Dissociation of cyclic GMP synthesis from cholinergic-stimulated secretion of protein from rat exocrine pancreas.

The phosphodiesterase inhibitors RO-201724/1 and 1-methyl-3-isobutylxanthine (MIX) stimulate a rapid increase in cyclic GMP content in rat pancreas; the latter agent also potentiates the stimulatory effect of carbachol on cyclic GMP synthesis. However, neither RO-201724/1 nor MIX alter basal secretion of 3H-labeled protein, nor do they affect the secretory response to carbachol used in either suboptimal or optimal concentrations. MIX as well does not alter the rate at which carbachol stimulates pancreatic enzyme release. The ability of carbachol to increase cyclic GMP synthesis is lost if extracellular calcium concentration is lowered to 0.05 mM; at this calcium concentration, however, the muscarinic agent still elicits a marked secretory effect. The dissociation between cyclic GMP synthesis and the secretory response suggests that the cyclic nucleotide does not play a major role in the stimulus--enzyme secretion coupling phenomenon of the exocrine pancreas.     

Effects of dibutyryl cyclic GMP on rabbit pancreas secretion

The isolated rabbit pancreas responds to the hormone cholecystokinin-pancreozymin and its C-terminal peptide with increases in protein secretion and in paracellular permeability. Dibutyryl cyclic GMP competitively inhibits these responses to the C-terminal octapeptide, but with different sensitivity. In low concentrations dibutyryl cyclic GMP lowers only the increase in the paracellular permeability, whereas in high concentrations it inhibits both the protein secretion and the permeability increase. The effect can be explained by assuming competition between dibutyryl cyclic GMP and the hormone at the level of the pancreozymin receptors.     

Effects of dibutyryl cyclic GMP on rabbit pancreas secretion

The isolated rabbit pancreas responds to the hormone cholecystokinin-pancreozymin and its C-terminal peptide with increases in protein secretion and in paracellular permeability. Dibutyryl cyclic GMP competitively inhibits these responses to the C-terminal octapeptide, but with different sensitivity. In low concentrations dibutyryl cyclic GMP lowers only the increase in the paracellular permeability, whereas in high concentrations it inhibits both the protein secretion and the permeability increase. The effect can be explained by assuming competition between dibutyryl cyclic GMP and the hormone at the level of the pancreozymin receptors.     

Stimulus-secretion coupling in exocrine pancreas; possible role of calmodulin

Many calcium-mediated effects in mammalian cells may be activated by calcium-calmodulin stimulated enzymes. These effects are inhibited by various antidepressant drugs which bind to and inactivate calmodulin. In the current study, calmodulin was identified by affinity chromatography and gel electrophoresis in the cytoplasm of dispersed rat pancreatic acinar cells. Its role in enzyme secretion was assessed by evaluating the effects of various antidepressants drugs on the enzyme secretory process. Chlorpromazine, trifluoperazine, thioridazine, chlorprothixene and amitriptyline inhibited amylase secretion stimulated by carbachol, A-23187, and cholecystokinin-pancreozymin but not that elicited by dibutyryl cyclic AMP secretin or vasoactive intestinal peptide (VIP). Haloperidol, sulpiride, phenobarbital, and ethanol were without effect on secretagogue-stimulated enzyme release. Only those agents which blocked secretion also inhibited 45Ca release stimulated by carbachol from isotope preloaded cells. The data suggest that calmodulin may have a functional role in pancreatic enzyme secretion.     

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.     

Receptor-mediated regulation of calcium mobilization and cyclic GMP synthesis in neuroblastoma cells

In neuroblastoma N1E 115 cells, carbachol, histamine and PGE1 elevated cyclic GMP content and, induced the efflux of preloaded 45Ca2+, the release of membrane-bound Ca2+ measured by fluorescent CTC, and the increase in [Ca2+]i as measured by Quin 2 fluorescence. The time course of the responses, the absolute requirement of extracellular Ca2+, the inhibition by receptor blockers, and the concentration dependency on histamine were all similar between these responses. The observation indicates that the mobilization of Ca2+, especially the increase of [Ca2+]i, may be intimately linked to the synthesis of cyclic GMP in the cells.     

Effect of endogenous LH secretion on ovarian cyclic AMP and cyclic GMP levels in the rat

Injection of LH (2 and 10 μg) into proestrus rats increased ovarian cyclic AMP levels and concomitantly decreased the levels of cyclic GMP. When injected into diestrus rats, cyclic AMP increases were even greater, whereas cyclic GMP levels were not significantly different from controls receiving saline injections. Ovarian cyclic nucleotide levels were also examined on different days of the cycle. On the afternoon of proestrus (1700 h), the time when circulating levels of LH are at their maximum, the concentration of cyclic AMP showed a moderate but insignificant increase. At the same time, cyclic GMP levels were significantly decreased. An inverse relation between cyclic AMP and cyclic GMP levels was seen on each day of the cycle. When rats were injected with pentobarbital (35 mg/kg) on the afternoon of proestrus (1300 h) to block the LH surge, the expected increases in ovarian cyclic AMP and decreases in cyclic GMP were effectively blocked. These results indicate that ovarian cyclic AMP and cyclic GMP levels are regulated by circulating LH. The apparent differences in direction of nucleotide response to LH, suggest divergent roles for the nucleotides in ovarian function.     

Pathways of secretion in the exocrine pancreas: the status of resting secretion

In the last decade the concept of two distinct pathways of secretion in the exocrine pancreas has slowly emerged. According to this concept, one pathway is involved in stimulated (regulated) conditions and another under resting (constitutive) conditions. This hypothesis was elaborated at first from the comparison of the specific radioactivities of secretory proteins released by the gland under resting and stimulated conditions. Analysis of the protein composition of the juice released under these two physiological conditions further supported that hypothesis. More recent studies compared the kinetic of accumulation of newly synthesized proteins in zymogen granule and their release in the gland lumen. The latter results are in agreement with a model in which secretory proteins are channelled in two separate pathways, one regulated, and one constitutive. Essentially, the constitutive pathway would correspond to a paragranular route in which the proteins would be immediately secreted instead of being stored in zymogen granules. In addition, some of the proteins released in the juice under "resting" conditions are associated to microvesicles. The term "microvesicular secretion" is used to designate that type of secretion.     

Constitutive protein secretion from the exocrine pancreas of fetal rats

Two general kinds of exocytotic secretion of proteins are known: that which is stimulated by secretagogues; and constitutive exocytosis, which is unable to be stimulated. The exocrine pancreas has often been cited as a model system for the first kind of secretion. However, the release of digestive enzymes from the exocrine pancreas of 1-day prenatal rats cannot be stimulated by secretagogues; therefore, its secretion is constitutive. To gain insight into the intracellular pathways which mediate secretion in the fetal gland, we examined the kinetics of release of newly synthesized proteins. We find that fetal pancreas in a steady state of secretion releases pulse-labeled secretory proteins in two kinetically distinct phases. The first phase occurring during 0-6.5 h of chase comprises approximately 12% of total incorporated radioactivity, the second phase beginning at greater than 7 h of chase comprises the remainder. Based on analysis by electron microscope autoradiography, radiolabel is localized during the first phase of secretion in immature granules/condensing vacuoles, Golgi compartments, and few mature granules. The second phase of secretion occurs when radiolabel is predominantly in mature granules. We propose that secretion occurs via (at least) 2 exocytotic routes, both of which are constitutive in fetal pancreatic tissue.     

The role of aquaporin water channels in fluid secretion by the exocrine pancreas

The mammalian exocrine pancreas secretes a near-isosmotic fluid over a wide osmolarity range. The role of aquaporin (AQP) water channels in this process is now becoming clearer. AQP8 water channels, which were initially cloned from rat pancreas, are expressed at the apical membrane of pancreatic acinar cells and contribute to their osmotic permeability. However, the acinar cells secrete relatively little fluid and there is no obvious defect in pancreatic function in AQP8 knockout mice. Most of the fluid secreted by the pancreas is generated by ductal epithelial cells, which comprise only a small fraction of the gland mass. In the human pancreas, secretion occurs mainly in the intercalated ducts, where the epithelial cells express abundant AQP1 and AQP5 at the apical membrane and AQP1 alone at the basolateral membrane. In the rat and mouse, fluid secretion occurs mainly in the interlobular ducts where AQP1 and AQP5 are again co-localized at the apical membrane but appear to be expressed at relatively low levels. Nonetheless, the transepithelial osmotic permeability of rat interlobular ducts is sufficient to support near-isosmotic fluid secretion at observed rates. Furthermore, apical, but not basolateral, application of Hg²⁺ significantly reduces the transepithelial osmotic permeability, suggesting that apical AQP1 and AQP5 may contribute significantly to fluid secretion. The apparently normal fluid output of the pancreas in AQP1 knockout mice may reflect the presence of AQP5 at the apical membrane.     

Role of GRPergic neurons in secretin-evoked exocrine secretion in isolated rat pancreas

Effects of intrapancreatic gastrin-releasing peptide (GRP)-containing neurons on secretin-induced pancreatic secretion were investigated in the totally isolated perfused rat pancreas. Electrical field stimulation (EFS) increased secretin (12 pM)-induced pancreatic secretions of fluid and amylase. EFS induced a twofold increase in GRP concentration in portal effluent, which was completely inhibited by tetrodotoxin but not modified by atropine. An anti-GRP antiserum inhibited the EFS-enhanced secretin-induced secretions of fluid and amylase by 12 and 43%, respectively, whereas a simultaneous infusion of the antiserum and atropine completely abolished them. Exogenous GRP dose-dependently increased the secretin-induced pancreatic secretion with an additive effect on fluid secretion and a potentiating effect on amylase secretion, which was not affected by atropine. In conclusion, excitation by EFS of GRPergic neurons in the isolated rat pancreas results in the release of GRP, which exerts an additive effect on fluid secretion and a potentiating effect on amylase secretion stimulated by secretin. The release and action of GRP in the rat pancreas are independent of cholinergic tone.     

Effect of lanthanum on 45Ca flux and secretion of protein from rat exocrine pancreas

The relationship between calcium flux and secretion of protein from rat exocrine pancreas was studied. The trivalent cation La³⁺ was used to measure ⁴⁵Ca uptake. Carbachol increased the uptake of ⁴⁵Ca into the tissue prior to onset of secretion of α-amylase. La³⁺ inhibited ⁴⁵Ca uptake in carbachol-stimulated tissue, and also inhibited secretion of protein from the pancreas. Carbachol increased the rate of efflux of ⁴⁵Ca from the pancreas independently of the presence or absence of La³⁺. In the presence of La³⁺, however, the net loss of ⁴⁵Ca from the tissue was reduced. The data suggest that La³⁺ inhibits ⁴⁵Ca influx into the pancreas and may additionally displace intracellular calcium.     

Involvement of cyclic AMP and calcium in exocrine protein secretion induced by vasoactive intestinal polypeptide in rat parotid cells

The effects of vasoactive intestinal polypeptide (VIP) on exocrine protein secretion were studied in enzymatically dispersed cell aggregates from rat parotid glands. VIP (10(-9) - 10(-7) M) stimulated secretion of alpha-amylase in a dose-dependent manner. The VIP-induced release of alpha-amylase was potentiated in the presence of a phosphodiesterase inhibitor. Basal levels of cyclic AMP of the dispersed cells were increased 6.7-fold after stimulation for 10 min by VIP (10(-7) M). The VIP-induced release of alpha-amylase was reduced by 40% when cells were incubated in a Ca2+-free medium in the presence of ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA). Efflux of 45Ca2+ was significantly increased over basal levels by stimulation with VIP (10(-8) and 10(-7) M), but this increased efflux was approximately only half the increased efflux induced by carbachol (10(-5) M). VIP had no effect on the incorporation of [14C]leucine into protein by parotid cells, whereas incorporation was reduced to 30% of the control value by carbachol (10(-5) M). Thus, the VIP-ergic secretory response in the rat parotid gland is associated with a raised intracellular cyclic AMP level and the mobilisation of a different intracellular Ca2+ pool than that mobilised by carbachol. It is, therefore, closely analogous to the beta-adrenergic response.