Stimulation of 45Ca efflux from rat lacrimal gland slices by carbachol and epinephrine

The effects of carbachol (10^?5M) and epinephrine (10^?5M) on efflux of ⁴⁵Ca from rat exorbital lacrimal gland slices were examined. Both carbachol and epinephrine stimulated a transient release of ⁴⁵Ca from the tissue. The quantity of Ca released was estimated to be of the order of 0.5 μmol/g. Release of ⁴⁵Ca by one agonist prevented subsequent release of ⁴⁵Ca by a different agonist. These data support the hypothesis put forth previously that in the lacrimal gland muscarinic or α-adrenergic receptor activation causes a transient increase in membrane permeability to K by triggering the release of a sizable intracellular pool of Ca common to both receptors.     

Calium, prostaglandins and the phosphatidylinositol effect in exocrine gland cells

The hypothesis that arachidonic acid metabolism might be involved in Ca-mobilization mechanisms in exocrine gland cells was investigated. Arachidonate (10⁻⁴M) failed to stimulate protein secretion from slices of pancreas, parotid or lacrimal glands and failed to stimulate ⁸⁶Rb efflux from parotid or lacrimal glands. The stimulation of protein secretion (all three glands) or ⁸⁶Rb efflux (parotid and lacrimal glands) by appropriate secretagogues was unaffected by 10⁻⁵M indomethacin. Eicosatetraynoic acid (2×10⁻⁵M) inhibited ⁸⁶Rb efflux due to carbachol but not that due to physalaemin or ionomycin. Nordihydroguaiaretic acid inhibited lacrimal and parotid gland responses only at high (10⁻⁴M) concentration. Collectively, these results argue against an obligatory role for arachidonate metabolites in Ca-mediated responses of these exocrine glands.In the exocrine glands activation by neurotransmitters (or analogs) of receptors that mobilize cellular Ca also stimulates the incorporation of ³²PO₄ into phosphatidylinositol (1–3). Michell (4,5) has suggested that in some manner this alteration in phospholipid metabolism may be functionally responsible for the opening of surface membrane Ca gates which presumably precedes the expression of a number of Ca-mediated responses by the exocrine cell. That this reaction probably preceeds Ca mobilization is deduced primarily from two experimental observations. First, receptor activation of phosphatidylinositol turnover is not prevented by Ca omission (6–8). Second, the effect is not mimicked by the divalent cationophore A-23187, while other effects of receptor activation are mimicked by this compound (7–9).There has also been some speculation as to the manner in which altered phosphatidylinositol metabolism might be involved in the Ca-gating mechanism (10–14). One such hypothesis suggests that receptor activation may lead to phosphatidylinositol breakdown which in turn leads to the release of free arachidonate (13, 14). As free arachidonate is generally believed to be the rate-limiting substrate for prostaglandin synthesis (15), the resulting prostaglandins might act to mobilize Ca or might act in concert with Ca (13, 14). There is evidence for this hypothesis for the mouse pancreas, where exogenous arachidonate and prostaglandins can stimulate amylase release (13). The effects of arachidonate, carbachol, caerulein and pancreozmin were all antagonized by sub-micromolar concentrations of indomethacin (13), a potent cyclooxygenase inhibitor (15). Additionally, recent reports have demonstrated stimulation by acetylcholine of prostaglandin E synthesis in mouse pancreas (16, 17).The purpose of this study was to examine the general applicability of this hypothesis by investigating the effects of arachidonate and substances that inhibit prostaglandin formation in two other exocrine tissues that show a prominent phosphatidylinositol turnover — the rat parotid and lacrimal glands.     

Effect of carbachol on ouabain-sensitive uptake of 86Rb by dispersed lacrimal gland cells.

Uptake of ⁸⁶Rb was measured in dispersed rat exorbital lacrimal gland cells. The uptake was inhibited by ouabain (0.9 mM) and stimulated by carbachol (10^?5M). In the presence of quabain, in the absence of Ca, or in the presence of decreased extracellular Na, carbachol failed to stimulate ⁸⁶Rb uptake. Cellular concentrations of Na and K were also determined. Cells treated with carbachol had elevated Na content and decreased K content. Omission of external Ca prevented both the K loss and Na gain. Decreasing extracellular Na prevented the Na gain but only partially inhibited the loss of cellular K. The conclusions to be reached from these data are: (1) in the resting lacrimal cell, a quabain sensitive pump actively maintains the intracellular concentration of K high and that of Na low, (2) carbachol acts, through Ca, to increase the passive membrane permeability to Na and K as well as the activity of the pump, and (3) the stimulus for the activation of the pump may be a rise in the intracellular concentration of Na.     

The secretory response in dissociated acini from the rat submandibular gland

Rat submandibular gland was dissociated by enzymatic digestion with collagenase and hyaluronidase, followed by mild mechanical shearing and filtration through a nylon mesh. The dissociated cell populations contained predominantly groups of acinar cells which maintained their acinar arrangement. The morphological and functional viability of the cells was confirmed by electron microscopic examination and a normal secretory response to β-adrenergic or cholinergic stimulation was observed. Both isoproterenol (IPR) and carbachol caused the fusion of secretory granules into large vacuoles which were also continuous with the lumen, and into which the secretory product was released. Secretion was assessed quantitatively from the incorporation of ¹⁴C-glucosamine into the acinar cells and its subsequent release into the culture medium as labelled glycoprotein. IPR stimulated secretion to 125% of untreated controls in the concentration range 5 × 10^?5 to 5 × 10^?7 M, and to 110% of controls at 5 × 10^?8 M, after 40 min incubation. Carbachol stimulated secretion to 131% of controls at 5 × 10^?5 M and to 115% at 5 × 10^?6 M but had no effect at 5 × 10^?7 or 5 × 10^?8 M. The secretory response was blocked by the respective β-adrenergic and cholinergic antagonists, propranolol and atropine. These findings show that dissociated rat submandibular acinar cells provide a useful in vitro model for the study of mucus synthesis and secretion.     

c-Jun NH2-terminal kinase mediates interleukin-1beta-induced inhibition of lacrimal gland secretion

Sjögren's syndrome, an inflammatory disease affecting the lacrimal and salivary glands, is the leading cause of aqueous tear‐deficient type of dry eye. We previously showed that interleukin‐1β (IL‐1β) protein is up regulated in the lacrimal gland of a murine model of Sjögren's syndrome and that exogenous addition of this cytokine inhibits neurotransmitter release and lacrimal gland protein secretion. In the present study we investigated the role of c‐Jun NH₂‐terminal kinase (JNK) in IL‐1β‐mediated inhibition of lacrimal gland secretion and tear production. In vitro, IL‐1β induced a time‐dependent activation of JNK with a maximum 7.5‐fold at 30 min. SP600125, a JNK inhibitor, inhibited, in a concentration‐dependent manner, IL‐1β‐induced activation of JNK with a maximum of 87% at 10^?4 m . In vivo, IL‐1β stimulated JNK and the expression of the inducible isoform of nitric oxide synthase (iNOS). IL‐1β inhibited high KCl and adrenergic agonist induced protein secretion by 85% and 66%, respectively. SP600125 alleviated the inhibitory effect of IL‐1β on KCl‐ and agonist‐induced protein secretion by 79% and 47%, respectively, and completely blocked the expression of iNOS. Treatment for 7 days with SP600125 increased tear production in a murine model of Sjögren's syndrome dry eye. We conclude that JNK plays a pivotal role in IL‐1β‐mediated inhibition of lacrimal gland secretion and subsequent dry eye.     

β-Adrenergic receptor stimulated peroxidase secretion from rat lacrimal gland

Incubation of rat extraorbital lacrimal gland slices with the β-agonist isoproterenol caused peroxidase secretion but no K⁺ release. The peroxidase secretion was inhibited by propranolol. Addition od dibutyryl cyclic AMP or adenosine 3′,5′-cyclic phosphorothioate to lacrimal slices produced peroxidase secretion at a higher rate than that obtained with optimal concentration of isoproterenol. Methyl isobutylxanthine is also a strong stimulator of peroxidase secretion. Peroxidase activity was determined by a modified sensitive guaiacol method. Membrane fraction of lacrimal cells was shown to contain an isoproterenol-stimulated adenylate cyclase activity. It is therefore suggested that there is a β-adrenergic receptor in the rat lacrimal gland and that its stimulation causes activation of an adenylate cyclase which leads to peroxidase secretion.     

Block by phencyclidine of acetylcholine and barium induced adrenal catecholamine secretion

Activation of the sympathetic system by phencyclidine (PCP) should result in catecholamine release from the adrenals. However, adrenalectomy does not reduce PCP-induced hypertension. In an attempt to rectify this inconsistency, the direct effects of PCP on the bovine adrenal medulla were examined. At (3×10^?6M), PCP reduced the acetylcholine-(ACh)-induced catecholamine release by 50%. Surprisingly, barium-induced secretion of catecholamines was also reduced by PCP. ACh-induced catecholamine release was not altered by 10^?3M 4-aminopyridine (4 AP), the potassium channel blocker. Thus, calcium antagonist actions of PCP and consequent block of catecholamine secretion from adrenal medulla may explain the lack of effect of adrenalectomy on PCP-induced hypertension. Possible contributions of calcium and/or potassium channel blockade to other manifestations of PCP overdosage are discussed.     

Amylase secretion by rabbit parotid gland role of cyclic AMP and cyclic GMP

Amylase secretion and changes in the levels of cyclic AMP and GMP were studied in rabbit parotid gland slices incubated in vitro with a variety of neurohumoral transmitters, their analogs and inhibitors. Cyclic GMP levels increased 8-fold 5 min after exposure to carbachol (10⁻⁴ M), without a change in cyclic AMP levels; amylase output also rose. These effects were completely inhibited by muscarinic blockade with atropine, but were unaffected by α-adrenergic blockade with phenoxybenzamine. Epinephrine (4 · 10⁻⁵ M) produced a rapid increase in the levels of both cyclic nucleotides and in amylase release. The increase in cyclic GMP level was inhibited by previous exposure of the slices to phenoxybenzamine, while the cyclic AMP rise was prevented by the β-blocking agent, propranolol. Pure α-adrenergic stimulation with methoxamine (4 · 10⁻⁴ M) produced modest elevations in cyclic GMP content and amylase output, effects blocked by pre-treatment of slices with either atropine or phenoxybenzamine. At a concentration of 4 · 10^{−6 M}, isoproterenol (a β-agonist) failed to affect cyclic GMP levels, but promptly stimulated increases in cyclic AMP levels, and after a short lag, amylase secretion. At a higher dose (4 · 10⁻⁵ M) isoproterenol produced elevations in the levels of both nucleotides. The carbachol-induced effects on cylcic GMP content and amylase release were greatly potentiated by the addition of isoproterenol (4 · 10⁻⁶ M).These data strongly suggest that cholinergic muscarinic agonists and α-adrenergic agonist stimulate amylase output in rabbit parotid gland by mechanisms involving cyclic GMP. The atropine-sensitive intracellular events effected by α-stimulation may be dependent upon endogenous generation of acetylcholine. Both cyclic nucleotides seem to be required for the early rapid secretion of amylase. The unique responses achieved by the combination of carbachol and isoproterenol suggest that isoproterenol may increase the sensitivity of this issue to the effects of cholinergic stimuli.     

Immunoprecipitation of insulin receptors by antibodies against Class 1 antigens of the murine H-2 major histocompatibility complex

In rat lacrimal gland, cholinergic, α- or β-adrenergic or methylxanthine stimulations of protein secretion are extracellular calcium dependent. 10 μM trifluoperazine (TFP) inhibited only cholinergic and α-adrenergic stimulations. Half maximal effect was observed at 30 μM, with all inducers except norepinephrine (3 μM). 10 or 30 μM TFP also suppressed the decrease of L-[³H]leucine incorporation into protein due to carbamylcholine. 100 μM TFP inhibited protein secretion and L-[³H]leucine incorporation. 500 μM TFP promoted cell lysis. It is suggested that: (a) at 100 μM TFP, inhibition is not specific for protein secretion; (b) at 30 μM TFP, inhibition could be related to a role of calmodulin in the secretory regulation process.     

Absence of (Na+, K+)-ATPase involvement in lactose production by lactating guinea pig mammary gland

The role of the (Na⁺, K⁺)-ATPase system in lactose production by the lactating guinea pig mammary gland has been studied in vitro with slices of the gland. In this system there is an initial fast lactose release, mainly representing secretion of preformed lactose, followed by a continuous slow lactose release, representing mainly lactose synthesis. The latter process occurs at a rate of 1.6 to 2.4 g lactose/kg wet wt/h, which value is about half of the lactose production in vivo (3.9 g/kg wet wt/h).Incubation of slices in the presence of 10⁻⁴ M ouabain does not influence the rate of overall lactose production. When determined separately, it does not change either the rate of secretion or that of synthesis. This pleads against a role of the (Na⁺, K⁺)-ATPase system in lactose secretion or synthesis, in particular it seems to rule out control of the rates of these processes by the intracellular potassium concentration. An explanation for the generally observed correlation between the lactose and potassium concentrations in milk, may be that both the maintenance of the intracellular potassium concentration and the lactose synthesis rate require the presence of ATP.     

Differential calcium dependence of contractile responses and 86Rb efflux from the rabbit aorta induced by vasoactive stimuli

⁸⁶Rb was used to monitor potassium movements in strips of rabbit aorta simultaneously with measurements of tension. Histamine, noradrenaline, the prostaglandin endoperoxide analogue U46619, angiotensin II, and 144 mM K⁺ each induced an increase in ⁸⁶Rb efflux concomitantly with contraction. For the first four agonists there was a rank-order correlation between the contractile response and ⁸⁶Rb efflux, but 144 mM K⁺ induced a massive increase in ⁸⁶Rb efflux although it was the weakest contractile stimulus. Contraction and increase in ⁸⁶Rb efflux-induced K⁺ were both reduced by verapamil, which blocks voltage-sensitive calcium channels, implying that both effects of K⁺ were mediated mainly by a depolarisation-induced influx of calcium. Noradrenaline increased both tension and ⁸⁶Rb efflux through an action on alpha-adrenoceptors, but its effect on efflux, unlike its effect on tension, was apparently totally dependent on the presence of extracellular calcium. Experiments performed in the presence of lanthanum, which blocks calcium influx, showed that the intracellular store of calcium released by noradrenaline apparently played no role in inducing ⁸⁶Rb efflux, although it could trigger contraction. Lanthanum also blocked contraction induced by K⁺ but less effect on the increase in ⁸⁶Rb efflux induced by K⁺. Thus, agonist-induced vascular contraction and ⁸⁶Rb efflux can be dissociated, but under normal conditions all the contractile stimuli tested induced ⁸⁶Rb efflux.     

Regulation of calcium fluxes in rat pancreatic islets: Quinine mimics the dual effect of glucose on calcium movements

The effects of quinine and 9-aminoacridine, two blockers of potassium conductance in islet cells, on ⁴⁵Ca efflux and insulin release from perifused islets were investigated in order to elucidate the mechanisms by which glucose initially reduces ⁴⁵Ca efflux and later stimulates calcium inflow in islet cells. In the absence of glucose, 100 μM quinine stimulated ⁴⁵Ca net uptake, ⁴⁵Ca outflow rate and insulin release. Quinine also dramatically enhanced the cationic and the secretory response to intermediate concentrations of glucose, but had little effect on ⁴⁵Ca net uptake, ⁴⁵Ca fractional outflow rate and insulin release at a high glucose concentration (16.7 mM). The ability of quinine to stimulate ⁴⁵Ca efflux depended on the presence of extracellular calcium, suggesting that it reflects a stimulation of calcium entry in the islet cells. In the absence of extracellular calcium, quinine provoked a sustained decrease in ⁴⁵Ca efflux. Such an inhibitory effect was not additive to that of glucose, and was reduced at low extracellular Na⁺ concentration. At a low concentration (5 μM), quinine, although reducing ⁸⁶Rb efflux from the islets to the same extent as a non-insulinotropic glucose concentration (4.4 mM), failed to inhibit ⁴⁵Ca efflux. In the presence of extracellular calcium, 9-aminoacridine produced an important but transient increase in ⁴⁵Ca outflow rate and insulin release from islets perifused in the absence of glucose. In the absence of extracellular calcium, 9-aminoacridine, however, failed to reduced ⁴⁵Ca efflux from perifused islets. It is concluded that quinine, by reducing K⁺ conductance, reproduces the effect of glucose to activate voltage-sensitive calcium channels and to stimulate the entry of calcium into the B-cell. However, the glucose-induced inhibition of calcium outflow rate, which may also participate in the intracellular accumulation of calcium, does not appear to be mediated by changes in K⁺ conductance.     

Spontaneous and potassium-evoked release of 3H-GABA newly synthesized from 3H-glutamine in slices of the rat substantia nigra.

A technique has been developed to measure ³H-GABA not only in tissues but also in medium of slices of the rat substantia nigra (SN) incubated for 15 min with ³H-glutamine. The quantity of ³H-GABA in tissues was about 30 to 35 times that released in the medium. Nevertheless, the amount of the ³H-transmitter spontaneously released was about 10 to 15 times the blank value. GAD activity in the SN was decreased by 40 and 80% respectively ten days after the kainic acid lesion of the ipsilateral striatum or hemitransection. These effects were associated with parallel reductions in the amounts of ³H-GABA accumulated in tissues and released in medium. The spontaneous release of newly synthesized ³H-GABA was increased in absence of calcium and reduced with an excess of calcium (10^?2M). Tetrodotoxin (10^?5, 5.10^?6M) reduced by 40% the spontaneous release of ³H-GABA. These various effects were not associated with significant change in the total accumulation of ³H-GABA in tissues + medium. Finally depolarization of the slices with potassium (30 mM) increased the release of ³H-GABA (300%). This effect was abolished in absence of calcium and was not associated with a significant change in the amount of ³H-GABA accumulated in tissues.     

Effect of local anesthetics on plasma protein secretion by rat hepatocytes

The effects of some local anesthetics on plasma protein secretion by rat liver slices have been studied and have been compared with those of colchicine. Rat liver slices were pulse-labelled with l-[¹⁴C]leucine for 9 min at 37°C, collected on filter paper, washed with non-radioactive leucine and reincubated in the presence or absence of the drug to be tested. The radioactive plasma proteins produced were obtained by immunoprecipitation from either the chase medium or from the washed slices. Chlorpomazine, (3 · 10^?5 M), dibucaine (10^?5 M), lidocaine (10^?3 M) and procaine (5 · 10^?5 M) inhibited both the synthesis and secretion of plasma protein but did not affect the uptake of l-leucine into the slices nor the incorporation of phosphate into intracellular nucleotide phosphates or into phopholipids. The inhibition of secretion elicited by these drugs is probably not due to the inhibition of protein synthesis since cycloheximide, when added to the chase medium at a concentration which completely inhibits protein synthesis, did not inhibit plasma protein secretion, while cycloheximide plus procaine did inhibit secretion and also caused a retention of non-secreted plasma proteins within the slices. Unlike colchicine, howover, procaine did not cause the retained plasma proteins to accumulate in Goli-derived secretory vesicles, but showed a more general effect causing a distribution among several cell fractions.     

Preliminary measurements of intracellular calcium in an insect salivary gland using a calcium-sensitive microelectrode

A calcium-sensitive microeletrode was used to measure free intracellular calcium in salivary gland cells of Calliphora during stimulation with 5-hydroxytryptamine (5-HT). The resting level of calcium was approximately 10^?7M or less but increased in a dose-dependent way sometimes to levels in excess of 10^?6M. The onset of the calcium signal was closely related to changes in membrane and transepithelial potential. This calcium response was greatly reduced when the extracellular calcium concentration was reduced from 10^?3 to 10^?4M. This dependence on external calcium is consistent with previous observations that 5-HT acts to increase the permeability of the basal plasma membrane to calcium. These observations indicate that an increase in the intracellular level of calcium is an early event associated with the onset of fluid secretion in this insect salivary gland.     

Effect of muscarinic agonists on the release of 3H-norepinephrine and 3H-dopamine by potassium and electrical stimulation from rat brain slices

The effect of acetylcholine (ACh) on the release of ³H-norepinephrine (NE) from the cerebellum and ³H-dopamine (DA) from the striatum following the administration of potassium chloride or electrical field stimulation was studied in superfused brain slices. ACh in conc. of 10^?6 and 10^?5M significantly inhibited the release of ³H-NE from cerebellar slices and ³H-DA from striatal slices following 2 min infusion of 50mM potassium chloride. In addition ACh produced a dose-dependent inhibition of the release of ³H-DA from striatal slices following electrical stimulation. The results obtained in the present study are quite consistent with the concept that a muscarinic inhibitory mechanism may be operative on noradrenergic and dopaminergic neurons in the brain.     

Intracellular calcium localization in stimulated and non-stimulated extraorbital lacrimal glands of rats

Acinar cells of extraorbital lacrimal glands from control, pilocarpinetreated, atropine-treated and atropine + pilocarpine-treated rats were studied using a potassium pyroantimonate technique and X-ray microanalysis for calcium localization at the ultrastructural level. This was done in order to identify intracellular compartmentalization of calcium and to elucidate any calcium translocation that might occur during the secretory process. Calcium-pyroantimonate complexes were identified in the mitochondria, plasma membrane and cytoplasmic vesicles of the untreated specimens and in the plasma membrane of atropine-treated specimens, these complexes decreased drastically in the actively-secreting cells. The function of calcium in lacrimal gland secretion and the action of pilocarpine and atropine on membrane calcium are discussed.     

Effects of papaverine on basal and on isoproterenol-stimulated renin secretion from rat kidney slices

Papaverine inhibited the basal renin secretion of rat kidney slices incubated in a physiological salt solution at 37°C. Inhibition was concentration-dependent; secretion was 99 ± 0.2 % inhibited by 5 × 10^?4 M papaverine, and 8 × 10^?5 M was the estimated ED₅₀. In contrast, 2 × 10^?4 M IBMx (3-isobutyl-1-methyl-xanthine) increased the rate of secretion from 215 ± 17 to 366 ± 30 ng hr^?1mg^?1/20 min (p < 0.001). Isoprotenol (4 × 10^?7, 8 × 10^?7, and 5 × 10^?6 M) stimulated renin secretion in a concentration-dependent manner; the stimulatory effects were antagonized by papaverine but unaffected by IBMx. Thus, two known inhibitors of phosphodiesterase--IBMx and papaverine--produce sharply contrasting effects on basal and on isoproterenol-stimulated renin secretion from rat kidney slices.     

Activation by calcium of membrane channels for potassium in exocrine gland cells

In the rat parotid salivary gland, fluid secretion is regulated by alterations in fluxes of monovalent ions. , stimulation of muscarinic, α-adrenergic or substance P receptors provokes a biphasic increase in membrane permeability to K⁺ which can be conveniently assayed as efflux of ⁸⁶Rb. The increased ⁸⁶Rb flux is thought to arise in response to a receptor mediated elevation in [Ca²⁺]_i which activates Ca²⁺-activated K⁺-channels. The biphasic nature of the response is presumably due to a biphasic mode of Ca²⁺ mobilization by secretagogues; a transient response reflects release of a finite pool of Ca from an intracellular store while a more sustained phase results from Ca entry through receptor operated Ca channels or gates. Calcium also mediates an increased Na⁺ entry which in turn activates the Na⁺, K⁺-pump. The mechanism involved in the regulation of monovalent ion channels by Ca²⁺ is not understood.     

Amylase secretion by rabbit parotid gland. Role of cyclic AMP and cyclic GMP.

Amylase secretion and changes in the levels of cyclic AMP and GMP were studied in rabbit parotid gland slices incubated in vitro with a variety of neurohumoral transmitters, their analogs and inhibitors. Cyclic GMP levels increased 8-fold 5 min after exposure to carbachol (10(-4) M), without a change in cyclic AMP levels; amylase output also rose. These effects were completely inhibited by muscarinic blockade with atropine, but were unaffected by alpha-adrenergic blockade with phenoxybenzamine. Epinephrine (4 - 10(-5) M) produced a rapid increase in the levels of both cyclic nucleotides and in amylase release. The increase in cyclic GMP level was inhibited by previous exposure of the slices to phenoxybenzamine, while the cyclic AMP rise was prevented by the beta-blocking agent, propranolol. Pure alpha-adrenergic stimulation with methoxamine (4 - 10(-4) M) produced modest elevations in cyclic GMP content and amylase output, effects blocked by pre-treatment of slices with either atropine or phenoxybenzamine. At a concentration of 4 - 10(-6) M, isoproterenol (a beta-agonist) failed to affect cyclic GMP levels, but promptly stimulated increases in cyclic AMP levels, and after a short lag, amylase secretion. At a higher dose (4 - 10(-5) M) isoproterenol produced elevations in the levels of both nucleotides. The carbachol-induced effects on cyclic GMP content and amylase release were greatly potentiated by the addition of isoproterenol (4 - 10(-6) M). These data strongly suggest that cholinergic muscarinic agonists and alpha-adrenergic agonists stimulate amylase output in rabit parotid gland by mechanisms involving cyclic GMP. The atropine-sensitive intracellular events effected by alpha-stimulation may be dependent upon endogenous generation of acetylcholine. Both cyclic nucleotides seem to be required for the early rapid secretion of amylase. The unique responses achieved by the combination of carbachol and isoproterenol suggest that isoproterenol may increase the sensitivity of this tissue to the effects of cholinergic stimuli.