Effect of hyperglycemia on the changes of intracellular [Ca2+]i in heart myoblast

A rise in cytosolic free Ca2+ is the immediate trigger for contraction in heart muscle. In the present study, we investigated changes of intracellular Ca2+ increased by potassium chloride (KCl) and phenylephrine (PE) under hyperglycemia in rat heart myoblast H9c2 cells (BCRC 60096), respectively. We employed the fluorescent Ca2+-indicator, fura-2, and digital imaging microscopy to measure [Ca2+]i in H9c2 cells. Cells were cultured in hyperglycemic (30 mM glucose) Dulbecco's Modified Eagle's Medium. The variation of [Ca2+]i induced by KCI and PE in hyperglycemia was examined, respectively. Moreover, tiron, one of the antioxidants, was pretreated in hyperglycemia-treated H9c2 cells to measure the role of free radicals in the changes of intracellular [Ca2+]i. An influx in intracellular Ca2+ induced by KCl or PE was observed in a dose-dependent manner and reached the highest concentration of 434 +/- 42.3 nM and 443 +/- 42.8 nM (n = 24 cells), respectively. Moreover, this increase of intracellular [Ca2+]i induced by KCl or PE was markedly reduced in cells exposed to hyperglycemia (434 +/- 42.3 vs. 1.26 +/- 0.21 nM and 443 +/- 42.8 vs. 2.54 +/- 0.25 nM, n = 24 cells, P < 0.001, respectively). Similar changes were not observed in cells received mannitol showing same osmolarity. However, the reduction of intracellular [Ca2+]i induced by hyperglycemia was abolished significantly in the presence of tiron. Our results suggest that an increase of intracellular Ca2+ by KCl or PE in heart cell was markedly reduced by hyperglycemic treatment; mediation of free radicals in this action can be considered because it was reversed in the presence of tiron.     

Effects of estrogens and selective estrogen receptor modulators on vascular reactivity in the perfused mesenteric vascular bed

Estrogens and selective estrogen receptor modulators (SERMs), such as raloxifene (RAL) and tamoxifen (TAM), acutely relax arteries, but the long-term effects of estrogens and SERMs on vascular reactivity in the mesenteric vasculature have not been well defined. In this study, we used an isolated, perfused mesenteric vascular bed technique to investigate the effect of chronic treatment of estrogens and SERMs on vascular reactivity of the mesenteric bed. Ovariectomized female Sprague-Dawley rats were treated by gavage with vehicle (control, 2-hydroxypropyl-beta-cyclodextrin), ethinyl estradiol, estradiol benzoate, equilin (EQ), TAM, or RAL for 3 wk. EQ and TAM increased vasoconstriction in response to all three vasoconstrictors tested (KCl, norepinephrine, and 5-HT). Ethinyl estradiol increased vasoconstriction in response to KCl and 5-HT, whereas responses to estradiol benzoate and RAL were less consistent. Only EQ (134 +/- 4 mmHg) and TAM (104 +/- 4 mmHg) changed mean arterial blood pressure compared with control (117 +/- 4 mmHg). These data demonstrate that 3-wk gavage treatment with estrogens and SERMs affects vascular reactivity in the mesenteric vascular bed. However, the three formulations of estrogen did not produce equivalent effects, and the effects of the SERMs were different from those of the estrogens.     

Dual effect of Ca2+ on ultrasonic ATPase activity and polymerization of muscle actin.

Millimolar concentrations of Ca2+ stimulate actin polymerization whereas micromolar concentrations of Ca2+ depress polymerization. This latter effect leads to a reduction of ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity of actin during sonication at low Mg2+ concentrations and in the absence of KCl. In the presence of KCl (90 mM) there is activation of ATPase activity by micromolar Ca2+ concentrations. These Ca2+ effects are half-maximal at a Ca2+ concentration of 2-10(-7) M. They can be explained by assuming that that ATPase activity is optimal in a medium range of actin polymer stability and that micromolar Ca2+ concentrations tend to labilize and depolymerize F-actin.     

K(Ca) channel blockers reveal hyperpolarization and relaxation to K+ in rat isolated mesenteric artery

Raising extracellular K+ concentration ([K+](o)) around mesenteric resistance arteries reverses depolarization and contraction to phenylephrine. As smooth muscle depolarizes and intracellular Ca(2+) and tension increase, this effect of K+ is suppressed, whereas efflux of cellular K+ through Ca(2+)-activated K+ (K(Ca)) channels is increased. We investigated whether K+ efflux through K(Ca) suppresses the action of exogenous K+ and whether it prestimulates smooth muscle Na(+)-K(+)-ATPase. Under isometric conditions, 10.8 mM [K+](o) had no effect on arteries contracted >10 mN, unless 100 nM iberiotoxin (IbTX), 100 nM charybdotoxin (ChTX), and/or 50 nM apamin were present. Simultaneous measurements of membrane potential and tension showed that phenylephrine depolarized and contracted arteries to -32.2 +/- 2.3 mV and 13.8 +/- 1.6 mN (n = 5) after blockade of K(Ca), but 10.8 mM K+ reversed fully the responses (107.6 +/- 8.6 and 98.8 +/- 0.6%, respectively). Under isobaric conditions and preconstriction with phenylephrine, 10.7 mM [K+](o) reversed contraction at both 50 mmHg (77.0 +/- 8.5%, n = 9) and 80 mmHg (83.7 +/- 5.5%, n = 5). However, in four additional vessels at 80 mmHg, raising K+ failed to reverse contraction unless ChTX was present. Increases in isometric and decreases in isobaric tension with phenylephrine were augmented by either ChTX or ouabain (100 microM), whereas neither inhibitor altered tension under resting conditions. Inhibition of cellular K+ efflux facilitates hyperpolarization and relaxation to exogenous K+, possibly by indirectly reducing the background activation of Na(+)-K(+)-ATPase.     

Ca2+-stimulated, Mg2+-dependent ATPase activity in neutrophil plasma membrane vesicles. Coupling to Ca2+ transport

Low concentrations of free Ca2+ stimulated the hydrolysis of ATP by plasma membrane vesicles purified from guinea pig neutrophils and incubated in 100 mM HEPES/triethanolamine, pH 7.25. In the absence of exogenous magnesium, apparent values obtained were 320 nM (EC50 for free Ca2+), 17.7 nmol of Pi/mg X min (Vmax), and 26 microM (Km for total ATP). Studies using trans- 1,2-diaminocyclohexane- N,N,N',N',-tetraacetic acid as a chelator showed this activity was dependent on 13 microM magnesium, endogenous to the medium plus membranes. Without added Mg2+, Ca2+ stimulated the hydrolysis of several other nucleotides: ATP congruent to GTP congruent to CTP congruent to ITP greater than UTP, but Ca2+-stimulated ATPase was not coupled to uptake of Ca2+, even in the presence of 5 mM oxalate. When 1 mM MgCl2 was added, the vesicles demonstrated oxalate and ATP-dependent calcium uptake at approximately 8 nmol of Ca2+/mg X min (based on total membrane protein). Ca2+ uptake increased to a maximum of approximately 17-20 nmol of Ca2+/mg X min when KCl replaced HEPES/triethanolamine in the buffer. In the presence of both KCl and MgCl2, Ca2+ stimulated the hydrolysis of ATP selectively over other nucleotides. Apparent values obtained for the Ca2+-stimulated ATPase were 440 nM (EC50 for free Ca2+), 17.5 nmol Pi/mg X min (Vmax) and 100 microM (Km for total ATP). Similar values were found for Ca2+ uptake which was coupled efficiently to Ca2+-stimulated ATPase with a molar ratio of 2.1 +/- 0.1. Exogenous calmodulin had no effect on the Vmax or EC50 for free Ca2+ of the Ca2+-stimulated ATPase, either in the presence or absence of added Mg2+, with or without an ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N',-tetraacetic acid pretreatment of the vesicles. The data demonstrate that calcium stimulates ATP hydrolysis by neutrophil plasma membranes that is coupled optimally to transport of Ca2+ in the presence of concentrations of K+ and Mg2+ that appear to mimic intracellular levels.     

Analysis of tert-butyl hydroperoxide induced constrictions of perfused vascular beds in vitro

tert-Butyl hydroperoxide (t-BOOH), an inducer of oxidative stress in vitro, elicits constrictor responses of the isolated, rat kidney and mesenteric arteries perfused (5 ml/min) with physiological salt solutions (PSS) at 37 degrees C gassed with carbogen. We hypothesized that generation of superoxide anions (O(2)(-)) accounts for these responses. We assessed responses to t-BOOH in preparations with/without endothelium, and in the absence/presence of antioxidant compounds, catalase and tempol, scavengers of hydroxyl (OH(-)) radical and O(2)(-), respectively. t-BOOH (0.01-50 micromol) induced (expressed as % of 50 micromol KCl vasoconstriction) were abolished by endothelium denudation, perfusion with Ca(2+)-free PSS and by nifedipine, (1 nM). Infusion of t-BOOH (0.1 microM) did not significantly (P > 0.05) affect phenylepherine E(max) in the mesenteric arteries, however it reduced phenylepherine E(max) responses in the kidney from 94.9 +/- 3.9 % to 64.7 +/- 4.7 %. Nitroblue tetrazolium, as well as alpha-phenyl N-tert-butyl nitrone, at 100 microM, but not catalase (500 IU) significantly attenuated t-BOOH (10 micromol) vasoconstrictor responses. Tempol (100 microM), a membrane permeable antioxidant, also significantly reduced t-BOOH (10 micromol) responses from 17.0 +/- 1.9 % (control) to 9.6 +/- 0.5 % (+tempol) in the mesenteric arteries and from 40.4 +/- 4.2 % (control) to 20.7 +/- 1.5 % (+tempol) in the kidney. Our data suggest that t-BOOH elicits vasoconstriction via two distinct mechanisms: (i) increased influx of extracellular Ca(2+), and (ii) generation of free radicals including O(2)(-) anions.     

Ca2+-activated K channel of the BK-type in the inner mitochondrial membrane of a human glioma cell line

A single channel current was recorded from mitoplasts (i.e., inner mitochondrial membrane) of the human glioma cell line LN229 using patch-clamp techniques in the mitoplast-attached mode. We frequently found a 295 +/- 18 pS channel that showed a straight i-E relation in the range +/-60 mV in 150 mM KCl solutions on either side of the mitoplast. If KCl in the bath was exchanged against NaCl, outward currents were undetectable, indicating potassium selectivity. Channel activity determined as open probability increased with increasing Ca2+ concentrations (EC50 = 0.9 microM at 60 mV). Open probability was voltage dependent. An e-fold increase of time spent in the open state was induced by a depolarization of 10.5 mV. Open probability was decreased by charybdotoxin concentration and voltage dependently (EC50 = 1.4 nM). In conclusion, we show for the first time that the inner mitochondrial membrane in human glioma cells contains a calcium-dependent K channel of the BK-type.     

Repetitive Action Potentials in Isolated Nerve Terminals in the Presence of 4-Aminopyridine: Effects on Cytosolic Free Ca2+ and Glutamate Release

The mechanisms by which an elevated KCl level and the K+-channel inhibitor 4-aminopyridine induce release of transmitter glutamate from guinea-pig cerebral cortical synaptosomes are contrasted. KCl at 30 mM caused an initial spike in the cytosolic free Ca2+ concentration ([Ca2+]c), followed by a partial recovery to a plateau 112 +/- 13 nM above the polarized control. The Ca2+-dependent release of endogenous glutamate, determined by continuous fluorimetry, was largely complete by 3 min, by which time 1.70 +/- 0.35 nmol/mg was released. [Ca2+]c elevation and glutamate release were both insensitive to tetrodotoxin. KCl-induced elevation in [Ca2+]c could be observed in both low-Na+ medium and in the presence of low concentrations of veratridine. 4-Aminopyridine at 1 mM increased [Ca2+]c by 143 +/- 18 nM to a plateau similar to that following 30 mM KCl. The initial rate of increase in [Ca2+]c following 4-aminopyridine administration was slower than that following 30 mM KCl, and a transient spike was less apparent. Consistent with this, the 4-aminopyridine-induced net uptake of 45Ca2+ is much lower than that following an elevated KCl level. 4-Aminopyridine induced the Ca2+-dependent release of glutamate, although with somewhat slower kinetics than that for KCl. The measured release was 0.81 nmol of glutamate/mg in the first 3 min of 4-aminopyridine action. In contrast to KCl, glutamate release and the increase in [Ca2+]c with 4-aminopyridine were almost entirely blocked by tetrodotoxin, a result indicating repetitive firing of Na+ channels. Basal [Ca2+]c and glutamate release from polarized synaptosomes were also significantly lowered by tetrodotoxin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Carbachol and KCl-induced changes in intracellular free calcium concentration in isolated, fura-2 loaded smooth-muscle cells from the anterior byssus retractor muscle of Mytilus edulis

Intracellular free calcium concentration [( Ca2+]1) was measured in suspensions of fura-2 loaded smooth-muscle cells isolated from the anterior byssus retractor muscle of Mytilus edulis. Successive application of 5mM carbachol (CCh) and 100mM KCl to the cells transiently elevated [Ca2+]1 from the resting value of 124 +/- 4.5nM (mean +/- S.E., n = 14) to 295 +/- 15.3 and 383 +/- 20.5 nM, respectively. The response to CCh was concentration-dependent with an ED50 of 10(-5) M. Under the microscope, 67 +/- 3.0 and 83 +/- 1.3 % of fura-2 loaded cells contracted on the addition of 5mM CCh and 100mM KCl, respectively. In Ca2+ -free sea water, the CCh induced change in [Ca2+]1 was partially suppressed whereas that induced by KCl was completely abolished, suggesting an agonist-evoked release of stored Ca2+.     

Vagal inhibitory effects on peripheral circulation in acute coronary occlusion.

The reflex adjustments of the peripheral circulation in response to acute coronary occlusion were studied in anesthetized dogs with isolated vascular beds perfused at constant flow. Coronary occlusion caused significant increases in perfusion pressure which averaged 27 +/- 4 mmHg in the hindlimb, 19 +/- 8 mmHg in skeletal muscle, and 13 + 5 mmHg in the mesenteric artery. These responses were less than half those caused by a similar decrease in aortic pressure obtained with hemorrhage. Coronary occlusion caused no significant changes in renal and paw circulations, while marked vasoconstriction resulted from hemorrhage. When aortic pressure was maintained constant throughout the duration of coronary occlusion, there was a significant vasodilatation in all beds studied. After vagotomy, coronary occlusion caused a constrictor response similar in magnitude to that caused by hemorrhage in each vascular bed and the dilator responses to occlusion at constant aortic pressure were abolished. Both constrictor and dilator changes were prevented by alpha-adrenergic blockade. Mechanical distension of the left ventricle in four dogs with carotid sinus nerves cut caused a significant reflexdilatation in the hindlimb. Thus, coronary occlusion initiates an inhibitory reflex mediated by vagal afferents which opposes peripheral vasoconstriction most effectively in the renal and paw circulations.     

Evidence that binding of Indo-1 to cardiac myocyte protein does not markedly change Kd for Ca2+

Quantitative measurement of [Ca2+]i with the fluorescent Ca(2+)-indicators Indo-1 and Fura-2 is complicated by the possibility that the value of the dissociation constant (Kd) may be influenced by binding to intracellular proteins. We investigated this question in cultured chick ventricular myocytes by use of two different Indo-1 calibration methods. First, the Indo-1 fluorescence ratio (R) (400/500 nm) was measured in beating myocytes loaded by exposure to Indo-1/AM. Then, cells were exposed to the Ca2+ ionophore Br A-23187 and fluorescence ratio was measured in the presence of 500 nM Ca2+ (EGTA-Ca2+ buffer). Subsequently cells were permeabilized to Ca2+ by a 1 min exposure to 25 microM digitonin in the presence of 'zero' Ca2+ (10 mM EGTA) and saturating 1 mM Ca2+ to obtain Rmin, Rmax and beta. We then calculated [Ca2+]i from the formula ([Ca2+]i = Kd [( R - Rmin)/(Rmax - R)]beta). With Kd = 250 nM, calculated systolic [Ca2+]i was 750 +/- 44 nM and diastolic 269 +/- 19 nM (means +/- SEM, n = 16). The R value calculated for an assumed [Ca2+]i = 500 nM using the above formula and digitonin derived constants was very similar to the value measured using Br A-23187 (digitonin, 0.67 +/- 0.03: Br A-23187, 0.66 +/- 0.03, ns). As the Br A-23187 method is independent of the value chosen for Kd, we conclude that the Kd of 250 nM for Indo-1 measured in free solutions closely approximates the Kd for intracellular Indo-1 in these cells, and that therefore the Kd of Indo-1 for Ca2+ does not appear to be markedly affected by binding to proteins or other intracellular molecules.     

Different effects of simple anoxic lactic acidosis and simulated in vivo anoxic acidosis on turtle heart.

We compared responses of turtle heart at 20 degrees C to an anoxic lactic acidosis solution (LA) containing 35 mM lactic acid in an otherwise normal turtle Ringers equilibrated with 3% CO2/97% N2 at pH 7.0) to a solution simulating in vivo anoxic acidosis (VA), with elevated concentrations of lactate, Ca2+, Mg2+, and K+, and decreased Cl-, equilibrated with 10.8% CO2/89.2% N2 at pH 7.0. We examined mechanical properties on cardiac muscle strips and determined intracellular pH (pHi) and high energy phosphates on perfused hearts using 31P-NMR. Maximum active force (Fmax) and the maximum rate of force development (dF/dtmax) of muscle strips were significantly higher during VA than during LA superfusion. An elevation of Ca2+ alone (to 6 mM) in LA significantly increased both Fmax and dF/dtmax but the effects diminished toward the end of the exposure; however, hypercapnic anoxic lactic acidosis (addition of 20 mM HCO3- to LA, equilibrated with 10.8% CO2/89.2% N2, pH 7.0) did not significantly affect Fmax or dF/dtmax. During VA perfusion, pHi (6.73 +/- 0.01) was significantly higher than that during LA perfusion (pHi 6.69 +/- 0.013), but the difference is probably too small to have physiological significance. ATP, creatine phosphate, and inorganic phosphate were not significantly different in the two anoxic solutions. We conclude that the reduction of cardiac mechanical function in vivo is minimized by the integrated effects of changes of ionic concentrations, but the observed changes in Ca2+ and pHi cannot fully explain the effect.     

Atrial natriuretic peptide prevents diabetes-induced endothelial dysfunction

Atrial natriuretic peptide (ANP) exerts beneficial effects on the cardiovascular system in part by exerting antioxidant activity. Given that oxidant stress is a key cause of endothelial dysfunction in diabetes, we investigated whether ANP improves endothelial function in rats with diabetes. Rats were injected with streptozotocin (55 mg/kg iv) to induce type 1 diabetes or the citrate vehicle as controls (n=12). After 4 weeks the diabetic rats were treated with ANP (10 pmol/kg/min sc, n=12) or the antioxidant tempol (1.5 mmol/kg/day sc, n=11), both by osmotic minipump, ramipril (1 mg/kg per day in the drinking water) or remained untreated (n=11). After a further 4 weeks, anaesthetised rats were killed by exsanguination and the thoracic aortae collected for examination of vascular activity and measurement of superoxide generation. Diabetic rats showed elevated plasma glucose concentration (45+/-3 mM) compared to controls (10+/-1 mM) and this was not affected by ANP (43+/-3 mM), ramipril (41+/-2 mM) or tempol (43+/-2 mM). Endothelium-dependent relaxation ex vivo in response to acetylcholine was impaired in diabetic rats (Rmax=66+/-4%) compared to control rats (Rmax=94+/-1%) but treatment with ANP (Rmax=80+/-4%), ramipril (Rmax=88+/-2%) or tempol (Rmax=81+/-5%) significantly improved those responses. Relaxant responses to the endothelium-independent vasodilator sodium nitroprusside were enhanced by treatment of diabetic rats with ANP or ramipril and their combination; but not by tempol. Superoxide generation was significantly elevated in aorta from untreated diabetic rats (649+/-146% of control). In diabetic rats, superoxide generation was significantly attenuated by ANP (to 229+/-78%) or tempol (to 186+/-64%). This study demonstrates that ANP improves vascular oxidant stress in concert with endothelial function, independent of any effect on plasma glucose levels. These studies may lead to new therapies, based on natriuretic peptide and/or antioxidant approaches, for ameliorating the vascular complications of diabetes.     

Voltage-sensitive dye RH421 increases contractility of cardiac muscle.

Voltage-sensitive dyes and imaging techniques have proved to be indispensable tools for use in in vitro electrophysiological studies. To avoid motion artifacts in optical recordings, electromechanical uncouplers such as 2,3-butanedione monoxime (BDM) are required. In this study, we sought to determine whether the voltage-sensitive dye RH421 had an effect on the contractility of heart muscle, either alone or in the presence of BDM. Ventricular contractility was studied in (i) isolated rat myocytes and (ii) Langendorff-perfused rat hearts under control conditions, and during perfusion with RH421 or RH421 + BDM. The following results were obtained. (i) The amplitude of cell shortening increased progressively from 6.24 +/- 0.64 to 9.95 +/- 1.02 microm during 15 min of superfusion with 5 microM RH421 (n = 11), and further increased to 12.54 +/- 0.97 microm during washout. In seven cells first perfused with 15 mM BDM and then with 15 mM BDM + 5 microM RH421, the amplitude of the cell shortening first decreased from 5.17 +/- 0.51 to 0.41 +/- 0.19 microm, then the amplitude increased to 2.63 +/- 0.25 microm. (ii) Left ventricular pressure (LVP) of the heart (n = 7) was reduced by 15 mM BDM from 60.7 +/- 2.5 to 2.8 +/- 0.5 mmHg (1 mmHg = 133.3 Pa). LVP increased to 12.8 +/- 1.1 mmHg during subsequent perfusion with 10 microM RH421 in the presence of BDM and did not change (LVP = 12.4 +/- 2.4 mmHg) during washout of the dye. Therefore, RH421 increased the contractility of rat hearts and isolated myocytes with and without BDM.     

Acute hypoxia increases intracellular [Ca2+] in pulmonary arterial smooth muscle by enhancing capacitative Ca2+ entry

Hypoxic pulmonary vasoconstriction (HPV) requires influx of extracellular Ca2+ in pulmonary arterial smooth muscle cells (PASMCs). To determine whether capacitative Ca2+ entry (CCE) through store-operated Ca2+ channels (SOCCs) contributes to this influx, we used fluorescent microscopy and the Ca2+-sensitive dye fura-2 to measure effects of 4% O2 on intracellular [Ca2+] ([Ca2+]i) and CCE in primary cultures of PASMCs from rat distal pulmonary arteries. In PASMCs perfused with Ca2+-free Krebs Ringer bicarbonate solution (KRBS) containing cyclopiazonic acid to deplete Ca2+ stores in sarcoplasmic reticulum and nifedipine to prevent Ca2+ entry through L-type voltage-operated Ca2+ channels (VOCCs), hypoxia markedly enhanced both the increase in [Ca2+]i caused by restoration of extracellular [Ca2+] and the rate at which extracellular Mn2+ quenched fura-2 fluorescence. These effects, as well as the increased [Ca2+]i caused by hypoxia in PASMCs perfused with normal salt solutions, were blocked by the SOCC antagonists SKF-96365, NiCl2, and LaCl3 at concentrations that inhibited CCE >80% but did not alter [Ca2+]i responses to 60 mM KCl. In contrast, the VOCC antagonist nifedipine inhibited [Ca2+]i responses to hypoxia by only 50% at concentrations that completely blocked responses to KCl. The increased [Ca2+]i caused by hypoxia was completely reversed by perfusion with Ca2+-free KRBS. LaCl3 increased basal [Ca2+]i during normoxia, indicating effects other than inhibition of SOCCs. Our results suggest that acute hypoxia enhances CCE through SOCCs in distal PASMCs, leading to depolarization, secondary activation of VOCCs, and increased [Ca2+]i. SOCCs and CCE may play important roles in HPV.     

The effects of extracellular K+ and angiotensin II on cytosolic Ca++ and steroidogenesis in adrenal glomerulosa cells

We evaluated changes in cytosolic calcium concentration (Ca++) and steroidogenesis in rat adrenal glomerulosa cells (GC) stimulated with potassium (K+) or angiotensin II (AII). Cytosolic Ca++ concentration was determined using the Ca++-sensitive, fluorescent dye QUIN 2. Raising extracellular K+ increased cytosolic Ca++ from 267 +/- 23 nM at 3.7 mM K+ to a maximum of 377 +/- 40 nM at 8.7 mM K+ (p less than 0.01, N = 23). AII also increased cytosolic Ca++ from 238 +/- 20 nM to a maximum of 427 +/- 42 nM at 10(-7) M (p less than 0.01, N = 16). In parallel studies, K+ and AII stimulated aldosterone secretion from QUIN 2-loaded GC at concentrations similar to those which raised cytosolic Ca++. QUIN 2-loaded cells were as responsive steroidogenically as unloaded cells and showed trypan blue exclusion of 98% suggesting that QUIN 2 did not compromise cellular viability. These results provide direct support for a role of cytosolic Ca++ as a second messenger during stimulation of aldosterone secretion by both K+ and AII.     

Cytochrome P-450 metabolites of 2-arachidonoylglycerol play a role in Ca2+-induced relaxation of rat mesenteric arteries

The perivascular sensory nerve (PvN) Ca(2+)-sensing receptor (CaR) is implicated in Ca(2+)-induced relaxation of isolated, phenylephrine (PE)-contracted mesenteric arteries, which involves the vascular endogenous cannabinoid system. We determined the effect of inhibition of diacylglycerol (DAG) lipase (DAGL), phospholipase A(2) (PLA(2)), and cytochrome P-450 (CYP) on Ca(2+)-induced relaxation of PE-contracted rat mesenteric arteries. Our findings indicate that Ca(2+)-induced vasorelaxation is not dependent on the endothelium. The DAGL inhibitor RHC 802675 (1 microM) and the CYP and PLA(2) inhibitors quinacrine (5 microM) (EC(50): RHC 802675 2.8 +/- 0.4 mM vs. control 1.4 +/- 0.3 mM; quinacrine 4.8 +/- 0.4 mM vs. control 2.0 +/- 0.3 mM; n = 5) and arachidonyltrifluoromethyl ketone (AACOCF(3), 1 microM) reduced Ca(2+)-induced relaxation of mesenteric arteries. Synthetic 2-arachidonoylglycerol (2-AG) and glycerated epoxyeicosatrienoic acids (GEETs) induced concentration-dependent relaxation of isolated arteries. 2-AG relaxations were blocked by iberiotoxin (IBTX) (EC(50): control 0.96 +/- 0.14 nM, IBTX 1.3 +/- 0.5 microM) and miconazole (48 +/- 3%), and 11,12-GEET responses were blocked by IBTX (EC(50): control 55 +/- 9 nM, IBTX 690 +/- 96 nM) and SR-141716A. The data suggest that activation of the CaR in the PvN network by Ca(2+) leads to synthesis and/or release of metabolites of the CYP epoxygenase pathway and metabolism of DAG to 2-AG and subsequently to GEETs. The findings indicate a role for 2-AG and its metabolites in Ca(2+)-induced relaxation of resistance arteries; therefore this receptor may be a potential target for the development of new vasodilator compounds for antihypertensive therapy.     

Effects of intracellular and extracellular concentrations of Ca2+, K+, and Cl- on the Na+-dependent Mg2+ efflux in rat ventricular myocytes

Intracellular Mg2+ concentration ([Mg2+]i) was measured in rat ventricular myocytes with the fluorescent indicator furaptra (25 degrees C). After the myocytes were loaded with Mg2+, the initial rate of decrease in [Mg2+]i (initial Delta[Mg2+]i/Deltat) was estimated upon introduction of extracellular Na+, as an index of the rate of Na+-dependent Mg2+ efflux. The initial Delta[Mg2+]i/Deltat values with 140 mM [Na+]o were essentially unchanged by the addition of extracellular Ca2+ up to 1 mM (107.3+/-8.7% of the control value measured at 0 mM [Ca2+]o in the presence of 0.1 mM EGTA, n=5). Intracellular loading of a Ca2+ chelator, either BAPTA or dimethyl BAPTA, by incubation with its acetoxymethyl ester form (5 microM for 3.5 h) did not significantly change the initial Delta[Mg2+]i/Deltat: 115.2+/-7.5% (seven BAPTA-loaded cells) and 109.5+/-10.9% (four dimethyl BAPTA loaded cells) of the control values measured in the absence of an intracellular chelator. Extracellular and/or intracellular concentrations of K+ and Cl- were modified under constant [Na+]o (70 mM), [Ca2+]o (0 mM with 0.1 mM EGTA), and membrane potential (-13 mV with the amphotericin-B-perforated patch-clamp technique). None of the following conditions significantly changed the initial Delta[Mg2+]i/Deltat: 1), changes in [K+]o between 0 mM and 75 mM (65.6+/-5.0% (n=11) and 79.0+/-6.0% (n=8), respectively, of the control values measured at 140 mM [Na+]o without any modification of extracellular and intracellular K+ and Cl-); 2), intracellular perfusion with K+-free (Cs+-substituted) solution from the patch pipette in combination with removal of extracellular K+ (77.7+/-8.2%, n=8); and 3), extracellular and intracellular perfusion with K+-free and Cl--free solutions (71.6+/-5.1%, n=5). These results suggest that Mg2+ is transported in exchange with Na+, but not with Ca2+, K+, or Cl-, in cardiac myocytes.     

Zinc has opposite effects on NMDA and non-NMDA receptors expressed in Xenopus oocytes

Pharmacological characterization of Zn2+ effects on glutamate ionotropic receptors was investigated in Xenopus oocytes injected with rat brain mRNA, using a double microelectrode, voltage-clamp technique. At low concentration, Zn2+ inhibited NMDA currents (IC50 = 42.9 +/- 1.3 microM) and potentiated both AMPA (EC50 = 30.0 +/- 1.2 microM) and desensitized kainate responses (EC50 = 13.0 +/- 0.1 microM). At higher concentrations, Zn2+ inhibited non-NMDA responses with IC50 values of 1.3 +/- 0.1 mM and 1.2 +/- 0.3 mM for AMPA and kainate, respectively. The potentiation of AMPA or quisqualate currents by Zn2+ was more than 2-fold, whereas that of the kainate current was only close to 30%. This potentiating effect of Zn2+ on AMPA current modified neither the affinity of the agonist for its site nor the current-voltage relationship. In addition, 500 microM Zn2+ differentially affected NMDA and non-NMDA components of the glutamate-induced response. The possible physiological relevance of Zn2+ modulation is discussed.     

Myocardial function and energy substrate metabolism in the insulin-resistant JCR:LA corpulent rat.

Alterations in myocardial energy substrate utilization contribute to the development of cardiomyopathic changes in insulin-dependent and non-insulin-dependent diabetic rats. Energy substrate utilization and contractile function, however, have not been characterized in insulin-resistant diabetes. In this study, we studied these parameters in the insulin-resistant obese JCR:LA-cp rat homozygous for the corpulent gene (cp/cp). Homozygous (+/+) or heterozygous (+/cp) lean non-insulin-resistant rats were used as controls. Isolated working hearts from cp/cp and lean control rats were perfused with Krebs-Henseleit buffer containing either 11 mM [U-14C]glucose and 0.4 mM palmitate or 11 mM glucose and 0.4 mM [1-14C]palmitate. Unlike control hearts, hearts from cp/cp rats were found to require high doses of insulin and Ca2+ concentrations of less than or equal to 1.75 mM to maintain mechanical function. In the presence of 2,000 microU/ml insulin, contractile function from cp/cp rat hearts was not depressed in the presence of either 1.25 or 1.75 mM Ca2+. Steady-state glucose oxidation rates in hearts perfused with 1.25 mM Ca2+ and 2,000 microU/ml insulin were 811 +/- 86 (SE) and 612 +/- 51 nmol.min-1.g dry wt-1 in cp/cp and control rats, respectively. Palmitate oxidation was 307 +/- 47 and 307 +/- 47 nmol.min-1.g dry wt-1 in cp/cp and lean control hearts, respectively. Under these perfusion conditions, 40% of myocardial ATP production was derived from glucose, whereas 60% was derived from palmitate in both cp/cp and control rats.(ABSTRACT TRUNCATED AT 250 WORDS)