Autonomic regulation of potassium release from human labial salivary glands in vitro

Labial salivary-gland slices from normal human subjects were incubated in vitro according to various protocols. The autonomic regulation of these was significantly different from that of most major salivary glands. K release was stimulated by incubation with a cholinergic agonist but not with alpha- or beta-adrenergic agonists. The cholinergically-induced K release was dependent on agonist concentration and was inhibited by the addition of atropine, and by the removal of Ca in the presence of EGTA.     

Relation of Rab26 to the amylase release from rat parotid acinar cells

Amylase secretion from rat parotid acinar cells is induced by the accumulation of cAMP in response to beta-adrenergic agonists as well as by the elevation of intracellular Ca2+ in response to muscarinic cholinergic stimulation. Several proteins including the low molecular weight GTP-binding protein Rab may participate in these exocytic processes. In the current studies, we investigated the role of Rab26 in the process of amylase secretion. Secretory granules were separated by centrifugation on a Percoll-sucrose density gradient into mature and immature granule fractions. Rab26 and two other type III Rab proteins, Rab3D and Rab27, were present in the mature granule membrane fraction. Also, Rab26 was absent in immature granule membrane fractions. Isoproterenol-induced amylase release from streptolysin-O-permeabilised acinar cells was inhibited by an anti-Rab26 antibody, but this antibody had no effect on the Ca2+-induced release of amylase. Finally, in the early stage of beta-adrenergic stimulation, Rab26 was condensed in the secretory granule membrane. These results indicate that Rab26 is involved in the recruitment of mature granules to the plasma membrane upon beta-adrenergic stimulation.     

Contribution of extracellular and intracellular calcium to the enhanced effect of an alpha-adrenergic agonist on amylase release from dispersed rat parotid cells

The role of calcium in the potentiation of amylase release by the alpha-adrenergic agonist methoxamine (MTX) was examined using dispersed rat parotid acinar cells. The stimulatory effect of MTX (10 microM) on the beta-adrenergic agonist isoproterenol (ISP, 1 microM)-induced amylase release was blocked by the alpha-adrenergic antagonist phentolamine (10 microM). In Ca-free (1 mM EGTA) medium, the increment of amylase release by MTX in normal medium was decreased by about 70%, but MTX still potentiated ISP-induced amylase release. MTX did not affect the cyclic AMP accumulation activated by ISP in either normal or Ca-free medium. MTX enhanced the ISP-stimulated uptake and efflux of 45Ca2+. These results suggest that both extracellular Ca2+ and intracellular stored Ca2+ may play an important role in the potentiation of amylase release from rat parotid acinar cells.     

Differential regulation of agonist-stimulated Ca2+ influx in acini of rat pancreas and submandibular gland.

In order to characterize agonist-dependent Ca2+ influx pathways, changes in cystolic Ca2+ (Ca2+i) during stimulation with multiple Ca(2+)-mobilizing agents were measured in rat pancreatic and submandibular gland acini loaded with fura-2. In pancreatic acini, maximal levels of carbachol and cholecystokinin octapeptide (CCK-8) produced virtually identical changes in Ca2+i when added alone or together: an immediate increase to 4-5 times resting Ca2+i followed by a decline to a steady-state level 2-3 times resting, which was unchanged by another stimulus. In submandibular gland acini, maximal carbachol stimulation increased Ca2+i 3-4-fold followed by a plateau at 2-3 times resting, which was further increased by epinephrine. Epinephrine alone increased steady-state Ca2+i to 53 +/- 18% (n = 21) of that observed with carbachol. Stimulation with both agents increased the steady-state plateau level of Ca2+i to 144 +/- 28% of that during exposure to carbachol alone (n = 11, p < 0.05). When changes in Ca2+i due solely to Ca2+ influx were measured, carbachol and epinephrine together increased Ca2+i during the steady-state phase to 149 +/- 31% of that measured with carbachol alone (n = 8, p < 0.05). Atropine blocked only responses to carbachol, prazosin blocked only responses to epinephrine and L 364,718 blocked only CCK-8-induced changes in Ca2+i. Thus, in pancreatic acini, a single agonist-sensitive Ca2+ influx pathway is linked independently to muscarinic cholinergic and peptidergic (CCK-8) receptors. In contrast, submandibular gland acini contain functionally separate agonist-sensitive Ca2+ influx pathways, which are independently linked to muscarinic and to alpha-1 adrenergic receptors.     

Evidence against a role for guanosine 3',5'-cyclic monophosphate in rat submandibular salivary gland potassium release

The role of guanosine 3',5'-cyclic monophosphate (cGMP) in the net K+ efflux following parasympathomimetic and sympathomimetic stimulation in dispersed rat submandibular cells was evaluated. In unstimulated cells, approx. 70 per cent of the total cGMP content was present in the incubation medium; the addition of 1-methyl-3-isobutylxanthine (MIX), a phosphodiesterase inhibitor, to the medium elevated both intracellular and extracellular levels of cGMP. Cholinergic receptor stimulation resulted in a rapid increase of the cGMP content in the cells and in the medium. Extracellular Ca2+ was necessary for an increased cGMP response and MIX potentiated the cGMP response. Adrenergic-receptor activation produced a slight increase in cGMP after 5 min of stimulation but only when MIX was present in the medium. cGMP analogues (8-Br-cGMP and Bt2cGMP) had no effect on the rate of maximal net K+ efflux, on submaximal net K+ efflux, or on K+ re-uptake following parasympathomimetic or sympathomimetic stimulation. The dose-response relationship for a cholinergic agonist, carbamylcholine, or an adrenergic agonist, norepinephrine, was unaffected by the presence of either 8-Br-cGMP or Bt2cGMP in the medium. MIX, at a concentration sufficient to increase intracellular and extracellular cGMP levels, had no effect on net K+ efflux. These data do not support a role for cGMP in modulating rat submandibular potassium release.     

Soluble guanylyl cyclase is localised in the acinar cells and participates in amylase secretion in rat parotid gland

It is well known that the muscarinic cholinergic agonists, carbachol and methacholine, enhance nitric oxide synthase (NOS) activity, and also stimulate salivary secretion. In the present study, we investigated whether salivary secretion by muscarinic cholinergic stimulation is mediated through the NO/cGMP signaling pathway in rat salivary glands. Since NO activates soluble guanylyl cyclase (sGC) and cGMP may function as a mediator, the localisation of sGC was investigated in the salivary glands. sGC was localized in both the acinar and duct cells of the rat parotid and sublingual glands, and localized only in the acinar cells of the submandibular glands. S-Nitroso-glutathione (NO generator; GSNO) and YC-1 (NO-independent sGC activator) stimulated sGC in the cytosol to synthesise cGMP. The combination of GSNO and YC-1 stimulated sGC synergistically. Carbachol, GSNO and YC-1 enhanced amylase release from the rat parotid glands. Amylase release stimulated by carbachol and GSNO was inhibited by addition of the sGC inhibitor, ODQ, and cGMP-dependent protein kinase inhibitor, KT-5823. These results indicate that amylase release may be mediated through the NO/cGMP signaling pathway.     

Radioimmunoassay of rat submandibular salivary gland mucin

A solid phase radioimmunoassay (RIA) for rat submandibular mucin (RSM) was developed and applied to studies of mucin concentration, antigenicity and secretion. The assay utilizes the affinity of 125I-Protein A for IgG to quantitate antigen-antibody immunocomplexes immobilized in the wells of plastic microtitre plates. The technique was highly reproducible and capable of detecting as little as 3 ng mucin protein. The submandibular glands of rats weighing 150-180 g contained 257 micrograms of mucin (dry wt) per mg protein, which is equivalent to about 3.6 mg of mucin per gland. The antiserum to RSM was cross-reactive with mucins from rat sublingual and pig submandibular glands, and rat, human and pig small intestine. No cross-reactivity was detected with mucins from mouse, canine or bovine submandibular glands, and there was no evidence that ABH blood-group sugars contributed to mucin antigenicity. The RIA was used to estimate secretion from dispersed rat submandibular gland cells and gave a more specific and accurate assay of mucin release than previous assays of precursor-labelled radioactive glycoproteins in the culture medium. The beta-adrenergic agonist, isoproterenol, stimulated immunoreactive mucin secretion from cultured cells to approximately twice the level of unstimulated or propranolol-inhibited controls. The RIA appears to offer promising new approaches for studies on mucin metabolism and secretion in health and disease.     

Actions of prostaglandins E2 and F2 alpha on release of 14C-labelled mucins from rat submandibular salivary acini in vitro

Mucin secretion was studied in vitro using well-characterized preparations of isolated acini. PGE2 significantly (p less than 0.05) increased release of [14C]-glucosamine labelled mucins at the highest concentration tested (10(-5) M), but was ineffective at lower doses (10(-9)-10(-6) M). PGF2 alpha had no effect on mucin secretion over this concentration range. PGE2 (10(-9)-10(-5) M) did not modify isoproterenol stimulated mucin secretion. The cyclooxygenase inhibitor indomethacin (10(-7)-10(-5) M) did not significantly inhibit either isoproterenol or noradrenaline stimulated mucin secretion. Thus it seems that, although PGE2 significantly increases mucin secretion at a high concentration, it is unlikely that prostaglandins play a major role in modulating beta-adrenergic stimulation of mucin secretion in rat submandibular acinar cells.     

Purines,a new class of agonists in salivary glands?

The response of rat submandibular glands to extracellular purines was tested. In crude cellular suspensions, ATP increased the [Ca²⁺]_i mostly by promoting uptake of extracellular calcium. ATP caused the pH_i to drop, a response blocked by chloride channel inhibitors. ATP also inhibited the basal and isoproterenol-stimulated activity of the Na⁺-K⁺-2Cl-cotransporter. These effects were reproduced by benzoyl-ATP, an agonist of ionotropic purinoceptors. In pure ductal suspensions, ATP activated a metabotropic P2Y₁ purinergic receptor coupled to phospholipase C and opened a non-specific cation channel coupled to a P2X₇ receptor. Activation of these receptors stimulated a Ca²⁺-dependent and a Ca²⁺-independent phospholipase A₂, the latter resulting in kallikrein secretion.We conclude that purinergic agonists can modulate the activity of both acinar and ductal phases of secretion. Activation of metabotropic receptors coupled to phospholipase C could lead to responses resembling those to muscarinic or adrenergic agonists. Activation of ionotropic receptors could stimulate new intracellular responses also involved in secretory function.     

Stimulation of glucose oxidation in rat submandibular gland cells in vitro by analogues of cyclic AMP

Secretion from salivary glands, following autonomic stimulation, is energy-dependent. Rat submandibular gland cells, when treated in vitro with both alpha- and beta-adrenergic agonists, showed increased glucose oxidation. The effects of alpha-adrenergic agents on glucose metabolism can be mimicked by non-receptor mobilization of Ca2+. Analogues of cyclic AMP were capable of elevating glucose metabolism to nearly the same extent as beta-adrenergic agonists.     

Modulation by somatostatin of rat submandibular salivary secretion

Although somatostatin (somatotrophin release inhibitory factor; SRIF) is a well-known inhibitory peptide, there are only a few reports of it acting as a positive modulator. In this work, the action of somatostatin upon rat submandibular protein secretion was studied. In vivo somatostatin infusion (35 microg/(kg h)) raised protein secretion stimulated by adrenergic and peptidergic agents. To rule out possible systemic effects of somatostatin, in vitro experiments were performed. Somatostatin (90 nmol/l) augmented protein release stimulated by noradrenaline (19 micromol/l) and substance P (10 micromol/l), but it did not affect isoprenaline (400 micromol/l)-induced protein release. Phenoxybenzamine (20 micromol/l) reduced the effect of somatostatin on noradrenaline-stimulated protein release. Propranolol (20 micromol/l) increased the noradrenaline-stimulated protein release and this effect was synergistic with the action of somatostatin. The absence of extracellular calcium did not significantly reduce somatostatin enhancement of agonist-induced secretion. Fluorescence measurements of the Ca(2+)-sensitive dye fluo3 showed that cytosolic calcium in acinar cells remained elevated during stimuli when somatostatin was present in the medium. It was concluded that somatostatin modulates rat submandibular protein secretion by prolonging the time that the cytosolic calcium signal remains high after stimulus.     

Cholinergic submandibular effects and muscarinic receptor expression in blood vessels of the rat

In order to functionally characterise the muscarinic vasodilator responses, effects of cholinergic agonists were studied on isolated preparations of the rat submandibular artery and vein and carotid and jugular vessels. Tentatively, a cholinergic regulatory mechanism having different effects on the arterial and venous vessels would enhance vascular fluid recruitment for the secretory response. In vitro functional findings were correlated to the expression and cellular location of the different receptors that were assessed by immunohistochemistry. In order to find in vivo correlates to the in vitro findings, the influence of muscarinic receptors on permeability was studied on the vasculature of the submandibular gland in anaesthetised rats. Staining for muscarinic M1 receptors occurred in the endothelium, and muscarinic M5 receptors, and possibly M3 also, were detected in the arterial smooth muscle. In venous endothelium, muscarinic M1 and M4 receptors occurred. In the jugular smooth muscle layer, staining for M1, and possibly also for M3, appeared. Muscarinic agonists caused arteries to relax and veins to contract. The nitric oxide synthase inhibitor Nomega-nitro-L-arginine (L-NNA; 10(-4)M) markedly reduced the cholinergic-evoked relaxation of pre-contracted carotid arterial preparations. In the presence of 4-DAMP (10(-7)M), the relaxation to cholinergic agonists was inhibited. Pirenzepine (10(-5)M) did not only inhibit the relaxatory effects, but even reversed the effects, while it in the jugular vein abolished the cholinergic effects. The arterial nitric oxide-dependent response to muscarinic receptor stimulation consisted of two parts -- one sensitive to pirenzepine and 4-DAMP and the other to 4-DAMP only. Inhibition of the former part only, resulted in cholinergic arterial contraction. Also, the submandibular artery and vein responses to muscarinic receptor stimulation show a resemblance with those of the carotid and jugular vessels, i.e. a pronounced arterial relaxation and a contractile component in the venous response. In vivo examination of submandibular glandular vasculature by studying glandular permeability to Evans blue, confirmed the in vitro observations indicating muscarinic M1 receptors preserving perfusion pressure during the secretory process.     

Electron microscopy of the adrenergic and cholinergic nerve terminals in the mouse salivary glands.

5-Hydroxydopamine was used as a marker of adrenergic nerve elements. In the submandibular gland the most numerous innervation by both adrenergic and cholinergic terminals was in the peri-acinar connective tissue, although no nerve fibres occurred within the acini of this gland. Only the cholinergic type of nerves was occasionally found in close contact with parenchymal cells of the intercalated ducts, granular convoluted tubules and striated ducts. The parotid gland received adrenergic and cholinergic terminals in the acini, intercalated ducts and peri-acinar connective tissue, and the number of these terminals was less than in the submandibular gland. Comparison of the relative numbers of adrenergic and cholinergic nerve terminals showed that about 70 per cent of terminals seen in both glands were adrenergic in nature; the remaining 30 per cent were cholinergic. The sublingual gland received less frequent innervation than in the parotid or submandibular gland. In the sublingual and Ebner glands, the majority of peri-acinar terminals was cholinergic and adrenergic terminals comprised only 10–20 per cent and only the cholinergic type was found within the acini. The reaction products due to acetylcholinesterase activity appeared around both the adrenergic and cholinergic terminals in all four of glands.     

Effects of forksolin, dibutyryl cAMP and H89 on Ca2+ mobilization in submandibular salivary cells of newborn rats.

The effects of substances which affect cAMP or the cAMP-dependent protein kinase (PKA) on the inositol 1,4,5-trisphosphate (IP3) and Ca2+ responses to acetylcholine or thapsigargin were investigated in submandibular gland cells of newborn rats. Exposure to forskolin, dibutyryl cAMP or the PKA inhibitor H89 did not affect the formation of IP3 or the release of Ca2+ from intracellular stores elicited by acetylcholine. However, the thapsigargin-induced Ca2+ release was reduced by dibutyryl cAMP and enhanced by H89 in immature cells. Ca2+ influx activated by acetylcholine and thapsigargin was additive in immature cells but not in mature cells, suggesting the presence of a separate Ca2+ entry pathway in immature cells. Moreover, the acetylcholine-stimulated Ca2+ influx was significantly potentiated by forskolin and dibutyrylcAMP, but not by H89 in immature cells. In contrast, the thapsigargin-activated Ca2+ influx was dramatically enhanced by H89, but not by forskolin and dibutyrylcAMP in these cells. This modulation of Ca2+ mobilization by the test substances is different from that observed in mature submandibular cells in which forskolin, dibutyrylcAMP and H89 affected both IP3 formation and Ca2+ release in response to acetylcholine. Therefore, these results suggest differences in the interaction between the cAMP-PKA and the phosphoinositide-Ca2+ signalling pathways of mature and immature salivary cells. The modulation of Ca2+ influx by the cAMP-PKA pathway in immature cells is likely to play a part in the maturation of salivary cells.     

Role of calcium and PKC in salivary mucous cell exocrine secretion

Fluid and exocrine secretion of mucins by salivary mucous glands is regulated predominantly by parasympathetic activation of muscarinic receptors. A direct role for subsequent putative signaling steps, phospholipase C (PLC), increased intracellular calcium ([Ca(2+)](i)), and isoforms of protein kinase C (PKC) in mediating muscarinic exocrine secretion has not been elucidated, and these are potential therapeutic targets to enhance mucin secretion in hyposalivary patients. We found that muscarinic-induced mucin secretion by rat sublingual tubulo-acini was dependent upon PLC activation and the subsequent increase in [Ca(2+)](i), and further identified a transient PKC-independent component of secretion dependent upon Ca(2+) release from intracellular stores, whereas sustained secretion required entry of extracellular Ca(2+). Interactions among carbachol, PKC inhibitors, phorbol 12-myristate 13-acetate, and thapsigargin to modulate [Ca(2+)](i) implicated conventional PKC isoforms in mediating sustained secretion. With increasing times during carbachol perfusion of glands, in situ, PKC-α redistributed across glandular membrane compartments and underwent a rapid and persistent accumulation near the luminal borders of mucous cells. PKC-β1 displayed transient localization near luminal borders, whereas the novel PKCs, PKC-δ or PKC-ε, displayed little or no redistribution in mucous cells. Collective results implicate synergistic interactions between diacylglycerol (DAG) and increasing [Ca(2+)](i) levels to activate cPKCs in mediating sustained muscarinic-induced secretion.     

Reduction in the rate of inositol 1,4,5-trisphosphate synthesis in rat parotid acini by lithium

Stimulation of muscarinic cholinergic receptors on rat parotid acinar cells causes a rapid production of inositol phosphates, with the key metabolic event being the breakdown of phosphatidylinositol 4,5-bisphosphate into inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and diacylglycerol. Here a high-performance liquid chromatographic technique was used to measure the effects of intracellular lithium ions on the amount of various inositol phosphates produced. When acini were stimulated maximally with acetylcholine (ACh), the sum of all inositol phosphates produced followed a monoexponential function with a production rate constant for Ins(1,4,5)P3 of 0.07 +/- 0.01 solidus/sec. The presence of 23 mM LiCl intracellularly reduced the production rate constant of Ins(1,4,5)P3 induced by ACh to 0.03 +/- 0.01 solidus/sec, resulting in a decrease in the Ins(1,4,5)P3 production as well as in the magnitude of the rise in the intracellular free Ca2+ concentration. The lithium ion (Li+) did not affect the rate of conversion of Ins(1,4,5)P3 to either inositol 1,4-bisphosphate or inositol 1,3,4,5-tetrakisphosphate. The rate of the inositol phosphate production after the addition of the Ca2+ ionophore ionomycin was unaffected by intracellular Li+ (23 mM), which implies that the action of Li+ was at the muscarinic cholinergic receptor, on G-protein or on the interactions between G-proteins and phospholipase C. Thus, in the early events after receptor stimulation with ACh, Li+ causes a reduction in the concentration of the cellular messengers Ins(1,4,5)P3 and Ca2+.     

Regulation of cytosolic Ca2+ in resting and stimulated rat submandibular salivary gland acini

To determine the range over which cytosolic Ca2+ concentration (Cai2+) is regulated, Cai2+ was measured in salivary acini loaded with Fura-2. With extracellular Ca2+ (Cao2+) = 2.5 mM, mean resting Cai2+ (+/- SD) was 103 +/- 20 nM (n = 29). Carbachol increased Cai2+ immediately to 288 +/- 81 nM, which then declined to a sustained level of 253 +/- 58 nM. When Cao2+ was less than 10 nM, the immediate response to carbachol was little affected but the sustained response was abolished. Atropine abolished all responses to carbachol. Changes in Cai2+ were not due to Ca2+-induced Ca2+ release or to Ca2+ influx via voltage-dependent channels. With Cao2+ = 2.5 mM, epinephrine raised Cai2+ initially to 196 +/- 57 nM (n = 6), which soon declined to stable levels of 178 +/- 40 nM. When Cao2+ was less than 10 nM the prolonged response to epinephrine was abolished but the initial increase was relatively unaffected. All epinephrine-induced changes in Cai2+ were abolished by prazosin. The range of Cai2+ measured corresponds to that over which non-mitochondrial Ca2+ transport is stimulated in permeabilized submandibular gland acini suggesting that such a Ca2+ transport system may regulate physiological changes in Cai2+ in these acini in the rat.     

Immunohistochemical localization of cannabinoid receptor 1 (CB1) in the submandibular gland of mice under normal conditions and when stimulated by isoproterenol or carbachol

ObjectiveWe wished to investigate the subcellular localization of CB1, a receptor for the endocannabinoids in mouse submandibular glands (SMGs) under normal conditions and when stimulated by adrenergic or cholinergic agonists.Materials and methodsSMGs of both male and female adult mice were utilized for immunoblotting and immuno-light and –electron microscopic analyses. Isoproterenol and carbachol were used as adrenergic and cholinergic stimulants, respectively. SMGs were examined at 15, 30, 60 and 120 min after intraperitoneal injection of these agents.ResultsSelective localization of intense immunoreactivity for CB1 in the granular convoluted ductal cells was confirmed by immunoblotting and the antigen absorption test. In SMGs of control male mice, CB1-immunoreactivity was evident on the basolateral plasma membranes, including the basal infoldings, but was absent on the apical membranes in the ductal cells. Localization and intensity of CB1-immunoreactivity were essentially the same in SMGs of female mice. The immunoreactivity was transiently localized in the apical plasmalemma of some acinar and granular ductal cells of male SMGs shortly after stimulation by isoproterenol, but not by carbachol.ConclusionThe present finding suggests that CB1 functions primarily in the basolateral membranes of the granular convoluted ductal cells of SMGs under normal conditions, and that the CB1 can function additionally in the apical membrane of acinar and granular ductal cells for modulation of the saliva secretory condition via adrenoceptors.     

The control of cytosolic Ca2+ concentration: studies of high affinity Ca2+ transport in permeabilized acini of rat submandibular glands

Active Ca2+ transport was studied in acini that had been permeabilized by incubation in buffer nominally free of Ca2+ in order to avoid any contribution to measured Ca2+ uptake by elements of the plasma membrane. In most experiments, ruthenium red was used to inhibit mitochondrial Ca2+ uptake. Non-mitochondrial Ca2+ transport was greatest at pH 7.0-7.5 and required Mg2+-ATP at concentrations typical of the cytosol (K0.5 = 1.36 +/- 0.53 mM). Other substrates were much less effective than ATP. Ca2+ uptake was stimulated by Ca2+ at concentrations near those measured in intact cells (K0.5 = 0.43 +/- 0.17 microM). Hill coefficients at subsaturating concentrations of Ca2+ (1.08 +/- 0.27) and Mg2+-ATP (0.81 +/- 0.22) indicated that cooperative interactions are not characteristic of the major cationic regulators of Ca2+ transporting activity. Na+ did not release Ca2+ from acinar mitochondria, but consistently reduced non-mitochondrial Ca2+ uptake by about 20% as compared to uptake in the presence of an equimolar K+ concentration. The properties of non-mitochondrial Ca2+ uptake in permeabilized acini are similar to those of high affinity Ca2+ uptake which, in broken cell preparations, has been found distributed in parallel with elements of the endoplasmic reticulum. The Ca2+-sequestering properties of a non-mitochondrial organelle in permeabilized submandibular gland acini are those expected of a principal regulator of cytosolic Ca2+ and could account for the ionized Ca2+ concentration measured in resting salivary acinar cells.     

Differences in the regulatory mechanism of amylase release by rat parotid and submandibular glands

It is not known whether the mechanisms involved in amylase release in submandibular and parotid glands are similar. Here, the participation of different signalling pathways in amylase release by the parotid and submandibular glands of the male rat was compared by studying the secretory response after beta-adrenergic stimulation. The beta-adrenergic agonist isoproterenol induced an increase of cAMP in both salivary glands, but while in the parotid it triggered amylase release, in the submandibular it was unable to increase amylase secretion. Parotid amylase release was dependent on adenylate cyclase activation, as SQ-22536 inhibited the secretory effect. In contrast, submandibular amylase secretion did not depend on the intracellular concentration of cAMP, as SQ-22536 did not modify its secretory response. Moreover, other activators of adenylate cyclase, such as forskolin and prostaglandin E2, also failed to modify amylase release by the submandibular gland. Neither ionophores nor calcium-blocking agents, as well as calcium-calmodulin and nitric oxide synthase inhibitors, were effective in modifying basal amylase release by the submandibular gland. However, the disruption of microfilaments with cytochalasin B, but not the disruption of microtubules with colchicine, prevented amylase release in that gland. It is concluded that amylase exocytosis in the submandibular gland is a constitutive non-regulated phenomenon, as it is independent of extracellular or intracellular signals. It depends only on the integrity of the microfilaments, probably used by the vesicles to travel from the Golgi apparatus to the plasma membrane.