Involvement of Ca2+-induced Ca2+ release in the biphasic Ca2+ response evoked by readdition of Ca2+ to the medium after UTP-induced store depletion in A431 cells

 We have recently shown that the Ca²⁺ response in endothelial cells evoked by readdition of Ca²⁺ to the medium after store depletion caused by a submaximal concentration of agonist can involve Ca²⁺ release from Ca²⁺ stores sensitive to both inositol 1,4,5-trisphosphate and ryanodine. The present experiments were performed to determine whether this mechanism might also exist in other types of cell. For this purpose, we used the human carcinoma cell line A431, which has a varied resting [Ca²⁺]_i. We found that the amplitude of the Ca²⁺ response evoked by Ca²⁺ readdition did not correlate with the amplitude of the preceding UTP-evoked Ca²⁺ release, but did positively correlate with the initial [Ca²⁺]_i. An inspection of the two patterns of response seen in this study (the large biphasic and small plateau-shaped Ca²⁺ responses) revealed that there is an accelerating rise in [Ca²⁺]_i during the biphasic response. Application of ryanodine during the plateau-shaped Ca²⁺ response reversibly transformed it into the biphasic type. Unlike ryanodine, caffeine did not itself evoke Ca²⁺ release, but it caused a further [Ca²⁺]_i rise when [Ca²⁺]_i had already been elevated by thapsigargin. These data suggest that in A431 cells, as in endothelial cells, the readdition of Ca²⁺ after agonist-evoked store depletion can evoke Ca²⁺-induced Ca²⁺ release. This indicates that Ca²⁺ entry may be overestimated by this widely used protocol. Received: 28 July 1997 / Received after revision: 25 November 1997 / Accepted: 26 November 1997     

Evidence for the existence of inositol (1,4,5)-trisphosphate- and ryanodine-sensitive pools in bovine endothelial cells. Ca2+ releases in cells with different basal level of intracellular Ca2+

In single bovine aortic endothelial (BAE) cells pre-loaded with Fura-2, Ca²⁺ transients in a Ca²⁺-free medium have been revealed, which evidently reflects Ca²⁺ release from intracellular stores. In cells with different levels of resting basal cytoplasmic Ca²⁺ ([Ca²⁺]_i) from about 50 to 110 nM, a biphasic dependence of the Ca²⁺ transients on resting [Ca²⁺]_i was shown and spontaneous Ca²⁺ oscillations were observed. At a [Ca²⁺]_i level over 110 nM, a pronounced rise in Ca²⁺ transients occurred and only single transients were observed. Ryanodine (10 μM) produced a transient [Ca²⁺]_i elevation, suggesting the presence of ryanodine receptors in intracellular store membranes. The results imply that both inositol 1,4,5-trisphosphate-sensitive Ca²⁺ release (IICR) and Ca²⁺-sensitive Ca²⁺ release (CICR) take place in BAE cells. Only IICR seems to be sufficient for generating baseline Ca²⁺ oscillations in BAE cells, whereas the ATP-induced (5–100 μM) Ca²⁺ response involves the CICR set in motion by an oscillatory IICR of high frequency. The completion of both the spontaneous and ATP-induced Ca²⁺ transients was associated with a [Ca²⁺]_i decrease to a level below the initial resting [Ca²⁺]_i (undershoot). Its depth biphasically depended on the resting [Ca²⁺]_i from 50 to 110 nM, suggesting that the lack of a Ca²⁺ leak from inositol 1,4,5-trisphosphate-sensitive stores is responsible for the undershoot in this range. The Ca²⁺ leak is concluded to play a key role in the initiation and termination of regenerative IICR both in spontaneous oscillations and in ATP-induced transients. Received: 13 November 1995/Received after revision and accepted 27 March 1996     

Presence of two Ca2+ influx components in internal Ca2+-pool-depleted rat parotid acinar cells

The molecular mechanism(s) involved in mediating Ca²⁺ entry into rat parotid acinar and other non-excitable cells is not known. In this study we have examined the kinetics of Ca²⁺ entry in fura-2-loaded parotid acinar cells, which were treated with thapsigargin to deplete internal Ca²⁺ pools (Ca²⁺-pool-depleted cells). The rate of Ca²⁺ entry was determined by measuring the initial increase in free cytosolic [Ca²⁺] ([Ca²⁺]_i) in Ca²⁺-pool-depleted, and control (untreated), cells upon addition of various [Ca²⁺] to the medium. In untreated cells, a low-affinity component was detected with K _Ca = 3.4 ± 0.7 mM (where K _Ca denotes affinity for Ca²⁺) and V _max = 9.8 ± 0.4 nM [Ca²⁺]_i/s. In thapsigargin-treated cells, two Ca²⁺ influx components were detected with K _Ca values of 152 ±  79 μM (V _max = 5.1 ± 1.9 nM [Ca²⁺]_i/s) and 2.4 ±  0.9 mM (V _max = 37.6 ± 13.6 nM [Ca²⁺]_i/s), respectively. We have also examined the effect of Ca²⁺ and depolarization on these two putative Ca²⁺ influx components. When cells were treated with thapsigargin in a Ca²⁺-free medium, Ca²⁺ influx was higher than into cells treated in a Ca²⁺-containing medium and, while there was a 46% increase in the V _max of the low-affinity component (no change in K _Ca), the high-affinity component was not clearly detected. In depolarized Ca²⁺-pool-depleted cells (with 50 mM KCl in the medium) the high-affinity component was considerably decreased while there was an apparent increase in the K _Ca of the low-affinity component, without any change in the V _max. These results demonstrate that Ca²⁺ influx into parotid acinar cells (1) is increased (four- to five-fold) upon internal Ca²⁺ pool depletion, and (2) is mediated via at least two components, with low and high affinities for Ca²⁺. Received: 30 October 1995/Received after revisionand accepted: 13 December 1995     

Effect of alkalinization of cytosolic pH by amines on intracellular Ca2+ activity in HT29 cells

The effect of secondary, tertiary and quaternary methyl- and ethylamines on intracellular pH (pH_i) and intracellular Ca²⁺ activity ([Ca²⁺]_i) of HT₂₉ cells was investigated microspectrofluorimetrically using pH- and Ca²⁺- sensitive fluorescent indicators, [i.e. 2′,7′-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) and fura-2 respectively]. Membrane voltage (V _m) was studied by the patch-clamp technique. Secondary and tertiary amines led to a rapid and stable concentration-dependent alkalinization which was independent of their pK _a value. Trimethylamine (20 mmol/l) increased pH_i by 0.78 ± 0.03 pH units (n = 9) and pH remained stable for the application time. Removal led to an undershoot of pH_i and a slow and incomplete recovery: pH_i stayed 0.26 ± 0.06 pH units more acid than the resting value. The quaternary amines, tetramethyl- and tetraethylamine were without influence on pH_i. All tested secondary and tertiary amines (dimethyl-, diethyl-, trimethyl-, and triethyl-amine) induced a [Ca²⁺]_i transient which reached a peak value within 10–25 s and then slowly declined to a [Ca²⁺]_i plateau. The initial Δ[Ca²⁺]_i induced by trimethylamine (20 mmol/l) was 160 ± 15 nmol/l (n = 17). The [Ca²⁺]_i peak was independent of the Ca²⁺ activity in the bath solution, but the [Ca²⁺]_i plateau was significantly lower under Ca²⁺-free conditions and could be immediately interrupted by application of CO₂ (10%; n = 6), a manoeuvre to acidify pH_i in HT₂₉ cells. Emptying of the carbachol- or neurotensin-sensitive intracellular Ca²⁺ stores completely abolished this [Ca²⁺]_i transient. Tetramethylamine led to higher [Ca²⁺]_i changes than the other amines tested and only this transient could be completely blocked by atropine (10⁻⁶ mol/l). Trimethylamine (20 mmol/l) hyperpolarized V _m by 22.5 ± 3.7 mV (n = 16) and increased the whole-cell conductance by 2.3 ± 0.5 nS (n = 16). We conclude that secondary and tertiary amines induce stable alkaline pH_i changes, release Ca²⁺ from intracellular, inositol-1,4,5-trisphosphate-sensitive Ca²⁺ stores and increase Ca²⁺ influx into HT₂₉ cells. The latter may be related to both the store depletion and the hyperpolarization. Received: 11 September 1995/Received after revision and accepted: 18 December 1995     

The effect of intracellular pH on cytosolic Ca2+ in HT29 cells

 The influence of intracellular pH (pH_i) on intracellular Ca²⁺ activity ([Ca²⁺]_i) in HT₂₉ cells was examined microspectrofluorometrically. pH_i was changed by replacing phosphate buffer by the diffusible buffers CO₂/HCO₃ ^–or NH₃/NH₄ ⁺ (pH 7.4). CO₂/HCO₃ ^–buffers at 2,5 or 10% acidified pH_i by 0.1, 0.32 and 0.38 pH units, respectively, and increased [Ca²⁺]_i by 8–15 nmol/l. This effect was independent of the extracellular Ca²⁺ activity and the filling state of thapsigargin-sensitive Ca²⁺ stores. Removing the CO₂/HCO₃ ^–buffer alkalinized pH_i by 0.14 (2%), 0.27 (5%), and 0.38 (10%) units and enhanced [Ca²⁺]_i to a peak value of 20, 65, and 143 nmol/l, respectively. Experiments carried out with Ca²⁺-free solution and with thapsigargin showed that the [Ca²⁺]_i transient was due to release from intracellular pools and stimulated Ca²⁺ entry. NH₃/NH₄ ⁺ (20 mmol/l) induced a transient intracellular alkalinization by 0.6 pHunits and increased [Ca²⁺]_i to a peak (Δ [Ca²⁺]_i = 164 nmol/l). The peak [Ca²⁺]_i increase was not influenced by removal of external Ca²⁺, but the decline to basal [Ca²⁺]_i was faster. Neither the phospholipase C inhibitor U73122 nor the inositol 1,4,5-trisphosphate (InsP ₃) antagonist theophylline had any influence on the NH₃/NH₄ ⁺-stimulated [Ca²⁺]_i increase, whereas carbachol-induced [Ca²⁺]_i transients were reduced by more than 80% and 30%, respectively. InsP ₃ measurements showed no change of InsP ₃ during exposure to NH₃/NH₄ ⁺, whereas carbachol enhanced the InsP ₃ concentration, and this effect was abolished by U73122. The pH_i influence on ”capacitative” Ca²⁺ influx was also examined. An acid pH_i attenuated, and an alkaline pH_i enhanced, carbachol- and thapsigargin-induced [Ca²⁺]_i influx. We conclude that: (1) an alkaline pH_i releases Ca²⁺ from InsP ₃-dependent intracellular stores; (2) the store release is InsP ₃ independent and occurs via an as yet unknown mechanism; (3) the store release stimulates capacitative Ca²⁺ influx; (4) the capacitative Ca²⁺ influx activated by InsP ₃ agonists is decreased by acidic and enhanced by alkaline pH_i. The effects of pH_i on [Ca²⁺]_i should be of relevance under many physiological conditions. Received: 17 June 1996 / Received after revision and accepted: 30 August 1996     

Ca2+-transients induced by K+ channel openers in isolated coronary capillaries

 To study the role of endothelial ATP-sensitive K⁺ channels in the regulation of vascular tone we examined the intracellular calcium concentration ([Ca²⁺]_i) in coronary capillaries consisting only of endothelial cells. Coronary capillary fragments were isolated enzymatically from the guinea-pig heart and [Ca²⁺]_i was determined by microfluorometry of fura-2 loaded cells. Low concentrations of the K⁺ channel opener diazoxide, which caused pronounced glibenclamide-sensitive hyperpolarization in capillaries, induced a rapid, transient rise in [Ca²⁺]_i followed by a sustained elevation of [Ca²⁺]_i (19 of 40 experiments). [Ca²⁺]_i in the endothelial cells increased from 32 ± 7 nM at rest to 66 ± 11 nM at the peak (n = 19). One third of the [Ca²⁺]_i-transients showed irregular oscillations of [Ca²⁺]_i. No significant difference in the [Ca²⁺]_i-response induced by 100 nM or 1 μM diazoxide was found. Similar results were obtained with the K⁺ channel opener rilmakalim. Simultaneous measurements of the membrane potential and [Ca²⁺]_i with fluorometric methods indicated that the hyperpolarization but not the [Ca²⁺]_i-transient could be repeatedly induced in a single capillary by the K⁺ channel openers. Electrophysiological recordings of the membrane potential using the ”perforated patch” method (n = 4), showed that rilmakalim (1 μM) induced hyperpolarization of capillaries towards the K⁺ equilibrium potential, confirming our fluorometric measurements. In conclusion, for the first time, these data indicate that K⁺ channel openers induce [Ca²⁺]_i-transients in microvascular endothelial cells. This raises the possibility that these drugs not only act as synthetic vasoactive factors via hyperpolarizing smooth muscle cells but also via NO release of microvascular endothelial cells. Interestingly, only 100 nM diazoxide was sufficient for a maximal response, suggesting the expression of a new type of K_ATP-channel in coronary capillaries characterised by high sensitivity to diazoxide. Received: 22 August 1997 / Received after revision and accepted: 7 November 1997     

Activity of protein kinase C is necessary for sustained thrombin-induced [Ca2+]i oscillations in rat glioma cells

 The aim of the present study was to examine the possible role of protein kinase C (PKC) in thrombin-induced Ca²⁺ signalling. As shown before, continuous superfusion of rat glioma cells with thrombin caused sustained [Ca²⁺]_i oscillations through activation of cell surface receptors [Czubayko U, Reiser G (1995) Neuroreport 6: 1249]. These oscillations were inhibited by protease nexin-1. Addition of PKC inhibitors, i. e. staurosporine (0.2–20 μM), bisindolylmaleimide (1 μM) or chelerythrine (1 μM), irreversibly suppressed thrombin-induced [Ca²⁺]_i oscillations. Thereafter application of 2,5-di(tert-butyl)-1,4-benzohydroquinone (t-BuBHQ, 20 μM) or thapsigargin (1 μM) (inhibitors of sarco/endoplasmic reticulum Ca²⁺-ATPase) caused no [Ca²⁺]_i response, indicating that intracellular Ca²⁺ stores were completely empty. We tested whether PKC affects the refilling of internal Ca²⁺ stores in thrombin-stimulated cells, by monitoring the amount of Ca²⁺ release caused by t-BuBHQ in the presence or absence of PKC inhibitors or activators. The amount of Ca²⁺ released by t-BuBHQ, which was normalized by comparison with the thrombin-induced Ca²⁺ response, was decreased by simultaneous incubation with staurosporine or chelerythrine, but enhanced with the PKC activator oleoyl acetyl glycerol. Furthermore, the capacitative Ca²⁺ entry was reduced by inhibition or downregulation, and increased by activation, of PKC. Capacitative Ca²⁺ entry was induced in these experiments by depletion of Ca²⁺ stores by the addition of thapsigargin or t-BuBHQ. In contrast, the inhibition of PKC during thrombin-induced depletion of intracellular stores did not influence the Ca²⁺ entry but nearly completely abolished the refilling of the internal stores. Thus we conclude that during thrombin receptor stimulation activation of PKC is required to maintain the refilling of intracellular Ca²⁺ stores for sustained [Ca²⁺]_i oscillations. Thus, the control by PKC of the capacitative Ca²⁺ entry is apparently different depending on whether it is induced by sarco/endoplasmic reticulum Ca²⁺-ATPase inhibition or by activation of the thrombin receptor. Received: 5 July 1996 / Received after revision and accepted: 7 October 1996     

Relationship between force and Ca2+ in anococcygeal and vas deferens smooth muscle cells of the mouse

We compared the changes of the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i), as measured with the fluorescent Ca²⁺ indicator fura-2, and the force development in intact smooth muscle of the tonic anococcygeus (AC) and the phasic vas deferens (VD) of the mouse, during activation by K²⁺ depolarization and by agonists. Resting [Ca²⁺]_i was observed to be 33% lower in AC (80 nM) than in VD (115 nM), while the Ca²⁺ threshold for contraction was found to be about 120 nM in AC and 160 nM in VD. For a similar [Ca²⁺]_i increase, the agonist stimulation induced a higher force development than the K⁺ depolarization in both muscle types. During prolonged depolarization, the force/calcium ratio increased in AC but strongly declined in VD. This decline of the force/calcium ratio in VD during depolarization was partially reversed by lowering [Ca²⁺]_o. Our results indicate that the Ca²⁺ threshold for force development was about 150% of the resting [Ca²⁺]_i in both cell types. The resting [Ca²⁺]_i was lower in the tonic AC than in the phasic VD. Agonist-induced sensitization to Ca²⁺ occurred in both muscle types. The tonic and phasic smooth muscles essentially differed in the respective modulation of their Ca²⁺ sensitivity during contraction. The desensitization to Ca²⁺ was specific for phasic muscle, in which it occurred as an early, timeand Ca²⁺-dependent process that was partially reversible.     

Effect of chloride on Ca2+ release from the sarcoplasmic reticulum of mechanically skinned skeletal muscle fibres

 The effect of intracellular Cl^– on Ca²⁺ release in mechanically skinned fibres of rat extensor digitorum longus (EDL) and toad iliofibularis muscles was examined under physiological conditions of myoplasmic [Mg²⁺] and [ATP] and sarcoplasmic reticulum (SR) Ca²⁺ loading. Both in rat and toad fibres, the presence of 20 mM Cl^–in the myoplasm increased Ca²⁺ leakage from the SR at pCa (i.e. –log₁₀ [Ca²⁺]) 6.7, but not at pCa 8. Ca²⁺ uptake was not significantly affected by the presence of Cl^–. This Ca²⁺-dependent effect of Cl^– on Ca²⁺ leakage was most likely due to a direct action on the ryanodine receptor/Ca²⁺ release channel, and could influence channel sensitivity and the resting [Ca²⁺] in muscle fibres in vivo. In contrast to this effect, acute addition of 20 mM Cl^– to the myoplasm caused a 40–50% reduction in Ca²⁺ release in response to a low caffeine concentration both in toad and rat fibres. One possible explanation for this latter effect is that the addition of Cl^– induces a potential across the SR (lumen negative) which might reduce Ca²⁺ release via several different mechanisms. Received: 20 October 1997 / Received after revision: 1 December 1997 / Accepted: 2 December 1997     

The thapsigargin-evoked increase in [Ca2+]i involves an InsP3-dependent Ca2+ release process in pancreatic acinar cells

In pancreatic acinar cells, as in many other cell types, the tumour promoter thapsigargin (TG) evokes a significant increase of intracellular free Ca²⁺ ([Ca²⁺]_i). The increases of [Ca²⁺]_i evoked by TG was associated with significant changes of plasma membrane Ca²⁺ permeability, with [Ca²⁺]_i values following changes in extracellular [Ca²⁺]. Plasma membrane Ca²⁺ extrusion is activated rapidly as a consequence of the rise in [Ca²⁺]_i evoked by TG and the rate of extrusion is linearly dependent on [Ca²⁺]_i up to 1 μM Ca²⁺. In contrast, the activation of the Ca²⁺ entry pathway is delayed and the apparent rate of Ca²⁺ entry is independent of [Ca²⁺]_i. In the presence of 20 mM caffeine, which reduces the resting levels of inositol trisphosphate (InsP₃), the increase of [Ca²⁺]_i evoked by TG was significantly reduced. The reduction was manifest both as a decrease of the amplitude of the [Ca²⁺]_i peak (30% reduction) and, more importantly, as a reduction of the apparent maximal rate of [Ca²⁺]_i increase (from 12.3±1.0 to 6.1±0.6 nM Ca²⁺/s). The inhibition evoked by caffeine was reversible and the removal of caffeine in the continuous presence of TG evoked a further increase of [Ca²⁺]_i. The amplitude of the [Ca²⁺]_i increase upon caffeine removal was reduced as a function of the time of TG exposure. Addition of TG in the presence of 1 mM La³⁺, which is known to inhibit the plasma membrane Ca²⁺-activated adenosine triphosphatase, induced a much higher peak of [Ca²⁺]_i. This increase was associated with an augmentation of the apparent rate of [Ca²⁺]_i increase (from 12.3±1.2 to 16.1±1.9 nM Ca²⁺/s). The inhibitory effect of caffeine, as well as the increase in [Ca²⁺]_i observed on caffeine removal was not affected by the presence of 1 mM La³⁺. These data indicate that an important component of the TG-evoked [Ca²⁺]_i increase is due to InsP₃-sensitive Ca²⁺ release which is probably mediated by the resting levels of InsP₃.     

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.     

Mechanisms of reduced mitochondrial Ca2+ accumulation in failing hamster heart

Mitochondrial Ca²⁺ plays important roles in the regulation of energy metabolism and cellular Ca²⁺ homeostasis. In this study, we characterized mitochondrial Ca²⁺ accumulation in Syrian hamster hearts with hereditary cardiomyopathy (strain BIO 14.6). Exposure of isolated mitochondria from 70 nM to 30 μM Ca²⁺ ([Ca²⁺]_o) caused a concentration-dependent increase in intramitochondrial Ca²⁺ concentrations ([Ca²⁺]_m). The [Ca²⁺]_m was significantly lower in cardiomyopathic (CMP) hamsters than in healthy hamsters when [Ca²⁺]_o was higher than 1 μM and a decrease of about 52% was detected at [Ca²⁺]_o of 30 μM (916 ± 67 nM vs 1,932 ± 132 nM in control). A possible mechanism responsible for the decreased mitochondrial Ca²⁺ uptake in CMP hamsters is the depolarization of mitochondrial membrane potential (Δψ _m). Using a tetraphenylphosphonium (TPP⁺) electrode, the measured Δψ _m in failing heart mitochondria was −136 ± 1.5 mV compared with −159 ± 1.3 mV in controls. Analyses of mitochondrial respiratory chain demonstrated a significant impairment of complex I and complex IV activities in failing heart mitochondria. In summary, a less negative Δψ _m resulting from defects in the respiratory chain may lead to attenuated mitochondrial Ca²⁺ accumulation, which in turn may contribute to the depressed energy production and myocardial contractility in this model of heart failure. In addition to other known impairments of ion transport in sarcoplasmic reticulum and plasma membrane, results from this paper on mitochondrial dysfunctions expand our understanding of the molecular mechanisms leading to heart failure.     

ATP-induced Ca2+ signals in bronchial epithelial cells

 Ca²⁺-dependent Cl^– secretion in the respiratory tract occurs physiologically or under pathophysiological conditions when inflammatory mediators are released. The mechanism of intracellular Ca²⁺ release was investigated in the immortalized bronchial epithelial cell line 16HBE14o-. Experiments on both intact and permeabilized cells revealed that only inositol 1,4,5-trisphosphate (InsP ₃) receptors and not ryanodine receptors are involved in intracellular Ca²⁺ release. The expression pattern of the three InsP ₃ receptor isoforms was assessed both at the mRNA and at the protein level. The level of expression at the mRNA level was type 3 (92.5%) >> type 2 (5.4%) > type 1 (2.1%) and this rank order was also observed at the protein level. The ATP-induced Ca²⁺ signals in the intact cell, consisting of abortive Ca²⁺ spikes or fully developed [Ca²⁺] rises and intracellular Ca²⁺ waves, were indicative of positive feedback of Ca²⁺ on the InsP ₃ receptors. Low Ca²⁺ concentrations stimulated and high Ca²⁺ concentrations inhibited InsP ₃-induced Ca²⁺ release in permeabilized 16HBE14o- cells. We localized a cytosolic Ca²⁺-binding site between amino acid residues 2077 and 2101 in the type-2 InsP ₃ receptor and between amino acids 2030 and 2050 in the type-3 InsP ₃ receptor by expressing the respective parts of these receptors as glutathione S-transferase fusion proteins in bacteria. We conclude that the InsP ₃ receptor isoforms expressed in 16HBE14o- cells (mainly type-3 and type-2) are stimulated by Ca²⁺ and that this phenomenon contributes to the ATP-induced Ca²⁺ signals in intact 16HBE14o- cells. Recieved: 11 September 1997 / Received after revision: 2 January 1998 / Accepted: 21 January 1998     

Effect of acidosis on Ca2+-activated K+ channels in cultured porcine coronary artery smooth muscle cells

 Although acidosis induces vasodilation, the vascular responses mediated by large-conductance Ca²⁺-activated K⁺ (K_Ca) channels have not been investigated in coronary artery smooth muscle cells. We therefore investigated the response of porcine coronary arteries and smooth muscle cells to acidosis, as well as the role of K_Ca channels in the regulation of muscular tone. Acidosis (pH 7.3–6.8), produced by adding HCl to the extravascular solution, elicited concentration-dependent relaxation of precontracted, endothelium-denuded arterial rings. Glibenclamide (20 μM) significantly inhibited the vasodilatory response to acidosis (pH 7.3-6.8). Charybdotoxin (100 nM) was effective only at pH 6.9–6.8. When we exposed porcine coronary artery smooth muscle cells to a low-pH solution, K_Ca channel activity in cell-attached patches increased. However, pretreatment of these cells with 10 or 30 μM O, O′-bis(2-aminophenyl)ethyleneglycol-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl)ester (BAPTA-AM), a Ca²⁺ chelator for which the cell membrane is permeable, abolished the H⁺-mediated activation of K_Ca channels in cell-attached patches. Under these circumstances H⁺ actually inhibited K_Ca channel activity. When inside-out patches were exposed to a [Ca²⁺] of 10^–6 M [adjusted with ethyleneglycolbis(β-aminoethylester)-N,N,N′,N′-tetraacetic acid (EGTA) at pH 7.3], K_Ca channels were activated by H⁺ concentration dependently. However, when these patches were exposed to a [Ca²⁺] of 10^–6 M adjusted with BAPTA at pH 7.3, H⁺ inhibited K_Ca channel activity. Extracellular acidosis had no significant direct effect on K_Ca channels, suggesting that extracellular H⁺ exerts its effects after transport into the cell, and that K_Ca channels are regulated by intracellular H⁺ and by cytosolic free Ca²⁺ modulated by acute acidosis. These results indicate that the modulation of K_Ca channel kinetics by acidosis plays an important role in the determination of membrane potential and, hence, coronary arterial tone. Received: 20 January 1998 / Received after revision: 9 April 1998 / Accepted: 22 April 1998     

Mechanisms responsible for quantal Ca2+ release from inositol trisphosphate-sensitive calcium stores

Activation of cells by hormones, growth factors or neurotransmitters leads to an increased production of inositol trisphosphate (InsP₃) and, after activation of the InsP₃ receptor (InsP₃R), to Ca²⁺ release from intracellular Ca²⁺ stores. The release of intracellular Ca²⁺ is characterised by a graded response when submaximal doses of agonists are used. The basic phenomenon, called “quantal Ca²⁺ release”, is that even the maintained presence of a submaximal dose of agonist or of InsP₃ for long time periods (up to 20 min) provokes only a partial release of Ca²⁺. This partial, or quantal, release phenomenon is due to the fact that the initially very rapid InsP₃-induced Ca²⁺ release eventually develops into a much slower release phase. Physiologically, quantal release allows the Ca²⁺ stores to function as increment detectors and to induce local Ca²⁺ responses. The basic mechanism for quantal release of Ca²⁺ is presently not known. Possible mechanisms to explain the quantal behaviour of InsP₃- induced Ca²⁺ release include the presence of InsP₃Rs with varying sensitivities for InsP₃, heterogeneous InsP₃R distribution, intrinsic inactivation of the InsP₃Rs, and regulation of the InsP₃Rs by Ca²⁺ store content. This article reviews critically the evidence for the various mechanisms and evaluates their functional importance. A Ca²⁺-mediated conformational change of the InsP₃R is most likely the key feature of the mechanism for quantal Ca²⁺ release, but the exact mode of operation remains unclear. It should also be pointed out that in intact cells more than one mechanism can be involved.     

Endothelin-B receptor-mediated Ca2+ signaling in human melanocytes

 The mechanism of an endothelin-1- (ET-1-) induced intracellular Ca²⁺ ([Ca²⁺]_i) increase and the receptor subtype(s) responsible for this effect in single human melanocytes were studied using fura-2/AM. ET-1 induced a transient increase in [Ca²⁺]_i in a concentration-dependent manner. The transient [Ca²⁺]_i increase was followed by a sustained plateau level of [Ca²⁺]_i which was higher than the initial [Ca²⁺]_i level. IRL-1620, a specific ET-B receptor agonist, increased [Ca²⁺]_i in a dose-dependent manner. BQ-788, a specific ET-B receptor antagonist, abolished the ET-1-induced [Ca²⁺]_i increase, but BQ-123, a specific ET-A receptor antagonist, failed to prevent it. U73122, an inhibitor of phospholipase C (PLC), inhibited the ET-1-induced [Ca²⁺]_i rise in a dose-dependent manner. Prior depletion of intracellular Ca²⁺ stores with thapsigargin, an inhibitor of Ca²⁺-ATPase of the endoplasmic reticulum, abolished the ET-1-induced Ca²⁺ transient, whereas removal of extracellular Ca²⁺ with EGTA eliminated the sustained rise. These results suggest that in cultured human melanocytes the binding of ET-1 to ET-B receptors and the subsequent activation of PLC mediate ET-1-induced [Ca²⁺]_i increase. The transient [Ca²⁺]_i increase is attributed to mobilization of Ca²⁺ from inositol 1,4,5-trisphosphate-sensitive intracellular Ca²⁺ stores, and the sustained [Ca²⁺]_i level may be related to the influx of extracellular Ca²⁺. Received: 21 July 1997 / Received after revision and accepted: 16 September 1997     

Toxin of the marine alga Prymnesium patelliferum enhances voltage dependent Ca2+-currents,elevates the cytosolic Ca2+-concentration and facilitates hormone release in clonal rat pituitary cells

The marine flagellate Prymnesium patelliferum produces toxins lethal to fish. The toxin extracted from the alga has haemolytic, cytotoxic and neurotoxic effects, but the action mechanisms of the toxin are not known in detail. We have examined the toxin effects on the voltage sensitive Ca²⁺-currents, the cytosolic Ca²⁺-level ([Ca²⁺]₁) and the prolactin release in clonal rat anterior pituitary GH₄C₁ cells, which possess T- and L-type Ca²⁺-channels. The trans-membrane Ca²⁺-current was recorded using whole-cell voltage clamp. After 5–15 min exposure to the algal toxin at a final concentration of 50000–100000 cells mL^-1, the Ca²⁺-currents through both the T- and L-channels showed a 2–3-fold enhancement. The voltage sensitivity of the Ca²⁺-currents was not affected by the algal toxin, and the toxin-induced currents were inhibited by 100 μM of the Ca²⁺-channel blocker D-600. In toxin-exposed cells microfluorometric measurements based on fura-2 revealed an increase of [Ca²⁺]₁ from 100–150 to 300–500 nM. This elevation was delayed and partially inhibited by 100 μM D-600. The algal toxin induced prolactin release in a dose-dependent manner, and this effect was inhibited by the Ca²⁺-channel blocker verapamil. We therefore conclude that the toxin of P. patelliferum affects the Ca²⁺ homeostasis of the pituitary cells by increasing the leak through voltage sensitive Ca²⁺-channels, resulting in increased [Ca²⁺]₁ and secretion of prolactin.     

Cyclic AMP potentiates glucose-induced insulin release from mouse pancreatic islets without increasing cytosolic free Ca2

RORSMAN P. & ABRAHAMSSON, H. 1985. Cyclic AMP potentiates glucose-induced insulin release from mouse pancreatic islets without increasing cytosolic free Ca²⁺. Acta Physiol Scand 125 , 639–647. Received 18 March 1985, accepted 28 May 1985. ISSN 0001–6772. Department of Medical Cell Biology, Biomedicum, University of Uppsala, Sweden. The effects of various stimulants of insulin release on cytosolic free Ca²⁺, [Ca²⁺]i, in dispersed and cultured pancreatic β-cells from ob/ob-mice were studied using the indicator quin-2, which in itself has only slight effects on the glucose-induced insulin release and the metabolism of the sugar. The resting [Ca²⁺]_i was 158 ± 7 nM. After increasing glucose to 20 mM there was a lag-period of 1–2 min before [Ca²⁺]_i gradually rose, reaching a new plateau 60% higher after 5–6 min. Increasing intracellular cyclic AMP by adding forskolin did not further increase [Ca²⁺]_i; on the contrary there was a slight temporary reduction despite a doubling of insulin secretion. The maintenance of the β-cell function was evident from a marked increase of cytosolic [Ca²⁺]_i after depolarization evoked by high extracellular K⁺. Also dibutyryl cyclic AMP and theophylline lacked the ability to raise [Ca²⁺]_i beyond that obtained by glucose. The results suggest that cyclic AMP potentiates glucose-induced insulin release by sensitizing the secretory machinery to changes of [Ca²⁺]_i rather than by increasing the cytosolic concentration of the ion.     

Induction of Ca2+ oscillations by selective,U73122-mediated,depletion of inositol-trisphosphate-sensitive Ca2+ stores in rabbit pancreatic acinar cells

The effect of the putative inhibitor of phospholipase C activity, U73122, on the Ca²⁺ sequestering and releasing properties of internal Ca²⁺ stores was studied in both permeabilized and intact rabbit pancreatic acinar cells. U73122 dose dependently inhibited ATP-dependent Ca²⁺ uptake in the inositol (1,4,5)-trisphosphate-[Ins(1,4,5)P ₃]-sensitive, but not the Ins(1,4,5)P ₃-insensitive, Ca²⁺ store in acinar cells permeabilized by saponin treatment. In a suspension of intact acinar cells, loaded with the fluorescent Ca²⁺ indicator, Fura-2, U73122 alone evoked a transient increase in average free cytosolic Ca²⁺ concentration ([Ca²⁺]_i,av), which was largely independent of external Ca²⁺. Addition of U73122 to cell suspensions prestimulated with either cholecystokinin octapeptide or JMV-180 revealed an inverse relationship in size between the U73122- and the agonistevoked [Ca²⁺]_i,av transient. Moreover, thapsigargin-induced inhibition of intracellular Ca²⁺-ATPase activity resulted in a [Ca²⁺]_i,av transient, the size of which was not different following maximal prestimulation with either U73122 or agonist. These observations suggest that U73122 selectively affects the Ins(1,4,5)P ₃- casu quo agonist-sensitive internal Ca²⁺ store, whereas thapsigargin affects both the Ins(1,4,5)P ₃-sensitive and -insensitive Ca²⁺ store. Digital-imaging microscopy of Fura-2-loaded acinar cells demonstrated that U73122, in contrast to thapsigargin, evoked sustained oscillatory changes in [Ca²⁺]_i. The U73122-evoked oscillations were abolished in the absence of external Ca²⁺. The ability of U73122 to generate external Ca²⁺-dependent Ca²⁺ oscillations suggests that depletion of the agonistsensitive store leads to an increase in Ca²⁺ permeability of the plasma membrane and that the Ins(1,4,5)P ₃-insensitive Ca²⁺ pool is necessary for the Ca²⁺ oscillations.     

Ca2+ entry through cardiac L-type Ca2+ channels modulates β-adrenergic stimulation in mouse ventricular myocytes

 β-adrenergic receptor (β-AR) stimulation increases cardiac L-type Ca²⁺ channel (CaCh) currents via cAMP-dependent phosphorylation. We report here that the affinity and maximum response of CaCh to isoproterenol (Iso), in mouse ventricular myocytes were significantly higher when Ba²⁺ was used as the charge carrier (I _Ba) instead of Ca²⁺ (I _Ca). The EC₅₀ and maximum increase of peak currents were 43.7 ± 7.9 nM and 1.8 ± 0.1-fold for I _Ca and 23.3 ± 4.7 nM and 2.4 ± 0.1-fold for I _Ba. When cells were dialyzed with the faster Ca²⁺ chelator, BAPTA, both sensitivity and maximum response of I _Ca to Iso were significantly augmented compared to cells with EGTA (EC₅₀ of 23.1 ± 5.2 nM and maximal increase of 2.2 ± 0.1-fold). Response of I _Ca to forskolin was also significantly increased when cells were dialyzed with BAPTA or when currents were measured in Ba²⁺. In contrast, depletion of the sarcoplasmic reticulum (SR) Ca²⁺ stores by ryanodine did not alter sensitivity of I _Ca to Iso or forskolin. These results suggest that the Ca²⁺ entering through CaCh regulates cAMP-dependent phosphorylation, and such negative feedback may play a significant role in cellular Ca²⁺ homeostasis and contraction in cardiac cells during β-AR stimulation. Received: 10 December 1997 / Received after revision: 19 January 1998 / Accepted: 21 January 1998