Calcium-Sensing Receptor Regulates Cytosolic [Ca 2+ ] and Plays a Major Role in the Development of Pulmonary Hypertension.

Pulmonary arterial hypertension (PAH) is a progressive disease characterized by elevated pulmonary vascular resistance (PVR) leading to right heart failure and premature death. The increased PVR results in part from pulmonary vascular remodeling and sustained pulmonary vasoconstriction. Excessive pulmonary vascular remodeling stems from increased pulmonary arterial smooth muscle cell (PASMC) proliferation and decreased PASMC apoptosis. A rise in cytosolic free Ca2+ concentration ([Ca2+]cyt) in PASMC is a major trigger for pulmonary vasoconstriction and a key stimulus for PASMC proliferation and migration, both contributing to the development of pulmonary vascular remodeling. PASMC from patients with idiopathic PAH (IPAH) have increased resting [Ca2+]cyt and enhanced Ca2+ influx. Enhanced Ca2+ entry into PASMC due to upregulation of membrane receptors and/or Ca2+ channels may contribute to PASMC contraction and proliferation and to pulmonary vasoconstriction and pulmonary vascular remodeling. We have shown that the extracellular Ca2+-sensing receptor (CaSR), which is a member of G protein-coupled receptor (GPCR) subfamily C, is upregulated, and the extracellular Ca2+-induced increase in [Ca2+]cyt is enhanced in PASMC from patients with IPAH in comparison to PASMC from normal subjects. Pharmacologically blockade of CaSR significantly attenuate the development and progression of experimental pulmonary hypertension in animals. Additionally, we have demonstrated that dihydropyridine Ca2+ channel blockers (e.g., nifedipine), which are used to treat PAH patients but are only effective in 15-20% of patients, activate CaSR resulting in an increase in [Ca2+]cyt in IPAH-PASMC, but not normal PASMC. Our data indicate that CaSR functionally couples with transient receptor potential canonical (TRPC) channels to mediate extracellular Ca2+-induced Ca2+ influx and increase in [Ca2+]cyt in IPAH-PASMC. Upregulated CaSR is necessary for the enhanced extracellular Ca2+-induced increase in [Ca2+]cyt and the augmented proliferation of PASMC in patients with IPAH. This review will highlight the pathogenic role of CaSR in the development and progression of PAH

Diabetes mellitus and ischemic heart disease. the role of ion channels

Diabetes mellitus is one the strongest risk factors for cardiovascular disease and, in particular, for ischemic heart disease (IHD). The pathophysiology of myocardial ischemia in diabetic patients is complex and not fully understood: some diabetic patients have mainly coronary stenosis obstructing blood flow to the myocardium; others present with coronary microvascular disease with an absence of plaques in the epicardial vessels. Ion channels acting in the cross-talk between the myocardial energy state and coronary blood flow may play a role in the pathophysiology of IHD in diabetic patients. In particular, some genetic variants for ATP-dependent potassium channels seem to be involved in the determinism of IH

Cardiovascular and hepatic toxicity of cocaine: potential beneficial effects of modulators of oxidative stress

Oxidative stress (OS) is thought to play an important role in the pharmacological and toxic effects of various drugs of abuse. Herein we review the literature on the mechanisms responsible for the cardiovascular and hepatic toxicity of cocaine with special focus on OS-related mechanisms. We also review the preclinical and clinical literature concerning the putative therapeutic effects of OS modulators (such as N-acetylcysteine, superoxide dismutase mimetics, nitroxides and nitrones, NADPH oxidase inhibitors, xanthine oxidase inhibitors, and mitochondriotropic antioxidants) for the treatment of cocaine toxicity. We conclude that available OS modulators do not appear to have clinical efficacy

The Effects of Smoking on the Calcification of the Aortic Valve

Calcific Aortic Valve Disease is responsible for approximately 28,000 deaths annually and the complete mechanism is not completely understood. The disease is known to be effected by an inflammatory response, osteogenic response and an oxidative stress response that leads to thickening and mineralization of the aortic valve. Originally, this disease was thought to be brought on by age and deterioration of the valve but these rapid stress responses when the valve is disrupted by biochemical and mechanical stress has led to its classification as an active disease. Smoking has been shown to have many of the same effects experienced by CAVD but whether the two are directly connected as not been determined. The focus of this Honors project is thus to identify if smoking will elicit a pathological response in vitro to better understand the pathways induced and to identify ways to mitigate smoking-related valve disease. It is hypothesized that cigarette smoke will lead to an inflammatory response, osteogenic-like response, and oxidative stress by analyzing the changes in TGF-B1, IL-6, osteopontin, osteocalcin, RUNX2, and LOX-1. To achieve this, VICs were subjected to 1% and 0.5% Marlboro Red and Silver cigarettes for 6, 24, and 48 hours and were analyzed via RT-PCR and Western Blot. This study concluded that smoking results in the upregulation of IL6 when examined with RT- PCR and the presence of LOX-1 when examined by Western Blot. The presence of these two factors proves that there was an increase of oxidized LDLs as is characteristic of oxidative stress. These results, in combination with the fact that osteopontin, osteocalcin, and RUNX2 gene expression were upregulated at the 6 hour and 48- hour time points, also suggest that there is still a possibility that smoking could induce an osteogenic response if the VICs were subjected to the smoking extracts for longer time periods. The degree to which smoking induces an inflammatory response and an osteogenic response will have to be further tested

The arteriolar vasodilatation model of vibrio cholerae induced diarrhoeal disease

Secretory diarrhoeal disease caused by enterotoxins produced by pathogenic bacteria is characterised by severe fluid loss into the intestine. A prevalent explanation for such high rates of loss, such as occur in episodes of cholera, is that intestinal epithelial cells (enterocytes) actively secrete chloride ion into the lumen. Fluid is drawn into the lumen because of the osmotic pressure difference that is created across the mucosa. Widely proposed as the cause of many forms of secretory diarrhoea, the enterocyte based paradigm displaced an earlier model of secretion i.e. fluid filtration caused by increased capillary hydrostatic pressure, possibly coupled with increased hydraulic conductivity. This would be aggravated by any concurrent inhibition of fluid absorption if it occurred. In the earlier and alternative paradigm, pathophysiological reductions in smooth muscle tone elevate capillary pressure, thereby increasing the hydrostatic pressure gradient that forces fluid from the capillary into the interstitial space and thence into the lumen. In this review, the present and historical evidence for the vasodilatation view of secretory diarrhoeal disease is presented, together with past challenges of this concept, particularly those involving the erroneous equating of solute permeability with hydraulic conductivity. It can be seen that the physical forces model of altered Starling forces combined with enhanced fluid permeation explains more experimental findings than the cellular based enterocyte model can. Several key past papers advocating enterocyte secretion in which the capillary vasodilatation model was also discounted, were examined for the inherent fallacies within the arguments that were proposed. Where possible, quantitative arguments are proposed that indicate that is it the combination of capillary vasodilatation combined with increased tight junctional hydraulic conductivity that causes profuse secretion, made worse by any concurrent inability to absorb fluid. To assist the general physiological reader, an appendix reviews Bernoulli’s principle of flow within tubes and explains the arguably counter-intuitive phenomenon that vasodilatation increases capillary pressure because of a velocity reduction within a dilated segment

A nitric oxide synthase transgene ameliorates muscular dystrophy in mdx mice.

Dystrophin-deficient muscles experience large reductions in expression of nitric oxide synthase (NOS), which suggests that NO deficiency may influence the dystrophic pathology. Because NO can function as an antiinflammatory and cytoprotective molecule, we propose that the loss of NOS from dystrophic muscle exacerbates muscle inflammation and fiber damage by inflammatory cells. Analysis of transgenic mdx mice that were null mutants for dystrophin, but expressed normal levels of NO in muscle, showed that the normalization of NO production caused large reductions in macrophage concentrations in the mdx muscle. Expression of the NOS transgene in mdx muscle also prevented the majority of muscle membrane injury that is detectable in vivo, and resulted in large decreases in serum creatine kinase concentrations. Furthermore, our data show that mdx muscle macrophages are cytolytic at concentrations that occur in dystrophic, NOS-deficient muscle, but are not cytolytic at concentrations that occur in dystrophic mice that express the NOS transgene in muscle. Finally, our data show that antibody depletions of macrophages from mdx mice cause significant reductions in muscle membrane injury. Together, these findings indicate that macrophages promote injury of dystrophin-deficient muscle, and the loss of normal levels of NO production by dystrophic muscle exacerbates inflammation and membrane injury in muscular dystrophy

Renal pericytes: regulators of medullary blood flow

Regulation of medullary blood flow (MBF) is essential in maintaining normal kidney function. Blood flow to the medulla is supplied by the descending vasa recta (DVR), which arise from the efferent arterioles of juxtamedullary glomeruli. DVR are composed of a continuous endothelium, intercalated with smooth muscle-like cells called pericytes. Pericytes have been shown to alter the diameter of isolated and in situ DVR in response to vasoactive stimuli that are transmitted via a network of autocrine and paracrine signalling pathways. Vasoactive stimuli can be released by neighbouring tubular epithelial, endothelial, red blood cells and neuronal cells in response to changes in NaCl transport and oxygen tension. The experimentally described sensitivity of pericytes to these stimuli strongly suggests their leading role in the phenomenon of MBF autoregulation. Because the debate on autoregulation of MBF fervently continues, we discuss the evidence favouring a physiological role for pericytes in the regulation of MBF and describe their potential role in tubulo-vascular cross-talk in this region of the kidney. Our review also considers current methods used to explore pericyte activity and function in the renal medulla

Protein Trafficking of BK Channel β1 Subunits in Cerebral Artery Myocytes

Rationale: Large-conductance calcium (Ca2+)-activated potassium channels (BK) are expressed in arterial myocytes to control arterial contractility. It is composed of pore- forming BKα and auxiliary β1 subunits. Auxiliary β1 subunits associate with BKα which modulate Ca2+ sensitivity of BK channel. Previous data showed that BKα locates at cell membrane, whereas β1 subunits are primarily intracellular which regulated by Rab11A- positive recycling endosomes. Endothelin-1 (ET-1), a vasoconstrictor, induces contraction of myocytes. ET-1 inhibits BK channel but mechanisms are not fully understood. It is unclear that vasoconstrictors regulate the cellular distribution of BK channels. Furthermore, BK channels are involved in hypertension. Hypertension increases risk of major cardiovascular and cerebrovascular events, such as stroke and mental dysfunction. During hypertension, cerebral arteries have high myogenic tone and are less responsive to vasodilators, including nitric oxide (NO). The regulation of arterial contractility by BK channels is altered during hypertension, although mechanisms involved are also unclear. Objective: Test the hypothesis that ET-1 inhibits β1 surface trafficking in myocytes via activation of PKC. Test the hypothesis that activation of PKC directly modulates Rab11A through phosphorylation. Furthermore, test the hypothesis that trafficking of pore- forming BK channel (BKα) and auxiliary β1 subunits contributes to pathological changes in contractility in cerebral arteries of stroke-prone spontaneously hypertensive rats (SP- SHRs). Methods and Results: ET-1 decreased NO-induced or depolarization-induced surface β1 expression and association with BKα in myocytes through activation of protein kinase C (PKC). Total β1, total BKα proteins or surface BKα was not altered by ET-1. Rab11A regulates β1 protein trafficking in Rab11A-positive recycling endosome. ET-1 reduced Rab11 activity via phosphorylation. Five probable phosphorylated sites on Rab11A were identified, among which Ser177 has highest probability. A phosphorylation-mute Rab11A construct (Rab11A S177A) or wild-type Rab11A construct similarly increased total Rab11A protein in transfected myocytes. Rab11A S177A inhibited ET-1-reduced Rab11A activity and decreased β1 protein trafficking. Rab11A S177A reversed PKC- dependent block of single BK channels and transient BK currents in myocytes. Rab11A S177A partially blocked ET-1-induced vasoconstriction. In contrast, NO-induced surface-trafficking of β1 subunits, BK current activity and vasodilation did not involve Rab11A S177. Our data also indicate that the amounts of total and surface BKα and β1 subunits were similar in unstimulated arteries of SP-SHRs and age-matched, normotensive Wistar-Kyoto rat controls. In contrast, the stimulated surface-trafficking of β1 subunits by either NO (sodium nitroprusside, SNP) or membrane depolarization was inhibited in SP-SHR arteries. BIM, a PKC inhibitor, and overexpression of a mutant Rab11A construct that cannot be phosphorylated by PKC at serine 177 (Rab11A S177A) restored the stimulated surface-trafficking of β1 subunits. PKC-mediated inhibition of β1 trafficking prevented BK channel activation by NO in arterial myocytes of SP-SHRs and this was restored by the expression of Rab11A S177A, but not by Rab11A. Vasodilation to NO and lithocholate, an activator of β1 subunit-containing BK channels, was inhibited in pressurized arteries of SP-SHRs. Vasodilation to these agents was reestablished by BIM in SP-SHR arteries. Conclusions: In smooth muscle cell, ET-1 activates protein kinase C which phosphorylates Rab11A at Ser177 to reduce Rab11A activity. Inhibition of Rab11A blocks anterograde trafficking of β1 subunits to associate with BKα on cell surface. Less β1 subunits reduces Ca2+ sensitivity of BK channel and transient BK channel currents which leads to vasoconstriction. Spontaneously active PKC inhibits β1 subunit trafficking in arterial myocytes and is responsible for dysfunctional NO-induced BK channel activation and vasodilation in cerebral arteries of SP-SHRs

Amiloride reduces portal hypertension in rat liver cirrhosis

Objective This study aimed to investigate the effect of amiloride on portal hypertension. Amiloride is known to inhibit Na(+)/H(+) exchangers on activated hepatic stellate cells. Methods Liver cirrhosis in rats was induced by bile duct ligation (BDL) or thioacetamide (TAA) administration. The effects of zymosan for Kupffer cell (KC) activation or a thromboxane (TX) analogue (U46619) were tested in isolated perfused livers of cirrhotic rats and in vivo. Downstream mechanisms were investigated using Rho kinase inhibitor (Y-27632) or amiloride. Acute and chronic effects of amiloride and canrenoate on portal pressure were compared in perfused livers and in vivo. TXB(2) efflux was measured by ELISA. The phosphorylation state of moesin (p-moesin) as an indicator of Rho kinase activity and expression of the thromboxane synthase were assessed by western blot analyses. The activity of hepatic stellate cells was analysed by western blot and staining for alpha-smooth muscle actin (alpha-SMA). Results In BDL rats, KC activation via zymosan increased portal pressure. This was attenuated by the Rho kinase inhibitor Y-27632. Increased thromboxane efflux following zymosan infusion remained unaltered by Y-27632. The infusion of amiloride attenuated zymosan- and U46619-induced increases in portal perfusion pressure. In vivo, direct administration of amiloride, but not of canrenoate, lowered portal pressure. In TAA and BDL rats, treatment with amiloride for 3 days reduced basal portal pressure and KC-induced increases in portal pressure whereas canrenoate had no effect. In livers of amiloride-treated animals, the phosphorylation state of moesin and the number of alpha-SMA positive cells were reduced. Conclusions Amiloride lowers portal pressure in rat liver cirrhosis by inhibition of intrahepatic vasocontraction. Therefore, patients with cirrhosis and portal hypertension may benefit from amiloride therapy