thapsigargin and Hyperlipidemias

No studies exist concerning the ability of the plasma membrane Ca(2+) pump (PMCA), sarcoplasmic reticulum Ca(2+) pump (SERCA) and Na(+)-Ca(2+) exchanger (NCX) to regulate myoplasmic Ca(2+) (Ca(m)) in vascular smooth muscle cells from diabetic individuals with dyslipidemia. We tested the hypothesis that diabetic dyslipidemia would increase vascular smooth muscle cells to buffer Ca(m). Cells were isolated from the coronary artery of male Yucatan pigs treated for 20 weeks with: (1) a low fat diet (control group); (2) a high fat/cholesterol diet (F group); or (3) alloxan-induced diabetic pigs fed the high fat diet (DF group). The maximum Ca(m) response to a depolarizing 80 mM KCl (80 K) solution was evaluated in the absence and presence of thapsigargin (TSG; inhibits SERCA) and low Na (inhibits NCX). In response to 80 K alone, there was no difference in the Ca(m) response between groups. In the presence of TSG, the 80 K response decreased by 43% in the DF group; TSG did not affect the 80 K response in the control and F groups. When exposed to both TSG and low Na, the 80 K response also decreased by 55% in the DF group. This suggests increased Ca(m) buffering by the PMCA and/or mitochondria in the DF group when SERCA and NCX are inhibited. Compared to the control and F groups, low Na alone elicited a 50% lower Ca(m) amplitude in the DF group, which was reversed with TSG treatment; this suggests that SERCA activity is increased in DF pigs. Western blots also indicated a 7-fold increase in the approximately 115 kDa band density of an anti-SERCA2 antibody in DF compared to control pigs. This is the first report to demonstrate increased Ca(2+) buffering, specifically by SERCA, in vascular smooth muscle cells from diabetic individuals with dyslipidemia.

Topics: Analysis of Variance; Animals; Blotting, Western; Buffers; Calcium-Transporting ATPases; Cells, Cultured; Coronary Vessels; Diabetes Mellitus, Experimental; Disease Models, Animal; Enzyme Inhibitors; Hyperlipidemias; Myocytes, Smooth Muscle; Probability; Reference Values; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Sensitivity and Specificity; Swine; Thapsigargin

Hyperlipidemia represents a major risk factor for development of vascular dysfunction and atherosclerosis. Although the unfortunate role of low-density lipoprotein has been clearly demonstrated, the mechanistic pathways through which triglyceride-derived free fatty acids (FFAs) contribute to vascular disorders are not completely understood. Thus, the present study was designed to elucidate the effects of FFAs on cultured endothelial cells. The Ca(2+) signaling, endothelial nitric oxide synthase (eNOS) activity, and production of superoxide anions (.O(2)(-)) were monitored in cells treated with bovine serum albumin-conjugated FFA. FFA-loaded cells showed enhanced intracellular Ca(2+) release in response to ATP, histamine, or the SERCA inhibitor thapsigargin. This effect corresponded to an overall increase in intracellularly stored Ca(2+). In contrast, autacoid-triggered elevation of cytosolic free Ca(2+) concentration was blunted in FFA-loaded cells due to inhibition of capacitative Ca(2+) entry. In agreement with the reduced Ca(2+) signaling, the Ca(2+)-dependent activity of eNOS was reduced under basal conditions and if cells were stimulated with ATP, histamine, or thapsigargin. The attenuated eNOS activity was associated with.O(2)(-) release in FFA-loaded cells. These data indicate that FFAs significantly affect endothelial Ca(2+) signaling, eNOS activity, and.O(2)(-) release and, thus, might contribute to vascular dysfunction in atherogenesis.

Topics: Adenosine Triphosphate; Albumins; Anions; Calcium; Calcium-Transporting ATPases; Cells, Cultured; Dose-Response Relationship, Drug; Endothelium, Vascular; Fatty Acids, Nonesterified; Histamine; Humans; Hyperlipidemias; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Oleic Acid; Oxygen; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Signal Transduction; Superoxides; Thapsigargin; Umbilical Veins

Intracellular Ca(2+) store loading has been shown to alter proliferation and apoptosis of several cell types. In addition, HMG-CoA reductase inhibitors (i.e. atorvastatin) are effective in treating diabetic dyslipidemic patients. Thus, we hypothesized that chronic atorvastatin treatment would prevent increased Ca(2+) uptake into intracellular Ca(2+) stores in vascular smooth muscle cells from diabetic dyslipidemic pigs. Male Yucatan pigs were divided into four groups for 20 weeks-- (1) low fat fed (control); (2) hyperlipidemic (F); (3) alloxan-induced diabetic dyslipidemic (DF); and (4) diabetic dyslipidemic pigs treated with atorvastatin (DFA). The F, DF, and DFA groups were fed a high fat/cholesterol diet. Cells were isolated from the coronary artery and the myoplasmic Ca(2+) (Ca(m)) response measured using single cell fura-2 imaging. The Ca(m) response to caffeine (5 mM to release Ca(2+) from the sarcoplasmic reticulum, SR) and ionomycin (10 microM; to release the total Ca(2+) store) was determined in either the presence of low Na (19Na; inhibits Na(+)-Ca(2+) exchange), thapsigargin (TSG; inhibits the SR Ca(2+) pump), and a 19Na+TSG solution. Low Na induced the uptake of Ca(2+) into both SR and non-SR Ca(2+) stores in the DF group, but not the DFA group. Furthermore, after depletion of the SR Ca(2+) store with TSG, 19Na evoked Ca(2+) uptake into non-SR Ca(2+) stores in all three groups except in the DFA group. In summary, this study demonstrates that atorvastatin prevents the enhanced uptake of Ca(2+) by SR and non-SR Ca(2+) stores in diabetic dyslipidemic pigs.

Topics: Animals; Atorvastatin; Caffeine; Calcium; Coronary Vessels; Cytoplasm; Diabetes Mellitus, Experimental; Diet, Atherogenic; Fluorescent Dyes; Fura-2; Heptanoic Acids; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Hyperlipidemias; In Vitro Techniques; Ionomycin; Ionophores; Male; Microscopy, Fluorescence; Muscle, Smooth, Vascular; Pyrroles; Sarcoplasmic Reticulum; Sodium; Swine; Swine, Miniature; Thapsigargin