Alterations In Cytosolic Ca2 Research Articles

Increased Mitochondrial Calcium Fluxes in Hypertrophic Right Ventricular Cardiomyocytes from a Rat Model of Pulmonary Artery Hypertension

Pulmonary artery hypertension causes right ventricular hypertrophy which rapidly progresses to heart failure with underlying cardiac mitochondrial dysfunction. Prior to failure, there are alterations in cytosolic Ca2+ handling that might impact mitochondrial function in the compensatory phase of RV hypertrophy. Our aims, therefore, were (i) to measure beat-to-beat mitochondrial Ca2+ fluxes, and (ii) to determine mitochondrial abundance and function in non-failing, hypertrophic cardiomyocytes. Male Wistar rats were injected with either saline (CON) or monocrotaline (MCT) to induce pulmonary artery hypertension and RV hypertrophy after four weeks. Cytosolic Ca2+ ([Ca2+]cyto) transients were obtained in isolated right ventricular (RV) cardiomyocytes, and mitochondrial Ca2+ ([Ca2+]mito) was recorded in separate RV cardiomyocytes. The distribution and abundance of key proteins was determined using confocal and stimulated emission depletion (STED) microscopy. The RV mitochondrial function was also assessed in RV homogenates using oxygraphy. The MCT cardiomyocytes had increased area, larger [Ca2+]cyto transients, increased Ca2+ store content, and faster trans-sarcolemmal Ca2+ extrusion relative to CON. The MCT cardiomyocytes also had larger [Ca2+]mito transients. STED images detected increased mitochondrial protein abundance (TOM20 clusters per μm2) in MCT, yet no difference was found when comparing mitochondrial respiration and membrane potential between the groups. We suggest that the larger [Ca2+]mito transients compensate to match ATP supply to the increased energy demands of hypertrophic cardiomyocytes.

Lithium protects against lipopolysaccharide‐induced cytotoxicity in SH‐SY5Y cells expressing Alzheimer’s presenilin 1 mutation by ameliorating calcium dysregulation

AbstractBackgroundLipopolysaccharide is a crucial mediator of neuroinflammation in sepsis. Neuroinflammation is implicated in normal brain aging and neurodegeneration in various neurodegenerative disorders including Alzheimer's disease. We examined the effect and mechanisms of lithium protection against lipopolysaccharide‐induced cytotoxicity in human neuroblastoma SH‐SY5Y cells expressing familial Alzheimer’s disease mutant presenilin‐1 (PS1) and compared to wide type control.MethodHuman neuroblastoma SH‐SY5Y cells transfected with human wild type or point mutant PS1 (M146L) were pretreated with lithium chloride (1 mM) for 1 week, and then challenged with lipopolysaccharide for 24 h. Cell viability was determined by the 3‐(4,5‐dimethylthiazol‐2yl)‐2,5‐diphenyltetrazolium bromide (MTT) reduction assay. Alterations of cytosolic Ca2+ concentrations were measured using jellyfish specific photo protein aequorin‐based probes.ResultLipopolysaccharide resulted in a dose‐dependent cytotoxic effects (starting from the concentration of 400 μg/ml) in both types of cells. Moreover, lipopolysaccharide (400 μg/ml) caused significantly more cytotoxicity in M146L than its corresponding wild type control cells, which could be inhibited significantly by lithium (1 mM) pretreatment. Lipopolysaccharide induced significantly more elevation of peak cytosolic Ca2+ concentrations in M146L cells than in wide type control cells, which could be abolished by lithium (1 mM) pretreatment.ConclusionsOur results suggest that lithium pretreatment at clinically relevant concentrations could effectively mitigates lipopolysaccharide mediated cytotoxicity, especially in Alzheimer’s PS 1 mutated cells, possibly by ameliorating the abnormally elevated cytosolic Ca2+ concentration.

NO-induced vasodilation correlates directly with BP in smooth muscle-Na/Ca exchanger-1-engineered mice: elevated BP does not attenuate endothelial function.

Arterial smooth muscle Na+/Ca2+ exchanger-1 (SM-NCX1) promotes vasoconstriction or vasodilation by mediating, respectively, Ca2+ influx or efflux. In vivo, SM-NCX1 mediates net Ca2+ influx to help maintain myogenic tone (MT) and neuronally activated constriction. SM-NCX1-TG (overexpressing transgenic) mice have increased MT and mean blood pressure (MBP; +13.5 mmHg); SM-NCX1-KO (knockout) mice have reduced MT and MBP (-11.1 mmHg). Endothelium-dependent vasodilation (EDV) is often impaired in hypertension. We tested whether genetically engineered SM-NCX1 expression and consequent BP changes similarly alter EDV. Isolated, pressurized mesenteric resistance arteries with MT from SM-NCX1-TG and conditional SM-NCX1-KO mice, and femoral arteries in vivo from TG mice were studied. Acetylcholine (ACh)-dilated TG arteries with MT slightly more than control or KO arteries, implying that SM-NCX1 overexpression does not impair EDV. In preconstricted KO, but not TG mouse arteries, however, ACh- and bradykinin-triggered vasodilation was markedly attenuated. To circumvent the endothelium, phenylephrine-constricted resistance arteries were tested with Na-nitroprusside [SNP; nitric oxide (NO) donor] and cGMP. This endothelium-independent vasodilation was augmented in TG but attenuated in KO arteries that lack NCX1-mediated Ca2+ clearance. Baseline cytosolic Ca2+ ([Ca2+]cyt) was elevated in TG femoral arteries in vivo, supporting the high BP; furthermore, SNP-triggered [Ca2+]cyt decline and vasodilation were augmented as NO and cGMP promote myocyte polarization thereby enhancing NCX1-mediated Ca2+ efflux. The TG mouse data indicate that BP elevation does not attenuate endothelium-dependent vasodilation. Thus, in essential hypertension and many models the endothelial impairment that supports the hypertension apparently is not triggered by BP elevation but by extravascular mechanisms.NEW & NOTEWORTHY Endothelium-dependent, ACh-induced vasodilation (EDV) is attenuated, and arterial myocyte Na+/Ca2+ exchangers (NCX1) are upregulated in many forms of hypertension. Surprisingly, mildly hypertensive smooth muscle-specific (SM)-NCX1 transgenic mice exhibited modestly enhanced EDV and augmented endothelium-independent vasodilation (EIV). Conversely, mildly hypotensive SM-NCX1-knockout mice had greatly attenuated EIV. These adaptations help compensate for NCX1 expression-induced alterations in cytosolic Ca2+ and blood pressure (BP) and belie the view that elevated BP, itself, causes the endothelial dysregulation in hypertension.

TLR-2 mediated cytosolic-Ca2+ surge activates ER-stress-superoxide-NO signalosome augmenting TNF-\u03b1 production leading to apoptosis of Mycobacterium smegmatis-infected fish macrophages

The implications of TLR-2 mediated alterations in cytosolic-Ca2+((Ca2+)c) levels in M. smegmatis infections is not well known. Using headkidney macrophages (HKM) from Clarias gariepinus, we observed TLR-2 signalling is required in the phagocytosis of M. smegmatis. M. smegmatis induced caspase-dependent HKM apoptosis in MOI, time and growth-phase dependent manner. RNAi and inhibitor studies demonstrated critical role of TLR-2 in eliciting (Ca2+)c-surge and c-Src-PI3K-PLC axis playing an intermediary role in the process. The (Ca2+)c-surge triggered downstream ER-stress and superoxide (O2−) generation. The cross-talk between ER-stress and O2− amplified TNF-α production, which led to HKM apoptosis and bacterial clearance. Release of nitric oxide (NO) was also observed and silencing the NOS2-NO axis enhanced intracellular bacterial survival and attenuated caspase activity. Pre-treatment with diphenyleneidonium chloride inhibited NO production implicating O2−–NO axis imperative in M. smegmatis-induced HKM apoptosis. NO positively impacted CHOP expression and TNF-α production in infected HKM. We conclude that, TLR-2 induced (Ca2+)c-surge and ensuing cross-talk between ER-stress and O2− potentiates HKM pathology by amplifying pro-inflammatory TNF-α production. Moreover, the pro-oxidant environment triggers NO release which prolonged ER-stress and TNF-α production, culminating in HKM apoptosis and bacterial clearance. Together, our study suggests HKM an alternate model to study macrophage-mycobacteria interactions.

The Role of Calcium Handling Mechanisms in Reperfusion Injury.

Cardiovascular diseases, such as stroke and myocardial infarction (MI) remain the major cause of death and disability worldwide. However, the mortality of MI has declined dramatically over the past several decades because of advances in medicines (thrombolytic agents, antiplatelet drugs, beta blockers, and angiotensin converting enzyme inhibitors) and approaches to restore tissue perfusion (percutaneous coronary intervention and cardiopulmonary bypass). Animal studies have been shown that these treatments have been effective in reducing acute myocardial ischemic injury and limiting MI size. The paradox is that the process of reperfusion can itself amplify cell injury and death, known as myocardial ischemia-reperfusion injury (I/R). Intensive research has uncovered several complex mechanisms of cardiomyocyte damage after reperfusion,and potential therapeutic targets for preventing I/R. Importantly, it is now recognized that excessive elevation of intracellular and mitochondrial Ca2+during reperfusion predisposes the cells to hypercontracture, proteolysis and mitochondrial failure and eventually to necrotic or apoptotic death. These enormous alterations in cytosolic Ca2+ levels are induced by the Ca2+ channels of the sarcolemma(L-Type Ca2+channels, sodium/calcium exchanger), the endoplasmic/ sarcoplasmic reticulum (SERCA ATPase) and ryanodine receptors, SOCE(store-operated calcium entry), lysosomes and others, which are modified by I/R injury. The overall goal of this review is to describe the different pathways that lead to I/R injury via Ca2+ overload, focus on recent discoveries and highlight prospects for therapeutic strategies for clinical benefit.

Role of SERCA Pump in Muscle Thermogenesis and Metabolism.

In muscle cells, the sarcoplasmic reticulum (SR) not only acts as a Ca2+ store, but also regulates the contractile characteristics of the muscle. Ca2+ release from the SR is the primary mechanism for activating muscle contraction and reuptake of Ca2+ by the sarcoplasmic reticulum Ca2+ ATPase (SERCA) pump causes muscle relaxation. The SERCA pump isoforms are encoded by three genes, SERCA 1, 2, and 3, which are differentially expressed in muscle and determine SR Ca2+ dynamics by affecting the rate and amount of Ca2+ uptake, thereby affecting SR store and release of Ca2+ in muscle. In muscle, small molecular weight proteins, including Phospholamban (PLB) and Sarcolipin (SLN), also regulate the SERCA pump. Regulation of the SERCA pump by PLB or SLN affects cytosolic Ca2+ dynamics and changes in cytosolic Ca2+ not only affect contractile function, but also mitochondrial ATP production. Recent studies have shown that alterations in cytosolic Ca2+ affects Ca2+ entry into mitochondria and ATP production; thus, Ca2+ serves as an integrating signal between muscle contraction-dependent energy demand and mitochondrial energy production. In addition, changes in cytosolic Ca2+ can affect Ca2+ signaling pathways modulating gene expression and muscle growth. An emerging area of research shows that SR Ca2+ cycling is also a player in muscle-based nonshivering thermogenesis. Recent data shows that SERCA uncoupling by SLN leads to increased ATP hydrolysis and heat production. Our studies, using genetically altered mouse models of SLN, show that SLN/SERCA interaction plays an important role in muscle thermogenesis and metabolism, which will be discussed here, in great length. © 2017 American Physiological Society. Compr Physiol 7:879-890, 2017.

Evidence in Favor of a Novel Excitation‐Contraction Calcium Release Mechanism in Skeletal Muscle L6 Cells

Our laboratory has provided evidence for the first half of a model to explain both store‐operated or capacitative Ca2+ entry (CCE) as well as excitation‐contraction coupled Ca2+ release (ECC) in skeletal muscle. Here we offer evidence in favor of a model that proposes ECC acts by a relative displacement of the sarcolemmal L‐channel (DHPR) with respect to the sarcoplasmic reticulum‐localized Ryanodine Receptor (RyR). We titrated cells with ryanodine (Ry) which is known to both active (low concentrations) and inhibit (high concentrations) the RyR. We then repeated this titration after two types of treatments: conditions to depolarize the sarcolemma; and alterations in cytosolic Ca2+ levels under non‐depolarizing conditions. Depolarizing conditions, including high extracellular [K+] and field depolarization with solution electrodes, caused a shift in the Ry titration such that lower concentrations elicited both stimulation and inhibition. Addition of the DHPR agonist BAY K‐8644 caused a similar shift, although of smaller magnitude. On the other hand, altering intracellular Ca2+ through direct activation of RyR with the caffeine‐like xx did not cause a shift in Ry sensitivity. Similarly, alterations in exogenous Ca2+ concentration did not change Ry sensitivity. We suggest that these findings are consistent with our “relative displacement” hypothesis for ECC, whereby excitation moves the DHPR to unoccupied RyR, releasing Ca2+ into the cytosol from previously occluded RyR channels.

The Mitogen-activated Protein Kinase p38 Links Shiga Toxin-dependent Signaling and Trafficking

Shiga toxin (Stx) binds to the cell, and it is transported via endosomes and the Golgi apparatus to the endoplasmic reticulum and cytosol, where it exerts its toxic effect. We have recently shown that Stx activates the tyrosine kinase Syk, which in turn induces clathrin phosphorylation and up-regulates Stx uptake. Here, we show that toxin-induced signaling can also regulate another step in intracellular Stx transport. We demonstrate that transport of Stx to the Golgi apparatus is dependent on the mitogen-activated protein kinase p38. Treatment of cells with chemical inhibitors or small interfering RNA targeting p38 inhibited Stx transport to the Golgi and reduced Stx toxicity. This p38 dependence is specific to Stx, because transport of the related toxin ricin was not affected by p38 inhibition. Stx rapidly activated p38, and recruited it to early endosomes in a Ca(2+)-dependent manner. Furthermore, agonist-induced oscillations in cytosolic Ca(2+) levels were inhibited upon Stx stimulation, possibly reflecting Stx-dependent local alterations in cytosolic Ca(2+) levels. Intracellular transport of Stx is Ca(2+) dependent, and we provide evidence that Stx activates a signaling cascade involving cross talk between Ca(2+) and p38, to regulate its trafficking to the Golgi apparatus.

Ion interaction at the pore of Lc‐type Ca2+ channel is sufficient to mediate depolarization‐induced exocytosis

The coupling of voltage-gated Ca2+ channel (VGCC) to exocytotic proteins suggests a regulatory function for the channel in depolarization-evoked exocytosis. To explore this possibility we have examined catecholamine secretion in PC12 and chromaffin cells. We found that replacing Ca2+ with La3+ or other lanthanide ions supported exocytosis in divalent ion-free solution. Cd2+, nifedipine, or verapamil inhibited depolarization-evoked secretion in La3+, indicating specific binding of La3+ at the pore of L-type VGCC, probably at the poly-glutamate (EEEE) locus. Lanthanide efficacy was stringently dependent on ionic radius with La3+>Ce3+>Pr3+, consistent with a size-selective binding interface of trivalent cations at the channel pore. La3+ inward currents were not detected and the highly sensitive La3+/fura-2 imaging assay (approximately 1 pm) detected no La3+ entry, cytosolic La3+ build-up, or alterations in cytosolic Ca2. These results provide strong evidence that occupancy of the pore of the channel by an impermeable cation leads to a conformational change that is transmitted to the exocytotic machinery upstream of intracellular cation build-up (intracellular Ca2+ concentration). Our model allows for a tight temporal and spatial coupling between the excitatory stimulation event and vesicle fusion. It challenges the conventional view that intracellular Ca2+ ion build-up via VGCC permeation is required to trigger secretion and establishes the VGCC as a plausible Ca2+ sensor protein in the process of neuroendocrine secretion.

Hyperglycemic conditions affect shape and Ca2+ homeostasis of mitochondria in endothelial cells.

In this study the contribution of alternating architecture and Ca2+ handling of mitochondria to cytosolic Ca2+ homeostasis was elucidated under normoglycemic and hyperglycemic (HGC) conditions in the human endothelial cell line EA.hy926. Exposure of endothelial cells to hyperglycemic medium elevated basal cytosolic free Ca2+ concentration ([Ca2+]cyto), the histamine-initiated cytosolic Ca2+ signaling, and the mitochondrial Ca2+ content after cell stimulation. The latter was possibly due to the prolonged mitochondrial Ca2+ elevation in response to agonists found in HGC-pretreated cells. Moreover, under HGC mitochondrial free radical production was increased and mitochondrial shape changed from a mainly tubular, highly interconnected network toward multiple, isolated singular structures. Such changes could not be correlated with HGC-induced alterations of cytosolic Ca2+ signaling that became normalized with antimycin A, an inhibitor of the respiratory chain. These data suggest that although mitochondrial structure changes considerably during HGC, alterations in cytosolic Ca2+ signaling are more likely due to the enhanced energy status/metabolism of the mitochondria. On the other hand, in normoglycemic cells of unforced fragmentation of mitochondria yielded elevated basal [Ca2+]cyto, while the global Ca2+ signaling in response to histamine remained unchanged. Thus, mitochondrial architecture (ie, tubular versus fragmented structure) per se does not have a detectable impact on agonist-initiated global cytosolic Ca2+ signaling, while this organelle represents an early target in hyperglycemia leading to alterations in cytosolic Ca2+ signaling.

Activation of human neutrophils with chemotactic peptide, opsonized zymosan and the calcium ionophore A23187, but not with a phorbol ester, is accompanied by efflux and store-operated influx of calcium.

The objective of this study was to monitor alterations in cellular Ca2+ metabolism following activation of neutrophils with receptor- (chemotactic peptide, FMLP, 1 microM; opsonized zymosan, OZ, 0.5 mg/ml) and non-receptor (calcium ionophore, A23187, 1 microM; phorbol 12-myristate 13-acetate, PMA, 25 ng/ml)-mediated stimuli of the pro-inflammatory functions of these cells. Ca2+ fluxes in activated neutrophils were measured using a fura-2-based spectrofluorimetric method in combination with radiometric (45Ca) procedures which facilitate distinction between net efflux and net influx of the cation. Exposure of neutrophils to receptor-mediated stimuli and to A23187 was associated with an abrupt increase in cytosolic Ca2+ coincident with a rapid efflux of the cation which terminated at around 30 s. In the case of FMLP and OZ, this was followed by a delayed (30-60 s), store-operated influx of Ca2+, which was complete at around 5 min after addition of the stimulus. With A23187, however, influx of Ca2+ occurred immediately following activation of the cells. There were no detectable alterations in cytosolic Ca2+ or measurable net efflux or influx of the cation above control levels in PMA-activated neutrophils. These data demonstrate that FMLP, OZ- and A23187-mediated alterations in neutrophil cytosolic Ca2+ are due to mobilization of both intracellular and extracellular cation, while activation of neutrophils by PMA is independent of alterations in cytosolic Ca2+.

Role of Ca2+ in apoptosis evoked by human amylin in pancreatic islet β-cells

The objective of these studies was to clarify the role of Ca(2+) in the mechanism of death evoked by human amylin (hA) in islet beta-cells. hA forms fibrils in vitro and islet amyloid in vivo. Here we show that pure synthetic hA aggregated in solution, formed fibrils and evoked death in cultured RINm5F islet beta-cells in a time-dependent (0-24 h) and concentration-dependent (0-20 microM) manner. Dying cells underwent shrinkage of the nucleus, with clumping and segregation of chromatin into masses that lay against the nuclear envelope, and internucleosomal DNA fragmentation. These cells therefore show many features of apoptosis, although aspects of the morphology might be characteristic of this particular cell type rather than of a general apoptotic nature. Aurintricarboxylic acid, an inhibitor of both Ca(2+)-dependent and Ca(2+)-independent nucleases, suppressed this DNA fragmentation and inhibited apoptosis at concentrations between 25 and 200 microM. Direct measurements of the cytoplasmic free Ca(2+) concentration ([Ca(2+)](i)) in fura-2 acetoxymethyl ester (AM)-loaded beta-cells showed that neither hA nor its non-cytotoxic homologue, rat amylin were effective in raising [Ca(2+)](i). Modulators of Ca(2+) regulation were tested for their effects on hA-induced beta-cell apoptosis. Ca(2+) ionophore (A23187) and thapsigargin (an inhibitor of endoplasmic reticular Ca(2+)-ATPase activity) by themselves evoked apoptosis accompanied by increased [Ca(2+)](i). Neither the Ca(2+) channel blocker verapamil, the extracellular Ca(2+) chelator EGTA nor the cytosolic Ca(2+) buffer bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid ('BAPTA')/AM protected beta-cells from hA-evoked apoptosis. Prolonged incubation of beta-cells with a lethal dose of hA altered neither the basal [Ca(2+)](i) nor the thapsigargin-induced release of Ca(2+) from intracellular stores. Furthermore, (45)CaCl(2) uptake by RINm5F cells did not differ in the presence or absence of hA. These results suggest that, whereas alterations in cytosolic Ca(2+) homoeostasis do have a significant role in certain forms of beta-cell death, they do not contribute to the pathway of apoptosis evoked by hA in islet beta-cells.

Modulation of intestinal paracellular permeability by intracellular mediators and cytoskeleton

The influence of cytoskeletal inhibitors (cytochalasin E and colchicine) and intracellular mediators (cAMP, Ca2+, and protein kinase C) in the control of paracellular permeability to mannitol has been examined in rat jejunum in Ussing-type chambers. Cytoskeletal inhibitors, cytochalasin E (20 mumol.L-1) or colchicine (0.5 mmol.L-1), when present in mucosal, serosal, or in both mediums, significantly increase unidirectional mannitol fluxes. Exposure of mucosal and serosal intestinal surface to 10 mmol.L-1 theophylline or 1 mmol.L-1 cyclic AMP analogue for raising the intracellular cAMP enhances paracellular permeability. In Ca(2+)-free physiological medium passive permeability strongly increases. Alterations of cytosolic Ca2+ levels induced by the Ca2+ ionophore A23187 (20 mumol.L-1) or by 0.3 mmol.L-1 TMB-8, a Ca2+ releasing inhibitor from the intracellular stores, enhance mannitol flux. Addition of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate, which activates protein kinase C (PKC), also induces a large increase in the intestinal permeability to mannitol. These results provide evidence that tight junctions and consequently epithelial paracellular permeability can be physiologically controlled by intracellular mediators (Ca2+, cAMP, and PKC) probably through modulation of the cytoskeleton activity.

Modulation of intestinal paracellular permeability by intracellular mediators and cytoskeleton

The influence of cytoskeletal inhibitors (cytochalasin E and colchicine) and intracellular mediators (cAMP, Ca2+, and protein kinase C) in the control of paracellular permeability to mannitol has been examined in rat jejunum in Ussing-type chambers. Cytoskeletal inhibitors, cytochalasin E (20 mumol.L-1) or colchicine (0.5 mmol.L-1), when present in mucosal, serosal, or in both mediums, significantly increase unidirectional mannitol fluxes. Exposure of mucosal and serosal intestinal surface to 10 mmol.L-1 theophylline or 1 mmol.L-1 cyclic AMP analogue for raising the intracellular cAMP enhances paracellular permeability. In Ca(2+)-free physiological medium passive permeability strongly increases. Alterations of cytosolic Ca2+ levels induced by the Ca2+ ionophore A23187 (20 mumol.L-1) or by 0.3 mmol.L-1 TMB-8, a Ca2+ releasing inhibitor from the intracellular stores, enhance mannitol flux. Addition of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate, which activates protein kinase C (PKC), also induces a large increase in the intestinal permeability to mannitol. These results provide evidence that tight junctions and consequently epithelial paracellular permeability can be physiologically controlled by intracellular mediators (Ca2+, cAMP, and PKC) probably through modulation of the cytoskeleton activity.

Inositol 1,4,5-trisphosphate-induced Ca2+ release is regulated by cytosolic Ca2+ in intact skeletal muscle

Microinjection of inositol 1,4,5-trisphosphate (InsP3) into intact skeletal muscle fibers isolated from frogs (Rana temporaria) increased resting cytosolic Ca2+ concentration ([Ca2+]i) as measured by double-barreled Ca2+-selective microelectrodes. In contrast, microinjection of inositol 1-phosphate, inositol 1,4-biphosphate, and inositol 1,4,5,6-tetrakisphosphate did not induce changes in [Ca2+]i. Incubation in low-Ca2+ solution, or in the presence of L-type Ca2+ channel blockers did not affect InsP3-induced release of cytosolic Ca2+. Neither ruthenium red, a blocker of ryanodine receptor Ca2+-release channels, nor cytosolic Mg2+, a known inhibitor of the Ca2+-induced Ca2+-release process, modified the InsP3-induced release of cytosolic Ca2+. However, heparin, a blocker of InsP3 receptors, inhibited InsP3-induced release of cytosolic Ca2+. Also, pretreatment with dantrolene or azumulene, two inhibitors of cytosolic Ca2+ release, reduced [Ca2+]i, and prevented InsP3 from inducing release of cytosolic Ca2+. Incubation in caffeine or lengthening of the muscle increased [Ca2+]i and enhanced the ability of InsP3 to induce release of cytosolic Ca2+. These results indicate that InsP3, at physiological concentrations, induces Ca2+ release in intact muscle fibers, and suggest that the InsP3-induced Ca2+ release is regulated by [Ca2+]i. A Ca2+-dependent effect of InsP3 on cytosolic Ca2+ release could be of importance under physiological or pathophysiological conditions associated with alterations in cytosolic Ca2+ homeostasis.

The role of calcium in the regulation of apoptosis

The recognition that apoptosis is regulated by an evolutionarily conserved set of polypeptides from the nematode Caenorhabditis elegans to humans suggests that a conserved set of biochemical mechanisms may also he involved in the response. Work from a number of independent laboratories suggests that alterations in cytosolic Ca2+ homeostasis represent one such candidate mechanism, and molecular targets for Ca2+ are now being identified. This review will summarize what is known about the role of Ca2+ in the regulation of apoptosis and discuss how Ca2+ might interact with some of the other biochemical signals implicated in cell death.

Characterization and regulation of apamin-binding K+ channels in skeletal muscle.

The Pattern of development and regulation of the apamin receptor (afterhyperpolarization channel) was studied in cultures of skeletal muscle prepared from 1-2-day-old rat pups. Expression was measured by the specific binding of (125)I-apamin. Apamin binding was virtually undetectable until the time of fusion (3-4 days in culture) of single myoblasts into myotubes. Mature myotubes (5-7 days in vitro) displayed a Bmax of 7.4 fmol/mg protein and a Kd of 376 pmol/L. When studied in mature muscle cells apamin binding was found to increase twofold in response to tetrodotoxin (TTX) and elevated Ko, which resulted in decreased Na(i). In contrast, treatments causing an increase in Na(i), such as monensin and veratridine, caused a decrease in apamin binding. The increase in apamin binding following TTX treatment was due mainly to synthesis of new channels, as the effect was blocked by cycloheximide. Alterations in cytosolic Ca2+ by calcium ionophore or Ca-channel blockers were without effect on apamin-sensitive channel expression. We conclude that afterhyperpolarization channel expression is regulated by the level of intracellular Na+ ions.

Cytosolic calcium transients in myocytes isolated from rats with ischemic heart failure

Mechanical performance and cytosolic Ca2+ dynamics were characterized in myocytes isolated from left and right ventricles of rats with ischemic heart failure. Seven days after coronary artery narrowing (CAN) in rats filling pressures were elevated, whereas systolic pressures and ejection of blood were depressed. Left ventricular myocytes increased 18% in length and 19% in width, whereas right myocytes expanded longitudinally by 23% and transversely by 24%. Contractile behavior of myocytes displayed reductions in myocyte shortening and velocity of shortening, despite prolongation of time to peak shortening. Diastolic Ca2+ increased by 32 and 39% in left and right myocytes of CAN animals, whereas peak systolic Ca2+ in left ventricular myocytes was depressed (22%). Time to peak Ca2+ was prolonged by 68% in left myocytes. Moreover, time required for peak Ca2+ to return to diastolic levels was prolonged in left myocytes. Regression analysis revealed correlations between end-diastolic pressure and diastolic Ca2+ and peak developed pressure and systolic Ca2+. Thus ischemic heart failure finds its cellular basis in a depression in myocyte contractility that may in turn be due to alterations in cytosolic Ca2+ handling.

Diastolic dysfunction in pressure-overload hypertrophy and its modification by angiotensin II: current concepts.

Cardiac hypertrophy is an adaptive response to an increased load imposed on the myocyte which allows the heart to perform increased work while maintaining normal myocardial fiber stress and shortening in systole. A deleterious consequence of pressure-overload hypertrophy is the prolongation of Ca(2+)-sensitive force inactivation (impaired myocardial relaxation) which is related to intrinsic alterations in cytosolic Ca2+ transport and reuptake in diastole. Additional factors appear to adversely modify myocardial relaxation in the hypertrophied heart, including the imposition of ischemia. There is also evidence that the expression and activity of the cardiac tissue renin angiotensin system (RAS) may be modified in the hypertrophied heart and contribute to diastolic dysfunction. Recent studies have demonstrated the presence of increased cardiac angiotensin converting enzyme (ACE) mRNA expression and activity in animal models of hypertrophy, including the aortic-banded rat with compensatory pressure-overload hypertrophy and rats with post-infarction remodeling. In the beating, isovolumic aortic-banded rat heart, the increased intracardiac activation of angiotensin I to II has been shown to be associated with a dose-dependent depression of diastolic relaxation. Preliminary studies suggest that the depression of diastolic function by angiotensin II in the hypertrophied heart can be prevented by the specific inhibition of cardiac ACE. In addition, the well-recognized susceptibility of the hypertrophied heart to severe ischemic diastolic dysfunction also appears to be favorably modified by the inhibition of cardiac ACE activity. The mechanisms responsible for the adverse effects of angiotensin II on diastolic relaxation in the hypertrophied heart are likely to be complex.(ABSTRACT TRUNCATED AT 250 WORDS)