The inhibitory effect of simvastatin and aspirin on histamine responsiveness in human vascular endothelial cells.

Statins and aspirin deliver well-established cardiovascular benefits resulting in their increased use as combined polypills to decrease risk of stroke and heart disease. However, the direct endothelial effect of combined statin/aspirin cotreatment remains unclear. Histamine is an inflammatory mediator that increases vascular permeability, and so we examined the effect of treating human umbilical vein endothelial cells (HUVECs) for 24 h with 1 µM simvastatin and 100 µM aspirin on histamine responsiveness. Subsequent histamine (1 µM) challenge increased intracellular calcium (Ca(2+)i) concentration, an effect that was significantly inhibited by combined simvastatin/aspirin pretreatment but not when then the compounds were given separately, even at 10-fold higher concentrations. In contrast, the Ca(2+)i mobilization response to ATP challenge (10 µM) was not inhibited by combined simvastatin/aspirin pretreatment. The H1 receptor antagonist pyrilamine significantly inhibited both histamine-induced Ca(2+)i mobilization and extracellular signal-regulated kinase (ERK) activation, whereas ranitidine (H2 receptor antagonist) was without effect. However, combined simvastatin/aspirin pretreatment failed to decrease H1 receptor protein expression ruling out receptor downregulation as the mechanism of action. Histamine-induced ERK activation was also inhibited by atorvastatin pretreatment, while simvastatin further inhibited histamine-induced vascular endothelial cadherin phosphorylation as well as altered HUVEC morphology and inhibited actin polymerization. Therefore, in addition to the known therapeutic benefits of statins and aspirin, here we provide initial cellular evidence that combined statin/aspirin treatment inhibits histamine responsiveness in HUVECs.

Increased endothelin-1 responsiveness in human coronary artery smooth muscle cells exposed to 1,25-dihydroxyvitamin D(3).

Low blood concentrations of 25-hydroxyvitamin D(3) are associated with increased mortality, while some studies suggest improved cardiovascular outcomes with vitamin D(3) supplementation in chronic kidney disease. However, the physiological effects of vitamin D(3) on the cardiovascular system remain poorly understood making it difficult to determine whether vitamin D(3) supplementation might provide cardiovascular benefit or even cause harm. Thus here we investigated the effects of chronic 1,25-dihydroxyvitamin D(3) treatment on intracellular signaling in human coronary artery smooth muscle cells (HCASMCs) and found that 1,25-dihydroxyvitamin D(3) significantly potentiated endothelin (ET-1) signaling. Specifically, 1,25-dihydroxyvitamin D(3) (24-h pretreatment) caused a more than threefold enhancement in both ET-1-induced intracellular calcium mobilization and extracellular signal-regulated kinase (ERK) activation. This 1,25-dihydroxyvitamin D(3)-elicited signaling enhancement was not observed for either vasopressin or carbachol. With the use of endothelin receptor (ETR) isoform-selective antagonists, ETRA was found to be primarily responsible for the 1,25-dihydroxyvitamin D(3)-induced ET-1 responsiveness and yet ETRA mRNA expression and protein abundance were unaltered following 1,25-dihydroxyvitamin D(3) treatment. While there was an increase in ETRB mRNA expression in response to 1,25-dihydroxyvitamin D(3), the protein abundance of ETRB was again unchanged. Finally, ETRA/ETRB heterodimerization was not detected in HCASMCs in either the absence or presence of 1,25-dihydroxyvitamin D(3). Together, these data show for the first time that 1,25-dihydroxyvitamin D(3) enhances endothelin responsiveness in HCASMCs and that the effect is mediated through ETRA.

Remodelling and dysfunction of the sinus node in pulmonary arterial hypertension.

Patients with pulmonary arterial hypertension (PAH) have a high burden of arrhythmias, including arrhythmias arising from sinus node dysfunction, and the aim of this study was to investigate the effects of PAH on the sinus node. In the rat, PAH was induced by an injection of monocrotaline. Three weeks after injection, there was a decrease of the intrinsic heart rate (heart rate in the absence of autonomic tone) as well as the normal heart rate, evidence of sinus node dysfunction. In the sinus node of PAH rats, there was a significant downregulation of many ion channels and Ca2+-handling genes that could explain the dysfunction: HCN1 and HCN4 (responsible for pacemaker current, If), Cav1.2, Cav1.3 and Cav3.1 (responsible for L- and T-type Ca2+ currents, ICa,L and ICa,T), NCX1 (responsible for Na+-Ca2+ exchanger) and SERCA2 and RYR2 (Ca2+-handling molecules). In the sinus node of PAH rats, there was also a significant upregulation of many fibrosis genes that could also help explain the dysfunction: vimentin, collagen type 1, elastin, fibronectin and transforming growth factor ß1. In summary, in PAH, there is a remodelling of ion channel, Ca2+-handling and fibrosis genes in the sinus node that is likely to be responsible for the sinus node dysfunction. This article is part of the theme issue 'The heartbeat: its molecular basis and physiological mechanisms'.

Simvastatin causes pulmonary artery relaxation by blocking smooth muscle ROCK and calcium channels: Evidence for an endothelium-independent mechanism.

Simvastatin reduces pulmonary arterial pressure and right ventricular hypertrophy in animal models of pulmonary arterial hypertension (PAH) and is thought to restore endothelial dysfunction. In vivo effects of drugs are complicated by several factors and little is known of the direct effects of statins on pulmonary arteries. This study investigated the direct effects of simvastatin on pulmonary arteries isolated from rats with or without monocrotaline-induced PAH. Simvastatin suppressed contractions evoked by the thromboxane A2 receptor agonist U46619 (30 nM), the α1-adrenergic agonist phenylephrine (5 µM) and KCl (50 mM) by ~50% in healthy and diseased arteries, but did not reduce contraction evoked by sarco/endoplasmic reticulum ATPase blockers. It relaxed hypertensive arteries in the absence of stimulation. Removing the endothelium or inhibiting eNOS did not prevent the inhibition by simvastatin. Inhibiting RhoA/rho kinase (ROCK) with Y27632 (10 µM) suppressed contractions to U46619 and phenylephrine by ~80% and prevented their inhibition by simvastatin. Y27632 reduced KCl-induced contraction by ~30%, but did not prevent simvastatin inhibition. Simvastatin suppressed Ca2+ entry into smooth muscle cells, as detected by Mn2+ quench of fura-2 fluorescence. The calcium antagonist, nifedipine (1 µM), almost abolished K+-induced contraction with less effect against U46619 and phenylephrine. We conclude that simvastatin relaxes pulmonary arteries by acting on smooth muscle to interfere with signalling through G-protein coupled receptors and voltage-dependent Ca2+ entry. Its actions likely include inhibition of ROCK-dependent Ca2+ sensitisation and voltage-gated Ca2+ channels. These are likely to contribute to the beneficial effects of simvastatin in animal models of PAH.

Evidence in favor of a calcium-sensing receptor in arterial endothelial cells: studies with calindol and Calhex 231.

Small increases in extracellular Ca2+ dilate isolated blood vessels. In the present study, the possibility that a vascular, extracellular Ca2+-sensing receptor (CaSR) could mediate these vasodilator actions was investigated. Novel ligands that interact with the CaSR were used in microelectrode recordings from rat isolated mesenteric and porcine coronary arteries. The major findings were that (1) raising extracellular Ca2+ or adding calindol, a CaSR agonist, produced concentration-dependent hyperpolarizations of vascular myocytes, actions attenuated by Calhex 231, a negative allosteric modulator of CaSR. (2) Calindol-induced hyperpolarizations were inhibited by the intermediate conductance, Ca2+-sensitive K+ (IKCa) channel inhibitors, TRAM-34, and TRAM-39. (3) The effects of calindol were not observed in the absence of endothelium. (4) CaSR mRNA and protein were present in rat mesenteric arteries and in porcine coronary artery endothelial cells. (5) CaSR and IKCa proteins were restricted to caveolin-poor membrane fractions. We conclude that activation of vascular endothelial CaSRs opens endothelial cell IKCa channels with subsequent myocyte hyperpolarization. The endothelial cell CaSR may have a physiological role in the control of arterial blood pressure.

Atrioventricular Node Dysfunction and Ion Channel Transcriptome in Pulmonary Hypertension.

BACKGROUND: Heart block is associated with pulmonary hypertension, and the aim of the study was to test the hypothesis that the heart block is the result of a change in the ion channel transcriptome of the atrioventricular (AV) node. METHODS AND RESULTS: The most commonly used animal model of pulmonary hypertension, the monocrotaline-injected rat, was used. The functional consequences of monocrotaline injection were determined by echocardiography, ECG recording, and electrophysiological experiments on the Langendorff-perfused heart and isolated AV node. The ion channel transcriptome was measured by quantitative PCR, and biophysically detailed computer modeling was used to explore the changes observed. After monocrotaline injection, echocardiography revealed the pattern of pulmonary artery blood flow characteristic of pulmonary hypertension and right-sided hypertrophy and failure; the Langendorff-perfused heart and isolated AV node revealed dysfunction of the AV node (eg, 50% incidence of heart block in isolated AV node); and quantitative PCR revealed a widespread downregulation of ion channel and related genes in the AV node (eg, >50% downregulation of Cav1.2/3 and HCN1/2/4 channels). Computer modeling predicted that the changes in the transcriptome if translated into protein and function would result in heart block. CONCLUSIONS: Pulmonary hypertension results in a derangement of the ion channel transcriptome in the AV node, and this is the likely cause of AV node dysfunction in this disease.

The effect of streptozotocin-induced diabetes on the EDHF-type relaxation and cardiac function in rats.

The endothelium-derived hyperpolarizing factor (EDHF) response is a critical for the functioning of small blood vessels. We investigated the effect of streptozotocin-induced diabetes on the EDHF response and its possible role in the regulation of cardiac function. The vasorelaxant response to ACh- or NS309- (direct opener endothelial small- (SKCa)- and intermediate-conductance (IKCa) calcium-activated potassium channels; main components of EDHF response) were measured in pressurized mesenteric arteries (diameter 300-350 µm). The response to 1 µM ACh was reduced in diabetes (84.8 ± 2.8% control vs 22.5 ± 5.8% diabetics; n â©¾ 8; P < 0.001). NS309 (1 µM) relaxations were also decreased in diabetic arteries (78.5 ± 8.7% control vs 32.1 ± 5.8% diabetics; n â©¾ 5; P < 0.001). SKCa and IKCa-mediated EDHF relaxations in response ACh or NS309 were also significantly reduced by diabetes. Ruthenium red, RuR, a blocker of TRP channels, strongly depress the response to ACh and NS309 in control and diabetic arteries. RuR decreased SKCa and IKCa-mediated EDHF vasodilatation in response to NS309 but not to ACh. An elevation in systolic blood pressure was observed in diabetic animals. ECG recording of control hearts showed shortening of PR interval. RuR reduced PR interval and R wave amplitude in diabetic hearts. In conclusion, the reduced EDHF-type relaxations in STZ-induced diabetes is due impairment of KCa channels function. TRP channels possibly contribute to EDHF vasodilatation via direct opening of endothelial KCa. It is possible that EDHF and TRP channels contribute to the regulation of cardiac function and therefore can be considered as therapeutic targets to improve cardiovascular complications of diabetes.

The effect of levofloxacin and moxifloxacin on cardiovascular functions of rats with streptozotocin-induced diabetes.

Fluoroquinolone antibiotics cause rare, but clinically important, adverse events including hyperglycaemia and hypoglycaemia. The present study focuses on the possible effect of levofloxacin and moxifloxacin on the cardiovascular functions of rats with type I diabetes. Both antibiotics caused bradycardia. Levofloxacin but not moxifloxacin caused hypoglycaemia in diabetic rats and an increase in amplitude of the ST segment revealed by electrocardiogram (ECG) analysis of isolated hearts. In pressurized mesenteric arteries, levofloxacin did not affect the endothelium-derived hyperpolarising factor (EDHF) pathway or its main components, the small-conductance Ca(2+) activated potassium (SK(Ca)) and intermediate-conductance Ca(2+) activated potassium (IK(Ca)) channels. In moxifloxacin-treated rats, an increase in the EDHF response was observed, which was largely attributed to SK(Ca)-activation. In conclusion, levofloxacin and moxifloxacin use appeared to vary but with no evidence of impairment of the cardiovascular function. However, it is still possible that these antibiotics may produce different effects if there are co-morbidities and therefore their use must be with care.