Thrombin is a multifunctional protease, which, in addition to its established role in blood coagulation and thrombus formation, induces a variety of cellular responses through the recently cloned thrombin receptor (for review, see Coughlin 4 ). 1 2 In cardiac myocytes, as in other cell types, the activity of the Na +-H + exchanger is regulated not only by pH i but also by a number of extracellular stimuli (such as exposure to adrenergic agonists, endothelin, angiotensin II, and adenosine triphosphate) through receptor-mediated mechanisms (for recent review, see Pucéat and Vassort 3 ). 1 Recent evidence suggests that the exchanger may also play an important role in determining the severity of the unfavorable sequelae of myocardial ischemia and reperfusion, such as arrhythmias, contractile dysfunction, and infarction. The sarcolemmal Na +-H + exchanger is a major acid extrusion system in cardiac myocytes and plays an important role in restoration of pH i following an acid load. We conclude that (1) adult rat ventricular myocytes express a functional thrombin receptor, whose stimulation results in increased activity of the sarcolemmal Na +-H + exchanger, and (2) this effect appears to occur through a protein kinase C–mediated mechanism. Neither 5 U/mL thrombin nor 25 μmol/L SFLLRN affected resting pH i and Ca 2+ or background acid loading. In the presence of 10 μmol/L HOE-694 (a Na +-H + exchanger inhibitor), pH i did not recover after an acid load, even during exposure to 5 U/mL thrombin or 25 μmol/L SFLLRN, confirming that the Na +-H + exchanger was the primary acid efflux mechanism under the conditions used. In cells pretreated with 100 nmol/L GF109203X or 5 μmol/L chelerythrine (protein kinase C inhibitors), neither 5 U/mL thrombin nor 25 μmol/L SFLLRN produced a significant increase in J H. In contrast, an inactive control peptide (FLLRN) was without effect at 25 μmol/L. A hexameric thrombin receptor–activating peptide (SFLLRN) mimicked the effect of thrombin and increased J H by 73% ( P<.05) at 25 μmol/L. However, when the second pulse occurred in the presence of 0.2, 1, or 5 U/mL thrombin, J H increased by 30%, 62% ( P<.05), and 87% ( P<.05), respectively. At a pH i of 6.9, J H did not change significantly during the second pulse relative to the first in control cells.
As an index of Na +-H + exchanger activity, acid efflux rates ( J Hs) were determined in single myocytes (n=4 to 11 per group) loaded with the pH-sensitive fluoroprobe carboxy-seminaphthorhodafluor-1 after two consecutive intracellular acid pulses (induced by transient exposure to 20 mmol/L NH 4Cl) in bicarbonate-free medium. Reverse-transcription polymerase chain reaction analysis revealed thrombin receptor mRNA expression in a myocyte-enriched cell preparation. We have studied (1) the effect of thrombin on the activity of the sarcolemmal Na +-H + exchanger in freshly isolated quiescent ventricular myocytes from the adult rat heart and (2) the signaling mechanism(s) underlying any effect. Thrombin can activate the plasma membrane Na +-H + exchanger in a variety of noncardiac cells. Customer Service and Ordering Information.Stroke: Vascular and Interventional Neurology.Journal of the American Heart Association (JAHA).Circ: Cardiovascular Quality & Outcomes.Arteriosclerosis, Thrombosis, and Vascular Biology (ATVB).This additional content includes new spells, weapons, units, heroes, buildings, quests and more.
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