Hypoxia and metabolic inhibitors alter the intracellular ATP:ADP ratio and membrane potential in human coronary artery smooth muscle cells.

ATP-sensitive potassium (KATP) channels couple cellular metabolism to excitability, making them ideal candidate sensors for hypoxic vasodilation. However, it is still unknown whether cellular nucleotide levels are affected sufficiently to activate vascular KATP channels during hypoxia. To address this fundamental issue, we measured changes in the intracellular ATP:ADP ratio using the biosensors Perceval/PercevalHR, and membrane potential using the fluorescent probe DiBAC4(3) in human coronary artery smooth muscle cells (HCASMCs). ATP:ADP ratio was significantly reduced by exposure to hypoxia. Application of metabolic inhibitors for oxidative phosphorylation also reduced ATP:ADP ratio. Hyperpolarization caused by inhibiting oxidative phosphorylation was blocked by either 10 µM glibenclamide or 60 mM K+. Hyperpolarization caused by hypoxia was abolished by 60 mM K+ but not by individual K+ channel inhibitors. Taken together, these results suggest hypoxia causes hyperpolarization in part by modulating K+ channels in SMCs.

Calcium/calmodulin-dependent kinase 2 mediates Epac-induced spontaneous transient outward currents in rat vascular smooth muscle.

KEY POINTS: The Ca2+ and redox-sensing enzyme Ca2+ /calmodulin-dependent kinase 2 (CaMKII) is a crucial and well-established signalling molecule in the heart and brain. In vascular smooth muscle, which controls blood flow by contracting and relaxing in response to complex Ca2+ signals and oxidative stress, surprisingly little is known about the role of CaMKII. The vasodilator-induced second messenger cAMP can relax vascular smooth muscle via its effector, exchange protein directly activated by cAMP (Epac), by activating spontaneous transient outward currents (STOCs) that hyperpolarize the cell membrane and reduce voltage-dependent Ca2+ influx. How Epac activates STOCs is unknown. In the present study, we map the pathway by which Epac increases STOC activity in contractile vascular smooth muscle and show that a critical step is the activation of CaMKII. To our knowledge, this is the first report of CaMKII activation triggering cellular activity known to induce vasorelaxation. ABSTRACT: Activation of the major cAMP effector, exchange protein directly activated by cAMP (Epac), induces vascular smooth muscle relaxation by increasing the activity of ryanodine (RyR)-sensitive release channels on the peripheral sarcoplasmic reticulum. Resultant Ca2+ sparks activate plasma membrane Ca2+ -activated K+ (BKCa ) channels, evoking spontaneous transient outward currents (STOCs) that hyperpolarize the cell and reduce voltage-dependent Ca2+ entry. In the present study, we investigate the mechanism by which Epac increases STOC activity. We show that the selective Epac activator 8-(4-chloro-phenylthio)-2'-O-methyladenosine-3', 5-cyclic monophosphate-AM (8-pCPT-AM) induces autophosphorylation (activation) of calcium/calmodulin-dependent kinase 2 (CaMKII) and also that inhibition of CaMKII abolishes 8-pCPT-AM-induced increases in STOC activity. Epac-induced CaMKII activation is probably initiated by inositol 1,4,5-trisphosphate (IP3 )-mobilized Ca2+ : 8-pCPT-AM fails to induce CaMKII activation following intracellular Ca2+ store depletion and inhibition of IP3 receptors blocks both 8-pCPT-AM-mediated CaMKII phosphorylation and STOC activity. 8-pCPT-AM does not directly activate BKCa channels, but STOCs cannot be generated by 8-pCPT-AM in the presence of ryanodine. Furthermore, exposure to 8-pCPT-AM significantly slows the initial rate of [Ca2+ ]i rise induced by the RyR activator caffeine without significantly affecting the caffeine-induced Ca2+ transient amplitude, a measure of Ca2+ store content. We conclude that Epac-mediated STOC activity (i) occurs via activation of CaMKII and (ii) is driven by changes in the underlying behaviour of RyR channels. To our knowledge, this is the first report of CaMKII initiating cellular activity linked to vasorelaxation and suggests novel roles for this Ca2+ and redox-sensing enzyme in the regulation of vascular tone and blood flow.

Bioenergetic profile of human coronary artery smooth muscle cells and effect of metabolic intervention.

Bioenergetics of artery smooth muscle cells is critical in cardiovascular health and disease. An acute rise in metabolic demand causes vasodilation in systemic circulation while a chronic shift in bioenergetic profile may lead to vascular diseases. A decrease in intracellular ATP level may trigger physiological responses while dedifferentiation of contractile smooth muscle cells to a proliferative and migratory phenotype is often observed during pathological processes. Although it is now possible to dissect multiple building blocks of bioenergetic components quantitatively, detailed cellular bioenergetics of artery smooth muscle cells is still largely unknown. Thus, we profiled cellular bioenergetics of human coronary artery smooth muscle cells and effects of metabolic intervention. Mitochondria and glycolysis stress tests utilizing Seahorse technology revealed that mitochondrial oxidative phosphorylation accounted for 54.5% of ATP production at rest with the remaining 45.5% due to glycolysis. Stress tests also showed that oxidative phosphorylation and glycolysis can increase to a maximum of 3.5 fold and 1.25 fold, respectively, indicating that the former has a high reserve capacity. Analysis of bioenergetic profile indicated that aging cells have lower resting oxidative phosphorylation and reduced reserve capacity. Intracellular ATP level of a single cell was estimated to be over 1.1 mM. Application of metabolic modulators caused significant changes in mitochondria membrane potential, intracellular ATP level and ATP:ADP ratio. The detailed breakdown of cellular bioenergetics showed that proliferating human coronary artery smooth muscle cells rely more or less equally on oxidative phosphorylation and glycolysis at rest. These cells have high respiratory reserve capacity and low glycolysis reserve capacity. Metabolic intervention influences both intracellular ATP concentration and ATP:ADP ratio, where subtler changes may be detected by the latter.

Exchange protein activated by cAMP (Epac) induces vascular relaxation by activating Ca2+-sensitive K+ channels in rat mesenteric artery.

Vasodilator-induced elevation of intracellular cyclic AMP (cAMP) is a central mechanism governing arterial relaxation but is incompletely understood due to the diversity of cAMP effectors. Here we investigate the role of the novel cAMP effector exchange protein directly activated by cAMP (Epac) in mediating vasorelaxation in rat mesenteric arteries. In myography experiments, the Epac-selective cAMP analogue 8-pCPT-2-O-Me-cAMP-AM (5 µM, subsequently referred to as 8-pCPT-AM) elicited a 77.6 ± 7.1% relaxation of phenylephrine-contracted arteries over a 5 min period (mean ± SEM; n = 6). 8-pCPT-AM induced only a 16.7 ± 2.4% relaxation in arteries pre-contracted with high extracellular K(+) over the same time period (n = 10), suggesting that some of Epac's relaxant effect relies upon vascular cell hyperpolarization. This involves Ca(2+)-sensitive, large-conductance K(+) (BK(Ca)) channel opening as iberiotoxin (100 nM) significantly reduced the ability of 8-pCPT-AM to reverse phenylephrine-induced contraction (arteries relaxed by only 35.0 ± 8.5% over a 5 min exposure to 8-pCPT-AM, n = 5; P < 0.05). 8-pCPT-AM increased Ca(2+) spark frequency in Fluo-4-AM-loaded mesenteric myocytes from 0.045 ± 0.008 to 0.103 ± 0.022 sparks s(-1) µm(-1) (P < 0.05) and reversibly increased both the frequency (0.94 ± 0.25 to 2.30 ± 0.72 s(-1)) and amplitude (23.9 ± 3.3 to 35.8 ± 7.7 pA) of spontaneous transient outward currents (STOCs) recorded in isolated mesenteric myocytes (n = 7; P < 0.05). 8-pCPT-AM-activated STOCs were sensitive to iberiotoxin (100 nM) and to ryanodine (30 µM). Current clamp recordings of isolated myocytes showed a 7.9 ± 1.0 mV (n = 10) hyperpolarization in response to 8-pCPT-AM that was sensitive to iberiotoxin (n = 5). Endothelial disruption suppressed 8-pCPT-AM-mediated relaxation in phenylephrine-contracted arteries (24.8 ± 4.9% relaxation after 5 min of exposure, n = 5; P < 0.05), as did apamin and TRAM-34, blockers of Ca(2+)-sensitive, small- and intermediate-conductance K(+) (SK(Ca) and IK(Ca)) channels, respectively, and N(G)-nitro-L-arginine methyl ester, an inhibitor of nitric oxide synthase (NOS). In Fluo-4-AM-loaded mesenteric endothelial cells, 8-pCPT-AM induced a sustained increase in global Ca(2+). Our data suggest that Epac hyperpolarizes smooth muscle by (1) increasing localized Ca(2+) release from ryanodine receptors (Ca(2+) sparks) to activate BK(Ca) channels, and (2) endothelial-dependent mechanisms involving the activation of SK(Ca)/IK(Ca) channels and NOS. Epac-mediated smooth muscle hyperpolarization will limit Ca(2+) entry via voltage-sensitive Ca(2+) channels and represents a novel mechanism of arterial relaxation.

Exchange protein activated by cAMP (Epac) mediates cAMP-dependent but protein kinase A-insensitive modulation of vascular ATP-sensitive potassium channels.

Exchange proteins directly activated by cyclic AMP (Epacs or cAMP-GEF) represent a family of novel cAMP-binding effector proteins. The identification of Epacs and the recent development of pharmacological tools that discriminate between cAMP-mediated pathways have revealed previously unrecognized roles for cAMP that are independent of its traditional target cAMP-dependent protein kinase (PKA). Here we show that Epac exists in a complex with vascular ATP-sensitive potassium (KATP) channel subunits and that cAMP-mediated activation of Epac modulates KATP channel activity via a Ca2+-dependent mechanism involving the activation of Ca2+-sensitive protein phosphatase 2B (PP-2B, calcineurin). Application of the Epac-specific cAMP analogue 8-pCPT-2'-O-Me-cAMP, at concentrations that activate Epac but not PKA, caused a 41.6 +/- 4.7% inhibition (mean +/- S.E.M.; n = 7) of pinacidil-evoked whole-cell KATP currents recorded in isolated rat aortic smooth muscle cells. Importantly, similar results were obtained when cAMP was elevated by addition of the adenylyl cyclase activator forskolin in the presence of the structurally distinct PKA inhibitors, Rp-cAMPS or KT5720. Activation of Epac by 8-pCPT-2'-O-Me-cAMP caused a transient 171.0 +/- 18.0 nM (n = 5) increase in intracellular Ca2+ in Fura-2-loaded aortic myocytes, which persisted in the absence of extracellular Ca2+. Inclusion of the Ca2+-specific chelator BAPTA in the pipette-filling solution or preincubation with the calcineurin inhibitors, cyclosporin A or ascomycin, significantly reduced the ability of 8-pCPT-2'-O-Me-cAMP to inhibit whole-cell KATP currents. These results highlight a previously undescribed cAMP-dependent regulatory mechanism that may be essential for understanding the physiological and pathophysiological roles ascribed to arterial KATP channels in the control of vascular tone and blood flow.

Human keratinocytes release ATP and utilize three mechanisms for nucleotide interconversion at the cell surface.

Nucleotide activation of P2 receptors is important in autocrine and paracrine regulation in many tissues. In the epidermis, nucleotides are involved in proliferation, differentiation, and apoptosis. In this study, we have used a combination of luciferin-luciferase luminometry, pharmacological inhibitors, and confocal microscopy to demonstrate that HaCaT keratinocytes release ATP into the culture medium, and that there are three mechanisms for nucleotide interconversion, resulting in ATP generation at the cell surface. Addition of ADP, GTP, or UTP to culture medium elevated the ATP concentration. ADP to ATP conversion was inhibited by diadenosine pentaphosphate, oligomycin, and UDP, suggesting the involvement of cell surface adenylate kinase, F(1)F(0) ATP synthase, and nucleoside diphosphokinase (NDPK), respectively, which was supported by immunohistochemistry. Simultaneous addition of ADP and GTP elevated ATP above that for each nucleotide alone indicating that GTP acts as a phosphate donor. However, the activity of NDPK, F(1)F(0) ATP synthase or the forward reaction of adenylate kinase could not fully account for the culture medium ATP content. We postulate that this discrepancy is due to the reverse reaction of adenylate kinase utilizing AMP. In normal human skin, F(1)F(0) ATP synthase and NDPK were differentially localized, with mitochondrial expression in the basal layer, and cell surface expression in the differentiated layers. We and others have previously demonstrated that keratinocytes express multiple P2 receptors. In this study we now identify the potential sources of extracellular ATP required to activate these receptors and provide better understanding of the role of nucleotides in normal epidermal homeostasis and wound healing.

P2 receptor-mediated Ca2+ transients in rat cerebral artery smooth muscle cells.

Significant Ca(2+) release was previously noted with the activation of L-type Ca(2+) current in rat superior cerebral artery smooth muscle cells. Here we examined whether the P(2X) current that is partly carried by Ca(2+) also triggers Ca(2+) release in this preparation. Application of P(2X) agonists evoked membrane currents and concomitant Ca(2+) transients in whole cell voltage-clamped single cells. The expected increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) was calculated from the time-integrated P(2X) current by assuming Ca(2+) is the only charge carrier. The measured increase in [Ca(2+)](i) was plotted as a function of the expected increase in [Ca(2+)](i), and Ca(2+)-buffering power was obtained as a reciprocal of the linear fit to this relationship. Both ryanodine, a Ca(2+)-induced Ca(2+)-release inhibitor, and cADP ribose, a putative activator of Ca(2+)-induced Ca(2+) release, had no significant effects on Ca(2+)-buffering power. These results suggest that Ca(2+) influx through P(2X) receptors does not trigger significant Ca(2+) release. We then examined whether P(2X) responses influence the subsequent P(2Y) response. P(2Y) responses were characterized by measuring the rate of [Ca(2+)](i) increase obtained as the slope of the linear regression to the rising phase of the Ca(2+) transient. During simultaneous application of the P(2X) and P(2Y) agonist, the rate of [Ca(2+)](i) increase was facilitated or suppressed depending on the size of the P(2X) receptor-mediated [Ca(2+)](i) increase. Membrane depolarization close to the Ca(2+) equilibrium potential significantly promoted the rate of [Ca(2+)](i) increase. Our results suggest that the [Ca(2+)](i) increase and membrane depolarization caused by the P(2X) current may regulate the subsequent P(2Y) response.

Mitochondrial Ca2+ uptake is important over low [Ca2+]i range in arterial smooth muscle.

Mitochondrial Ca(2+) uptake is usually thought to occur only when intracellular Ca(2+) concentration ([Ca(2+)](i)) is high. We investigated whether mitochondrial Ca(2+) removal participates in shaping [Ca(2+)](i) signals in arterial smooth muscle over a low [Ca(2+)](i) range. [Ca(2+)](i) was measured using fura 2-loaded, voltage-clamped cells from rat femoral arteries. Both diazoxide and carbonyl cyanide m-chlorophenylhydrazone (CCCP) depolarized the mitochondria. Diazoxide application increased resting [Ca(2+)](i), suggesting that Ca(2+) is sequestered in mitochondria. Over a low [Ca(2+)](i) range, diazoxide and CCCP slowed Ca(2+) removal rate, determined after a brief depolarization. When [Ca(2+)](i) was measured during sustained depolarization to -30 mV, CCCP application increased [Ca(2+)](i). When Ca(2+) transients were repeatedly evoked by caffeine applications, CCCP application elevated resting [Ca(2+)](i). Caffeine-induced Ca(2+) transients were compared before and after CCCP application using the half decay time, or time required to reduce increase in [Ca(2+)](i) by 50% (t((1/2))). CCCP treatment significantly increased t((1/2)). These results suggest that Ca(2+) removal to mitochondria in arterial smooth muscle cells may be important at a low [Ca(2+)](i).