Vascular Smooth Muscle Contractility Research Articles

Adaptation of Pharmacomechanical Coupling of Vascular Smooth Muscle to Chronic Hypoxia

Hypoxia is one of the most common stresses that affect an organism's homeostasis. Although much is known of the mechanisms of the cellular and biochemical responses to acute hypoxia, relatively little is known of the mechanisms of the responses to prolonged or chronic hypoxia. Chronic hypoxia suppresses vascular smooth muscle contractility in many vascular beds. While the endothelium is likely to play a role, part of the mechanisms underlying chronic hypoxic-induced changes in vascular responses resides in the changes in receptor-mediated excitation-contraction coupling and/or signal transduction in the vascular smooth muscle. Recent studies have demonstrated that chronic hypoxia attenuates both receptor-second messenger and second messenger-contraction coupling efficiencies in the vascular smooth muscle. This suppression of pharmacomechanical coupling is likely to represent one of the adaptive mechanisms of vascular smooth muscle and to play an important role in an adjustment of vascular tone and blood flow under the stress of moderate chronic hypoxia.

Fibroblast growth factor 2 control of vascular tone.

Vascular tone control is essential in blood pressure regulation, shock, ischemia-reperfusion, inflammation, vessel injury/repair, wound healing, temperature regulation, digestion, exercise physiology, and metabolism. Here we show that a well-known growth factor, FGF2, long thought to be involved in many developmental and homeostatic processes, including growth of the tissue layers of vessel walls, functions in vascular tone control. Fgf2 knockout mice are morphologically normal and display decreased vascular smooth muscle contractility, low blood pressure and thrombocytosis. Following intra-arterial mechanical injury, FGF2-deficient vessels undergo a normal hyperplastic response. These results force us to reconsider the function of FGF2 in vascular development and homeostasis in terms of vascular tone control.

AE anion exchanger mRNA and protein expression in vascular smooth muscle cells, aorta, and renal microvessels.

Intracellular pH (pHi) is an important regulator of vascular smooth muscle cell (VSMC) tone, contractility, and intracellular Ca2+ concentration. Among the multiple transport processes that regulate VSMC pHi, Na(+)-independent Cl-/HCO3- exchange is the major process that acidifies VSMCs in response to an alkaline load. Here, we characterize, in native and cultured VSMCs, the expression of the AE family of band 3-related anion exchangers, the best studied of these Cl-/HCO3- exchangers. A 4.2-kb AE2 mRNA was present in aorta and in all cultured VSMCs tested. Cultured VSMCs and aorta both expressed a approximately 165-kDa AE2 polypeptide, but a approximately 115-kDa polypeptide was the major AE2-related protein in aorta. AE3 mRNA levels in VSMCs and in arterial tissue were significantly lower than those for AE2, but AE3 or related polypeptides were readily detected by immunoblot and immunolocalization experiments. The approximately 125-kDa AE3 polypeptide was present in an immortalized aortic VSMC line, but the predominant AE3 epitope in aorta and most cultured cells was associated with a polypeptide of M(r) approximately 80 kDa. These data demonstrate the expression in native arteries and in VSMCs of products of the AE2 and AE3 genes, which may contribute to Na(+)-independent Cl-/HCO3- exchange activity in these tissues and cells.

Carbon Monoxide Controls the Proliferation of Hypoxic Vascular Smooth Muscle Cells

Excess vascular smooth muscle cell (VSMC) proliferation and contractility are key events in the pathophysiology of vascular disorders induced by hypoxia. We have recently reported that carbon monoxide (CO), produced by VSMC under conditions of hypoxia, can be a modulator of cGMP levels in both endothelial and smooth muscle cells. In this respect, some of the physiologic effects of CO in the vasculature parallel those of nitric oxide (NO), a well characterized regulator of vascular tone. We report here that under hypoxia, VSMC-derived CO is an important regulator of VSMC proliferation. Inhibiting CO formation or scavenging CO with hemoglobin increased VSMC proliferation in response to serum or to mitogens such as endothelin, whereas increasing CO production or exposing cells to exogenous CO lead to a markedly attenuated growth response. The effects of CO on VSMC proliferation correlated with changes in E2F-1 expression, the prototype member of a family of transcription factors that participate in the control of cell cycle progression. CO significantly suppressed E2F-1 expression, whereas, removal of CO from the cultures with hemoglobin lead to increased E2F-1 gene transcription, mRNA, and protein production as well as mRNA levels of c-myc, a target gene of E2F-1. Moreover, the actions of CO were mediated by the second messenger molecule, cGMP. Limiting VSMC growth by increasing the release of CO may represent a key event in the body's compensatory responses to hypoxia.

Effects of 4-Methyl-2-aminopyridine on [ 3H]-Noradrenaline Overflow and Contractility of Isolated Rabbit Arteries

1. The effects of 4-methyl-2-aminopyridine (4M2AP) and 4-aminopyridine (4AP) on spontaneous and evoked [ 3H]-noradrenaline overflow were compared in rabbit ear artery strips. The effects of 4M2AP on smooth muscle contractility were also investigated in isolated perfused ear arteries. 2. Both 4M2AP and 4AP enhanced spontaneous [ 3H] overflow from arterial strips in a concentration-dependent manner (10–1000 μM). A bell-shaped dose-response relation was obtained for evoked [ 3H] overflow over the same concentration range, with maximum effects occurring at 10 μM for 4M2AP (163±31% increase) and 100 μM for 4AP (154±16% increase). 3. 4M2AP did not significantly affect evoked tension in the 1- to 100-μM range but clearly depressed it at 1,000 μM (by 65±11%). In contrast, 4AP enhanced evoked tension in the 10- to 100- μM range (by 30–50%). 4. 4M2AP (10–100 μM) enhanced vasoconstrictor responses to exogenous noradrenaline injections in isolated perfused rabbit ear arteries, whereas higher concentrations (1,000 μM) caused significant depression. 5. 4M2AP (1,000 μM) markedly potentiated vasoconstrictor responses induced by perfusion with a high extracellular K + solution. When 4M2AP was present during the reloading of noradrenaline-sensitive Ca 2+ stores, it enhanced the subsequent vasoconstrictor responses to noradrenaline obtained in a Ca 2+-free medium. 6. The results show that 4M2AP, like 4AP, enhances [ 3H] overflow from sympathetic nerve terminals and has complex effects on vascular smooth muscle contractility, indicating the ability of these compounds to affect the Ca 2+ permeability of both extracellular and intracellular membrane systems.

Interleukin-1β-induced, nitric oxide-dependent and -independent inhibition of vascular smooth muscle contraction

Stimulation of vascular smooth muscle by bacterial lipopolysaccharide has been shown to produce interleukin-1β and to induce vasodilation in septic shock. To understand the mechanisms of interleukin-1β-induced relaxation, we examined the effects of interleukin-1β on contractility and cyclic GMP contents of vascular smooth muscle. After treatment of the rat aorta with interleukin-1β (20 ng/ml) for 6 h, the cyclic GMP content increased and the contraction induced by phenylephrine (1 μM) was partially inhibited. An inhibitor of nitric oxide (NO) synthase, N G-monomethyl- l-arginine ( l-NMMA, 100 μM), prevented the inhibitory effect of interleukin-1β. After treatment with interleukin-1β for 24 h, the phenylephrine-induced contraction was inhibited more strongly. Neither l-NMMA (100 μM) nor aminoguanidine (100 μM) reversed the inhibition, whereas methylene blue (10 μM) partially reversed the inhibition. After treatment with interleukin-1β for 12 or 24 h, the cyclic GMP content increased but to a level lower than that obtained with a 6-h treatment. The effects of sodium nitroprusside (1 μM) to inhibit the phenylephrine-induced contraction and to increase the cyclic GMP content were markedly suppressed by the 24-h interleukin-1β treatment. In contrast, the 24-h interleukin-1β treatment did not change the ability of 8-bromo-cGMP to relax the phenylephrine-stimulated aorta. Addition of l-NMMA (1 mM) during the 24 h treatment prevented NO production and preserved the sodium nitroprusside-induced cGMP generation by interleukin-1β. The 24 h interleukin-1β treatment increased the threshold concentration of KCl needed to induce contraction without changing the maximum contraction. In the presence of 25.4 mM KCl or the non-selective K + channel inhibitor, tetraethylammonium, the inhibitory effect of the 24-h interleukin-1β treatment on phenylephrine-induced contraction was restored. These results suggest that interleukin-1β inhibits vascular smooth muscle contraction by a time-dependent, dual mechanism. After a 6-h treatment with interleukin-1β, the NO/cyclic GMP system is activated. After a 24-h interleukin-1β treatment, in contrast, the NO/cyclic GMP system may be desensitized and the contraction of vascular smooth muscle is inhibited by another mechanism, possibly membrane hyperpolarization.

Cerebral cortex blood flow and vascular smooth muscle contractility in a rat model of ischemia: a correlative laser Doppler flowmetric and scanning electron microscopic study.

The present study was undertaken to ascertain the role of smooth muscles and pericytes in the microcirculation during hyperperfusion and hypoperfusion following ischemia in rats. Paired external carotids, the pterygopalatine branch of the internal carotids and the basilar artery were exposed and divided. Reversible inflatable occluders were placed around the common carotids. After 24 h, the unanesthetized rat underwent 10-min ischemia by inflating the occluders. Continuous cortical cerebral blood flow (c-CBF) was monitored by laser Doppler flowmetry. The measured c-CBF was below 20% of control (P < 0.001) during ischemia. A c-CBF of 227.5 +/- 54.1% (P < 0.001) was obtained during reperfusion hyperemia. A c-CBF of 59.7 +/- 8.8% (P < 0.001) occurred at the nadir of postischemic hypoperfusion, and this was followed by a second hyperemia. The cytoarchitecture of the vascular smooth muscles and pericytes was assessed by scanning electron microscopy. Samples were prepared using a KOH-collagenase digestion method. In control rats, arteriolar muscle cells showed smooth surfaces. Capillary pericytes were closely apposed to the endothelium. Immediately after reperfusion, transverse membrane creases were observed on the smooth muscle surfaces. During maximal hyperemia the creases disappeared. When c-CBF started to decrease the creases became visible again. Throughout the postischemic hypoperfusion the creases remained. Capillary endothelial walls became tortuous in the late phase of hypoperfusion. During the second hyperemia most arteriolar muscle cells showed smooth surfaces. Some pericytes appeared to have migrated from the vascular wall. The morphological changes of smooth muscle membranes suggest that they are related to specific perfusional disturbances during ischemia and reperfusion.

Targeted ablation of the phospholamban gene is associated with a marked decrease in sensitivity in aortic smooth muscle.

Phospholamban (PLB) is a protein associated with the Ca(2+)-ATPase of the sarcoplasmic reticulum (SR) in cardiac, slow-twitch skeletal, and smooth muscle. PLB inhibits the SR Ca(2+)-ATPase in cardiac muscle; this inhibition is relieved on phosphorylation. The role of PLB in smooth muscle contractility is less clear. To elucidate the role of PLB in vascular smooth muscle contractility in vivo, we used a model in which the PLB gene was targeted in murine embryonic stem cells, generating mice deficient in PLB (PLB-). The PLB- mice exhibited no gross developmental abnormalities, but marked changes in aortic contractility were observed. The time course of force development with phenylephrine stimulation was faster in the PLB- aorta, suggesting changes in SR Ca2+ release. No differences were observed for KCl contractures between tissue types for either maximum forces observed or time course of force production; relaxation was faster in 7 of 11 arteries, but this trend did not attain statistical significance. The cumulative concentration-isometric force relations for the PLB- aorta were to the right of the wild-type for both KCl and phenylephrine stimulation, indicating a less sensitive tissue. To investigate whether the observed changes were related to SR function, we inhibited the SR Ca(2+)-ATPase with cyclopiazonic acid (CPA). CPA treatment resulted in a leftward shift of the concentration-isometric force relations for both aorta types, as expected after removal of a major Ca2+ uptake system. Most interestingly, the differences between PLB and wild-type aorta were abolished by SR inhibition. Our results suggest that PLB is a regulator of the SR Ca2+ pump in mouse aorta and plays a regulatory role in both KCl-induced and receptor-mediated contractility in vascular smooth muscle.

Mesenteric Artery Vasoconstriction During Hypoxia is Mediated by a Loss of Basal Nitric Oxide (NO) Production † 983

The newborn intestinal circulation is unique in that its intrinsic response to hypoxemia is pronounced vasoconstriction. One mechanism for this unique response might be an acute reduction in endothelial cell NO production, inasmuch as newborn gut vascular tone is highly contingent on the NO/cGMP axis(Am J Physiol 268:G949, '95). To test this hypothesis, mesenteric artery rings from perinatal swine were suspended in buffer-filled myographs and tension recorded continuously; buffer hypoxia (pO2 <10 mmHg) was induced in phenylephrine (PE) precontracted rings by aeration with 95% N2 - 5% CO2 The initial response to hypoxia was brief relaxation(<15sec; -16±2% in both 3- and 35d rings), which was absent in endothelium-denuded rings (E-), but still present following pretreatment with LNMMA (blocks NO production) or indomethacin. Precontraction by depolarization(40 mM KCl) in place of PE significantly blunted this early dilation, suggesting its mediation by an endothelium-derived hyperpolarizing factor. The second, and principal response to hypoxia was pronounced constriction (to 36±4% of maximal KCl-induced constriction in rings from 3d, but only 12±3% in rings from 35d old subjects, p<0.01). Hypoxic vasoconstriction (HVC) was sustained until ATP depletion impaired the contractility of vascular smooth muscle. HVC was absent in E- rings and eliminated by LNMMA, but not by indomethacin, phenidone (lipoxygenase inhibitor), or BQ610 (endothelin ETA receptor antagonist). Addition of superoxide dismutase significantly enhanced HVC (O2- inactivates NO; O2- removal would thus accentuate NO-based responses). Impairment of ATP production with 2,4-dinitrophenol in the presence of adequate O2 did not induce HVC; thus, HVC was initiated by reduced pO2 and not by hypoxia-induced ATP depletion, consistent with the known requirement for molecular O2 in the oxidation of L-arg to L-cit+NO. Speculation: These data support the pivotal role of endothelial NO production in setting basal vascular tone in newborn intestine. Factors which attenuate NO production, such as hypoxia, should thus exert a particularly deleterious effect on newborn gut perfusion. This fact may explain, in part, the unique susceptibility of the newborn intestinal circulation to profound ischemia and the development of necrotizing enterocolitis.

Polyubiquitin is a new phenotypic marker of contractile vascular smooth muscle cells.

Medial vascular smooth muscle cells (VSMCs) in healthy vessels are phenotypically distinct from their intimal counterparts in vascular disease. To compare the genes expressed in these phenotypes we have previously performed a differential cDNA library screen on cultured rat VSMCs. The aim of this study was to identify and characterise a 2.8 kb transcript, 2E10, which was highly expressed in freshly dispersed rat aortic VSMCs and downregulated in multiply passaged cultured VSMCs. Sequence analysis was used to identify the 2.8 kb rat cDNA. After trypsinisation of proliferating cultured rat and human VSMCs, or enzymatic digestion of aortic tunica media, total cytoplasmic RNA was isolated from VSMCs by lysis in Nonidet P-40 and extraction in phenol; 15 micrograms of total cytoplasmic RNA was used in Northern blot analysis with a 32P-[dCTP]-labelled 2E10 cDNA probe. 35S-[dATP]-labelled 2E10 riboprobe was hybridised in situ to frozen sections of normal and diseased human coronary arteries. DNA sequencing identified 2E10 as a rat polyubiquitin which is homologous to the human polyubiquitin, UbC. Northern blot analysis showed that this polyubiquitin was more highly expressed in differentiated, freshly dispersed rat and human aortic VSMCs compared with their dedifferentiated proliferating counterparts. This also identified a 3.2 kb transcript cross-reacting with the polyubiquitin probe which is specific to differentiated rat VSMCs only. However, expression in growth arrested and proliferating VSMCs was identical, suggesting that UbC does not have a role in VSMC growth arrest. In situ hybridisation of the polyubiquitin riboprobe to sections of diseased human coronary arteries indicated much higher expression in medial than in intimal VSMCs. Northern blot analysis of RNA from the developing rat aorta showed that polyubiquitin expression increased substantially after week 2 of neonatal life, coincident with expression of VSMC-specific contractile proteins. The greater expression of a UbC polyubiquitin transcript in contractile, differentiated VSMCs compared with proliferating, synthetic VSMCs provides a new gene marker for the phenotypic characterisation of VSMCs in vivo. This, and the finding that the developmental induction of expression of polyubiquitin (UbC) mirrors that of VSMC contractile proteins, suggests that ubiquitin, a protein known to associate with and degrade contractile proteins in skeletal muscle, is involved in the function or maintenance of the contractile phenotype of VSMCs.

ACE inhibition, endothelial function and coronary artery lesions. Role of kinins and nitric oxide.

In healthy coronary arteries, the endothelium plays an important role in the regulation of vascular smooth muscle growth and contractility. Furthermore, the endothelium inhibits overt platelet aggregation and prevents the adhesion of white blood cells to, and their infiltration into, the vascular wall. Among the mediators of these functions of endothelial cells, nitric oxide (NO) plays a central role. Moreover, the presence of local kinin-generating enzymatic systems associated with endothelial cells, vascular smooth muscle, platelets, neutrophils and monocytes suggests that bradykinin stimulates endothelial cells to release NO locally. The activation of endothelial cells by bradykinin is inhibited by kininase II, best known as angiotensin converting enzyme (ACE). Hence, ACE inhibitors, in addition to reducing the levels of angiotensin II (a potent stimulus to vascular smooth muscle growth and contraction), cause an amplification of the release of NO and other endothelial mediators that is induced by bradykinin. Independent risk factors for coronary artery disease such as hypertension, diabetes and hypercholesterolaemia reduce the NO-dependent regulation of vascular smooth muscle contractility and growth in otherwise normal coronary arteries. This endothelial dysfunction probably also affects the inhibitory role of NO with regard to platelet aggregation and monocyte infiltration into the vascular wall. In atherosclerotic vessels, the role of NO is severely reduced. In animal models, as well as in patients with coronary artery disease, endothelial dysfunction is improved by treatment with ACE inhibitors. Although in humans the mechanism of the restoration of endothelial function is not known, in animals endogenous kinins and NO are involved. However, it is clear that this process is multifactorial, and thus probably involves both the prevention of the deleterious actions of angiotensin II and the potentiation of bradykinin.

Changes in plasma membrane ionic permeability and related contractile responses in vascular smooth muscle at hypoxia

Present study was designed to examine the effects of low P O 2 (9.1-3.9 kPa) on Ca 2+ and K + permeability in plasma membrane and related contractile responses of vascular smooth muscle (VSM). Recordings of VSM contractile force were done by the force displacement transducer coupled to a polygraph. Cellular 45 Ca uptake was determined by the lanthanum method. Ionic current measurements were made using patch-clamp technique (whole cell configuration). It was shown that the gradual decrease in bath P O 2 below 14.3-13 kPa as a rule resulted in the inhibition of VSM myogenic activity (portal vein) and the relaxation of VSM preconstricted with noradrenaline and high-K + solution or transmural electrical stimulation (aorta and portal vein). In muscles from aorta, hypoxia often led to biphasic response: a pronounced dilation following phasic (transient) contraction; in portal vein hypoxia induced the dilation only. The constrictor component of aorta response to hypoxia was endothelium-dependent, but dilations in both cases were independent of intact endothelium. The similar hypoxia-like effects in VSM were produced by a Ca 2+ -free solution and Ca 2+ channel blockers. 45 Ca uptake was significantly decreased in muscles from portal vein at hypoxia. The lowering of bath P O 2 led to decrease of peak amplitude of Ca 2+ current ( I Ca ) in both kinds of VSM cells. Hypoxia also increased the transient [ K(Ca)t ] and oscillatory [ I K(Ca)o ] components of Ca 2+ -activated K + current [ I K(Ca) ] and was able to increase the amplitude and frequency of spontaneous transient outward currents (STOCs) in freshly dispersed single cells from portal vein. Intracellular ‘injection’ of phosphocreatine (PCr), but not adenosine triphosphate (ATP), to VSM cell myoplasm using positively charged liposomes prevented the inhibitor effects of low P O 2 on VSM contractile activity. The effects of hypoxia were completely reversible as evident from contractile and electrophysiological measurements. Thus, low P O 2 (hypoxia) may affect Ca 2+ permeability and hence the VSM contractility for at least two mechanisms: (i) by the decrease of a number of open Ca 2+ channels as a result of the damage in their phosphorylation; (ii) by Ca 2+ channel inactivation resultant from a rise in intracellular Ca 2+ which could be induced by a failure of active Ca 2+ outward transport or/and intracellular Ca 2+ sequestration.

Insulin and hypertension: a causal relationship?

Although considerable evidence lends credence to the association between insulin and hypertension, the precise nature of this link remains elusive. Recent observations indicating that insulin may modulate vascular smooth muscle contractility has given yet another interesting twist to this intriguing association. The finding that insulin can directly alter smooth muscle calcium transients as well as attenuate the effect of other vasoconstrictor amines suggests that this metabolic hormone may also play an important hemodynamic role under pathophysiologic conditions. However, the independent contribution of insulin resistance toward an increase in BP is probably smaller and more complex than is often emphasized and to assume that insulin is directly linked to a rise in BP in hypertensive subjects is perhaps oversimplistic and incorrect. Future research efforts should be targeted at examining the effects of insulin on vascular smooth muscle contractility in hypertensive subjects and the interaction of insulin with other vasoactive peptides.

Serotonin stimulates protein tyrosyl phosphorylation and vascular contraction via tyrosine kinase.

Serotonin (5-HT, 5-hydroxytryptamine) is a mitogen in vascular smooth muscle and vascular reactivity to 5-HT is significantly enhanced in hypertension and atherosclerosis. We have tested the hypothesis that tyrosine kinases, enzymes important for mitogenesis, may play a role in 5-HT-induced vascular smooth muscle contractility. Helical strips of rat carotid artery and aorta denuded of endothelium were mounted in tissue baths for measurement of contractile force. The tyrosine kinase inhibitor genistein (5 x 10(-6) M) decreased the potency of 5-HT approximately 4-fold and reduced maximal contraction to 5-HT in carotid arterial strips denuded of endothelium (58% control). Genistein's inactive congener daidzein (5 x 10(-6) M) did not reduce maximal contraction to 5-HT in carotid arteries but did shift the 5-HT concentration response curve 3-fold to the right. Tyrphostin 23 (5 x 10(-5) M), another tyrosine kinase inhibitor, decreased the potency of 5-HT 4-fold and reduced the maximal contraction to 5-HT in the carotid artery (10% control). Contractions induced by phorbol-12,13-dibutyrate (10(-9) to 10(-5) M) were not reduced or shifted by either tyrosine kinase inhibitor, indicating that phorbolester-sensitive protein kinase C isoforms were not affected. KCl-induced contraction was shifted 2-fold and the maximum significantly inhibited by tyrphostin 23 (38.6% control) but not genistein or daidzein, indicating that tyrphostin 23 but not genistein may inhibit voltage-gated calcium channels to reduce contractility. Western blot analysis using antiphosphotyrosine antibody confirmed that 5-HT produced a time- and concentration-dependent increase in the phosphotyrosine immunoreactivity of a 42-kD protein in cultured aortic smooth muscle cells. Lysate immunoprecipitation with an antimitogen-activated-protein (MAP)-kinase antibody indicated that the 42-kD protein was most likely a MAP kinase. 5-HT (10(-5) M) stimulated contraction and increased antiphosphotyrosine immunoreactivity in whole aorta mounted in tissue baths. Importantly, aortic contraction to 5-HT was shifted (5-fold rightward) and reduced (69% control) by genistein but not daidzein. These findings demonstrate that (1) tyrosine kinase activation may partially mediate contractility to 5-HT in arterial smooth muscle, (2) tyrphostin 23 is somewhat nonselective and (3) 5-HT stimulates tyrosine kinase as documented by increased tyrosyl phosphorylation of proteins in cultured aortic smooth muscle cells and aortic tissue in active contraction of 5-HT. These findings have significant implications not only in understanding a novel pathway of 5-HT signal transduction but also in vascular diseases in which growth and/or contractility to 5-HT is increased (e.g. hypertension, atherosclerosis).

Chronic L-arginine administration attenuates cardiac hypertrophy in spontaneously hypertensive rats.

Nitric oxide inhibits proliferation and migration of vascular smooth muscle cells and contractility of cardiomyocytes in vitro. In spontaneously hypertensive rats (SHR), evidence suggests intrinsic abnormalities of the L-arginine-nitric oxide axis, such as low cGMP-dependent protein kinase in the heart and abnormal L-arginine metabolism. To investigate the in vivo effect of L-arginine on cardiac hypertrophy, 30 SHR and 30 Wistar-Kyoto rats (WKY) were randomly grouped to receive L-arginine (7.5 g/L in drinking water) or vehicle for 12 weeks. L-Arginine treatment did not affect body weight or arterial pressure in either strain. In vehicle-treated animals, the heart/body weight ratio was significantly higher in SHR than in WKY (P < .01). L-Arginine treatment decreased the heart/body weight ratio in SHR (P < .05) but did not affect it in WKY. Expression of skeletal alpha-actin mRNA, known to be expressed in the hypertrophied myocardium, was attenuated in L-arginine-treated SHR compared with vehicle-treated SHR. Cardiac cGMP content and nitrate/nitrite content were less in SHR than WKY. L-Arginine treatment increased these levels only in SHR, suggesting enhanced nitric oxide production. Thus, chronic L-arginine administration attenuated cardiac hypertrophy independently of blood pressure and increased myocardial content of cGMP and nitrate/nitrite. Our results suggest that abnormality of the cardiac L-arginine-nitric oxide axis may play an important role in the pathogenesis of cardiac hypertrophy in SHR.

Diadenoine polyphosphate-induced increase in cytosolic free calcium in vascular smooth muscle cells

To evaluate the effects of two new endogenous vasoconstricting substances, diadenosine pentaphosphate (AP5A) and diadenosine hexaphosphate (AP6A) on the cytosolic free calcium concentration in vascular smooth muscle cells. Spectrofluorophotometric measurements of cytosolic Ca2+ were conducted in monolayers of cultured rat vascular smooth muscle cells using the calcium-sensitive fluorescent dye fura-2. The resting Ca2+ concentration in vascular smooth muscle cells was 80 +/- 5 nmol/l (mean +/- SEM; n = 39). The addition of 10 mu mol/l AP5A or AP6A significantly increased Ca2+ in vascular smooth muscle cells to 248 +/- 55 and 358 +/- 124 nmol/l, respectively. The sustained increase in Ca2+ after administration of AP5A or AP6A was 143 +/- 40 and 148 +/- 57 nmol/l, respectively. Diadenosine polyphosphates induce a transplasmamembrane calcium influx as detected by experiments in the absence of external calcium or by using the manganese quenching technique to report unidirectional calcium fluxes. The effects of diadenosine polyphosphates were compared to those of the well known vasoconstrictor angiotensin II, which increased Ca2+ in vascular smooth muscle cells by 1053 +/- 174 nmol/l. Diadenosine polyphosphates increase Ca2+ in vascular smooth muscle cells, thereby regulating the contractility of vascular smooth muscle and subsequently blood pressure.

Vasodilator effects of visnagin in isolated rat vascular smooth muscle

Visnagin (4-methoxy-7-methyl-5 H-furo [3,2- g][1]-benzopyran-5-one) is an active principle of the fruit of Ammi visnaga, a plant traditionally used in cardiovascular disorders. We have studied its vasodilator effects in rat vascular smooth muscle. The results demonstrated that visnagin inhibited the contractile responses induced in rat aortic rings by: (a) KCl or increases of extracellular Ca 2+ in KCl depolarized aortic rings, its effects being more potent against low (20 mM) than high (80 mM) KCl-induced contractions, (b) noradrenaline in Ca 2+-containing solution and less effectively those in Ca 2+-free solution and (c) phorbol 12-myristate 13-acetate (PMA) in a Ca 2+-containing and with a lower potency in Ca 2+-free medium. The relaxation induced by visnagin in aorta precontracted with noradrenaline was not affected by endothelium removal. Additionally, visnagin inhibited the spontaneous myogenic contractions of portal veins. The results showed that visnagin inhibited vascular smooth muscle contractility by acting at multiple sites. In the range of 10 −6 M to 5 × 10 −5 M visnagin appears to inhibit only the contractions mediated by Ca 2+ entry through pathways with low sensitivity to classical Ca 2+-entry blockers, i.e. agonist-, PMA-or mild depolarization-induced Ca 2+ entry. Therefore, the vasodilator profile of visnagin, is not that of typical Ca 2+-entry blockers which preferentially inhibit the contractions induced by strong depolarizations. At higher concentrations (> 5 × 10 −5 M) visnagin causes non-specific inhibition of vascular smooth muscle contractility.

Characteristics and possible mechanisms of low-Na+ induced contractions in rat aorta.

The influence of reducing external Na+ concentration ([Na+]ex) upon vascular smooth muscle contractility was investigated using the rat isolated aorta. NaCl from the physiological saline solution (PSS) was replaced with either choline-Cl, sucrose, or LiCl to give the following [Na+]ex (mM): 115, 85, 55, and 25 (115NaPSS to 25NaPSS). Small reductions in [Na+]ex (115NaPSS) induced a biphasic contraction, comparable in amplitude with the control one induced by phenylephrine 10(-6) M. Elimination of the endogenous catecholamine participation using either phentolamine 10(-5) M or guanethidine 3.10(-6) M similarly reduces these contractions to 25% (sucrose replacement). A similar relaxing effect was obtained with D600 10(-5) M, an antagonist of the voltage operated Ca2+ channels (25-30% residual tension for all the substitutes). Large reductions in [Na+]ex (25NaPSS) induced contractions comparable in amplitude and shape, but less sensitive to phentolamine and guanethidine (residual tension 65-75%, sucrose replacement) and insensitive to D600 (all the substitutes). The Na+/K+ ATPase inhibitor ouabain (10(-4) M) elicited slowly developing contractions, the amplitude being 115% of the phenylephrine 10(-6) M control. Phenylephrine further contracted the 115NaPSS precontracted preparations, but was significantly less effective in 25NaPSS, although the precontraction levels were similar for the same substitute used. The amplitude of the superimposed phenylephrine contractions exhibited [Na+]ex dependence. Phenylephrine 10(-6) M failed to further contract the ouabain 10(-4) M precontracted rings. We conclude that relatively small reductions in [Na+]ex are able to induce contractions of rat aorta primarily through release of endogenous catecholamines, probably through neural Na+/Ca2+ exchange. Larger reductions in [Na+]ex appear to cause contraction through muscular Na+/Ca2+ exchange.

Pulmonary Hypertension in Acute Lung Injury Is Due to Impaired Vasodilation with Intact Vascular Contractility

The major hemodynamic feature of acute lung injury (ALI) is pulmonary hypertension. Both endothelial-dependent and -independent pulmonary vasorelaxation is impaired in ALI due to endotoxemia. We hypothesized that endotoxemia selectively impairs relaxation of the pulmonary artery but does not impair contractility of pulmonary vascular smooth muscle (VSM). Our purpose was to determine the effect of endotoxemia (ETX) on the contractile response of pulmonary VSM to (1) cellular depolarization (KCI), (2) α 1-adrenoreceptor stimulation (phenylephrine, PE), (3) 5HT 2 receptor stimulation (serotonin, 5HT), and (4) prostaglandin F 2α receptor stimulation. Pulmonary artery rings were isolated from rats 6 hr after injection of ETX, 20 mg/kg ip ( n ⩾ 6), or saline ( n ⩾ 6) and suspended on tensiometers in individual organ baths. Endothelial-dependent cGMP-mediated relaxation was determined using the receptor agonist acetylcholine (ACh) in rings preconstricted with PE. Dose-response curves were generated to each contractile agonist. Statistical comparison was performed using one-way ANOVA with post hoc Bonferonni-Dunn, P < 0.05 accepted as significant. Relaxation to ACh was 96.4 ± 1.3% in controls vs 21.4 ± 3.1% ( P ± 0.05) in endotoxin-treated rats. Endotoxin did not affect the maximal tension in response to the contractile agonists nor did it change the concentration required to produce 50% contraction (EC 50). From these data we conclude that endotoxemia causes a decrease in vasorelaxation to the endothelial-dependent receptor agonist acetylcholine but does not impair agonist-induced contractility of pulmonary VSM. This suggests that pulmonary hypertension in ALI is mediated by impairment of pulmonary vasodilation with preservation of VSM contractility.

Steroid regulation of parathyroid hormone-related protein expression and action in the rat uterus.

The gene encoding parathyroid hormone-related protein (PTHrP), an autocrine/paracrine inhibitor of vascular and nonvascular smooth muscle contractility, is regulated by hormonal steroids including estrogens (E2), 1,25-dihydroxy vitamin D (Vit D3) and glucocorticoids. While E2 increases PTHrP gene expression, Vit D3 and glucocorticoids inhibit transcriptional activity of this gene. In the uterus of ovariectomized rats, E2-treatment increases both PTHrP mRNA levels and smooth muscle sensitivity to the action of PTHrP(1-34). To examine the action(s) of Vit D3 and glucocorticoids on these parameters, OVX rats were treated with E2, Vit D3 or the synthetic glucocorticoid, dexamethasone (Dex), alone, or with E2 following a 1 h pretreatment with Vit D3 or Dex. PTHrP and PTH/PTHrP receptor mRNA were measured by blot hybridization analysis of RNA prepared from uteri collected 2, 4 and 24 h after treatment. Uterine horns were used to measure the effect of the steroids on the ability of PTHrP(1-34) to inhibit spontaneous myometrial contraction. When E2, Vit D3 and Dex were given alone, only E2 altered PTHrP mRNA levels in the uterus, however, a 1 h pretreatment with Dex but not Vit D3 markedly diminished this effect of E2. The temporal decline in uterine PTH/PTHrP receptor mRNA levels measured 2 and 4 h after E2 treatment inversely correlated to changes in sensitivity of the tissue to PTHrP(1-34) measured at 24 h after E2 administration. In comparison to E2 alone, treatment with Vit D3 and E2 augmented the uterine responsiveness to PTHrP(1-34) while pretreatment with Dex (1 mg/kg) and E2 decreased this response. These data indicate that in the uterus, Dex opposes the positive effect of E2 on PTHrP gene activity and differentially modulates the action of PTHrP on myometrial tone. Moreover, elevations in the circulating levels of cortisol at term may serve to decrease both the uterine expression of PTHrP and the local action of PTHrP on the myometrium prior to parturition, therefore promoting myometrial contraction associated with labor.