Regulation of Particulate Matter-Induced Mucin Secretion by Transient Receptor Potential Vanilloid 1 Receptors

Exposure to airborne particulate matter (PM) is a worldwide health problem. Previous studies have reported that PMs induced depolarizing currents and increased intracellular Ca²⁺ in human bronchial epithelial cells. Ca²⁺ plays important role in the regulation of mucus exocytosis, and mucin hypersecretion is a key pathological feature of inflammatory respiratory diseases. To explore more mechanisms underlying PM toxicity, we measured PM-induced mucin secretion in human bronchial epithelial (16HBE) cells. MUC5AC secretion and cyclic adenosine monophosphate (cAMP) level were detected by ELISA. Transient receptor potential vanilloid (TRPV)1 inward currents were examined by electrophysiology. Ca²⁺ concentration was assessed by laser scanning confocal microscope. Exposure of PMs to 16HBE cells was found to induce mucin secretion, as a consequence of sustained Ca²⁺ influx and cAMP increase through TRPV1 receptors. Mucin secretion was completely inhibited by TRPV1 receptor antagonist capsazepine. Removal of Ca²⁺ by Ca²⁺ chelator BAPTA or inhibition of protein kinase A (PKA) by the PKA inhibitors H-89 each partially reduced PC₂s-induced mucin secretion. The combination of BAPTA and H-89 completely prevented mucin secretion mediated by PMs. These results suggest that PM induces mucin secretion through Ca²⁺ influx and cAMP/PKA pathway by TRPV1 receptors in human bronchial epithelial cells, thereby providing a potential mechanism to reduce PM toxicity.     

Muscarinic, α1 and peptidergic agonists stimulate phosphoinositide hydrolysis and regulate mucin secretion in rat submandibular gland cells

Three classes of agonists, associated with Ca²⁺-mobilization — ₁-adrenergic (methoxamine), muscarinic (carbachol) and peptidergic (substance P, SP) — significantly stimulated the secretion of mucin from enzymatically-dispersed rat submandibular gland acinar cells. The same three secretagogues also caused the hydrolysis of membrane inositol phospholipids, resulting in elevated cellular levels of inositol phosphates, particularly inositol 1,4,5-trisphosphate (IP₃). Exogenous IP₃ elicited the dose-dependent release of mucin in dispersed cells suggesting that agonist-generated endogenous IP₃ may provoke a secretory response. IP₃ and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) in combination, stimulated an additive secretion of mucin in the model. The potential use of these two agents as specific probes of the IP₃- and diacylglycerol-associated legs of the polyphosphoinositide (PPI) breakdown pathway is indicated. Although all three agonists shared a common action in stimulating PPI hydrolysis, their effects on the -adrenergic mucosecretory response were inconsistent. A brief preincubation of cells with carbachol or SP significantly reduced the subsequent isoproterenol (IPR)-provoked secretion of mucin, whereas methoxamine plus IPR stimulated an additive response. The mechanisms underlying these opposite effects are not known. Failure of IP₃ or TPA to modify IPR responses suggests that modulation of the response may operate at a locus before the generation of diacylglycerol and IP₃, possibily at the level of signal transduction. The study indicates a role for Ca²⁺-mobilizing, agonists in controlling submandibular mucin secretion and provides evidence that receptor-linked phosphoinositide hydrolysis is an early stage in their stimulus-secretion coupling mechanism.     

Tetraethylammonium-insetisitive,Ca2+-activated whole-cell K+ currents in rat submandibular acinar cells

Using whole-cell patch-clamp techniques, we demonstrate, for the first time, that rat submandibular acinar cells contain a tetraethylammonium (TEA)-insensitive, Ca²⁺-activated K⁺ conductance which is not attributable to large conductance, voltage-sensitive, Ca²⁺-dependent K⁺ channels (maxi-K⁺ channels). Taken together with our recent K⁺ efflux and fluid secretion studies in intact rat submandibular gland, we postulate that the K⁺ conductance reported here may be involved in the basolateral K⁺ efflux pathway activated by cytosolic Ca²⁺ concentration during secretion by this gland.     

Effects of 2,5-di(tert-butyl)-1,4-hydroquinone on intracellular free Ca2+ levels and histamine secretion in RBL-2H3 cells

The effects of 2,5-di(tert-butyl)-1,4-hydroquinone (DTBHQ) on the intracellular free Ca²⁺ level ([Ca²⁺]_i) and histamine secretion of rat basophilic leukemia (RBL-2H3) cells were examined. DTBHQ (0.1–10 µmol/l) alone induced rapid and sustained increases in [Ca²⁺]_i in a concentration-dependent manner. In cells sensitized with anti-dinitrophenyl IgE, DTBHQ (10 µmol/1) further increased the antigen (dinitrophenylated BSA)-induced Ca²⁺ response. In the absence of external Ca²⁺ with addition of 1 mmol/1 EGTA, both DTBHQ (10 µmol/l) and the antigen (10 µg/ml) induced transient increase in [Ca²⁺]_i. In sensitized cells, both DTBHQ (10 µmol/1) and antigen (10 µg/ml) elicited histamine secretion, although the response was far stronger in the latter case. The DTBHQ-induced histamine secretion was markedly enhanced by addition of the protein kinase C activator, phorbol 12-myristate 13-acetate (TPA) (10 ng/ml) whereas TPA alone did not cause any increase. Moreover, DTBHQ enhanced the antigen-induced histamine secretion. The results suggest that DTBHQ increases [Ca²⁺]_i and enhances antigeninduced histamine secretion while DTBHQ alone does not cause as much histamine secretion as antigen, which support the idea that calcium signals are necessary but are not sufficient for maximum histamine secretion in RBL-2H3 cells.     

Exogenous nitric oxide stimulates mucin secretion from LS174T colonic adenocarcinoma cells

The effect of exogenous and endogenous nitric oxide on the secretion of mucins from the human colonic adenocarcinoma cell-line LS174T was studied. Mucin secretion was followed by measuring the release of [³H]-glucosamine metabolically labelled glycoproteins eluted in the void volume of Sepharose 4B column chromatography. In response to exogenously produced nitric oxide from sodium nitroprusside, mucin secretion occured in a time- and dose-dependent fashion that preceded epithelial cell damage. However, in the presence of the nitric oxide scavenger myoglobin, mucin secretion and cell damage were abrogated. Endogenously produced nitric oxide did not affect mucin secretion as the addition of excess L-arginine, the substrate for nitric oxide synthase, the removal of arginine from the culture medium with arginase or the inhibition of nitric oxide synthase with the competitive inhibitor N^G-monomethyl-L-arginine had no effect on basal mucin release. These results suggest that exogenously produced nitric oxide can directly affect mucin secretion as a cytoprotective mechanism.accepted by I. Ahnfelt-Rønne     

Muscarinic regulation of phospholipase D and its role in arachidonic acid release in rat submandibular acinar cells

The characteristics of muscarinic cholinergic-induced phospholipase D (PLD) activation, and the involvement of the enzyme in the release of arachidonic acid were examined in rat submandibular acinar cells. Carbachol produced a dose-related activation of PLD to around fivefold control values at 100 M agonist concentration. This was associated with the appearance of free choline, phosphatidic acid and arachidonic acid, indicating that the PLD substrate was phosphatidylcholine. The response to carbachol was inhibited by 60% by U73122, a blocker of a phospholipase C (PLC) specific to phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P ₂], suggesting that the cleavage of phosphatidylcholine by PLD was, at least in part, secondary to agonist-coupled hydrolysis of PtdIns(4,5)P ₂ by PLC. Consistent with this, PLD was also activated to levels comparable to those induced by carbachol, by the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), and the Ca²⁺ mobilizer, thapsigargin, two agents that respectively mimic the activation of protein kinase C (PKC) by diacylglycerol and the elevation of cytosolic Ca²⁺by inositol 1,4,5-triphosphate [Ins(1,4,5)P ₃] in the phosphoinositide effect. The cell-permeant Ca²⁺chelator 1,2-bis-(O-aminophenoxy)-ethane-N, N,N,N-tetraacetic acid, tetraacetoxymethyl ester (BAPTA/AM) abolished the thapsigargin-induced activation of PLD and inhibited the responses of PLD to carbachol and TPA by 60%. The PKC inhibitor, Ro-31-8220, also inhibited the activation of PLD by carbacol and TPA to a level of approximately double control values, but had no effect on the thapsigargin-induced elevation of PLD. A role for both the PKC-associated and Ca²⁺-mobilizing arms of the PtdIns(4,5)P ₂-PLC pathway in PLD regulation is thus suggested. Pretreatment of cells with the phosphatidate phosphohydrolase blocker, propranolol, significantly enhanced the carbachol-induced elevation of phosphatidic acid, but decreased agonist-stimulated production of diacylglycerol and arachidonic acid, indicating that phosphatidlycholine was the likely source of arachidonic acid. We therefore propose that, in submandibular mucous acinar cells, muscarinic activation of the PtdIns(4,5)P ₂-PLC pathway regulates phosphatidylcholine-specific PLD through both the PKC- and Ca²⁺-mobilizing arms of the phosphoinositide response, and that diacylglycerol, derived from phosphatidylcholine via phosphatidic acid, is a source of free arachidonic acid.     

Vasopressin responses in electrically coupled A7r5 cells

Changes in cytosolic Ca²⁺ concentration ([Ca²⁺]_i) and in membrane potential were monitored in single A7r5 smooth-muscle cells during spontaneous spiking and after arginine vasopressin stimulation. Spontaneous Ca²⁺ oscillations, which were associated with the occurrence of action potentials, occurred in about 90% of the confluent monolayers investigated. This spontaneous activity was synchronized amongst all the cells of the monolayer, indicating that the cells were electrically coupled. Arginine vasopressin stimulation produced a [Ca²⁺]_i rise that was about 5 times higher than the amplitude of the spontaneous Ca²⁺ oscillations and resulted in a subsequent cessation of spontaneous electrical activity and associated Ca²⁺ spiking, which persisted after [Ca²⁺]_i returned to baseline. Individual cells in the monolayer responded to arginine vasopressin with a different latency. Agonist-induced Ca²⁺ waves within one cell propagated much more slowly than spontaneous [Ca²⁺]_i rises. We conclude that agonist-induced [Ca²⁺]_i increases in an electrically coupled cell monolayer can be asynchronous.     

Different mechanisms of lysophosphatidylcholine-induced Ca mobilization in N2a mouse and SH-SY5Y human neuroblastoma cells

In mice, lysophosphatidylcholine (LPC) was found to be a physiological substrate of neuropathy target esterase, which is also bound by organophosphates that cause a delayed neuropathy in human and some animals. However, the mechanism responsible for causing the different symptoms in mice and humans that are exposed to neuropathic organophosphates still remains unknown. In the present study, we examined and compared the effect of exogenous LPC on intracellular Ca²⁺ overload in mouse N2a and human SH-SY5Y neuroblastoma cells. LPC caused an intracellular Ca²⁺ level ([Ca²⁺]_i) increase in both N2a and SH-SY5Y cells; moreover, the amplitude was higher in N2a cells than that in SH-SY5Y cells. Preincubation of the cells with verapamil, an L-type Ca²⁺ channel blocker, did not affect the LPC-induced Ca²⁺ increase in N2a cells, verapamil inhibited the response by 23% in SH-SY5Y cells. In Ca²⁺-free medium, LPC produced a significant [Ca²⁺]_i decrease in N2a cells, while it caused 64% of total [Ca²⁺]_i increase in SH-SY5Y cells. The results of a cell viability test suggest that N2a cells were more sensitive to LPC than were SH-SY5Y cells. These data suggested that the LPC-induced [Ca²⁺]_i increase was produced in each cell line through different mechanisms. In particular, the [Ca²⁺]_i increase occurred via entry through a permeabilized membrane in N2a cells, but through L-type Ca²⁺ channels as well as by Ca²⁺ release from intracellular Ca²⁺ stores in SH-SY5Y cells. Thus, the symptomatic differences of organophosphate-induced neurotoxicity between mice and humans are probably not related to the diverse amplitudes of intracellular Ca²⁺ overload produced by LPC. Moreover, the demyelination effect induced by LPC in mice may be a consequence of its detergent effect on membranes.     

G-Protein- and capacitatively regulated Ca2+ entry pathways are activated by muscarinic receptor stimulation in a human submandibular ductal cell line

In the human submandibular ductal cell line (HSG) thapsigargin and carbachol stimulated Ca²⁺ release from the internal Ca²⁺ pool, resulting in the activation of capacitatively regulated Ca²⁺ entry (CRCE). This entry pathway was permeant to both Ca²⁺ and Mn²⁺, blocked by Ni²⁺ and insensitive to the muscarinic antagonist, atropine. Carbachol also stimulated an increase in cytosolic [Ca²⁺] in internal Ca²⁺-pool-depleted (i.e.thapsigargin-treated) cells which was dependent on the presence of external Ca²⁺ and blocked by Ni²⁺, demonstrating that it was due to Ca²⁺ entry. However, under the same experimental conditions, carbachol was unable to stimulate Mn²⁺ entry. Additionally, this latter carbachol-stimulated Ca²⁺ entry pathway was blocked by atropine. Pretreatment of HSG cells with AlF₄-increased basal rates of Mn²⁺ entry due to CRCE activation, but attenuated carbachol-stimulated Ca²⁺ entry into thapsigargin-treated cells. The data suggest that two distinct divalent cation entry pathways are activated in muscarinic-receptor-stimulated HSG cells; a CRCE mechanism, permeable to both Mn²⁺ and Ca²⁺, and a second entry mechanism, permeable only to Ca²⁺. The latter does not depend on internal pool depletion, but appears to be regulated via G-protein activation.We thank Dr. Bruce Baum for his encouragement and support during the course of this work. We also thank our colleagues for their cooperation and assistance.     

Measurement of Ca2+-release-dependent inward current reveals two distinct components of Ca2+ release from sarcoplasmic reticulum in guinea-pig atrial myocytes

Ca²⁺ current (L-type) and inward current caused by Ca²⁺ release from the sarcoplasmic reticulum and carried by electrogenic Na⁺/Ca²⁺ exchange have been measured in cultured atrial myocytes from hearts of adult guinea-pigs using whole-cell voltage clamp techniques. The pipette solution, used for internal dialysis of the cells, contained a high concentration, 60 mM or 25 mM, of citrate as a non-saturable low-affinity Ca²⁺-chelating compound. It has been shown previously that Ca²⁺-release-dependent inward current under these conditions is carried by electrogenic Na⁺/Ca²⁺ exchange. Furthermore, Ca²⁺-release-dependent inward current (the release signal) can be completely separated from triggering Ca²⁺ current if brief depolarizations for activating I _Ca are used. In the majority of cells that did not produce spontaneous Ca²⁺ release, conditions could be found that caused the release signal to be split into two components: an early component of variable amplitude and a late component of rather constant amplitude. The delay of the late component with regard to triggering I _Ca was inversely related to the amplitude of the first one. Below a certain amplitude of the first component, the second one failed to be elicited. This suggests the second component to be triggered by the first one. Weakly Ca²⁺-buffered cells produced spontaneous Ca²⁺ release, resulting in irregular transient inward currents at constant membrane-holding potential. Synchronization by trains of step depolarizations unmasked two components also in the spontaneous release signals. In none of the cells studied was any indication of more than two components of the release signal detected. The results are discussed in terms of two distinct compartments of sarcoplasmic reticulum with different properties of Ca²⁺ release.Supported by the Deutsche Forschungsgemeinschaft (FG Konzell)     

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.     

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.     

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.     

Effects of metabolic inhibition by sodium azide on stimulus-secretion coupling in B cells of human islets of Langerhans

Sodium azide (NaN₃), a reversible inhibitor of mitochondrial respiration, blocks glucose-induced electrical activity and insulin secretion in human pancreatic islet B cells. Here we show that brief (10–15 min) application followed by removal of 3 mM NaN₃ results in transient overshoot of electrical activity and insulin secretion even at substimulatory levels of glucose (3–5 mM). In addition, application of NaN₃, even at very low [Ca²⁺]_o, reversibly increases cytosolic Ca²⁺ to levels usually associated with substantial insulin release. These results suggest that (i) metabolic inhibition may reset B cell stimulus-secretion coupling and (ii) a rise in free cytosolic Ca²⁺, by itself, is not sufficient to trigger insulin secretion.Deceased     

Action of stimulatory and inhibitory α-MSH secretagogues on spontaneous calcium oscillations in melanotrope cells of Xenopus laevis

The secretion of α-melanophore-stimulating hormone (α-MSH) from melanotrope cells in the pituitary gland of Xenopus laevis is regulated by various neural factors, both classical neurotransmitters and neuropeptides. The majority of these cells (80%) display spontaneous Ca²⁺ oscillations. In order to gain a better understanding of the external regulation of intracellular Ca²⁺ ([Ca²⁺]_i) in the melanotrope cell, we have examined the action of well known α-MSH secretagogues on the Ca²⁺ oscillations. It is shown that all secretagogues tested also control the oscillatory state of Xenopus melanotropes, that is, the secreto-inhibitors dopamine, isoguvacine (γ-aminobutyric acid, GABA_A agonist), baclofen (GABA_B agonist) and neuropeptide Y evoked a rapid quenching of the spontaneous Ca²⁺ oscillations, whereas the secreto-stimulant sauvagine, an amphibian peptide related to corticotropin releasing hormone, induced oscillatory activity in non-oscillating cells. Supporting argument is given for the idea that the regulation of Ca²⁺ oscillations is a focal point in the regulation of secretory activity of melanotrope cells. There was considerable heterogeneity among melanotrope cells in the threshold of their Ca²⁺ response to secretagogue treatment. This heterogeneity may be the basis for melanotrope cell recruitment observed during physiological adaptations of the animal to the light intensity of its background.     

Mitochondria and chromaffin cell function

Chromaffin cells are an excellent model for stimulus?Csecretion coupling. Ca²⁺ entry through plasma membrane voltage-operated Ca²⁺ channels (VOCC) is the trigger for secretion, but the intracellular organelles contribute subtle nuances to the Ca²⁺ signal. The endoplasmic reticulum amplifies the cytosolic Ca²⁺ ([Ca²⁺]_C) signal by Ca²⁺-induced Ca²⁺ release (CICR) and helps generation of microdomains with high [Ca²⁺]_C (HCMD) at the subplasmalemmal region. These HCMD induce exocytosis of the docked secretory vesicles. Mitochondria close to VOCC take up large amounts of Ca²⁺ from HCMD and stop progression of the Ca²⁺ wave towards the cell core. On the other hand, the increase of [Ca²⁺] at the mitochondrial matrix stimulates respiration and tunes energy production to the increased needs of the exocytic activity. At the end of stimulation, [Ca²⁺]_C decreases rapidly and mitochondria release the Ca²⁺ accumulated in the matrix through the Na⁺/Ca²⁺ exchanger. VOCC, CICR sites and nearby mitochondria form functional triads that co-localize at the subplasmalemmal area, where secretory vesicles wait ready for exocytosis. These triads optimize stimulus?Csecretion coupling while avoiding propagation of the Ca²⁺ signal to the cell core. Perturbation of their functioning in neurons may contribute to the genesis of excitotoxicity, ageing mental retardation and/or neurodegenerative disorders.     

Receptor-evoked Ca2+ mobilization in pancreatic acinar cells: Evidence for a regulatory role of protein kinase C by a mechanism involving the transition of high-affinity receptors to a low-affinity state

In order to establish a regulatory role for phosphoproteins in the process of receptor-stimulated Ca²⁺ mobilization, isolated pancreatic acinar cells, loaded with fura-2, were stimulated with cholecystokin-in-octapeptide (CCK₈) in the presence of either staurosporine, a general inhibitor of protein kinase activity, or 12-O-tetradecanoylphorbol 13-acetate (TPA), an activator of protein kinase C. Staurosporine alone did not affect the average free cytosolic Ca²⁺ concentration ([Ca²⁺]_i,av) in a suspension of acinar cells. However, in the presence of 1.0 M Staurosporine the stimulatory effect of submaximal concentrations of CCK₈ was significantly enhanced. The potentiating effect of the inhibitor was paralleled by the increased production of inositol 1,4,5-trisphosphate. In addition, staurosporine evoked a transient increase in [Ca²⁺]_i,av in cells prestimulated with a submaximal concentration of CCK₈. The data obtained with staurosporine indicate that CCK₈-stimulated phosphorylations exert a negative feedback role in the process of receptor-mediated Ca²⁺ mobilization. The involvement of protein kinase C was investigated by studying the effects of TPA on CCK₈-induced Ca²⁺ mobilization. The phorbol ester induced a rightward shift of the dose/response curve for the CCK₈-evoked increase in [Ca²⁺]_i,av, which, in contrast to the unlimited shift obtained with the receptor antagonist D-lorglumide, reached a maximum of approximately one order of a magnitude at 10 nM TPA. The inhibitory effect of TPA was completely overcome by CCK₈ at concentrations at or beyond 10 nM. This observation has led to the hypothesis that protein kinase C, directly or indirectly, converts the CCK receptor from a high-affinity state to a low-affinity state. Substantial evidence in favour of this hypothesis was provided by the observation that the increase in [Ca²⁺]_i,av evoked by the CCK₈ analogue JMV-180, which acts as an agonist at the high-affinity receptor, was completely blocked by TPA pretreatment. TPA also evoked a rightward shift of the dose/response curve for the carbachol-induced increase in [Ca²⁺]_i,av, indicating that the protein-kinase-C-mediated transition of the affinity state of receptors is a more general phenomenon. In the presence of submaximal CCK₈ concentrations, TPA dose-dependently decreased the poststimulatory elevated [Ca²⁺]_i,av to the prestimulatory level, indicating that protein kinase C also inhibits the process of sustained Ca²⁺ mobilization. The effects of TPA were counteracted by staurosporine, suggesting that the effects of the inhibitor itself were indeed due to inhibition of the receptor-mediated activation of protein kinase C. The data presented are in support of a negative-feedback role for protein kinase C in the process of receptor-mediated Ca²⁺ mobilization by a process that involves phosphorylation of the CCK receptor, thereby transforming it from a high-affinity state into a low-affinity state.     

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.     

Caffeine-induced oscillations of cytosolic Ca2+ in GH3 pituitary cells are not due to Ca2+ release from intracellular stores but to enhanced Ca2+ influx through voltage-gated Ca2+ channels

Caffeine, a well known facilitator of Ca²⁺-induced Ca²⁺ release, induced oscillations of cytosolic free Ca²⁺ ([Ca²⁺]_i) in GH₃ pituitary cells. These oscillations were dependent on the presence of extracellular Ca²⁺ and blocked by dihydropyridines, suggesting that they are due to Ca²⁺ entry through L-type Ca²⁺ channels, rather than to Ca²⁺ release from the intracellular Ca²⁺ stores. Emptying the stores by treatment with ionomycin or thapsigargin did not prevent the caffeine-induced [Ca²⁺]_i oscillations. Treatment with caffeine occluded phase 2 ([Ca²⁺]_i oscillations) of the action of thyrotropin-releasing hormone (TRH) without modifying phase 1 (Ca²⁺ release from the intracellular stores). Caffeine also inhibited the [Ca²⁺]_i increase induced by depolarization with high-K⁺ solutions (56% at 20 mM), suggesting direct inhibition of the Ca²⁺ entry through voltage-gated Ca²⁺ channels. We propose that the [Ca²⁺]_i increase induced by caffeine in GH₃ cells takes place by a mechanism similar to that of TRH, i.e. membrane depolarization that increases the firing frequency of action potentials. The increase of the electrical activity overcomes the direct inhibitory effect on voltage-gated Ca²⁺ channels with the result of increased Ca²⁺ entry and a rise in [Ca²⁺]_i. Consideration of this action cautions interpretation of previous experiments in which caffeine was assumed to increase [Ca²⁺]_i only by facilitating the release of Ca²⁺ from intracellular Ca²⁺ stores.     

Monitoring transient Ca dynamics with large-conductance Ca-dependent K channels at active zones in frog saccular hair cells

Neurotransmitter release from the basolateral surface of auditory and vestibular hair cells is mediated by Ca²⁺ influx through voltage-gated Ca²⁺ channels. Co-localization of large-conductance Ca²⁺-activated K⁺ (BK) channels at the active zones of these cells affords them with an optimal location to act as reporters of the Ca²⁺ concentration changes at active zones of transmitter release. In this report we use BK channels in frog (Rana pipiens) hair cells to monitor dynamic changes in intracellular Ca²⁺ concentration during transient influxes of Ca²⁺, showing that BK current magnitude and delay to onset are correlated with the rate and duration of Ca²⁺ entry through Ca²⁺ channels. We also show that BK channels exhibit a much higher Ca²⁺ binding affinity in the open state than in the closed state.