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     

The pathway for refilling intracellular Ca2+ stores passes through the cytosol in human leukaemia cells

The pathway for refilling the intracellular Ca²⁺ stores of HL60 and U937 human leukaemia cells loaded with fura-2 has been investigated. On addition of external Ca²⁺ to cells with empty stores there was an increase in the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) which preceded the refilling of the stores. The increase in [Ca²⁺]_i was faster than the refilling, by 3-to 15-fold, depending on the cell type. In measurements in single HL60 cells we found that the refilling of the stores correlated with the extent of the [Ca²⁺]_i increase on addition of external Ca²⁺. The cells showing no [Ca²⁺]_i increase were unable to refill their stores. The addition of Ni²⁺ to the extracellular medium prevented both the [Ca²⁺]_i increase and the refilling of the stores. These results indicate that the limiting step for store refilling is the entry of Ca²⁺ from the extracellular medium to the cytosol. Hence, we conclude that extracellular Ca²⁺ cannot gain access directly to the intracellular Ca²⁺ stores in these cells, but must first enter the cytosol and be taken up from there into the stores.     

Capacitative Ca2+ entry contributes to the Ca2+ influx induced by thyrotropin-releasing hormone (TRH) in GH3 pituitary cells

Treatment of GH₃ cells with either hypothalamic peptide thyrotropin-releasing hormone (TRH), the endomembrane Ca²⁺-ATPase inhibitor thapsigargin or the Ca²⁺ ionophore ionomycin mobilized, with different kinetics, essentially all of the Ca²⁺ pool from the intracellular Ca²⁺ stores. Any of the above-described treatments induced a sustained increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i), which was dependent on extracellular Ca²⁺ and was prevented by Ni²⁺ but not by dihydropyridines (DHPs), suggesting that it was due to capacitative Ca²⁺ entry via activation of a plasma membrane pathway which opened upon the emptying of the intracellular Ca²⁺ stores. The increase of the plasma membrane permeability to Ca²⁺ correlated negatively with the filling degree of the intracellular Ca²⁺ stores and was reversed by refilling of the stores. The mechanism of capacitative Ca²⁺ entry into GH₃ cells differed from similar mechanisms described in several types of blood cells in that the pathway was poorly permeable to Mn²⁺ and not sensitive to cytochrome P₄₅₀ inhibitors. In GH₃ cells, TRH induced a transient [Ca²⁺]_i increase due to Ca²⁺ release from the stores (phase 1) followed by a sustained [Ca²⁺]_i increase due to Ca²⁺ entry (phase 2). At the single-cell level, phase 2 was composed of a DHP-insensitive sustained [Ca²⁺]_i increase, due to activation of capacitative Ca²⁺ entry, superimposed upon which DHP-sensitive [Ca²⁺]_i oscillations took place. The two components of the TRH-induced Ca²⁺ entry differed also in that [Ca²⁺]_i oscillations remained for several minutes after TRH removal, whereas the sustained [Ca²⁺]_i increase dropped quickly to prestimulatory levels, following the same time course as the refilling of the stores. The drop was prevented when the refilling was inhibited by thapsigargin. It is concluded that, even though the mechanisms of capacitative Ca²⁺ entry may show differences from cell to cell, it is also present and may contribute to the regulation of physiological functions in excitable cells such as GH₃. There, capacitative Ca²⁺ entry cooperates with voltage-gated Ca²⁺ channels to generate the [Ca²⁺]_i increase seen during phase 2 of TRH action. This contribution of capacitative Ca²⁺ entry may be relevant to the enhancement of prolactin secretion induced by TRH.     

[Ca2+]i elevation evoked by Ca2+ readdition to the medium after agonist-induced Ca2+ release can involve both IP 3-, and ryanodine-sensitive Ca2+ release

 Sustained Ca²⁺ elevation (”Ca²⁺ response”), caused by subsequent readdition of Ca²⁺ to the medium after application of adenosine 5’-triphosphate (ATP, 15 μM) in a Ca²⁺-free medium, was studied using single bovine aortic endothelial (BAE) cells. In cells in which the resting intracellular Ca²⁺ concentration ([Ca²⁺]_i) was between about 50 and 110 nM, a massive Ca²⁺ response occurred and consisted of phasic and sustained components, whereas cells with a resting [Ca²⁺]_i of over 110 nM displayed small plateau-like Ca²⁺ responses. An increase of internal store depletion resulted in loss of the phasic component. When the store was partly depleted, the dependence of the Ca²⁺ response amplitude on resting [Ca²⁺]_i was biphasic over the range of 50 to 110 nM. The greatest degree of store depletion was associated with small monophasic Ca²⁺ responses, the amplitudes of which were almost constant and in the same range as resting [Ca²⁺]_i. Ni²⁺, known to partly block Ca²⁺ entry, caused no change in the half-decay time of [Ca²⁺]_i down to the level of the sustained phase [57 ± 4 s in control and 54 ± 3 s (n = 13) in the presence of 10 mM Ni²⁺] when added at the peak of the phasic component of the Ca²⁺ response. However, it lowered the sustained phase of the Ca²⁺ response by 42%. When applied at the start of the readdition of Ca²⁺, Ni²⁺ blocked the phasic component of the Ca²⁺ response, there being a threefold decrease in the initial rate of [Ca²⁺]_i rise. In cells with a resting [Ca²⁺]_i of 75–80 nM, pre-treatment with ryanodine (10 μM) did not affect the peak amplitude of the Ca²⁺ response, but it did increase the level of the sustained component. In some cells, ryanodine caused an oscillatory Ca²⁺ response. In conclusion, partial depletion of the inositol 1,4,5-trisphosphate-(IP ₃-) sensitive store by a submaximal concentration of agonist (in Ca²⁺-free medium) was followed, on readdition of Ca²⁺, by Ca²⁺ entry, which appeared to trigger IP ₃-sensitive Ca²⁺ release (IICR) which, in turn, initiated Ca²⁺-sensitive Ca²⁺ release (CICR), thus resulting in a massive elevation of [Ca²⁺]_i. Received: 3 July 1996 / Received after revision and accepted: 9 September 1996     

Calcium channel subtypes in porcine adrenal chromaffin cells

 The effects of nifedipine, ω-conotoxin GVIA (ω-CgTx) and ω-agatoxin IVA (ω-AgTx) on Ca²⁺ currents, a 60-mM-K⁺-induced increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) and catecholamine secretion were examined to clarify the subtypes of Ca²⁺ channels in cultured adrenal chromaffin cells from the pig. Nifedipine, ω-CgTx, and ω-AgTx inhibited Ca²⁺ currents in a dose-dependent manner, suggesting the presence of L-, N- and P-type Ca²⁺ channels. The maximal doses of nifedipine (10 μM), ω-CgTx (1 μM), and ω-AgTx (0.1 μM) inhibited Ca²⁺ currents to 85%, 22%, and 94% of control currents, respectively. The inhibitory effects of these three blockers were observed in the same cell, indicating that at least three subtypes of Ca²⁺ channels are present in porcine chromaffin cells. The increase in [Ca²⁺]_i and catecholamine secretion induced by 60 mM K⁺ were inhibited equally by nifedipine (10 μM) and ω-CgTx (1 μM), but not by ω-AgTx (0.1 μM). These results suggest that L-, N- and P-type Ca²⁺ channels are present in porcine adrenal chromaffin cells, and that the major pathways of Ca²⁺ entry evoked by a high concentration of K⁺ are L- and N-type Ca²⁺ channels. Received: 6 September 1996 / Received after revision: 3 February 1997 / Accepted: 18 February 1997     

Effect of uncoupling NO/cGMP pathways on carbachol- and CCK-stimulated Ca2+ entry and amylase secretion from the rat pancreas

 Nitric oxide (NO) production reportedly regulates guanosine 3′,5′-cyclic monophosphate (cGMP) formation and Ca²⁺ influx in pancreatic acini. We have investigated the functional roles of the NO/cGMP messenger system in rat pancreatic acini. In dispersed acini, the levels of amylase secretion, cytosolic [Ca²⁺]([Ca²⁺]_i), NO synthase, and cGMP were measured. The NO synthase inhibitor N ^G-nitro-L-arginine methyl ester (L-NAME, 0.01–100 μM) had no effect on amylase secretion induced by various concentrations of carbachol, cholecystokinin octapeptide (CCK-8) or the high affinity CCK agonist, JMV-180. Similarly, L-NAME up to 100 μM did not affect the changes in Ca²⁺ spiking evoked by these secretagogues; nor was Ca²⁺ entry, refilling or oscillation altered by L-NAME. Sub- and supramaximal concentrations of these secretagogues did not change NO synthase activities compared with basal levels. While sodium nitroprusside (SNP), a NO donor, caused a 9.4-fold increase in cGMP levels compared with basal levels, carbachol, CCK-8 and JMV-180 had no effect. In addition, the guanylate cyclase inhibitor LY 83583 (10 nM to 10 μM) altered neither amylase secretion nor Ca²⁺ signaling induced by these secretagogues. These findings indicate that the stimulatory action of carbachol or CCK-8 is not mediated by NO or cGMP. To investigate whether cGMP stimulates pancreatic secretion we showed that both SNP and a cell-permeant cGMP analog at 0.1–1 mM stimulated amylase secretion and Ca²⁺ transients to a level equal to 10–15% and 13–24%, respectively, of those observed with maximal concentrations of secretagogues. The guanylate cyclase activator guanylin (1–10 μM), which increased cGMP levels 2.4-fold compared with basal levels, elicited a small amount of amylase secretion and a small Ca²⁺ transient. In conclusion, exogenous NO is capable of increasing endogenous cGMP, which results in a modest increase in the [Ca²⁺]_i transient and pancreatic amylase secretion. However, the NO/cGMP system does not appear to be involved significantly in the mediation of Ca²⁺ signaling and amylase secretion stimulated by carbachol and CCK-8. Received: 30 October 1996 / Received after revision and accepted: 13 January 1997     

Suppression of Ca2+ oscillations induced by cholecystokinin (CCK) and its analog OPE in rat pancreatic acinar cells by low-level protein kinase C activation without transition of the CCK receptor from a high- to low-affinity state

Cholecystokinin (CCK) analogs, JMV-180 and OPE, release Ca²⁺ from intracellular stores and induce oscillations in the concentration of cytosolic Ca²⁺ ([Ca²⁺]_i), but do not generate a detectable rise in inositol 1,4,5-trisphosphate (InsP ₃) levels. In contrast, high concentrations of CCK elevate InsP ₃, as well [Ca²⁺]_i, to a peak which decreases to near basal levels without oscillations. The mechanisms which underlie inhibition of [Ca²⁺]_i oscillations observed with high CCK concentrations are unclear, but are believed to involve a low-affinity CCK receptor state. Alternately, CCK analogs may be weak partial agonists of the phospholipase C pathway, whereas native CCK, as a full agonist of this pathway, stimulates low levels of protein kinase C (PKC) activity. Preincubation of acini with 1 nM 12 O-tetradecanoyl-phorbol 13-acetate (TPA) for 15 min at 37°C did not affect OPE binding to acini, but abolished OPE-induced (at 1 M) [Ca²⁺]_i oscillations without affecting the initial [Ca²⁺]_i spike. These transformed OPE-induced [Ca²⁺]_i responses mimicked those induced by supramaximal CCK octapeptide (CCK-8) concentrations. Inhibition of [Ca²⁺]_i oscillations by 1 nM TPA was reversed by the PKC inhibitor staurosporine (0.2 M). After [Ca²⁺]_i oscillations were induced with OPE or low concentrations of CCK-8 (20 pM), 1 nM TPA caused a gradual slowing of oscillation frequency over 15–20 min without affecting [Ca²⁺]_i spike amplitude. In contrast, 1 M TPA inhibited OPE binding and caused a more generalized inhibition of OPE- and CCK-evoked Ca²⁺ signals. These data suggest that inhibitory effects of low-level PKC activation on agonist-evoked Ca²⁺ signalling are distinct from the effects of high-level PKC activation by 1 M TPA, and do not require the transition of the CCK receptor from a high-affinity to a low-affinity state.     

Effects of thapsigargin and cyclopiazonic acid on sarcoplasmic reticulum Ca2+ uptake,spontaneous force oscillations and myofilament Ca2+ sensitivity in skinned rat ventricular trabeculae

Thapsigargin (TG) and cyclopiazonic acid (CPA) have been reported to be potent inhibitors of the sarcoplasmic reticulum (SR) Ca²⁺ uptake in isolated SR vesicles and cells. We have examined the effect of TG and CPA on (1) the Ca²⁺ uptake by the SR in saponin-skinned rat ventricular trabeculae, using the amplitude of the caffeine-induced contraction to estimate the Ca²⁺ content loaded into the SR, (2) the spontaneous Ca²⁺ oscillations at pCa 6.6 using force oscillation as the indicator, and (3) the myofilament Ca²⁺ sensitivity in Triton X-100-treated preparations. Inhibition of Ca²⁺ loading by TG and CPA increased with time of exposure to the inhibitor over 18–24 min. TG and CPA produced half inhibition of Ca²⁺ loading at 34.9 and 35.7 μM respectively, when 18–24 min were allowed for diffusion. The spontaneous force oscillations were more sensitive to the inhibitors: 10 μM TG and 30 μM CPA both abolished the oscillations in this time. The myofilament Ca²⁺ sensitivity was not affected by 10 and 300 μM TG or CPA. The results show that the concentrations of TG and CPA necessary to inhibit the SR Ca²⁺ uptake of skinned ventricular trabeculae are much higher than the reported values for single intact myocytes. One reason for this may be slow diffusion of the inhibitors into the multicellular trabecula preparation. Received: 28 July 1995/Received after revision: 11 December 1995/Accepted: 18 December 1995     

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     

Q-type Ca2+ channels are located closer to secretory sites than L-type channels: functional evidence in chromaffin cells

 This study uses a new strategy to investigate the hypothesis that, of the various Ca²⁺ channels expressed by a neurosecretory cell, a given channel subtype is coupled more tightly to the exocytotic apparatus than others. The approach is based on the prediction that the degree of inhibition of the secretory response by various Ca²⁺ channel blockers will differ at low (0.5 mM) and high (5 mM) extracellular Ca²⁺ concentrations ([Ca²⁺]_o). So, at low [Ca²⁺]_o the K⁺-evoked catecholamine release from superfused bovine chromaffin cells was depressed 60–70% by 2 μM ω-agatoxin IVA (P/Q-type Ca²⁺ channel blockade), by 3 μM ω-conotoxin MVIIC (N/P/Q-type Ca²⁺ channel blockade), or by 3 μM lubeluzole (N/P/Q-type Ca²⁺ channel blockade); in high [Ca²⁺]_o these blockers inhibited the responses by only 20–35%. At 1–3 μM ω-conotoxin GVIA (N-type Ca²⁺ channel blockade) or 3 μM furnidipine (L-type Ca²⁺ channel blockade), secretion was inhibited by 30 and 50%, respectively; such inhibitory effects were similar in low or high [Ca²⁺]o. Combined furnidipine plus ω-conotoxin MVIIC, ω-agatoxin IVA or ω-conotoxin GVIA exhibited additive blocking effects at both Ca²⁺ concentrations. The results suggest that Q-type Ca²⁺ channels are coupled more tightly to exocytotic active sites, as compared to L-type channels. This hypothesis if founded in the fact that external Ca²⁺ that enters the cell through a Ca²⁺ channel located near to chromaffin vesicles will saturate the K⁺ secretory response at both [Ca²⁺]_o, i.e. 0.5 mM and 5 mM. In contrast, Ca²⁺ ions entering through more distant channels will be sequestered by intracellular buffers and, thus, will not saturate the secretory machinery at lower [Ca²⁺]_o. Received: 23 September 1997 / Received after revision: 29 October 1997 / Accepted: 30 October 1997     

Simultaneous presence of cAMP and cGMP exert a co-ordinated inhibitory effect on the agonist-evoked Ca2+ signal in pancreatic acinar cells

The stimulation of the pancreatic acinar cells by physiological secretagogues, such as acetycholine (ACh), activates a well-established intracellular signalling pathway, which involves the generation of Inositol 1,4,5-trisphosphate (InsP₃) and the release of Ca²⁺ from intracellular stores. Caffeine, which inhibits this agonist-evoked Ca²⁺ response reversibly and competitively also blocks the Ca²⁺ signal generated by the non-specific activation of the membrane guanine nucleotide-binding proteins (G-proteins). Removal of caffeine is associated with an increase of intracellular [Ca²⁺] ([Ca²⁺]_i) and the spatial and temporal characteristics of this Ca²⁺ signal are identical to those of the signal generated by the initial agonist stimulation. Caffeine is also a potent non-specific inhibitor of various cellular phosphodiesterases (PDE) and its inhibitory effect can be reproduced by other PDE inhibitors, chemically related (theophylline) or not (papaverine). Various protocols designed to increase the concentration of either of the major intracellular cyclic nucleotides [adenosine 3′,5′-cyclic monophosphate (cAMP) and guanosine 3′,5′-cyclic monophosphate (cGMP)] failed to reproduce the full extent of the caffeine inhibition: at maximal agonist concentration (1 μM ACh) increases of either cAMP or cGMP did not affect the Ca²⁺ signal, whereas at submaximal doses of agonist (0.1–0.3 μM ACh) they induced partial inhibition. Here we show that only the simultaneous increase of the cellular concentrations of both cyclic nucleotides (either simultaneous or sequential) are effective in mimicking the blocking effect of caffeine and other non-specific PDE inhibitors. These data indicate, thus, that, in addition to other independent intracellular effects, cAMP and cGMP can exert a co-ordinated inhibitory effect of the agonist-evoked Ca²⁺ signal in pancreatic acinar cells. Received: 25 March 1996/Accepted: 23 May 1996     

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.     

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     

Regulation of calcium release by calcium inside the sarcoplasmic reticulum in ventricular myocytes

 To study the effects of changes in sarcoplasmic reticulum (SR) intraluminal Ca²⁺ on the Ca²⁺ release mechanism, we correlated the activity of single cardiac ryanodine receptor (RyR) channels, monitored in planar bilayers, with the properties of spontaneous elementary Ca²⁺ release events (sparks) in intact ventricular myocytes, monitored by scanning confocal microfluorimetry. Under both normal conditions and Ca²⁺ overload, induced by elevation of extracellular [Ca²⁺], Ca²⁺ sparks represented single populations of events. During Ca²⁺ overload, the frequency of sparks increased from 0.8 to 3.1 events per second per 100 μm line scanned, and their amplitude increased from 100 nM to 400 nM. The duration of the Ca²⁺ sparks, however, was not altered. Changes in the properties of Ca²⁺ sparks were accompanied by only an ≈ 30% increase in the SR Ca²⁺ content, as determined by emptying the intracellular Ca²⁺ stores using caffeine. When single Ca²⁺ release channels were incorporated into lipid bilayers and activated by cytoplasmic Ca²⁺ (≈ 100 nM) and ATP (3 mM), elevation of Ca²⁺ on the luminal side from 20 μM to 0.2–20 mM resulted in a 1.2-fold to 7-fold increase, respectively, in open probability (P _o). This potentiation of P _o was due to an increase in mean open time and frequency of events. The relative effect of luminal Ca²⁺ was greater at low levels of cytoplasmic [Ca²⁺] than at high levels of cytoplasmic [Ca²⁺], and no effect of luminal Ca²⁺ was observed to occur in channels activated by 0.5–50 μM cytoplasmic Ca²⁺ in the absence of ATP. Our results suggest that SR Ca²⁺ release channels are modulated by SR intraluminal Ca²⁺. These alterations in properties of release channels may account for, or contribute to, the mechanism of spontaneous Ca²⁺ release in cardiac myocytes Received: 15 May 1996 / Received after revision: 5 June 1996 / Accepted: 8 July 1996     

Attenuation of stimulated Ca2+ influx in colonie epithelial (HT29) cells by cAMP

In HT₂₉ colonic epithelial cells agonists such as carbachol (CCH) or ATP increase cytosolic Ca²⁺ activity ([Ca²⁺]_i) in a biphasic manner. The first phase is caused by inositol 1,4,5-trisphophate-(Ins P ₃-) mediated Ca²⁺ release from their respective stores and the second plateau phase is mainly due to stimulated transmembraneous Ca²⁺ influx. The present study was undertaken to examine the effect of increased adenosine 3′,5′-cyclic monophasphate (cAMP) (forskolin 10 μmol/l = FOR) on the Ca²⁺ transient in the presence of CCH (100 μmol/l). In unpaired experiments it was found that FOR induced a depolarization and reduced cytosolic Ca²⁺ ([Ca²⁺]_i, measured as the fura-2 fluorescence ratio 340/380 nm) significantly. Dideoxyforskolin had no such effect. The effect of FOR was abolished when the cells were depolarized by a high-K⁺ solution. In further paired experiments utilizing video imaging in conjunction with whole-cell patch-clamp, [Ca²⁺]_i was monitored separately for the patch-clamped cell and three to seven neighbouring cells. In the presence of CCH, FOR reduced [Ca²⁺]_i uniformly from a fluorescence ratio (345/380) of 2.9 ± 0.12 to 1.8 ± 0.07 in the patch-clamped cell and its neighbours (n = 48) and depolarized the membrane voltage (V _m) of the patch-clamped cells significantly and reversibly from −54 ± 7.4 to −27 ± 5.9 mV (n = 6). In additional experiments V _m was depolarized by 15–54 mV by various increments in the bath K⁺ concentration. This led to corresponding reductions in [Ca²⁺]_i. Irrespective of the cause of depolarization (high K⁺ or FOR) there was a significant correlation between the change in V _m and change in [Ca²⁺]_i. These data indicate that the cAMP-mediated attenuation of Ca²⁺ influx is caused by the depolarization produced by this second messenger. Received: 12 March 1996/Accepted: 2 April 1996     

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     

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     

Effects of nicotine on human nasal epithelium: evidence for nicotinic receptors in non-excitable cells

 We investigated the effects of nicotine and its derivate nicotine di-d-tartrate on primary cultured human nasal epithelial cells. Both substances evoked an in-crease in the intracellular free calcium concentration. In the presence of extracellular Ca²⁺ the cytosolic Ca²⁺ ([Ca²⁺]_i) increase was long lasting, whereas in the absence of external Ca²⁺ there was a transient increase of [Ca²⁺]_i indicating that nicotine has an influence on Ca²⁺ conductances across the membranes and on intracellular Ca²⁺ stores. Both effects could be blocked by the nicotinic receptor antagonist methyllycaconitine (MLA). Apical or basolateral application of nicotine during transepithelial transport measurements with confluent monolayers of cultured human nasal cells resulted in a significant, reversible decrease of amiloride-sensitive sodium absorption with an apparent half-maximal blocker concentration of about 950 μM. To exclude the possibility that remnant neuronal components were responsible for the observed effects we used tetrodotoxin and verapamil to block putative neuronal channels and 4-(4-diethyla- mino)styryl-N-methylpyridinium iodide (4-di-2-Asp) to stain neuronal tissue. Both experimental approaches demonstrated that there were no neuronal-mediated effects. These results indicate the direct effects of nicotine on human nasal epithelium, giving the first evidence of the existence of nicotinic receptors in non-excitable cells. Received: 6 December 1996 / Received after revision: 7 April 1997 / Accepted: 12 May 1997     

Factors affecting SOCE activation in mammalian skeletal muscle fibers

Enzymatically dissociated mouse FDB muscle fibers, loaded with Fura-2 AM, were used to study the effect of mitochondrial uncoupling on the capacitative Ca²⁺ entry, SOCE. Sarcoplasmic reticulum (SR) Ca²⁺ stores were depleted by repetitive exposures to high K⁺ or 4-chloro-m-Cresol (4-CmC) in the absence of extracellular Ca²⁺. SR Ca²⁺ store replenishment was substantially reduced using 5 μM cyclopiazonic acid (CPA). Readmission of external Ca²⁺ (5 mM) increased basal [Ca²⁺]_i under two modalities. In mode 1 [Ca²⁺]_i initially increased at a rate of 0.8 ± 0.1 nM/s and later at a rate of 12.3 ± 2.6 nM/s, reaching a final value of 477.8 ± 36.8 nM in 215.7 ± 25.9 s. In mode 2, [Ca²⁺]_i increased at a rate of 0.8 ± 0.1 nM/s to a value of 204.9 ± 20.6 nM in 185.4 ± 21.1 s. FCCP, 2 μM, reduced this Ca²⁺ entry. In nine FCCP-poisoned fibers, the initial rate of Ca²⁺ increase was 0.34 ± 0.1 nM/s (mean ± SEM), reaching a plateau of 149.2 ± 14.1 nM in 217 ± 19 s. The results may likely be explained by the hypothesis that SOCE is inhibited by mitochondrial uncouplers, pointing to a possible mitochondrial role in its activation. Using time-scan confocal microscopy and the dyes CaOr-5N AM or Rhod-2 AM to label mitochondrial Ca²⁺, we show that during depletion [Ca²⁺]_mito initially increases and later diminishes. Finally, we show that the increase in basal [Ca²⁺]_i, associated with SOCE activation, diminishes upon external Na⁺ withdrawal. Na⁺ entry through the SOCE pathway and activation of the reversal of Na⁺/Ca²⁺ exchanger could explain this SOCE modulation by Na⁺.     

Role of PKC in the effects of α1-adrenergic stimulation on Ca2+ transients, contraction and Ca2+ current in guinea-pig ventricular myocytes

 The effects of α₁-adrenoceptor stimulation on intracellular Ca²⁺ transients, contractility and L-type Ca²⁺ current (I _Ca,L) were studied in single cells isolated from ventricles of guinea-pig hearts. The aim of our study was to elucidate the mechanisms of the positive inotropic effect of α₁-adrenergic stimulation by focussing on the role of protein kinase C (PKC). Phenylephrine, an α₁-adrenergic agonist, at concentrations of 50–100 μM elicited a biphasic inotropic response: a transient negative inotropic response (22.9±6.0% of control) followed by a sustained positive inotropic response (61.0±8.4%, mean±SE, n=12). The Ca²⁺ transient decreased by 10.2±3.9% during the negative inotropic phase, while it increased by 67.7±10% (n=12) during the positive inotropic phase. These effects were inhibited by prazosin (1 μM), a α₁-adrenergic antagonist. Phenylephrine increased the I _Ca,L by 60.8±21% (n=5) during the positive inotropic phase. To determine whether activation of PKC is responsible for the increases in Ca²⁺ transients, contractile amplitude and I _Ca,L during α₁-adrenoceptor stimulation, we tested the effects of 4β-phorbol 12-myristate 13-acetate (PMA), a PKC activator, and of bisindolylmaleimide I (GF109203X) and staurosporine, both of which are PKC inhibitors. PMA mimicked phenylephrine’s effects on Ca²⁺ transients, contractile amplitude and I _Ca,L. PMA (100 nM) increased the Ca²⁺ transient, contractile amplitude and I _Ca,L by 131±17%, 137±25% (n=8), and 81.1±26% (n=5), respectively. Prior exposure to GF109203X (1 μM) or staurosporine (10 nM) prevented the phenylephrine-induced increases in Ca²⁺ transients, contractile amplitude and I _Ca,L. Our study suggests that during α₁-adrenoceptor stimulation increase in I _Ca,L via PKC causes an increase in Ca²⁺ transients and thereby in the contractile force of the ventricular myocytes. Received: 16 July 1998 / Received after revision and accepted: 20 October 1998