John M. Quayle

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, F1F0 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, F1F0 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, F1F0 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.

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.

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

•  Relaxation of vascular smooth muscle, which increases blood vessel diameter, is often mediated through vasodilator‐induced elevations of intracellular 3′‐5′‐cyclic adenosine monophosphate (cAMP), although the mechanisms are incompletely understood. •  In this study we investigate the role of the novel cAMP effector exchange protein directly activated by cAMP (Epac) in mediating vasorelaxation in rat mesenteric arteries. •  We show that Epac mediates vasorelaxation in mesenteric arteries by facilitating the opening of several subtypes of Ca2+‐sensitive K+ channel within the endothelium and on vascular smooth muscle. •  Epac‐mediated hyperpolarization of the smooth muscle membrane brought about by opening of these channels acts to limit Ca2+ entry via voltage‐gated Ca2+ channels leading to vasorelaxation. •  This represents a potentially important, previously uncharacterised mechanism through which vasodilator‐induced elevation of cAMP can regulate vascular tone and thus blood flow.

TREK-1 K+ Channels in the Cardiovascular System: Their Significance and Potential as a Therapeutic Target

Potassium (K(+) ) channels are important in cardiovascular disease both as drug targets and as a cause of underlying pathology. Voltage-dependent K(+) (K(V) ) channels are inhibited by the class III antiarrhythmic agents. Certain vasodilators work by opening K(+) channels in vascular smooth muscle cells (VSMCs), and K(+) channel activation may also be a route to improving endothelial function. The two-pore domain K(+) (K(2P) ) channels form a group of 15 known channels with an expanding list of functions in the cardiovascular system. One of these K(2P) channels, TREK-1, is the focus of this review. TREK-1 channel activity is tightly regulated by intracellular and extracellular pH, membrane stretch, polyunsaturated fatty acids (PUFAs), temperature, and receptor-coupled second messenger systems. TREK-1 channels are also activated by volatile anesthetics and some neuroprotectant agents, and they are inhibited by selective serotonin reuptake inhibitors (SSRIs) as well as amide local anesthetics. Some of the clinical cardiovascular effects and side effects of these drugs may be through their actions on TREK-1 channels. It has recently been suggested that TREK-1 channels have a role in mechano-electrical coupling in the heart. They also seem important in the vascular responses to PUFAs, and this may underlie some of the beneficial cardiovascular effects of the essential dietary fatty acids. Development of selective TREK-1 openers and inhibitors may provide promising routes for intervention in cardiovascular diseases.

Cholesterol depletion alters coronary artery myocyte Ca2+ signalling in a stimulus-specific manner

Although there is evidence that caveolae and cholesterol play an important role in myocyte signalling processes, details of the mechanisms involved remain sparse. In this paper we have studied for the first time the clinically relevant intact coronary artery and measured in situ Ca2+ signals in individual myocytes using confocal microscopy. We have examined the effect of the cholesterol-depleting agents, methyl-cyclodextrin (MCD) and cholesterol oxidase, on high K+, caffeine and agonist-induced Ca2+ signals. We find that cholesterol depletion produces a stimulus-specific alteration in Ca2+ responses; with 5-HT (10 μM) and endothelin-1 (10 nM) responses being selectively decreased, the phenylephrine response (100 μM) increased and the responses to high K+ (60 mM) and caffeine (10 mM) unaffected. Agonist-induced Ca2+ signals were restored when cholesterol was replenished using cholesterol-saturated MCD. In additional experiments, enzymatically isolated myocytes were patch clamped. We found that cholesterol depletion caused a selective modification of ion channel function, with whole cell inward Ca2+ current being unaltered, whereas outward K+ current was increased, due to BKCa channel activation. There was also a significant decrease in cell capacitance. These data are discussed in terms of the involvement of caveolae in receptor localisation, Ca2+ entry pathways and SR Ca2+ release, and the role of these in agonist signalling.

Atherosclerosis differentially affects calcium signalling in endothelial cells from aortic arch and thoracic aorta in Apolipoprotein E knockout mice

Apolipoprotein‐E knockout (ApoE−/−) mice develop hypercholesterolemia and are a useful model of atherosclerosis. Hypercholesterolemia alters intracellular Ca2+ signalling in vascular endothelial cells but our understanding of these changes, especially in the early stages of the disease process, is limited. We therefore determined whether carbachol‐mediated endothelial Ca2+ signals differ in plaque‐prone aortic arch compared to plaque‐resistant thoracic aorta, of wild‐type and ApoE−/− mice, and how this is affected by age and the presence of hypercholesterolemia. The extent of plaque development was determined using en‐face staining with Sudan IV. Tissues were obtained from wild‐type and ApoE−/− mice at 10 weeks (pre‐plaques) and 24 weeks (established plaques). We found that even before development of plaques, significantly increased Ca2+ responses were observed in arch endothelial cells. Even with aging and plaque formation, ApoE−/− thoracic responses were little changed, however a significantly enhanced Ca2+ response was observed in arch, both adjacent to and away from lesions. In wild‐type mice of any age, 1–2% of cells had oscillatory Ca2+ responses. In young ApoE−/− and plaque‐free regions of older ApoE−/−, this is unchanged. However a significant increase in oscillations (~13–15%) occurred in thoracic and arch cells adjacent to lesions in older mice. Our data suggest that Ca2+ signals in endothelial cells show specific changes both before and with plaque formation, that these changes are greatest in plaque‐prone aortic arch cells, and that these changes will contribute to the reported deterioration of endothelium in atherosclerosis.

Atherosclerosis affects calcium signalling in endothelial cells from apolipoprotein E knockout mice before plaque formation

Little is known about how hypercholesterolaemia affects Ca2+ signalling in the vasculature of ApoE−/− mice, a model of atherosclerosis. Our objectives were therefore to determine (i) if hypercholesterolaemia alters Ca2+ signalling in aortic endothelial cells before overt atherosclerotic lesions occur, (ii) how Ca2+ signals are affected in older plaque-containing mice, and (iii) whether Ca2+ signalling changes were translated into contractility differences. Using confocal microscopy we found agonist-specific Ca2+ changes in endothelial cells. ATP responses were unchanged in ApoE−/− cells and methyl-β-cyclodextrin, which lowers cholesterol, was without effect. In contrast, Ca2+ signals to carbachol were significantly increased in ApoE−/− cells, an effect methyl-β-cyclodextrin reversed. Ca2+ signals were more oscillatory and store-operated Ca2+ entry decreased as mice aged and plaques formed. Despite clearly increased Ca2+ signals, aortic rings pre-contracted with phenylephrine had impaired relaxation to carbachol. This functional deficit increased with age, was not related to ROS generation, and could be partially rescued by methyl-β-cyclodextrin. In conclusion, carbachol-induced calcium signalling and handling are significantly altered in endothelial cells of ApoE−/− mice before plaque development. We speculate that reduction in store-operated Ca2+ entry may result in less efficient activation of eNOS and thus explain the reduced relaxatory response to CCh, despite the enhanced Ca2+ response.

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.

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

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.

Caveolar disruption causes contraction of rat femoral arteries via reduced basal NO release and subsequent closure of BKCa channels

Background and Purpose. Caveolae act as signalling hubs in endothelial and smooth muscle cells. Caveolar disruption by the membrane cholesterol depleting agent methyl-β-cyclodextrin (M-β-CD) has various functional effects on arteries including (i) impairment of endothelium-dependent relaxation, and (ii) alteration of smooth muscle cell (SMC) contraction independently of the endothelium. The aim of this study was to explore the effects of M-β-CD on rat femoral arteries. Methods. Isometric force was measured in rat femoral arteries stimulated to contract with a solution containing 20 mM K+ and 200 nM Bay K 8644 (20 K/Bay K) or with one containing 80 mM K+(80 K). Results. Incubation of arteries with M-β-CD (5 mM, 60 min) increased force in response to 20 K/Bay K but not that induced by 80 K. Application of cholesterol saturated M-β-CD (Ch-MCD, 5 mM, 50 min) reversed the effects of M-β-CD. After mechanical removal of endothelial cells M-β-CD caused only a small enhancement of contractions to 20 K/Bay K. This result suggests M-β-CD acts via altering release of an endothelial-derived vasodilator or vasoconstrictor. When nitric oxide synthase was blocked by pre-incubation of arteries with L-NAME (250 µM) the contraction of arteries to 20 K/Bay K was enhanced, and this effect was abolished by pre-treatment with M-β-CD. This suggests M-β-CD is inhibiting endothelial NO release. Inhibition of large conductance voltage- and Ca2+-activated (BKCa) channels with 2 mM TEA+ or 100 nM Iberiotoxin (IbTX) enhanced 20 K/Bay K contractions. L-NAME attenuated the contractile effect of IbTX, as did endothelial removal. Conclusions. Our results suggest caveolar disruption results in decreased release of endothelial-derived nitric oxide in rat femoral artery, resulting in a reduced contribution of BKCa channels to the smooth muscle cell membrane potential, causing depolarisation and contraction.