Relationship between amylase and fluid secretion in the isolated perfused whole parotid gland of the rat

Whole gland perfusion technique was applied to rat parotid glands to assess whether amylase affects fluid secretion. Control perfusion without any secretagogue evoked no spontaneous secretion. Carbachol (CCh 1 microM) induced both amylase and fluid secretion with distinctive kinetics. Fluid secretion occurred constantly at 40-120 microliter/g-min (average plateau was 60 microliter/g-min), whereas amylase secretion exhibited an initial peak (10 mg maltose/30 s per g wet w. of the gland), followed by a rapid decrease to reach a plateau level of 1 mg maltose/30 s later than 1.5-2 min. Isoproterenol (Isop 1 microM) alone did not induce fluid secretion although it evoked amylase secretion as measured in isolated perfused acini. Addition of Isop during CCh stimulation evoked a rapid and large rise in amylase secretion to 15 mg maltose/30 s accompanied by the increase in oxygen consumption. However, the fluid secretion exhibited a rather gradual decrease. These findings suggest that control of salivary fluid secretion is independent of the amylase secretion system induced by CCh and/or Isop. Morphological observations carried out by HR SEM and TEM revealed exocytotic profiles following Isop stimulation. CCh stimulation alone seldom showed -exocytotic profiles, suggesting a low incidence of amylase secretion during copious fluid secretion. Combined stimulation of CCh and Isop induced both vacuolation and exocytosis along intercellular canaliculi. During washout of secretagogues, lysosomal digestion of excess membrane took place.     

Dose-dependent morphological changes of intercellular canaliculi during stimulation with carbachol and isoproterenol in the isolated rat submandibular gland

Intercellular canaliculi (IC) form a primary mixing reservoir for transcellularly and paracellularly secreted saliva whose composition depends on the degree of elevation of cytosolic Ca2+ and of cytosolic cyclic AMP concentrations caused by the secretagogues employed. In perfused rat submandibular gland (SMG), appearance of exocytosis on IC reflected the quantity of secreted mucin. Morphological observations were carried out by HR-SEM using a modified osmium maceration method on specimens treated with CCh and/or ISP. Mild secretory stimulation revealed that exocytosis did not occur simultaneously, even along the same intercellular canaliculus. Higher doses did not alter the spatial distribution of exocytosis along intercellular canaliculi but increased its temporal frequency, dose dependently. These findings lead us to conclude that, under low levels of secretory stimulation, exocytosis does not show a dose-dependent change, but that its spatial and temporal frequency changes in a dose-dependent manner.     

Calcium antagonists cause dry mouth by inhibiting resting saliva secretion

Ca2+ antagonists cause dry mouth by inhibiting saliva secretion. The present study was undertaken to elucidate the mechanism by which Ca2+ antagonists cause dry mouth. Since the intracellular Ca2+ concentration ([Ca2+]i) is closely related to saliva secretion, [Ca2+]i was measured with a video-imaging analysis system by using human submandibular gland (HSG) cells as the material. The Ca2+ antagonist, nifedipine, inhibited the elevation in [Ca2+]i induced by 1-10 microM carbachol (CCh), but had no inhibitory effect on that induced by 30 and 100 microM CCh. The other kinds of Ca2+ antagonists, verapamil (10 microM), diltiazem (10 microM), and the inorganic Ca2+ channel blocker, CdCl2 (50 microM), also inhibited the [Ca2+]i elevation induced by 10 microM CCh. The Ca2+ channel activator, Bay K 8644 (5 microM), significantly enhanced the CCh (10 microM)-induced [Ca2+]i elevation. Endothelin-1 and norepinephrine also increased the CCh (10 microM)-induced [Ca2+]i elevation. SKF-96365 reversed the enhancement of the CCh (10 microM)-induced [Ca2+]i elevation caused by AlF4- and phenylephrine. The phospholipase Cbeta (PLCbeta) inhibitor, U-73122 (5 microM), significantly inhibited the [Ca2+]i elevation induced by 100 microM CCh compared with that induced by 10 microM CCh, while the PLCbeta activator, m-3M3FBS (20 microM), significantly increased the [Ca2+]i elevation induced by 100 microM CCh compared with that induced by 10 microM CCh. We therefore conclude that non-selective cation and voltage-dependent Ca2+ channels are involved in resting salivation and that Ca2+ antagonists depress H2O secretion by blocking the Ca2+ channels and thereby cause dry mouth.     

Secretory responses of rat peritoneal mast cells to high intracellular calcium

The patch-clamp technique was used to investigate the secretory responses of rat peritoneal mast cells at various intracellular calcium concentrations ([Ca2+]i). When Calcium was introduced into the cell with pipette-loaded dibromo-BAPTA, elevation of [Ca2+]i into the range 1-10 microM induced membrane capacitance increases indicative of exocytosis in a concentration-dependent manner. At higher concentrations a decrease of the response was observed. Cells that were exposed to micromolar [Ca2+]i underwent morphological alterations resulting in swelling, which is indicative of cytoskeletal alterations. The presence of dibromo-BAPTA (4 mM) strongly inhibited secretion induced by GTP-gamma-S, thus hampering the contribution of G-protein-mediated stimulation. Application of the Ca2+ ionophore ionomycin resulted in transient increases in [Ca2+]i which were parallelled by Ca2+-dependent secretion. Effective buffering of the cytosolic calcium level below 1 microM abolished the secretory response. Our results show that an increase in [Ca2+]i can trigger secretion, but only if it is high and sustained. During physiological stimulation, however, secretion proceeds at [Ca2+]i below 1 microM. It is, therefore, concluded that mast cell degranulation under physiological conditions is not simply a result of an increase in [Ca2+]i, but that other second messenger systems in conjunction with calcium act synergistically in order to ensure fast and efficient secretion.     

The T84 human colonic adenocarcinoma cell line produces mucin in culture and releases it in response to various secretagogues.

The T84 colonic adenocarcinoma cell line, which has been used extensively as a model for studies of epithelial chloride secretion, also produces mucin and secretes it in culture. Electron microscopy of fixed sections of cultured cells, along with Immunogold labelling with an antibody to human small intestine (SI) mucin, revealed the presence of goblet-like cells with mucin-containing secretory granules. The mucin was of high molecular mass, had an amino acid composition similar to that of purified human SI and colonic mucins, and competed effectively with SI mucin for binding to the anti-(SI mucin) antibody. A sensitive solid-phase immunoassay specific for intestinal mucins was developed and used to measure mucin secretion by T84 cells. Cultures were treated for 30 min at 37 degrees C with a number of agents known to cause chloride secretion by T84 cell monolayers and the amount of mucin appearing in the medium was measured. Carbachol (1 mM), A23187 (10 microM), prostaglandin E1 (PGE1) (1 microM) and vasoactive intestinal polypeptide (VIP) (0.1 microM) all stimulated mucin release, but histamine (1 mM) had no effect. Whereas VIP is reported to stimulate chloride secretion more strongly than carbachol, it was less effective than carbachol in stimulating mucin secretion. Phorbol 12-myristate 13-acetate (PMA) (0.1-10 microM) also stimulated mucin release strongly, implicating a responsive protein-kinase C-dependent pathway. Additive secretory responses were obtained with combined stimulation by VIP (10 nM-1 microM) and carbachol (1 mM). Responses to stimulation with A23187 (1-10 microM) together with PMA (10 nM-10 microM) suggest that cytosolic Ca2+ concentration is a modulator of PMA activity.     

The role of protein kinase C in cholinergic stimulation of insulin secretion from rat islets of Langerhans.

The role of the Ca2+/phospholipid-dependent protein kinase C (PKC) in cholinergic potentiation of insulin release was investigated by measuring islet PKC activity and insulin secretion in response to carbachol (CCh), a cholinergic agonist. CCh caused a dose-dependent increase in insulin secretion from cultured rat islets at stimulatory glucose concentrations (greater than or equal to 7 mM), with maximal effects observed at 100 microM. Short-term exposure (5 min) of islets to 500 microM-CCh at 2 mM- or 20 mM-glucose resulted in redistribution of islet PKC activity from a predominantly cytosolic location to a membrane-associated form. Prolonged exposure (greater than 20 h) of islets to 200 nM-phorbol myristate acetate caused a virtual depletion of PKC activity associated with the islet cytosolic fraction. Under these conditions of PKC down-regulation, the potentiation of glucose-stimulated insulin secretion by CCh (500 microM) was significantly decreased, but not abolished. CCh stimulated the hydrolysis of inositol phospholipids in both normal and PKC-depleted islets, as assessed by the generation of radiolabelled inositol phosphates. These results suggest that the potentiation of glucose-induced insulin secretion by cholinergic agonists is partly mediated by activation of PKC as a consequence of phospholipid hydrolysis.     

Octanol blocks fluid secretion by inhibition of capacitative calcium entry in rat mandibular salivary acinar cells

The aliphatic alcohol octanol is thought to modulate enzyme secretion from the exocrine pancreas by the inhibition of gap junction permeability. We have now investigated the effects of octanol on salivary secretion and intracellular calcium concentration ([Ca2+]i), measured in isolated perfused rat mandibular glands and in isolated mandibular acinar cells respectively. Stimulation of perfused glands with 10 microM carbachol (CCh) evoked a rapid increase in fluid secretion followed by a decrease to a sustained elevated level. Application of 1 mM octanol during CCh stimulation inhibited fluid secretion reversibly. In isolated acini, the CCh-induced [Ca2+]i increase was reversibly inhibited by the same concentration of octanol. However, octanol also inhibited the increase in [Ca2+]i in single acinar cells where gap junctions were no longer functional, indicating that octanol directly affected the intracellular Ca2+ signalling pathway. The initial increase in [Ca2+]i induced by 0.5-10 microM CCh, which is due to Ca2+ release from IP3-sensitive Ca2+ stores, was not affected by pretreatment with octanol. In contrast, CCh-, phenylephrine- or thapsigargin-induced Ca2+ entry was almost completely and reversibly inhibited by octanol. Octanol also blocked agonist-evoked Ca2+ entry in pancreatic acinar cells, and thapsigargin-evoked Ca2+ entry in fibroblasts. These data strongly suggest that octanol blocks salivary secretion from mandibular gland by the inhibition of capacitative Ca2+ entry, and raise the possibility that octanol may be a useful tool for inhibiting agonist-evoked Ca2+ entry pathways.     

Changes in cytosolic free calcium concentration in isolated rat parotid cells by cholinergic and beta-adrenergic agonists

The alteration in the concentration of cytosolic free calcium ([Ca2+]i) in isolated rat parotid cells caused by autonomic agents was directly measured using the Ca-sensitive fluorescent probe, quin2. [Ca2+]i of unstimulated cells was estimated to be 162.7 +/- 3.2 nM in normal medium. Carbachol (CCh) and isoproterenol (ISP) caused a rapid rise in [Ca2+]i in a dose-dependent manner. Maximum increases in [Ca2+]i induced by CCh and ISP were approximately 100% and 25% of resting level, respectively. In Ca-free medium, CCh produced a small, rapid rise in [Ca2+]i, followed by a slow decay and a return to resting level within 3-4 min, while all doses of ISP tested failed to change [Ca2+]i. These results suggest that CCh mobilizes Ca2+ from both extracellular and intracellular pools and then results in a rise in [Ca2+]i, whereas ISP may slightly mobilize only the extracellular Ca pool.     

Secretagogue response of goblet cells and columnar cells in human colonic crypts

Crypts of Lieberkühn were isolated from human colon, and differential interference contrast microscopy distinguished goblet and columnar cells. Activation with carbachol (CCh, 100 microM) or histamine (10 microM) released contents from goblet granules. Stimulation with prostaglandin E(2) (PGE(2), 5 microM) or adenosine (10 microM) did not release goblet granules but caused the apical margin of columnar cells to recede. Goblet volume was lost during stimulation with CCh or histamine ( approximately 160 fl/cell), but not with PGE(2) or adenosine. Three-quarters of goblet cells were responsive to CCh but released only 30% of goblet volume. Half-time for goblet volume release was 3.7 min. PGE(2) stimulated a prolonged fluid secretion that attained a rate of approximately 350 pl/min. Columnar cells lost approximately 50% of apical volume during maximal PGE(2) stimulation, with a half-time of 3.3 min. In crypts from individuals with ulcerative colitis, goblet cells were hypersensitive to CCh for release of goblet volume. These results support separate regulation for mucus secretions from goblet cells and from columnar cells, with control mechanisms restricting total release of mucus stores.     

Nematocytes’ activation in Pelagia noctiluca (Cnidaria, Scyphozoa) oral arms

Nematocytes' discharge is triggered to perform both defense and predation strategies in cnidarians and occurs under chemico-physical stimulation. In this study, different compounds such as amino acids and proteins (mucin, albumin, poly-L: -lysine, trypsin), sugars and N-acetylate sugars (N-acetyl neuraminic acid, N-acetyl galactosamine, sucrose, glucose, agarose and trehalose), nucleotides (ATP and cAMP), were tested as chemosensitizers of nematocyte discharge in the oral arms of the scyphozoan Pelagia noctiluca, particularly abundant in the Strait of Messina (Italy). Excised oral arms were submitted to a combined chemico-physical stimulation by treatment with different compounds followed by mechanical stimulation by a non-vibrating test probe. Discharge induced by a chemico-physical stimulation was more significant than that obtained after mechanical stimulation alone. A chemosensitizing mechanism, with a dose-dependent effect, was observed after treatment with sugars, amino compounds such as glutathione, nucleotides and mucin, according to that already seen in sea anemones. Such findings suggest that, though Anthozoa and Scyphozoa exhibit different divergence times during the evolutionary process, the discharge activation exhibits common features, probably derived from their last common ancestor.     

Inositol polyphosphate production and regulation of cytosolic calcium during the biphasic activation of adrenal glomerulosa cells by angiotensin II

Stimulation of aldosterone production by angiotensin II in the adrenal glomerulosa cell is mediated by increased phosphoinositide turnover and elevation of intracellular Ca2+ concentration. In cultured bovine glomerulosa cells, angiotensin II caused rapid increases in inositol-1,4,5-trisphosphate (Ins-1,4,5-P3) levels and cytosolic Ca2+ during the first minute of stimulation, when both responses peaked between 5 and 10 s and subsequently declined to above-baseline levels. In addition to this temporal correlation, the dose-response relationships of the angiotensin-induced peak increases in cytosolic Ca2+ concentrations and Ins-1,4,5-P3 levels measured at 10 s were closely similar. However, at later times (greater than 1 min) there was a secondary elevation of Ins-1,4,5-P3, paralleled by increased formation of inositol 1,3,4,5-tetrakisphosphate that was associated with cytosolic Ca2+ concentrations only slightly above the resting value. These results are consistent with the primary role of Ins-1,4,5-P3 in calcium mobilization during activation of the glomerulosa cell by angiotensin II. They also suggest that Ins-1,4,5-P3 participates in the later phase of the target-cell response, possibly by acting alone or in conjunction with its phosphorylated metabolites to promote calcium entry and elevation of cytosolic Ca2+ during the sustained phase of aldosterone secretion.     

Effect of staurosporine on fMet-Leu-Phe-stimulated human neutrophils: dissociated release of inositol 1,4,5-trisphosphate, diacylglycerol and intracellular calcium.

Staurosporine, a microbial alkaloid, enhances inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol (DG) production rapidly and dose-dependently in fMet-Leu-Phe (FMLP)-stimulated human neutrophils showing maximal effects at 1 microM concentration. The IP3 increase was specific for staurosporine as three other putative protein kinase C (PKC) inhibitors, H7, sphingosine and palmitoylcarnitine were unable to enhance the IP3 generation in FMLP-stimulated human neutrophils. Staurosporine, at concentrations 0.3-1.0 microM, did not affect the initial mobilization of FMLP-induced intracellular Ca2+ (Ca2+i), although a sustained elevation of cytosolic Ca2+ level was observed within 5 min. This effect could not be suppressed, even by 1 microM phorbol-myristate 12,13-acetate (PMA). Whereas lower concentrations of staurosporine (less than or equal to 100 nM) were unable to affect FMLP-induced IP3 production, DG accumulation and Ca2+i, the PMA-inhibited initial Ca2+i signal and IP3 formation triggered by FMLP were almost completely restored. At higher concentrations (greater than or equal to 300 nM) staurosporine reversed the inhibitory effect of other protein kinases, distinct from the PMA-inducible one, which may be responsible for the phosphatidyl inositol 4,5-bisphosphate (PIP2) breakdown, thus causing accumulation of IP3 and DG and an elevation of C2+i level. Whereas IP3 declined to basal level within 5 min, the DG level remained elevated during the same period. This phenomenon is attributed to phospholipase D (PLD) stimulation by staurosporine, which augments the DG synthesis, in part through PA degradation via phosphatidic acid (PA) phosphohydrolase.     

The thapsigargin-sensitive intracellular Ca2+ pool is more important in plasma membrane Ca2+ entry than the IP3-sensitive intracellular Ca2+ pool in neuronal cell lines

In NG108-15 cells, bradykinin (BK) and thapsigargin (TG) caused transient increases in a cytosolic free Ca2+ concentration ([Ca2+]i), after which [Ca2+]i elevated by TG only declined to a higher, sustained level than an unstimulated level. In PC12 cells, carbachol (CCh) evoked a transient increase in [Ca2+]i followed by a sustained rise of [Ca2+]i, whereas [Ca2+]i elevated by TG almost maintained its higher level. In the absence of extracellular Ca2+, the sustained elevation of [Ca2+]i induced by each drug we used was abolished. In addition, the rise in [Ca2+]i stimulated by TG was less affected after CCh or BK, whereas CCh or BK caused no increase in [Ca2+]i after TG. TG neither increased cellular inositol phosphates nor modified the inositol phosphates format on stimulated by CCh or BK. We conclude that TG may release Ca2+ from both IP3-sensitive and -insensitive intracellular pools and that some kinds of signalling to link the intracellular Ca2+ pools and Ca2+ entry seem to exist in neuronal cells.     

HCO3(-) secretion by murine nasal submucosal gland serous acinar cells during Ca2+-stimulated fluid secretion

Airway submucosal glands contribute to airway surface liquid (ASL) composition and volume, both important for lung mucociliary clearance. Serous acini generate most of the fluid secreted by glands, but the molecular mechanisms remain poorly characterized. We previously described cholinergic-regulated fluid secretion driven by Ca(2+)-activated Cl(-) secretion in primary murine serous acinar cells revealed by simultaneous differential interference contrast (DIC) and fluorescence microscopy. Here, we evaluated whether Ca(2+)-activated Cl(-) secretion was accompanied by secretion of HCO(3)(-), possibly a critical ASL component, by simultaneous measurements of intracellular pH (pH(i)) and cell volume. Resting pH(i) was 7.17 +/- 0.01 in physiological medium (5% CO(2)-25 mM HCO(3)(-)). During carbachol (CCh) stimulation, pH(i) fell transiently by 0.08 +/- 0.01 U concomitantly with a fall in Cl(-) content revealed by cell shrinkage, reflecting Cl(-) secretion. A subsequent alkalinization elevated pH(i) to above resting levels until agonist removal, whereupon it returned to prestimulation values. In nominally CO(2)-HCO(3)(-)-free media, the CCh-induced acidification was reduced, whereas the alkalinization remained intact. Elimination of driving forces for conductive HCO(3)(-) efflux by ion substitution or exposure to the Cl(-) channel inhibitor niflumic acid (100 microM) strongly inhibited agonist-induced acidification by >80% and >70%, respectively. The Na(+)/H(+) exchanger (NHE) inhibitor dimethylamiloride (DMA) increased the magnitude (greater than twofold) and duration of the CCh-induced acidification. Gene expression profiling suggested that serous cells express NHE isoforms 1-4 and 6-9, but pharmacological sensitivities demonstrated that alkalinization observed during both CCh stimulation and pH(i) recovery from agonist-induced acidification was primarily due to NHE1, localized to the basolateral membrane. These results suggest that serous acinar cells secrete HCO(3)(-) during Ca(2+)-evoked fluid secretion by a mechanism that involves the apical membrane secretory Cl(-) channel, with HCO(3)(-) secretion sustained by activation of NHE1 in the basolateral membrane. In addition, other Na(+)-dependent pH(i) regulatory mechanisms exist, as evidenced by stronger inhibition of alkalinization in Na(+)-free media.     

Muscarinic stimulation of inositol phosphate accumulation and acid secretion in gastric fundic mucosal cells

The muscarinic agonist, carbachol (CCh), was shown to stimulate the production of inositol phosphates (IP) in isolated cells from rabbit fundic mucosa. This stimulatory effect was time- and dose-dependent: EC50 values for IP1, IP2 and IP3 accumulation were not statistically different. The mean value was 30 +/- 8 microM (n = 6). The corresponding maximal stimulation (% of basal value) observed after 20 min incubation in the presence of 100 microM CCh was 160 +/- 15%. CCh-induced IP accumulation was abolished by atropine (Ki = 0.32 +/- 0.18 nM (n = 3)). The CCh concentrations leading to half-maximal inhibition of N-[3H]methylscopolamine binding and half-maximal IP accumulation were similar. The half-maximal value for CCh-induced aminopyrine accumulation was 8-times lower. These results indicate that IP3-mediated mobilization of intracellular Ca2+ might be involved in CCh-induced acid secretion by parietal cells.     

Dose-dependency in spatial dynamics of [Ca2+]c in adrenal chromaffin cells

The spatial dynamics of cytosolic Ca2+ concentration, [Ca2+]c, in guinea pig adrenal chromaffin cells was monitored by a digital image analysing technique using Fura-2. When a freshly isolated cluster of cells was stimulated with lower concentrations of carbachol (CCh; 0.3-1 microM), the [Ca2+]c began to increase in the region beneath the plasma membrane facing the extracellular environment. The [Ca2+]c increase depended on the presence of extracellular Ca2+ ([Ca2+]o). CCh at a higher concentration (100 microM), however, caused [Ca2+]c increase even in the absence of [Ca2+]o. These results are compatible with the view that the receptor activation with a physiological concentration of secretagogue accelerates Ca2+ entry, and that stimulation with a higher concentration of the secretagogue induces small transient Ca2+ release from intracellular stores and predominant continuous Ca2+ entry.     

Calcium mobilization and catecholamine secretion in adrenal chromaffin cells. A Quin-2 fluorescence study

To better understand the relation between cell calcium and exocytotic secretion, a quantitative dependence of adrenal catecholamine secretion on cytosolic free calcium has been determined for isolated, intact, bovine chromaffin cells, using the fluorescent probe Quin-2. The cells required a threshold of 250-300 nM cytosolic calcium to be reached before detectable secretion occurred and half-maximal secretion occurred near 2 microM cytosolic calcium. Nicotinic receptors mediated an increase of cytosolic calcium from resting levels near 100 nM to levels in the 1-10 microM range within seconds followed by a decay back to resting levels over several minutes. Muscarinic receptors mediated a smaller rise in cytosolic free calcium from 100 to about 200 nM, within seconds. The nicotinic response required extracellular calcium, while the muscarinic response was largely independent of extracellular calcium, suggesting the latter mobilizes intracellular calcium. The acetylcholine-evoked rise in cytosolic calcium decayed by at least two kinetically distinct processes with half-time constants: t1 = 0.6 min and t2 = 3.2 min. Extracellular Na+ deprivation caused a more prolonged elevation of the acetylcholine-evoked calcium transient, suggesting a possible role of Na+/Ca2+ exchange and/or other Na+ -dependent processes in lowering cytosolic calcium following stimulation. The possible perturbing effects of Quin-2 on resting and stimulated cytosolic calcium levels and on secretion were examined and a novel use of Quin-2 to measure membrane calcium flux was demonstrated.     

Characterization of an endo-alpha-N-acetyl galactosaminidase from Diplococcus pneumoniae.

Evidence is presented for the presence in filtrates of Diplococcus pneumoniae of an endo-glycosidase capable of acting on the O-glycosidic linkage between N-acetyl galactosamine and serine or threonine residues. The glycosidase was partially purified by chromatography on Affi-Gel 202. The resulting preparation acted on glycopeptides from mouse melanoma, fetuin and pig submaxillary mucin to release a disaccharide characterized as galactosyl-N-acetyl galactosamine. The enzyme had no action on phenyl α-N-acetyl-D-galactosaminide, asialo ovine submaxillary mucin or monosialoganglioside. A similar activity was detected in a commercial preparation of Clostridium perfringens neuraminidase.     

Lan-1: A Human Neuroblastoma Cell Line With M1 and M3 Muscarinic Receptor Subtypes Coupled to Intracellular Ca2+ Elevation and Lacking Ca2+ Channels Activated by Membrane Depolarization

The LAN-1 clone, a cell line derived from a human neuroblastoma, possesses muscarinic receptors. The stimulation of these receptors with increasing concentrations of carbachol (CCh; 1-1,000 microM) caused a dose-dependent increase of the intracellular free Ca2+ concentration ([Ca2+]i). This increase was characterized by an early peak phase (10 s) and a late plateau phase. The removal of extracellular Ca2+ reduced the magnitude of the peak phase to approximately 70% but completely abolished the plateau phase. The muscarinic-activated Ca2+ channel was gadolinium (Gd3+) blockade and nimodipine and omega-conotoxin insensitive. In addition, membrane depolarization did not cause any increase in [Ca2+]i. The CCh-induced [Ca2+]i elevation was concentration-dependently inhibited by pirenzepine and 4-diphenylacetoxy-N-methylpiperidine methiodide, two rather selective antagonists of M1 and M3 muscarinic receptor subtypes, respectively, whereas methoctramine, an M2 antagonist, was ineffective. The coupling of M1 and M3 receptor activation with [Ca2+]i elevation does not seem to be mediated by a pertussis toxin-sensitive guanine nucleotide-binding protein or by the diacylglycerol-protein kinase C system. The mobilization of [Ca2+]i elicited by M1 and M3 muscarinic receptor stimulation seems to be dependent on an inositol trisphosphate-sensitive intracellular store. In addition, ryanodine did not prevent CCh-induced [Ca2+]i mobilization, and, finally, LAN-1 cells appear to lack caffeine-sensitive Ca2+ stores, because the methylxanthine was unable to elicit intracellular Ca2+ mobilization, under basal conditions, after a subthreshold concentration of CCh (0.3 microM), or after thapsigargin.