[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     

Bradykinin and inositol 1,4,5-trisphosphate-stimulated calcium release from intracellular stores in cultured bovine endothelial cells

The relative importance of intracellular and extracellular Ca²⁺ in the release of endothelium-derived relaxing factor (EDRF) and the mechanisms involved in the release of intracellular Ca²⁺ were investigated in cultured bovine endothelial cells. The release of EDRF by bradykinin, determined by bioassay, was dose-dependent showing an EC₅₀ of 4×10^–10 M. The bradykinin-induced EDRF release from endothelial cells was maintained in the presence of extracellular Ca²⁺. However, in the absence of external Ca²⁺, bradykinin-induced EDRF release was both attenuated and transient. In cells loaded to isotopic equilibrium with⁴⁵Ca, bradykinin increased the⁴⁵Ca efflux into both calcium-containing and calcium-free solutions, with an EC₅₀ for the increase in⁴⁵Ca efflux induced by bradykinin of 1.3×10^–9 M. The involvement of an intracellular Ca²⁺ store and the participation of a second messenger in its release were investigated in saponin-permeabilized endothelial cells. In saponin-permeabilized cells, ATP-sensitive calcium uptake was Ca²⁺,Mg²⁺-ATPase-dependent. The ATP-sensitive uptake of calcium at different free Ca²⁺ concentrations showed at least two compartments involved in the uptake of Ca²⁺. The⁴⁵Ca uptake into the compartment with the lowest affinity and highest capacity could be inhibited by sodium azide, suggesting that this uptake was into mitochondria. The majority of the⁴⁵Ca uptake into the azide-insensitive store could be released by inositol-1,4,5-trisphosphate (IP₃). The IP₃-induced release was not affected by apyrase or exogenous GTP. The EC₅₀ for the release of Ca²⁺ by IP₃ was 1.0 M and was unaffected by an inhibitor of IP₃ breakdown (2,3-diphosphoglyceric acid). The results suggest that the release of EDRF is dependent on extracellular Ca²⁺ influx and the release of intracellular Ca²⁺. The release of calcium from one of the high affinity intracellular Ca²⁺ stores is mediated by the intracellular second messenger, IP₃.     

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     

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     

Single-cell fura-2 microfluorometry reveals different purinoceptor subtypes coupled to Ca2+ influx and intracellular Ca2+ release in bovine adrenal chromaffin and endothelial cells

ATP and adenosine(5)tetraphospho(5)adenosine (Ap₄A), released from adrenal chromaffin cells, are potent stimulators of endothelial cell function. Using single-cell fura-2 fluorescence recording techniques to measure free cytosolic Ca²⁺ concentration ([Ca²⁺]_i), we have investigated the role of purinoceptor subtypes in the activation of cocultured chromaffin and endothelial cells. ATP evoked concentration-dependent [Ca²⁺]_i rises (EC₅₀=3.8 M) in a subpopulation of chromaffin cells. Both ATP-sensitive and -insensitive cells were potently activated by nicotine, bradykinin and muscarine. Reducing extracellular free Ca²⁺ concentration to around 100 nM suppressed the [Ca²⁺]_i transient evoked by ATP but not the [Ca²⁺]_i response to bradykinin. ATP-sensitive chromaffin cells were also potently stimulated by 2-methylthioadenosine triphosphate (2MeSATP; EC₅₀= 12.5 M) and UTP, but did not respond to either adenosine 5-[-thio]diphosphate (ADP[S]), a P_2Y receptor agonist, adenosine 5-[,-methylene]triphosphate (pp[CH₂]pA), a P_2X agonist or AMP. Adrenal endothelial cells displayed concentration-dependent [Ca²⁺]_i responses when stimulated with ATP (EC₅₀=0.86 M), UTP (EC₅₀=1.6 M) and 2MeSATP (EC₅₀= 0.38 M). 2MeSATP behaved as a partial agonist. Ap₄A and ADP[S] also raised the [Ca²⁺]_i in endothelial cells, whereas AMP and pp[CH₂]pA were ineffective. Lowering extracellular free Ca²⁺ to around 100 nM did not affect the peak ATP-evoked [Ca²⁺]_i rise in these cells. It is concluded that different purinoceptor subtypes are heterogeneously distributed among the major cell types of the adrenal medulla. An intracellular Ca²⁺-releasing P_2U-type purinoceptor is specifically localized to adrenal endothelial cells, while a subpopulation of chromaffin cells expresses a non-P_2X, non-P_2Y subtype exclusively coupled to Ca²⁺ influx.     

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     

The increase in the intracellular Ca2+ concentration induced by mechanical stimulation is propagated via release of pyrophosphorylated nucleotides in mammary epithelial cells

Mechanical stimulation of one mammary tumor cell in culture induced an increase in its intracellular calcium concentration which spread to surrounding cells. The increase in calcium can also be induced by addition of a solution in which cultured mammary tumor cells were stimulated by repeated pipetting (solution after pipetting cells, SAPC). The activity of the SAPC was completely abolished by treatment with snake venom phosphodiesterase or pyrophosphatase. Uridine triphosphate (UTP), uridine diphosphate (UDP) and ATP (1 M each) were detected in the SAPC, whereas 5-UMP and 5-AMP were produced by phosphodiesterase digestion. A mixture of UTP, UDP and ATP (1 M each) elicited a calcium response which was comparable to that induced by SAPC, while UTP, UDP or ATP alone at 1 M elicited a small increase in calcium concentration in mammary tumor cells. Suramin, a competitive antagonist of P₂ purinoceptors, diminished the spreading of the calcium wave induced by mechanical stimulation. It also blocked the responses to SAPC, UTP, UDP and ATP. These findings suggest that the mechanical stimulation results in the release of UTP, UDP and ATP into the extracellular space which mediates induction of the spreading calcium response via P_2U-type purinoceptors.     

Involvement of P2-purinergic receptors in intracellular Ca2+ responses and the contraction of mammary myoepithelial cells

 Mammary myoepithelial cells were isolated and cultured to characterize their properties. The intracellular calcium concentration (Ca_i ²⁺) was measured using the ratio of fura-2 fluorescence at 340 nm to that at 360 nm (F ₃₄₀/F ₃₆₀), and the contraction was simultaneously monitored by the change of fluorescence intensity at 360 nm (F ₃₆₀). Cultured myoepithelial cells retained their contractile machinery as in the intact tissue. NBD-phallacidin fluorescence, which marks F-actin, and electron microscopy showed abundant bundles of microfilaments in the cytoplasm. Oxytocin (> 0.1 nM) induced an increase in Ca_i ²⁺ and contraction. The amplitude and time course of the Ca_i ²⁺ increase were not markedly affected in the Ca²⁺-free solution. Nifedipine (10 μM) did not affect the response to oxytocin. ATP (>1 μM) induced an increase in Ca_i ²⁺ and contraction. The response to ATP was not affected by Ca²⁺ removal, but was suppressed by suramin (100 μM), an antagonist of P₂-purinergic receptors. The order of potency of nucleotides to increase Ca_i ²⁺ was ATP = ADP > UTP > UDP. These findings indicate the presence of P₂-purinergic receptors in mammary myoepithelial cells. The results suggest that stimulant-induced Ca_i ²⁺ increases and contractions are due to release of Ca²⁺ from intracellular stores in these cells. In addition to the hormonal action of oxytocin, extracellular nucleotides may function as paracrine agents to contract myoepithelial cells in the mammary gland. Received: 17 March 1997 / Received after revision: 10 July 1997 / Accepted: 21 July 1997     

Calcium fluxes in rat thyroid FRTL-5 cells. Evidence for a functional Na+/Ca2+ exchange mechanism

Törnquist , K. 1992. Calcium fluxes in rat thyroid FRTL-5 cells. Evidence for a functional Na⁺/Ca²⁺ exchange mechanism. Acta Physzol Scand 144 , 341–348. Received 28 April 1 991 , accepted 30 October 1991. ISSN 0001–6772. Endocrine Research Laboratory, University of Helsinki, Minerva Foundation Institute for Medical Research, Helsinki, Finland. The effect of extracellular Na⁺ on cytosolic free Ca²⁺ and on influx and efflux of Ca²⁺ was investigated in FRTL-5 thyroid cells. Stimulating the cells with the purinergic agonist ATP induced a rapid efflux of ⁴⁵Ca²⁺ from cells loaded with ^4aCa²⁺. Replacement of extracellular Na⁺ with choline⁺, significantly decreased the adenosine triphosphate-induced efflux of ⁴⁵Ca²⁺. Furthermore, adenosine triphosphate-induced uptake of ⁴⁵Ca²⁺ was increased when extracellular Na⁺ was replaced with choline⁺, compared with the uptake seen in Na⁺ buffer. Replacing extracellular Na⁺ with choline⁺, increased resting levels of cytosolic free Ca²⁺ from 50 ± 2 nM (mean ± SE) to 81 ± 3 nM (P < 0.05) in Fura 2 loaded cells. In cells preincubated with 1 mM ouabain for 30 min, resting cytosolic free Ca²⁺ increased to 73 ± 3 nM (P < 0.05). In a Na⁺ buffer, the adenosine triphosphate-induced transient increase in cytosolic free Ca²⁺ was 872 ± 59 nM, compared with 1070 ±63 nM in choline' buffer (P < 0.05). The plateau level of cytosolic free Ca²⁺ in response to adenosine triphosphate was 130±16 nM in Na⁺ buffer, compared with 209±9 nM in choline⁺ buffer (P < 0.05). Readdition of Na⁺ to the plateau phase decreased cytosolic free Ca⁺² to 152 ± 5 nM. Stimulating the cells with 10 μM of the Na^+-selective monovalent ionophore monensin increased cytosolic free Ca²⁺ from 53 ± 9 nM to 12416 nM (P < 0.05). This increase in cytosolic free Ca²⁺ was dependent on both extracellular Na⁺ and extracellular Ca²⁺ If cells were first stimulated with monensin, and then with adenosine triphosphate, the transient increase in cytosolic free Ca²⁺ was 1027 ± 24 nM (P < 0. 05 , compared with control cells). The results thus indicate, that FRTL-5 cells have a functional Na⁺/Ca²⁺ exchange mechanism and that this mechanism is of importance in restoring adenosine triphosphate-induced transient increase in cytosolic free Ca²⁺ to resting cytosolic free Ca²⁺ levels.     

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     

Plasma membrane Ca2+ ATPase 1 (PMCA1) but not PMCA4 is critical for B-cell development and Ca2+ homeostasis in mice

The amplitude and duration of Ca²⁺ signaling is crucial for B-cell development and self-tolerance; however, the mechanisms for terminating Ca²⁺ signals in B cells have not been determined. In lymphocytes, plasma membrane Ca²⁺ ATPase (PMCA) isoforms 1 and 4 (PMCA1 and PMCA4, aka ATP2B1 and ATP2B4) are the main candidates for expelling Ca²⁺ from the cell through the plasma membrane. We report here that Pmca4 (Atp2b4) KO mice had normal B-cell development, while mice with a conditional KO of Pmca1 (Atp2b1) had greatly reduced numbers of B cells, particularly splenic follicular B cells, marginal zone B cells, and peritoneal B-1a cells. Mouse and naïve human B cells showed only PMCA1 expression and no PMCA4 by western blot, in contrast to T cells, which did express PMCA4. Calcium handling was normal in Pmca4^−/− B cells, but Pmca1 KO B cells had elevated basal levels of Ca²⁺, elevated levels in ER stores, and reduced Ca²⁺ clearance. These findings show that the PMCA1 isoform alone is required to ensure normal B-cell Ca²⁺ signaling and development, which may have implications for therapeutic targeting of PMCAs and Ca²⁺ in B cells.     

Evidence for agonist-induced export of intracellular Ca2+ in epithelial cells

There is increasing evidence that some agonists not only induce intracellular Ca²⁺ increases, due to store release and transmembranous influx, but also that they stimulate Ca²⁺ efflux. We have investigated the agonist-stimulated response on the intracellular Ca²⁺ activity ([Ca²⁺]_i) in the presence of thapsigargin (10^–8 mol/l, TG) in HT₂₉ and CFPAC-1 cells. For CFPAC-1 the agonists ATP (10^–7–10^–3 mol/l, n=9), carbachol (10^–6–10^–3 mol/l, n=5) and neurotensin (10^–10–10^–7 mol/l, n=6) all induced a concentration-dependent decrease in [Ca²⁺]_i in the presence of TG. Similar results were obtained with HT₂₉ cells. This decrease of [Ca²⁺]_i could be caused by a reduced Ca²⁺ influx, either due to a reduced driving force for Ca²⁺ in the presence of depolarizing agonists or due to agonist-regulated decrease in Ca²⁺ permeability. Using the fura-2 Mn²⁺ quenching technique we demonstrated that ATP did not slow the TG-induced Mn²⁺ quench. This indicates that the agonist-induced [Ca²⁺]_i decrease in the presence of TG was not due to a reduced influx of Ca²⁺ into the cell, but rather due to stimulation of Ca²⁺ export. We used the cell attached nystatin patch clamp technique in CFPAC-1 cells to examine whether, in the presence of TG, the above agonists still led to the previously described electrical changes. The cells had a mean membrane voltage of –49±3.6 mV (n=9). Within the first 3 min ATP was still able to induce a depolarization which could be attributed to an increase in Cl^– conductance. This was expected, since at this time after TG stimulation all Ca²⁺ agonists still liberated some [Ca²⁺]_i. When TG incubation was prolonged, agonist application led to strongly attenuated or to no electrical responses. Therefore, the agonist-stimulated [Ca²⁺]_i decrease cannot be explained by the reduction of the driving force for Ca²⁺ into the cell. In the same cells hypotonic swelling (160 mosmol/l, n=15) still induced a further [Ca²⁺]_i increase in the presence of TG and concomitantly induced Cl^– and K⁺ conductances. We conclude that the agonist-induced decrease of [Ca²⁺]_i in the presence of TG probably unmasks a stimulation of [Ca²⁺]_i export.     

Intracellular Ca2+ signals in human-derived pancreatic somatostatin-secreting cells (QGP-1N)

Single-cell microfluorimetry techniques have been used to examine the effects of acetylcholine (0.1–100 M) on the intracellular free calcium ion concentration ([Ca²⁺]_i) in a human-derived pancreatic somatostatin-secreting cell line, QGP-1N. When applied to the bath solution, acetylcholine was found to evoke a marked and rapid increase in [Ca²⁺]_i at all concentrations tested. These responses were either sustained, or associated with the generation of complex patterns of [Ca²⁺]_i transients. Overall, the pattern of response was concentration related. In general, 0.1–10 M acetylcholine initiated a series of repetitive oscillations in cytoplasmic Ca²⁺, whilst at higher concentrations the responses consisted of a rapid rise in [Ca²⁺]_i followed by a smaller more sustained increase. Without external Ca²⁺, 100 M acetylcholine caused only a transient rise in [Ca²⁺]_i, whereas lower concentrations of the agonist were able to initiate, but not maintain, [Ca²⁺]_i oscillations. Acetylcholine-evoked Ca²⁺ signals were abolished by atropine (1–10 M), verapamil (100 M) and caffeine (20 mM). Nifedipine failed to have any significant effect upon agonist-evoked increases in [Ca²⁺]_i, whilst 50 mM KCl, used to depolarise the cell membrane, only elicited a transient increase in [Ca²⁺]_i. Ryanodine (50–500 nM) and caffeine (1–20 mM) did not increase basal Ca²⁺ levels, but the Ca²⁺-ATPase inhibitors 2,5-di(tert-butyl)-hydroquinone (TBQ) and thapsigargin both elevated [Ca²⁺]_i levels. These data demonstrate for the first time cytosolic Ca²⁺ signals in single isolated somatostatin-secreting cells of the pancreas. We have demonstrated that acetylcholine will evoke both Ca²⁺ influx and Ca²⁺ mobilisation, and we have partially addressed the subcellular mechanism responsible for these events.     

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.     

An examination of the role of intracellular ATP in the activation of store-operated Ca2+ influx and Ca2+-dependent capacitance increases in rat basophilic leukaemia cells

 The role of ATP in both the activation of store-operated Ca²⁺ current I _CRAC and in Ca²⁺-dependent vesicular fusion was examined in a study of rat basophilic leukaemia (RBL) cells using the whole-cell patch-clamp technique. Fusion was monitored via changes in plasma membrane capacitance. Following a decrease in the levels of intracellular ATP, achieved using the mitochondrial poison antimycin and the ATP synthase inhibitor oligomycin, as well as a reduction of glycolysis by removal of external glucose, I _CRAC activated in a manner similar to control cells when stores are depleted by dialysis with a pipette solution containing either inositol 1,4,5-trisphosphate (InsP ₃) or ionomycin together with a high concentration of EGTA. Dialysis of cells for 150 s with the non-hydrolysable ATP analogue 5′-adenylylimidodiphosphate (AMP-PNP) (2 mM) in addition to the mitochondrial inhibitors also failed to prevent activation of I _CRAC following external application of ionomycin and thapsigargin, when compared with control recordings obtained with 2 mM ATP instead. Ca²⁺-dependent vesicular fusion was triggered by dialysing cells with 10 μM Ca²⁺ and guanosine-5′-O-(3-thiotriphosphate (GTP[γ-S]). The capacitance increase was unaffected by inhibition of glycolysis, mitochondrial inhibitors or dialysis with either AMP-PNP or adenosine 5′-O-(3-thiotriphosphate) (ATP[γ-S]) instead of ATP. We conclude that ATP hydrolysis does not seem to be necessary for the activation of I _CRAC or for the capacitance increases elicited by high concentrations of intracellular Ca²⁺. Received: 1 May 1998 / Received after revision: 16 July 1998 / Accepted: 16 July 1998     

Fura-2 imaging of spontaneous and electrically induced oscillations of intracellular free Ca2+ in rat myotubes

Rat myotubes have a resting [Ca²⁺]_i of about 82 nM. Myotubes 3–5 days old (quiescent myotubes) display electrically induced and spontaneous transients in the intracellular concentration of free Ca²⁺ ions ([Ca²⁺]_i) uncoupled to any detectable contraction. By contrast, 1-to 2-day-old myotubes are insensitive to electrical stimuli and, after 6 days in culture, stimulated myotubes always show [Ca²⁺]_i transients and twitch contractions. The spatial distribution of [Ca²⁺]_i variations in quiescent myotubes is heterogeneous, local increases in [Ca²⁺]_i being mainly observed near the periphery of the cell. The small effect of different external Ca²⁺ concentrations and of Cd²⁺ on the amplitude of the [Ca²⁺]_i oscillation indicates that the main source of Ca²⁺ may be the sarcoplasmic reticulum. This conclusion is supported by the close similarity between electrically induced and caffeine-induced [Ca²⁺]_i maps. These findings suggest that, at an early stage of myotube ontogenesis, a part of the excitation/contraction coupling, as membrane ionic channels, voltage sensors and Ca²⁺ release and reuptake mechanisms, is functional but, apparently, still uncoupled to the contractile machinery.This work was supported by the Centre National de la Recherche Scientifique, the Ministère de la Recherche et de la Technologie, the Conseil Régional de l'Aquitaine and the Association Française contre les Myopathies     

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     

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.     

Effects of aging on Ca2+ signaling in murine mesenteric arterial smooth muscle cells

Pathophysiological changes in arterial smooth muscle structure and function occur with aging and there are a number of reports illustrating reductions in vascular responsiveness with aging. While much is known about arterial remodeling and functional adaptations with aging, very little is known about the biophysical adaptations in individual arterial myocytes. Cytosolic Ca2+ signaling, involving activation of L-type Ca2+ channels on the plasma membrane as well as InsP3 and ryanodine receptors on the sarcoplasmic reticulum, is integral to vascular tone and reactivity. Thus, we tested the hypothesis that aging results in reductions in the functional expression of L-type channels and temporal aspects of ryanodine receptor and InsP3 receptor Ca2+ signaling, in mesenteric arterial smooth muscle cells isolated from 6 and 30 months old C57Bl/6 mice. Comparisons of L-type current activity were made using dialyzed, whole-cell voltage-clamp techniques and Ba2+ as charge carrier. Ca2+ signaling was measured using fura-2 fluorescence microscopy techniques. Cell morphological changes were also investigated using electrophysiological and immunocytochemical approaches. The amplitudes of L-type Ca2+ currents were increased in older mice, but this was associated with membrane surface area increases of approximately 50%, due to increases in cell length not cell width. Consequently, L-type Ca2+ current densities were preserved with age, indicating functional channel expression was unchanged. In contrast, aging was associated with decrements in Ca2+ signaling in response to either ryanodine receptor stimulation by caffeine or InsP3 receptor activation with phenylephrine. These changes with aging may be related to the previously reported depression in myogenic reactivity.