The effect of L-arginine/nitric oxide pathway on salivary amylase secretion in conscious rats

In a recent study we have demonstrated the presence of nitric oxide synthase immunoreactive neurons and also perivascular, periacinar and periductal nerve fibres in feline submandibular salivary gland. The role of nitric oxide (NO) in salivary vasoregulation has been suggested by other data too, but the effect of NO on salivary amylase secretion has not been investigated yet. Under ether anaesthesia a catheter was introduced into the oesophagus for salivary juice collections, and a cannula was inserted into the jugular vein for infusions. After postanaesthesia recovery, submaximal carbachol infusion was given as a background to obtain steady secretion because of the low basal secretory rate. Then different groups of animals received NO synthase inhibitor NOLA (N^G-nitro-L-arginine), L-arginine, D-arginine or NO donor SIN-1 (3-morpholinosydnonimine). Volume and amylase activity were determined in mixed saliva samples collected for 30 min. Carbachol background infusion alone induced an elevated, sustained salivary secretion. NOLA (30 mg/kg) increased both volume and amylase output (P < 0.001). This effect was prevented by L-arginine but not by D-arginine. SIN-1 did not change either volume or amylase secretion. The present results suggest that the L-arginine/NO pathway has a modulatory effect on salivary fluid and amylase secretion, which is probably not related to its effect on salivary blood flow. NO may block certain presently unidentified secretagogue mechanisms and/or may relax myoepithelial cells.     

Salivary secretion induced by substance P

Secretion of fluid, ions, and amylase from parotid and submaxillary glands of rat, induced by intravenous injection of substance P (SP), was examined. The action of SP on salivary glands, like physalaemin, resembled that of cholinergic stimulation. While SP-evoked salivary flow from both glands was blocked by atropine, atropine did not modify composition of SP-evoked saliva. The present study suggests that salivary secretion and secretion of ions and amylase evoked by SP are mediated via SP-sensitive cholinergic receptors and specific SP receptors, respectively.     

Effect of centrally administered nitric oxide modulators in Brewer's yeast-induced nociception in rats

Possible modulation of Brewer's yeast-induced nociception by centrally (icv) administered nitric oxide (NO) modulators, viz., NO synthase (NOS) inhibitors, NO precursor, donors, scavengers and co-administration of NO donor (SIN-1) with NOS inhibitor (L-NAME) and NO scavenger (Hb) was investigated in rats. Administration of NOS inhibitors and NO scavenger Hb increased the pain threshold capacity significantly, whereas NO donors SIN-1, SNP and NO precursor L-arginine were found to be hyperalgesic. D-arginine, the inactive isomer of L-arginine and methylene blue, inhibitor of soluble guanylate cyclase failed to alter the nociceptive behaviour in rats. Co-administration of SIN-1 with L-NAME and Hb found to increase the nociceptive threshold. The results indicate, that centrally administered NO modulators alter the nociceptive transmission induced by Brewer's yeast in rats.     

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.     

An immunohistochemical study of secretagogue-induced secretion of epidermal growth factor in the submandibular salivary glands of mice

Routine histological and indirect immunofluorescence techniques were used to examine the histological details of changes in the distribution of epidermal growth factor (EGF) in the submandibular salivary glands of mice during secretion. Comparisons were made bewteen glands of normal mice and those of mice given one of a number of secretagogues at various times prior to sampling. Normal submandibular salivary glands in male mice had an extensive system of convoluted granular tubules (CGT), the cells of which contained EGF. When adrenaline of alpha-phenylephrine was administered, the CGT cells degranulated, and there was a concomitant loss of intracellular EGF-positive immunofluorescence. The excretory ducts were engorged with immunofluorescent material, indicating secretion of EGF into saliva, while the ductal cells themselves remained EGF-negative. The degranulation response could be blocked by phentolamine, but not by propranolol, and no changes in EGF distribution followed the administration of pilocarpine. It was concluded that EGF is secreted, at least partly into the saliva, following an alpha-adrenergic response, and that this occurs with degranulation of the cells of the CGT.     

Vasoactive intestinal polypeptide and acetylcholine stimulate exocrine secretion of epidermal growth factor from the rat submandibular gland

The effect of vasoactive intestinal polypeptide (VIP) and acetylcholine on secretion of epidermal growth factor (EGF) from the rat salivary glands was investigated. VIP in doses of 3 X 10(-10) to 3 X 10(-8) mol/kg per h stimulated secretion of saliva and total output of EGF dose-dependently. Acetylcholine also stimulated salivation and output of EGF. VIP in a dose of 3 X 10(-11) to 3 X 10(-10) mol/kg per h enhanced the stimulatory effect of acetylcholine, but this effect disappeared when the dose of VIP was increased. Adrenalectomy decreased acetylcholine stimulated total output of EGF by approximately 50%, but only by 20% when acetylcholine plus VIP was administered. EGF was localized to the convoluted granular tubules in the submandibular gland, whereas EGF could not be detected in the remaining salivary glands. The results suggest that VIP and acetylcholine cooperate in the control of exocrine secretion from the rat salivary glands. The effect of acetylcholine, however, seems to be partly dependent on circulating catecholamines.     

Response of the parotid gland of red kangaroos,Macropus rufus,to phenylephrine stimulation

Intracarotid infusions of l-phenylephrine at 1.0 or 10 nmol kg(-1) min(-1) were accompanied by increases in salivary amylase activity, protein, potassium, magnesium and chloride relative to cholinergically-stimulated saliva. Intravenous infusions of phenylephrine at the same dose rates had a lesser effect on salivary composition particularly protein. Propranolol administered with phenylephrine via the carotid artery, at an antagonist/agonist ratio of 10:1, was much more effective in blocking the phenylephrine-induced changes in salivary composition than equimolar infusion of phentolamine with phenylephrine. It was concluded that alpha(1)-adrenoreceptors were not present in functionally significant numbers in the gland and that the effect of phenylephrine on the kangaroo parotid was mediated by beta-adrenoreceptors. As the phenylephrine dose rates in the kangaroos were comparable with those used to determine alpha-adrenergic responses of eutherian salivary glands and as phentolamine appeared to have minor beta-sympathomimetic activity, at least one subtype of beta-adrenoreceptors in macropods may not be identical to its eutherian counterpart.     

Sympathetic control on salivary secretion by the parotid gland in rabbit. I. Effects on the unstimulated gland (author's transl)]

The sympathetic influences on the rabbit unstimulated parotid gland were studied. The experiments were carried out in anaesthetized rabbits with the Stenon aduct cannulated. Direct stimulation of the superior cervical ganglion elicits variable salivary flows. The high amylase content in the saliva points to a sympathetic secretory action upon acinar cells. The administration of alpha-adrenergic blocking agents (dihydroergotamine, phentolamine and phenoxybenzamine) clearly reduces and even abolishes the effect of the sympathetic stimulation upon flow. The administration of a beta-adrenergic blocking agent (propranolol) slightly reduces the sympathetic action. However the amylase activity is greatly reduced. All this suggests that the secretory effects on the fluid fraction should predominantly be alpha-adrenergic while on the secretion of enzymes the beta-receptors should play an important role.     

Protease-activated receptor-2 (PAR-2) in the pancreas and parotid gland: Immunolocalization and involvement of nitric oxide in the evoked amylase secretion

Protease-activated receptor-2, a G protein-coupled receptor activated by serine proteases such as trypsin, tryptase and coagulation factors VIIa and Xa, modulates pancreatic and salivary exocrine secretion. In the present study, we examined the distribution of PAR-2 in the pancreas and parotid gland, and characterized the PAR-2-mediated secretion of amylase by these tissues in vivo. Immunohistochemical analyses using the polyclonal antibody against rat PAR-2 clearly showed abundant expression of PAR-2 in rat pancreatic and parotid acini. The PAR-2 agonist SLIGRL-NH2, administered intraperitoneally (i.p.) at 1-10 micromol/kg and 1.5-15 micromol/kg, in combination with amastatin, an aminopeptidase inhibitor, facilitated in vivo secretion of pancreatic and salivary amylase in a dose-dependent manner, respectively, in the mouse. The PAR-2-mediated secretion of pancreatic amylase was abolished by pretreatment with N(G)-nitro-L-arginine methyl ester (L-NAME), an NO synthase inhibitor. The secretion of salivary amylase in response to the PAR-2 agonist at a large dose, 15 micromol/kg, but not at a smaller dose, 5 micromol/kg, was partially reduced by L-NAME. Pretreatment with capsaicin for ablation of the sensory neurons did not modify the PAR-2-mediated secretion of pancreatic and salivary amylase in the mouse. In conclusion, our study demonstrates expression of PAR-2 in rat pancreatic acini as well as parotid acini and indicates that nitric oxide participates in the PAR-2-mediated in vivo secretion of pancreatic amylase, and, to a certain extent, of salivary amylase, although capsaicin-sensitive sensory neurons, known to be activated by PAR-2, are not involved in the evoked pancreatic or salivary amylase secretion.     

Effects of chronic clonidine administration on parasympathetic-evoked rat saliva.

Effects of chronic administration of clonidine on parasympathetic-evoked saliva from both parotid and submandibular glands were investigated. Clonidine at 1 mg/kg/day for 5 or 7 days caused a significant reduction in the salivary secretion (flow rate and total volume) evoked by parasympathetic nerve stimulation of parotid but not submandibular glands. Ion concentrations (Na, K and Ca) of parasympathetically nerve-evoked parotid saliva were not altered. However, the total protein concentration as well as output, amylase activity, and output of such saliva were markedly increased. Possible mechanisms for clonidine-induced increase in nerve-elicited salivary protein concentration include release of neuropeptides, and changes in adrenergic receptor binding which need further study.     

A comparison of the effects of vasoactive intestinal polypeptide on secretion from the submaxillary gland of the sheep and pig

The effects, on secretion of fluid and protein from the submaxillary gland of intracarotid injections of acetylcholine or vasoactive intestinal polypeptide (VIP), and intracarotid infusions of VIP during a background of muscarinic stimulation, were examined in sheep and pigs. Intracarotid injections of VIP produced secretion of saliva from the ovine gland which continued after administration of atropine, phentolamine and propranolol. The protein concentration of this saliva was over 5-fold greater than that secreted in response to acetylcholine. Intracarotid injection of VIP did not evoke secretion from the porcine submaxillary gland but increased 3-fold the protein concentration in saliva evoked by subsequent intracarotid injection of acetylcholine. Intracarotid infusions of VIP in sheep produced dose-related increases in both flow (up to 1.9-fold) and protein concentration (up to 42-fold) of submaxillary saliva secreted in response to a background infusion of bethanechol. In pigs, intracarotid infusions of VIP at 0.015, 0.15 and 1.5 nmol/min produced increases in both flow and protein concentration of bethanechol-evoked saliva. The increases in protein concentration (up to 2.8-fold) were dose-related, but the increases in flow were not, being ca. 25% with each dose of VIP. The experiments provide evidence that VIP may effect mobilization of protein into saliva even in a species (pig) in which VIP does not evoke secretion of fluid.     

Exocrine secretion of epidermal growth factor from Brunner's glands. Stimulation by VIP and acetylcholine

Brunner's glands of the duodenum are innervated by cholinergic and VIP-ergic nerves, and the glands have been shown to contain epidermal growth factor (EGF). In this study the effect of VIP and acetylcholine (Ach) on secretion of EGF from Brunner's glands was investigated in the rat. Intravenous infusion of VIP stimulated the flow rate of duodenal secretion, an effect which was inhibited by atropine. Ach alone did not significantly increase flow rate, and combined infusion of VIP and Ach induced the same flow as VIP alone. Concentration of EGF in duodenal secretion was increased by infusion of Ach, and this effect was potentiated by VIP. Infusion of VIP alone did not influence EGF concentration. EGF output from Brunner's glands was significantly stimulated by i.v. infusion of VIP and of Ach and combined infusion further increased EGF output. The study has demonstrated exocrine secretion of EGF from Brunner's glands, and it is suggested that stimulation is mediated by interaction of neuronal VIP and Ach.     

Relationships between plasma composition and parotid salivary composition and secretion rates in the potoroine marsupials,Aepyprymnus rufescens and Potorous tridactylus

Summary Parotid salivation was investigated in two species of potoroine marsupial, Aepyprymnus rufescens and Potorous tridactylus to ascertain flow rates and composition, the buffer capacity of the saliva with respect to possible dependence of these animals on foregut fermentation, and the similarity of anion excretion patterns to those of the kangaroo parotid. Under anaesthesia neither species secreted spontaneously and secretion was stimulated by intravenous infusion of carbachol, bethanechol and isoprenaline. Under cholinergic stimulation in Aepyprymnus, the concentrations of Na, Cl, HCO₃ and osmolality were positively correlated with flow rate, whereas K, Mg, PO₄, H⁺ and urea were negatively correlated with flow. Amylase activity and the concentrations of protein and Ca showed no consistent relation to flow. Relative to Aepyprymnus, saliva of Potorous had much lower amylase activity and amylase activity per gram protein, lower concentrations of urea and Ca, and higher Na. Protein, K and HCO₃ concentrations were similar in both species. The plasma of both species had similar electrolyte concentrations, but Potorous had lower protein, urea, osmolality and amylase activity. Plasma amylase activity in Aepyprymnus rose during cholinergic stimulation to levels in excess of rodent plasma. Isoprenaline infusion in Aepyprymnus increased salivary amylase activity and concentrations of protein, Ca, HCO₃ and PO₄, and reduced the concentrations of Cl and H⁺. The patterns of anion excretion in the two potoroine marsupials were dissimilar to those of the kangaroo parotid suggesting that parotid fluid secretion is not HCO₃ driven to the same extent as that of kangaroos. Buffer anion concentrations and secretion rates were similar to koalas and low relative to kangaroos, indicating that these potoroines do not rely on foregut fermentation.Abbreviations bw body weight - SEM standard error of mean - VFA volatile fatty acids     

Evidence nitric oxide mediates the vasodepressor response to hypoxia in sino-denervated rats.

Systemic hypoxia, produced in deeply anesthetized, paralyzed rats in which arterial chemoreceptors were denervated, elicited a decrease in arterial pressure (AP) averaging -47 mmHg. Systemic administration of NG-nitro-L-arginine (L-NO2Arg), an inhibitor of nitric oxide (NO) synthase, attenuated the hypoxic depressor response by 79% and elevated AP by 21 mmHg. The effects of L-NO2Arg on the hypoxic depressor response and arterial pressure were reversed by systemic administration of L- but not D-arginine. Elevation of AP with arginine-vasopressin or reduction of AP with nitroprusside to the pre-L-NO2Arg levels did not modify the fall of AP to hypoxia. Endogenous NO synthesized in vivo from L-arginine, mediates most of the hypoxic depressor response.     

Sympathetic control on salivary secretion by the parotid gland in rabbit. II. Effects upon the gland submitted to a weak parasympathetic stimulation (author's transl)]

Superior cervical ganglion stimulation significantly increases both, flow and amylase activity of saliva. Stimulation provokes two markedly different periods of flow: during the first half, flow is very high and differs significantly from the flow of the second half which closely resembles the one previous to stimulation. Alpha-adrenergic blocking agents, when administered intravenously, abolish the hypersecretion induced by sympathetic stimulation; beta-adrenergic blocking agents do not. These facts strengthen the hypothesis that alpha-adrenoceptors are most important in fluid secretion. The infusion of epinephrine acts similarly on cervical ganglion stimulation, but it differs because of its more diffuse effects and deeper cardiovascular alterations. Isoproterenol, after a long latency period, slightly increases salivary flow, which seemingly indicates that beta-adrenoceptors are involved in the fluid secretory processes although in a lesser degree than alpha-adrenoceptors.     

Exocrine and endocrine secretion of renin and epidermal growth factor from the mouse submandibular glands

Renin and epidermal growth factor (EGF) are synthesized in large amounts by the male mouse submandibular glands. We report the peptides to be secreted mainly in an exocrine manner. The highest values in saliva are obtained upon stimulation with the alpha-adrenergic agonist phenylephrine. The median value for renin is 54 700 nmol/l and the median value for EGF is 211 800 nmol/l. Aggressive behaviour and beta-adrenergic stimulation also increase salivary output of both peptides, while vasoactive intestinal polypeptide (VIP) plus pilocarpine selectively stimulate the secretion of renin. The pattern of increase in plasma is comparable to that in saliva though the substance concentration is lower by a factor of 2 to 70 for renin and a factor of 280 to 12 000 for EGF.     

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.     

Depletion of neuropeptides in rat parotid glands and declining atropine-resistant salivary secretion upon continuous parasympathetic nerve stimulation

In rats the parasympathetic auriculo-temporal nerve on one side was continuously stimulated at 40 Hz for 20-80 min in the presence of adrenergic blockers (dihydroergotamine and propranolol) +/- atropine. During the first 10 min this gave rise to a flow of saliva from the parotid gland that in the atropinized rats amounted to 35% of that found in rats not treated with atropine, while the protein and amylase outputs were 75% of those in non-atropinized rats. The atropine-resistant secretion of fluid and proteins declined to 5-10% of the initial value within 40 min but did not cease completely even after 80 min. The marked reduction in secretory responses was not due to desensitization or exhaustion of the gland cells. The nerve stimulation reduced the parotid gland content of vasoactive intestinal peptide (VIP) and substance P (SP) to approximately 60 and 25% of that of contralateral glands after 20 and 60 min, respectively. The probable explanation for the decline in secretory response seems to be depletion of non-adrenergic, non-cholinergic transmitter(s). The present results suggest that neuropeptides are involved in the regulation of salivary secretion but provide no direct evidence that either VIP or SP is responsible for the atropine-resistant salivary secretion.     

Secretagogue-stimulated pancreatic secretion is differentially regulated by constitutive NOS isoforms in mice

Nitric oxide (NO) and NO synthase (NOS) play controversial roles in pancreatic secretion. NOS inhibition reduces CCK-stimulated in vivo pancreatic secretion, but it is unclear which NOS isoform is responsible, because NOS inhibitors lack specificity and three NOS isoforms exist: neuronal (nNOS), endothelial (eNOS), and inducible (iNOS). Mice having individual NOS gene deletions were used to clarify the NOS species and cellular interactions influencing pancreatic secretion. In vivo secretion was performed in anesthetized mice by collecting extraduodenal pancreatic duct juice and measuring protein output. Nonselective NOS blockade was induced with N(omega)-nitro-L-arginine (L-NNA; 10 mg/kg). In vivo pancreatic secretion was maximal at 160 pmol.kg(-1).h(-1) CCK octapeptide (CCK-8) and was reduced by NOS blockade (45%) and eNOS deletion (44%). Secretion was unaffected by iNOS deletion but was increased by nNOS deletion (91%). To determine whether the influence of NOS on secretion involved nonacinar events, in vitro CCK-8-stimulated secretion of amylase from isolated acini was studied and found to be unaltered by NOS blockade and eNOS deletion. Influence of NOS on in vivo secretion was further examined with carbachol. Protein secretion, which was maximal at 100 nmol.kg(-1).h(-1) carbachol, was reduced by NOS blockade and eNOS deletion but unaffected by nNOS deletion. NOS blockade by L-NNA had no effect on carbachol-stimulated amylase secretion in vitro. Thus constitutive NOS isoforms can exert opposite effects on in vivo pancreatic secretion. eNOS likely plays a dominant role, because eNOS deletion mimics NOS blockade by inhibiting CCK-8 and carbachol-stimulated secretion, whereas nNOS deletion augments CCK-8 but not carbachol-stimulated secretion.