Soft tissue calcification in the Ossabaw miniature pig: experimental and kinetic modeling studies.

UNLABELLED: Calcium (Ca) deposition into vascular tissue was measured in Ossabaw miniature pigs with and without metabolic syndrome (MetS) using Ca tracer kinetics and coronary atherosclerosis measured with intravascular ultrasound. Pigs with MetS had higher Ca uptake into coronary arteries than lean pigs. INTRODUCTION: Ca deposition into arteries is a common disease in humans. The Ossabaw pig develops MetS when fed an atherogenic diet. The aim of this study was to measure Ca deposition into arteries of lean vs. MetS pigs. METHODS: Male pigs were fed for 5 months with chow diet (healthy, lean; n = 7) or atherogenic diet (n = 8) consisting of chow supplemented with 2 % cholesterol, 43 % kcal from fat, and 20 % kcal from fructose. Pigs were verified to have MetS by obesity, insulin resistance, impaired glucose tolerance, dyslipidemia, and hypertension. Two pigs received 50 nCi of (41)Ca i.v. and blood was drawn frequently for 24 h, and 2, 3, 6, 8, 10, 15, 20, and at sacrifice at 28 days after injection. Peripheral arteries were biopsied four times per pig over the 28th day and coronary artery sampled at sacrifice. Tissues were analyzed for (41)Ca:Ca. A compartmental model was used to estimate rates of Ca deposition into the arteries. RESULTS: The MetS swine had higher (41)Ca and atherosclerosis in coronary arteries than lean pigs. CONCLUSIONS: This pig model is a suitable model for studying vascular calcification in humans.

Fat-induced membrane cholesterol accrual provokes cortical filamentous actin destabilisation and glucose transport dysfunction in skeletal muscle.

AIMS/HYPOTHESIS: Diminished cortical filamentous actin (F-actin) has been implicated in skeletal muscle insulin resistance, yet the mechanism(s) is unknown. Here we tested the hypothesis that changes in membrane cholesterol could be a causative factor, as organised F-actin structure emanates from cholesterol-enriched raft microdomains at the plasma membrane. METHODS: Skeletal muscle samples from high-fat-fed animals and insulin-sensitive and insulin-resistant human participants were evaluated. The study also used L6 myotubes to directly determine the impact of fatty acids (FAs) on membrane/cytoskeletal variables and insulin action. RESULTS: High-fat-fed insulin-resistant animals displayed elevated levels of membrane cholesterol and reduced F-actin structure compared with normal chow-fed animals. Moreover, human muscle biopsies revealed an inverse correlation between membrane cholesterol and whole-body glucose disposal. Palmitate-induced insulin-resistant myotubes displayed membrane cholesterol accrual and F-actin loss. Cholesterol lowering protected against the palmitate-induced defects, whereas characteristically measured defects in insulin signalling were not corrected. Conversely, cholesterol loading of L6 myotube membranes provoked a palmitate-like cytoskeletal/GLUT4 derangement. Mechanistically, we observed a palmitate-induced increase in O-linked glycosylation, an end-product of the hexosamine biosynthesis pathway (HBP). Consistent with HBP activity affecting the transcription of various genes, we observed an increase in Hmgcr, a gene that encodes 3-hydroxy-3-methyl-glutaryl coenzyme A reductase, the rate-limiting enzyme in cholesterol synthesis. In line with increased HBP activity transcriptionally provoking a membrane cholesterol-based insulin-resistant state, HBP inhibition attenuated Hmgcr expression and prevented membrane cholesterol accrual, F-actin loss and GLUT4/glucose transport dysfunction. CONCLUSIONS/INTERPRETATION: Our results suggest a novel cholesterolgenic-based mechanism of FA-induced membrane/cytoskeletal disorder and insulin resistance.

Tuning in to the 'right' calcium channel regulation in experimental models of diabetes.

Elucidation of cellular and molecular mechanisms underlying vascular disease is of fundamental importance to the development of pharmacological agents to target these pathways. Pinho et al. in this issue of the BJP provide highly compelling evidence that the δ isoform of phosphatidyl inositol 3-kinase (PI3K δ) was upregulated and accounted for the increase in L-type, voltage-gated, Ca channel current in aortic vascular smooth muscle (VSM) cells of a mouse model of type 1 diabetes. There are several key issues of broad fundamental significance to this work. Firstly, what is the 'right' answer about calcium channel regulation in diabetes? Conflicting reports of increased and decreased Ca channel current may be due to specificity of the vascular bed and species. Then, the time course of diabetic vasculopathy may influence the expression of contractile versus proliferative phenotypes of VSM. Also the metabolic characterization of diabetes may enlighten or confound any study of diabetic vascular disease. These issues need attention to move forward work in this area.

Diabetic dyslipidemia and exercise affect coronary tone and differential regulation of conduit and microvessel K+ current.

Spontaneous transient outward K(+) currents (STOCs) elicited by Ca(2+) sparks and steady-state K(+) currents modulate vascular reactivity, but effects of artery size, diabetic dyslipidemia, and exercise on these differentially regulated K(+) currents are unclear. We studied the conduit arteries and microvessels of male Yucatan swine assigned to one of three groups for 20 wk: control (C, n = 7), diabetic dyslipidemic (DD, n = 6), or treadmill-trained DD animals (DDX, n = 7). Circumflex artery blood flow velocity obtained with intracoronary Doppler and lumen diameters obtained by intravascular ultrasound enabled calculation of absolute coronary blood flow (CBF). Ca(2+) sparks were determined in pressurized microvessels, and perforated patch clamp assessed K(+) current in smooth muscle cells isolated from conduits and microvessels. Baseline CBF in DD was decreased versus C. In pressurized microvessels, Ca(2+) spark activity was significantly lower in DD versus C and DDX (P < 0.05 vs. DDX). STOCs were pronounced in microvessel (approximately 35 STOCs/min) in sharp contrast to conduit cells ( approximately 2 STOCs/min). STOCs were decreased by 86% in DD versus C and DDX in microvessels; in contrast, there was no difference in STOCs across groups in conduit cells. Steady-state K(+) current in microvessels was decreased in DD and DDX versus C; in contrast, steady-state K(+) current in conduit cells was decreased in DDX versus DD and C. We conclude that steady-state K(+) current and STOCs are differentially regulated in conduit versus microvessels in health and diabetic dyslipidemia. Exercise prevented diabetic dyslipidemia-induced decreases in baseline CBF, possibly via STOC-regulated basal microvascular tone.

Exercise prevents diabetes-induced impairment in superficial buffer barrier in porcine coronary smooth muscle.

In healthy coronary smooth muscle cells, the superficial sarcoplasmic reticulum (SR) buffers rise in intracellular Ca(2+) levels. In diabetic dyslipidemia, basal Ca(2+) levels are increased, yet Ca(2+) influx is decreased and SR Ca(2+) uptake is increased. Exercise prevents diabetic dyslipidemia-induced increases in basal Ca(2+) levels and decreases in Ca(2+) influx. We tested the hypothesis that diabetic dyslipidemia impairs Ca(2+) extrusion via a decrease in superficial SR and that exercise will prevent these losses. Male Yucatan swine were maintained in four treatment groups: control, hyperlipidemic, diabetic dyslipidemic, and diabetic dyslipidemic plus aerobically exercise trained. Intracellular Ca(2+) levels were measured during depolarization-induced Ca(2+) influx and caffeine-induced SR Ca(2+) release. Na(+)/Ca(2+) exchanger and plasmalemmal Ca(2+)-ATPase activity were assessed by inhibition with low extracellular Na(+) and 5,6-carboxyeosin, respectively. Superficial SR was quantified using the internal membrane dye 3,3'-dihexyloxacarbocyanine iodide (DiOC(6)) and novel analysis techniques. We found that, in diabetic dyslipidemia, Ca(2+) extrusion was impaired and superficial SR was decreased. Exercise prevented the diabetic dyslipidemia-induced decrease in superficial SR and restored plasmalemmal Ca(2+) extrusion. On the basis of these results, we conclude exercise attenuates the diabetic dyslipidemia-induced impairment in intracellular Ca(2+) regulation.

Altered functional coupling of coronary K+ channels in diabetic dyslipidemic pigs is prevented by exercise.

Chronic hyperglycemia and hypercholesterolemia have been shown to alter ionic currents in vascular smooth muscle. We tested the hypothesis that the combined effect of hyperglycemia and hyperlipidemia (diabetic dyslipidemia) would increase the Ca2+-sensitive K+ (KCa) current as a compensatory response to an increase in intracellular Ca2+ concentration. We also hypothesized that exercise training would prevent this elevation in KCa current. Miniature Yucatan swine were randomly assigned to five groups: control, standard pig chow (C, n = 6); hyperlipidemic, high-fat pig chow (H, n = 5); diabetic, standard pig chow (D, n = 7); diabetic, high-fat pig chow ("diabetic dyslipidemic," DD, n = 12); and exercise-trained DD (DDX, n = 9). High-fat chow consisted of standard minipig chow supplemented with cholesterol (2%) and coconut oil. Increased coronary vasoconstriction assessed in vivo and in vitro in DD was prevented by exercise. Patch-clamp experiments performed on right coronary artery smooth muscle cells resulted in greater K+ current densities in the H, D, and DD groups vs. the DDX group between -10 and 40 mV. In fura 2-loaded cells, current activated by caffeine-induced Ca2+ release was greater in H, D, and DD compared with C and DDX (P < 0.05), whereas intracellular Ca2+ concentration was not different across groups. Finally, there were no differences in the KCa or Kv channel protein content between groups. These data indicate that hyperglycemia, hyperlipidemia, and diabetic dyslipidemia lead to elevated whole cell K+ current and increased functional coupling of KCa and Ca2+ release. Endurance exercise prevented increased coupling of Ca2+ release to KCa channel activation in diabetic dyslipidemia.

Increased endothelin-induced Ca2+ signaling, tyrosine phosphorylation, and coronary artery disease in diabetic dyslipidemic Swine are prevented by atorvastatin.

Endothelin-1 (ET-1) signaling mechanisms have been implicated in the pathogenesis of excess coronary artery disease in diabetic dyslipidemia. We hypothesized that in diabetic dyslipidemia ET-1-induced coronary smooth muscle calcium (Ca2+m) and tyrosine phosphorylation would be increased, and the lipid lowering agent, atorvastatin, would inhibit these increases. Male Yucatan miniature swine groups were treated for 20 weeks: normal low-fat fed control, high-fat/cholesterol fed (hyperlipidemic), hyperlipidemic made diabetic with alloxan (diabetic dyslipidemic), and diabetic dyslipidemic treated with atorvastatin (atorvastatin-treated). Blood glucose values were 5-fold greater in diabetic dyslipidemic and atorvastatin-treated versus control and hyperlipidemic. Total and low-density lipoprotein (LDL) plasma cholesterol in hyperlipidemic, diabetic dyslipidemic, and atorvastatin-treated were approximately 5-fold greater than control. Intravascular ultrasound detectable coronary disease and hypertriglyceridemia were only observed in diabetic dyslipidemic and were abolished by atorvastatin. In freshly isolated cells, the Ca2+m response to ET-1 in diabetic dyslipidemic was greater than in control, hyperlipidemic, and atorvastatin-treated groups. Selective ET-1 receptor antagonists showed in the control group that the ETB subtype inhibits ETA regulation of Ca2+m. There was almost a complete switch of receptor subtype regulation of Ca2+m from largely ETA in control to an increased inhibitory interaction between ETA and ETB in hyperlipidemic and diabetic dyslipidemic groups, such that neither ETA nor ETB antagonist alone could block the ET-1-induced Ca2+m response. The inhibitory interaction was attenuated in the atorvastatin-treated group. In single cells, basal and ET-1-induced tyrosine phosphorylation in diabetic dyslipidemic were more than 3- and 6-fold greater, respectively, than in control, hyperlipidemic, and atorvastatin-treated. Attenuation by atorvastatin of coronary disease and ET-1-induced Ca2+m and tyrosine phosphorylation signaling with no change in cholesterol provides strong evidence for direct actions of atorvastatin and/or triglycerides on the vascular wall.

Exercise training attenuates coronary smooth muscle phenotypic modulation and nuclear Ca2+ signaling.

Physical inactivity is an independent risk factor for coronary heart disease, yet the mechanism(s) of exercise-related cardioprotection remains unknown. We tested the hypothesis that coronary smooth muscle after exercise training would have decreased mitogen-induced phenotypic modulation and enhanced regulation of nuclear Ca(2+). Yucatan swine were endurance exercise trained (EX) on a treadmill for 16-20 wk. EX reduced endothelin-1-induced DNA content by 40% compared with sedentary (SED) swine (P < 0.01). EX decreased single cell peak endothelin-1-induced cytosolic Ca(2+) responses compared with SED by 16% and peak nuclear Ca(2+) responses by 33% (P < 0.05), as determined by confocal microscopy. On the basis of these results, we hypothesized that sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) and intracellular Ca(2+) stores in native smooth muscle are spatially localized to dissociate cytosolic Ca(2+) and nuclear Ca(2+). Subcellular localization of SERCA in living and fixed cells revealed a distribution of SERCA near the sarcolemma and on the nuclear envelope. These results show that EX enhances nuclear Ca(2+) regulation, possibly via SERCA, which may be one mechanism by which coronary smooth muscle cells from EX are less responsive to mitogen-induced phenotypic modulation.

Atorvastatin treatment prevents alterations in coronary smooth muscle nuclear Ca2+ signaling in diabetic dyslipidemia.

Atorvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, alters bulk myoplasmic Ca2+ regulation and inhibits phenotypic modulation and proliferation of vascular smooth muscle in culture. Nuclear Ca2+ (Ca(n)) signaling is tightly coupled to transcriptional events and cell growth. Therefore, we hypothesized that in vivo treatment with atorvastatin would attenuate alterations in mitogen-induced Ca(n) signaling associated with coronary atherosclerosis. Three groups of male Yucatan pigs were treated for 20 weeks: controls, alloxan-induced diabetics fed an atherogenic diet and diabetics fed an atherogenic diet plus atorvastatin (80 mg/day). Right coronary artery single-cell cytosolic Ca2+ (Ca(c)) and Ca(n) responses to the mitogen endothelin-1 (5 x 10(-8) M) were measured by laser confocal microscopy using the calcium indicator Fluo-4. We observed a 39% increase in Ca(c) and a 52% increase in Ca(n) responses to endothelin-1 in cells from diabetic dyslipidemic arteries compared to control. These alterations were prevented in animals treated with atorvastatin. We show that during proliferation, the nucleus of a smooth muscle cell becomes rounded and loses the characteristic multilobular shape, clefts and invaginations. Consistent with this, a redistribution of Ca2+ stores from a transnuclear morphology in controls to a more perinuclear morphology occurred in cells from diabetic dyslipidemic arteries and was prevented by atorvastatin. In addition, the peak Ca(n) responses to endothelin-1 were inversely correlated (r = 0.712) with the extent of the transnuclear distribution of Ca2+ stores and directly correlated (r = 0.874) with the extent of atherosclerosis, as assessed in vivo by intravascular ultrasound. These findings indicate that chronic treatment with atorvastatin directly decreases mitogen-induced Ca(n) mobilization, which we suggest is related to the spatial localization of Ca(n) stores.

Effect of atorvastatin on intracellular calcium uptake in coronary smooth muscle cells from diabetic pigs fed an atherogenic diet.

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.

Hydrogen peroxide activates Na(+)-dependent Ca(2+) influx in coronary endothelial cells.

The purpose of the present study was to examine the effect of short duration H(2)O(2) exposure on coronary artery endothelial cell [Ca(2+)](i) regulation. Freshly dispersed cells from porcine coronary artery were exposed to H(2)O(2) (300 micromol/L) for 3 min while monitoring [Ca(2+)](i) using fura-2 microfluorometry. H(2)O(2) increased [Ca(2+)](i) from 0.86 +/- 0.03 to 2.19 +/- 0.41 ratio units at 3 min of H(2)O(2) (P < 0.05). Intracellular Ca(2+) remained elevated 3 min following removal of H(2)O(2), yet H(2)O(2) had no effect on the subsequent [Ca(2+)](i) response to bradykinin (0.1 micromol/L). The H(2)O(2)-induced [Ca(2+)](i) increase was completely abolished either by removal of extracellular Ca(2+) or lowering extracellular Na(+). Cells exposed to the Na(+) ionophore, monensin, showed an increase in [Ca(2+)](i) with a time course similar to that seen with H(2)O(2). Furthermore, H(2)O(2)-induced Ca(2+) influx was not attenuated by either Ni(2+) (300 micromol/L) or econazole (10 micromol/L), excluding Ca(2+) influx via the agonist-sensitive pathway. Thus, in coronary arterial endothelial cells, H(2)O(2) increases Ca(2+) influx in an extracellular Na(+)-dependent manner via an agonist-insensitive pathway.

Functional nucleotide receptor expression and sarcoplasmic reticulum morphology in dedifferentiated porcine coronary smooth muscle cells.

The phenotypic dedifferentiation of vascular smooth muscle cells (SMCs) is an early event associated with cell culturing and vascular injury. The purpose of this study was to evaluate the SMC phenotype underlying the functional responsiveness of SMCs to nucleotides in organ culture. Porcine coronary arteries were either used fresh, cold stored (5 degrees C) 4 days, or organ cultured (37 degrees C) 4 days. Fura-2 digital imaging of single SMCs was used to measure the myoplasmic calcium (Ca(m)) response to 10 microM of the following nucleotide receptor agonists: UTP, UDP, ATP, ADP, and 2-MeSATP. In contrast to the nucleotides UDP, ATP, ADP, and 2-MeSATP, the Ca(m) response increased 10-fold and the number of cells that responded to UTP increased 5-fold in SMCs from organ culture compared to SMCs from fresh or cold-stored arteries. Simultaneous imaging of Ca(m), DNA content, and SR distribution in SMCs from organ culture indicated that the UTP-induced Ca(m) increase occurred exclusively in SMCs that had a dedifferentiated cell phenotype. Three-dimensional image reconstruction of the nucleus and sarcoplasmic reticulum (SR) revealed a novel transnuclear SR distribution that intertwined with the nucleus in fresh SMCs, while in SMCs from organ culture the SR was predominantly perinuclear and cytoplasmic. This study demonstrates that the functional up-regulation of UTP-sensitive receptors and the disappearance of the transnuclear SR distribution are novel features of dedifferentiated coronary SMCs.

Calcium buffering in coronary smooth muscle after chronic occlusion and exercise training.

OBJECTIVE: Exercise promotes "sarcoplasmic reticulum (SR) Ca2+ unloading" in porcine coronary smooth muscle, resulting in decreased agonist-induced Ca2+ release. We studied Ca2+ handling in healthy, non-occluded right coronary artery cells from hearts chronically occluded at the circumflex artery. METHODS: Myoplasmic free Ca2+ (Ca(m)) was assessed with fura-2 in cells from sedentary (n=8) and aerobically exercise-trained (n=6) female Yucatan pigs after 6-month circumflex artery ameroid occlusion (OCC) and in cells from non-occluded, sedentary pigs (SED, n=5). First, Ca influx was induced by 80 mM KCl depolarization (priming step) followed by 5 mM caffeine to elicit maximal Ca2+ release and depletion. The SR was Ca-loaded again by depolarization and then exposed to caffeine after 2- or 11-min recovery to compare SR Ca2+ unloading. RESULTS: Baseline Ca(m), caffeine-induced peak Ca(m), and depolarization-induced maximum Ca(m) were decreased, and depolarization-induced time-to-half-maximum was increased in OCC vs. SED pigs, suggesting a tonic Ca2+ buffering (lowering) effect of occlusion. Exercise did not alter these effects. SR Ca2+ unloading occurred only in SED, as evidenced by decreased caffeine-induced Ca2+ release after 11 min of recovery, and was inhibited by low extracellular Na+. CONCLUSIONS: SR Ca2+ unloading can be demonstrated in coronary smooth muscle from sedentary pigs using a novel SR Ca2+ unloading protocol, and Ca2+ unloading partly depends on Na+-Ca2+ exchange activity. Furthermore, SR Ca2+ unloading in cells from non-occluded right coronary arteries of chronically circumflex-occluded pig hearts was not altered by exercise, perhaps due to enhanced tonic Ca2+ extrusion versus cells from normal, sedentary animals.

Sarcoplasmic reticulum Ca(2+) uptake is impaired in coronary smooth muscle distal to coronary occlusion.

After chronic occlusion, collateral-dependent coronary arteries exhibit alterations in both vasomotor reactivity and associated myoplasmic free Ca(2+) levels that are prevented by chronic exercise training. We tested the hypotheses that coronary occlusion diminishes Ca(2+) uptake by the sarcoplasmic reticulum (SR) and that exercise training would prevent impaired SR Ca(2+) uptake. Ameroid constrictors were surgically placed around the proximal left circumflex (LCx) artery of female swine 8 wk before initiating 16-wk sedentary (pen confined) or exercise-training (treadmill run) protocols. Twenty-four weeks after Ameroid placement, smooth muscles cells were enzymatically dissociated from both the LCx and nonoccluded left anterior descending (LAD) arteries of sedentary and exercise-trained pigs, and myoplasmic free Ca(2+) was studied using fura 2 microfluorometry. After the SR Ca(2+) store was partially depleted with caffeine (5 mM), KCl-induced membrane depolarization produced a significant decrease in the time to half-maximal (t(1/2)) myoplasmic free Ca(2+) accumulation in LCx versus LAD cells of sedentary pigs. Furthermore, inhibition of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA; 10 microM cyclopiazonic acid) significantly reduced t(1/2) in cells isolated from the LAD but not from the LCx. Exercise training did not prevent the differences in t(1/2) myoplasmic free Ca(2+) accumulation observed between LCx and LAD cells. Occlusion or exercise training did not alter SERCA protein levels. These results support our hypothesis of impaired SR Ca(2+) uptake in coronary smooth muscle cells isolated distal to chronic occlusion. Impaired SR Ca(2+) uptake was independent of SERCA protein levels and was not prevented by exercise training.

Endotoxin impairs agonist-stimulated intracellular free calcium (Ca(i)) responses in freshly dispersed aortic endothelial cells.

Impairment in endothelial cell intracellular free calcium (Ca(i)) mobilization mechanisms may contribute to decreased nitric oxide (NO) biosynthesis and impaired vasorelaxation responses of endotoxemic guinea pigs to endothelium-dependent vasodilators. We tested this hypothesis using fura-2 microfluorometry to compare agonist-stimulated Ca(i) responses of aortic endothelial cells freshly dispersed from guinea pigs 16 h after intraperitoneal injection of Escherichia coli endotoxin (lipopolysaccharide, LPS; 4 mg/kg) or saline (CON). In the presence of normal extracellular Ca2+ (2 mmol/L), basal (non-stimulated) endothelial Ca(i) (340/380 nm fluorescence ratio, R) was not different between CON and LPS cells (1.1 +/- 0.03 and 1.1 +/- 0.03, respectively). However, exposure to ADP (10 micromol/L) produced a biphasic increase in Ca(i) that was markedly decreased in cells from LPS-treated animals (P < 0.0001). Peak ADP-stimulated Ca(i) responses averaged 2.2 +/- 0.21 in CON cells and 1.5 +/- 0.11 (P < 0.01) in cells dispersed from LPS-treated animals. Exposure to acetylcholine (ACh; 10 micromol/L) produced sustained increases in Ca(i) (R = 1.4 +/- 0.13) in CON cells; however, LPS abolished Ca(i) responses to ACh. Exposure of endothelial cells to substance P (100 nmol/L) produced a biphasic increase in Ca(i) that was not different between groups. In the absence of extracellular Ca2+ (plus 10 micromol/L EGTA), exposure to ADP (10 micromol/L) produced transient increases in Ca(i) (Ca2+ release) that were decreased in cells from LPS-treated versus CON animals. Exposure to ACh in zero Ca2+ (10 micromol/L) produced smaller increases in Ca(i) (peak R = 1.3 +/- 0.12) in CON cells (when compared to ADP); however, Ca(i) responses to ACh remained absent in cells from LPS-treated animals. Re-exposure to Ca2+ produced sustained ACh-induced Ca(i) responses (Ca2+ influx) in cells from CON, but not LPS-treated animals; LPS markedly impaired (P< 0.05) ADP-induced sustained Ca(i) responses. Our data demonstrate that in vivo LPS exposure elicits decreased agonist-stimulated endothelial Ca(i) responses primarily involving impaired Ca2+ influx mechanisms. Known dependence of endothelial agonist-stimulated NO synthesis on Ca(i) suggests that defects in cell Ca2+ mobilization may contribute to LPS-induced impaired NO biosynthesis and decreased endothelium-dependent relaxation.

Alterations in the oxidative metabolic profile in vascular smooth muscle from hyperlipidemic and diabetic swine.

High cholesterol, especially LDL cholesterol, has been associated with the development of atherosclerotic plaques in arteries. To investigate the changes in cellular substrate metabolism early in the atherogenic process, Sinclair miniature swine were treated for 12 weeks with either a control diet, a high fat diet, or a high fat diet with the addition of alloxan to induce diabetes. The fractional entry into the TCA cycle of 1,2-(13)C-acetate (5 mM), 1-(13)C-glucose (5 mM), and unlabeled, endogenous lipids was determined in control, hyperlipidemic, and diabetic/hyperlipidemic pigs using 13C-isotopomer analysis of glutamate. The diabetic state of the pigs was validated by plasma glucose measurements made after 10 weeks of alloxan treatment for control (65 +/- 6 mg/dL), hyperlipidemic (63 +/- 5 mg/dL), and diabetic/hyperlipidemic (333 +/- 52 mg/dL) pigs. Plasma glucose values did not correlate with the percentage of glucose entry into the TCA cycle (R2 = 0.0819, n = 10). Alterations in the pattern of substrate oxidation were better correlated with changes in plasma lipids (cholesterol and triglycerides) than with changes in plasma glucose. Plasma total cholesterol and total triglyceride levels significantly correlated with changes in acetate metabolism (R2 = 0.7768 and R2 = 0.4787, respectively) and with changes in glucose metabolism (R2 = 0.6067 and R2 = 0.4506, respectively). We conclude that alterations in lipid profile, especially those that were observed in the diabetic milieu, are associated with early changes in vascular smooth muscle oxidative metabolism. These changes in oxidative metabolism may precede alterations in smooth muscle phenotype and, therefore, may play an important role in the early pathogenesis of atherosclerosis.

Enhanced L-type Ca2+ channel current density in coronary smooth muscle of exercise-trained pigs is compensated to limit myoplasmic free Ca2+ accumulation.

We hypothesized that enhanced voltage-gated Ca2+ channel current (VGCC) density in coronary smooth muscle cells of exercise-trained miniature Yucatan pigs is compensated by other cellular Ca2+ regulatory mechanisms to limit net myoplasmic free Ca2+ accumulation. Whole-cell voltage clamp experiments demonstrated enhanced VGCC density in smooth muscle cells freshly dispersed from coronary arteries of exercise-trained vs. sedentary animals. In separate experiments using fura-2 microfluorometry, we measured depolarization-induced (80 mM KCl) accumulation of myoplasmic free Ba2+ and free Ca2+. Both maximal rate and net accumulation of free Ba2+ in response to membrane depolarization were increased in smooth muscle cells isolated from exercise-trained pigs, consistent with an increased VGCC density. Depolarization also produced an enhanced maximal rate of free Ca2+ accumulation in cells of exercise-trained pigs; however, net accumulation of free Ca2+ was not significantly increased suggesting enhanced Ca2+ influx was compensated to limit net free Ca2+ accumulation. Inhibition of sarco-endoplasmic reticulum Ca2+-transporting ATPase (SERCA; 10 microM cyclopiazonic acid) and/or sarcolemmal Na+-Ca2+ exchange (low extracellular Na+) suggested neither mechanism compensated the enhanced VGCC in cells of exercise-trained animals. Local Ca2+-dependent inactivation of VGCC, assessed by buffering myoplasmic Ca2+ with EGTA in the pipette and using Ca2+ and Ba2+ as charge carriers, was not different between cells of sedentary and exercise-trained animals. Our findings indicate that increased VGCC density is compensated by other cellular Ca2+ regulatory mechanisms to limit net myoplasmic free Ca2+ accumulation in smooth muscle cells of exercise-trained animals. Further, SERCA, Na+-Ca2+ exchange and local Ca2+-dependent inactivation of VGCC do not appear to function as compensatory mechanisms. Additional potential compensatory mechanisms include Ca2+ extrusion via plasma membrane Ca2+-ATPase, mitochondrial uptake, myoplasmic Ca2+-binding proteins and other sources of VGCC inactivation.

Enhanced endothelin(A) receptor-mediated calcium mobilization and contraction in organ cultured porcine coronary arteries.

Arterial injury models for coronary artery disease have demonstrated an enhanced expression and function of either the endothelin(A) or endothelin(B) (ET(A) or ET(B)) receptor subtype. We hypothesized that organ culture would enhance the physiological function of ET receptors in the porcine right coronary artery. Arteries were either cold stored (4 degrees C) or organ cultured (37 degrees C) for 4 days. After 4 days, the artery was either 1) sectioned into rings to measure the ET-1-induced isometric tension response (3 x 10(-10)-3 x 10(-7) M), or 2) enzymatically dispersed and the isolated smooth muscle cells imaged using fura-2 to measure the myoplasmic calcium (Ca(m)) response to 3 x 10(-8) M ET-1 ( approximately EC(50)). Isometric tension and Ca(m) to ET-1 were measured in the absence and presence of bosentan (nonselective ET(A) or ET(B) receptor antagonist), BQ788 (ET(B)-selective antagonist), and BQ123 (ET(A)-selective antagonist). Compared with cold storage, organ culture induced a 2-fold increase in tension development (3 x 10(-7) M ET-1) and Ca(m) (3 x 10(-8) M ET-1), which was inhibited with bosentan, thus confirming the enhanced responses to ET-1 were due to ET receptor activation. BQ123 also inhibited the enhanced contraction and Ca(m) responses to ET-1. In contrast, BQ788 failed to inhibit tension development and Ca(m) responses to ET-1 in organ culture and cold storage. Sarafotoxin 6C (ET(B) agonist) failed to elicit an increased Ca(m) response in organ culture compared with cold storage. Our results indicate the increased tension development and Ca(m) responses to ET-1 in organ culture are attributable to ET(A) receptors, and not ET(B) receptors.

Exercise training restores adenosine-induced relaxation in coronary arteries distal to chronic occlusion.

We previously reported that canine collateral-dependent coronary arteries exhibit impaired relaxation to adenosine but not sodium nitroprusside. In contrast, exercise training enhances adenosine sensitivity of normal porcine coronary arteries. These results stimulated the hypothesis that chronic coronary occlusion and exercise training produce differential effects on cAMP- versus cGMP-mediated relaxation. To test this hypothesis, Ameroid occluders were surgically placed around the proximal left circumflex coronary artery (LCx) of female Yucatan miniature swine 8 wk before initiating sedentary or exercise training (treadmill run, 16 wk) protocols. Relaxation to the cAMP-dependent vasodilators adenosine (10(-7) to 10(-3) M) and isoproterenol (3 x 10(-8) to 3 x 10(-5) M) were impaired in collateral-dependent LCx versus nonoccluded left anterior descending (LAD) arterial rings isolated from sedentary but not exercise-trained pigs. Furthermore, adenosine-mediated reductions in simultaneous tension and myoplasmic free Ca(2+) were impaired in LCx versus LAD arteries isolated from sedentary but not exercise-trained pigs. In contrast, relaxation in response to the cAMP-dependent vasodilator forskolin (10(-9) to 10(-5) M) and the cGMP-dependent vasodilator sodium nitroprusside (10(-9) to 10(-4) M) was not different in LCx versus LAD arteries of sedentary or exercise-trained animals. These data suggest that chronic occlusion impairs receptor-dependent, cAMP-mediated relaxation; receptor-independent cAMP- and cGMP-mediated relaxation were unimpaired. Importantly, exercise training restores cAMP-mediated relaxation of collateral-dependent coronary arteries.