Kenneth L. Byron

Hydrogen Peroxide Activates Mitogen-Activated Protein Kinases and Na+-H+ Exchange in Neonatal Rat Cardiac Myocytes

Reperfusion of cardiac tissue after an ischemic episode is associated with metabolic and contractile dysfunction, including reduced tension development and activation of the Na+-H+ exchanger (NHE). Oxygen-derived free radicals are key mediators of reperfusion abnormalities, although the cellular mechanisms involved have not been fully defined. In the present study, the effects of free radicals on mitogen-activated protein (MAP) kinase function were investigated using cultured neonatal rat ventricular myocytes. Acute exposure of spontaneously beating myocytes to 50 micromol/L hydrogen peroxide (H2O2) caused a sustained decrease in contraction amplitude (80% of control). MAP kinase activity was measured by in-gel kinase assays and Western blot analysis. Acute exposure to H2O2 (100 micromol/L, 5 minutes) resulted in sustained MAP kinase activation that persisted for 60 minutes. Catalase, but not superoxide dismutase, completely inhibited MAP kinase activation by H2O2. Pretreatment with chelerythrine (10 micromol/L, 45 minutes), a protein kinase C inhibitor, or genistein (75 micromol/L, 45 minutes) or herbimycin A (3 micromol/L, 45 minutes), tyrosine kinase inhibitors, caused significant inhibition of H2O2-stimulated MAP kinase activity (51%, 78%, and 45%, respectively, at 20 minutes). Brief exposure to H2O2 also stimulated NHE activity. This effect was completely abolished by pretreatment with the MAP kinase kinase inhibitor PD 98059 (30 micromol/L, 60 minutes). These results suggest that low doses of H2O2 induce MAP kinase-dependent pathways that regulate NHE activity during reperfusion injury.

Calcium- and Protein Kinase C–Dependent Activation of the Tyrosine Kinase PYK2 by Angiotensin II in Vascular Smooth Muscle

Angiotensin II (Ang II) induces vascular smooth muscle cell (VSMC) growth by activating Gq-protein-coupled AT1 receptors, which leads to elevation of cytosolic Ca2+ ([Ca2+]i) and activation of protein kinase C (PKC) and mitogen-activated protein kinases. To assess the link between these Ang II-induced signaling events, we examined the effect of Ang II on the proline-rich tyrosine kinase (PYK2), previously found to be activated by a variety of stimuli that increase [Ca2+]i or activate PKC. PYK2 distribution was demonstrated in rat aortic tissue and in cultured VSMC by immunohistochemistry, revealing a cytosolic distribution distinct from smooth muscle alpha-actin, focal adhesion kinase, or paxillin. The involvement of PYK2 in Ang II signaling was measured by immunoprecipitation and immune complex kinase assays. Treatment of quiescent VSMC with Ang II resulted in a concentration- and time-dependent increase in PYK2 tyrosine phosphorylation and kinase activity in PYK2 immunoprecipitates. PYK2 phosphorylation was inhibited by AT1 receptor blockade and was attenuated by downregulation of PKC or the chelation of [Ca2+]i. Treatment with either phorbol ester or Ca2+ ionophore also increased PYK2 phosphorylation, suggesting that PKC activation and/or increased [Ca2+]i are both necessary and sufficient to activate PYK2. Activation of PYK2 by Ang II was also associated with increased PYK2-src complex formation, suggesting that PYK2 activation represents a potential link between Ang II-stimulated [Ca2+]i and PKC activation with downstream signaling events such as mitogen-activated protein kinase activation involved in the regulation of VSMC growth.

Vascular KCNQ Potassium Channels as Novel Targets for the Control of Mesenteric Artery Constriction by Vasopressin, Based on Studies in Single Cells, Pressurized Arteries, and in Vivo Measurements of Mesenteric Vascular Resistance

Pressor effects of the vasoconstrictor hormone arginine vasopressin (AVP), observed when systemic AVP concentrations are less than 100 pM, are important for the physiological maintenance of blood pressure, and they are also the basis for therapeutic use of vasopressin to restore blood pressure in hypotensive patients. However, the mechanisms by which circulating AVP induces arterial constriction are unclear. We examined the novel hypothesis that KCNQ potassium channels mediate the physiological vasoconstrictor actions of AVP. Reverse transcriptase polymerase chain reaction revealed expression of KCNQ1, KCNQ4, and KCNQ5 in rat mesenteric artery smooth muscle cells (MASMCs). Whole-cell perforated patch recordings of voltage-sensitive K+ (Kv) currents in freshly isolated MASMCs revealed 1,3-dihydro-1-phenyl-3,3-bis(4-pyridinylmethyl)-2H-indol-2-one (linopirdine)- and 10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone (XE-991)-sensitive KCNQ currents that were electrophysiologically and pharmacologically distinct from other Kv currents. Suppression of KCNQ currents by AVP (100 pM) was associated with significant membrane depolarization, and it was abolished by the protein kinase C (PKC) inhibitor calphostin C (250 nM). The KCNQ channel blocker linopirdine (10 μM) inhibited KCNQ currents in MASMCs, and it induced constriction of isolated rat mesenteric arteries. The vasoconstrictor responses were not additive when combined with 30 pM AVP, and they were prevented by the L-type Ca2+ channel blocker verapamil. Ethyl-N-[2-amino-6-(4-fluorophenylmethylamino)pyridin-3-yl] carbamic acid (flupirtine) significantly enhanced KCNQ currents, and it reversed constrictor responses to 30 pM AVP. In vivo, i.v. administration of linopirdine induced a dose-dependent increase in mesenteric artery resistance and blood pressure, whereas flupirtine had the opposite effects. We conclude that physiological concentrations of AVP induce mesenteric artery constriction via PKC-dependent suppression of KCNQ currents and L-type Ca2+ channel activation in MASMCs.

Pharmacological and Electrophysiological Characterization of Store-operated Currents and Capacitative Ca 2+ Entry in Vascular Smooth Muscle Cells

Capacitative Ca2+ entry (CCE) in vascular smooth muscle cells contributes to vasoconstrictor and mitogenic effects of vasoactive hormones. In A7r5 rat aortic smooth muscle cells, measurements of cytosolic free Ca2+ concentration ([Ca2+]i) have demonstrated that depletion of intracellular Ca2+ stores activates CCE. However, there is disagreement in published studies regarding the regulation of this mechanism by the vasoconstrictor hormone [Arg8]-vasopressin (AVP). We have employed electrophysiological methods to characterize the membrane currents activated by store depletion [store-operated current (ISOC)]. Because of different recording conditions, it has not been previously determined whether ISOC corresponds to CCE measured using fura-2; nor has the channel protein responsible for CCE been identified. In the present study, the pharmacological characteristics of ISOC, including its sensitivity to blockade by 2-aminoethoxydiphenylborane, diethylstilbestrol, or micromolar Gd3+, were found to parallel the effects of these drugs on thapsigargin- or AVP-activated CCE measured under identical external ionic conditions using fura-2. Thapsigargin-stimulated ISOC was also measured in freshly isolated rat mesenteric artery smooth muscle cells (MASMC). Members of the transient receptor potential (TRP) family of nonselective cation channels, TRPC1, TRPC4, and TRPC6, were detected by reverse transcription-polymerase chain reaction and Western blot in both A7r5 cells and MASMC. TRPC1 expression was reduced in a stable A7r5 cell line expressing a small interfering RNA (siRNA) or by infection of A7r5 cells with an adenovirus expressing a TRPC1 antisense nucleotide sequence. Thapsigargin-stimulated ISOC was reduced in both the TRPC1 siRNA- and TRPC1 antisense-expressing cells, suggesting that the TRPC1 channel contributes to the ISOC/CCE pathway.

Vasopressin stimulation of Ca2+ mobilization, two bivalent cation entry pathways and Ca2+ efflux in A7r5 rat smooth muscle cells.

1. Arg8‐vasopressin (AVP)‐regulated Ca2+ transport were investigated in fura‐2‐loaded A7r5 cells using both single cell and population measurements. 2. AVP evokes an initial concentration‐dependent rise in cytosolic free Ca2+ concentration ([Ca2+ ]i) to a peak which is independent of extracellular Ca2+, and a sustained Ca2+ signal that results from a balance between stimulation of Ca2+ entry and efflux. 3. Depletion of intracellular Ca2+ stores with thapsigargin, ionomycin, or prior treatment with AVP in Ca2(+)‐free medium activates ‘capacitative’ entry of Ca2+, Ba2+ or Mn2+. Capacitative Mn2+ entry is inhibited by refilling stores with Ca2+; neither Sr2+ nor Ba2+ substitute for Ca2+ to give this effect. 4. In cells with empty stores, AVP stimulates further bivalent cation entry, and the effect persists when extracellular Na+ is replaced by N‐methyl‐D‐glucamine or under depolarizing condition (extracellular KCl concentration ([KCl]o), 135 mM). This effect of AVP is not therefore merely a consequence of AVP causing membrane hyperpolarization or stimulation of Na(+)‐Ca2+ exchange, but results from opening of a bivalent cation influx pathway. 5. Several lines of evidence indicate that AVP‐stimulated bivalent cation entry is not a consequence of more complete emptying of the intracellular stores and consequent further activation of the capacitative pathway. AVP stimulates Ba2+ entry when the intracellular Ca2+ stores have been both emptied by ionomycin and prevented from refilling by thapsigargin. Mn2+ permeates the capacitative pathway, but AVP does not further increase Mn2+ entry, confirming that AVP does not further activate the capacitative pathway and that the two pathways differ in their permeability to Mn2+. When the extracellular [Sr2+] is low, empty stores do not stimulate detectable Sr2+ entry, but addition of AVP causes substantial Sr2+ entry. 6. A decrease in [Ca2+]i occurs when 50 nM AVP is added during a sustained elevation of [Ca2+]i evoked by thapsigargin. Since AVP does not inhibit the capacitative pathway, this result suggests that AVP stimulates Ca2+ extrusion. 7. We conclude that stimulation of Ca2+ mobilization, two modes of bivalent cation entry, and Ca2+ efflux all contribute to the complex concentration‐dependent effects of AVP in A7r5 smooth muscle cells.

Cardiovascular KCNQ (Kv7) Potassium Channels: Physiological Regulators and New Targets for Therapeutic Intervention

Potassium channels play an important role in electrical signaling of excitable cells such as neurons, cardiac myocytes, and vascular smooth muscle cells (VSMCs). In particular, the KCNQ (Kv7) family of voltage-activated K+ channels functions to stabilize negative resting membrane potentials and thereby opposes electrical excitability. Of the five known members of the mammalian Kv7 family, Kv7.1 was originally recognized for its role in cardiac myocytes, where it contributes to repolarization of the cardiac action potential. Kv7.2 to Kv7.5 were first discovered in neurons, in which they play a well characterized role in neurotransmitter-stimulated action potential firing. Over the past 5 years, important new roles for Kv7 channels have been identified. Kv7 channels have been found to be expressed in VSMCs from several vascular beds where they contribute to the regulation of vascular tone. There is evidence that Kv7.5 channels in VSMCs are targeted by the hormone vasopressin to mediate its physiological vasoconstrictor actions and evidence that neuronal Kv7 channels in the baroreceptors of the aortic arch adjust the sensitivity of the mechanosensitive neurons to changes in arterial blood pressure. These newly identified physiological roles for Kv7 channels in the cardiovascular system warrant increased attention because pharmacological modulators of this family of channels are being used clinically to treat a variety of neurological disorders. This raises questions about the cardiovascular side effects associated with existing therapies, but there is also obvious potential to capitalize on the established and evolving pharmacology of these channels to develop new therapies for cardiovascular diseases.

Ca2+-Dependent Mitogen-Activated Protein Kinase Activation in Spontaneously Hypertensive Rat Vascular Smooth Muscle Defines a Hypertensive Signal Transduction Phenotype

The mechanisms responsible for altered vascular smooth muscle cell (VSMC) function in hypertension remain unknown. In the spontaneously hypertensive rat (SHR) model of genetic hypertension, there are multiple abnormalities in VSMC function, including increased growth, Na(+)-H+ exchange, and increased signal transduction by protein kinase C. The family of kinases termed mitogen-activated protein (MAP) kinases has recently been shown to be essential mediators of growth factor signal transduction. In the present study, alterations in MAP kinase function in the hypertensive phenotype were investigated using early-passage SHR and Wistar-Kyoto (WKY) VSMCs stimulated with angiotensin II (Ang II, 100 nmol/L) or platelet-derived growth factor-BB (PDGF-BB, 10 ng/mL). MAP kinase activity was measured by in-gel kinase assays and Western blot analysis. Two differences between SHR and WKY rats were observed for Ang II-mediated MAP kinase activation: (1) Inactivation after Ang II stimulation was more rapid in SHR than WKY VSMCs. (2) Activity in SHR VSMCs showed a greater dependence on Ca2+ mobilization, since chelation of intracellular Ca2+ with BAPTA inhibited maximal activity by 95% in SHR VSMCs but by only 50% in WKY VSMCs. In contrast to the results with Ang II, no differences in PDGF-stimulated MAP kinase activity were observed. These findings establish activation of MAP kinase by Ang II as a feature that distinguishes SHR VSMCs from WKY VSMCs and suggest that differences in regulation of MAP kinase signaling may alter cellular events that are increased in the SHR genetic model of hypertension.

Differential effects of selective cyclooxygenase-2 inhibitors on vascular smooth muscle ion channels may account for differences in cardiovascular risk profiles.

Celecoxib, rofecoxib, and diclofenac are clinically used cyclooxygenase-2 (COX-2) inhibitors, which have been under intense scrutiny because long-term rofecoxib (Vioxx; Merck, Whitehouse Station, NJ) treatment was found to increase the risk of adverse cardiovascular events. A differential risk profile for these drugs has emerged, but the underlying mechanisms have not been fully elucidated. We investigated the effects of celecoxib, rofecoxib, and diclofenac on ionic currents and calcium signaling in vascular smooth muscle cells (VSMCs) using patch-clamp techniques and fura-2 fluorescence and on arterial constriction using pressure myography. Celecoxib, but not rofecoxib or diclofenac, dramatically enhanced KCNQ (Kv7) potassium currents and suppressed L-type voltage-sensitive calcium currents in A7r5 rat aortic smooth muscle cells (native KCNQ currents or overexpressed human KCNQ5 currents) and freshly isolated rat mesenteric artery myocytes. The effects of celecoxib were concentration-dependent within the therapeutic concentration range, and were reversed on washout. Celecoxib, but not rofecoxib, also inhibited calcium responses to vasopressin in A7r5 cells and dilated intact or endothelium-denuded rat mesenteric arteries. A celecoxib analog, 2,5-dimethyl-celecoxib, which does not inhibit COX-2, mimicked celecoxib in its enhancement of vascular KCNQ5 currents, suppression of L-type calcium currents, and vasodilation. We conclude that celecoxib inhibits calcium responses in VSMCs by enhancing KCNQ5 currents and suppressing L-type calcium currents, which ultimately reduces vascular tone. These effects are independent of its COX-2 inhibitory actions and may explain the differential risk of cardiovascular events in patients taking different drugs of this class.

Myosin heavy chain turnover in cultured neonatal rat heart cells: effects of [Ca2+]i and contractile activity

Blockade of L-type Ca2+ channels in spontaneously contracting cultured neonatal rat ventricular myocytes causes contractile arrest, myofibrillar disassembly, and accelerated myofibrillar protein turnover. To determine whether myofibrillar protein turnover. To determine whether myofibrillar atrophy results indirectly from loss of mechanical signals or directly from alterations in intracellular Ca2+ concentration ([Ca2+]i), contractile activity was inhibited with verapamil (10 microM) or 2,3-butanedione monoxime (BDM), and their effects on cell shortening, [Ca2+]i, and myosin heavy chain (MHC) turnover were assessed. Control cells demonstrated spontaneous [Ca2+]i transients (peak amplitude 232 +/- 15 nM, 1-2 Hz) and vigorous contractile activity. Verapamil inhibited shortening by eliminating spontaneous [Ca2+]i transients. Low concentrations of BDM (5.0-7.5 mM) had no effect on basal or peak [Ca2+]i transient amplitude but reduced cell shortening, whereas 10 mM BDM reduced both [Ca2+]i transient amplitude and shortening. Both agents inhibited MHC synthesis, but only verapamil accelerated MHC degradation. Thus MHC half-life does not change in parallel with contractile activity but rather more closely follows changes in [Ca2+]i. [Ca2+]i transients appear critical in maintaining myofibrillar assembly and preventing accelerated MHC proteolysis.

Contractile activity is required for sarcomeric assembly in phenylephrine-induced cardiac myocyte hypertrophy

Agonist-induced hypertrophy of cultured neonatal rat ventricular myocytes (NRVM) has been attributed to biochemical signals generated during receptor activation. However, NRVM hypertrophy can also be induced by spontaneous or electrically stimulated contractile activity in the absence of exogenous neurohormonal stimuli. Using single-cell imaging of fura 2-loaded myocytes, we found that low-density, noncontracting NRVM begin to generate intracellular Ca2+ concentration ([Ca2+]i) transients and contractile activity within minutes of exposure to the α1-adrenergic agonist phenylephrine (PE; 50 μM). However, NRVM pretreated with verapamil and then stimulated with PE failed to elicit [Ca2+]itransients and beating. We therefore examined whether PE-induced [Ca2+]itransients and contractile activity were required to elicit specific aspects of the hypertrophic phenotype. PE treatment (48-72 h) increased cell size, total protein content, total protein-to-DNA ratio, and myosin heavy chain (MHC) isoenzyme content. PE also stimulated sarcomeric protein assembly and prolonged MHC half-life. However, blockade of voltage-gated L-type Ca2+ channels with verapamil, diltiazem, or nifedipine (10 μM) blocked PE-induced total protein and MHC accumulation and prevented the time-dependent assembly of myofibrillar proteins into sarcomeres. Inhibition of actin-myosin cross-bridge cycling with 2,3-butanedione monoxime (7.5 mM) also prevented PE-induced total protein and MHC accumulation, indicating that mechanical activity, rather than [Ca2+]itransients per se, was required. In contrast, blockade of [Ca2+]itransients and contractile activity did not prevent the PE-induced increase in cell surface area, activation of the mitogen-activated protein kinases ERK1 and ERK2, or upregulation of atrial natriuretic factor gene expression. Thus contractile activity is required to elicit some but not all aspects of the the hypertrophic phenotype induced by α1-adrenergic receptor activation.Agonist-induced hypertrophy of cultured neonatal rat ventricular myocytes (NRVM) has been attributed to biochemical signals generated during receptor activation. However, NRVM hypertrophy can also be induced by spontaneous or electrically stimulated contractile activity in the absence of exogenous neurohormonal stimuli. Using single-cell imaging of fura 2-loaded myocytes, we found that low-density, noncontracting NRVM begin to generate intracellular Ca2+ concentration ([Ca2+]i) transients and contractile activity within minutes of exposure to the alpha 1-adrenergic agonist phenylephrine (PE; 50 microM). However, NRVM pretreated with verapamil and then stimulated with PE failed to elicit [Ca2+]i transients and beating. We therefore examined whether PE-induced [Ca2+]i transients and contractile activity were required to elicit specific aspects of the hypertrophic phenotype. PE treatment (48-72 h) increased cell size, total protein content, total protein-to-DNA ratio, and myosin heavy chain (MHC) isoenzyme content. PE also stimulated sarcomeric protein assembly and prolonged MHC half-life. However, blockade of voltage-gated L-type Ca2+ channels with verapamil, diltiazem, or nifedipine (10 microM) blocked PE-induced total protein and MHC accumulation and prevented the time-dependent assembly of myofibrillar proteins into sarcomeres. Inhibition of actin-myosin cross-bridge cycling with 2,3-butanedione monoxime (7.5 mM) also prevented PE-induced total protein and MHC accumulation, indicating that mechanical activity, rather than [Ca2+]i transients per se, was required. In contrast, blockade of [Ca2+]i transients and contractile activity did not prevent the PE-induced increase in cell surface area, activation of the mitogen-activated protein kinases ERK1 and ERK2, or upregulation of atrial natriuretic factor gene expression. Thus contractile activity is required to elicit some but not all aspects of the the hypertrophic phenotype induced by alpha 1-adrenergic receptor activation.