Adenosine 3',5'-monophosphate-dependent protein kinase and amylase secretion from rat parotid gland

Tolbutamide at a concentration of 10 mM inhibited cyclic AMP-dependent protein kinase in cell-free preparations of rat parotid glands as reported in rat adipose tissues. Incubation of rat parotid slices with 10 mM tolbutamide markedly interfered with the isoproterenol stimulation of amylase secretion. A carboxy derivative of tolbutamide, 1-butyl-3-$\text{p}$-carboxyphenylsulfonylurea, had minimal inhibitory effects both on protein kinase activity and on amylase secretion. These evidences strongly suggest the participation of cyclic AMP-dependent protein kinase in amylase secretion.     

Inhibitory effect of colchicines on amylase secretion by rat parotid glands: possible localization in the golgi area

Colchicine inhibited amylase secretion by isolated rat parotid glands only 6 h after administration of the drug in vivo. This delayed effect was not the result of the inability of the drug to reach its reaction site. When parotid glands were emptied of their secretory granules by isoproterenol treatment, the subsequent replenishment of cells with granules was inhibited by colchicines. Colchicine concomitantly produced alterations of the Golgi complexes, the cisternae of which were reduced in size and surrounded by clusters of microvesicles. Incubation of parotid glands with colchicines for prolonged durations failed to alter stored amylase secretion as stimulated by isoproterenol, but it inhibited the release of de novo synthesized enzyme. Another colchicines-binding activity, firmly bound to the particular fraction of homogenates, was found, of which a part may represent membrane located microtubular protein. An assembly-disassembly cycle of microtubules appears to exist in the parotid gland, as in the liver. However, only 14 percent of tubulin was found to be polymerized as microtubules in parotid glands as opposed to 40 percent in the liver. The present data suggest that colchicine primarily inhibits the transfer of secretory material towards or away from the Golgi complexes but not the hormone-stimulated secretion of stored amylase.     

SECRETORY PROTEIN SYNTHESIS IN THE STIMULATED RAT PAROTID GLAND : Temporal Dissociation of the Maximal Response from Secretion

Administration of the β-adrenergic drug, isoproterenol (IPR), affects the release of 98% of stored amylase from rat parotid gland acinar cells. A period of 6 h elapses from the onset of secretion to the maximum [¹⁴C]phenylalanine (Phe) incorporation into total protein and amylase. 10 h after IPR administration the rate of [¹⁴C]Phe incorporation into total protein was no longer elevated above that of control. Incorporation into amylase, however, remained elevated above the control by 2.3 times. This latent period may reflect: (a) reduced amounts of available ATP which occurs as a result of the process of secretion as well as (b) the time required for reorganization of cellular organelles and membranes after secretion. The latent period after IPR-induced secretion appears similar to the latent period which has recently been reported to occur after physiologic release of amylase from the parotid gland during the diurnal feeding cycle of the rat. These observations support the existence of a positive feedback system operant in the parotid acinar cell linking the release of secretory proteins with their synthesis. The period of greatest protein synthesis is, however, temporally dissociated from the secretory process.     

Phorbol ester stimulates amylase secretion from rat parotid cells

Phorbol myristate acetate (PMA), a potent activator of Ca2+- and phospholipid-dependent protein kinase (protein kinase C), evoked amylase release from rat parotid cells. In dose-response studies, PMA stimulated amylase release independently of db-cAMP, but potentiated the effect of carbachol. PMA and A23187, a Ca2+ ionophore, synergistically increased amylase release. The maximum effect of carbachol was further enhanced by PMA but not by A23187, suggesting that protein kinase C is not fully activated by the muscarinic-cholinergic agonist under the condition where calcium is fully utilized for amylase secretion.     

Effect of beta-antagonists on isoprenaline-induced secretion of fluid, amylase and protein by the parotid gland of the red kangaroo, Macropus rufus

Selective and non-selective beta-adrenoceptor antagonists were used to block the increases in fluid, protein and amylase secretion caused by sympathomimetic stimulation of the parotid gland of red kangaroos during intracarotid infusion of isoprenaline. ICI118551 at antagonist/agonist ratios up to 300:1 caused increasing but incomplete blockade of fluid secretion, and protein/amylase release. Atenolol at antagonist/agonist ratios up to 300:1 was only marginally more potent than ICI118551 at blocking the fluid, protein and amylase responses. Propranolol at antagonist/agonist ratios of 30:1 was as effective at blocking fluid and protein secretion as the highest ratios of either atenolol or ICI118551. Simultaneous administration of atenolol (30:1) with ICI118551 (30:1) was not as potent as propranolol (30:1). Thus, the beta-adrenoceptor/s in the acini of the kangaroo parotid gland appear to have antagonist-binding affinities atypical of those found for eutherian tissues. The data are consistent with the gland possessing either a single anomalous beta-adrenoceptor or functional beta(2)-receptors in addition to the beta(1)-receptors which are characteristic of eutherian salivary glands.     

Influence of superior cervical ganglion stimulation frequency on salivary secretion in the rabbit. Comparative study of parotid and mandibular glands

Saliva secretion in response to the stimulation of the superior cervical ganglion (S.C.G.) at different frequencies (2, 3, 5, 10, 15, 20 Hz) has been studied in anaesthetized rabbits. The differences between the two major glands in this species were analyzed, with respect to the flow response, potassium, amylase and total protein content during the sympathetic stimulation. The stimulation of S.C.G. increased the salivary flow rate at all frequencies, on both parotid and mandibular gland. In the parotid gland the flow and stimulation frequency show a positive linear correlation which does not appear in the mandibular gland. In conclusion, the differences observed in the response to sympathetic stimulation in both glands seem to be due to distinct patterns of sympathetic innervation on different glandular elements.     

Cholecystokinin- and gastrin-induced protein and amylase secretion from the parotid gland of the anaesthetized rat

I.V. infusion of pentagastrin (20 microg/kg/h) or cholecystokinin (CCK)-8 (1 microg/kg/h) for 10 min caused secretion of salivary proteins from the parotid gland in the anaesthetized rat without any accompanying overt fluid secretion. This "occult" response was revealed by a subsequent wash-out injection of methacholine (5 microg/kg, I.V.) 10 min after the end of the infusion period (aiming at avoiding synergistic interactions). While the fluid response to methacholine was unaffected by the preceding infusion of pentagastrin and CCK-8, the output of protein increased by 147% (pentagastrin) and 74% (CCK-8) and that of amylase by 45% (CCK-8) compared to the responses to methacholine upon saline infusion. Those increases were abolished by the CCK-A receptor blocker (lorglumide), but not by the CCK-B receptor blocker (itriglumide). Evisceration, combined sympathetic and parasympathetic denervation of the glands and assay under adrenoceptor blockade excluded contribution from the gastro-intestinal tract, central or ganglionic mechanisms and circulating catecholamines to the increase in protein/amylase. Furthermore, Western blot demonstrated CCK receptors for both A and B subtypes in normal and chronically denervated glands. In the submandibular gland, both pentagastrin and CCK-8 evoked a trace secretion of saliva but, under the present experimental set-up, no statistically significant increase in protein output. Thus, in addition to the autonomic nervous system, gastrointestinal hormones may, in some types of glands, be involved in the secretion of salivary gland proteins.     

Protein tyrosine phosphorylation in cardiovascular system

Treatment of rat parotid acinar cells with sodium orthovanadate (an inhibitor of protein tyrosine phosphatase) caused a dose-dependent inhibition of phosphatase activity as measured by the hydrolysis of para nitrophenylphosphate (pNPP). Inclusion of 50 M sodium orthovanadate inin vitro gland cultures prevented the amylase secretion from both untreated control and isoproterenol-stimulated parotid acinar cells. Four different tyrosine-phosphorylated proteins with M_r 40, 45, 70 and 95 kDa, respectively, were identified in secretory granule preparations from rat parotid glands by immunoblot using a monospecific antibody for phosphotyrosine. An increase in the phosphorylation levels of these phosphoproteins was noted in the presence of 50 M sodium orthovanadate, suggesting that a protein tyrosine phosphatase (PTPase) is involved in parotid gland protein dephosphorylation reactions. Using antibody to Syp (a PTPase belonging to class 1D), a major fraction of subcellular activity was found to be associated with secretory granule membranes. These results suggest the possible involvement of a PTPase (Syp) in parotid gland secretory mechanisms.     

Parasympathetic denervation increases responses to VIP in isolated rat parotid acini

Vasoactive intestinal peptide (VIP) is a putative neurotransmitter found in the salivary glands of many species, including the rat parotid gland. Parasympathetic denervation has been reported to deplete VIP in the rat parotid gland and to lead to supersensitivity to this peptide in vivo. We have compared the effects of VIP on acini isolated from parasympathetically denervated and unoperated parotid glands to examine possible supersensitivity to the peptide in vitro. VIP normally produced responses similar to those obtained with a low concentration of the beta adrenergic agonist isoproterenol (ISO), but strikingly different from the effects obtained with the muscarinic agonist carbachol (CARB). In parotid membrane preparations, VIP stimulated adenylate cyclase activity. Dissociated acini treated with VIP showed increases in cAMP accumulation and amylase release which were potentiated by forskolin and also by inhibition of phosphodiesterase. After parasympathetic denervation, maximal effects of VIP on adenylate cyclase, cAMP accumulation and amylase release in intact cells were increased two- to five-fold over contralateral control (or unoperated) parotid responses. The increase in adenylate cyclase-mediated responses after denervation was specific to VIP; there was no increased response nor increased sensitivity of any of these responses to ISO. Specific [125I]VIP binding to parotid acini increased two-fold per gland and three-fold per mg of protein after denervation; this probably explains the observed increases in the response to VIP.     

H(1)-Receptor activation triggers the endogenous nitric oxide signalling system in the rat submandibular gland

BACKGROUND: Histamine is released from mast cells by immunologic and non-immunologic stimuli during salivary gland inflammation, regulating salivary secretion. The receptor-secretory mechanism has not been studied in detail. AIMS: The studies reported were directed toward elucidating signal transduction/second messenger pathways within the rat submandibular gland associated with 2-thiazolylethylamine (ThEA)-induced H(1)-receptor responses. MATERIALS AND METHODS: To assess the H(1) receptor subtype expression in the rat submandibular gland, a radioligand binding assay was performed. The study also included inositolphosphates and cyclic GMP accumulation, protein kinase C and nitric oxide synthase activities, and amylase release. RESULTS: The histamine H(1) receptor subtype is expressed on the rat submandibular gland with high-affinity binding sites. The ThEA effect was associated with activation of phosphoinositide-specific phospholipase C, translocation of protein kinase C, stimulation of nitric oxide synthase activity and increased production of cyclic GMP. ThEA stimulation of nitric oxide synthase and cyclic GMP was blunted by agents able to interfere with calcium movilization, while a protein kinase C inhibitor was able to stimulate ThEA action. On the other hand, ThEA stimulation evoked amylase release via the H1 receptor but was not followed by the L-arginine/nitric oxide pathway activation. CONCLUSIONS: These results suggest that, apart from the effect of ThEA on amylase release, it also appears to be a vasoactive chemical mediator that triggers vasodilatation, modulating the course of inflammation.     

Comparison of the secretory processes in the parotid and sublingual glands of the mouse. 1. Regulation of the secretory processes.

1. Secretion from the mucous sublingual gland of the mouse has been investigated and compared with the serous parotid gland. The influence of acetylcholine, noradrenalin and adrenalin on the secretion of glycoproteins (e.g. mucins) and proteins (e.g. amylase) from these glands in vitro, and the involvement of cyclic AMP and Ca2+ has been studied. 2. Secretion from the parotid gland could be stimulated by both acetylcholine and the catecholamines. It appears that cyclic AMP plays an important role in the adrenergic secretory process, but not in the cholinergic-induced secretion. In the latter case, exogenous Ca2+ strongly increased the secretion. 3. Mucin secretion from the sublingual gland could be affected by acetylcholine in the presence of exogenous Ca2+. Noradrenalin and adrenalin induced only a slow mucin secretion and, for this secretory process, exogenous Ca2+ is also required. Though cyclic AMP is present in the sublingual gland, no influence on its level could be detected in this gland after stimulation of the adrenergic beta-receptor, whereas, in contrast to the parotid gland, dibutyryl cyclic AMP induced only a slow secretion. Because it was observed that the sublingual gland of the mouse is not innervated sympathetically, it seems reasonable to suppose that the catecholamines stimulate the mucin secretion from this gland via hormonal receptors and not via the adrenergic beta-receptor. 4. The protein secretion from the sublingual gland could be stimulated by both acetylcholine and the catecholamines. An involvement of cyclic AMP in this process was not observed. Addition of exogenous Ca2+ is less important, as was found for the mucin secretion. So it has been concluded that protein and mucin secretion from the sublingual gland are regulated via different pathways.     

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.     

Amblyomma americanum (L.): protein kinase C-independent fluid secretion by isolated salivary glands.

Protein kinase C activity was partially purified from tick salivary glands by fast protein liquid chromatography anion-exchange chromatography. Enzyme activity was stimulated by Ca2+, phosphatidylserine, and diacylglycerol with the highest activity observed in the presence of all three modulators. Enzyme activity was inhibited by a synthetic pseudosubstrate peptide with an amino acid sequence resembling the protein kinase C substrate phosphorylation site. The protein kinase C activator, 1-oleoyl-2-acetyl-sn-glycerol (OAG), when added to whole in vitro salivary glands previously prelabeled with 32P, stimulated the phosphorylation of salivary gland proteins. Activators of protein kinase C (phorbol ester or OAG) did not stimulate fluid secretion by isolated tick salivary glands. OAG and phorbol ester had only minimal affects on the ability of dopamine to stimulate secretion by isolated salivary glands and dopamine's ability to increase salivary gland cyclic AMP.     

Isoproterenol increases sorting of parotid gland cargo proteins to the basolateral pathway

Exocrine cells have an essential function of sorting secreted proteins into the correct secretory pathway. A clear understanding of sorting in salivary glands would contribute to the correct targeting of therapeutic transgenes. The present work investigated whether there is a change in the relative proportions of basic proline-rich protein (PRP) and acidic PRPs in secretory granules in response to chronic isoproterenol treatment, and whether this alters the sorting of endogenous cargo proteins. Immunoblot analysis of secretory granules from rat parotids found a large increase of basic PRP over acidic PRPs in response to chronic isoproterenol treatment. Pulse chase experiments demonstrated that isoproterenol also decreased regulated secretion of newly synthesized secretory proteins, including PRPs, amylase and parotid secretory protein. This decreased efficiency of the apical regulated pathway may be mediated by alkalization of the secretory granules since it was reversed by treatment with mild acid. We also investigated changes in secretion through the basolateral (endocrine) pathways. A significant increase in parotid secretory protein and salivary amylase was detected in sera of isoproterenol-treated animals, suggesting increased routing of the regulated secretory proteins to the basolateral pathway. These studies demonstrate that shifts of endogenous proteins can modulate regulated secretion and sorting of cargo proteins. amylase; parotid secretory protein; polarized secretion     

Parasympathetic non-adrenergic, non-cholinergic transmission in rat parotid glands: effects of cholecystokinin-A and -B receptor antagonists on the secretory response

Recent studies show i.v. administered pentagastrin and cholecystokinin to evoke protein/amylase secretion from the rat parotid gland and to stimulate gland protein synthesis, the two phenomena being abolished by cholecystokinin receptor antagonists. In the rat parotid gland, non-adrenergic, non-cholinergic transmission mechanisms contribute to secretion of fluid and protein/amylase. Since cholecystokinin may act as a neurotransmitter, activation of cholecystokinin receptors of the gland might contribute to the parasympathetic nerve-evoked secretion. In this study, the parasympathetic innervation was stimulated in non-atropinized (in periods of 2 min) or atropinized (in periods of 3 min) pentobarbitone-anaesthetized rats before and after administration of the cholecystokinin-A receptor antagonist lorglumide (48 mg/kg, i.v.) and the cholecystokinin-B receptor antagonist itriglumide (5.5 mg/kg, i.v.). The non-adrenergic, non-cholinergic transmission fatigues rapidly resulting in declining responses. Therefore, atropinized rats, not receiving the cholecystokinin receptor antagonists, had to serve as controls. Neither at a stimulation frequency of 10 Hz nor of 40 Hz were the secretory responses of the atropinized rats affected by the receptor antagonists. After lorglumide, the saliva volume and the amylase output were (expressed as percentage of the response to the stimulation period before the administration of the antagonist) 98.0+/-3.8% (vs. control 91.1+/-4.0%) and 91.9+/-4.9% (vs. 87.7+/-3.7%) at 10 Hz, respectively, and 79.8+/-4.5% (vs. 77.3+/-2.1%) and 73.6+/-5.3% (vs. 71.7+/-2.3%) at 40 Hz, respectively. After itriglumide, the corresponding percentage figures for saliva volume and amylase output were, at 10 Hz, 99.5+/-8.9% (vs. 92.0+/-2.8%) and 95.8+/-11.8% (vs. 89.2+/-6.4%), respectively, and, at 40 Hz, 74.0+/-3.1% (vs. 79.6+/-2.2%) and 66.6+/-3.3% (vs. 63.9+/-6.0%), respectively. Similarly, the antagonists were without effect on the parotid secretory responses of non-atropinized rats subjected to stimulation at 10 Hz. Thus, under physiological conditions, the cholecystokinin receptors of the parotid gland are likely to be stimulated by circulating hormones rather than by nervous activity.     

Electrophoretic behavior of amylase in mouse: the parotid gland is major source of serum amylase

Amylase activity in the saliva, salivary glands, serum, liver (perfused and non perfused) and pancreas was assayed and isoamylases were separated by electrophoresis in these organs using C57BR/cdJ and M. m. molossinus (Kor) mice. Amylase isozymes in the saliva, parotid gland, serum and liver were identical in both strains, respectively. Amylase activity in the liver was lower than that in the serum and liver amylase disappeared almost by perfusion. Major serum amylase was released from the parotid gland in intact animals.