Jeff Angermann

Mechanism of the inhibition of Ca2+-activated Cl- currents by phosphorylation in pulmonary arterial smooth muscle cells

The aim of the present study was to provide a mechanistic insight into how phosphatase activity influences calcium-activated chloride channels in rabbit pulmonary artery myocytes. Calcium-dependent Cl− currents (IClCa) were evoked by pipette solutions containing concentrations between 20 and 1000 nM Ca2+ and the calcium and voltage dependence was determined. Under control conditions with pipette solutions containing ATP and 500 nM Ca2+, IClCa was evoked immediately upon membrane rupture but then exhibited marked rundown to ∼20% of initial values. In contrast, when phosphorylation was prohibited by using pipette solutions containing adenosine 5′-(β,γ-imido)-triphosphate (AMP-PNP) or with ATP omitted, the rundown was severely impaired, and after 20 min dialysis, IClCa was ∼100% of initial levels. IClCa recorded with AMP-PNP–containing pipette solutions were significantly larger than control currents and had faster kinetics at positive potentials and slower deactivation kinetics at negative potentials. The marked increase in IClCa was due to a negative shift in the voltage dependence of activation and not due to an increase in the apparent binding affinity for Ca2+. Mathematical simulations were carried out based on gating schemes involving voltage-independent binding of three Ca2+, each binding step resulting in channel opening at fixed calcium but progressively greater “on” rates, and voltage-dependent closing steps (“off” rates). Our model reproduced well the Ca2+ and voltage dependence of IClCa as well as its kinetic properties. The impact of global phosphorylation could be well mimicked by alterations in the magnitude, voltage dependence, and state of the gating variable of the channel closure rates. These data reveal that the phosphorylation status of the Ca2+-activated Cl− channel complex influences current generation dramatically through one or more critical voltage-dependent steps.

Stimulation of Ca2+-Gated Cl- Currents by the Calcium-Dependent K+ Channel Modulators NS1619 [1,3-Dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one] and Isopimaric Acid

Because chloride (Cl-) channel blockers such as niflumic acid enhance large-conductance Ca2+-activated potassium channels (BKCa), the aim of this study was to determine whether there is a reciprocal modification of Ca2+-activated chloride Cl- currents (IClCa) by two selective activators of BKCa. Single smooth muscle cells were isolated by enzymatic digestion from murine portal vein and rabbit pulmonary artery. The BKCa activators NS1619 [1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl-)phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one] and isopimaric acid (IpA) augmented macroscopic IClCa elicited by pipette solutions containing [Ca2+]i > 100 nM without any alteration in current kinetics. Enhanced currents recorded in the presence of NS1619 or IpA reversed at the theoretical Cl- equilibrium potential, which was shifted by approximately -40 mV upon replacement of the external anion with the more permeable thiocyanate anion. NS1619 increased the sensitivity of calcium-activated chloride channel (ClCa) to Ca2+ (∼100 nM at +60 mV) and induced a leftward shift in their voltage dependence (∼80 mV with 1 μMCa2+). Single-channel experiments revealed that NS1619 increased the number of open channels times the open probability of small-conductance (1.8–3.1 pS) ClCa without any alteration in their unitary amplitude or number of observable unitary levels of activity. These data, in addition to the established stimulatory effects of niflumic acid on BKCa, show that there is similarity in the pharmacology of calcium-activated chloride and potassium channels. Although nonspecific interactions are possible, one alternative hypothesis is that the channel underlying vascular IClCa shares some structural similarity to the BKCa or that the latter K+ channel physically interacts with ClCa.

Monomethylarsonous acid, but not inorganic arsenic, is a mitochondria-specific toxicant in vascular smooth muscle cells.

Arsenic exposure has been implicated as a risk factor for cardiovascular diseases, metabolic disorders, and cancer, yet the role mitochondrial dysfunction plays in the cellular mechanisms of pathology is largely unknown. To investigate arsenic-induced mitochondrial dysfunction in vascular smooth muscle cells (VSMCs), we exposed rat aortic smooth muscle cells (A7r5) to inorganic arsenic (iAs(III)) and its metabolite monomethylarsonous acid (MMA(III)) and compared their effects on mitochondrial function and oxidative stress. Our results indicate that MMA(III) is significantly more toxic to mitochondria than iAs(III). Exposure of VSMCs to MMA(III), but not iAs(III), significantly decreased basal and maximal oxygen consumption rates and concomitantly increased compensatory extracellular acidification rates, a proxy for glycolysis. Treatment with MMA(III) significantly increased hydrogen peroxide and superoxide levels compared to iAs(III). Exposure to MMA(III) resulted in significant decreases in mitochondrial ATP, aberrant perinuclear clustering of mitochondria, and decreased mitochondrial content. Mechanistically, we observed that mitochondrial superoxide and hydrogen peroxide contribute to mitochondrial toxicity, as treatment of cells with MnTBAP (a mitochondrial superoxide dismutase mimetic) and catalase significantly reduced mitochondrial respiration deficits and cell death induced by both arsenic compounds. Overall, our data demonstrates that MMA(III) is a mitochondria-specific toxicant that elevates mitochondrial and non-mitochondrial sources of ROS.

Molecular and functional significance of Ca2+-activated Cl− channels in pulmonary arterial smooth muscle

Increased peripheral resistance of small distal pulmonary arteries is a hallmark signature of pulmonary hypertension (PH) and is believed to be the consequence of enhanced vasoconstriction to agonists, thickening of the arterial wall due to remodeling, and increased thrombosis. The elevation in arterial tone in PH is attributable, at least in part, to smooth muscle cells of PH patients being more depolarized and displaying higher intracellular Ca2+ levels than cells from normal subjects. It is now clear that downregulation of voltage-dependent K+ channels (e.g., Kv1.5) and increased expression and activity of voltage-dependent (Cav1.2) and voltage-independent (e.g., canonical and vanilloid transient receptor potential [TRPC and TRPV]) Ca2+ channels play an important role in the functional remodeling of pulmonary arteries in PH. This review focuses on an anion-permeable channel that is now considered a novel excitatory mechanism in the systemic and pulmonary circulations. It is permeable to Cl− and is activated by a rise in intracellular Ca2+ concentration (Ca2+-activated Cl− channel, or CaCC). The first section outlines the biophysical and pharmacological properties of the channel and ends with a description of the molecular candidate genes postulated to encode for CaCCs, with particular emphasis on the bestrophin and the newly discovered TMEM16 and anoctamin families of genes. The second section provides a review of the various sources of Ca2+ activating CaCCs, which include stimulation by mobilization from intracellular Ca2+ stores and Ca2+ entry through voltage-dependent and voltage-independent Ca2+ channels. The third and final section summarizes recent findings that suggest a potentially important role for CaCCs and the gene TMEM16A in PH.

Activation of Ca2+-activated Cl- channels by store-operated Ca2+ entry in arterial smooth muscle cells does not require reverse-mode Na+/Ca2+ exchange.

The main purpose of this study was to characterize the stimulation of Ca(2+)-activated Cl(-) (Cl(Ca)) by store-operated Ca(2+) entry (SOCE) channels in rabbit pulmonary arterial smooth muscle cells (PASMCs) and determine if this process requires reverse-mode Na(+)/Ca(2+) exchange (NCX). In whole-cell voltage clamped PASMCs incubated with 1 μmol/L nifedipine (Nif) to inhibit Ca(2+) channels, 30 μmol/L cyclopiazonic acid (CPA), a SERCA pump inhibitor, activated a nonselective cation conductance permeable to Na(+) (I(SOC)) during an initial 1-3 s step, ranging from-120 to +60 mV, and Ca(2+)-activated Cl(-) current (I(Cl(Ca))) during a second step to +90 mV that increased with the level of the preceding hyperpolarizing step. Niflumic acid (100 μmol/L), a Cl(Ca) channel blocker, abolished I(Cl(Ca)) but had no effect on I(SOC), whereas the I(SOC) blocker SKF-96365 (50 μmol/L) suppressed both currents. Dual patch clamp and Fluo-4 fluorescence measurements revealed the appearance of CPA-induced Ca(2+) transients of increasing magnitude with increasing hyperpolarizing steps, which correlated with I(Cl(Ca)) amplitude. The absence of Ca(2+) transients at positive potentials following a hyperpolarizing step combined with the observation that SOCE-stimulated I(Cl(Ca)) was unaffected by the NCX blocker KB-R7943 (1 μmol/L) suggest that the SOCE/Cl(Ca) interaction does not require reverse-mode NCX in our conditions.

Enhanced capacitative calcium entry and sarcoplasmic-reticulum calcium storage capacity with advanced age in murine mesenteric arterial smooth muscle cells.

Intracellular Ca(2+) signaling is important to perfusion pressure related arterial reactivity and to vascular disorders including hypertension, angina and ischemic stroke. We have recently shown that advancing-age leads to calcium signaling adaptations in mesenteric arterial myocytes from C57 BL/6 mice [Corsso, C.D., Ostrovskaya, O., McAllister, C.E., Murray, K., Hatton, W.J., Gurney, A.M., Spencer, N.J., Wilson, S.M., 2006. Effects of aging on Ca(2+) signaling in murine mesenteric arterial smooth muscle cells. Mech. Ageing Dev. 127, 315-323)] which may contribute to decrements in perfusion pressure related arterial contractility others have shown occur. Even still, the mechanisms underlying the changes in Ca(2+) signaling and arterial reactivity are unresolved. Ca(2+) transport and storage capabilities are thought to contribute to age-related Ca(2+) signaling dysfunctions in other cell types. The present studies were therefore designed to test the hypothesis that cytosolic and compartmental Ca(2+) homeostasis in mesenteric arterial myocytes changes with advanced age. The hypothesis was tested by performing digitalized fluorescence microscopy on mesenteric arterial myocytes isolated from 5- to 6-month and 29- to 30-month-old C57Bl/6 mice. The data provide evidence that with advanced age capacitative Ca(2+) entry and sarcoplasmic reticulum Ca(2+) storage are increased although sarcoplasmic reticulum Ca(2+) uptake and plasma membrane Ca(2+) extrusion are unaltered. Overall, the studies begin to resolve the mechanisms associated with age-related alterations in mesenteric arterial smooth muscle Ca(2+) signaling and their physiological consequences.

Antioxidants Protect against Arsenic Induced Mitochondrial Cardio-Toxicity

Arsenic is a potent cardiovascular toxicant associated with numerous biomarkers of cardiovascular diseases in exposed human populations. Arsenic is also a carcinogen, yet arsenic trioxide is used as a therapeutic agent in the treatment of acute promyelotic leukemia (APL). The therapeutic use of arsenic is limited due to its severe cardiovascular side effects. Many of the toxic effects of arsenic are mediated by mitochondrial dysfunction and related to arsenic’s effect on oxidative stress. Therefore, we investigated the effectiveness of antioxidants against arsenic induced cardiovascular dysfunction. A growing body of evidence suggests that antioxidant phytonutrients may ameliorate the toxic effects of arsenic on mitochondria by scavenging free radicals. This review identifies 21 antioxidants that can effectively reverse mitochondrial dysfunction and oxidative stress in cardiovascular cells and tissues. In addition, we propose that antioxidants have the potential to improve the cardiovascular health of millions of people chronically exposed to elevated arsenic concentrations through contaminated water supplies or used to treat certain types of leukemias. Importantly, we identify conceptual gaps in research and development of new mito-protective antioxidants and suggest avenues for future research to improve bioavailability of antioxidants and distribution to target tissues in order reduce arsenic-induced cardiovascular toxicity in a real-world context.

Arsenic Methylation Capacity and Metabolic Syndrome in the 2013–2014 U.S. National Health and Nutrition Examination Survey (NHANES)

Arsenic methylation capacity is associated with metabolic syndrome and its components among highly exposed populations. However, this association has not been investigated in low to moderately exposed populations. Therefore, we investigated arsenic methylation capacity in relation to the clinical diagnosis of metabolic syndrome in a low arsenic exposure population. Additionally, we compared arsenic methylation patterns present in our sample to those of more highly exposed populations. Using logistic regression models adjusted for relevant biological and lifestyle covariates, we report no association between increased arsenic methylation and metabolic syndrome in a population in which arsenic is regulated at 10 ppb in drinking water. However, we cannot rule out the possibility of a positive association between arsenic methylation and metabolic syndrome in a subsample of women with normal body mass index (BMI). To our knowledge this is the first investigation of arsenic methylation capacity with respect to metabolic syndrome in a low exposure population. We also report that methylation patterns in our sample are similar to those found in highly exposed populations. Additionally, we report that gender and BMI significantly modify the effect of arsenic methylation on metabolic syndrome. Future studies should evaluate the effectiveness of arsenic policy enforcement on subclinical biomarkers of cardiovascular disease.