vigna radiata, in the case of Saccharomyces cerevisae. Vicilins from cowpea seeds inhibited the glucose stimulated acidification of the medium by S. cerevisae up to 60%. We have also observed that vicilins bind to yeast cells. We suggest that vicilins bind to chitin-containing structures of yeast cells and that such association could result in inhibition of H+ pumping, cell growth and spore formation. A final consequence of the yeast growth inhibition by vicilins is (probably) the formation of spores.     

Vasoinhibitory effect of leminoprazole, a H+,K(+)-ATPase inhibitor, on rat aortic rings

The effects of the marine ascidian compound bistratene A on in vitro cultures of Plasmodium falciparum were assessed. Concentrations from 0 to 1.5 micrograms ml(-1) of the compound were tested. The parasitaemia in asynchronous cultures treated with bistratene A increased normally over the first 40 h, then decreased leaving only gametocytes. When synchronized cultures were treated with a constant dose of 50 ng ml(-1), gametocytes developed more rapidly than they did in control cultures. In addition, gametocytes developed in drug-treated cultures of P. falciparum that normally do not produce gametocytes. Bistratene A appears to inhibit merozoite invasion as well as to induce gametocytogenesis.     

Amplification of alpha 1D-adrenoceptor mediated contractions in rat aortic rings partially depolarised with KCl

Partial depolarisation of smooth muscle in endothelium-denuded rat aortic ring preparations, by increasing physiological buffer KC1 concentrations from 4.7 to 14.7 mM, produced a leftward shift of concentration response curves (CRCs) to the alpha 1-adrenoceptor agonist noradrenaline (NA), phenylephrine and methoxamine, without changing maximal responses, whereas maximal responses to clonidine (CLON), also an alpha 1-agonist in this tissue were considerably increased. Partial depolarisation did not alter responses to 10 nM NA or 100 nM CLON in Ca2+(-free) buffer, but significantly increased the contractions obtained on adding Ca2+ back in the presence of the agonists. The potentiation of NA (2.5 and 5 nm) contractions by partial depolarisation was prevented by the voltage-operated Ca2+ channel (VOCC) antagonist nifedipine (NIF, 1 microM). NIF did not significantly affect NA CRCs in 4.7 mM KCl, whereas responses in 14.7 mM KCl were significantly decreased, indicating VOCC recruitment by NA only in the latter condition. Initial depletion of intracellular Ca2+ stores with 1 microM thapsigargin (THAP) in Ca2+(-free) buffer did not alter NA CRCs subsequently obtained in normal Ca2+. However, after THAP-pretreatment, these NA responses (in both 4.7 and 14.7 mM KC1) were attenuated by NIF, indicating that VOCCs were activated by NA in THAP-treated tissues. SKF 96365 (SKF, 30 microM), which can block VOCC and non-VOCC routes of extracellular Ca2+ influx, inhibited NA responses in 4.7 mM and 14.7 mM KCl, possibly implying a role for both types of Ca2+ entry in contractions. However, the greater inhibitory effects of SKF in THAP-pretreated tissues, probably reflected the mobilisation of VOCCs by NA following THAP exposure, because SKF was shown separately to block VOCC-mediated contractions in tissues depolarised with 100 mM KCl alone. 10 microM niflumic acid, an inhibitor of Ca2+(-activated) Cl- channels, did not affect responses to NA in 4.7 mM or 14.7 mM KC1, suggesting that VOCC opening induced by NA in 14.7 mM KCl was not due to depolarisation produced by alpha 1-adrenoceptor induced Cl- efflux. CRCs for NA were unaffected by pretreatment of rings with 100 ng ml-1 pertussis toxin (PT), suggesting a lack of involvement of PT-sensitive G proteins in the contractions obtained either in 4.7 or 14.7 mM KCl. BMY 7378 (100 microM), a selective antagonist for alpha 1D-adrenoceptors, competitively inhibited NA contractions with apparent pKB values of 8.7 +/- 0.2 and 8.4 +/- 0.1 in 4.7 mM and 14.7 mM KCl, respectively. Pretreatment of rings with chloroethylclonidine (100 microM), an irreversible antagonist of alpha 1B-and alpha 1D-adrenoceptors, produced similar rightward shifts in CRCs to NA by 3.2 +/- 0.2 and 3.7 +/- 0.3 log concentration units in 4.7 mM and 14.7 mM KCl, respectively, without changing maximal responses. Inositol phosphate (IP) turnover produced by NA in aortic rings was not significantly different in 4.7 mM compared with 14.7 mM KCl. As a whole, these results suggest that partial depolarisation of the rat aorta with KCl enhances alpha 1-adrenoceptor mediated contractions predominantly via the alpha 1D-subtype, and by a mechanism to be identified which allows greater recruitment of VOCCs by NA. In addition, the ability of THAP-pretreatment also to enhance VOCC activation by NA suggests that Ca2+ release from, or prevention of its reuptake into, intracellular stores may contribute to those processes leading to VOCC opening.     

DC photoplethysmography in the evaluation of sympathetic vasomotor responses

The d.c. component of the photoplethsmographic signal was used to determine the response of the finger vasculature to three standard tests of vasomotor function: (1) an inspiratory gasp (IG), (2) immersion of the contralateral hand in ice water (IW), and (3) the Valsalva manoeuvre. The vasoconstrictor response to the first two of these stimuli could be measured in all of 25 normal subjects. The response to the Valsalva manoeuvre could not be detected consistently. Seven patients with known sympathetic autonomic dysfunction showed no response to either IG or IW. In 30 patients with diabetes mellitus of over 10 years duration, 46.7% had no response to IG, and 20% had no response to IW. Absent responses correlated with abnormal autonomic cardiovascular reflexes, with absent sympathetic skin responses and with the severity of peripheral somatic neuropathy. The d.c. photoplethysmographic determination of the vasoconstrictor response in the finger after a deep inspiratory gasp and after ice water immersion offers an additional measure of the function of small (2 mu-6 mu) peripheral nerve fibres. Because of variability in the amplitude of the responses in normals, only an absent response should be accepted as abnormal.     

Local actions of acetylcholine on vasomotor regulation in rat incisor pulp

Local actions of acetylcholine on vasomotor regulation in rat incisor pulp were investigated in anaesthetized animals. Pulpal blood flow was measured with laser Doppler flowmetry. lontophoresis (40 or 60 muA for 20 s) with acetylcholine or carbachol on an exposed dentin surface caused a biphasic response: an initial vasoconstriction was followed by an atropinesensitive vasodilation. The vasoconstrictor response was enhanced by 171% in the presence of atropine, whereas in control experiments, using isotonic saline as a medium for the direct current, similar vasoconstriction was unaffected by atropine. Carbachol-induced vasodilation was significantly reduced by 30% after intravenous injection of the nitric oxide synthesis blocker N omega-nitro-L-arginine methyl ester (10 mg kg-1), and the remaining vasodilation was abolished by atropine. The present results support the concept of a cholinergic modulation of sympathetic vasoconstrictor function and of a partial contribution of nitric oxide in carbachol-induced vasodilation in rat incisor pulp.     

The cytological mechanisms of the postnatal growth of aortic endothelium

Contribution of proliferation and hypertrophy of endotheliocytes to the postnatal growth of the internal aortic lining in rats was estimated using organo- and cytometry, cytophotometry of nuclear DNA, radioautographic analysis of 3H-thymidine incorporation, determination of the mitotic index and the number of binucleated cells. Mitotic divisions of diploid endotheliocytes (in adult rats, their proportion attains 97-98%) is the principal cytological mechanism that, throughout the life of an animal, provides for an increase in the area of endothelial layer. Cell hypertrophy, which is unrelated to polyploidization, develops only during the first month of postnatal life. In old rats (2.5 years of age), an increased proportion of binucleate cells (2c x 2) and cells with pycnotic nuclei is observed.     

Effects of dietary NaCl deprivation during early development on behavioral and neurophysiological taste responses.

Investigated whether the gustatory system can be modified by restricting dietary NaCl during early development by recording neurophysiological taste responses in Sprague-Dawley rats at various times after deprivation (Exp I), and by measuring behavioral taste preferences in 3 groups of 7 NaCl deprived adult rats (Exp II). Overall findings indicate that Ss deprived of dietary NaCl from the 3rd day of gestation to 12 days postnatally and then placed on a NaCl-replete diet had chorda tympani nerve responses similar to those of nondeprived Ss when recordings were made at 28 days of age and older; however, preferences for NaCl solutions over water were significantly less than those of controls when tested at adulthood. NaCl deprivation in Ss from the 3rd day of gestation to approximately 35 days postnatally resulted in altered chorda tympani nerve responses to NaCl but not to other stimuli such as NH?Cl and KCl. Thus, it is concluded that restriction of dietary NaCl at a period in the rat's development when peripheral and central taste responses are changing results in short-term alterations in peripheral neural responses and in long-term changes in preference behaviors. (36 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Effects of dietary anticarcinogens on rat gastrointestinal glutathione S-transferase theta 1-1 levels

Several naturally occurring food components or non-steroidal anti-inflammatory drugs (NSAIDs) may reduce gastrointestinal cancer rates. Recently we have shown that dietary administration of such compounds enhanced the glutathione S-transferase (GST) enzyme activity and class alpha, mu and pi isoenzyme levels in the rat gastrointestinal tract. Elevation of the levels of GSTs, a family of biotransformation enzymes with many functions such as detoxification of carcinogens, might be one of the mechanisms that lead to cancer prevention. We therefore investigated whether the anticarcinogens alpha-angelicalactone, alpha-tocopherol, beta-carotene, coumarin, ellagic acid, flavone, indole-3-carbinol, d-limonene, oltipraz, phenethylisothiocyanate (PEITC) and the sulphoraphane analogue compound-30 affect gastrointestinal rGSTT1-1 protein levels in male Wistar rats. rGSTT1-1 protein levels were determined in cytosolic fractions of liver and oesophageal-, gastric-, small intestinal- and colonic mucosa by densitometrical analyses of western blots after immunodetection with an anti human GSTT1-1 monoclonal antibody, that cross-reacts with rGSTT1-1. In control Wistar rats, gastrointestinal rGSTT1-1 protein levels were highest in the liver and decreased in the order liver > stomach > colon > oesophagus > small intestine. Gastric rGSTT1-1 protein levels were enhanced by alpha-angelicalactone, alpha-tocopherol, coumarin, ellagic acid, oltipraz, PEITC and the sulphoraphane analogue compound-30. Oesophageal rGSTT1-1 protein levels were elevated by a-angelicalactone and coumarin, whereas colonic rGSTT1-1 protein levels were elevated by coumarin. Ellagic acid, on the other hand, reduced hepatic rGSTT1-1 protein levels to 53% of the control. In conclusion, dietary anticarcinogens are capable of inducing rGSTT1-1 protein levels in the rat gastrointestinal tract, and are most pronounced in the stomach. Enhanced rGSTT1-1 protein levels might lead to an increase of enzyme activity and to a more efficient detoxification of carcinogens and thus could contribute to prevention of carcinogenesis.