Guinea-pig Aorta Research Articles

Synthesis and Anti-histaminic Evaluation of N-Phenyl-(alkyl)-5-(dialkylamino)methyl-2-amino-2-oxazolines

New N- phenyl(alkyl)-5-(dialkylamino)methyl-2-amino-2-oxazolines, 5a–e, have been synthesized from the corresponding 3-phenyl(alkyl)carbamoyl-2-iminooxazolidines 2. A two-stage hydrolysis reaction led finally to the corresponding ring-opened N-phenyl(alkyl)-N′-[1-{3-(dialkylamino)-propan-2-ol}]ureas 4. The oxazoline ring was regenerated through an intramolecular nucleophilic substitution involving an halogen atom introduced by the reaction of thionyl chloride on 4. Pharmacological properties of 5a–e were evaluated on histaminic and adrenergic receptors in guinea-pig trachea and rat aorta. Compounds 5b and 5e showed a selective anti-histaminic effect on guinea-pig airways, but a significant response was obtained for a concentration >10-6 M. No pharmacological activity was obtained with oxazoline 5c whereas oxazolines 5a and 5d seemed to present a non-selective effect on the contractile mechanism of the smooth muscle cell.

Dyslipidogenic microangiopathy in guinea pigs at early stages of atherogenesis.

We studied the effect of dyslipidemia on lipid metabolism, state of microcirculatory system, and morphological alterations in the aorta and liver of guinea pigs at the early stages of experimental atherogenesis. The important role of microcirculatory disorders in the development of regional pathology and atherosclerosis is confirmed. The proposed alimentary model can be used in the development of novel methods for prevention and treatment of atherosclerosis.

Investigation of the pharmaceutical and pharmacological equivalence of different Hawthorn extracts.

Seven Hawthorn extracts were tested in isolated guinea pig aorta rings. The effect on noradrenaline- (10 microM) induced contraction was investigated. The extracts were prepared using ethanol (40 to 70% v/v), methanol (40 to 70% v/v), and water as the extraction solvents. The aqueous-alcoholic extracts displayed similar spectra of constituents. They were characterised by similar procyanidin, flavonoid, total vitexin and total phenols content and by similar TLC fingerprint chromatograms. The aqueous extract, however, showed a different fingerprint and a noticeably lower concentration of procyanidins, flavonoids and total phenols but a similar total vitexin content. All 7 extracts had a relaxant effect on the aorta precontracted by noradrenaline and led to relaxations to 44 until 29% of the initial values. The EC50 values of the aqueous-alcoholic extracts varied between 4.16 and 9.8 mg/l. The aqueous extract produced a similarly strong maximal relaxation as the other extracts, but the EC50, at 22.39 mg/l, was markedly higher. The results show that Hawthorn extracts with comparable quality profiles were obtained by using aqueous-alcoholic extraction solvents (40 to 70% ethanol or methanol). The extracts exerted comparable pharmacological effects. When using water as the extraction solvent, both, the spectrum of constituents and the pharmacological effect, deviated remarkably. It is thus possible to obtain bioequivalent extracts with comparable effect profiles by using 40 to 70% ethanol or methanol as the extraction solvent.

Mechanisms underlying the hydrogen peroxide-induced, endothelium-independent relaxation of the norepinephrine-contraction in guinea-pig aorta

The mechanisms underlying the hydrogen peroxide-induced relaxation of the norepinephrine-contraction were studied by measuring isometric force, myosin light chain (MLC 20) phosphorylation and cyclic GMP in endothelium-denuded muscle from the guinea-pig aorta. Norepinephrine (5.2±1.3 μM) produced a phasic, followed by a tonic contraction. Hydrogen peroxide (10 and 100 μM), glyceryl trinitrate (30 and 300 nM) and 8-bromo cyclic GMP (30 and 100 μM) did not change the basal tone, but reduced the norepinephrine-induced contraction. Phosphorylation of MLC 20 (percentage of phosphorylated to total MLC 20) was increased 1 min (5.9±1.0% vs. 35.9±4.9%) and, to a lesser extent, 20 min (3.7±1.7% vs. 13.9±1.6%) after the addition of norepinephrine. Hydrogen peroxide (100 μM) did not modify basal MLC 20 phosphorylation, but reduced the increase in MLC 20 phosphorylation induced by 1-min exposure to norepinephrine (20.9±4.1%). Its effect was abolished by catalase. When the tissue was incubated for 20 min with norepinephrine in the presence of hydrogen peroxide, norepinephrine-induced MLC 20 phosphorylation was not changed (13.6±1.5%), as compared to that in the absence of hydrogen peroxide. Hydrogen peroxide relaxed norepinephrine-stimulated aortas in a concentration-dependent fashion with EC 50 values of 5.9±0.2 μM. The relaxation was inhibited by soluble guanylate cyclase inhibitors and increased by an inhibitor of cyclic GMP-selective phosphodiesterase. In aorta precontracted with norepinephrine, hydrogen peroxide (100 μM) relaxed the tissue by 89±11% and almost doubled tissue concentrations of cyclic GMP, whereas sodium nitroprusside (1 μM) relaxed the tissue by 100% and increased cyclic GMP concentrations 30-fold. It is suggested that the inhibitory effects of hydrogen peroxide on the norepinephrine-induced phasic and sustained contractions are explained by a decrease in MLC 20 phosphorylation and by an alteration in MLC 20 phosphorylation-independent mechanisms, respectively. The effects of hydrogen peroxide were in part mediated by cyclic GMP.

Paracrine effect of vascular smooth muscle cells in the prevention of aortic aneurysm formation.

Inflammation and elastinolysis are observed in the media of abdominal aortic aneurysms (AAAs) where vascular smooth muscle cell (VSMC) density is decreased. In contrast, elastin and VSMCs are preserved in the noninflammatory media of stenotic atherosclerotic lesions. We have tested the hypothesis that VSMCs exert a protective effect against inflammation and proteolysis in a model of AAA in rats, in which medial elastin degradation is driven by inflammation and matrix metalloproteinases. Decellularized guinea pig aortas (xenografts) were implanted orthotopically into Fischer-344 rats and seeded with a suspension of rat VSMCs syngeneic to the rat recipient, or were infused with culture medium as a control. Diameter and elastin in the media were quantified 8 weeks after implantation. Inflammation, matrix metalloproteinase (MMP) and tissue inhibitor of matrix metalloproteinase (TIMP) expression were analyzed 1 and 2 weeks after implantation. VSMC addition prevented AAA formation (mean +/- standard deviation diameter increase: 198.2% +/- 106.6% vs 35.3% +/- 17.8%, P =.009), elastin degradation, and decreased infiltration by monocyte-macrophages. Reverse-transcriptase polymerase chain reaction, zymography and reverse zymography for MMP-2, MMP-9, TIMP-1, TIMP-2, and TIMP-3 demonstrated a shift of the proteolytic-antiproteolytic balance upon addition of VSMCs. Transcriptional changes were observed in the adventitia, although seeded VSMCs remained located in the intima. VSMCs exert a paracrine effect on the adventitia that participate in artery wall homeostasis against inflammation and proteolysis. Failure of this protective mechanism results in AAA formation. The understanding of the molecular mechanisms underlying VSMC protective effect may represent a new approach in the treatment of aneurysm and plaque rupture.

The effects of vasodilators on the relaxation of guinea-pig aorta during acute recoil

Background: We have investigated the effects of various vasodilators on smooth muscle relaxation during acute recoil with guinea-pig aorta to find effective therapies to prevent acute recoil at percutaneous transluminal coronary angioplasty (PTCA). Methods: Muscle strips from guinea-pig aorta without endothelium were placed in a bath filled with modified Krebs solution. The strip was isotonically stretched with a tension of 9 mN for 1 min, and then isometric tension was measured until the tension reached a steady state. Various vasodilators were applied during isometric tension measurement. Results: When no drug was applied (control), isometric tension reached a steady state within 20 min. The steady state was approximately 10% lower (more relaxed) than the stretched level. When isosorbide dinitrate (ISDN, 1 mM) was applied during isometric measurement, 18.7±5.3% greater relaxation occurred than in control relaxation ( P<0.05, n=5). When nicorandil (1 mM) was applied, the following relaxation was 18.6±5.7% greater than control ( P<0.05, n=8). The simultaneous application of glibenclamide (1 mM) completely inhibited such additional relaxation by nicorandil. Application of nifedipine (10 μM) caused a 12.9±2.5% greater relaxation than control ( P=0.066, n=4). The first metabolite of sarpogrelate (BP984, 10 μM) caused much greater relaxation than control (23.7±7.9%, P<0.05, n=6). Amlodipine (10 μM), phentolamine (10 μM), and sarpogrelate (10 μM) had no influence on the relaxation of the strip. Conclusions: ISDN, nicorandil and BP984 significantly increase relaxation of the muscle strips after stretching compared to the control. These vasodilators may reduce acute recoil of the smooth muscle after PTCA.

Effect of Hexasulfobutylated C60 on the Isolated Aortic Ring of Guinea Pig

The effects of hexasulfobutylated C₆₀ (FC₄S) and monomalonic acid C₆₀ (MMA C₆₀), the fullerene C₆₀ derivatives, on isolated endothelium-containing or endothelium-denuded aorta of guinea pig were studied pharmacologically in vitro. In the endothelium-containing preparation of the aortic rings, phenylephrine (PHE) elicited contracture and acetylcholine (ACh) elicited a relaxing effect on the PHE-precontracted preparation. In the PHE-precontracted preparation, MMA C₆₀ (10 µmol/l) did not elicit a relaxing effect on the PHE-precontracted preparation. However, MMA C₆₀ (10 µmol/l) significantly reduced the maximum response of the relaxation elicited by ACh. FC₄S itself significantly relaxed the PHE-precontracted aortic rings. ACh-induced relaxation in PHE-precontracted endothelium-containing strips of the aortic rings was significantly potentiated if pretreated with FC₄S (10 µmol/l). In the denuded aortic rings, FC₄S did not elicit the relaxing effect in the PHE-precontracted preparation. The relaxing effect of FC₄S on the PHE-precontracted preparation was not altered when superoxide dismutase (250 units/ml) was pretreated. However, the relaxing effect was reduced when N(G)-nitro-L-arginine methyl ester (L-NAME, 1 mmol/l) or methylene blue (1 µmol/l) was pretreated. These results demonstrated that the vasorelaxation effect of FC₄S on the PHE-precontracted aortic ring is partly dependent on the release of nitric oxide (NO) or an NO-derived substance from the vascular endothelium.

Functional contribution of voltage-dependent and Ca2+ activated K+ (BK(Ca)) channels to the relaxation of guinea-pig aorta in response to natriuretic peptides.

We examined the relaxant effects of natriuretic peptide family on the isolated guinea-pig aorta to determine the receptor subtype which primarily mediates this vascular relaxation, with particular attention to the apparent contribution of voltage-dependent and Ca2+-activated KS (BK(Ca)) channels to the response. Three endogenous natriuretic peptide ligands (natriuretic peptide, ANP; brain natriuretic peptide, BNP; C-type natriuretic peptide, CNP) produced a concentration-dependent relaxation in de-endothelialized guinea-pig aorta pre-contracted by noradrenaline (NA), with a potency order of ANP > or = BNP >> CNP. Although the relaxations elicited by these three natriuretic peptide ligands were significantly diminished by iberiotoxin (IbTx, 10(-7) M), a selective BK(Ca) channel blocker, the inhibitory effect of IbTx was most pronounced for the CNP-induced relaxation; when estimated at 10(-7) M of each peptide, the apparent extent of BK(Ca) channel contribution to the total relaxant response was approximately 60% for CNP > approximately 20% for either ANP or BNP. Supporting the substantial role of BK(Ca) channels in the vascular responses, high-KCl (80 mM) potently suppressed the relaxations induced by these natriuretic peptide ligands. The relaxant response to 8-Bromo-cyclic GMP, a membrane permeable cyclic GMP analogue, was also diminished by IbTx (10(-7) M) and high-KCl (80 mM), which indicates the key role of cyclic GMP in the BK(Ca) channel-mediated, natriuretic peptide-elicited vascular relaxation. These results indicate that the A-type receptor (NPR-A, which is more selective for ANP and BNP) rather than the B-type receptor (NPR-B, which is more selective for CNP) predominates in the guinea-pig aorta as the natriuretic peptide receptor which mediates this vascular smooth muscle relaxation. Although activation of BK(Ca) channels substantially contributes to both NPR-A- and NPR-B-activated relaxations, particularly in the NPR-B-activated relaxation, this K channel may function as a primary relaxant mediator in this conduit artery.

Nucleotide‐mediated relaxation in guinea‐pig aorta: selective inhibition by MRS2179

The vasodilatory effects of nucleotides in the guinea-pig thoracic aorta were examined to determine the relationship between molecular expression and function of P2Y receptors. In aortic rings precontracted with norepinephrine, vasodilatory responses to purine nucleotides exhibited a rank-order of potency of 2-methylthio-ATP>ADP>ATP. Responses to UTP, but not UDP suggested a functional role for P2Y4 but not P2Y6 receptors. Aortic endothelial cells express at least four P2Y receptors; P2Y1, P2Y2, P2Y4 and P2Y6. In primary culture, these cells exhibit desensitizing transient calcium responses characteristic of P2Y1, P2Y2 and P2Y4, but not P2Y6 receptors. UDP had no effect on endothelial cell calcium. The pyrimidinergic receptor agonist UTP is capable of eliciting robust vasodilation in aortic rings and causing calcium responses in cultured guineapig aortic endothelial cells. These responses are equivalent to the maximum responses observed to ATP and ADP. Measurement of intracellular calcium release in response to ATP and 2-methylthio-ATP were similar, however only the 2-methylthio-ATP response was sensitive to the P2Y1 antagonist N(6)-methyl-2'-deoxyadenosine-3',5'-bisphosphate (MRS2179). In aortic rings, vasodilatory responses to 2-methylthio-ATP, ATP and ADP were all blocked by pre-incubation of tissues with MRS2179. MRS2179 pretreatment had no effect of the ability of UTP to cause relaxation of norepinephrine responses in aortic rings or the ability of UTP to cause calcium release in aortic endothelial cells. We demonstrate robust effects of purine and pyrimidine nucleotides in guineapig aorta and provide functional and biochemical evidence that MRS2179 is a selective P2Y1 antagonist.

MaxiK channel-mediated relaxation of guinea-pig aorta following stimulation of IP receptor with beraprost via cyclic AMP-dependent and -independent mechanisms.

The present study was aimed to elucidate the cellular pathway(s) controlling vascular relaxation triggered by stimulation of prostaglandin I2 (PGI2, IP) receptor with a stable PGI2 analog, beraprost. Beraprost caused a concentration-dependent relaxation in de-endothelialized guinea-pig aorta contracted with prostaglandin F2alpha (PGF2alpha). Beraprost-induced relaxation was almost abolished in high-KCl-contracted tissue, indicating a major role of K+ conductances. In contrast to other PGI2 analogs (e.g. cicaprost and iloprost), beraprost-induced relaxation was practically abolished by a selective voltage and Ca2+-activated K+ (MaxiK, BK) channel blocker Iberiotoxin (10(-7) M) or by tetraethylammonium (2 x 10(-3) M). The relaxation induced by beraprost was not significantly affected by other K+ channel blockers glibenclamide (10(-6) M) or Ba2+ (10(-5) M), but was slightly attenuated by 4-aminopyridine (10(-4) M). Beraprost increased intracellular cyclic AMP levels, suggesting a role for cyclic AMP-dependent pathways. A selective inhibitor of cyclic AMP-specific phosphodiesterase, RO-20-1724 (10(-4) M), significantly potentiated beraprost-induced relaxation. Iberiotoxin (10(-7) M) completely counteracted this potentiation. Moreover, tension decrement due to forskolin (3 x 10(-7) M) or 8-bromo-cyclic AMP (10(-2) M) was thoroughly restored by Iberiotoxin (10(-7) M), confirming a role for a cyclic AMP-dependent mechanism. However, SQ 22,536 (10(-4) M), an adenylyl cyclase inhibitor, did not affect beraprost-induced relaxation though it almost totally inhibited the elevation of cyclic AMP contents induced by beraprost, suggesting the existence of an additional mechanism that is cyclic AMP-independent. Moreover, cholera toxin (CTX, 1 microg/ml for 6 h), which activates the stimulatory G protein of adenylyl cyclase (Gs), significantly suppressed PGF2alpha-induced contraction both in the absence and presence of SQ 22,536 (10(-4) M). Iberiotoxin (10(-7) M) was also capable of restoring the relaxation induced by CTX. These findings suggest that MaxiK channel plays a primary role in mediating smooth muscle relaxation following stimulation of IP receptor with beraprost in guinea-pig aorta. Both cyclic AMP-dependent and -independent pathways contribute to the MaxiK channel-mediated relaxation following IP receptor stimulation in this vascular tissue. Direct regulation of MaxiK channels by Gs may partly account for the cyclic AMP-independent relaxant mechanism.

Distinction between relaxations induced via prostanoid EP(4) and IP(1) receptors in pig and rabbit blood vessels.

1. Our study shows that the prostacyclin analogues AFP-07 and cicaprost are moderately potent agonists for prostanoid EP(4) receptors, in addition to being highly potent IP(1) receptor agonists. Both activities were demonstrated on piglet and rabbit saphenous veins, which are established EP(4) preparations. 2. On piglet saphenous vein, PGE(2) was 6.1, 24, 96, 138, 168 and 285 times respectively more potent than AFP-07, cicaprost, PGI(2), iloprost, carbacyclin and TEI-9063 in causing relaxation. Another prostacyclin analogue taprostene did not induce maximum relaxation (21 - 74%), and did not oppose the action of PGE(2). The EP(4) receptor antagonist AH 23848 (30 microM) blocked relaxant responses to PGE(2) (dose ratio=8.6+/-1.3, s.e.mean) to a greater extent than cicaprost (4.9+/-0.7) and AFP-07 (3.8+/-0.8), had variable effects on TEI-9063-induced relaxation (3.7+/-1.5), and had no effect on taprostene responses (<2.0). 3. On rabbit saphenous vein, AH 23848 blocked the relaxant actions of PGE(2), AFP-07, cicaprost, iloprost and carbacyclin to similar extents. 4. AFP-07, cicaprost and TEI-9063 showed high IP(1) relaxant potency on piglet carotid artery, rabbit mesenteric artery and guinea-pig aorta, with AFP-07 confirmed as the most potent IP(1) agonist reported to date. AH 23848 did not block cicaprost-induced relaxation of piglet carotid artery. EP(3) contractile systems in these preparations can confound IP(1) agonist potency estimations. 5. Caution is urged when using AFP-07 and cicaprost to characterize IP(1) receptors in the presence of EP(4) receptors. Taprostene may be a lead to a highly selective IP(1) receptor agonist.

Intracellular alkalinization augments α 1-adrenoceptor-mediated vasoconstriction by promotion of Ca 2+ entry through the non-L-type Ca 2+ channels

Modulation by intracellular pH of the vasoconstriction induced by α-adrenoceptor agonists was investigated in isolated guinea pig aorta. NH 4Cl (15 mM) increased intracellular pH of aortic smooth muscle cells by about 0.2 pH unit and significantly augmented KCl-induced contraction of aortic strips, whereas simultaneous administration of NH 4Cl (15 mM) plus Na + propionate (30 mM) failed to affect intracellular pH or contractility. NH 4Cl (15 mM) potentiated contractions induced by α-adrenoceptor agonists, norepinephrine, phenylephrine and clonidine. Contraction induced by α 1-selective adrenoceptor agonist, phenylephrine, but not that induced by norepinephrine or clonidine, was insensitive to inhibition by verapamil (1 μM). Phenylephrine-induced contraction was not affected by NH 4Cl in Ca 2+-free medium whereas extracellular Ca 2+-induced contraction of phenylephrine-stimulated aorta was significantly augmented by NH 4Cl. Consistently, 45Ca 2+uptake into phenylephrine (1 μM)-stimulated aortic strips was increased by incubation with NH 4Cl. The potentiating effects of NH 4Cl on both phenylephrine-induced Ca 2+ entry and contraction were antagonized by Na + propionate. These results suggest that intracellular alkalinization facilitates α 1-adrenoceptor-mediated vasoconstriction by facilitation of an agonist-induced Ca 2+ entry pathway that is independent of L-type Ca 2+ channels.

Lowering of lipid composition in aorta of guinea pigs by Curcuma domestica.

BackgroundA short-term study was carried out using guinea pigs to determine the effects of Curcuma domestica on lipid composition in the serum and aorta.MethodsAnimals were given food pellets containing 4% (w/w) powdered rhizome of C. domestica in order to determine its effect on cholesterol, triglyceride and phospholipid levels in the aorta and serum. The animals were fed either a cholesterol free diet or a high cholesterol diet (2% cholesterol, w/w, in food pellet) in order to induce hypercholesterolemia.. After five weeks of this diet treatment, blood and aorta were taken for biochemical analysis and histological studies.ResultsC. domestica in the diet showed no significant effect on the levels of cholesterol, triglyceride and phospholipid in the serum and aorta of the cholesterol free diet animals. However, addition of C. domestica to a high cholesterol diet counteracted increases in the levels of cholesterol, triglyceride and phospholipid in the aorta. Histology studies showed less cholesterol deposits in the aorta of high cholesterol diet animals given C. domestica compared to the high cholesterol diet animals not given C. domestica supplement. C. domestica also had a lowering effect on triglyceride level in the serum of high cholesterol diet animals but showed no effect on serum cholesterol and phospholipid levels.ConclusionThis study has shown that dietary intake of C. domestica decreased all lipid composition levels in the aorta and also the serum triglyceride level. In addition, C. domestica also reduced cholesterol deposition in the aorta of high cholesterol diet animals.

In vitro and in vivo pharmacology of olmesartan medoxomil, an angiotensin II type AT1 receptor antagonist.

Olmesartan medoxomil is a new non-peptide angiotensin (A) II antagonist under development for treating hypertension. It is a pro-drug containing an ester moiety that, after oral administration, is rapidly cleaved to release the active form olmesartan (RNH-6270). In vitro, olmesartan is a highly potent, competitive and selective All AT1 receptor antagonist with almost no antagonistic activity on AT2 and AT4 receptors. Olmesartan produces selective insurmountable inhibition of All-induced contractions of the guinea-pig aorta and is much more potent than losartan in reducing maximal responses. In vivo, intravenous olmesartan produces a rapid and long-lasting inhibition of All-induced pressor responses in conscious rats. Oral olmesartan medoxomil also inhibits All-pressor response but onset of the action is slower compared with intravenous administration. Following oral administration, olmesartan has a faster onset but similar potency when compared with candesartan cilexetil, and clearly exceeds losartan in both respects. Oral olmesartan medoxomil exhibits dose-dependent antihypertensive effects in several rat and dog models, with the most marked effects seen in high plasma renin models, when compared with normal or low renin types. Haemodynamic studies in spontaneously hypertensive rats and normotensive dogs showed intravenous olmesartan selectively reduces renal vascular resistance, which suggests that vasodilatation in the renal vascular bed contributes most to the antihypertensive action of the drug. Long-term treatment with olmesartan medoxomil exhibits, beside antihypertensive effects, beneficial effects in animal models of various types of nephrosis and heart failure, and anti-atherogenic effects in hyperlipidaemic animals. Olmesartan medoxomil is worthy of clinical development in essential and renal hypertension, particularly where renal function is threatened by underlying diabetic disease.

Capillary filtration is reduced in lungs adapted to chronic heart failure: morphological and haemodynamic correlates.

To determine pulmonary capillary filtration in experimental chronic heart failure and to investigate some morphological and haemodynamic mechanisms that could account for reduced filtration in lungs adapted to chronic heart failure. We studied pulmonary capillary filtration, vascular resistances and morphology in lungs from guinea-pigs adapted to chronic heart failure. Heart failure was induced by banding of the ascending aorta (n=66) or sham control operation (n=78) in guinea-pigs which were studied at 150+/-8 days post-operation. Reduced cardiac output, increased systemic vascular resistance and LV end diastolic pressure and increased LV and RV weight:body weight ratio (all P<0.05) indicated chronic heart failure at 5 months following aortic banding in guinea-pigs. Lung weight was increased (61%, P<0.05) in heart failure compared with controls, but lung water content was reduced (5.5%, P<0.05), a reversal of the pattern seen acutely. Studies in isolated perfused lungs demonstrated a reduced capillary filtration coefficient (0. 018+/-0.003 vs. 0.003+/-0.002 ml min(-1)mmHg(-1)g(-1), P<0.001), increased arterial (61%) and venous resistance (50%) in heart failure lungs, P<0.05. Wall thickness:lumen ratio was increased in small (<250 microm) pulmonary arterioles (0.15+/-0.02 vs. 0.08+/-0. 01) and venules (0.06+/-0.005 vs. 0.04+/-0.002) in heart failure, P<0.01. Alveolar septal volume fractions (35.2+/-5.1 vs. 23.1+/-2.7) and septal:air-space volume ratios (60.5+/-13.6 vs. 31.9+/-5.3) were also increased in heart failure, P<0.05. Pulmonary adaptation to chronic heart failure is associated with vascular and alveolar remodelling that contributes to increased vascular resistance and reduced capillary filtration. These changes are likely to be important in mediating resistance to pulmonary oedema in chronic heart failure.

Tyrosine phosphorylation increases Ca 2+ sensitivity of vascular smooth muscle contraction

In order to elucidate the role of tyrosine phosphorylation in vasoconstriction, we investigated the effects of inhibitors of tyrosine kinase (genistein, 30 μM) and phosphatase (sodium o-vanadate, 5 μM) on the contraction of aorta isolated from guinea pig. Genistein significantly inhibited norepinephrine-induced contraction, but it did not affect that induced by KCl. Thus, tyrosine phosphorylation may not be involved in the contractile response to KCl alone. The aortic contraction elicited by KCl was significantly augmented by sodium o-vanadate, which increased both the maximum force and pD 2 values of KCl contraction. In the presence of verapamil, KCl-induced contraction was abolished even after pretreatment with sodium o-vanadate. Sodium o-vanadate also augmented Ca 2+-induced contraction in the aortic strips depolarized with KCl, increasing both its maximum force and pD 2 values. Neither basal 45Ca 2+ uptake nor verapamil-sensitive 45Ca 2+ uptake induced by KCl were affected by pretreatment with sodium o-vanadate. These results suggest that tyrosine phosphorylation is involved in the contraction of guinea-pig aorta not through transplasmalemmal Ca 2+ entry but through increased Ca 2+ sensitivity of the intracellular contractile pathway.

Ascorbic acid and glutathione modulate the biological activity of S-nitrosoglutathione.

Ascorbic acid and glutathione (GSH) are important determinants of the intracellular redox state, and both are known to accelerate the decomposition of S-nitrosoglutathione (GSNO), an endogenous adduct of nitric oxide (NO). The implications of these observations for GSNO bioactivity are not yet clear. We investigated the effect of ascorbic acid and GSH on GSNO bioactivity by using a bioassay with isolated segments of guinea pig aorta suspended in organ chambers. Arterial segments demonstrated relaxation to GSNO (0.1 micromol/L) that was significantly enhanced by 300 micromol/L ascorbic acid (71+/-6% versus 53+/-6%, P<0.05) but not GSH. Both ascorbic acid and GSH significantly shortened the duration of arterial relaxation in response to 0.1 micromol/L GSNO (from >120 minutes to 22.5+/-3.5 and 36.3+/-4.3 minutes, respectively; P<0.05), consistent with accelerated decomposition of GSNO that was confirmed spectrophotometrically. The effect of ascorbic acid was abrogated by either DTPA or the copper(I)-specific agent bathocuproine but not deferoxamine, indicating a dependence on the availability of redox-active copper. Consistent with this notion, the action of ascorbic acid on GSNO bioactivity was also supported by copper-zinc superoxide dismutase, a physiologically relevant source of copper. In contrast, the effect of GSH on GSNO degradation and GSNO-mediated arterial relaxation was independent of transition metal ions, because DTPA had no effect. These data indicate that both ascorbic acid and GSH modulate GSNO bioactivity and suggest a distinction between the mechanism of GSNO degradation by ascorbic acid or GSH. Whereas both ascorbic acid and GSH accelerate the degradation of GSNO, only ascorbic acid is dependent on the presence of transition metal ions.

Chronic Hypoxia Diminishes Pregnancy-associated DNA Synthesis in Guinea Pig Uteroplacental Arteries

Enlargement of the uterine artery (UA) during pregnancy is diminished in women residing at a high altitude. We asked whether chronic hypoxia alters the rise in DNA synthesis in uteroplacental vessels and, if so, whether the reduction is related to the intrauterine growth retardation (IUGR) observed under conditions of chronic hypoxia. We used bromodeoxyuridine (BrdU) labelling to measure DNA synthesis in all vascular layers of the UA, mesometrial arteries (MA), thoracic aorta and mesenteric artery of guinea pigs, residing throughout pregnancy at a low (1600m) or high (3962m) altitude. Pregnancy increased DNA synthesis throughout the UA at both altitudes, yet the maximal value was less at high than low altitude (P< 0.05). Likewise, pregnancy increased DNA synthesis throughout the MA, yet at high altitude pregnancy elevated levels returned to non-pregnant values after 42 days of gestation, whereas at low altitude DNA synthesis continued to be elevated until near term. Fetal weights were lower (P=0.01) and placental/fetal weight ratios tended to be greater in high than low altitude, near term pups (P=0.09). We conclude that a diminished growth response by the uteroplacental vasculature to pregnancy may contribute to the previously reported reduced uterine artery blood flow and resulting IUGR at high altitude.

Lateral zone of cell-cell adhesion as the major fluid shear stress-related signal transduction site.

It has been proposed previously that actin filaments and cell adhesion sites are involved in mechanosignal transduction. In this study, we present certain morphological evidence that supports this hypothesis. The 3D disposition of actin filaments and phosphotyrosine-containing proteins in endothelial cells in situ was analyzed by using confocal microscopy and image reconstruction techniques. Surgical coarctations were made in guinea pig aortas, and the same 3D studies were conducted on such areas 1 week later. Stress fibers (SFs) were present at both basal and apical regions of endothelial cells regardless of coarctation, and several phosphotyrosine-containing proteins were associated with SF ends. Apical SFs had one end attached to the apical cell membrane and the other attached to either the basal membrane or the lateral cell border. Within the coarctation area, the actin filament-containing and vinculin-containing structures became prominent, especially at the apical and the lateral regions. Substantially higher levels of anti-phosphotyrosine and anti-Src staining were detected in the constricted area, particularly at the cell-cell apposition, whereas the anti-focal adhesion kinase, anti-CT10-related kinase, anti-platelet endothelial cell adhesion molecule-l, anti-vinculin, and phalloidin staining intensities increased only slightly after coarctation. We propose that apical SFs directly transmit the mechanical force of flow from the cell apex to the lateral and/or basal SF anchoring sites and that the SF ends associated with signaling molecules are sites of signal transduction. Our results support the idea that the cell apposition area is the major fluid shear stress-dependent mechanosignal transduction site in endothelial cells.

NO-mediated MaxiK Ca channel activation produces relaxation of guinea pig aorta independently of voltage-dependent L-type Ca 2+ channels

(1) The role of L-type Ca 2+ channels in the relaxation to nitric oxide (NO)-mediated MaxiK Ca channel activation was examined in guinea pig aorta. (2) Acetylcholine (ACh) produced an endothelium-dependent relaxation of guinea pig aorta precontracted with noradrenaline (NA), which was abolished by an NO synthase inhibitor, N G-nitro- l-arginine ( l-NNA). (3) Both endothelium-dependent relaxation by ACh and endothelium-independent relaxation by an NO donor, (±)-( E)-ethyl-2-[( E)-hydroxyimino]-5-nitro-3-hexeneamide (NOR3), were strongly suppressed by a soluble guanylate cyclase (sGC) inhibitor, 1 H-[1,2,4]-oxadiazolo-[4,3- a]-quinoxalin-1-one (ODQ), suggesting that increased intracellular cGMP plays the key role in both responses. (4) ACh- and NOR3-induced relaxations were significantly suppressed by iberiotoxin (IbTX), a selective blocker of MaxiK Ca channels. (5) ACh- and NOR3-induced relaxations were greatly attenuated when arteries were precontracted with high KCl instead of NA, supporting the idea that K + channel activation mediates the relaxant responses. (6) NOR3-induced relaxations were not affected by a L-type Ca 2+ channel blocker, diltiazem. Furthermore, endothelium-independent relaxation by a K ATP channel opener, (+)-7,8-dihydro-6,6-dimethyl-7-hyroxy-8-(2-oxo-1-piperidinyl)-6 H-pyrano[2,3- f]benz-2,1,3-oxadiazole (NIP-121) was not affected by diltiazem and nicardipine. (7) These findings suggest that blockade of L-type Ca 2+ channels is not a major mechanism responsible for the vascular relaxation due to NO-mediated MaxiK Ca channel activation in guinea pig aorta.