Ischemia-induced structural change in SR Ca2+-ATPase is associated with reduced enzyme activity in rat muscle

In this study, we employed an in vivo model of prolonged ischemia in rat skeletal muscle to investigate the hypothesis that structural modifications to the sarcoplasmic reticulum (SR) Ca2+-ATPase can explain the alterations in Ca2+-ATPase activity that occur with ischemia. To induce total ischemia, a tourniquet was placed around the upper hindlimb in 27 female Sprague-Dawley rats weighing 256 +/- 6.7 g (mean +/- SE) and was inflated to 350 mmHg for 4 h. The contralateral limb served as control (C) to the ischemic limb (I), and the limbs of animals killed immediately after anesthetization served as a double control (CC). Mixed gastrocnemius and tibialis anterior muscles were sampled and used for SR vesicle preparation. Maximal Ca2+-ATPase activity (micromol x g protein(-1) x min(-1)) of C (15,802 +/- 1,246) and I (11,609 +/- 1,029) was 90 and 73% (P < 0.05) of CC (17,562 +/- 1,682), respectively. No differences were found between groups in either the Hill coefficient or the free Ca2+ at half-maximal activity. The fluorescent probes, FITC and N-cyclohexyl-N'-(dimethylamino-alpha-naphthyl) carbodiimide, used to assess structural alterations in the regions of the ATP binding site and the Ca2+ binding sites of the Ca2+-ATPase, respectively, indicated a 26% reduction (P < 0.05) in FITC binding capacity (absolute units) in I (0.22 +/- 0.01) compared with CC (0.29 +/- 0.02) and C (0.29 +/- 0.03). Our results suggest that the reduction in maximal SR Ca2+-ATPase activity in SR vesicles with ischemia is related to structural modification in the region of the nucleotide binding domain by mechanisms that are as yet unclear.     

Thrombin-stimulated increases in cytosolic Ca2+ level and gonadotropin-releasing hormone release in GT1-7 neurons.

The effects of thrombin on cytosolic calcium levels ([Ca2+]cyt), and on gonadotropin-releasing hormone (GnRH) release, were characterized in cultured GT1-7 neurons. GnRH release from GT1-7 neurons was pulsatile with an average pulse amplitude of 14.3+/-5.8 pg x min x ml(-1) and an average pulse duration of 21.3+/-4.2 min. The [Ca2+]cyt response to 0.005 to 0.2 U/ml thrombin was saturable and concentration dependent (EC50 = 0.0268 U/ml). Ethyleneglycotetraacetic acid (EGTA) chelation of extracellular Ca2+ resulted in an approximately 70% attenuation of thrombin-stimulated increase in [Ca2+]cyt. By use of a special superfusion system, a 5-min exposure to 0.1 U/ml thrombin significantly increased the amplitude (193.2+/-67.8 pg x min x ml(-1); P = 0.001) but not the duration (22.5+/-2.4 min; P = 0.8) of GnRH release. These results suggest that thrombin increases [Ca2+]cyt and GnRH release from GT1-7 neurons via specific membrane-bound receptors.     

Effects of low-calcium reperfusion and adenosine on diastolic behavior during the transitory systolic overshoot of the stunned myocardium in the rabbit

The aims of the present study were to determine whether the transitory systolic overshoot (TSO) that occurs in the early reperfusion (R) of the stunned myocardium is accompanied by diastolic alterations, and to determine whether the R with low Ca2+ Krebs-Henseleit's solution or with adenosine modifies these alterations. Isolated-isovolumic rabbit hearts were divided in 3 groups (G). G1 (n = 11) was perfused with Krebs-Henseleit's solution, subjected to 15 min of global ischemia and 30 min R; G2 (n = 10) was reperfused during the first 10 min with Krebs-Henseleit's solution [Ca2+] = 1 mmol/L, which was increased in the perfusate to 1.5 mmol/L up to 20 min R and at 2.5 mmol/L from 20 to 30 min R. G3 (n = 12) was perfused with Krebs-Henseleit's solution with adenosine (0.03 microg x kg(-1) x min(-1)) from 10 min before ischemia and during all R. Left ventricular (LV) +dP/dtmax (mmHg/s), LV end diastolic pressure (LVEDP, mmHg), and 1 relaxation index (t(1/2)) were measured in preischemic state, at 30, 50, 60, 70, 90, and 120 s R, and then at 5 and 30 min R. The +dP/dtmax recovered to 621 +/- 77 mmHg/s (p > 0.05), 346 +/- 31 mmHg/s (p < 0.05 vs. G1), and 533 +/- 76 mmHg/s (p > 0.05) from preischemic value of 730 +/- 39, 690 +/- 32, and 758 +/- 57 in G1, G2, and G3, respectively. The LVEDP in G1 and G3 increased early in the R, and it was negatively correlated with the +dP/dtmax (r = -0.63, p = 0.0369; and r = -0.71, p = 0.0090, respectively). The R with low Ca2+ abolished this correlation and attenuated the TSO phase. The correlation between LVEDP and +dP/dtmax in G1 and G3 and the lack of correlation in G2 suggests there are common mechanisms for the systolic and diastolic alterations during the TSO phase that are possibly related to Ca2+ overload but not with the vascular tone.     

Limiting sarcolemmal Na+ entry during resuscitation from ventricular fibrillation prevents excess mitochondrial Ca2+ accumulation and attenuates myocardial injury.

BACKGROUND: intracellular Na+ accumulation during ischemia and reperfusion leads to cytosolic Ca2+ overload through reverse-mode operation of the sarcolemmal Na+ -Ca2+ exchanger. Cytosolic Ca2+ accumulation promotes mitochondrial Ca2+ (Ca2+ m) overload, leading to mitochondrial injury. We investigated whether limiting sarcolemmal Na+ entry during resuscitation from ventricular fibrillation (VF) attenuates Ca2+ m overload and lessens myocardial dysfunction in a rat model of VF and closed-chest resuscitation. METHODS: hearts were harvested from 10 groups of 6 rats each representing baseline, 15 min of untreated VF, 15 min of VF with chest compression given for the last 5 min (VF/CC), and 60 min postresuscitation (PR). VF/CC and PR included four groups each randomized to receive before starting chest compression the new NHE-1 inhibitor AVE4454B (1.0 mg/kg), the Na+ channel blocker lidocaine (5.0 mg/kg), their combination, or vehicle control. The left ventricle was processed for intracellular Na+ and Ca2+ m measurements. RESULTS: limiting sarcolemmal Na+ entry attenuated cytosolic Na+ increase during VF/CC and the PR phase and prevented Ca2+ m overload yielding levels that corresponded to 77% and 71% of control hearts at VF/CC and PR, without differences among specific Na+ -limiting interventions. Limiting sarcolemmal Na+ entry attenuated reductions in left ventricular compliance during VF and prompted higher mean aortic pressure (110 +/- 7 vs. 95 +/- 11 mmHg, P < 0.001) and higher cardiac work index (159 +/- 34 vs. 126 +/- 29 g x m x min(-1) x kg(-1), P < 0.05) with lesser increases in circulating cardiac troponin I at 60 min PR. CONCLUSIONS: Na+ -limiting interventions prevented excess Ca2+ m accumulation induced by ischemia and reperfusion and ameliorated myocardial injury and dysfunction.     

Effects of divalent cations on voltage-gated Ca2+ channels and depolarization-induced [Ca2+], transients of freshly isolated pyramidal cells of the rat dorsal cochlear nucleus

The effects of divalent cations on voltage-activated Ca2+ channels and depolarization-evoked cytoplasmic [Ca2+] elevations were studied in pyramidal neurones isolated from the dorsal cochlear nucleus of the rat. Ca2+ currents were recorded using the whole-cell configuration of the patch-clamp technique. 10 micromol x l(-1) Cd2+ exerted a greater blocking effect on the high-voltage activated (HVA) currents than on the low-voltage activated (LVA) ones (decrease to 26.6+/-2.5% and to 87.8+/-2.1%, respectively). The blocking effect of 200 micromol x l(-1) Cd2+ was more pronounced and the difference between the effect on the HVA and LVA currents became smaller (decrease to 11.7+/-2.1% and to 32.4+/-2.7%, respectively). 200 micromol x l(-1) Ni2+ reduced the LVA component more effectively (to 77.6+/-5.4%) than the HVA one (to 86.9+/-2.6%). Cytoplasmic [Ca2+] changes were measured applying a fluorimetric technique (Fura-2). 10 micromol x l(-1) Cd2+ decreased the peak values of 50 mmol x l(-1) K+ depolarization-induced [Ca2]+i transients to 30.4+/-1.4% while 200 micromol x l(-1) Cd2+ caused a drop to 2.5+/-0.2%. 200 micromol x l(-1) Ni2+ decreased the peak of the transients to 69.6+/-2.9%. Comparison of the blocking effects of divalent cations on Ca2+ currents and [Ca2+]i transients supports further the conclusion that the depolarization-induced [Ca2+]i changes are produced mainly by the activation of the HVA Ca2+ channels.     

Attenuated renovascular constrictor responses to angiotensin II in adenosine 1 receptor knockout mice

In the present experiments we examined the renovascular constrictor effects of ANG II in the chronic and complete absence of A1 adenosine receptors (A1AR) using mice with targeted deletion of the A1AR gene. Glomerular filtration rate (GFR) was not different between A1AR +/+ and A1AR -/- mice under control conditions (450.5 +/- 60 vs. 475.2 +/- 62.5 microl/min) but fell significantly less in A1AR -/- mice during infusion of ANG II at 1.5 ng/min (A1AR +/+: 242 +/- 32.5 microl/min, A1AR -/-: 371 +/- 42 microl/min; P = 0.03). Bolus injection of 1, 10, and 100 ng of ANG II reduced renal blood flow and increased renal vascular resistance significantly more in A1AR +/+ than in A1AR -/- mice. Perfused afferent arterioles isolated from A1AR +/+ mice constricted in response to bath ANG II with an EC50 of 1.5 +/- 0.4 x 10(-10) mol/l, whereas a right shift in the dose-response relationship with an EC50 of 7.3 +/- 1.2 x 10(-10) mol/l (P < 0.05) was obtained in arterioles from A1AR -/- mice (P < 0.05). The expression of AT1A receptor mRNA was not different in kidney RNA from A1AR +/+ or A1AR -/- mice. We conclude that chronic A1AR deficiency diminishes the effectiveness of ANG II to constrict renal resistance vessels and to reduce GFR.     

Silver ions induce Ca2+ release from the SR in vitro by acting on the Ca2+ release channel and the Ca2+ pump.

Silver nitrate (AgNO3) is a sulfhydryl oxidizing agent that induces a biphasic Ca2+ release from isolated sarcoplasmic reticulum (SR) vesicles by presumably oxidizing critical sulfhydryl groups in the Ca2+ release channel (CRC), causing the channel to open. To further examine the effects of AgNO3 on the CRC and the Ca2+-ATPase, Ca2+ release was measured in muscle homogenates prepared from rat hindlimb muscle using indo 1. Cyclopiazonic acid (CPA) and ruthenium red (RR) were used to inhibit the Ca2+-ATPase and block the CRC, respectively, before inducing Ca2+ release with both AgNO3 and 4-chloro-m-cresol (4-CMC), a releasing agent specific for the CRC. With AgNO3 and CPA, the early rapid rate of release (phase 1) was increased (P < 0.05) by 42% (314 +/- 5 vs. 446 +/- 39 micromol x g protein(-1) x min(-1)), whereas the slower, more prolonged rate of release (phase 2) was decreased (P < 0.05) by 72% (267 +/- 39 vs. 74 +/- 7.7 micromol x g protein(-1) x min(-1)). RR, in combination with AgNO3, had no effect on phase 1 (P > 0.05) (314 +/- 51 vs. 334 +/- 43 micromol x g protein(-1) x min(-1)) and decreased phase 2 (P < 0.05) by 65% (245 +/- 34 vs. 105 +/- 8.2 micromol x g protein(-1) x min(-1)). With 4-CMC, CPA had no effect (P > 0.05) on either phase 1 or 2. With addition of RR, phase 1 was reduced (P < 0.05) by 59% (2,468 +/- 279 vs. 1,004 +/- 87 micromol x g protein(-1) x min(-1)), and RR completely blocked phase 2. Both AgNO3 and 4-CMC fully inhibited Ca2+-ATPase activity measured in homogenates. These findings indicate that AgNO3, but not 4-CMC, induces Ca2+ release by acting on both the CRC and the Ca2+-ATPase.     

Intrinsic A(1) adenosine receptor activation during ischemia or reperfusion improves recovery in mouse hearts

We assessed the role of A(1) adenosine receptor (A(1)AR) activation by endogenous adenosine in the modulation of ischemic contracture and postischemic recovery in Langendorff-perfused mouse hearts subjected to 20 min of total ischemia and 30 min of reperfusion. In control hearts, the rate-pressure product (RPP) and first derivative of pressure development over time (+dP/dt) recovered to 57 +/- 3 and 58 +/- 3% of preischemia, respectively. Diastolic pressure remained elevated at 20 +/- 2 mmHg (compared with 3 +/- 1 mmHg preischemia). Interstitial adenosine, assessed by microdialysis, rose from approximately 0.3 to 1.9 microM during ischemia compared with approximately 15 microM in rat heart. Nonetheless, these levels will near maximally activate A(1)ARs on the basis of effects of exogenous adenosine and 2-chloroadenosine. Neither A(1)AR blockade with 200 nM 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) during the ischemic period alone nor A(1)AR activation with 50 nM N(6)-cyclopentyladenosine altered rapidity or extent of ischemic contracture. However, ischemic DPCPX treatment significantly depressed postischemic recovery of RPP and +dP/dt (44 +/- 3 and 40 +/- 4% of preischemia, respectively). DPCPX treatment during the reperfusion period alone also reduced recovery of RPP and +dP/dt (to 44 +/- 2 and 47 +/- 2% of preischemia, respectively). These data indicate that 1) interstitial adenosine is lower in mouse versus rat myocardium during ischemia, 2) A(1)AR activation by endogenous adenosine or exogenous agonists does not modify ischemic contracture in murine myocardium, 3) A(1)AR activation by endogenous adenosine during ischemia attenuates postischemic stunning, and 4) A(1)AR activation by endogenous adenosine during the reperfusion period also improves postischemic contractile recovery.     

Biphasic effect of hydrogen peroxide on skeletal muscle arteriolar tone via activation of endothelial and smooth muscle signaling pathways.

We hypothesized that hydrogen peroxide (H2O2) has a role in the local regulation of skeletal muscle blood flow, thus significantly affecting the myogenic tone of arterioles. In our study, we investigated the effects of exogenous H2O2 on the diameter of isolated, pressurized (at 80 mmHg) rat gracilis skeletal muscle arterioles (diameter of approximately 150 microm). Lower concentrations of H2O2 (10(-6)-3 x 10(-5) M) elicited constrictions, whereas higher concentrations of H2O2 (6 x 10(-5)-3 x 10(-4) M), after initial constrictions, caused dilations of arterioles (at 10(-4) M H2O2, -19 +/- 1% constriction and 66 +/- 4% dilation). Endothelium removal reduced both constrictions (to -10 +/- 1%) and dilations (to 33 +/- 3%) due to H2O2. Constrictions due to H2O2 were completely abolished by indomethacin and the prostaglandin H2/thromboxane A2 (PGH2/TxA2) receptor antagonist SQ-29548. Dilations due to H2O2 were significantly reduced by inhibition of nitric oxide synthase (to 38 +/- 7%) but were unaffected by clotrimazole or sulfaphenazole (inhibitors of cytochrome P-450 enzymes), indomethacin, or SQ-29548. In endothelium-denuded arterioles, clotrimazole had no effect, whereas H2O2-induced dilations were significantly reduced by charybdotoxin plus apamin, inhibitors of Ca(2+)-activated K+ channels (to 24 +/- 3%), the selective blocker of ATP-sensitive K+ channels glybenclamide (to 14 +/- 2%), and the nonselective K(+)-channel inhibitor tetrabutylammonium (to -1 +/- 1%). Thus exogenous administration of H2O2 elicits 1) release of PGH2/TxA2 from both endothelium and smooth muscle, 2) release of nitric oxide from the endothelium, and 3) activation of K+ channels, such as Ca(2+)-activated and ATP-sensitive K+ channels in the smooth muscle resulting in biphasic changes of arteriolar diameter. Because H2O2 at low micromolar concentrations activates several intrinsic mechanisms, we suggest that H2O2 contributes to the local regulation of skeletal muscle blood flow in various physiological and pathophysiological conditions.     

Manipulation of dietary carbohydrates after prolonged effort modifies muscle sarcoplasmic reticulum responses in exercising males

The hypothesis tested was that disturbances in the sarcoplasmic reticulum (SR) Ca2+-cycling responses to exercise would associate with muscle glycogen reserves. Ten untrained males [peak O2 consumption (VO2 peak) = 3.41 +/- 0.20 (SE) l/min] performed a standardized cycle test (approximately 70% VO2 peak) on two occasions, namely, following 4 days of a high (Hi CHO)- and 4 days of a low (Lo CHO)-carbohydrate diet. Both Hi CHO and Lo CHO were preceded by a session of prolonged exercise designed to deplete muscle glycogen. SR Ca2+ cycling in crude homogenates prepared from vastus lateralis samples indicated higher (P < 0.05) Ca2+ uptake (microM x g protein(-1) x min(-1)) in Hi CHO compared with Lo CHO at 30 min (2.93 +/- 0.10 vs. 2.23 +/- 0.12) and at 67 min (2.77 +/- 0.16 vs. 2.10 +/- 0.12) of exercise, the point of fatigue in Lo CHO. Similar effects (P < 0.05) were noted between conditions for maximal Ca2+-ATPase (microM x g protein(-1) x min(-1)) at 30 min (142 +/- 8.5 vs. 107 +/- 5.0) and at 67 min (130 +/- 4.5 vs. 101 +/- 4.7). Both phase 1 and phase 2 Ca2+ release were 23 and 37% higher (P < 0.05) at 30 min of exercise and 15 and 34% higher (P < 0.05), at 67 min during Hi CHO compared with Lo CHO, respectively. No differences between conditions were observed at rest for any of these SR properties. Total muscle glycogen (mmol glucosyl units/kg dry wt) was higher (P < 0.05) in Hi CHO compared with Lo CHO at rest (+36%), 30 min (+53%), and at 67 min (+44%) of cycling. These results indicate that exercise-induced reductions in SR Ca2+-cycling properties occur earlier in exercise during low glycogen states compared with high glycogen states.     

Effects of adenosine and acadesine on interstitial nucleosides and myocardial stunning in the pig.

5-Amino-4-imidazolecarboxamide riboside (AICAr) or acadesine has been proposed to exert cardioprotection by enhancing adenosine production in ischemic myocardium. However, there are conflicting reports on acadesine's effects in ischemic myocardium and few studies in which myocardial adenosine levels have been measured. The purpose of this study was to determine whether acadesine increases interstitial fluid adenosine levels and attenuates myocardial stunning or potentiates the effects of adenosine in the intact pig. In pentobarbital-anesthetized pigs, myocardial stunning was induced by 10 min left anterior descending coronary artery occlusion and 90 min reperfusion. Regional ventricular function was assessed by measuring systolic wall thickening, and interstitial nucleosides were estimated by cardiac microdialysis. Control hearts were compared with hearts treated with acadesine, adenosine, and adenosine plus acadesine. Adenosine pretreatment (100 microg x kg(-1) x min(-1), intracoronary) immediately prior to ischemia increased interstitial adenosine levels 9-fold and improved postischemic functional recovery from a control value of 17.6 +/- 4.1% to 43.6 +/- 3.4% of preischemic systolic wall thickening. In contrast, acadesine (20 mg/kg i.v. bolus 10 min prior to ischemia + 0.5 mg x kg (-1) x min(-1), i.v. infusion through 60 min reperfusion) had no effect on interstitial fluid adenosine levels or the recovery of regional function (21.5 +/- 5.9% recovery), nor were the functional effects of adenosine potentiated by acadesine. These findings indicate that acadesine does not enhance myocardial adenosine levels, attenuate myocardial stunning, or potentiate the cardioprotective effects of adenosine in the pig.     

Lymphocyte adhesion-dependent calcium signaling in human endothelial cells

Vascular endothelial cells (ECs) can undergo dramatic phenotypic and functional alterations in response to humoral and cellular stimuli. These changes promote endothelial participation in the inflammatory response through active recruitment of immune effector cells, increased vascular permeability, and alteration in vascular tone. In an attempt to define early events in lymphocyte-mediated EC signaling, we investigated cytosolic-free calcium (Ca2+) changes in single, Fluo-3- labeled human umbilical vein ECs (HUVECs), using an ACAS interactive laser cytometer. Of all lymphocyte subsets tested, allogeneic CD3-, CD56+ natural killer (NK) cells uniquely elicited oscillatory EC Ca2+ signals in cytokine (interleukin [IL]-1- or tumor necrosis factor [TNF])-treated ECs. The induction of these signals required avid intercellular adhesion, consisted of both Ca2+ mobilization and extracellular influx, and was associated with EC inositol phosphate (IP) generation. Simultaneous recording of NK and EC Ca2+ signals using two-color fluorescence detection revealed that, upon adhesion, NK cells flux prior to EC. Lymphocyte Ca2+ buffering with 1,2-bis-5-methyl-amino- phenoxylethane-N,N,N'-tetra-acetoxymethyl acetate (MAPTAM) demonstrated that lymphocyte fluxes are, in fact, prerequisites for the adhesion- dependent EC signals. mAb studies indicate that the beta 2 integrin- intercellular adhesion molecule (ICAM)-1 adhesion pathway is critically involved. However, ICAM-1 antisense oligonucleotide inhibition of IL-1- mediated ICAM-1 hyperinduction had no effect on EC Ca2+ signaling in lymphocyte-EC conjugates, indicating that additional cytokine-induced EC alteration is required. These experiments combine features of lymphocyte-endothelial interactions, intercellular adhesion, EC cytokine activation and transmembrane signaling. The results implicate the IP/Ca2+ second messenger pathway in EC outside-in signaling induced by cytotoxic lymphocytes, and suggest that these signals may play a role in EC alteration by lymphocyte adhesion.     

Changes in excitation-contraction coupling in an isolated ventricular myocyte model of cardiac stunning

To investigate cardiac stunning, we recorded intracellular [Ca(2+)], contractions, and electrical activity in isolated guinea pig ventricular myocytes exposed to simulated ischemia and reperfusion. After equilibration, ischemia was simulated by exposing myocytes to hypoxia, acidosis, hyperkalemia, hypercapnia, lactate accumulation, and substrate deprivation for 30 min at 37 degrees C. Reperfusion was simulated by exposure to Tyrode solution. Field-stimulated myocytes exhibited stunning upon reperfusion. By 10 min of reperfusion, contraction amplitude decreased to 43.0 +/- 5.5% of preischemic values (n = 15, P < 0.05), although action potential configuration and sarcoplasmic reticulum Ca(2+) stores, assessed with caffeine, were normal. Diastolic [Ca(2+)] and Ca(2+) transients (fura 2) were also normal in stunned myocytes. In voltage-clamped cells, peak L-type Ca(2+) current was reduced to 47.4 +/- 4.5% of preischemic values at 10 min of reperfusion (n = 21, P < 0.05). Contractions elicited by Ca(2+)-induced Ca(2+) release and the voltage-sensitive release mechanism were both depressed in reperfusion. Our observations suggest that stunning is associated with reduced L-type Ca(2+) current but that alterations in Ca(2+) homeostasis and release are not directly responsible for stunning.     

Protein kinase C and preconditioning: role of the sarcoplasmic reticulum

Activation of protein kinase C (PKC) is cardioprotective, but the mechanism(s) by which PKC mediates protection is not fully understood. Inasmuch as PKC has been well documented to modulate sarcoplasmic reticulum (SR) Ca2+ and because altered SR Ca2+ handling during ischemia is involved in cardioprotection, we examined the role of PKC-mediated alterations of SR Ca2+ in cardioprotection. Using isolated adult rat ventricular myocytes, we found that addition of 1,2-dioctanoyl-sn-glycerol (DOG), to activate PKC under conditions that reduced myocyte death associated with simulated ischemia and reperfusion, also reduced SR Ca2+. Cell death was 57.9 +/- 2.9% and 47.3 +/- 1.8% in untreated and DOG-treated myocytes, respectively (P < 0.05). Using fura 2 fluorescence to monitor Ca2+ transients and caffeine-releasable SR Ca2+, we examined the effect of DOG on SR Ca2+. Caffeine-releasable SR Ca2+ was significantly reduced (by approximately 65%) after 10 min of DOG treatment compared with untreated myocytes (P < 0.05). From our examination of the mechanism by which PKC alters SR Ca2+, we present the novel finding that DOG treatment reduced the phosphorylation of phospholamban (PLB) at Ser16. This effect is mediated by PKC-epsilon, because a PKC-epsilon-selective inhibitory peptide blocked the DOG-mediated decrease in phosphorylation of PLB and abolished the DOG-induced reduction in caffeine-releasable SR Ca2+. Using immunoprecipitation, we further demonstrated that DOG increased the association between protein phosphatase 1 and PLB. These data suggest that activated PKC-epsilon reduces SR Ca2+ content through PLB dephosphorylation and that reduced SR Ca2+ may be important in cardioprotection.     

Inosine released after hypoxia activates hepatic glucose liberation through A3 adenosine receptors

Inosine, an endogenous nucleoside, has recently been shown to exert potent effects on the immune, neural, and cardiovascular systems. This work addresses modulation of intermediary metabolism by inosine through adenosine receptors (ARs) in isolated rat hepatocytes. We conducted an in silico search in the GenBank and complete genomic sequence databases for additional adenosine/inosine receptors and for a feasible physiological role of inosine in homeostasis. Inosine stimulated glycogenolysis (approximately 40%, EC50 4.2 x 10(-9) M), gluconeogenesis (approximately 40%, EC50 7.8 x 10(-9) M), and ureagenesis (approximately 130%, EC50 7.0 x 10(-8) M) compared with basal values; these effects were blunted by the selective A3 AR antagonist 9-chloro-2-(2-furanyl)-5-[(phenylacetyl)amino][1,2,4]-triazolo[1,5-c]quinazoline (MRS 1220) but not by selective A1, A2A, and A2B AR antagonists. In addition, MRS 1220 antagonized inosine-induced transient increase (40%) in cytosolic Ca2+ and enhanced (90%) glycogen phosphorylase activity. Inosine-induced Ca2+ mobilization was desensitized by adenosine; in a reciprocal manner, inosine desensitized adenosine action. Inosine decreased the cAMP pool in hepatocytes when A1, A2A, and A2B AR were blocked by a mixture of selective antagonists. Inosine-promoted metabolic changes were unrelated to cAMP decrease but were Ca2+ dependent because they were absent in hepatocytes incubated in EGTA- or BAPTA-AM-supplemented Ca2+-free medium. After in silico analysis, no additional cognate adenosine/inosine receptors were found in human, mouse, and rat. In both perfused rat liver and isolated hepatocytes, hypoxia/reoxygenation produced an increase in inosine, adenosine, and glucose release; these actions were quantitatively greater in perfused rat liver than in isolated cells. Moreover, all of these effects were impaired by the antagonist MRS 1220. On the basis of results obtained, known higher extracellular inosine levels under ischemic conditions, and inosine's higher sensitivity for stimulating hepatic gluconeogenesis, it is suggested that, after tissular ischemia, inosine contributes to the maintenance of homeostasis by releasing glucose from the liver through stimulation of A3 ARs.     

Relationship between myoplasmic calcium transients and calcium currents in frog skeletal muscle

Ca2+ currents (ICa) and myoplasmic Ca2+ transients were simultaneously recorded in single muscle fibers from the semitendinosus muscle of Rana pipiens. The vaseline-gap voltage-clamp technique was used. Ca2+ transients were recorded with the metallochromic indicator dye antipyrylazo III. Ca2+ transients consisted of an early fast rising phase followed by a late slower one. The second phase was increased by experimental maneuvers that enlarged ICa, such as augmenting [Ca2+]o (from 2 to 10 mM) or adding (-)-Bay K 8644 (2 microM). When [Ca2+]o was increased, the second phase of the Ca2+ transients and ICa showed an average increase at 0 mV of 2 +/- 0.9 microM (4) and 1.4 +/- 0.3 mA/ml (4), respectively. (-)-Bay K 8644 increased the late phase of the Ca2+ transients and ICa at 0 mV by 0.8 +/- 0.3 microM (3) and 6.7 +/- 2.0 mA/ml (4), respectively. The initial fast rising phase of the Ca2+ transients was not modified. (-)-Bay K 8644 slowed the time constant of decay of the transients by 57 +/- 6 ms. In other experimental conditions, Ca2+ release from the sarcoplasmic reticulum (SR) was impaired with repetitive stimulation in 1 mM [EGTA]i-containing fibers. Under those circumstances, Ca2+ transients directly followed the time integral of ICa. Pulses to 0 mV caused a large Ca2+ transient that became suppressed when large pulses to 100 mV were applied. In fibers with functioning SR, pulses to 100 mV elicited somewhat smaller or similar amplitude Ca2+ transients when compared with those elicited by pulses to 0 mV. The increase in ICa after raising [Ca2+]o or adding (-)-Bay K 8644 cannot directly explain the change in Ca2+ transients in fibers with functioning SR. On the other hand, when Ca2+ release from the SR is impaired Ca2+ transients depend on ICa.     

Insulin-dependent contractility of glomerular mesangial cells in response to angiotensin II, platelet-activating factor and endothelin is attenuated by prostaglandin E2.

Culture of glomerular mesangial cells in the absence of insulin decreased the degree of contraction of individual cells in response to vasoconstrictive agonists, angiotensin II, platelet-activating factor and endothelin 1, as compared with cells cultured in the presence of insulin (0.7 nM). This change was associated with a decreased sensitivity of the intracellular Ca2+ response to vasoactive agents in fura-2-loaded cells and with an increase in the basal level of prostanoid [prostaglandins (PG) E1 and E2] production estimated by radioimmunoassay. Addition of exogenous PGE2 to insulin-exposed cells decreased the contractile response to that observed in insulin-deficient cells. Inclusion of 8-bromo cyclic AMP had a similar effect. In 45Ca2(+)-release studies it was shown that, in saponin-permeabilized insulin-exposed cells, preincubation with exogenous PGE2 or 8-bromo cyclic AMP decreased the sensitivity of 45Ca2+ release in response to Ins(1,4,5)P3, as demonstrated by an increase in the EC50 (concn. giving half-maximal effect) to 0.182 +/- 0.024 microM and 0.457 +/- 0.031 microM respectively, as compared with untreated permeabilized cells (EC50 0.091 +/- 0.021 microM). A similar decrease in Ins(1,4,5)P3-sensitive 45Ca2+ release was seen in permeabilized cells from insulin-free conditions of culture (EC50 0.20 +/- 0.061 microM). As altered glomerular haemodynamics are found in insulinopaenic diabetic conditions, it is proposed that a decrease in intracellular Ca2+ availability in response to vasoactive agonists and consequent decrease in mesangial-cell contractility contributes to the hyperfiltration seen in this condition.     

Conducted dilations initiated by purines in arterioles are endothelium dependent and require endothelial Ca2+

The signaling pathways underlying the regulation of vascular resistance by purines in intact microvessels and particularly in communication of remote vasomotor responses are unclear. One process by which remote regions of arterioles communicate is via transmission of signals axially along the vessel wall. In this study, we identified a pathway for local and conducted dilations initiated by purines. Adenosine (Ado) or ATP (bind P1 and P2 purinergic receptors, respectively) was micropipette applied to arterioles (maximum diameter approximately 40 microm) in the cheek pouch of anesthetized hamsters. Observations were made at the site of stimulation (local) or approximately 1200 microm upstream along the same vessel. P2 antagonists (pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid tetrasodium and suramin) inhibited local constriction to ATP, whereas local and upstream dilations were unaffected. In contrast, during inhibition of P1 receptors (with xanthine amine congener) the local constriction was unchanged, whereas both local and upstream dilations to ATP were inhibited. Hydrolysis of ATP to Ado is implicated in the dilator response as blocking 5'-ectonucleotidase (with alpha,beta-methyleneadenosine 5'-diphosphate) attenuated ATP-induced dilations. After endothelium denudation, constriction to ATP was unchanged, but dilations to both ATP and Ado were inhibited, identifying endothelial cells (ECs) as the primary target for P1-mediated dilation. Purines increased EC Ca2+ locally and upstream. Chelation of EC Ca2+ (with BAPTA) abolished the local and upstream dilations to P1 receptor stimulation. Collectively, these data demonstrate that stimulation of P1 receptors on ECs produces a vasodilation that spreads to remote regions. There is an associated increase in EC Ca2+, which is a required signaling intermediate in the manifestation of both the local and axially communicated arteriolar dilations.     

Differences of Ca2+ regulation in skin fibroblasts from blacks and whites

Black people have a higher propensity than caucasians toward essential hypertension. To explore the possibility that this racial difference relates to cellular Ca2+ metabolism, we measured 45Ca2+ washout and uptake and cytosolic free concentration of Ca2+ [Ca2+]i in serially passed skin fibroblasts from normotensive black and white males. Depending on the experimental conditions, 45Ca2+ washout in these cells was described by either two or three exponential functions, whereas 45Ca2+ uptake was described only by a two-exponent function. There were no racial differences in 45Ca2+ uptake and washout of unstimulated fibroblasts. However, stimulation by human serum resulted in an increase in the 45Ca2+ washout that was higher in fibroblasts from blacks than from whites. The racial differences were expressed primarily by higher values of the apparent washout rate constant (k1) of 45Ca2+ from the largest and most rapidly exchangeable cellular pool. The effect of human serum was not related to its origin (blacks vs. whites). In 2 mM Ca2+ medium and 10% serum from blacks, the respective k1 (mean +/- SEM; x 10(-2)/min) values for fibroblasts from blacks and whites were 89.68 +/- 5.23 and 73.29 +/- 4.0; in the presence of 10% serum from whites, the k1 values for cells from blacks and whites were 84.14 +/- 2.80 and 76.36 +/- 3.23 (overall significance of P less than .01). In Ca2+-deficient medium in the presence of 10% human serum, the k1 for fibroblasts from blacks and whites were 115.57 +/- 3.76 and 102.15 +/- 3.30 (P less than .05). Serum substantially increased the 45Ca2+ uptake in fibroblasts from both blacks and whites; however, racial differences were not observed. Basal levels of [Ca2+]i were not different in fibroblasts of blacks vs. whites (46.8 +/- 6.8 and 43.2 +/- 7.1 nM for blacks and whites, respectively). However, the peak response of Cai2+ transients for cell stimulated by 5% human serum was significantly higher in blacks than whites (blacks = 963 +/- 213, whites = 481 +/- 162 nM; P = .0286). We conclude that Ca2+ regulation is different in serum-stimulated fibroblasts from blacks and whites and that, at least in part, this difference may relate to a greater agonist-induced mobilization of Ca2+ in fibroblasts from blacks.     

Creatine kinase-deficient hearts exhibit increased susceptibility to ischemia-reperfusion injury and impaired calcium homeostasis

The creatine kinase (CK) system is involved in the rapid transport of high-energy phosphates from the mitochondria to the sites of maximal energy requirements such as myofibrils and sarcolemmal ion pumps. Hearts of mice with a combined knockout of cytosolic M-CK and mitochondrial CK (M/Mito-CK(-/-)) show unchanged basal left ventricular (LV) performance but reduced myocardial high-energy phosphate concentrations. Moreover, skeletal muscle from M/Mito-CK(-/-) mice demonstrates altered Ca2+ homeostasis. Our hypothesis was that in CK-deficient hearts, a cardiac phenotype can be unmasked during acute stress conditions and that susceptibility to ischemia-reperfusion injury is increased because of altered Ca2+ homeostasis. We simultaneously studied LV performance and myocardial Ca2+ metabolism in isolated, perfused hearts of M/Mito-CK(-/-) (n = 6) and wild-type (WT, n = 8) mice during baseline, 20 min of no-flow ischemia, and recovery. Whereas LV performance was not different during baseline conditions, LV contracture during ischemia developed significantly earlier (408 +/- 72 vs. 678 +/- 54 s) and to a greater extent (50 +/- 2 vs. 36 +/- 3 mmHg) in M/Mito-CK(-/-) mice. During reperfusion, recovery of diastolic function was impaired (LV end-diastolic pressure: 22 +/- 3 vs. 10 +/- 2 mmHg), whereas recovery of systolic performance was delayed, in M/Mito-CK(-/-) mice. In parallel, Ca2+ transients were similar during baseline conditions; however, M/Mito-CK(-/-) mice showed a greater increase in diastolic Ca2+ concentration ([Ca2+]) during ischemia (237 +/- 54% vs. 167 +/- 25% of basal [Ca2+]) compared with WT mice. In conclusion, CK-deficient hearts show an increased susceptibility of LV performance and Ca2+ homeostasis to ischemic injury, associated with a blunted postischemic recovery. This demonstrates a key function of an intact CK system for maintenance of Ca2+ homeostasis and LV mechanics under metabolic stress conditions.