Effects on rat parotid amylase and Ca of alpha- and beta-adrenergic sympathetic stimulation.

The roles of alpha- and beta-adrenergic receptors in the regulation of the secretion of amylase and calcium from rat parotid gland were studied by using direct electrical stimulation of the sympathetic innervation to the gland in the presence of selective adrenergic blocking agents. When phenoxybenzamine was administered intraperitoneally 25 min prior to nerve stimulation, the highest [Ca] (12-14 meq/liter) and amylase activity (1,000 mg/microliter) in the evoked saliva were observed. On the other hand, stimulation of the nerve in the presence of propranolol evoked a saliva that contained the lowest [Ca] (5-6 meq/liter) and amylase activity (170 mg/microliter). Furthermore, salivary flow (4.27 +/- 0.42 microliter/min X g) induced by sympathetic nerve stimulation in the presence of phenoxybenzamine was higher than that induced by sympathetic nerve stimulation in the presence of propranolol (2.55 +/- 0.39 microliter/min X g). Therefore, it was concluded that beta-adrenergic receptors play the major role in the regulation of salivary flow and the secretion of amylase and calcium, whereas alpha-adrenergic receptors play a minor role in the regulation of these parameters.     

Augmentation and inhibition of beta-adrenergic agonists-stimulated tissue cyclic AMP accumulation by alpha-adrenergic agonists in rat parotid gland

It was found that phenylephrine and methoxamine had two effects (one was inhibitory and the other was augmentative) on isoproterenol-stimulated cyclic AMP in rat parotid slices. The augmentation was abolished by alpha-adrenergic antagonists or by omission of calcium in the medium. Cyclic AMP accumulation by norepinephrine (NE) was significantly decreased by omission of calcium in the medium. Calmodulin antagonists, trifluoperazine and W-7, decreased NE-induced cyclic AMP accumulation, but another calmodulin antagonist, carmidazolium, did not. Phorbol ester such as 4 beta-phorbol 12-myristate, 13-acetate and phorbol 12, 13-dibutyrate, did not augment the effect of isoproterenol. These results suggest that although the influx of calcium is required in the alpha-adrenergic agonists-induced augmentation, calmodulin and protein kinase C may not be intermediates in this process. Calcium ions (10(-7) and 10(-6) M) slightly increased the activity of adenylate cyclase, but calcium (10(-6)-10(-4) M) dose-dependently inhibited the effect of isoproterenol. Therefore, calcium ions do not participate in the augmentation by directly modulating the activity of adenylate cyclase. The inhibitory effect was not affected by alpha-adrenergic antagonists. The activation of adenylate cyclase by isoproterenol was inhibited by phenylephrine with higher inhibition being obtained in lower concentrations of isoproterenol. Phenylephrine in the presence of isobutylmethylxanthine increased the amount of cyclic AMP and this effect was inhibited by propranolol, but not by phentolamine. [3H]-CGP 12177 binding of the parotid membrane was inhibited by alpha-adrenergic antagonists. These results suggest that the inhibitory effect of phenylephrine and methoxamine may be mediated by beta-adrenergic receptor.     

alpha-Adrenergic, beta-adrenergic and cholinergic mechanisms for amylase secretion by rat parotid gland in vitro.

1. Rat parotid gland slices, incubated in a balanced, buffered salt solution, were found to be physiologically stable for up to 2 hr with respect to O2 consumption, water content, extracellular space and cation content. 2. The slices could be stimulated to secrete amylase by activation of alpha-adrenergic, beta-adrenergic or muscarinic cholinergic receptors. 3. The secretion elicited through all three receptors appeared to involve exocytosis as revealed by electron microscopy. 4. The beta-agonist, isoprenaline, increased tissue content of cyclic adenosine 3',5'-monophosphate (cyclic AMP); alpha-adrenergic and cholinergic agents had no effect on the level of this cyclic nucleotide. 5. Secretion via cholinergic or alpha-adrenergic mechanisms required extra-cellular calcium; the beta-adrenergic mechanism did not. 6. It was concluded that stimulation of rat parotid cells activates distinctly separate pathways leading ultimately to exocytosis, one pathway involving cyclic AMP, and the other, external Ca2+ ion.     

Recepto-secretory mechanism in histamine-stimulated amylase release from rat parotid gland

The recepto-secretory mechanism in histamine-stimulated amylase release from rat parotid slices was studied using blockers of receptors and inhibitors of the intracellular messenger systems. Amylase release stimulated by histamine was inhibited by pyrilamine, an H₁-receptor blocker, but not by cimetidine, an H₂-receptor blocker. Atropine, prazosin and yohimbine had no effect on the release. Histamine-stimulated amylase release was inhibited by W-7, ML-9 and H-7, inhibitors of a calmodulin, a myosin light chain kinase (MLCK) and protein kinase C, respectively, while H-8, an inhibitor of protein kinase A, did not inhibit the release. These results suggest that histamine stimulation evokes amylase release via H₁-receptors, followed by the Ca²⁺-dependent systems involving calmodulin, MLCK and protein kinase C.     

Effect of extracellular magnesium on nerve-mediated and acetylcholine-evoked in vitro amylase release in rat parotid gland tissue

In this study the effects of changes in extracellular magnesium ([Mg(2+)](o)) and calcium ([Ca(2+)](o)) concentrations on basal and on nerve-mediated and acetylcholine (ACh)-evoked in vitro amylase release and calcium mobilization were investigated in rat parotid gland tissue. In the presence of a normal (2.56 mM) [Ca(2+)](o), both zero (0 mM) and an elevated (10 mM) [Mg(2+)](o) significantly attenuated basal and ACh-evoked amylase release compared to the response obtained in normal (1.1 mM) [Mg(2+)](o). During electrical field stimulation (EFS) of parotid tissues, only elevated [Mg(2+)](o) reduced amylase release. In a Ca(2+)-free medium, both basal and ACh-evoked amylase output were markedly reduced compared to the responses obtained under similar conditions in normal [Ca(2+)](o). Again, the ACh-induced amylase release in a Ca(2+)-free solution was larger in normal [Mg(2+)](o) than when the [Mg(2+)](o) was either zero or was elevated to 10 mM. Perturbation of [Mg(2+)](o) had no significant effect on basal intracellular free calcium concentration ([Ca(2+)](i)) in parotid acinar cells loaded with the fluorescent Ca(2+) indicator fura-2. Both zero Mg(2+) and an elevated [Mg(2+)](o) significantly reduced the ACh-induced rise in the peak and the plateau phase of the Ca(2+) transient that was seen in normal [Mg(2+)](o). In parotid acinar cells loaded with the fluorescent Mg(2+) indicator magfura-2, ACh elicited a gradual decrease in intracellular free Mg(2+) concentration ([Mg(2+)](i)) to below the basal level. The results indicate that both hypo- and hypermagnesaemia may reduce both basal and ACh-evoked amylase secretion from the salivary gland. As far as the ACh-evoked response is concerned, the effect may be exerted by a decrease in cellular Ca(2+) transport.     

Augmented amylase release from rat parotid gland slices,in vitro

Using a continuous amylase assay the effect of simultaneous stimulation of adrenergic and cholinergic receptors on amylase release by perfused rat parotid slices was investigated. Super imposing adrenergic stimulation (isoprenaline) on continual submaximal cholinergic stimulation (acetyl--methylcholine) resulted in an augmented amylase release compared with the sum of the two separate effects. This could also be shown with continual adrenergic stimulation with cholinergic stimulation superimposed on this. Possible explanations of this effect are discussed with particular respect to the role of Ca⁺⁺.     

Augmentation of isoproterenol-stimulated tissue cyclic AMP level by cholinergic agonists in rat parotid gland

It was found that methacholine and carbamylcholine, in addition to their known inhibitory effect, augmented the effect of isoproterenol on tissue cyclic AMP accumulation. The effect of methacholine was dose dependent, and significant augmentation was obtained at 0.1 microM with the maximum being attained at about 0.5 microM, whereas more than 10 microM were required to obtain the inhibitory effect. Atropine completely blocked the effect of methacholine. Similar augmentation of isoproterenol effect was obtained by oxotremorine and pilocarpine. Oxotremorine, however, did not inhibit the effect of isoproterenol. Difference in the effect between methacholine or carbamylcholine and oxotremorine was observed in their binding property to cholinergic receptors. A23187 augmented the effect of isoproterenol in a dose-dependent manner. Oxotremorine and A23187 augmented the effect of isoproterenol in the presence of isobutylmethylxanthine, but they did not augment the effect of forskolin and isobutylmethylxanthine on tissue cyclic AMP accumulation. Cholinergic agonist- and A23187-induced augmentation was abolished by omission of calcium in the medium. These results suggest that the augmentation is due to activation of adenylate cyclase, which is mediated by an increase in concentration of intracellular calcium.     

Interrelatinoship between alpha- and beta-adrenergic agonists and histamine in canine airways

To determine the interrelationship between adrenergic receptors in airway smooth muscle and histamine-induced airway constriction, we studied the responses of 26 parasympathectomized dogs to selective and mixed alpha- and beta-adrenergic stimulation in situ by means of an isometric tracheal smoth-muscle preparation. Intra-arterial (IA) phenylephrine (PE) caused dose-related tracheal contraction beginning at 10^?8 mol; maximal active tension was 9.47 ± 2.2 gm F/cm (mean ± SD) at 10^?5 mol. Pretreatment with propranolol augmented tracheal contraction to PE (maximum 35.5 ± 3.2 gm F/cm). The contractile resposne to PE was blocked with a dose of phentolamine (200 μg/kg IA), which did not alter the respionse to acetylcholine. Isoproterenol (ISO) caused dose-related tarcheal relaxation beginning at 4.2 × 10^?11 mol (maximum 43.2 ± 19.6 gm F/cm). Norepinephrine (NE) also caused tracheal relaxation beginning at 1.2 × 10^?6 mol, which was less than relaxation caused by ISO. Prestimulation with histamine did not augment the contractile response to PE but reduced maximal tracheal relaxation to NE (p < 0.001). It is concluded that selective alpha-adrenergic stimulation causes tracheal contraction. However, nonselective stimulation results in tracheal relaxation, even with a weak beat-agonist such as NE. Histamine does not augment alpha-adrenergic contraction as previously suggested but causes physiologic antagonism of beta-adrenergic relaxation of tracheal muscle.     

The effect of calmodulin antagonists on amylase release from the rat parotid gland in vitro

The effect of several calmodulin antagonists on the release of alpha-amylase (EC 3.2.1.1) from rat parotid gland minces was investigated as an approach to determine whether calmodulin has a role in the stimulus-secretion coupling mechanism in this tissue. The phenothiazines, trifluoperazine, chlorpromazine, and thioridazine, failed to inhibit amylase release induced by N6,O2'-dibutyryl adenosine 3':5'-cyclic monophosphate. All three phenothiazines increased basal amylase release at high concentrations. This release was independent of cellular energy, indicating that the release was probably due to the membrane perturbing properties of these compounds rather than their ability to antagonize calmodulin. R24571, a more potent calmodulin antagonist, also failed to inhibit amylase release induced by N6,O2'-dibutyryl adenosine 3':5'-cyclic monophosphate but increased basal amylase release. A different calmodulin antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, partially inhibited release while 8-(N,N-diethylamino)-octyl 3,4,5-trimethoxybenzoate hydrochloride (TMB-8) completely blocked the amylase release induced by the cyclic AMP derivative. However, concentrations of TMB-8 inhibiting amylase release drastically reduced the ATP concentration of rat parotid minces, suggesting that the inhibition of secretion was due to toxic effects of TMB-8 on parotid cells rather than the ability of the compound to antagonize calmodulin. The use of calmodulin antagonists has therefore failed to implicate calmodulin as an intermediate in the stimulus-secretion coupling mechanism of the rat parotid gland.     

Involvement of cytoskeletal integrity in the regulation of Cl- and amylase secretion from rat parotid acinar cells

The cytoskeleton serves as a signal modulator for Ca2+ and cAMP-regulated cell functions including the secretion of ions and granule contents. The interaction between Ca2+ and cAMP signaling systems potentiates amylase secretion and suppresses Cl- secretion in the parotid glands. In this study, we investigated the role of the cytoskeleton in the modulation of Cl- and amylase secretion from rat parotid acinar cells upon activation of each intracellular signaling system and their interaction. Cytochalasin D markedly inhibited the Ca2+-activated outwardly rectifying Cl- current at positive membrane potentials and carbachol (CCh)-induced Cl- currents in the whole-cell configuration at -80 mV, whereas colchicine enhanced Cl- currents. Cytochalasin D, but not colchicine, markedly inhibited CCh-induced Cl- secretion. Synergistic actions of CCh and forskolin on Cl- and amylase secretion were observed even in the presence of cytochalasin D. These results suggest that the synergistic effects of Ca2+ and cAMP signaling systems on amylase and Cl- secretion do not require actin filament integrity but that secretion by the two signals themselves does require actin filament integrity.     

Effects of beta-adrenergic agonists in the parotid gland of the rat--an electrophysiological study

The effects of some beta-adrenergic agonists were studied in the parotid gland of the rat by electrophysiological techniques. In the unoperated gland, isoprenaline caused depolarizations which were slowly developing, long-lasting and of low amplitude. The same response was seen when noradrenaline was combined with alpha-adrenoceptor blocking drugs. A greater number of cells responded to this combination than to isoprenaline. After either parasympathetic or sympathetic denervation 1-3 weeks in advance, to induce supersensitivity, the number of cells responding to beta-adrenoceptor stimulating drugs was significantly increased. In the latter case the threshold dose required to evoke a response was also significantly lowered. Atropine did not have any effect on the isoprenaline-evoked response. The combined parasympathetic and sympathetic denervation did not further increase the responsiveness. It is concluded that beta-adrenoceptor stimulation in the parotid gland of the rat may cause membrane depolarizations. The response is mediated by beta 1-adrenoceptors. The responsiveness is increased in the denervated gland. Secretory studies have demonstrated a supersensitivity to beta-adrenergic agonists as a result of denervation. On the other hand, beta-adrenoceptor stimulation is believed mainly to activate the adenylate cyclase/cyclic AMP system independent of membrane potential changes. It is thus not known if the present 'supersensitivity' is correlated to the increased secretory response earlier demonstrated in this gland.     

Melatonin release by exocytosis in the rat parotid gland

Several beneficial effects on oral health are ascribed to melatonin. Due to its lipophilic nature, non‐protein‐bound circulating melatonin is usually thought to enter the saliva by passive diffusion through salivary acinar gland cells. Recently, however, using transmission electron microscopy (TEM), melatonin was found in acinar secretory granules of human salivary glands. To test the hypothesis that granular located melatonin is actively discharged into the saliva by exocytosis, i.e. contrary to the general belief, the β‐adrenergic receptor agonist isoprenaline, which causes the degranulation of acinar parotid serous cells, was administered to anaesthetised rats. Sixty minutes after an intravenous bolus injection of isoprenaline (5 mg kg^?1), the right parotid gland was removed; pre‐administration, the left control gland had been removed. Samples were processed to demonstrate melatonin reactivity using the immunogold staining method. Morphometric assessment was made using TEM. Gold particles labelling melatonin appeared to be preferentially associated with secretory granules, occurring in their matrix and at membrane level but, notably, it was also associated with vesicles, mitochondria and nuclei. Twenty‐six per cent of the total granular population (per 100 μm² per cell area) displayed melatonin labelling in the matrix; three‐quarters of this fraction disappeared (P < 0.01) in response to isoprenaline, and melatonin reactivity appeared in dilated lumina. Thus, evidence is provided of an alternative route for melatonin to reach the gland lumen and the oral cavity by active release through exocytosis, a process which is under the influence of parasympathetic and sympathetic nervous activity and is the final event along the so‐called regulated secretory pathway. During its stay in granules, anti‐oxidant melatonin may protect their protein/peptide constituents from damage.     

alpha- and beta-adrenergic receptors in regulation of ionic transport in frog cornea.

The effect of catecholamines and adrenergic blocking agents on the transport of Cl- and Na+ of the isolated frog cornea was studied. Cl- transport was stimulated by the three catecholamines tested at 10(-6) to 10(-5) M concentration. The stimulation produced by 10(-6) M epinephrine was partially inhibited by both the alpha-blocker phentolamine and the beta blocker propranolol. The stimulation produced by 10(-6) M isoproterenol was not affected by 10(-5) M phentolamine but was completely prevented by 10(-5) M propranolol. The stimulation produced by 10(-5) M norepinephrine was completely blocked by the 10(-5) M propranolol and partially by 10(-5) M phentolamine. Catecholamines had no effect on the normally small Na+ transport. When the Na+ transport was previously increased by amphotericin B, epinephrine and isoproterenol produced a small and inconsistent stimulatory effect. Norepinephrine was without effect. It is concluded that both alpha- and beta-adrenergic receptors are present in the frog cornea and that the Cl- transport is increased by the stimulation of either receptor. The minimal effect on the Na+ transport further suggests the separation of pathways of the Cl- and Na+ transport in the frog cornea.     

Effects of cholinergic and adrenergic nerve stimulations on amylase release from superfused small segment of rat parotid gland

The effects of the cholinergic and adrenergic nerve stimulations on amylase release from the segment isolated from the rat parotid gland were investigated, employing the combined techniques of electrical field stimulation (FS) and tyramine application with automated fluorescence method for measuring amylase. The maximum amylase release in response to FS for a short period (1 min) was attained at 16 Hz frequency, 2 ms pulse width and 8 V strength stimulation in the absence of any autonomic antagonist. Periodic short-lasting FS using these parameters at intervals of about 15 min could reproduce similar sizes of amylase release for about 2 h. Continuous long-lasting FS (3 V, 2 ms, 16 Hz) caused a transient sharp increase in amylase release followed by a sustained one. The FS-evoked amylase release was completely abolished by tetrodotoxin (TTX) (10(-7) g/ml) and markedly reduced by atropine (7 X 10(-6) M) or by propranolol (10(-5) M), while it was scarcely affected by hexamethonium (3 X 10(-4) M) and phentolamine (10(-5) M). The maximum stimulus frequency of short-lasting FS for amylase release in the presence of propranolol was similar to that (16 Hz) in the control, but it was higher (32 Hz) in the presence of atropine. Reduced response in amylase release to FS by propranolol was completely restored by the superimposed addition of dibutyryl cyclic AMP (10(-4) M). Application of tyramine (5 X 10(-4) M) evoked amylase release in the presence of atropine, which was blocked mostly by propranolol and partly by phentolamine, while tyramine was ineffective in the tissue segment from rats pretreated with 6-hydroxydopamine. From these results the following were suggested; that the intrinsic cholinergic neurotransmitter activates a muscarinic receptor of the acinar cell, while the adrenergic neurotransmitter stimulates mainly a beta-adrenergic and partly an alpha-adrenergic receptor, resulting in amylase release.     

Dynamic changes in the rate of amylase release induced by various secretagogues examined in isolated rat parotid cells by using column perifusion.

Isolated parotid acinar cells were perifused in small columns by embedding them in Bio-Gel P-2 beads as an inert supporting matrix, and the effect of carbamylcholine, substance P, and isoproterenol on the rate of amylase release was examined by measuring amylase activity in the effluent. Amylase release by continuous stimulation with carbamylcholine and substance P was biphasic. They caused a rapid and large increase in the rate of amylase release that reached maximum 30 to 60s after the onset of stimulation, followed by a rapid decline to a lower sustained level that was maintained as long as the agonists were present. The rapid decline in the rate of amylase release was due to rapid development of refractoriness. Repeated 1 min pulse stimulation with these secretagogues showed that recovery from refractoriness was also rapid in onset, and 1 min of washout was sufficient to cause significant recovery from refractoriness for both carbamylcholine and substance P. Recovery, however, was not complete after 10 min of washout. Amylase release by continuous stimulation with isoproterenol, on the other hand, developed more slowly with the peak rate being attained at about 6 min after the onset of stimulation. Refractoriness was not observed in the effect of isoproterenol. The maximum effect in the rate of amylase release attained by carbamylcholine or substance P was higher than that by isoproterenol. These results suggest that the apparent small effect of carbamylcholine and substance P on amylase release reported earlier by using batch systems is probably due to the rapid development of refractoriness to these secretagogues, but not to isoproterenol.     

Calcium mediation of cholinergic-stimulated amylase release from mouse parotid gland.

Amylase release from mouse parotid fragments was stimulated independently by cholinergic and beta-adrenergic agents. The cholinergic agonist, carbachol, significantly increased release of amylase only in Ca2+ containing medium whereas isoproterenol-stimulated amylase release was unaffected by Ca2+ removal. The ionophore, A23187, mimicked the effect of cholinergic stimulation when Ca2+ was present in the medium. Uptake of 45Ca2+ into tissue fragments was enhanced by carbachol and A23187 but not by isoproterenol; atropine blocked the effect of carbachol. Diphenylhydantoin (DPH) and verapamil partially inhibited carbachol-stimulated amylase release and 45Ca2+ uptake, whereas diazoxide potentiated these effects; in all cases there was good parallelism between 45Ca2+ uptake and amylase release. It was concluded that the primary step in the release of amylase from mouse parotid gland in response to cholinergic agents is an increased influx of Ca2+.     

The relationship between stimulation-induced potassium release and amylase secretion in the mouse parotid

1. The output of amylase from superfused mouse parotid segments in response to stimulation with acetylcholine (ACh), phenylephrine and isoprenaline during exposure to solutions with varying potassium concentrations was monitored by an on line automated fluorometric method. 2. During stimulation with ACh or phenylephrine a 10-fold increase in superfusion fluid potassium concentration caused an immediate very marked reduction in amylase output which was fully reversible. A 10-fold reduction in potassium concentration resulted in a prominent rise in amylase output. During stimulation with isoprenaline there was no effect on the amylase output of varying the extracellular potassium concentration. Acetylcholine and phenylephrine caused potassium release from the mouse parotid whereas isoprenaline had no such effect. 3. It appears that under conditions where stimulation-induced potassium release is enhanced there is also an enhanced amylase secretion and vice cersa. There may therefore be a link between passive potassium transport and amylase secretion.     

Influence of castration on isoprenaline-induced amylase release in parotid gland from male rats

The purpose of this study was to determine the influence of testosterone, the male sex hormone, on beta-adrenergic agonist-induced amylase secretion from rat parotid glands. Isoprenaline (isoproterenol)-induced amylase secretion was measured in vitro from the parotid glands of control and castrated rats with and without testosterone replacement. The isoprenaline-induced amylase release was reduced in parotid glands from castrated rats compared to controls. The reduction of amylase release by isoprenaline in parotid glands of castrated rats, could be reversed by administration of testosterone. Furthermore, beta-adrenergic receptor density and the level of isoprenaline-evoked cAMP in parotid glands from castrated rats was lower compared to intact rats. Using SQ-22536 (an adenylyl cyclase inhibitor), dibutyryl cAMP (a cAMP analogue) and verapamil (a calcium channel blocker), we conclude that the impairment of amylase release from parotid glands after castration was not related to either adenylyl cyclase activity or cAMP accumulation. Amylase release from the parotid glands of castrated rats appears to be mediated by an increase in calcium ion influx.     

Mechanism of negative feed-back inhibition of norepinephrine release by alpha-adrenergic agonists

The mechanism of alpha-adrenergic receptor-mediated feedback regulation of [3H]norepinephrine release was studied in the guinea-pig heart. The overflow of [3H]norepinephrine evoked by stimulation (1 Hz for 60 s) was inhibited up to 90% in a dose-dependent manner by norepinephrine (58.5 and 177 nM) and epinephrine (54 and 164 nM). These inhibitory effects were antagonized by 20 mM tetraethylammonium. The overflow of [3H]norepinephrine was near normal when the calcium concentration of the perfusion medium was reduced from a control value of 2.5 to 0.25 mM in the presence of 20 mM tetraethylammonium. Norepinephrine exerted its typical inhibitory effect on the overflow induced in low [Ca2+] and high [tetraethylammonium] solution. Similarly, the inhibitory effects of epinephrine became evident in the presence of 20 mM tetraethylammonium and 0.25 mM calcium ions. Another K-channel blocker, 4-aminopyridine, was also effective in antagonizing the presynaptic actions of norepinephrine; the effect was more pronounced at lower than higher concentrations of norepinephrine. However, after reduction of the calcium ion concentration to 0.2 mM in the presence of 1 mM 4-aminopyridine, all concentrations of norepinephrine significantly reduced the overflow of [3H]norepinephrine. We conclude that the alpha-adrenergic agonists directly influence the availability of calcium ions needed for the transmitter release, and that this effect is brought about without altering the electrical properties (i.e. nerve conduction, action potential size, resting membrane potential of the nerves, etc.). This is in contrast to the actions of acetylcholine and adenosine which, in an earlier study, were shown to inhibit [3H]norepinephrine release primarily by modifying the electrical properties of the nerves.