Activation of Ca2+-dependent Cl- and K+ conductances in rat and mouse parotid acinar cells

Isolated acinar cells from rat and mouse parotid glands were studied with patch-clamp whole-cell current recordings. Acetylcholine (ACh) stimulation caused a transient inward current at a membrane potential of -70 mV, and a sustained outward current at a membrane potential of 0 mV, in quasi physiological Na+, K+ ion gradients, except the zero-Cl- ion gradient condition across the membrane. The reversal potential obtained from the ACh-evoked steady current was about -75 mV, in this ionic condition. When major Cl- ions of both the pipette and the bath solution were replaced, either by glutamate or by sulphate, only a large outward current was observed, at a membrane potential of -60 mV, in the presence of ACh. The addition of Ca2+-ionophore A23187 caused responses similar to those evoked by ACh. The reversal potential of A23187-induced current was close to the K+ equilibrium potential of -90 mV, in a Cl- -free condition. When K+-free NaCl solution was used in the pipette and the bath, A23187 caused only a large inward current, at a membrane potential of -60 mV. The reversal potential of A23187-evoked current was about -15 mV, in a symmetrical K+-free, NaCl condition. These results suggest that the ACh and A23187 activate Cl- as well as K+ conducting pathways via an increase in [Ca2+]i in the parotid acinar cells. The A23187-evoked large K+ current could not be explained solely by a rise in open probability of the channels.     

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.     

Alteration of expression of Ca2+ signaling proteins and adaptation of Ca2+ signaling in SERCA2+/- mouse parotid acini

PURPOSE: The sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), encoded by ATP2A2, is an essential component for G-protein coupled receptor (GPCR)-dependent Ca2+ signaling. However, whether the changes in Ca2+ signaling and Ca2+ signaling proteins in parotid acinar cells are affected by a partial loss of SERCA2 are not known. MATERIALS AND METHODS: In SERCA2+/- mouse parotid gland acinar cells, Ca2+ signaling, expression levels of Ca2+ signaling proteins, and amylase secretion were investigated. RESULTS: SERCA2+/- mice showed decreased SERCA2 expression and an upregulation of the plasma membrane Ca2+ ATPase. A partial loss of SERCA2 changed the expression level of 1, 4, 5-tris-inositolphosphate receptors (IP3Rs), but the localization and activities of IP3Rs were not altered. In SERCA2+/- mice, muscarinic stimulation resulted in greater amylase release, and the expression of synaptotagmin was increased compared to wild type mice. CONCLUSION: These results suggest that a partial loss of SERCA2 affects the expression and activity of Ca2+ signaling proteins in the parotid gland acini, however, overall Ca2+ signaling is unchanged.     

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     

Synaptic modulation by astrocytes via Ca2+-dependent glutamate release

In the past 15 years the classical view that astrocytes play a relatively passive role in brain function has been overturned and it has become increasingly clear that signaling between neurons and astrocytes may play a crucial role in the information processing that the brain carries out. This new view stems from two seminal observations made in the early 1990s: 1. astrocytes respond to neurotransmitters released during synaptic activity with elevation of their intracellular Ca2+ concentration ([Ca2+]i); 2. astrocytes release chemical transmitters, including glutamate, in response to [Ca2+]i elevations. The simultaneous recognition that astrocytes sense neuronal activity and release neuroactive agents has been instrumental for understanding previously unknown roles of these cells in the control of synapse formation, function and plasticity. These findings open a conceptual revolution, leading to rethink how brain communication works, as they imply that information travels (and is processed) not just in the neuronal circuitry but in an expanded neuron-glia network. In this review we critically discuss the available information concerning: 1. the characteristics of the astrocytic Ca2+ responses to synaptic activity; 2. the basis of Ca2+-dependent glutamate exocytosis from astrocytes; 3. the modes of action of astrocytic glutamate on synaptic function.     

Initiation site of Ca(2+) entry evoked by endoplasmic reticulum Ca(2+) depletion in mouse parotid and pancreatic acinar cells

Purpose

In non-excitable cells, which include parotid and pancreatic acinar cells, Ca²⁺ entry is triggered via a mechanism known as capacitative Ca²⁺ entry, or store-operated Ca²⁺ entry. This process is initiated by the perception of the filling state of endoplasmic reticulum (ER) and the depletion of internal Ca²⁺ stores, which acts as an important factor triggering Ca²⁺ entry. However, both the mechanism of store-mediated Ca²⁺ entry and the molecular identity of store-operated Ca²⁺ channel (SOCC) remain uncertain.

Materials and Methods

In the present study we investigated the Ca²⁺ entry initiation site evoked by depletion of ER to identify the localization of SOCC in mouse parotid and pancreatic acinar cells with microfluorometeric imaging system.

Results

Treatment with thapsigargin (Tg), an inhibitor of sarco/ endoplasmic reticulum Ca²⁺-ATPase, in an extracellular Ca²⁺ free state, and subsequent exposure to a high external calcium state evoked Ca²⁺ entry, while treatment with lanthanum, a non-specific blocker of plasma Ca²⁺ channel, completely blocked Tg-induced Ca²⁺ entry. Microfluorometric imaging showed that Tg-induced Ca²⁺ entry started at a basal membrane, not a apical membrane.

Conclusion

These results suggest that Ca²⁺ entry by depletion of the ER initiates at the basal pole in polarized exocrine cells and may help to characterize the nature of SOCC.     

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

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

Excess divalent cations activate Ca2+-mobilizing receptors in pancreatic acinar cells

In single, enzymatically dissociated, rat pancreatic acinar cells, external application of excess divalent cations (Ca²⁺, Sr²⁺, Ba²⁺, Ni²⁺ and Mg²⁺ over 50 mM) induced Ca²⁺-dependent current responses monitored with the whole-cell recording technique. Inclusion of either EGTA, heparin or GDP[S] in the internal solution or treatment of acinar cells with a phorbol ester abolished the divalent-cation-induced responses. In contrast, internal inositol trisphosphate (InsP ₃) or GTP[S] potentiated the responses. The results indicate that excess divalent cations activate membrane surface receptors or receptor/effector complexes, thereby inducing InsP ₃-mediated Ca²⁺ mobilization. The mechanism may be due to modulation of the receptors by changes in electrical profile through indirect action of divalent cations on membrane surface charges, i. e. neutralization of anionic charges. This proposal was supported by the evidence that the trivalent cation, La³⁺, and the polyvalent cation, protamine, both at much lower concentrations, could induce Ca²⁺-dependent responses, which were abolished by internal application of heparin, GDP[S] or a high concentration of EGTA or by protein kinase C activation with a phorbol ester.     

Formation of post-confluence structure in human parotid gland acinar cells on PLGA through regulation of E-cadherin

As a potential solution for patients to retrieve their lost salivary gland functions, tissue engineering of an auto-secretory device is profoundly needed. Under serum-free environment, primary human parotid gland acinar (PGAC) cells can be obtained. After reaching confluence, PGAC cells spontaneously form three-dimension (3D) cell aggregations, termed post-confluence structure (PCS), and change their behaviors. Poly (lactic-co-glycolic acid) (PLGA) has been widely used in the field of biomedical applications because of its biodegradable properties for desired functions. Nonetheless, the role of PLGA in facilitating PGAC cells to form PCS has seldom been explored to recover epithelial characteristics. In this study, PGAC cells were found to have a greater tendency to form PCS on PLGA than on tissue culture polystyrene (TCPS). By tracing cell migration paths and modulating E-cadherin activity with specific inhibitor or antibody, we demonstrated that the static force of homophilic interaction on surfaces of individual cells, but not the dynamics of cell migration, played a more important role in PCS formation. Thus, PLGA was successfully confirmed to support PGAC cells to form more PCS through the effects on enhancing E-cadherin expression, which is associated with FAK/ILK/Snail expression in PGAC cells. This result indicates that selective appropriate biomaterials may be potentially useful in generating 3D PCS on two-dimension (2D) substrate without fabricating a complex 3D scaffold.     

Acetylcholine stimulates a Ca2+-dependent Cl− conductance in mouse lacrimal acinar cells

Patch-clamp whole-cell current recordings under voltage-clamp conditions were carried out on isolated mouse exorbital lacrimal acinar cells. Acetylcholine evoked outward current at a membrane potential of –20 mV whereas an inward current was observed at –80 mV. The outward current is due to the well-known calcium-activated K⁺ channels whereas the inward current was Cl^– dependent. The acetylcholine-evoked Cl^– current was abolished when the intracellular Ca²⁺ concentration was clamped at very low levels by a high intracellular EGTA concentration. Acetylcholine therefore activates a Ca²⁺-dependent Cl^– conductance in mouse lacrimal acinar cells.     

2-Aminoethoxydiphenyl borate inhibits agonist-induced Ca2+ signals by blocking inositol trisphosphate formation in acutely dissociated mouse pancreatic acinar cells

Evidence suggests that 2-aminoethoxydiphenyl borate (2-APB) modulates intracellular Ca²⁺ signals in a complex manner. 2-APB inhibits or potentiates intracellular Ca²⁺ signals in different cell types, perhaps through different mechanisms. Here, we report a novel mechanism underlying 2-APB-induced inhibition of agonist-activated Ca²⁺ oscillations in mouse pancreatic acinar cells, using patch-clamp and biochemical techniques. Pre-treatment of the cells with 100 µM 2-APB completely abolished ACh- but not inositol trisphosphate (InsP₃)-induced Ca²⁺ oscillations, suggesting that the mechanism of inhibition occurs between cytoplasmic receptors and InsP₃ receptor activation. In addition, 100 µM 2-APB significantly inhibited ACh-induced phospholipase C (PLC) activation. These findings indicate that, in mouse pancreatic acinar cells, in addition to modulating InsP₃ receptors and blocking the store-operated Ca²⁺ pathway, high concentrations of 2-APB also inhibit agonist-induced Ca²⁺ signals by reducing InsP₃ formation.     

α1-Adrenergic regulation of Cl− and Ca2+ movements in rat parotid acinar cells

In rat parotid acinar cells, maximal ₁-adrenergic receptor stimulation (10^–5 M epinephrine +10^–5 M propranolol) leads to a rapid (<10 s), 4–5-fold elevation in cytosolic Ca²⁺ ( 800 nM at peak) which decreases to 50% of peak Ca²⁺ by 3–4 min. Similarly, cells preloaded with³⁶Cl^– show a rapid (<10 s) 35–50% loss of³⁶Cl^– which returns to 80% of resting values in 3–4 min. Both responses are dependent on epinephrine, with half-maximal effects achieved at 2×10^–7 M and 2×10^–6 M agonist for Cl^– and Ca²⁺, respectively. In the presence of low extracellular Ca²⁺ (i.e. with EGTA), the initial rapid changes in cellular Ca²⁺ and Cl^– are unaltered. However, cellular Ca²⁺ and Cl^– levels return to basal values sooner than when extracellular Ca²⁺ is present (within 2 and 3 min, respectively). Maximal epinephrine-induced Ca²⁺ and Cl^– responses are unaffected by the ₂-adrenergic antagonist, yohimbine, are completely blocked by the ₁-adrenergic antagonist, SZL-49, and are similar to ion fluxes induced by maximal muscarinic-cholinergic receptor stimulation (10^–5 M carbachol). The data suggest that a close association exists between mobilization of intracellular Ca²⁺ and Cl^– content in rat parotid acinar cells after ₁-adrenocetor stimulation.     

Nitric oxide synthesis causes inositol phosphate production and Ca2+ release in rat parotid acinar cells

The activity of nitric oxide synthase (NOS) in rat parotid acinar cells was measured using a newly synthesized fluorescent NO indicator DAF-2/DA. Our results show that NO production is most effectively stimulated by activation of the beta-adrenergic receptor, and to a minor extent by substance P (SP). NO activates the production of cGMP, an intracellular messenger that has been shown to release Ca2+ from ryanodine-sensitive intracellular stores. We found that cGMP is also able to release Ca2+ from ryanodine-insensitive intracellular stores. Our data show that a rise in the cGMP concentration induces inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] synthesis and Ca2+ release from intracellular stores.     

Ca2+-activated K+-channels in the nuclear envelope isolated from single pancreatic acinar cells

The patch-clamp techniques are applied to the outer membrane of the nuclear envelope isolated from rat pancreatic acinar cells. The nucleus identified under an inverted microscope was removed by cell surgery from enzymatically dispersed single cells. All the patch-clamp techniques, in situ, excised, and whole-material recordings were applied to the envelope. We have found voltage- and Ca²⁺-activated K⁺-channels with an unitary conductance of 200 pS in the outer membrane. The channels are activated by lumen positive potentials and by an increase in luminal Ca²⁺ concentration. They may play a role for controlling Ca²⁺-release from the lumen of the nuclear envelope (endoplasmic reticulum) to the nucleoplasm and the perinuclear cytoplasm.     

A hypo-osmotically induced increase in intracellular Ca2+ in lactating mouse mammary epithelial cells involving Ca2+ influx

 Using a fluorescent Ca²⁺-sensitive dye, we studied the effect of hypo-osmotic stress on the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) in acini freshly isolated from lactating mouse mammary gland. The basal [Ca²⁺]_i of mammary acini was unaffected by a 50% (v/v) dilution of suspensions with isotonic or hypertonic buffer, or after ionic (iso-osmotic) dilution (external Ca²⁺ was 3 mM). Hypo-osmotic dilution (50%) elicited a rapid increase in [Ca²⁺]_i comprising a large, transient elevation, followed by a maintained plateau phase. No hypo-osmotically induced rise in [Ca²⁺]_i was observed in the absence of extracellular Ca²⁺. Neither microtubule disassembly using nocodazole nor actin disruption with cytochalasin D prevented hypo-osmotically evoked stimulation of [Ca²⁺]_i. Pre-incubation of acini with nifedipine did not prevent hypo-osmotically induced stimulation of [Ca²⁺]_i, whereas a non-specific cation channel blocker, gadolinium, partially inhibited the increases in [Ca²⁺]_i induced by hypo-osmotic stress. Furthermore, the transient component was still apparent, and not diminished in magnitude, after [Ca²⁺]_i had been elevated by mobilisation of Ca²⁺ from intracellular stores using thapsigargin. The results demonstrate that hypo-osmotic stress generates an increase in [Ca²⁺]_i in lactating mammary epithelial cells, the major, transient component of which appears to be due to influx of extracellular Ca²⁺. Received: 15 October 1996 / Received after revision and accepted: 1 November 1996     

Capacitative Ca2+ influx and a Ca2u+-dependent nonselective cation pathway are discriminated by genistein in mouse pancreatic acinar cells

We have investigated the effect of genistein on the hormone-stimulated Ca²⁺ influx and on a 28 pS nonselective cation channel in mouse pancreatic acinar cells using the Ca²⁺ indicator fluo-3 and the patch-clamp technique. The identity of the Ca^2u+ influx pathway has not been established in this cell type so far. Therefore we have investigated the Ca²⁺-dependent nonselective cation channel as a potential pathway for Ca²⁺ influx. Capacitative Ca²⁺ entry was induced by depletion of intracellular Ca²⁺ stores with 500 nM acetylcholine or with the Ca²⁺ ATPase inhibitor 2,5-di-tert- butylhydroquinone. In the presence of 100 M genistein, Ca²⁺ release was unimpaired, whereas Ca²⁺ influx was reversibly suppressed. Patch-clamp experiments demonstrated that genistein had no effect on Ca²⁺-activated nonselective cation channels, the activity of which was measured in excised membrane patches (inside/out) or in the whole-cell configuration. Therefore we conclude that this 28 pS nonselective cation channel does not contribute to Ca²⁺ influx into mouse exocrine pancreatic cells. With the exception of genistein and tyrphostin 25, other tyrosine kinase inhibitors such as methyl-2,5-dihydroxycinnamate, lavendustin A, herbimycin A, and tyrphostin B56 were without effect on Ca²⁺ signalling. Thus, the involvement of tyrosine phosphorylation in the activation of the Ca²⁺ entry mechanism in mouse pancreatic acinar cells is unclear.     

Menkes protein localization in rat parotid acinar cells

The aim was to study the subcellular localization of the Menkes protein (MNK; ATP7A) in the rat parotid acinar cell. MNK protein is a copper transporting P-type ATPase whose absence or dysfunction causes a fatal neurodegenerative disorder, MNK disease. Rat parotid glands were fixed and low-temperature embedded in Lowicryl K4M resin, and ultrathin sections were prepared for immunocytochemical analysis. Immunolocalization of MNK was demonstrated mainly over the trans Golgi network (TGN) area. Immature and mature secretory granules were also labelled, indicating that MNK protein could be involved here in copper secretion from acinar cells into saliva, consistent with a proposed cariostatic role for copper.     

Effects of external calcium on acetylcholine-evoked increases in membrane capacitance in rat pancreatic acinar cells

Effects of external calcium on acetylcholine-induced increases in membrane capacitance and conductance were investigated with the patch-clamp technique in combination with the phase-sensitive detection method, in single dialysed pancreatic acinar cells of rats. Both increases depended on an increase in [Ca²⁺]_i, and a high concentration of EGTA in the cell-dialysing solution made ACh ineffective. In acinar cells exposed to a bathing solution containing the normal concentration of Ca²⁺ (2.5 mM CaCl₂), the increase in membrane capacitance was transient and synchronous with that in membrane conductance (current) in response to 0.2 M acetylcholine. However, in a bathing solution without CaCl₂ and with EGTA (0.2 mM), the increase in membrane capacitance was sustained after the membrane conductance recovered to the original level during the ACh-stimulation. The evidence suggests that external calcium facilitates either the resealing of the fusion- or fission-pores formed at the contact between the secretory granule and the luminal cell membrane, or the membrane retrieval (endocytosis) in Ca²⁺-dependent exocytosis.     

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

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

Differential variations in Ca2+ entry, cytosolic Ca2+ and membrane capacitance upon steady or action potential depolarizing stimulation of bovine chromaffin cells

Aims: This study looks into the physiology of the exocytosis of catecholamines released by adrenal medullary chromaffin cells. We have comparatively explored the exocytotic responses elicited by two different patterns of depolarizing stimulation: the widely employed square depolarizing pulses (DPs) and trains of acetylcholine‐like action potentials (APs), likely the physiological mode of stimulation in the intact innervated adrenal medulla. APs were applied at 30 Hz, a frequency similar to that produced in a stressful situation. Methods: Patch‐clamp, cell membrane capacitance, single cell amperometry and fluorescence were the techniques used. The variations of calcium entry measured as the integral of the calcium current, cytosolic calcium (measured with the calcium‐sensitive fluorescent probe fluo‐4) and exo‐endocytosis (membrane capacitance variations) were the parameters measured. Results: Trains of AP depolarizations produced distinct responses compared to those of square depolarizations: (1) Calcium current amplitude decreased to a lesser extent along the AP train; (2) calcium entry and capacitance increments raised linearly with stimulation time whereas they deviated from linearity when square depolarizations were used; (3) slower activation and faster delayed decay phase of cytosolic calcium transients; (4) capacitance increments varied linearly with calcium entry with APs and deviated from linearity with longer depolarizations; (5) little endocytosis after stimulation with longer trains of APs and pronounced endocytosis with longer square depolarizations. Conclusions: Stimulation of chromaffin cells with trains of APs produced patterns of cytosolic calcium transients, exocytotic and endocytotic responses quite different from those elicited by the widely employed DPs. Our study is relevant from the methodological and physiological points of view.