Effects of intracellular Ca2+ chelating compounds on inward currents caused by Ca2+ release from sarcoplasmic reticulum in guinea-pig atrial myocytes

Ca²⁺ release from the sarcoplasmic reticulum (SR) of mammalian cardiac myocytes occuring either due to activation by a depolarization or the resulting transmembrane Ca²⁺ current (I _Ca), or spontaneously due to Ca²⁺ overload has been shown to cause inward current(s) at negative membrane potentials. In this study, the effects of different intracellular Ca²⁺ chelating compounds on I _Ca-evoked or spontaneous Ca²⁺-release-dependent inward currents were examined in dialysed atrial myocytes from hearts of adult guinea-pigs by means of whole-cell voltage-clamp. As compared to dialysis with solutions containing only a low concentration of a high affinity ethylene glycol-bis(-aminoethylether) N,N,N,N-tetraacetic acid (EGTA) like chelator (50–200 M), inward membrane currents (at –50 mV) due to evoked Ca²⁺ release, spontaneous Ca²⁺ release or Ca²⁺ overload following long-lasting depolarizations to very positive membrane potentials are prolonged if the dialysing fluid contains a high concentration of a low affinity Ca²⁺ chelating compound such as citrate or free adenosine 5-triphosphate (ATP). Without such a non-saturable Ca²⁺ chelator in the dialysing fluid, Ca²⁺-release-dependent inward currents are often oscillatory and show an irregular amplitude. With a low affinity chelator in a non-saturable concentration, discrete inward currents with constant properties can be recorded. We conclude that the variability in Ca²⁺-release-dependent inward current seen in single cells arises from spatial inhomogeneities of intracellular Ca²⁺ concentration ([Ca²⁺]_i) due to localized saturation of endogenous and exogenous high affinity Ca²⁺ buffers (e.g. [2]). This can be avoided experimentally by addition of a non-saturable buffer to the intracellular solution. This condition might be useful, if properties of Ca²⁺ release from the SR and/ or the resulting membrane current, like for example arrhythmogenic transient inward current, are to be investigated on the single cell level.     

Functional characterization of the Ca2+-gated Ca2+ release channel of vascular smooth muscle sarcoplasmic reticulum

The Ca²⁺-gated Ca²⁺ release channel of aortic sarcoplasmic reticulum (SR) was partially purified and reconstituted into planar lipid bilayers. Canine and porcine aorta microsomal protein fractions were solubilized in the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulphonate (CHAPS) in the presence and absence of ³[H]-ryanodine and centrifuged through linear sucrose gradients. A single ³[H]-ryanodine receptor peak with an apparent sedimentation coefficient of 30 s was obtained. Upon reconstitution into planar lipid bilayers, the unlabelled 30 s protein fraction induced the formation of a Ca²⁺- and monovalent-ion-conducting channel (110 pS in 100 mM Ca²⁺, 360 pS in 250 mM K⁺). The channel was activated by micromolar Ca²⁺, modulated by millimolar adenosine triphosphate, Mg²⁺ and the Ca²⁺-releasing drug caffeine, and inhibited by micromolar ruthenium red. Micro- to millimolar concentrations of the plant alkaloid ryanodine induced a permanently closed state of the channel. Our results suggest that smooth muscle SR contains a Ca²⁺-gated Ca²⁺ release pathway, with properties similar to those observed for the skeletal and cardiac ryanodine receptor/Ca²⁺ release channel complexes.     

Effects of Mg2+ on Ca2+ handling by the sarcoplasmic reticulum in skinned skeletal and cardiac muscle fibres

The influence of myoplasmic Mg²⁺ (0.05–10 mM) on Ca²⁺ accumulation (net Ca²⁺ flux) and Ca²⁺ uptake (pump-driven Ca²⁺ influx) by the intact sarcoplasmic reticulum (SR) was studied in skinned fibres from the toad iliofibularis muscle (twitch portion), rat extensor digitorum longus (EDL) muscle (fast twitch), rat soleus muscle (slow twitch) and rat cardiac trabeculae. Ca²⁺ accumulation was optimal between 1 and 3 mM Mg²⁺ in toad fibres and reached a plateau between 1 and 10 mM Mg²⁺ in the rat EDL fibres and between 3 and 10 mM Mg²⁺ in the rat cardiac fibres. In soleus fibres, optimal Ca²⁺ accumulation occurred at 10 mM Mg²⁺. The same trend was obtained with all preparations at 0.3 and 1 M Ca²⁺. Experiments with 2,5-di-(tert-butyl)-1,4-benzohydroquinone, a specific inhibitor of the Ca²⁺ pump, revealed a marked Ca²⁺ efflux from the SR of toad iliofibularis fibres in the presence of 0.2 M Ca²⁺ and 1 mM Mg²⁺. Further experiments indicated that the SR Ca²⁺ leak could be blocked by 10 M ruthenium red without affecting the SR Ca²⁺ pump and this allowed separation between SR Ca²⁺ uptake and SR Ca²⁺ accumulation. At 0.3 M Ca²⁺, Ca²⁺ uptake was optimal with 1 mM Mg²⁺ in the toad iliofibularis and rat EDL fibres and between 1 and 10 mM Mg²⁺ in the rat soleus and trabeculae preparations. At higher [Ca²⁺] (1 M), Ca²⁺ uptake was optimal with 1 mM Mg²⁺ in the iliofibularis fibres and between 1 and 3 mM Mg²⁺ in the EDL fibres. In the soleus and cardiac preparations Ca²⁺ uptake was optimal between 1 and 10 mM Mg²⁺. The results of this study demonstrate that SR Ca²⁺ accumulation is different from SR Ca²⁺ uptake and that these two important determinants of muscle function are differently affected by Mg²⁺ in different muscle fibre types.     

Measurement of Ca2+-release-dependent inward current reveals two distinct components of Ca2+ release from sarcoplasmic reticulum in guinea-pig atrial myocytes

Ca²⁺ current (L-type) and inward current caused by Ca²⁺ release from the sarcoplasmic reticulum and carried by electrogenic Na⁺/Ca²⁺ exchange have been measured in cultured atrial myocytes from hearts of adult guinea-pigs using whole-cell voltage clamp techniques. The pipette solution, used for internal dialysis of the cells, contained a high concentration, 60 mM or 25 mM, of citrate as a non-saturable low-affinity Ca²⁺-chelating compound. It has been shown previously that Ca²⁺-release-dependent inward current under these conditions is carried by electrogenic Na⁺/Ca²⁺ exchange. Furthermore, Ca²⁺-release-dependent inward current (the release signal) can be completely separated from triggering Ca²⁺ current if brief depolarizations for activating I _Ca are used. In the majority of cells that did not produce spontaneous Ca²⁺ release, conditions could be found that caused the release signal to be split into two components: an early component of variable amplitude and a late component of rather constant amplitude. The delay of the late component with regard to triggering I _Ca was inversely related to the amplitude of the first one. Below a certain amplitude of the first component, the second one failed to be elicited. This suggests the second component to be triggered by the first one. Weakly Ca²⁺-buffered cells produced spontaneous Ca²⁺ release, resulting in irregular transient inward currents at constant membrane-holding potential. Synchronization by trains of step depolarizations unmasked two components also in the spontaneous release signals. In none of the cells studied was any indication of more than two components of the release signal detected. The results are discussed in terms of two distinct compartments of sarcoplasmic reticulum with different properties of Ca²⁺ release.Supported by the Deutsche Forschungsgemeinschaft (FG Konzell)     

Effect of activation of Ca2+, Mg2+-dependent endonuclease on proliferative response of human peripheral blood lymphocytes

Activity of Ca²⁺, Mg²⁺-dependent endonuclease in human peripheral blood mononuclears is studied. Intact cells exhibit extremely low activity of the enzyme. Treatment with the synthetic hexapeptide imunofan considerably stimulates endonuclease activity in mononuclears. This activation does not depend on additional protein synthesis. An 1-h incubation in the presence of cycloheximide also activates Ca²⁺, Mg²⁺-dependent endonuclease. These data suggest that imunofant and/or cycloheximide activate the apoptotic cascade. This leads to activation of endonuclease, which is not synthesizedde novo but persists in the cell in the form of inactive precursor. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 123, No. 5, pp. 535–537, May, 1997     

Effect of verapamil on activity of Ca2+,Mg2+-dependent endonucleases in dog liver nuclei in long-term hypovolemic shock

Laboratory of Experimental Therapy of Terminal States, Institute of General Resuscitation, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR V. A. Negovskii.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 112, No. 12, pp. 583–584, December, 1991.     

Prolonged exercise reduces Ca2+ release in rat skeletal muscle sarcoplasmic reticulum

Prolonged exercise decreased the rate of Ca⁺ release in sarcoplasmic reticulum (SR) vesicles isolated from rat muscle by 20–30% when release was initiated by 5, 10, and 20 M AgNO₃. [³H]Ryanodine binding was also depressed by 20% in SR vesicles isolated from the exercised animals. In contrast, the maximum amount of Ca²⁺ released by Ag⁺ remained unaffected by exercise. The passive permeability of SR vesicles and the rate of Ca²⁺ release in the presence of ruthenium red, a known inhibitor of the Ca²⁺ release mechanism, was not affected by prolonged exercise. These results suggest that exercise depressed Ca²⁺ release from SR by directly modifying the Ca²⁺ release channel. Current address: Department of Physics, Portland State University, Portland, OR 97207, USA     

Potential activity of Ca2+/Mg2+-dependent endonuclease in endometrial hyperplasia and cancer

Potential activity of Ca²⁺/Mg²⁺-dependent endonuclease in hyperplastic endometrial tissues is lower than in normal endometrium and practically absent in endometrial cancer tissue. Thus, molecular mechanisms of apoptosis regulation are disturbed even at the stage of hyperplasia. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 127, No. 6, pp. 683–687, June, 1999     

Potentiation of sarcoplasmic reticulum Ca2+ release by 2,3-butanedione monoxime in crustacean muscle

The effect of the chemical phosphatase 2,3-butanedione monoxime (BDM) on various aspects of excitation/contraction coupling in crustacean muscle was investigated. Despite having a depressant effect on vertebrate skeletal and cardiac muscle, BDM was a potentiator of contraction in crustacean muscle. At concentrations of 1–3 mM BDM caused an increase of potassium contractures in bundles of fibers isolated from crayfish muscle. At higher concentrations BDM caused oscillatory contractions by itself. In single voltage-clamped cut muscle fibers loaded with rhod-2, BDM (0.5–2 mM) potentiated the magnitude and duration of intracellular Ca²⁺ transients elicited by depolarization. At the same time BDM did not affect the rate of Ca²⁺ removal from the myoplasm under conditions where Ca²⁺ release was blocked by tetracaine. Nor did BDM increase Ca²⁺ entry; in fact it caused a decrease in the amplitude of the inward Ca²⁺ current (I _Ca). In microsomes isolated from lobster muscle, BDM also potentiated Ca²⁺ release induced by caffeine and at higher concentrations (above 3 mM) induced release by itself. At the same time it had little effect on Ca²⁺ uptake. These results indicate that BDM potentiates Ca²⁺ release in crustacean muscle possibly by dephosphorylation of the Ca²⁺-release channel.     

A possible role of sarcoplasmic Ca2+ release in modulating the slow Ca2+ current of skeletal muscle

Ca²⁺ channels are regulated in a variety of different ways, one of which is modulation by the Ca²⁺ ion itself. In skeletal muscle, Ca²⁺ release sites are presumably located in the vicinity of the dihydropyridine-sensitive Ca²⁺ channel. In this study, we have tried to investigate the effects of Ca²⁺ release from the sarcoplasmic reticulum on the L-type Ca²⁺ channel in frog skeletal muscle, using the double Vaseline gap technique. We found an increase in Ca²⁺ current amplitude on application of caffeine, a well-known potentiator of Ca²⁺ release. Addition of the fast Ca²⁺ buffer BAPTA to the intracellular solution led to a gradual decline in Ca²⁺ current amplitude and eventually caused complete inhibition. Similar observations were made when the muscle fibre was perfused internally with the Ca²⁺ release channel blocker ruthenium red. The time course of Ca²⁺ current decline followed closely the increase in ruthenium red concentration. This suggests that Ca²⁺ release from the sarcoplasmic reticulum is involved in the regulation of L-type Ca²⁺ channels in frog skeletal muscle.     

Influence of Ca2+-binding proteins and the cytoskeleton on Ca2+-dependent inactivation of high-voltage activated Ca2+ currents in thalamocortical relay neurons

Ca²⁺-dependent inactivation (CDI) of high-voltage activated (HVA) Ca²⁺ channels was investigated in acutely isolated and identified thalamocortical relay neurons of the dorsal lateral geniculate nucleus (dLGN) by combining electrophysiological and immunological techniques. The influence of Ca²⁺-binding proteins, calmodulin and the cytoskeleton on CDI was monitored using double-pulse protocols (a constant post-pulse applied shortly after the end of conditioning pre-pulses of increasing magnitude). Under control conditions the degree of inactivation (34±9%) revealed a U-shaped and a sigmoid dependency of the post-pulse current amplitude on pre-pulse voltage and charge influx, respectively. In contrast to a high concentration (5.5 mM) of EGTA (31±3%), a low concentration (3 µM) of parvalbumin (20±2%) and calbindin_D28K (24±4%) significantly reduced CDI. Subtype-specific Ca²⁺ channel blockers indicated that L-type, but not N-type Ca²⁺ channels are governed by CDI and modulated by Ca²⁺-binding proteins. These results point to the possibility that activity-dependent changes in the intracellular Ca²⁺-binding capacity can influence CDI substantially. Furthermore, calmodulin antagonists (phenoxybenzamine, 22±2%; calmodulin binding domain, 17±1%) and cytoskeleton stabilizers (taxol, 23±5%; phalloidin, 15±3%) reduced CDI. Taken together, these findings indicate the concurrent occurrence of different CDI mechanisms in a specific neuronal cell type, thereby supporting an integrated model of this feedback mechanism and adding further to the elucidation of the role of HVA Ca²⁺ channels in thalamic physiology.     

Ca2+ channel Ca2+-dependent inactivation in a mammalian central neuron involves the cytoskeleton

Ca²⁺ channel inactivation was investigated in acutely isolated hippocampal pyramidal neurons from adult rats and found to have a component dependent on intracellular Ca²⁺. Ca²⁺-dependent inactivation was identified as the additional inactivation of channel current observed when Ca²⁺ replaced Ba²⁺ as the current carrying ion, and was found to be an independent process from that of Ba²⁺ current inactivation based on three lines of evidence: (1) no correlation between Ca²⁺-dependent inactivation and Ba²⁺ current inactivation was found, (2) only Ca²⁺-dependent inactivation was reduced by intracellular application of Ca²⁺ chelators, and (3) only Ca²⁺-dependent inactivation was sensitive to compounds which alter the cytoskeleton. Drugs which stabilize (taxol and phalloidin) and destabilize (colchicine and cytochalasin B) the cytoskeleton altered the development and recovery from Ca²⁺-dependent inactivation, indicating that the neuronal cytoskeleton may mediate Ca²⁺ channel sensitivity to intracellular Ca²⁺. Ca²⁺-dependent inactivation was not associated with a particular subset of Ca²⁺ channels, suggesting that all Ca²⁺ channels in these neurons are inactivated by intracellular Ca²⁺.     

Capacitative Ca2+ influx and a Ca2u+-dependent nonselective cation pathway are discriminated by genistein in mouse pancreatic acinar cells

We have investigated the effect of genistein on the hormone-stimulated Ca²⁺ influx and on a 28 pS nonselective cation channel in mouse pancreatic acinar cells using the Ca²⁺ indicator fluo-3 and the patch-clamp technique. The identity of the Ca^2u+ influx pathway has not been established in this cell type so far. Therefore we have investigated the Ca²⁺-dependent nonselective cation channel as a potential pathway for Ca²⁺ influx. Capacitative Ca²⁺ entry was induced by depletion of intracellular Ca²⁺ stores with 500 nM acetylcholine or with the Ca²⁺ ATPase inhibitor 2,5-di-tert- butylhydroquinone. In the presence of 100 M genistein, Ca²⁺ release was unimpaired, whereas Ca²⁺ influx was reversibly suppressed. Patch-clamp experiments demonstrated that genistein had no effect on Ca²⁺-activated nonselective cation channels, the activity of which was measured in excised membrane patches (inside/out) or in the whole-cell configuration. Therefore we conclude that this 28 pS nonselective cation channel does not contribute to Ca²⁺ influx into mouse exocrine pancreatic cells. With the exception of genistein and tyrphostin 25, other tyrosine kinase inhibitors such as methyl-2,5-dihydroxycinnamate, lavendustin A, herbimycin A, and tyrphostin B56 were without effect on Ca²⁺ signalling. Thus, the involvement of tyrosine phosphorylation in the activation of the Ca²⁺ entry mechanism in mouse pancreatic acinar cells is unclear.     

Effect of cytosolic Mg2+ on mitochondrial Ca2+ signaling

Cytosolic Ca²⁺ signals are followed by mitochondrial Ca²⁺ uptake, which, in turn, modifies several biological processes. Mg²⁺ is known to inhibit Ca²⁺ uptake by isolated mitochondria, but its significance in intact cells has not been elucidated. In HEK293T cells, activation of purinergic receptors with extracellular ATP caused cytosolic Ca²⁺ signals associated with parallel changes in cytosolic [Mg²⁺]. Neither signals were affected by omitting bivalent cations from the extracellular medium. The effect of store-operated Ca²⁺ influx on cytosolic Mg²⁺ concentration ([Mg²⁺]_c) was negligible. Uncaged Ca²⁺ displaced Mg²⁺ from cytosolic binding sites, but for an equivalent Ca²⁺ signal, the change in [Mg²⁺] was significantly smaller than that measured after adding extracellular ATP. Inositol 1,4,5-trisphosphate mobilized Ca²⁺ and Mg²⁺ from internal stores in permeabilized cells. The increase of [Mg²⁺] in the range that occurred in ATP-stimulated cells inhibited mitochondrial Ca²⁺ uptake in permeabilized cells without affecting mitochondrial Ca²⁺ efflux. Therefore, the Mg²⁺ signal generated by Ca²⁺ mobilizing agonists may attenuate mitochondrial Ca²⁺ uptake.     

Ca2+ transport in the myocardial sarcoplasmic reticulum of rats during antiorthostatic off-loading and during readaptation

The rate of Ca²⁺ accumulation in the myocardial sarcoplasmic reticulum is studied in experiments on rats under conditions of hind paw functional off-loading by suspending animals by the tail for 1 to 40 days, as well as during a 2-week period of readaptation after a 40-day load relief. The rate of Ca²⁺ transport in the myocardial sarcoplasmic reticulum reliably drops (by 33%) after 40 days of suspension. At earlier stages of off-loading Ca-pump activity in the sarcoplasmic reticulum does not change reliably. After resumption of the loads in animals suspended for 40 days, the transporting function of the myocardial sarcoplasmic reticulum rapidly reverts to the control level, which is indicative of a reversible pattern of load relief-induced changes in the rate of Ca²⁺ transport. Translated fromByulleten' Eksperimental'noi Biologii i Meditsity, Vol. 118, N ^o 12, pp. 596–599, December, 1994 Presented by G. N. Kryzhanovskii, Member of the Russian Academy of Medical Sciences     

Mitochondria and Ca2+ signaling: old guests, new functions

Mitochondria are ancient endosymbiotic guests that joined the cells in the evolution of complex life. While the unique ability of mitochondria to produce adenosine triphosphate (ATP) and their contribution to cellular nutrition metabolism received condign attention, our understanding of the organelle’s contribution to Ca²⁺ homeostasis was restricted to serve as passive Ca²⁺ sinks that accumulate Ca²⁺ along the organelle’s negative membrane potential. This paradigm has changed radically. Nowadays, mitochondria are known to respond to environmental Ca²⁺ and to contribute actively to the regulation of spatial and temporal patterns of intracellular Ca²⁺ signaling. Accordingly, mitochondria contribute to many signal transduction pathways and are actively involved in the maintenance of capacitative Ca²⁺ entry, the accomplishment of Ca²⁺ refilling of the endoplasmic reticulum and Ca²⁺-dependent protein folding. Mitochondrial Ca²⁺ homeostasis is complex and regulated by numerous, so far, genetically unidentified Ca²⁺ channels, pumps and exchangers that concertedly accomplish the organelle’s Ca²⁺ demand. Notably, mitochondrial Ca²⁺ homeostasis and functions are crucially influenced by the organelle’s structural organization and motility that, in turn, is controlled by matrix/cytosolic Ca²⁺. This review intends to provide a condensed overview on the molecular mechanisms of mitochondrial Ca²⁺ homeostasis (uptake, buffering and storage, extrusion), its modulation by other ions, kinases and small molecules, and its contribution to cellular processes as fundamental basis for the organelle’s contribution to signaling pathways. Hence, emphasis is given to the structure-to-function and mobility-to-function relationship of the mitochondria and, thereby, bridging our most recent knowledge on mitochondria with the best-established mitochondrial function: metabolism and ATP production.     

Enhanced expression of sarcoplasmic reticulum Ca2+-ATPase gene plays a role in protective effects of nitric oxide

This study verifies the hypothesis that NO-dependent regulation of sarcoplasmic reticulum Ca²⁺-ATPase gene (SERCA) plays an important role in preventing Ca²⁺ overload after exposure to damaging factors. The data confirmed NO-dependent activation of SERCA expression and important role of this mechanism in the reduction of calcium overload. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol 128, No. 10, pp. 375–379, October, 1999     

Caffeine-induced oscillations of cytosolic Ca2+ in GH3 pituitary cells are not due to Ca2+ release from intracellular stores but to enhanced Ca2+ influx through voltage-gated Ca2+ channels

Caffeine, a well known facilitator of Ca²⁺-induced Ca²⁺ release, induced oscillations of cytosolic free Ca²⁺ ([Ca²⁺]_i) in GH₃ pituitary cells. These oscillations were dependent on the presence of extracellular Ca²⁺ and blocked by dihydropyridines, suggesting that they are due to Ca²⁺ entry through L-type Ca²⁺ channels, rather than to Ca²⁺ release from the intracellular Ca²⁺ stores. Emptying the stores by treatment with ionomycin or thapsigargin did not prevent the caffeine-induced [Ca²⁺]_i oscillations. Treatment with caffeine occluded phase 2 ([Ca²⁺]_i oscillations) of the action of thyrotropin-releasing hormone (TRH) without modifying phase 1 (Ca²⁺ release from the intracellular stores). Caffeine also inhibited the [Ca²⁺]_i increase induced by depolarization with high-K⁺ solutions (56% at 20 mM), suggesting direct inhibition of the Ca²⁺ entry through voltage-gated Ca²⁺ channels. We propose that the [Ca²⁺]_i increase induced by caffeine in GH₃ cells takes place by a mechanism similar to that of TRH, i.e. membrane depolarization that increases the firing frequency of action potentials. The increase of the electrical activity overcomes the direct inhibitory effect on voltage-gated Ca²⁺ channels with the result of increased Ca²⁺ entry and a rise in [Ca²⁺]_i. Consideration of this action cautions interpretation of previous experiments in which caffeine was assumed to increase [Ca²⁺]_i only by facilitating the release of Ca²⁺ from intracellular Ca²⁺ stores.