thapsigargin and cariporide

Conditions of ischemia-reperfusion disturb the homoeostasis of cytosolic Ca2+ in cardiac microvascular endothelial cells (CMEC), leading to numerous malfunctions of the endothelium. Reperfusion specifically aggravates the Ca2+ overload developed during sustained ischemia. The aim of this study was to identify the origin of the reperfusion-induced part of the Ca2+ overload. Our hypotheses were that this is either due to a Na+-dependent process, e.g. involving the Na+/H+ exchanger (NHE) and/or the Na+/Ca2+ exchanger (NCX), or a process involving the endoplasmic reticulum (ER) and store-operated channels (SOC).. Cultured CMEC from rats were exposed to conditions of simulated ischemia (hypoxia, pH 6.4) and reperfusion (reoxygenation, pH 7.4). Cytosolic Ca2+ ([Ca2+]i) and cytosolic Na+ ([Na+]i) concentrations and cytosolic pH (pHi) were measured with the use of fluorescent indicators. Removal of Ca2+ from the extracellular media during reoxygenation prevented the [Ca2+]i rise. Neither the activation of the NHE nor of the NCX in reoxygenated CMEC caused a change in this [Ca2+]i rise. Complete or partial removal of Na+ from the external media also had no effect on the [Ca2+]i rise. In contrast, specific inhibition of the inositol trisphosphate (InsP3) receptor by xestospongin C (3 micromol/l), of phospholipase (PLC) by U73122 (1 micromol/l), or of SOC by the inhibitors gadolinium (10 micromol/l) or 2-APB (50 micromol/l) lowered or abolished the reoxygenation-induced [Ca2+]i rise.. In CMEC exposed to reperfusion conditions, the enhanced Ca2+ overload is due to Ca2+ influx. The influx is not mediated by a Na+-dependent mechanism, but rather is due to activation of the InsP3 receptor of the ER and activation of SOC.

Topics: Animals; Biological Transport; Calcium; Cell Hypoxia; Cells, Cultured; Cytosol; Endoplasmic Reticulum; Endothelial Cells; Guanidines; Hydrogen-Ion Concentration; Ion Channels; Male; Microcirculation; Myocardial Reperfusion Injury; Myocardium; Ouabain; Rats; Rats, Wistar; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Sodium; Sodium-Hydrogen Exchangers; Sodium-Potassium-Exchanging ATPase; Sulfones; Thapsigargin

Several studies have shown that myocardial ischemia leads to functional failure of endothelial cells (EC) whereby disturbance of Ca(2+) homeostasis may play an important role. The mechanisms leading to Ca(2+) disbalance in ischemic EC are not fully understood. The aim of this study was to test effects of different components of simulated ischemia (glucose deprivation, anoxia, low extracellular pH (pH(o)) and lactate) on Ca(2+) homeostasis in EC.. Cytosolic Ca(2+) (Ca(i)), cytosolic pH (pH(i)) and ATP content were measured in cultured rat coronary EC.. In normoxic cells 60 min glucose deprivation at pH(o) 7.4 had no effect on pH(i). It only slightly increased Ca(i) and decreased ATP content. Reduction of pH(o) to 6.5 under these conditions led to marked cytosolic acidosis and Ca(i) overload, but had no effect on ATP content. Anoxia at pH(o) 6.5 had no additional effect on Ca(i) overload, but significantly reduced cellular ATP. Addition of 20 mmol/l lactate to anoxia at pH(o) 6.5 accelerated Ca(i) overload due to faster cytosolic acidification. Acidosis-induced Ca(i) overload was prevented by inhibition of Ca(2+) release channels of endoplasmic reticulum (ER) with 3 micromol/l ryanodine or by pre-emptying the ER with thapsigargin. Re-normalisation of pH(o) for 30 min led to recovery of pH(i), but not of Ca(i).. The ischemic factors leading to cytosolic acidosis (low pH(o) and lactate) cause Ca(i) overload in endothelial cells, while anoxia and glucose deprivation play only a minor role. The ER is the main source for this Ca(i) rise. Ca(i) overload is not readily reversible.

Topics: Adenosine Triphosphate; Analysis of Variance; Animals; Calcium; Calcium Channel Blockers; Cell Size; Cells, Cultured; Coronary Vessels; Cytosol; Endoplasmic Reticulum; Endothelium, Vascular; Enzyme Inhibitors; Guanidines; Hydrogen-Ion Concentration; Lactic Acid; Male; Manganese; Myocardial Ischemia; Rats; Rats, Wistar; Ryanodine; Sodium-Hydrogen Exchangers; Sulfones; Thapsigargin

We examined the regulation of the Na+/H+ exchangers (NHEs) NHE2 and NHE3 by expressing them in human intestinal C2/bbe cells, which spontaneously differentiate and have little basal apical NHE activity. Unidirectional apical membrane 22Na+ influxes were measured in NHE2-transfected (C2N2) and NHE3-transfected (C2N3) cells under basal and stimulated conditions, and their activities were distinguished as the HOE-642-sensitive and -insensitive components of 5-(N,N-dimethyl)amiloride-inhibitable flux. Both C2N2 and C2N3 cells exhibited increased apical membrane NHE activity under non-acid-loaded conditions compared with nontransfected control cells. NHE2 was inhibited by 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate and thapsigargin, was stimulated by serum, and was unaffected by cGMP- and protein kinase C-dependent pathways. In contrast, NHE3 was inhibited by all regulatory pathways examined. Under acid-loaded conditions (which increase apical Na+ influx), NHE2 and NHE3 exhibited similar patterns of regulation, suggesting that the second messenger effects observed were not secondary to effects on cell pH. Thus, in contrast to their expression in nonepithelial cells, NHE2 and NHE3 expressed in an epithelial cell line behave similarly to endogenously expressed intestinal apical membrane NHEs. We conclude that physiological regulation and function of epithelium-specific NHEs are dependent on tissue-specific factors and/or conditional requirements.

Topics: Amiloride; Cell Line; Cell Membrane; Cyclic AMP; Cyclic GMP; Guanidines; Humans; Intestinal Mucosa; Kinetics; Phenotype; Recombinant Proteins; Second Messenger Systems; Sodium; Sodium-Hydrogen Exchanger 3; Sodium-Hydrogen Exchangers; Sulfones; Tetradecanoylphorbol Acetate; Thapsigargin; Thionucleotides; Time Factors; Transfection