Novel role of phospholipase C-delta1: regulation of liver mitochondrial Ca2+ uptake

Mitochondrial Ca2+ (mCa2+) handling is an important regulator of liver cell function that controls events ranging from cellular respiration and signal transduction to apoptosis. Cytosolic Ca2+ enters mitochondria through the ruthenium red-sensitive mCa2+ uniporter, but the mechanisms governing uniporter activity are unknown. Activation of many Ca2+ channels in the cell membrane requires PLC. This activation commonly occurs through phosphitidylinositol-4,5-biphosphate (PIP2) hydrolysis and the production of the second messengers inositol 1,4,5-trisphosphate [I(1,4,5)P3] and 1,2-diacylglycerol (DAG). PIP2 was recently identified in mitochondria. We hypothesized that PLC exists in liver mitochondria and regulates mCa2+ uptake through the uniporter. Western blot analysis with anti-PLC antibodies demonstrated the presence of PLC-delta1 in pure preparations of mitochondrial membranes isolated from rat liver. In addition, the selective PLC inhibitor U-73122 dose-dependently blocked mCa2+ uptake when whole mitochondria were incubated at 37 degrees C with 45Ca2+. Increasing extra mCa2+ concentration significantly stimulated mCa2+ uptake, and U-73122 inhibited this effect. Spermine, a uniporter agonist, significantly increased mCa2+ uptake, whereas U-73122 dose-dependently blocked this effect. The inactive analog of U-73122, U-73343, did not affect mCa2+ uptake in any experimental condition. Membrane-permeable I(1,4,5)P3 receptor antagonists 2-aminoethoxydiphenylborate and xestospongin C also inhibited mCa2+ uptake. Although extra mitochondrial I(1,4,5)P3 had no effect on mCa2+ uptake, membrane-permeable DAG analogs 1-oleoyl-2-acetyl-sn-glycerol and DAG-lactone, which inhibit PLC activity, dose-dependently inhibited mCa2+ uptake. These data indicate that PLC-delta1 exists in liver mitochondria and is involved in regulating mCa2+ uptake through the uniporter.     

Adenosine induces ATP release via an inositol 1,4,5-trisphosphate signaling pathway in MDCK cells

ATP is released into extracellular space as an autocrine/paracrine molecule by mechanical stress and pharmacological-receptor activation. Released ATP is partly metabolized by ectoenzymes to adenosine. In the present study, we found that adenosine causes ATP release in Madin-Darby canine kidney cells. This release was completely inhibited by CPT (an A1 receptor antagonist), U-73122 (a phospholipase C inhibitor), 2-APB (an inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) receptor blocker), thapsigargin (a Ca2+-ATPase inhibitor), and BAPTA/AM (an intracellular Ca2+ chelator), but not by DMPX (an A2 receptor antagonist). However, forskolin, epinephrine, and isoproterenol, inducers of cAMP accumulation, failed to release ATP. Adenosine increased intracellular Ca2+ concentrations that were strongly blocked by CPT, U-73122, 2-APB, and thapsigargin. Moreover, adenosine enhanced accumulations of Ins(1,4,5)P3 that were significantly reduced by U-73122 and CPT. These data suggest that adenosine induces the release of ATP by activating an Ins(1,4,5)P3 sensitive-Ca2+ pathway through the stimulation of A1 receptors.     

Calcium waves in intact guinea pig gallbladder smooth muscle cells

Intracellular Ca(2+) waves and spontaneous transient depolarizations were investigated in gallbladder smooth muscle (GBSM) whole mount preparations with intact mucosal layer [full thickness (FT)] by laser confocal imaging of intracellular Ca(2+) and voltage recordings with microelectrodes, respectively. Spontaneous Ca(2+) waves arose most often near the center, but sometimes from the extremities, of GBSM cells. They propagated regeneratively by Ca(2+)-induced Ca(2+) release involving inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] receptors and were not affected by TTX and atropine (ATS). Spontaneous Ca(2+) waves and spontaneous transient depolarizations were more prevalent in FT than in isolated muscularis layer preparations and occurred with similar pattern in GBSM bundles. Ca(2+) waves were abolished by the Ins(1,4,5)P(3) receptor inhibitors 2-aminoethoxydiphenyl borate and xestospongin C and by caffeine and cyclopiazonic acid. These events were reduced by voltage-dependent calcium channels (VDCCs) inhibitors diltiazem and nifedipine, by PLC inhibitor U-73122, and by thapsigargin and ryanodine. ACh, CCK, and carbachol augmented Ca(2+) waves and induced Ca(2+) flashes. The actions of these agonists were inhibited by U-73122. These results indicate that in GBSM, discharge and propagation of Ca(2+) waves depend on sarco(endo)plasmic reticulum (SR) Ca(2+) release via Ins(1,4,5)P(3) receptors, PLC activity, Ca(2+) influx via VDCCs, and SR Ca(2+) concentration. Neurohormonal enhancement of GBSM excitability involves PLC-dependent augmentation and synchronization of SR Ca(2+) release via Ins(1,4,5)P(3) receptors. Ca(2+) waves likely reflect the activity of a fundamental unit of spontaneous activity and play an important role in the excitability of GBSM.     

Effects of halothane on mechanical response of skeletal muscle from malignant hyperthermia susceptible patients.

The purpose of this investigation was to compare the effects of halothane on malignant hyperthermia (MH) and normal isolated muscle bundle performance during isometric contraction and relaxation phases. Mechanical parameters were measured: peak tension (PT), time to peak tension (TPT) and positive peak of isometric tension derivative (+dP/dtmax) characterized the contraction phase. Half-relaxation time (RT1/2) and negative peak of isometric tension derivative (-dP/dtmax) characterized the relaxation phase. The ratio R = (+dP/dtmax)/(-dP/dtmax) was used to study the coupling between contraction and relaxation under isometric condition. In normal muscle, halothane increased PT by nearly 40% without altering TPT. The +dP/dtmax value increased concomitantly with the -dP/dtmax values, thus no changes in R was observed. In MH muscle, PT was first potentiated (0.5-1.0 vol% halothane) and then depressed (2.0-3.0 vol% halothane). TPT and +dP/dtmax were not altered whereas RT1/2 increased progressively with concomitant decrease in -dP/dtmax, thus R increased by nearly 40%. The amplitude of MH muscle contracture with stepwise concentrations of halothane was correlated with the increase of RT1/2 and R, and the decrease of -dP/dtmax. These results suggest that halothane alters the relaxation phase more than the contraction phase in MH human skeletal muscle compared to normal muscle.     

Pollen tube tip growth depends on plasma membrane polarization mediated by tobacco PLC3 activity and endocytic membrane recycling

Phosphatidyl inositol 4,5-bisphosphate (PI 4,5-P2) accumulates in a Rac/Rop-dependent manner in the pollen tube tip plasma membrane, where it may control actin organization and membrane traffic. PI 4,5-P2 is hydrolyzed by phospholipase C (PLC) activity to the signaling molecules inositol 1,4,5-trisphosphate and diacyl glycerol (DAG). To investigate PLC activity during tip growth, we cloned Nt PLC3, specifically expressed in tobacco (Nicotiana tabacum) pollen tubes. Recombinant Nt PLC3 displayed Ca2+-dependent PI 4,5-P2-hydrolyzing activity sensitive to U-73122 and to mutations in the active site. Nt PLC3 overexpression, but not that of inactive mutants, inhibited pollen tube growth. Yellow fluorescent protein (YFP) fused to Nt PLC3, or to its EF and C2 domains, accumulated laterally at the pollen tube tip plasma membrane in a pattern complementary to the distribution of PI 4,5-P2. The DAG marker Cys1:YFP displayed a similar intracellular localization as PI 4,5-P2. Blocking endocytic membrane recycling affected the intracellular distribution of DAG but not of PI 4,5-P2. U-73122 at low micromolar concentrations inhibited and partially depolarized pollen tube growth, caused PI 4,5-P2 spreading at the apex, and abolished DAG membrane accumulation. We show that Nt PLC3 is targeted by its EF and C2 domains to the plasma membrane laterally at the pollen tube tip and that it maintains, together with endocytic membrane recycling, an apical domain enriched in PI 4,5-P2 and DAG required for polar cell growth.     

Short-term exercise improves myocardial tolerance to in vivo ischemia-reperfusion in the rat.

These experiments examined the independent effects of short-term exercise and heat stress on myocardial responses during in vivo ischemia-reperfusion (I/R). Female Sprague-Dawley rats (4 mo old) were randomly assigned to one of four experimental groups: 1) control, 2) 3 consecutive days of treadmill exercise [60 min/day at 60-70% maximal O2 uptake (VO2 max)], 3) 5 consecutive days of treadmill exercise (60 min/day at 60-70% VO2 max), and 4) whole body heat stress (15 min at 42 degrees C). Twenty-four hours after heat stress or exercise, animals were anesthetized and mechanically ventilated, and the chest was opened by thoracotomy. Coronary occlusion was maintained for 30-min followed by a 30-min period of reperfusion. Compared with control, both heat-stressed animals and exercised animals (3 and 5 days) maintained higher (P < 0.05) left ventricular developed pressure (LVDP), maximum rate of left ventricular pressure development (+dP/dt), and maximum rate of left ventricular pressure decline (-dP/dt) at all measurement periods during both ischemia and reperfusion. No differences existed between heat-stressed and exercise groups in LVDP, +dP/dt, and -dP/dt at any time during ischemia or reperfusion. Both heat stress and exercise resulted in an increase (P < 0.05) in the relative levels of left ventricular heat shock protein 72 (HSP72). Furthermore, exercise (3 and 5 days) increased (P < 0.05) myocardial glutathione levels and manganese superoxide dismutase activity. These data indicate that 3-5 consecutive days of exercise improves myocardial contractile performance during in vivo I/R and that this exercise-induced myocardial protection is associated with an increase in both myocardial HSP72 and cardiac antioxidant defenses.     

Positive inotropic, positive chronotropic and coronary vasodilatory effects of rat amylin: mechanisms of amylin-induced positive inotropy

Even though there are a few studies dealing with the cardiac effects of amylin, the mechanisms of amylin-induced positive inotropy are not known well. Therefore, we investigated the possible signaling pathways underlying the amylin-induced positive inotropy and compared the cardiac effects of rat amylin (rAmylin) and human amylin (hAmylin).Isolated rat hearts were perfused under constant flow condition and rAmylin or hAmylin was infused to the hearts. Coronary perfusion pressure, heart rate, left ventricular developed pressure and the maximum rate of increase of left ventricular pressure (+dP/dtmax) and the maximum rate of pressure decrease of left ventricle (-dP/dtmin) were measured.rAmylin at concentrations of 1, 10 or 100 nM markedly decreased coronary perfusion pressure, but increased heart rate, left ventricular developed pressure, +dP/dtmax and -dP/dtmin. The infusion of H-89 (50 μM), a protein kinase A (PKA) inhibitor did not change the rAmylin (100 nM)-induced positive inotropic effect. Both diltiazem (1 μM), an L-type Ca2+ channel blocker and ryanodine (10 nM), a sarcoplasmic reticulum (SR) Ca2+ release channel opener completely suppressed the rAmylin-induced positive inotropic effect, but staurosporine (100 nM), a potent protein kinase C (PKC) inhibitor suppressed it partially. hAmylin (1, 10 and 100 nM) had no significant effect on coronary perfusion pressure, heart rate and developed pressure, +dP/dtmax and -dP/dtmin.We concluded that rAmylin might have been produced vasodilatory, positive chronotropic and positive inotropic effects on rat hearts. Ca2+ entry via L-type Ca2+ channels, activation of PKC and Ca2+ release from SR through ryanodine-sensitive Ca2+ channels may be involved in this positive inotropic effect. hAmylin may not produce any significant effect on perfusion pressure, heart rate and contractility in isolated, perfused rat hearts.     

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.     

Short-term exercise training can improve myocardial tolerance to I/R without elevation in heat shock proteins

We examined the effects of 3 days of exercise in a cold environment on the expression of left ventricular (LV) heat shock proteins (HSPs) and contractile performance during in vivo ischemia-reperfusion (I/R). Sprague-Dawley rats were divided into the following three groups (n = 12/group): 1) control, 2) exercise (60 min/day) at 4 degrees C (E-Cold), and 3) exercise (60 min/day) at 25 degrees C (E-Warm). Left anterior descending coronary occlusion was maintained for 20 min, followed by 30 min of reperfusion. Compared with the control group, both the E-Cold and E-Warm groups maintained higher (P < 0.05) LV developed pressure, first derivative of pressure development over time (+dP/dt), and pressure relaxation over time (-dP/dt) throughout I/R. Relative levels of HSP90, HSP72, and HSP40 were higher (P < 0.05) in E-Warm animals compared with both control and E-Cold. HSP10, HSP60, and HSP73 did not differ between groups. Exercise increased manganese superoxide dismutase (MnSOD) activity in both E-Warm and E-Cold hearts (P < 0.05). Protection against I/R-induced lipid peroxidation in the LV paralleled the increase in MnSOD activity whereas lower levels of lipid peroxidation were observed in both E-Warm and E-Cold groups compared with control. We conclude that exercise-induced myocardial protection against a moderate duration I/R insult is not dependent on increases in myocardial HSPs. We postulate that exercise-associated cardioprotection may depend, in part, on increases in myocardial antioxidant defenses.     

Hydrogen sulfide contributes to cardioprotection during ischemia-reperfusion injury by opening K ATP channels

The present study was undertaken to investigate the protective effect of H2S against myocardial ischemia-reperfusion (I/R) injury and its possible mechanism by using isolated heart perfusion and patch clamp recordings. Rat isolated hearts were Langendorff-perfused and subjected to a 30-minute ischemia insult followed by a 30-minute reperfusion. The heart function was assessed by measuring the LVDP, +/-dP/dt max, and the arrhythmia score. The results showed that the treatment of hearts with a H2S donor (40 micromol/L NaHS) during reperfusion resulted in significant improvement in heart function compared with the I/R group (LVDP recovered to 85.0% +/- 6.4% vs. 35.0% +/- 6.1%, +dP/dt max recovered to 80.9% +/- 4.2% vs. 43.0% +/- 6.4%, and -dP/dt max recovered to 87.4% +/- 7.3% vs. 53.8% +/- 4.9%; p < 0.01). The arrhythmia scores also improved in the NaHS group compared with the I/R group (1.5 +/- 0.2 vs. 4.0 +/- 0.4, respectively; p < 0.001). The cardioprotective effect of NaHS during reperfusion could be blocked by an ATP-sensitive potassium channel (K ATP) blocker (10 micromol/L glibenclamide). In single cardiac myocytes, NaHS increased the open probability of K ATP channels from 0.07 +/- 0.03 to 0.15 +/- 0.08 after application of 40 mumol/L NaHS and from 0.07 +/- 0.03 to 0.36 +/- 0.15 after application of 100 mumol/L NaHS. These findings provide the first evidence that H2S increases the open probability of K ATP in cardiac myocytes, which may be responsible for cardioprotection against I/R injury during reperfusion.     

Inositol(1,4,5)trisphosphate signal triggers a receptor-mediated ATP release

Intracellular signal transduction pathways involved in ATP release evoked by angiotensin II (Ang II) were investigated in cultured guinea pig Taenia coli smooth muscle cells. Ang II (0.3-1 microM) elicited substantial release of ATP from the cells, but not from a human fibroblast cell line. However, Ang II even at 10 microM failed to cause a leakage of lactate dehydrogenase (LDH) from the smooth muscle cells. The release of ATP by Ang II was suppressed by 10 microM SC52458, an AT1 receptor antagonist, not by 10 microM PD123319, an AT2 receptor antagonist. The evoked release of ATP was almost completely inhibited in the presence of 10 microM U73122, a phospholipase C inhibitor, and 0.5 microM thapsigargin, a Ca2+-ATPase inhibitor. Furthermore, the release was hampered by 50 microM BAPTA/AM, an intracellular Ca2+ chelator, but not by 0.1 microM nifedipine, a voltage gated Ca2+ channel inhibitor. The basal release of ATP was increased by BAPTA/AM, but was reduced by U-73122. Ang II enhanced instantaneously inositol(1,4,5)trisphosphate (Ins(1,4,5)P3) accumulation in the cells. The enhancing effect was perfectly antagonized by SC52458. These findings suggest that intracellular Ca2+ signals activated via stimulation of Ins(1,4,5)P3 receptor are involved in the release of ATP evoked by Ang II.     

Expression of functional receptor-coupled TRPC3 channels in DT40 triple receptor InsP3 knockout cells

The TRPC3 channel, an intensively studied member of the widely expressed transient receptor potential (TRP) family, is a Ca(2+)-conducting channel activated in response to phospholipase C-coupled receptors. Despite scrutiny, the receptor-induced mechanism to activate TRPC3 channels remains unclear. Evidence indicates TRPC3 channels interact directly with intracellular inositol 1,4,5-trisphosphate receptors (InsP(3)Rs) and that channel activation is mediated through coupling to InsP(3)Rs. TRPC3 channels were expressed in DT40 chicken B lymphocytes in which all three InsP(3)R genes were deleted (DT40InsP(3)R-k/o). Endogenous B-cell receptors (BCR) coupled through Syk kinase to phospholipase C-gamma (PLC-gamma) activated the expressed TRPC3 channels in both DT40w/t and DT40InsP(3)R-k/o cells. The diacylglycerol (DAG) analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) also activated TRPC3 channels independently of InsP(3)Rs. BCR-induced TRPC3 activation was blocked by the PLC enzymic inhibitor, U-73122, and also blocked by wortmannin-induced PLC substrate depletion. Neither U-73122 nor wortmannin modified either OAG-induced TRPC3 activation or store-operated channel activation in DT40 cells. Cotransfection of cells with both G protein-coupled M5 muscarinic receptors and TRPC3 channels resulted in successful M5 coupling to open TRPC3 channels mediated by PLC-beta. We conclude that TRPC3 channels are activated independently of InsP(3)Rs through DAG production resulting from receptor-mediated activation of either PLC-gamma or PLC-beta.     

U-73122 does not specifically inhibit phospholipase C in rat pancreatic islets and insulin-secreting β-cell lines

Phospholipase C is activated in insulin secretion by islets of Langerhans and insulin-secreting β-cells such as RINm5F and β-TC3. We have examined the effects of the aminosteroid U-73122, a phospholipase C inhibitor, on insulin secretion and phospholipase C activation. U-73122 slightly inhibited glucose-induced insulin secretion from islets, but this effect was not specific since the structural “inactive” analogue U-73343 also inhibited insulin secretion. Likewise, in RINm5F cells, U-73122 did not inhibit glyceraldehyde-induced insulin secretion. Phospholipase C activity was assessed as the accumulation of inositol-1,4,5-trisphosphate (Ins(1,4,5)P₃) measured with a competitive binding assay: U-73122 failed to inhibit glucose-induced increase in Ins(1,4,5)P₃. Similarly, when the effects of U-73122 and U-73343 were measured on [³H]phosphatidylinositol hydrolysis of islets, both compounds caused a slight, non-specific inhibition of phospholipase C activity. These observations suggest that U-73122 does not specifically inhibit phospholipase C in insulin-secreting cells.     

Single cell analysis and temporal profiling of agonist-mediated inositol 1,4,5-trisphosphate, Ca2+, diacylglycerol, and protein kinase C signaling using fluorescent biosensors

The magnitude and temporal nature of intracellular signaling cascades can now be visualized directly in single cells by the use of protein domains tagged with enhanced green fluorescent protein (eGFP). In this study, signaling downstream of G protein-coupled receptor-mediated phospholipase C (PLC) activation has been investigated in a cell line coexpressing recombinant M(3) muscarinic acetylcholine and alpha(1B) -adrenergic receptors. Confocal measurements of changes in inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)), using the pleckstrin homology domain of PLCdelta1 tagged to eGFP (eGFP-PH(PLCdelta)), and 1,2-diacylglycerol (DAG), using the C1 domain of protein kinase Cgamma (PKCgamma) (eGFP-C1(2)-PKCgamma), demonstrated clear translocation responses to methacholine and noradrenaline. Single cell EC(50) values calculated for each agonist indicated that responses to downstream signaling targets (Ca(2+) mobilization and PKC activation) were approximately 10-fold lower compared with respective Ins(1,4,5)P(3) and DAG EC(50) values. Examining the temporal profile of second messenger responses to sub-EC(50) concentrations of noradrenaline revealed oscillatory Ins(1,4,5)P(3), DAG, and Ca(2+) responses. Oscillatory recruitments of conventional (PKCbetaII) and novel (PKCepsilon) PKC isoenzymes were also observed which were synchronous with the Ca(2+) response measured simultaneously in the same cell. However, oscillatory PKC activity (as determined by translocation of eGFP-tagged myristoylated alanine-rich C kinase substrate protein) required oscillatory DAG production. We suggest a model that uses regenerative Ca(2+) release via Ins(1,4,5)P(3) receptors to initiate oscillatory second messenger production through a positive feedback effect on PLC. By acting on various components of the PLC signaling pathway the frequency-encoded Ca(2+) response is able to maintain signal specificity at a level downstream of PKC activation.     

Role of Ca2+ feedback on single cell inositol 1,4,5-trisphosphate oscillations mediated by G-protein-coupled receptors

The dynamics of inositol 1,4,5-trisphosphate (Ins (1,4,5)P3) production during periods of G-protein-coupled receptor-mediated Ca2+ oscillations have been investigated using the pleckstrin homology (PH) domain of phospholipase C (PLC) delta1 tagged with enhanced green fluorescent protein (eGFP-PHPLCdelta1). Activation of noradrenergic alpha1B and muscarinic M3 receptors recombinantly expressed in the same Chinese hamster ovary cell indicates that Ca2+ responses to these G-protein-coupled receptors are stimulus strength-dependent. Thus, activation of alpha1B receptors produced transient base-line Ca2+ oscillations, sinusoidal Ca2+ oscillations, and then a steady-state plateau level of Ca2+ as the level of agonist stimulation increased. Activation of M3 receptors, which have a higher coupling efficiency than alpha1B receptors, produced a sustained increase in intracellular Ca2+ even at low levels of agonist stimulation. Confocal imaging of eGFP-PHPLCdelta1 visualized periodic increases in Ins(1,4,5)P3 production underlying the base-line Ca2+ oscillations. Ins(1,4,5)P3 oscillations were blocked by thapsigargin but not by protein kinase C down-regulation. The net effect of increasing intracellular Ca2+ was stimulatory to Ins(1,4,5)P3 production, and dual imaging experiments indicated that receptor-mediated Ins(1,4,5)P3 production was sensitive to changes in intracellular Ca2+ between basal and approximately 200 nM. Together, these data suggest that alpha1B receptor-mediated Ins(1,4,5)P3 oscillations result from a positive feedback effect of Ca2+ onto phospholipase C. The mechanisms underlying alpha1B receptor-mediated Ca2+ responses are therefore different from those for the metabotropic glutamate receptor 5a, where Ins(1,4,5)P3 oscillations are the primary driving force for oscillatory Ca2+ responses (Nash, M. S., Young, K. W., Challiss, R. A. J., and Nahorski, S. R. (2001) Nature 413, 381-382). For alpha1B receptors the Ca2+-dependent Ins(1,4,5)P3 production may serve to augment the existing regenerative Ca2+-induced Ca2+-release process; however, the sensitivity to Ca2+ feedback is such that only transient base-line Ca2+ spikes may be capable of causing Ins(1,4,5)P3 oscillations.     

Disruption of protein kinase A interaction with A-kinase-anchoring proteins in the heart in vivo: effects on cardiac contractility, protein kinase A phosphorylation, and troponin I proteolysis

Protein kinase A (PKA)-dependent phosphorylation is regulated by targeting of PKA to its substrate as a result of binding of regulatory subunit, R, to A-kinase-anchoring proteins (AKAPs). We investigated the effects of disrupting PKA targeting to AKAPs in the heart by expressing the 24-amino acid regulatory subunit RII-binding peptide, Ht31, its inactive analog, Ht31P, or enhanced green fluorescent protein by adenoviral gene transfer into rat hearts in vivo. Ht31 expression resulted in loss of the striated staining pattern of type II PKA (RII), indicating loss of PKA from binding sites on endogenous AKAPs. In the absence of isoproterenol stimulation, Ht31-expressing hearts had decreased +dP/dtmax and -dP/dtmin but no change in left ventricular ejection fraction or stroke volume and decreased end diastolic pressure versus controls. This suggests that cardiac output is unchanged despite decreased +dP/dt and -dP/dt. There was also no difference in PKA phosphorylation of cardiac troponin I (cTnI), phospholamban, or ryanodine receptor (RyR2). Upon isoproterenol infusion, +dP/dtmax and -dP/dtmin did not differ between Ht31 hearts and controls. At higher doses of isoproterenol, left ventricular ejection fraction and stroke volume increased versus isoproterenol-stimulated controls. This occurred in the context of decreased PKA phosphorylation of cTnI, RyR2, and phospholamban versus controls. We previously showed that expression of N-terminal-cleaved cTnI (cTnI-ND) in transgenic mice improves cardiac function. Increased cTnI N-terminal truncation was also observed in Ht31-expressing hearts versus controls. Increased cTnI-ND may help compensate for reduced PKA phosphorylation as occurs in heart failure.     

Glibenclamide improves postischemic recovery of myocardial contractile function in trained and sedentary rats.

In this study, we sought to determine whether there was any evidence for the idea that cardiac ATP-sensitive K+ (K(ATP)) channels play a role in the training-induced increase in the resistance of the heart to ischemia-reperfusion (I/R) injury. To do so, the effects of training and an K(ATP) channel blocker, glibenclamide (Glib), on the recovery of left ventricular (LV) contractile function after 45 min of ischemia and 45 min of reperfusion were examined. Female Sprague-Dawley rats were sedentary (Sed; n = 18) or were trained (Tr; n = 17) for >20 wk by treadmill running, and the hearts from these animals used in a Langendorff-perfused isovolumic LV preparation to assess contractile function. A significant increase in the amount of 72-kDa class of heat shock protein was observed in hearts isolated from Tr rats. The I/R protocol elicited significant and substantial decrements in LV developed pressure (LVDP), minimum pressure (MP), rate of pressure development, and rate of pressure decline and elevations in myocardial Ca(2+) content in both Sed and Tr hearts. In addition, I/R elicited a significant increase in LV diastolic stiffness in Sed, but not Tr, hearts. When administered in the perfusate, Glib (1 microM) elicited a normalization of all indexes of LV contractile function and reductions in myocardial Ca(2+) content in both Sed and Tr hearts. Training increased the functional sensitivity of the heart to Glib because LVDP and MP values normalized more quickly with Glib treatment in the Tr than the Sed group. The increased sensitivity of Tr hearts to Glib is a novel finding that may implicate a role for cardiac K(ATP) channels in the training-induced protection of the heart from I/R injury.     

Isolation, characterization, and localization of the inositol 1,4,5-trisphosphate receptor protein in Xenopus laevis oocytes.

Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) induces Ca2+ oscillations and waves in Xenopus laevis oocytes. Microsomes from oocytes exhibit high-affinity binding for Ins(1,4,5)P3, and demonstrate Ins(1,4,5)P3-induced Ca2+ release. The Ins(1,4,5)P3 receptor (InsP3R) was purified from oocyte microsomes as a large tetrameric complex and shown to have a monomer molecular mass of 256 kDa, compared with 273 kDa for the brain InsP3R. Binding to the oocyte receptor is highly specific for Ins(1,4,5)P3 and is inhibited by heparin (IC50, 2 micrograms/ml). Immunoblot analysis revealed that an antibody against the C-terminal sequence of the brain receptor recognized the oocyte receptor. These results, in addition to the difference in pattern obtained after limited proteolysis, suggest that the oocyte InsP3R is a new shorter isoform of the mammalian brain type I InsP3R. Immunofluorescence experiments indicated the presence of the InsP3R in the cortical layer and the perinuclear endoplasmic reticulum of the oocyte. However, immunological and biochemical experiments did not reveal the presence of the ryanodine receptor. The presence of an InsP3R and the absence of a ryanodine receptor support the importance of Ins(1,4,5)P3 in Ca2+ handling by oocytes and particularly in the induction of Ca2+ oscillations and waves.     

Spontaneous electrical rhythmicity and the role of the sarcoplasmic reticulum in the excitability of guinea pig gallbladder smooth muscle cells

Spontaneous action potentials and Ca(2+) transients were investigated in intact gallbladder preparations to determine how electrical events propagate and the cellular mechanisms that modulate these events. Rhythmic phasic contractions were preceded by Ca(2+) flashes that were either focal (limited to one or a few bundles), multifocal (occurring asynchronously in several bundles), or global (simultaneous flashes throughout the field). Ca(2+) flashes and action potentials were abolished by inhibiting sarcoplasmic reticulum (SR) Ca(2+) release via inositol (1,4,5)-trisphosphate [Ins(1,4,5)P(3)] channels with 2-aminoethoxydiphenyl borate and xestospongin C or by inhibiting voltage-dependent Ca(2+) channels (VDCCs) with nifedipine or diltiazem or nisoldipine. Inhibiting ryanodine channels with ryanodine caused multiple spikes superimposed upon plateaus of action potentials and extended quiescent periods. Depletion of SR Ca(2+) stores with thapsigargin or cyclopiazonic acid increased the frequency and duration of Ca(2+) flashes and action potentials. Acetylcholine, carbachol, or cholecystokinin increased synchronized and increased the frequency of Ca(2+) flashes and action potentials. The phospholipase C (PLC) inhibitor U-73122 did not affect Ca(2+) flash or action potential activity but inhibited the excitatory effects of acetylcholine on these events. These results indicate that Ca(2+) flashes correspond to action potentials and that rhythmic excitation in the gallbladder is multifocal among gallbladder smooth muscle bundles and can be synchronized by excitatory agonists. These events do not depend on PLC activation, but agonist stimulation involves activation of PLC. Generation of these events depends on Ca(2+) entry via VDCCs and on Ca(2+) mobilization from the SR via Ins(1,4,5)P(3) channels.     

Ins(1,4,5)P3 receptor-mediated Ca2+ signaling and autophagy induction are interrelated

The role of intracellular Ca2+ signaling in starvation-induced autophagy remains unclear. Here, we examined Ca2+ dynamics during starvation-induced autophagy and the underlying molecular mechanisms. Tightly correlating with autophagy stimulation, we observed a remodeling of the Ca2+ signalosome. First, short periods of starvation (1 to 3 h) caused a prominent increase of the ER Ca2+-store content and enhanced agonist-induced Ca2+ release. The mechanism involved the upregulation of intralumenal ER Ca2+-binding proteins, calreticulin and Grp78/BiP, which increased the ER Ca2+-buffering capacity and reduced the ER Ca2+ leak. Second, starvation led to Ins(1,4,5)P3R sensitization. Immunoprecipitation experiments showed that during starvation Beclin 1, released from Bcl-2, first bound with increasing efficiency to Ins(1,4,5)P3Rs; after reaching a maximal binding after 3 h, binding, however, decreased again. The interaction site of Beclin 1 was determined to be present in the N-terminal Ins(1,4,5)P3-binding domain of the Ins(1,4,5)P3R. The starvation-induced Ins(1,4,5)P3R sensitization was abolished in cells treated with BECN1 siRNA, but not with ATG5 siRNA, pointing toward an essential role of Beclin 1 in this process. Moreover, recombinant Beclin 1 sensitized Ins(1,4,5)P3Rs in 45Ca2+-flux assays, indicating a direct regulation of Ins(1,4,5)P3R activity by Beclin 1. Finally, we found that Ins(1,4,5)P3R-mediated Ca2+ signaling was critical for starvation-induced autophagy stimulation, since the Ca2+ chelator BAPTA-AM as well as the Ins(1,4,5)P3R inhibitor xestospongin B abolished the increase in LC3 lipidation and GFP-LC3-puncta formation. Hence, our results indicate a tight and essential interrelation between intracellular Ca2+ signaling and autophagy stimulation as a proximal event in response to starvation.