Influence of hyperglycemia on Ca2+-Mg2+-ATPase of red blood cells from diabetic patients

Summary The influence of hyperglycemia on calmodulin-stimulated Ca²⁺-Mg²⁺-ATPase activity was studied in 20 diabetic subjects. A clear inverse relationship between short-term hyperglycemia as well as chronic hyperglycemia and calmodulin-stimulated Ca²⁺-Mg²⁺-ATPase activity could be demonstrated. Such a relationship could not be shown for basal enzyme activity.Abbreviations ATPase Adenosine 5-triphosphatase - Pi Inorganic phosphate - TBA 2-Thiobarbituric acid This work was supported by the Bundesminister für Jugend, Frauen, Familie und Gesundheit, Bonn, and the Minister für Wissenschaft und Forschung des Landes Nordrhein-Westfalen, Düsseldorf, Federal Republic of GermanySome results are part of the thesis of J. Beeker     

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.     

Ca2+-Mg2+-ATPase activity of human red blood cells in healthy and diabetic volunteers

Summary The present investigation was dedicated to support biochemical interpretations of well-known long-term microvascular complications in diabetes. Provided the hypothetical correlation between erythrocyte membrane rigidity and increased intracellular calcium content holds true, a reduced Ca²⁺-Mg²⁺-ATPase activity in diabetic subjects could represent the underlying biochemical mechanism. Thus, we have compared basal and calmodulin-activated ATPase activity in healthy and diabetic volunteers. We could demonstrate a significant reduction of basal and stimulated enzyme activity in diabetic subjects. Furthermore, partial purification of calmodulin from erythrocytes of diabetic patients and healthy subjects yielded experimental evidence that reduced enzyme activity in diabetic volunteers is due to an altered basal activity as well as to a reduced stimulation by calmodulin.Abbreviations ATP Adenosine 5-triphosphate - ATPase Adenosine 5-triphosphatase - DEAE Diethylaminoethyl - EDTA Ethylenediamine tetraacetate - EGTA Ethyleneglycol-bis-(-aminoethyl ether)N,N-tetraacetic acid - P_i Inorganic phosphate - Tris Tris(hydroxymethyl)aminomethane     

ATP suppresses activity of Ca2+ -activated K+ channels by Ca2+ chelation

Ca²⁺-activated maxi K⁺ channels were studied in inside-out patches from smooth muscle cells isolated from either porcine coronary arteries or guinea-pig urinary bladder. As described by Groschner et al. (Pflügers Arch 417:517, 1990), channel activity (NP _o) was stimulated by 3 M [Ca²⁺]_c (1 mM Ca-EGTA adjusted to a calculated pCa of 5.5) and was suppressed by the addition of 1 mM Na₂ATP. The following results suggest that suppression of NP _o by Na₂ATP is due to Ca²⁺ chelation and hence reduction of [Ca²⁺]_c and reduced Ca²⁺ activation of the channel. The effect was absent when Mg ATP was used instead of Na₂ATP. The effect was diminished by increasing the [EGTA] from 1 to 10 mM. The effect was absent when [Ca²⁺]_c was buffered with 10 mM HDTA (apparent pK _Ca 5.58) instead of EGTA (pK _Ca 6.8). A Ca²⁺-sensitive electrode system indicated that 1 mM Na₂ATP reduced [Ca²⁺]_c in 1 mM Ca-EGTA from 3 M to 1.4 M. Na₂ATP, Na₂GTP, Li₄AMP-PNP and NaADP reduced measured [Ca²⁺]_c in parallel with their suppression of NP _o. After the Na₂ATP-induced reduction of [Ca²⁺]_c was re-adjusted by adding either CaCl₂ or MgCl₂, the effect of Na₂ATP on NP _o disappeared. In vivo, intracellular [Mg²⁺] exceeds free [ATP^4–], hence ATP modulation of maxi K⁺ channels due to Ca²⁺ chelation is without biological relevance.     

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.     

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+ and Sr2+ activation: Comparison of cardiac and skeletal muscle contraction models

The mechanism of contraction in rabbit fast-twitch, and bovine and rabbit cardiac muscle was examined using functionally skinned fibers, ATPase activity of myofibrils, and cardiac or skeletal troponintropomyosin regulated actin heavy meromyosin. The Ca²⁺ and Sr²⁺ activation properties for the different measures of contraction were evaluated. (1) Tension in rabbit and bovine cardiac skinned fibers and rabbit cardiac myofibrillar ATPase were activated equally well by either Ca²⁺ or Sr²⁺. By contrast, rabbit adductor magnus (fast-twitch) skinned fibers required substantially higher [Sr²⁺] than [Ca²⁺] for activation, as did rabbit myofibrils from back muscle (fast-twitch). (2) Substantially more Sr²⁺ than Ca²⁺ was also required for activation of skeletal muscle actin heavy meromyosin ATPase, controlled by either the skeletal or cardiac troponin-tropomyosin complex, similar to the activation of fast-twitch muscle. (3) The absence of correlation between the divalent cation selectivity properties of actin heavy meromyosin ATPase controlled by cardiac troponin-tropomyosin and cardiac muscle tension or myofibrillar ATPase activation by Ca²⁺ and Sr²⁺ suggests that troponin, if primarily responsible for the activation of cardiac muscle, has very different in vivo and in vitro binding properties. (4) The close correlation between percentage of maximal Ca²⁺- and Sr²⁺-activated myofibrillar ATPase and tension in skinned fibers strongly justifies the use of myofibrillar ATPase, in contrast to a reconstituted troponin-tropomyosin actin heavy meromyosin ATPase system, as a biochemical measure of contraction.     

Initiation sites for Ca2+ signals in endothelial cells

Intracellular Ca²⁺ signals in response to inositol 1,4,5-trisphosphate-producing agents often present themselves as Ca²⁺ oscillations and propagating Ca²⁺ waves originating at discrete initiation sites. We studied the spatial organization of the Ca²⁺ signal in single CPAE endothelial cells stimulated with adenosine triphosphate. The long, thin processes presented a higher agonist sensitivity and, for the same agonist concentration, a faster rise in cytoplasmic Ca²⁺ concentration and rate of wave propagation than the cell body. Ca²⁺ waves originated preferentially in one of these processes and then invaded the cell body. Removal of external Ca²⁺ induced a progressive inhibition up to blockade of the response in the process but not in the cell body. These findings suggest that CPAE cells contain many individual store units, each of which has the inherent ability to set the stage for Ca²⁺ release. A diffusing messenger originating from the initiation zone then coordinates the events leading to Ca²⁺ release in the individual store units to produce a Ca²⁺ wave.     

T-type Ca2+ channels mediate propagation of odor-induced Ca2+ transients in rat olfactory receptor neurons

Propagation of odor-induced Ca(2+) transients from the cilia/knob to the soma in mammalian olfactory receptor neurons (ORNs) is thought to be mediated exclusively by high-voltage-activated Ca(2+) channels. However, using confocal Ca(2+) imaging and immunocytochemistry we identified functional T-type Ca(2+) channels in rat ORNs. Here we show that T-type Ca(2+) channels in ORNs also mediate propagation of odor-induced Ca(2+) transients from the knob to the soma. In the presence of the selective inhibitor of T-type Ca(2+) channels mibefradil (10-15 microM) or Ni(2+) (100 microM), odor- and forskolin/3-isobutyl-1-methyl-xanthine (IBMX)-induced Ca(2+) transients in the soma and dendrite were either strongly inhibited or abolished. The percentage of inhibition of the Ca(2+) transients in the knob, however, was 40-50% less than that in the soma. Ca(2+) transients induced by 30 mM K(+) were partially inhibited by mibefradil, but without a significant difference in the extent of inhibition between the knob and soma. Furthermore, an increase of as little as 2.5 mM in the extracellular K(+) concentration (7.5 mM K(+)) was found to induce Ca(2+) transients in ORNs, and such responses were completely inhibited by mibefradil or Ni(2+). Total replacement of extracellular Na(+) with N-methyl-d-glutamate inhibited none of the odor-, forskolin/IBMX- or 7.5 mM K(+)-induced Ca(2+) transients. Positive immunoreactivity to the Ca(v)3.1, Ca(v)3.2 and Ca(v)3.3 subunits of the T-type Ca(2+) channel was observed throughout the soma, dendrite and knob. These data suggest that involvement of T-type Ca(2+) channels in the propagation of odor-induced Ca(2+) transients in ORNs may contribute to signal transduction and odor sensitivity.     

Blocking actions of Ca2+ antagonists on the Ca2+ channels in the smooth muscle cell membrane of rabbit small intestine

Actions of Ca²⁺ antagonists, verapamil, nicardipine and diltiazem, were investigated on the Ca²⁺ inward current in the fragmented smooth muscle cell membrane (smooth muscle ball; SMB) obtained from the longitudinal muscle layer of the rabbit ileum, by enzymatic dispersion. All Ca²⁺ antagonists inhibited the inward current, in a dose-dependent manner. The ID₅₀ value on the maximum amplitude of the inward current of nicardipine was 24 nM, and this value was roughly 50 times lower than values obtained with verapamil and diltiazem, when the inward current was provoked by 0 mV command pulse from the holding potential of –60 mV. Lowering the holding potential to –80 mV shifted the dose-response curve to the right. When depolarizing pulses (100 ms, stepped up to 0 mV from –60 mV or –80 mV) were applied every 20 s, the peak amplitude of the inward current remained unchanged, but nicardipine immediately, and diltiazem and verapamil slowly reduced the peak amplitude. These slow inhibitions by the latter two drugs depended on the frequency or number of stimulations. Nicardipine but not diltiazem and verapamil shifted the voltage-dependent inactivation curve to the left (3 s duration of the conditioning pulse). However, with a longer conditioning pulse (10 s) verapamil and diltiazem shifted the voltage-dependent inactivation curves to the left. Therefore, the inhibitory actions of these Ca²⁺ antagonists differ. Namely, diltiazem and verapamil inhibit the Ca²⁺ channels, mainly in a frequency-or use-dependent manner while nicardipine does so in a voltage-dependent manner.     

Role of Na+/Ca2+ exchange in transcellular Ca2+ transport across primary cultures of rabbit kidney collecting system

Cells from connecting tubule and cortical collecting duct of rabbit kidney were isolated by immunodissection with mAb R2G9 and cultured on permeable filters. Confluent monolayers developed an amiloride-sensitive transepithelial potential difference of –50±1 mV (lumen negative) and a transepithelial resistance of 507±18 cm². Transepithelial Ca²⁺ transport increased dose-dependently with apical [Ca²⁺] and, in solutions containing 1 mM Ca²⁺, the active transcellular Ca²⁺ transport rate was 92±2 nmol h^–1 cm^–2. Transcellular Ca²⁺ transport was dependent on basolateral Na⁺ (Na _b ⁺ ). Isoosmotic substitution of Na _b ⁺ for N-methylglucamine resulted in a concentration-dependent decrease in Ca²⁺ absorption, with maximal inhibition of 67±5%. A Hill plot of the Na⁺-dependence yielded a coefficient of 1.9±0.4, indicating more than one Na⁺ site on a Na⁺-dependent Ca²⁺ transport system. In addition, the absence of Ca _b ²⁺ resulted in a significant increase in Ca²⁺ transport both in the presence and absence of Na _b ⁺ . Added basolaterally, ouabain (0.1 mM) inhibited Ca²⁺ transport to the same extent as did Na⁺-free solutions, while bepridil (0.1 mM), an inhibitor of Na⁺/Ca²⁺ exchange, reduced Ca²⁺ transport by 32±6%. Methoxyverapamil, felodipine, flunarizine and diltiazem (10 M) were without effect. Depolarisation of the basolateral membrane, by raising [K⁺]_b to 60 mM, significantly decreased transcellular Ca²⁺ transport, which is indicative of electrogenic Na⁺/Ca²⁺ exchange. In conclusion, active Ca²⁺ transport in the collecting system of rabbit kidney is largely driven by basolateral Na⁺/Ca²⁺ exchange. However, a residual Ca²⁺ absorption of about 30% was always observed, suggesting that other Ca²⁺ transport mechanisms, presumably a Ca²⁺-ATPase, participate as well.     

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⁺.     

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)     

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.     

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.     

Effect of thapsigargin and caffeine on Ca2+ homeostasis in HeLa cells: implications for histamine-induced Ca2+ oscillations

Several studies have already established that the stimulation of H1 receptors by exogenous histamine induces intracellular Ca²⁺ oscillations in HeLa cells. The molecular mechanism underlying this oscillatory process remains, however, unclear. A series of fura-2 experiments was undertaken in which the nature of the Ca²⁺ pools involved in the histamine-induced Ca²⁺ oscillations was investigated using the tumour promoter agent thapsigargin (TG) and the Ca²⁺-induced Ca²⁺-release promoter, caffeine. The results obtained indicate first that TG causes a gradual increase in cytosolic Ca²⁺ without inducing internal Ca²⁺ oscillations, and second that TG and histamine share common internal Ca²⁺ storage sites. The latter conclusion was derived from experiments performed in the absence of external Ca²⁺, where the addition of TG before histamine resulted in a total inhibition of the Ca²⁺ response linked to H1 receptor stimulation, whereas the addition of histamine before TG decreased by more than 90% the TG-induced Ca²⁺ release. Finally, TG was found to inhibit irreversibly histamine-induced Ca²⁺ oscillations when added to the bathing medium during the oscillatory process. The effect of caffeine at concentrations ranging from 1 mM to 10 mM on intracellular Ca²⁺ homeostasis was also investigated. The results obtained show that caffeine does not affect systematically the internal Ca²⁺ concentration in resting and TG-stimulated HeLa cells, but increases the Ca²⁺ sequestration ability of inositol-trisphosphate (InsP ₃)-related Ca²⁺ stores. These results suggest either that TG acts on InsP ₃-sensitive as well as InsP ₃-insensitive Ca²⁺ pools, so that no final conclusion on the nature of the pools involved in Ca²⁺ spike generation can be currently drawn, or that the contribution of an InsP ₃-insensitive Ca²⁺-induced Ca²⁺-release process is not essential to the Ca²⁺ oscillation machinery in these cells. It is also concluded that a release of Ca²⁺ by caffeine may not be directly accessible to fura-2 measurements in this cellular preparation, but that the inhibitory effect of caffeine on the Ca²⁺ mobilization process triggered by InsP ₃ can be clearly documented using this experimental approach.     

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.     

Cytoplasmic Ca2+ determines the rate of Ca2+ entry into Mardin-Darby canine kidney-focus (MDCK-F) cells

Transformed Mardin-Darby canine kidney-focus (MDCK-F) cells exhibit spontaneous Ca²⁺ oscillations from an inositol 1,4,5-trisphosphate-sensitive cytoplasmic Ca²⁺ store. In this study, Ca²⁺ entry from the extracellular space and its role in generation of oscillations were investigated by means of Ca²⁺ video imaging and the Fura-2/Mn²⁺ quenching technique. Oscillations were dependent on extracellular Ca²⁺ concentration and were inhibited by extracellularly applied La³⁺, Co²⁺ and Ni²⁺. Depolarization of the cell membrane with high K⁺ concentrations and the L-type Ca²⁺ channel blocker nifedipine had no effect on oscillations, indicating the lack of involvement of voltage-gated Ca²⁺ channels. Mn²⁺ quenching experiments disclosed significant Ca²⁺ influx into MDCK-F cells. The rate of this influx was constant between Ca²⁺ spikes, but markedly increased during the spontaneous Ca²⁺ spikes. Similar transient increases in Ca²⁺ entry could be mimicked by agents triggering intracellular Ca²⁺ release such as bradykinin and thapsigargin. We conclude that the plasma membrane of MDCK-F cells exhibits a marked voltage-independent Ca²⁺ permeability permitting Ca²⁺ entry into the cytoplasm. The rate of Ca²⁺ entry which determines the frequency of oscillations is most likely to be regulated by the cytoplasmic Ca²⁺ concentration.     

AlF 4 − induces Ca2+ oscillations in guinea-pig ileal smooth muscle

The effects of different compounds that inhibit the isolated plasma-membrane Ca²⁺/Mg²⁺-ATPase on the cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) and on the corresponding force development have been examined in smooth muscle of the longitudinal layer of the guinea-pig ileum. F^–, in the presence of Al³⁺, induced an increase of the resting force and of the amplitude of the superimposed phasic contractions. The increase of resting force was associated with an increased level of basal [Ca²⁺]_i while the phasic contractions were accompanied by concomitant oscillations in [Ca²⁺]_i. Comparable contractions could be induced by vanadate and the calmodulin antagonist calmidazolium. The oscillations of [Ca²⁺]_i and of force elicited by AlF ₄ ^– were not modified by adrenergic or cholinergic blocking agents but were inhibited by verapamil. These phasic contractions were not affected by depleting the intracellular Ca²⁺ stores with ryanodine. This finding excludes a cytosolic origin of these oscillations. However, hyperpolarization and complete depolarization of the cells inhibited the oscillations. It is concluded that AlF ₄ ^– , vanadate and calmidazolium induce cytoplasmic Ca²⁺ oscillations possibly by acting at the plasma membrane. Indeed all these substances affect by different mechanisms the isolated plasma-membrane Ca²⁺/Mg²⁺-ATPase. The generation of membrane-linked Ca²⁺ oscillations could therefore be related to an inhibition of the plasma-membrane Ca²⁺ pump resulting in an increase of [Ca²⁺]_i. This change in [Ca²⁺]_i could be responsible for the pronounced changes of the electrical and mechanical activity of this tissue.