Thyrotropin releasing hormone

Summary Thyrotropin releasing hormone (TRH) acutely stimulates release of thyrotropin (TSH) and prolactin from anterior pituitary cells. A considerable number of studies have been performed with neoplastic and nonneoplastic pituitary cells in culture to elucidate the sequence of intracellular events involved in this action. Although cyclic AMP was suggested as an intracellular messenger, it has been demonstrated that TRH stimulation of hormone release can be dissociated from changes in cyclic AMP concentration, thereby supporting the contention that cyclic AMP is not a required mediator. In contrast, stimulation of hormone release by TRH requires Ca²⁺ and it seems likely that Ca²⁺ is the intracellular coupling factor between TRH stimulation and hormone secretion. TRH has been shown to stimulate ⁴⁵Ca²⁺ efflux from preloaded pituitary cells. Enhanced ⁴⁵Ca²⁺ efflux is thought to reflect an increase in the free intracellular Ca²⁺ concentration which leads to hormone release; however, the source of this Ca^2– is uncertain. Results are reviewed from a series of experiments in pituitary cells which attempt to determine the pool (or pools) of Ca²⁺ that is affected by TRH. These include the following: the effects of decreasing the extracellular Ca^2– concentration on hormone release stimulated by TRH; the effect of TRH on cellular Ca²⁺ as monitored by chlortetracycline; the effects of TRH on Ca²⁺ influx; the effects of the organic Ca²⁺ channel blocking agents, verapamil and methoxyverapamil, on TRH-stimulated hormone release; and the effects of TRH on plasma membrane potential difference and on Ca²⁺-dependent action potentials. Based on these data, separate hypotheses of the early events in TRH stimulation of hormone release in mammotropes and thyrotropes are proposed. In mammotropes, TRH is thought to stimulate prolactin release optimally by elevating the free intracellular Cat+ concentration by mobilizing cellular Ca^2– only. In contrast, in thyrotropes under normal physiological conditions, TRH is thought to stimulate TSH release by mobilizing Ca² from a cellular pool (or pools) and to augment this effect by also inducing influx of extracellular Ca²⁺ through voltage-dependent channels in the plasma membrane.     

Calcium movements during pigment aggregation in freshwater shrimp chromatophores

Pigment granule migration within crustacean chromatophores provides an excellent model with which to investigate cytoplasmic movements, given the antagonistic, neurosecretory peptide regulation of granule translocation, and the absence of innervation in these large, brightly colored cells. Red pigment-concentrating hormone (RPCH) induces pigment aggregation in shrimp chromatophores via an increase in intracellular Ca2+; however, how this increase is brought about is not known. To examine the putative Ca2+ movements leading to pigment translocation in red, ovarian chromatophores of the freshwater shrimp, Macrobrachium olfersii, this study manipulates intra- and extracellular Ca2+ employing ER Ca2+-ATPase inhibitors, ryanodine-sensitive, ER Ca2+ channel blockers, and EDTA/EGTA-buffered A23187/Ca2+-containing salines. Our findings reveal that during pigment aggregation, cytosolic Ca2+ apparently increases from an intracellular source, the abundant SER, loaded by the SERCA and released through ryanodine-sensitive receptor/channels, triggered by capacitative calcium influx and/or calcium-induced calcium release mechanisms. Aggregation also depends on external calcium, which may modulate RPCH/receptor coupling. Such calcium-regulated pigment movements form the basis of a complex system of chromatic adaptation, which confers selective advantages like camouflage and protection against ultra-violet radiation to this palaemonid shrimp.     

Regulation of total mitochondrial Ca2+ in perfused liver is independent of the permeability transition pore

Triggering ofthe permeability transition pore (PTP) in isolated mitochondria causesrelease of matrix Ca²⁺, ions, andmetabolites, and it has been proposed that the PTP mediatesmitochondrial Ca²⁺ release inintact cells. To study the role of the PTP in mitochondrial energymetabolism, the mitochondrial content ofCa²⁺,Mg²⁺, ATP, and ADP was determinedin hormonally stimulated rat livers perfused with cyclosporin A (CsA).Stimulation of livers perfused in the absence of CsA with glucagon andphenylephrine induced an extensive uptake ofCa²⁺,Mg²⁺, and ATP plus ADP by themitochondria, followed by a release on omission of hormones. In thepresence of CsA, the PTP was fully inhibited, but neither thehormone-induced uptake of Ca²⁺,ATP, or ADP by mitochondria nor their release after washout of hormoneswas significantly changed. We conclude that the regulation of sustainedchanges in mitochondrial Ca²⁺content induced by hormonal stimulation is independent of the PTP.

     

The mechanism of histamine secretion from gastric enterochromaffin-like cells

Enterochromaffin-like (ECL) cells play a pivotal role in theperipheral regulation of gastric acid secretion as they respond to thefunctionally important gastrointestinal hormones gastrin andsomatostatin and neural mediators such as pituitary adenylate cyclase-activating peptide and galanin. Gastrin is the keystimulus of histamine release from ECL cells in vivo and in vitro.Voltage-gated K⁺ andCa²⁺ channels have been detectedon isolated ECL cells. Exocytosis of histamine following gastrinstimulation and Ca²⁺ entry acrossthe plasma membrane is catalyzed by synaptobrevin andsynaptosomal-associated protein of 25 kDa, both characterized as asoluble N-ethylmaleimide-sensitivefactor attachment protein receptor protein. Histamine release occursfrom different cellular pools: preexisting vacuolar histamineimmediately released by Ca²⁺ entryor newly synthesized histamine following induction of histidine decarboxylase (HDC) by gastrin stimulation. Histamine is synthesized bycytoplasmic HDC and accumulated in secretory vesicles byproton-histamine countertransport via the vesicular monoaminetransporter subtype 2 (VMAT-2). The promoter region of HDC containsCa²⁺-, cAMP-, and protein kinaseC-responsive elements. The gene promoter for VMAT-2, however, lacksTATA boxes but contains regulatory elements for the hormones glucagonand somatostatin. Histamine secretion from ECL cells is thereby under acomplex regulation of hormonal signals and can be targeted at severalsteps during the process of exocytosis.

     

Involvement of JAK2 and Src kinase tyrosine phosphorylation in human growth hormone-stimulated increases in cytosolic free Ca2+ and insulin secretion

We previously reported that human growth hormone (hGH) increases cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) and proliferation in pancreatic -cells (Sjöholm Å, Zhang Q, Welsh N, Hansson A, Larsson O, Tally M, and Berggren PO. J Biol Chem 275: 21033–21040, 2000) and that the hGH-induced rise in [Ca²⁺]_i involves Ca²⁺-induced Ca²⁺ release facilitated by tyrosine phosphorylation of ryanodine receptors (Zhang Q, Kohler M, Yang SN, Zhang F, Larsson O, and Berggren PO. Mol Endocrinol 18: 1658–1669, 2004). Here we investigated the tyrosine kinases that convey the hGH-induced rise in [Ca²⁺]_i and insulin release in BRIN-BD11 -cells. hGH caused tyrosine phosphorylation of Janus kinase (JAK)2 and c-Src, events inhibited by the JAK2 inhibitor AG490 or the Src kinase inhibitor PP2. Although hGH-stimulated rises in [Ca²⁺]_i and insulin secretion were completely abolished by AG490 and JAK2 inhibitor II, the inhibitors had no effect on insulin secretion stimulated by a high K⁺ concentration. Similarly, Src kinase inhibitor-1 and PP2, but not its inactive analog PP3, suppressed [Ca²⁺]_i elevation and completely abolished insulin secretion stimulated by hGH but did not affect responses to K⁺. Ovine prolactin increased [Ca²⁺]_i and insulin secretion to a similar extent as hGH, effects prevented by the JAK2 and Src kinase inhibitors. In contrast, bovine GH evoked a rise in [Ca²⁺]_i but did not stimulate insulin secretion. Neither JAK2 nor Src kinase inhibitors influenced the effect of bovine GH on [Ca²⁺]_i. Our study indicates that hGH stimulates rise in [Ca²⁺]_i and insulin secretion mainly through activation of the prolactin receptor and JAK2 and Src kinases in rat insulin-secreting cells. c-Src; growth hormone receptor; prolactin receptor; Ca²⁺-induced Ca²⁺ release     

Plasma and intracellular membrane inositol 1,4,5-trisphosphate receptors mediate the Ca(2+) increase associated with the ATP-induced increase in ciliary beat frequency

An increase in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) has been shown to be involved in the increase in ciliary beat frequency (CBF) in response to ATP; however, the signaling pathways associated with inositol 1,4,5-trisphosphate (IP₃) receptor-dependent Ca²⁺ mobilization remain unresolved. Using radioimmunoassay techniques, we have demonstrated the appearance of two IP₃ peaks occurring 10 and 60 s after ATP addition, which was strongly correlated with a release of intracellular Ca²⁺ from internal stores and an influx of extracellular Ca²⁺, respectively. In addition, ATP-dependent Ca²⁺ mobilization required protein kinase C (PKC) and Ca²⁺/calmodulin-dependent protein kinase II activation. We found an increase in PKC activity in response to ATP, with a peak at 60 s after ATP addition. Xestospongin C, an IP₃ receptor blocker, significantly diminished both the ATP-induced increase in CBF and the initial transient [Ca²⁺]_i component. ATP addition in the presence of xestospongin C or thapsigargin revealed that the Ca²⁺ influx is also dependent on IP₃ receptor activation. Immunofluorescence and confocal microscopic studies showed the presence of IP₃ receptor types 1 and 3 in cultured ciliated cells. Immunogold electron microscopy localized IP₃ receptor type 3 to the nucleus, the endoplasmic reticulum, and, interestingly, the plasma membrane. In contrast, IP₃ receptor type 1 was found exclusively in the nucleus and the endoplasmic reticulum. Our study demonstrates for the first time the presence of IP₃ receptor type 3 in the plasma membrane in ciliated cells and leads us to postulate that the IP₃ receptor can directly trigger Ca²⁺ influx in response to ATP. transduction mechanisms; P2Y receptor; calcium influx     

Role of glycolytically generated ATP for CaMKII-mediated regulation of intracellular Ca2+ signaling in bovine vascular endothelial cells

The role of glycolytically generated ATP in Ca²⁺/calmodulin-dependent kinase II (CaMKII)-mediated regulation of intracellular Ca²⁺ signaling was examined in cultured calf pulmonary artery endothelial (CPAE) cells. Exposure of cells (extracellular Ca²⁺ concentration = 2 mM) to glycolytic inhibitors 2-deoxy-D-glucose (2-DG), pyruvate (pyr) + -hydroxybutyrate (-HB), or iodoacetic acid (IAA) caused an increase of intracellular Ca²⁺ concentration ([Ca²⁺]_i). CaMKII inhibitors (KN-93, W-7) triggered a similar increase of [Ca²⁺]_i. The rise of [Ca²⁺]_i was characterized by a transient spike followed by a small sustained plateau of elevated [Ca²⁺]_i. In the absence of extracellular Ca²⁺ 2-DG caused an increase in [Ca²⁺]_i, suggesting that inhibition of glycolysis directly triggered release of Ca²⁺ from intracellular endoplasmic reticulum (ER) Ca²⁺ stores. The inositol-1,4,5-trisphosphate receptor (IP₃R) inhibitor 2-aminoethoxydiphenyl borate abolished the KN-93- and 2-DG-induced Ca²⁺ response. Ca²⁺ release was initiated in peripheral cytoplasmic processes from which activation propagated as a [Ca²⁺]_i wave toward the central region of the cell. Focal application of 2-DG resulted in spatially confined elevations of [Ca²⁺]_i. Propagating [Ca²⁺]_i waves were preceded by [Ca²⁺]_i oscillations and small, highly localized elevations of [Ca²⁺]_i (Ca²⁺ puffs). Inhibition of glycolysis with 2-DG reduced the KN-93-induced Ca²⁺ response, and vice versa during inhibition of CaMKII 2-DG-induced Ca²⁺ release was attenuated. Similar results were obtained with pyr + -HB and W-7. Furthermore, 2-DG and IAA caused a rapid increase of intracellular Mg²⁺ concentration, indicating a concomitant drop of cellular ATP levels. In conclusion, CaMKII exerts a profound inhibition of ER Ca²⁺ release in CPAE cells, which is mediated by glycolytically generated ATP, possibly through ATP-dependent phosphorylation of the IP₃R. Ca²⁺/calmodulin-dependent kinase II; glycolysis; calcium regulation     

Oxidant-impaired intracellular Ca2+ signaling in pancreatic acinar cells: role of the plasma membrane Ca2+-ATPase

Pancreatitis is an inflammatory disease of pancreatic acinar cells whereby intracellular calcium concentration ([Ca²⁺]_i) signaling and enzyme secretion are impaired. Increased oxidative stress has been suggested to mediate the associated cell injury. The present study tested the effects of the oxidant, hydrogen peroxide, on [Ca²⁺]_i signaling in rat pancreatic acinar cells by simultaneously imaging fura-2, to measure [Ca²⁺]_i, and dichlorofluorescein, to measure oxidative stress. Millimolar concentrations of hydrogen peroxide increased cellular oxidative stress and irreversibly increased [Ca²⁺]_i, which was sensitive to antioxidants and removal of external Ca²⁺, and ultimately led to cell lysis. Responses were also abolished by pretreatment with (sarco)endoplasmic reticulum Ca²⁺-ATPase inhibitors, unless cells were prestimulated with cholecystokinin to promote mitochondrial Ca²⁺ uptake. This suggests that hydrogen peroxide promotes Ca²⁺ release from the endoplasmic reticulum and the mitochondria and that it promotes Ca²⁺ influx. Lower concentrations of hydrogen peroxide (10–100 µM) increased [Ca²⁺]_i and altered cholecystokinin-evoked [Ca²⁺]_i oscillations with marked heterogeneity, the severity of which was directly related to oxidative stress, suggesting differences in cellular antioxidant capacity. These changes in [Ca²⁺]_i also upregulated the activity of the plasma membrane Ca²⁺-ATPase in a Ca²⁺-dependent manner, whereas higher concentrations (0.1–1 mM) inactivated the plasma membrane Ca²⁺-ATPase. This may be important in facilitating "Ca²⁺ overload," resulting in cell injury associated with pancreatitis. oxidant stress; pancreatitis; calcium pump     

Synaptotagmin I increases the probability of vesicle fusion at low [Ca2+] in pituitary cells

Synaptotagmin I (Syt I),a low-affinity Ca²⁺-binding protein, is thought to serve asthe Ca²⁺ sensor in the release of neurotransmitter.However, functional studies on the calyx of Held synapse revealed thatthe rapid release of neurotransmitter requires only approximatelymicromolar [Ca²⁺], suggesting that Syt I may play a morecomplex role in determining the high-affinity Ca²⁺dependence of exocytosis. Here we tested this hypothesis by studying pituitary cells, which possess high- and low-affinityCa²⁺-dependent exocytic pathways and express Syt I. Usingpatch-clamp capacitance measurements to monitor secretion and the acuteantisense deletion of Syt I from differentiated cells, we have shownthat the rapid and the most Ca²⁺-sensitive pathway ofexocytosis in rat melanotrophs requires Syt I. Furthermore, stimulationof the Ca²⁺-dependent exocytosis by cytosol dialysis withsolutions containing 1 µM [Ca²⁺] was completelyabolished in the absence of Syt I. Similar results were obtained by thepreinjection of antibodies against the CAPS (Ca²⁺-dependentactivator protein for secretion) protein. These results indicate thatsynaptotagmin I and CAPS proteins increase the probability of vesiclefusion at low cytosolic [Ca²⁺].

     

ATP regulates anion channel-mediated organic osmolyte release from cultured rat astrocytes via multiple Ca2+-sensitive mechanisms

Ubiquitously expressed volume-regulated anion channels (VRACs) are activated in response to cell swelling but may also show limited activity in nonswollen cells. VRACs are permeable to inorganic anions and small organic osmolytes, including the amino acids aspartate, glutamate, and taurine. Several recent reports have demonstrated that neurotransmitters or hormones, such as ATP and vasopressin, induce or strongly potentiate astrocytic whole cell Cl^– currents and amino acid release, which are inhibited by VRAC blockers. In the present study, we explored the intracellular signaling mechanisms mediating the effects of ATP on D-[³H]aspartate release via the putative VRAC pathway in rat primary astrocyte cultures. Cells were exposed to moderate (5%) or substantial (30%) reductions in medium osmolarity. ATP strongly potentiated D-[³H]aspartate release in both moderately swollen and substantially swollen cells. These ATP effects were blocked (80% inhibition) by intracellular Ca²⁺ chelation with BAPTA-AM, calmodulin inhibitors, or a combination of the inhibitors of protein kinase C (PKC) and calmodulin-dependent kinase II (CaMK II). In contrast, control D-[³H]aspartate release activated by the substantial hyposmotic swelling showed little (25% inhibition) sensitivity to the same pharmacological agents. These data indicate that ATP regulates VRAC activity via two separate Ca²⁺-sensitive signaling cascades involving PKC and CaMK II and that cell swelling per se activates VRACs via a separate Ca²⁺/calmodulin-independent signaling mechanism. Ca²⁺-dependent organic osmolyte release via VRACs may contribute to the physiological functions of these channels in the brain, including astrocyte-to-neuron intercellular communication. volume-regulated anion channels; protein kinase C; calcium/calmodulin-dependent kinase II; glutamate release; neuron-glia communication     

Metabolic inhibition with cyanide induces calcium release in pulmonary artery myocytes and Xenopus oocytes

We examined the effectsof metabolic inhibition on intracellular Ca²⁺ release insingle pulmonary arterial smooth muscle cells (PASMCs). Severemetabolic inhibition with cyanide (CN, 10 mM) increased intracellularcalcium concentration ([Ca²⁺]_i) and activatedCa²⁺-activated Cl currents[I_Cl(Ca)] in PASMCs, responses that were greatlyinhibited by BAPTA-AM or caffeine. Mild metabolic inhibition with CN (1 mM) increased spontaneous transient inward currents andCa²⁺ sparks in PASMCs. In Xenopus oocytes, CNalso induced Ca²⁺ release and activatedI_Cl(Ca), and these responses were inhibited by thapsigarginand cyclopiazonic acid to deplete sarcoplasmic reticulum (SR)Ca²⁺, whereas neither heparin nor anti-inositol1,4,5-trisphosphate receptor (IP₃R) antibodies affected CNresponses. In both PASMCs and oocytes, CN-evoked Ca²⁺release was inhibited by carbonyl cyanidem-chlorophenylhydrazone (CCCP) and oligomycin or CCCP andthapsigargin. Whereas hypoxic stimuli resulted in Ca²⁺release in pulmonary but not mesenteric artery myocytes, CN induced release in both cell types. We conclude that metabolic inhibition withCN increases [Ca²⁺]_i in both pulmonary andsystemic artery myocytes by stimulating Ca²⁺ release fromthe SR and mitochondria.

     

Mitochondrial reactive oxygen species and Ca2+ signaling

Mitochondria are an important source of reactive oxygen species (ROS) formed as a side product of oxidative phosphorylation. The main sites of oxidant production are complex I and complex III, where electrons flowing from reduced substrates are occasionally transferred to oxygen to form superoxide anion and derived products. These highly reactive compounds have a well-known role in pathological states and in some cellular responses. However, although their link with Ca²⁺ is well studied in cell death, it has been hardly investigated in normal cytosolic calcium concentration ([Ca²⁺]_i) signals. Several Ca²⁺ transport systems are modulated by oxidation. Oxidation increases the activity of inositol 1,4,5-trisphosphate and ryanodine receptors, the main channels releasing Ca²⁺ from intracellular stores in response to cellular stimulation. On the other hand, mitochondria are known to control [Ca²⁺]_i signals by Ca²⁺ uptake and release during cytosolic calcium mobilization, specially in mitochondria situated close to Ca²⁺ release channels. Mitochondrial inhibitors modify calcium signals in numerous cell types, including oscillations evoked by physiological stimulus. Although these inhibitors reduce mitochondrial Ca²⁺ uptake, they also impair ROS production in several systems. In keeping with this effect, recent reports show that antioxidants or oxidant scavengers also inhibit physiological calcium signals. Furthermore, there is evidence that mitochondria generate ROS in response to cell stimulation, an effect suppressed by mitochondrial inhibitors that simultaneously block [Ca²⁺]_i signals. Together, the data reviewed here indicate that Ca²⁺-mobilizing stimulus generates mitochondrial ROS, which, in turn, facilitate [Ca²⁺]_i signals, a new aspect in the biology of mitochondria. Finally, the potential implications for biological modeling are discussed. mitochondria; calcium     

Bidirectional Ca2+ coupling of mitochondria with the endoplasmic reticulum and regulation of multimodal Ca2+ entries in rat brown adipocytes

How the endoplasmic reticulum (ER) and mitochondria communicate with each other and how they regulate plasmalemmal Ca²⁺ entry were studied in cultured rat brown adipocytes. Cytoplasmic Ca²⁺ or Mg²⁺ and mitochondrial membrane potential were measured by fluorometry. The sustained component of rises in cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) produced by thapsigargin was abolished by removing extracellular Ca²⁺, depressed by depleting extracellular Na⁺, and enhanced by raising extracellular pH. FCCP, dinitrophenol, and rotenone caused bi- or triphasic rises in [Ca²⁺]_i, in which the first phase was accompanied by mitochondrial depolarization. The FCCP-induced first phase was partially inhibited by oligomycin but not by ruthenium red, cyclosporine A, U-73122, a Ca²⁺-free EGTA solution, and an Na⁺-free solution. The FCCP-induced second phase paralleling mitochondrial repolarization was partially blocked by removing extracellular Ca²⁺ and fully blocked by oligomycin but not by thapsigargin or an Na⁺-deficient solution, was accompanied by a rise in cytoplasmic Mg²⁺ concentration, and was summated with a high pH-induced rise in [Ca²⁺]_i, whereas the extracellular Ca²⁺-independent component was blocked by U-73122 and cyclopiazonic acid. The FCCP-induced third phase was blocked by removing Ca²⁺ but not by thapsigargin, depressed by decreasing Na⁺, and enhanced by raising pH. Cyclopiazonic acid-evoked rises in [Ca²⁺]_i in a Ca²⁺-free solution were depressed after FCCP actions. Thus mitochondrial uncoupling causes Ca²⁺ release, activating Ca²⁺ release from the ER and store-operated Ca²⁺ entry, and directly elicits a novel plasmalemmal Ca²⁺ entry, whereas Ca²⁺ release from the ER activates Ca²⁺ accumulation in, or release from, mitochondria, indicating bidirectional mitochondria-ER couplings in rat brown adipocytes. plasmalemmal calcium entry; calcium release; mitochondrial depolarization; FCCP     

Characteristics of phosphate-induced Ca2+ efflux from the SR in mechanically skinned rat skeletal muscle fibers

The effects of P_i onsarcoplasmic reticulum (SR) Ca²⁺ regulation were studied inmechanically skinned rat skeletal muscle fibers. Brief application ofcaffeine was used to assess the SR Ca²⁺ content, andchanges in concentration of Ca²⁺([Ca²⁺]) within the cytosol were detected withfura 2 fluorescence. Introduction of P_i (1-40 mM)induced a concentration-dependent Ca²⁺ efflux from the SR.In solutions lacking creatine phosphate (CP), the amplitude of theP_i-induced Ca²⁺ transient approximatelydoubled. A similar potentiation of P_i-induced Ca²⁺ release occurred after inhibition of creatine kinase(CK) with 2,4-dinitrofluorobenzene. In the presence of ruthenium red or ryanodine, caffeine-induced Ca²⁺ release was almostabolished, whereas P_i-induced Ca²⁺ release wasunaffected. However, introduction of the SR Ca²⁺ ATPaseinhibitor cyclopiazonic acid effectively abolishedP_i-induced Ca²⁺ release. These data suggestthat P_i induces Ca²⁺ release from the SR byreversal of the SR Ca²⁺ pump but not via the SRCa²⁺ channel under these conditions. If this occurs inintact skeletal muscle during fatigue, activation of a Ca²⁺efflux pathway by P_i may contribute to the reporteddecrease in net Ca²⁺ uptake and increase in resting[Ca²⁺].

     

Effects of ATP, Mg2+, and redox agents on the Ca2+ dependence of RyR channels from rat brain cortex

Despite their relevance for neuronal Ca²⁺-induced Ca²⁺ release (CICR), activation by Ca²⁺ of ryanodine receptor (RyR) channels of brain endoplasmic reticulum at the [ATP], [Mg²⁺], and redox conditions present in neurons has not been reported. Here, we studied the effects of varying cis-(cytoplasmic) free ATP concentration ([ATP]), [Mg²⁺], and RyR redox state on the Ca²⁺ dependence of endoplasmic reticulum RyR channels from rat brain cortex. At pCa 4.9 and 0.5 mM adenylylimidodiphosphate (AMP-PNP), increasing free [Mg²⁺] up to 1 mM inhibited vesicular [³H]ryanodine binding; incubation with thimerosal or dithiothreitol decreased or enhanced Mg²⁺ inhibition, respectively. Single RyR channels incorporated into lipid bilayers displayed three different Ca²⁺ dependencies, defined by low, moderate, or high maximal fractional open time (P_o), that depend on RyR redox state, as we have previously reported. In all cases, cis-ATP addition (3 mM) decreased threshold [Ca²⁺] for activation, increased maximal P_o, and shifted channel inhibition to higher [Ca²⁺]. Conversely, at pCa 4.5 and 3 mM ATP, increasing cis-[Mg²⁺] up to 1 mM inhibited low activity channels more than moderate activity channels but barely modified high activity channels. Addition of 0.5 mM free [ATP] plus 0.8 mM free [Mg²⁺] induced a right shift in Ca²⁺ dependence for all channels so that [Ca²⁺] <30 µM activated only high activity channels. These results strongly suggest that channel redox state determines RyR activation by Ca²⁺ at physiological [ATP] and [Mg²⁺]. If RyR behave similarly in living neurons, cellular redox state should affect RyR-mediated CICR. Ca²⁺-induced Ca²⁺ release; Ca²⁺ release channels; endoplasmic reticulum; thimerosal; 2,4-dithiothreitol; ryanodine receptor     

Calcium Accumulation by Corn Mitochondria

Corn mitochondria carry out energy-linked Ca^{2 +} accumulationwhen energized in KC1, and Ca^{2 +} release occurs when respirationceases. Stoichiometric H⁺ ejection occurs giving a H⁺: Ca^{2 +}ratio of 0.9 but insensitivity to holmium suggests lack of aCa^{2 +}-specific carrier. Non-energized corn mitochondria contain168 nmol endogenous inorganic phosphate per mg protein, about70 per cent of which is labile to Millipore filtration but stableto centrifugal filtration. Energized mitochondria, collectedby Millipore filtration, show an apparent increase in endogenousinorganic phosphate content concurrent with Ca²⁺ accumulation,both of which are inhibited by mersalyl. Accumulation of Ca²⁺also occurs in a potassium acetate medium but without involvementof endogenous Pi. Subsequent Ca²⁺ release occurs despite continuingsubstrate oxidation and is associated with a reduced abilityto synthesize adenosine triphosphate.     

Ca(2+) regulates fluid shear-induced cytoskeletal reorganization and gene expression in osteoblasts

Osteoblasts subjected to fluid shearincrease the expression of the early response gene, c-fos, andthe inducible isoform of cyclooxygenase, COX-2, two proteins linked tothe anabolic response of bone to mechanical stimulation, in vivo. Theseincreases in gene expression are dependent on shear-induced actinstress fiber formation. Here, we demonstrate that MC3T3-E1osteoblast-like cells respond to shear with a rapid increase inintracellular Ca²⁺ concentration([Ca²⁺]_i) that wepostulate is important to subsequent cellular responses to shear. Totest this hypothesis, MC3T3-E1 cells were grown on glass slides coatedwith fibronectin and subjected to laminar fluid flow (12 dyn/cm²). Before application of shear, cells were treatedwith two Ca²⁺ channel inhibitors or various blockers ofintracellular Ca²⁺ release for 0.5-1 h. Althoughgadolinium, a mechanosensitive channel blocker, significantly reducedthe [Ca²⁺]_i response, neithergadolinium nor nifedipine, an L-type channel Ca²⁺ channelblocker, were able to block shear-induced stress fiber formation andincrease in c-fos and COX-2 in MC3T3-E1 cells. However, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraaceticacid-AM, an intracellular Ca²⁺ chelator, or thapsigargin,which empties intracellular Ca²⁺ stores, completelyinhibited stress fiber formation and c-fos/COX-2 production in shearedosteoblasts. Neomycin or U-73122 inhibition of phospholipase C, whichmediates D-myo-inositol 1,4,5-trisphosphate (IP₃)-induced intracellular Ca²⁺ release, alsocompletely suppressed actin reorganization and c-fos/COX-2 production.Pretreatment of MC3T3-E1 cells with U-73343, the inactive isoform ofU-73122, did not inhibit these shear-induced responses. These resultssuggest that IP₃-mediated intracellular Ca²⁺release is required for modulating flow-induced responses in MC3T3-E1 cells.

     

Regulation of dynamic behavior of cardiac ryanodine receptor by Mg2+ under simulated physiological conditions

Mg²⁺, an important constituent of the intracellular milieu in cardiac myocytes, is known to inhibit ryanodine receptor (RyR) Ca²⁺ release channels by competing with Ca²⁺ at the cytosolic activation sites of the channel. However, the significance of this competition for local, dynamic Ca²⁺-signaling processes thought to govern cardiac excitation-contraction (EC) coupling remains largely unknown. In the present study, Ca²⁺ stimuli of different waveforms (i.e., sustained and brief) were generated by photolysis of the caged Ca²⁺ compound nitrophenyl (NP)-EGTA. The evoked RyR activity was measured in planar lipid bilayers in the presence of 0.6-1.3 mM free Mg²⁺ at the background of 3 mM total ATP in the presence or absence of 1 mM luminal Ca²⁺. Mg²⁺ dramatically slowed the rate of activation of RyRs in response to sustained (=" BORDER="0">10-ms) elevations in Ca²⁺ concentration. Paradoxically, Mg²⁺ had no measurable impact on the kinetics of the RyR response induced by physiologically relevant, brief (<1-ms) Ca²⁺ stimuli. Instead, the changes in activation rate observed with sustained stimuli were translated into a drastic reduction in the probability of responses. Luminal Ca²⁺ did not affect the peak open probability or the probability of responses to brief Ca²⁺ signals; however, it slowed the transition to steady state and increased the steady-state open probability of the channel. Our results indicate that Mg²⁺ is a critical physiological determinant of the dynamic behavior of the RyR channel, which is expected to profoundly influence the fidelity of coupling between L-type Ca²⁺ channels and RyRs in heart cells. excitation-contraction coupling; cardiac myocytes; magnesium; calcium signaling     

Modulation of intracellular Ca2+ release and capacitative Ca2+ entry by CaMKII inhibitors in bovine vascular endothelial cells

The effects of inhibitors of CaMKII on intracellular Ca²⁺ signaling were examined in single calf pulmonary artery endothelial (CPAE) cells using indo-1 microfluorometry to measure cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i). The three CaMKII inhibitors, KN-93, KN-62, and autocamtide-2-related inhibitory peptide (AIP), all reduced the plateau phase of the [Ca²⁺]_i transient evoked by stimulation with extracellular ATP. Exposure to KN-93 or AIP alone in the presence of 2 mM extracellular Ca²⁺ resulted in a dose-dependent increase of [Ca²⁺]_i consisting of a rapid and transient Ca²⁺ spike followed by a small sustained plateau phase of elevated [Ca²⁺]_i. Exposure to KN-93 in the absence of extracellular Ca²⁺ caused a transient rise of [Ca²⁺]_i, suggesting that exposure to CaMKII inhibitors directly triggered release of Ca²⁺ from intracellular endoplasmic reticulum (ER) Ca²⁺ stores. Repetitive stimulation with KN-93 and ATP, respectively, revealed that both components released Ca²⁺ largely from the same store. Pretreatment of CPAE cells with the membrane-permeable inositol 1,4,5-trisphosphate (IP₃) receptor blocker 2-aminoethoxydiphenyl borate caused a significant inhibition of the KN-93-induced Ca²⁺ response, suggesting that exposure to KN-93 affects Ca²⁺ release from an IP₃-sensitive store. Depletion of Ca²⁺ stores by exposure to ATP or to the ER Ca²⁺ pump inhibitor thapsigargin triggered robust capacitative Ca²⁺ entry (CCE) signals in CPAE cells that could be blocked effectively with KN-93. The data suggest that in CPAE cells, CaMKII modulates Ca²⁺ handling at different levels. The use of CaMKII inhibitors revealed that in CPAE cells, the most profound effects of CaMKII are inhibition of release of Ca²⁺ from intracellular stores and activation of CCE. Ca²⁺/calmodulin-dependent kinase II; calcium regulation; capacitative calcium entry     

Effect of lactate on depolarization-induced Ca(2+) release in mechanically skinned skeletal muscle fibers

Itis unclear whether accumulation of lactate in skeletal muscle fibersduring intense activity contributes to muscle fatigue. Usingmechanically skinned fibers from rat and toad muscle, we were able toexamine the effect of L(+)-lactate onexcitation-contraction coupling independently of other metabolicchanges. We investigated the effects of lactate on the contractileapparatus, caffeine-induced Ca²⁺ release from thesarcoplasmic reticulum, and depolarization-induced Ca²⁺release. Lactate (15 or 30 mM) had only a small inhibitory effect directly on the contractile apparatus and caused appreciable(20-35%) inhibition of caffeine-induced Ca²⁺ release,seemingly by a direct effect on the Ca²⁺ release channels.However, 15 mM lactate had no detectable effect on Ca²⁺release when it was triggered by the normal voltage sensor mechanism, and 30 mM lactate reduced such release by only <10%. These results indicate that lactate has only a relatively small inhibitory effect onnormal excitation-contraction coupling, indicating that lactate accumulation per se is not a major factor in muscle fatigue.