Activation of protein kinase C does not cause desensitization in rat and rabbit mandibular acinar cells

We have examined whether activation of protein kinase C by phorbol esters decreases the responsiveness of rat and rabbit mandibular, and rat lacrimal, acinar cells to muscarinic stimulation. Intracellular free calcium concentration ([Ca²⁺]_i) was measured in isolated single acini and cell clusters by fura-2 microspectrofluorimetry. Accumulation of inositol phosphates was measured in acinar cell suspensions. All three cell types showed very similar changes in [Ca²⁺]_i in response to acetylcholine (ACh), although mobilization of Ca²⁺ required somewhat higher ACh concentrations in rat lacrimal acinar cells than in mandibular acinar cells. There was no evidence for different dose dependencies of the peak and plateau phases of the [Ca²⁺]_i response. The ACh-evoked [Ca²⁺]_i increase in rabbit mandibular acinar cells exhibited desensitization, since it declined in magnitude when cells were stimulated repeatedly with a maximal dose of agonist. The phorbol ester 12-O-tetradecanoylphorbo-l13-acetate (TPA) rapidly and irreversibly decreased the ACh-evoked [Ca²⁺]_i signals in rat lacrimal acinar cells and reduced ACh-stimulated inositol phosphate accumulation. This inhibitory effect of TPA was most marked in cells stimulated with low doses of ACh, implying that TPA treatment shifted the ACh dose response curve to higher ACh concentrations. In contrast to the results obtained with lacrimal acinar cells, TPA had no effect on the [Ca²⁺]i and inositol phosphate responses to ACh in either rat or rabbit mandibular acinar cells. These results suggest that, although ACh-evoked [Ca²⁺]_i signals, and hence presumably the stimulus-response coupling machinery, are very similar between different acinar cell types, acinar cells show marked differences in their sensitivity to phorbol esters. The insensitivity of mandibular acinar cell [Ca²⁺]_i signals to TPA also suggests that the secretory tachyphylaxis observed in perfused rat and rabbit mandibular salivary glands is unlikely to be a consequence of negative feedback mediated by protein kinase C.     

Acetylcholine-evoked calcium mobilization and ion channel activation in human labial gland acinar cells from patients with primary Sjögren's syndrome

Recent evidence has indicated that the salivary gland dysfunction associated with Sjögren's syndrome (SjS) is not necessarily due to immune‐mediated destruction of acinar tissue. SjS sufferers may possess substantial reserves of acinar tissue but nevertheless be incapable of maintaining salivary flow rates in the normal range. We have investigated the ability of isolated labial gland acinar cells from SjS patients to fluid secrete by measuring agonist‐evoked changes in intracellular Ca²⁺ ([Ca²⁺]_i) using fura‐2 microfluorimetry and activation of K⁺ and Cl^? channels using the patch‐clamp whole cell technique. We can confirm that stimulation with a super‐maximal dose of acetylcholine (ACh) increased [Ca²⁺]_i equally in both control acinar cells and those derived from SjS patients. However, at submaximal concentrations, the dose–response curve for ACh was shifted to the right by approximately one order of magnitude in acinar cells from SjS patients compared to control acinar cells. Patch‐clamp measurements consistent with the presence of Ca²⁺‐activated K⁺ and Cl^? conductances were obtained from both control acinar cells and those obtained from SjS patients. Dose‐dependent activation of the ion channels by acetylcholine was also right‐shifted in acinar cells from SjS patients compared to control cells. Our data show that labial gland acinar cells from SjS patients were capable of responding to agonist stimulation by mobilizing [Ca²⁺]_i and activating K⁺ and Cl^? channels consistent with the requirements of fluid secretion. However, the persistent loss of sensitivity to ACh observed in from SjS patients may account for the lack of saliva production observed in these patients in vivo.     

Role of protein kinase C in the regulation of inositol phosphate production and Ca2+ mobilization evoked by ATP and acetylcholine in rat lacrimal acini

Stimulation of rat lacrimal acinar cells with ATP and acetylcholine (ACh) induced a rapid accumulation of inositol 1,4,5-trisphosphate [Ins(1,4,5)P ₃] and its degradation products, resulting in an initial release of Ca²⁺ from intracellular stores. However, after pretreating the acini with U73122 no increase in the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) or Ins(1,4,5)P ₃ production was observed. A short pre-treatment with the phorbol ester 4--phorbol-12--myristate-13--acetate (PMA) significantly attenuated the ATP- and ACh-induced increase in [Ca²⁺]_i and over- all inositol phosphate production. In contrast, staurosporine enhanced Ins(1,4,5)P ₃ and inositol 1,3,4-trisphosphate [Ins(1,3,4)P ₃] production and [Ca²⁺]_i above control values in ATP- and ACh-stimulated cells. Stimulation of phospholipase C by iono-mycin-evoked changes in [Ca²⁺]_i were unaltered by pretreatment with staurosporine and PMA. The data show that a change in protein kinase C activity during cell stimulation affects the inositol phosphate metabolism and thereby the cellular Ca²⁺ signalling processes in lacrimal acinar cells.     

Effect of α1‐adrenergic stimulation of Cl− secretion and signal transduction in exocrine glands (Rana esculenta)

In the present work, the effect of stimulation of α‐adrenergic receptors on Cl^? secretion via exocrine frog skin glands was investigated. The α‐adrenergic stimulation was performed by addition of the adrenergic agonist noradrenaline in the presence of the β‐adrenergic antagonist propranolol. In the presence of propranolol, noradrenaline had no effect on the cellular cAMP content. The Cl^? secretion was measured as the amiloride‐insensitive short circuit current (I_SC). Addition of noradrenaline induced a biphasic increase in the I_SC. The increase in I_SC coincided with an increase in the net ³⁶Cl^? secretion. The noradrenaline‐induced increase in I_SC was dose‐dependent with an EC₅₀ of 13 ± 0.3 μM . Epifluorescence microscopic measurements of isolated, fura‐2‐loaded frog skin gland acini were used to characterize the intracellular calcium ([Ca²⁺]_i) response. Application of noradrenaline induced a biphasic [Ca²⁺]_i response, which was dose‐dependent with an EC₅₀ of 11 ± 6 μM . The Ca²⁺ plateau unlike the peak‐response was sensitive to removal of Ca²⁺ from the extracellular medium. The noradrenaline‐induced increase in the Cl^? secretion as well as in [Ca²⁺]_i was sensitive to the α₁‐adrenergic antagonist prazosine. Ryanodine and caffeine had no effect on [Ca²⁺]_i indicating that the release was independent of ryanodine‐sensitive Ca²⁺ stores. Noradrenaline mediated a significant increase in the cellular inositol 1,4,5‐trisphosphate (IP₃) content suggesting that the signal transduction pathway leading to the noradrenaline‐induced increase in Ca²⁺ involved IP₃ and a release of Ca²⁺ from IP₃‐sensitive stores.     

Subcellular gradients of intracellular free calcium concentration in isolated lacrimal acinar cells

The spatial distribution of intracellular free calcium concentration ([Ca²⁺]_i) was measured in small clusters of isolated rat lacrimal acinar cells by imaging the fluorescence of the Ca²⁺-sensitive dye fura-2. In the absence of extracellular Ca²⁺, stimulation with acetylcholine (ACh) caused an increase in [Ca²⁺]_i, due to release of intracellular Ca²⁺ stores, which was maximal at the luminal pole of the cell. In contrast, the organellar Ca²⁺-ATPase inhibitor 2,5-di(tert-butyl)-hydroquinone caused an increase in [Ca²⁺]_i, which was most marked in the basolateral region of the cell. When the cells were stimulated with ACh in a medium containing Ca²⁺, the gradients of [Ca²⁺]_i (with [Ca²⁺]_i most elevated at the luminal pole) were maintained for the duration of agonist stimulation. The possible implications of these results concerning the location and identity of intracellular Ca²⁺ stores, and the location of the sites that underlie agonist-stimulated Ca²⁺ influx, are considered. In particular, it seems likely that intracellular inositol-1,4,5-trisphosphate (InsP₃) binding sites may be concentrated in the luminal region of the cell. It is not clear, however, whether this implies that there is a distinct luminally located InsP ₃-sensitive organelle.     

Cell Signal Transduction Mechanism for Nitric Oxide Regulating Lymphatic Stomata and its Draining Capability

We have previously demonstrated that nitric oxide (NO) is involved in the regulation of the lymphatic stomata. However, the related mechanisms are still unknown. The present study was designed to test the hypothesis that NO–cyclic guanosine monophosphate (cGMP) ‐mediated cytosolic Ca²⁺ concentration ([Ca²⁺]i) signaling may contribute to the regulation of the lymphatic stomata and lymph drainage. Using trypan blue as a tracer, the effects of NO‐cGMP‐Ca²⁺ signal cascade on the lymphatic stomata and lymph absorption were examined by means of scanning electron microscopy. Then, the role of NO in cGMP and [Ca²⁺]i of rat peritoneal mesothelial cells (RPMCs) was measured by radioimmunoassay and a confocal laser scanning microscope. Our results showed that NO‐donor spermine/nitric oxide complex (Sper/NO) could broaden the opening area of the lymphatic stomata and enhance lymph absorption in a dose‐dependent manner. These NO‐mediated changes could be blocked by 1H‐[1,2,4] oxadiazolo [4,3‐a] quinoxalin‐1‐one (ODQ), a specific inhibitor of soluble guanylyl cyclase, and mimicked by calcium channel blocker nifedipine. Furthermore, Sper/NO enhanced the cGMP level and lessened [Ca²⁺]_i in RPMCs, which was completely abrogated at the presence of ODQ. Nifedipine induced an immediate and marked decrease of [Ca²⁺]_i in the RPMCs, which was not attenuated by addition of Sper/NO, indicating that the Sper/NO‐cGMP signaling system induced [Ca²⁺]_i change was related to the L‐type voltage‐gated calcium channel in the RPMCs. Our results suggest that NO enlarges the opening area of the lymphatic stomata to strengthen the lymph drainage of tracer by means of NO‐cGMP‐[Ca²⁺]i signal transduction pathway in the RPMCs. Anat Rec, 291:216–223, 2008. © 2008 Wiley‐Liss, Inc.     

Expression and function of Na+/Ca2+ exchangers 1 and 3 in human macrophages and monocytes

The Na⁺/Ca²⁺ exchanger (NCX) is a membrane transporter that can switch Na⁺ and Ca²⁺ in either direction to maintain the homeostasis of intracellular Ca²⁺. Three isoforms (NCX1, NCX2, and NCX3) have been characterized in excitable cells, e.g. neurons and muscle cells. We examined the expression of these NCX isoforms in primary human lung macrophages (HLM) and blood monocytes. NCX1 and NCX3, but not NCX2, are expressed in HLM and monocytes at both mRNA and protein levels. Na⁺‐free medium induced a significant increase in intracellular calcium concentration ([Ca²⁺]_i) in both cell types. This response was completely abolished by the NCX inhibitor 5‐(N‐4‐chlorobenzyl)‐20,40‐dimethylbenzamil (CB‐DMB). Moreover, inhibition of NCX activity during Ca²⁺‐signaling induced by histamine caused a delay in restoring baseline [Ca²⁺]_i. Na⁺‐free medium induced TNF‐α expression and release in HLM comparable to that caused by LPS. TNF‐α release induced by Na⁺‐free medium was blocked by CB‐DMB and greatly reduced by RNAi‐mediated knockdown of NCX1. These results indicate that human macrophages and monocytes express NCX1 and NCX3 that operate in a bidirectional manner to restore [Ca²⁺]_i, to generate Ca²⁺‐signals, and to induce TNF‐α production. Therefore, NCX may contribute to regulate Ca²⁺ homeostasis and proinflammatory functions in human macrophages and monocytes.     

Fibrinogen induces chemotactic activity in endothelial cells

The aim of the present study was to investigate the physiological role of nitric oxide (NO) in mediating secretory processes in rat lacrimal acinar cells. In addition, we wanted to determine whether the acinar cells possess endogenous nitric oxide synthase (NOS) activity by measuring NO production using the fluorescent NO indicator 4,5-diaminofluorescein (DAF-2). We initiated investigations by adding NO from an external source by means of the NO-donor, S-nitroso-N-acetyl-penicillamine (SNAP). Cellular concentrations of cyclic guanosine 5'-phosphate (cGMP) ([cGMP]) were measured by radioimmunoassay (RIA), and we found that SNAP induced a fast increase in the [cGMP], amounting to 350% of the [cGMP] in resting cells. Moreover, addition of SNAP and elevating [cGMP] in fura-2 loaded lacrimal acinar cells, resulted in a cGMP-dependent protein kinase-mediated release of Ca2+ from intracellular stores, leading to a rise in the intracellular free Ca2+ concentration ([Ca2+]i). The Mn2+ quenching studies revealed that the Ca2+ release was not accompanied by Ca2+ influx. Finally, we demonstrate that lacrimal acinar cells possess endogenous NOS activity, which is activated by beta-adrenergic stimulation and not by a rise in [Ca2+]i alone. We show that in rat lacrimal acinar cells, NO and cGMP induce Ca2+ release from intracellular stores via G kinase activation. However, the changes in [Ca2+]i are relatively small, suggesting that this pathway plays a modulatory role in Ca2+ signalling, thus not by itself causing fast transient increases in [Ca2+]i. In addition, we suggest that endogenously produced NO activated by beta-adrenergic receptor stimulation, plays an important role in signalling to the surrounding tissue.     

Integrin stimulation induces calcium signalling in rat cardiomyocytes by a NO-dependent mechanism

The myocardial stretch-induced increase in intracellular [Ca²⁺] ([Ca²⁺]_i) is considered to be caused by integrin stimulation. Myocardial stretch is also associated with increased nitric oxide (NO) formation. We hypothesised that NO is implicated in calcium signalling following integrin stimulation. Integrins of neonatal rat cardiomyocytes were stimulated with a pentapeptide containing the Arg-Gly-Asp (RGD) sequence. [Ca²⁺]_i was measured with Fura2, [NO]_i was measured with DAF2 and phosphorylation of focal adhesion kinase (FAK) was monitored with immunofluorescence techniques. Integrin stimulation increased both [NO]_i and [Ca²⁺]_i, the latter response being inhibited by ryanodine receptor-2 (RyR2) blockers and by N^G-monomethyl-L-arginine (L-NMMA), an inhibitor of NOS, but resistant to GdCl₃, diltiazem and wortmannin. Integrin-induced intracellular Ca²⁺ release thus appears to be independent of the influx of extracellular Ca²⁺ and phosphatidylinositol-3 kinase activity. In addition, integrin stimulation induced phosphorylation of FAK. Our results provide evidence for an integrin-induced Ca²⁺ release from RyR2 which is mediated by NO formation, probably via FAK-induced NOS activation.     

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     

Mathematical modelling of the calcium-left ventricular pressure relationship in the intact diabetic rat heart

Aim: The objective was to characterize cross‐bridge kinetics from the cytoplasmic calcium ion concentration ([Ca²⁺]_i) and the left ventricular pressure (LVP) in the early‐stage diabetic rat heart under baseline conditions and upon β‐adrenergic stimulation. Methods: Four weeks after the induction of diabetes in rats by the injection of streptozotocin, the hearts were perfused according to Langendorff, and [Ca²⁺]_i was obtained by epifluorescence measurements using Indo‐1 AM. [Ca²⁺]_i and LVP were measured simultaneously at a temporal resolution of 200 Hz. The input/output relationship between the Ca²⁺ and the pressure transients was described by a mathematical model representing the chemical binding of Ca²⁺ to troponin C on the actin myofilament (TnCA), and the subsequent cooperative force‐producing cross‐bridge formation of the Ca²⁺–TnCA complex with myosin. The kinetic parameters of this model were evaluated using a numerical optimization algorithm to fit the model equations to the experimental data. β‐adrenergic stimulation of the hearts with increasing doses of isoproterenol allowed quantification of the model parameters over an extended dynamic range, because isoproterenol administration increased developed pressure, heart rate, as well as [Ca²⁺]_i amplitude in a dose‐dependent manner. Results: Model analysis of the experimental data indicates that β‐adrenergic stimulation of healthy hearts resulted in a decreased sensitivity of TnCA for Ca²⁺, increased rates of cross‐bridge cycling and decreased cooperativity. By contrast, the responses in cross‐bridge kinetic parameters to isoproterenol stimulation were blunted in the 4‐week diabetic heart. Conclusion: We conclude from our modelling results that myocardial cross‐bridge cycling is impaired at the early stage of diabetes.     

Cytoplasmic and mitochondrial Ca2+ levels in brown adipocytes

Aim: We elucidated the mitochondrial functions of brown adipocytes in intracellular signalling, paying attention to mitochondrial activity and noradrenaline‐ and forskolin‐induced Ca²⁺ mobilizations in cold‐acclimated rats. Methods: A confocal laser‐scanning microscope of brown adipocytes from warm‐ or cold‐acclimated rats was employed using probes rhodamine 123 which is a mitochondria‐specific cationic dye, and the cytoplasmic and mitochondrial Ca²⁺ probes fluo‐3 and rhod‐2. X‐ray microanalysis was also studied. Results: The signal of rhodamine 123 in the cells was decreased by antimycin A which effect was less in cold‐acclimated cells than warm‐acclimated cells. Cytoplasmic and mitochondrial Ca²⁺ in cold‐acclimated brown adipocytes double‐loaded with fluo‐3 and rhod‐2 were measured. Noradrenaline induced the rise in cytoplasmic Ca²⁺ ([Ca²⁺]_cyto) followed by mitochondrial Ca²⁺ ([Ca²⁺]_mito), the effect being transformed into an increase in [Ca²⁺]_cyto whereas a decrease in [Ca²⁺]_mito by antimycin A or carbonyl cyanide m‐chlorophenylhydrazone (CCCP). Antimycin A induced small Ca²⁺ release from mitochondria. CCCP induced Ca²⁺ release from mitochondria only after the cells were stimulated with noradrenaline. Further, forskolin also elicited an elevation in [Ca²⁺]_cyto followed by [Ca²⁺]_mito in the cells. The Ca measured by X‐ray microanalysis was higher both in the cytoplasm and mitochondria whereas K was higher in the mitochondria of cold‐acclimated cells in comparison to warm‐acclimated cells. Conclusions: These results suggest that noradrenaline and forskolin evoked an elevation in [Ca²⁺]_cyto followed by [Ca²⁺]_mito, in which H⁺ gradient across the inner membrane is responsible for the accumulation of calcium on mitochondria. Moreover, cAMP also plays a role in intracellular and mitochondrial Ca²⁺ signalling in cold‐acclimated brown adipocytes.     

Spatial distribution of intracellular,free Ca2+ in isolated rat parotid acini

The spatial distribution of intracellular, free Ca²⁺ ([Ca²⁺]_i) in rat parotid acini was measured by imaging fura-2 fluorescence from individual acinar cells by means of a digital imaging microscope. Upon cholinergic stimulation in a Krebs-Ringer bicarbonate buffer at (37° C), [Ca²⁺]_i increased synchronously at both the basolateral and luminal membranes as well as in all cells of the secretory endpiece, reaching peak [Ca²⁺]_i levels 1 s after stimulation. Atropine addition caused a rapid down-regulation of [Ca²⁺]_i, which, however, never reached prestimulatory levels. When acini were stimulated in a medium containing 5 nM Ca²⁺, the Ca²⁺ mobilization arising from internal pools caused an increase in [Ca²⁺]_i predominantly near the basolateral area, where the endoplasmic reticulum is located, and standing Ca²⁺ gradients were observed for up to 10 s. A mathematical model is developed to simulate the time courses of the Ca²⁺ profiles through the cytoplasm using estimated values of the Ca²⁺ diffusion coefficients and the cytosolic Ca²⁺ buffering capacity. It is concluded that under physiological conditions, the Ca²⁺ release from the endoplasmic reticulum is responsible for the activation of the basolaterally located K⁺ channels. Furthermore, Ca²⁺ influx from the interstitium is responsible for much of the rise in [Ca²⁺]_i near the luminal membranes, where the Cl^– channels are supposed to be located.     

Interference with Ca2+ release activated Ca2+ (CRAC) channel function delays T‐cell arrest in vivo

Entry of lymphocytes into secondary lymphoid organs (SLOs) involves intravascular arrest and intracellular calcium ion ([Ca²⁺]_i) elevation. TCR activation triggers increased [Ca²⁺]_i and can arrest T‐cell motility in vitro. However, the requirement for [Ca²⁺]_i elevation in arresting T cells in vivo has not been tested. Here, we have manipulated the Ca²⁺ release‐activated Ca²⁺ (CRAC) channel pathway required for [Ca²⁺]_i elevation in T cells through genetic deletion of stromal interaction molecule (STIM) 1 or by expression of a dominant‐negative ORAI1 channel subunit (ORAI1‐DN). Interestingly, the absence of CRAC did not interfere with homing of naïve CD4⁺ T cells to SLOs and only moderately reduced crawling speeds in vivo. T cells expressing ORAI1‐DN lacked TCR activation induced [Ca²⁺]_i elevation, yet arrested motility similar to control T cells in vitro. In contrast, antigen‐specific ORAI1‐DN T cells had a twofold delayed onset of arrest following injection of OVA peptide in vivo. CRAC channel function is not required for homing to SLOs, but enhances spatiotemporal coordination of TCR signaling and motility arrest.     

Ca2+-dependent unidirectional vesicular release detected with a carbon-fibre electrode in rat pancreatic acinar cell triplets

An amperometric constant-voltage method for detection of serotonin oxidation currents was applied to pancreatic acinar cell triplets to determine the site of release of granular content following an increase in [Ca²⁺]_i. The carbon fibre electrode, fabricated to be compatible with a conventional patch-clamp amplifier, was voltage-clamped at 600 mV exceeding the serotonin oxidation voltage, 300 mV. The electrode was placed on the different regions of cell surface of acinar cell triplets loaded with exogenous serotonin. Transient oxidation currents were detected only when the electrode was placed on the acinar lumen after stimulation with a Ca²⁺ ionophore, A23187, but never observed on the basal or lateral cell surface, or paracellular clefts. No such current responses were observed in the acinar cells without serotonin loading. The results indicate that the A23187-induced sustained increase in [Ca²⁺]_i discharges serotonin specifically into the lumen, and provides direct evidence for the presence of Ca²⁺-dependent unidirectional release of granular contents in pancreatic acinar cells.     

Intracellular calcium in mammalian brain cells: fluorescence measurements with quin2

Summary Dispersed brain cells from 12–14 day old mouse embryos were loaded with the Ca²⁺-sensitive fluorescent probe, quin2 and shown to have a resting intracellular Ca²⁺ concentration ([Ca²⁺]_i) of 158 nM (SE ± 5) in the presence of 1 mM [Ca²⁺]_o. When external [Ca²⁺] was raised from 0 to 1 mM there was an increase of [Ca²⁺]_i of 70 nM; with further additions of Ca to >10 mM [Ca²⁺]_o the level of [Ca²⁺]_i increased by <25 nM. Releasable intracellular Ca²⁺ stores, estimated from the increase in [Ca²⁺] produced by 4Br A23187 in the absence of extracellular Ca²⁺, were 24 fmol/10⁶ cells. A small increase in [Ca²⁺]_i could be produced by the mitochondrial inhibitor, carbonyl cyanide m-chlorophenylhydrazone (CCCP). When extracellular K⁺ was raised by 10–20 mM, intracellular Ca²⁺ levels increased from 152 (SE ± 7) to 204 nM (SE ± 10). These K⁺-induced increases in [Ca²⁺]_i were blocked by verapamil, did not occur in the absence of extracellular Ca²⁺, and presumably reflect the activation of voltage-dependent Ca²⁺ channels. N-methyl-D-aspartic acid (NMDA) evoked an increase in [Ca²⁺]_i, while the kainate-like lathyrus sativus neurotoxin, L-3-oxalyl-amino-2aminopropionic acid (L-3,2-OAP) did not; this is consistent with previous observations of different and respectively Ca²⁺-dependent and -independent mechanisms of action of these excitatory amino acids.     

Carboxyeosin decreases the rate of decay of the [Ca2+]i transient in uterine smooth muscle cells isolated from pregnant rats

 In myometrial smooth muscle cells the rate of decline of intracellular calcium ([Ca²⁺]_i) is determined by Ca²⁺ extrusion from the cell and uptake into intracellular stores. The relative quantitative contribution of these processes however, has not been established. We therefore examined the effect of the sarcolemmal Ca²⁺ pump inhibitor, carboxyeosin, on the rate of the [Ca²⁺]_i transient decline in myocytes isolated from pregnant rat uterus. Indo-1 was used in conjunction with the whole-cell patch-clamp technique to measure [Ca²⁺]_i simultaneously with transmembrane calcium current (I _Ca). [Ca²⁺]_i transients were elicited by repetitive membrane depolarization to simulate the natural pattern of uterine electrical activity. The rate of [Ca²⁺]_i removal was calculated from the falling phase of the [Ca²⁺]_i transient. Pre-treatment of the cells with 2 μM carboxyeosin led to a marked decrease in the rate of [Ca²⁺]_i transient decay, suggesting that the sarcolemmal Ca²⁺ pump is involved in the calcium extrusion process. Removal of the extracellular Na also decreased the rate of [Ca²⁺]_i decay, indicating an important role for the Na⁺/Ca²⁺ exchange. When both the sarcolemmal Ca²⁺ pump and Na⁺/Ca²⁺ exchange were inhibited the cell failed to restore [Ca²⁺]_i after the stimulation. Comparison of the rate constants of [Ca²⁺]_i decay in control conditions and after carboxyeosin treatment shows that approximately 30% of [Ca²⁺]_i decay is due to the sarcolemmal calcium pump activity. The remaining 70% can be attributed to the activity of Na⁺/Ca²⁺ exchanger and the intracellular calcium stores. Received: 17 July 1998 / Received after revision: 23 September 1998 / Accepted: 25 September 1998     

Intracellular Ca2+ transients in HT29 cells induced by hypotonic cell swelling

Cell swelling induced by hypotonic solution led to an osmolality-dependent increase in intracellular Ca²⁺ activity ([Ca²⁺]_i) in HT₂₉ cells. At moderate reductions in osmolality from 290 to 240 or 225 mosmol/l in most cases only a small monophasic increase of [Ca²⁺]_i to a stable plateau of 10–20 nmol/l above resting [Ca²⁺]_i was observed. Lower osmolalities resulted in a triphasic increase of [Ca²⁺]_i to a peak value. In a first phase after the volume change, lasting 20–40 s, [Ca²⁺]_i increased slowly by about 30 nmol/l. Thereafter [Ca²⁺]_i increased more rapidly within 20–30 s to a peak value. This peak was 189±45 nmol/l (190 mosmol/l, n=9) and 243±41 nmol/l (160 mosmol/l, n=20) above resting [Ca²⁺]_i. The peak was then followed by a decline of [Ca²⁺]_i over the next 2–3 min to a stable plateau value of 28±6 (n=6) and 32±11 nmol/l (n=11) above resting [Ca²⁺]_i at 190 and 160 mosmol/l, respectively. The plateau lasted as long as the hypotonic solution was present. Under Ca²⁺-free bath conditions the peak value for the cell-swelling-induced [Ca²⁺]_i transient was reached significantly later (60–100 s, compared to 40–60 s under control conditions). The peak values under Ca²⁺-free conditions were not significantly lower. This indicates that the [Ca²⁺]_i peak was mostly of intracellular origin. No [Ca²⁺]_i plateau phase was observed under Ca²⁺-free bath conditions. With the use of the fura-2-Mn ²⁺ quenching technique an increased Ca²⁺ influx induced by hypotonic cell swelling was shown (160 mosmol/l; n=4). This influx started immediately after or simultaneously with the cell swelling and preceded the [Ca²⁺]_i peak for more than 50 s.This study was supported by DFG grant Gr 480/10.     

Mechanisms underlying changes of tetanic [Ca2+]i and force in skeletal muscle

Force development in skeletal muscle is driven by an increase in myoplasmic free [Ca²⁺] ([Ca²⁺]_i) due to Ca²⁺ release from the sarcoplasmic reticulum (SR). The magnitude of [Ca²⁺]_i elevation during stimulation depends on: (a) the rate of Ca²⁺ release from the SR, (b) the rate of Ca²⁺ uptake by the SR, and (c) the myoplasmic Ca²⁺ buffering. We have used fluorescent Ca²⁺ indicators to measure [Ca²⁺]_i in intact, single fibres from mouse and Xenopus muscles under conditions where one or more of the above factors are changed. The following interventions resulted in increased tetanic [Ca²⁺]_i: β-adrenergic stimulation, which potentiates the SR Ca²⁺ release; application of 2,5-di(tert-butyl)-1,4-benzohydroquinone, which inhibits SR Ca²⁺ pumps; application of caffeine, which facilitates SR Ca²⁺ release and inhibits SR Ca²⁺ uptake; early fatigue, where the rate of SR Ca²⁺ uptake is reduced; acidosis, which reduces both the myoplasmic Ca²⁺ buffering and the rate of SR Ca²⁺ uptake. Reduced tetanic [Ca²⁺]_i was observed in late fatigue, due to reduced SR Ca²⁺ release, and in alkalosis, due to increased myoplasmic Ca²⁺ buffering. Force is monotonically related to [Ca²⁺]_i, but depends also on the myofibrillar Ca²⁺ sensitivity and the maximum force cross-bridges can produce. This is clearly illustrated by changes of intracellular pH where, despite a lower tetanic [Ca²⁺]_i, tetanic force is higher in alkalosis than acidosis due to increases of myofibrillar Ca²⁺ sensitivity and maximum cross-bridge force.     

Involvement of intracellular calcium ions in the release of the fluorescent dye calcein by cholinergic and α-adrenergic agonists from rat parotid acinar cells

Effects of cholinergic and adrenergic agonists on the secretion of the fluorescent dye calcein were examined to clarify the involvement of calcium ions in the secretion of calcein from acinar cells dispersed from the rat parotid gland. Addition of carbachol (CCh) and noradrenalin (NA), but not isoproterenol (IPR), enhanced the net release of calcein from acinar cells during the subsequent 10 min in a dose range from 10^–8 M to 10^–6 M. The net release of calcein reached a maximum 7 min after the addition of CCh. The release of calcein was suppressed by the simultaneous additions of atropine with CCh, or phenoxybenzamine with NA. Addition of CCh induced a sustained dosedependent increase in the intracellular levels of calcium ions, ([Ca²⁺]_i). Addition of NA at 10^–6 M increased [Ca²⁺]_i. Phenoxybenzamine completely inhibited the NA-induced increase, but propranolol did not. The removal of extracellular calcium ions did not influence the release of calcein induced by 10^–6 M CCh, but it abolished the sustained increase in [Ca²⁺]_i. The transient increase in [Ca²⁺]_i induced by CCh was observed in the absence of extracellular calcium ions. A calcium ion chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid (BAPTA) inhibited the CCh-induced release of calcein. The calcium ionophore, A23187 (2.5×10^–6 M), but not 10^–3 M dibutyryl cAMP, evoked the release of calcein. It also increased [Ca²⁺]_i. Removal of extracellular calcium ions suppressed the A23187-induced release of calcein. These results suggest that the release of calcein from parotid acinar cells is transiently induced through an increase in [Ca²⁺]_i by muscarinic and -adrenergic agonists and may represent the initial process of salivary secretion.