Two Possibly Distinct Prostaglandin E1 Receptors in N1E-115 Clone: One Mediating Inositol Trisphosphate Formation, Cyclic GMP Formation, and Intracellular Calcium Mobilization and the Other Mediating Cyclic AMP Formation

Prostaglandin E1 (PGE1)-mediated transmembrane signal control systems were investigated in intact murine neuroblastoma cells (clone N1E-115). PGE1 increased intracellular levels of total inositol phosphates (IP), cyclic GMP, cyclic AMP, and calcium ([Ca2+]i). PGE1 transiently increased inositol 1,4,5-trisphosphate formation, peaking at 20 s. There was more than a 10-fold difference between the ED50 for PGE1 at cyclic AMP formation (70 nM) and its ED50 values at IP accumulation (1 microM), cyclic GMP formation (2 microM), and [Ca2+]i increase (5 microM). PGE1-mediated IP accumulation, cyclic GMP formation, and [Ca2+]i increase depended on both the concentration of PGE1 and extracellular calcium ions. PGE1 had more potent intrinsic activity in cyclic AMP formation, IP accumulation, and cyclic GMP formation than did PGE2, PGF2 alpha, or PGD2. A protein kinase C activator, 4 beta-phorbol 12 beta-myristate 13 alpha-acetate, had opposite effects on PGE1-mediated IP release and cyclic GMP formation (inhibitory) and cyclic AMP formation (stimulatory). These data suggest that there may be subtypes of the PGE1 receptor in this clone: a high-affinity receptor mediating cyclic AMP formation, and a low-affinity receptor mediating IP accumulation, cyclic GMP formation, and intracellular calcium mobilization.     

Formation of pyrophosphate by soluble guanylate cyclase from rat lung.

The guanylate cyclase reaction was studied to determine the identity of the product(s) formed other than guanosine-3′,5′-monophosphate (cyclic GMP). Partially purified guanylate cyclase preparations from rat lung catalyzed the formation of nearly equal amounts of PP₁ and of cyclic GMP from GTP. Column chromatography of the enzyme preparation on DEAE-Sephadex or Bio-Gel A-5m failed to separate the enzyme(s) involved in formation of cyclic GMP and of PP₁. Nucleotide inhibitors of cyclic GMP formation also inhibited PP₁ formation, and Ca²⁺, a stimulant of cyclic GMP formation in the presence of Mn²⁺, also stimulated PP₁ formation. Detectable PP₁ formation was not observed when ATP was present instead of GTP.The results show that guanylate cyclase, in vitro, catalyzes the formation of pyrophosphate from GTP concomitant with the synthesis of cyclic GMP.     

Cyclic nucleotides and their relationship to complement-component-C2 synthesis by human monocytes.

The time courses of changes in cyclic nucleotide levels in monocytes have been studied. Histamine and prostaglandin E2 (PGE2) produced a rapid rise in cyclic AMP (peak 15 min) levels, which returned to normal within 4h, whereas cholera toxin, NaF and phosphodiesterase inhibitors produced slow sustained rises lasting over 24h. With the exception of isobutylmethylxanthine (10 mumol X 1(-1), none of these reagents altered cyclic GMP levels. alpha 1-Adrenergic and nicotinic cholinergic receptor-ligand interactions and imidazole produced rapid and relatively short-lived falls in cyclic AMP, and rises in cyclic GMP. In contrast, prostaglandin synthetase inhibitors produced delayed but more sustained falls in cyclic AMP but no rises in cyclic GMP. Agents that increased cyclic AMP decreased complement-component-C2 production, and those that decreased cyclic AMP increased C2 production. Agents that increased cyclic GMP alone (ascorbate, nitroprusside and prostaglandin F2 alpha) did not affect C2 production. Antigen-antibody complexes that stimulate C2 synthesis produced falls in cyclic AMP and rises in cyclic GMP similar to those produced by adrenergic and cholinergic ligands. Serum-treated complexes and anaphylatoxins, which inhibited C2 production, were associated with changes in cyclic AMP similar to those produced by histamine and PGE2. These data suggest that there are two transmembrane signals involved in the regulation of C2 production by monocytes. The inhibitory signal is adenylyl cyclase activation. The stimulatory signal is not so obvious, but may be Ca2+ influx, since the time courses of changes in cyclic nucleotides produced by agents that stimulate C2 synthesis are identical, and alpha 1-adrenergic agonists cause the formation of Ca2+ channels.     

Inhibition of alpha 2-adrenergic receptor-mediated cyclic GMP formation by a phorbol ester, a protein kinase C activator

alpha 2-adrenergic receptor-mediated signal transduction in rat adrenocortical carcinoma cells occurs through the opposing regulation of two second messengers, cyclic GMP and cyclic AMP, in which guanylate cyclase is coupled positively and adenylate cyclase negatively to the receptor signal. We now show that in these cells phorbol 12-myristate 13-acetate (PMA), a known activator of protein kinase C, inhibits the alpha 2-agonist (p-aminoclodine)-dependent production of cyclic GMP in a dose-dependent and time-dependent fashion. The half-maximal inhibitory concentration of PMA was 10(-10) M. A protein kinase C inhibitor, 1-(5-isoquinolinyl-sulfonyl)-2-methyl piperazine (H-7), caused the release of the PMA-dependent attenuation of p-aminoclodine-stimulated cyclic GMP formation. These results suggest that protein kinase C negatively regulates the alpha 2-receptor coupled cyclic GMP system in these cells, a feature apparently shared with the other cyclic GMP-coupled receptors such as those of muscarine, histamine, and atrial natriuretic factor.     

Cyclic GMP regulates cromakalim-induced relaxation in the rat aortic smooth muscle: role of cyclic GMP in K(ATP)-channels.

Recent studies have shown that nitric oxide (NO) modulates K+-channel activity which play an important role in controlling vascular tone. The formation of cyclic guanosine 3',5'-monophosphate (cyclic GMP) has also been recognized to be associated with the vasodilatory effect of NO. Both cyclic GMP and NO increase whole-cell K+-current by activating Ca2+-activated K+-channels (K(Ca)-channels). Here, we show evidence that activators of soluble guanylyl cyclase sodium nitroprusside or 3-morpholino-sydnonimine (SIN-1), and an analogue of cyclic GMP 8-bromo-cyclic GMP enhance the relaxation induced by cromakalim which is blocked by glibenclamide (a specific inhibitor of ATP-sensitive K+-channels [K(ATP)-channels]), and partially attenuated by methylene blue (an inhibitor of cyclic GMP formation). However, this is not due to the increase of cyclic GMP level by cromakalim itself because the relaxation induced by cromakalim is not associated with the changes of cyclic GMP level formed in the aortic smooth muscle. Thus, it is most likely that cyclic GMP also modulates activity of K(ATP)-channels, in addition to K(Ca)-channels, in the rat aorta.     

Steroidogenic effect of atrial natriuretic factor in isolated mouse Leydig cells is mediated by cyclic GMP.

The effects of different atrial natriuretic peptides on cyclic GMP formation and steroidogenesis have been studied in Percoll-purified mouse Leydig cells. Rat atrial peptides rANP (rat atrial natriuretic peptide), rAP-I (rat atriopeptin I) and rAP-II (rat atriopeptin II), in the presence of a phosphodiesterase inhibitor, stimulated cyclic GMP formation in a concentration-dependent manner. In the presence of saturating concentrations of the peptides, a 400-600 fold stimulation of cyclic GMP accumulation was observed. Among the peptides, rAP-II appeared to be the most potent. ED50 values (concentration causing half-maximal effect) for rAP-II, rANP and rAP-I were 1 X 10(-9) M, 2 X 10(-9) M and 2 X 10(-8) M respectively. A parallel stimulation of cyclic GMP formation and testosterone production by the cells was observed after incubation of the cells with various concentrations of rAP-II. In the presence of a saturating concentration of rAP-II (2 X 10(-8) M), maximum stimulation of intracellular cyclic GMP content was obtained within 5 min of incubation. Testosterone production by mouse Leydig cells could be stimulated by 8-bromo cyclic GMP in a concentration-related manner. At a 10 mM concentration of the cyclic nucleotide, steroidogenesis was stimulated to a similar extent as that obtained with a saturating concentration of human chorionic gonadotrophin (5 ng/ml). On the basis of these results we conclude that cyclic GMP acts as a second messenger in atrial-peptide-stimulated steroidogenesis in mouse Leydig cells. The steroidogenic effect of atrial peptides appears to be species-specific, since none of these peptides stimulated testosterone production by purified Leydig cells of rats, though in these cells a 40-60-fold stimulation of cyclic GMP formation in response to each of the three peptides was observed. However, 8-bromo cyclic GMP could stimulate testosterone production in rat Leydig cells. Therefore we conclude that the lack of steroidogenic response in rat Leydig cells to atrial-natriuretic-factor-stimulation results from an insufficient formation of cyclic GMP in these cells. This species difference would appear to result from a lower guanylate cyclase activity in rat Leydig cells.     

Atrial natriuretic factor regulation of cyclic GMP levels and steroidogenesis in isolated fasciculata cells of rat adrenal cortex

Isolated fasciculata cells of rat adrenal cortex, when incubated with atrial natriuretic factor (ANF), stimulated the levels of cyclic GMP and corticosterone production in a concentration-dependent manner without a rise in the levels of cyclic AMP. The ANF-dependent elevation of cyclic GMP was rapid, with a detectable increment in 30 s. ANF also stimulated the particulate guanylate cyclase. These results not only indicate the coupling of cyclic GMP and corticosterone production with ANF signal, but also demonstrate that, like the ACTH signal, cyclic AMP is not the mediator of ANF-induced adrenocortical steroidogenesis.     

Streamers: chemotactic mutants of Dictyostelium discoideum with altered cyclic GMP metabolism

Mutants of Dictyostelium discoideum that developed huge aggregation streams in expanding clones were investigated using optical and biochemical techniques. Representatives of the six complementation groups previously identified (stmA-stmF) were found to be similar to the parental wild-type strain XP55 in both the extent and timing of their ability to initiate and relay chemotactic signals and in the formation of cyclic AMP receptors and phosphodiesterases. The mutants differed from the wild-type in producing an abnormal chemotactic (movement) response visible using both dark-field optics with synchronously aggregating amoebae on solid substrata and light scattering techniques with oxygenated cell suspensions. Mutants of complementation group stmF showed chemotactic movement responses lasting up to 520 s, rather than 100 s as seen in the parental and other strains. Measurements of cyclic GMP formed intracellularly in response to chemotactic pulses of cyclic AMP in stmF mutants showed that abnormally high concentrations of this nucleotide were formed within 10 s and were not rapidly degraded. A causal correlation between defective cyclic GMP metabolism and the altered chemotactic response is suggested, and a model is proposed that accounts for the formation of huge aggregation streams in clones of these mutants.U     

Blockade by NS-7, a neuroprotective compound, of both L-type and P/Q-type Ca2+ channels involving depolarization-stimulated nitric oxide synthase activity in primary neuronal culture

The effect of 4-(4-fluorophenyl)-2-methyl-6-(5-piperidinopentyloxy)pyrimidine hydrochloride (NS-7), a neuroprotective compound, on Ca2+ channels involving the activation of nitric oxide synthase (NOS) was investigated in primary neuronal culture. The NOS activity was estimated from the cyclic GMP formation. The KCl (25 mM)-stimulated cyclic GMP formation was totally abolished by a combined treatment with nifedipine and omega-agatoxin IVA (omega-Aga), whereas spontaneous cyclic GMP formation was partially but significantly reduced by nifedipine. In contrast to nifedipine, NS-7 blocked KCl-stimulated cyclic GMP formation without affecting spontaneous cyclic GMP formation. Subsequently, the effects of nifedipine and NS-7 on L-type Ca2+ channels were compared. Nifedipine blocked equally the cyclic GMP formation stimulated by various concentrations of (+/-)-Bay K 8644, whereas NS-7 inhibited the maximal response without affecting the responses induced by low concentrations of (+/-)-Bay K 8644. The effects of NS-7 on L-type and P/Q-type Ca2+ channels involving KCl-stimulated cyclic GMP formation were subsequently examined. NS-7 suppressed the KCl-stimulated cyclic GMP formation measured in the presence of omega-Aga to almost the same extent as that determined in the presence of nifedipine. In contrast, NS-7 had no influence on ionomycin-induced enhancement of cyclic GMP formation. Finally, NS-7 reversed KCl-induced elevation of the intracellular free Ca2+ concentration. These findings suggest that NS-7 inhibits NOS activation in primary neuronal culture by reducing Ca2+ entry through L-type and P/Q-type Ca2+ channels, in which the inhibition is largely dependent on Ca2+ channel activity.     

Atrial natriuretic factor stimulates luteal guanylate cyclase

Effect of a synthetic atrial natriuretic peptide, rat atriopeptin II (rAP-II) on the formation of cyclic nucleotides and progesterone production in Percoll-purified rat luteal cells was investigated. Incubation of luteal cells with varying concentrations of rAP-II resulted in a dose-related stimulation of intracellular cyclic GMP content; maximum stimulation being achieved with 10 nM rAP-II. The increase in cyclic GMP formation was extremely rapid and a 12-fold increase in the cyclic GMP content over basal level was attained within 5 min of incubation of the cells with 10 nM rAP-II. In the presence of phosphodiesterase inhibitor, 3-isobutyl-1-methyl-xanthine, both basal and rAP-II-stimulated levels of cyclic GMP were increased approximately 10 times, but the magnitude of stimulation remained similar in the presence or absence of the inhibitor. The atrial peptide at the concentration of 1-100 nM, however, had no effect on either basal or gonadotropin-stimulated progesterone production and cyclic AMP formation by the luteal cells. Furthermore, the increase in the level of cellular cyclic GMP content of rAP-II was demonstrated to result from a selective activation of particulate guanylate cyclase.     

Influence of calcium on guanosine 3'',5''-cyclic monophosphate levels in frog rod outer segments

In the presence of 10(-9) M calcium, rod outer segments freshly detached from dark-adapted frog retinas contain between 0.01 and 0.02 moles of guanosine 3',5'-cyclic monophosphate (cyclic GMP) per mole of rhodopsin. The dark level of cyclic GMP is reduced approximately 50% by illumination that bleaches 5 x 10(5) rhodopsin molecules/outer segments. The dark levels of cyclic GMP also can be suppressed to approximately 0.007 mol/mol of rhodopsin by increasing the concentration of calcium from 10(-9) M to 2 x 10(-9) M, and they remain at this level as calcium concentration is raised to 10(-3) M. The final level to which illumination reduces cyclic GMP in unaffected by the calcium concentration between 10(-9) and 10(-3) M. The maximal light-induced decrease in cyclic GMP occurs within 1 s from the onset of illumination at all calcium concentrations. The magnitude and time-course of the light-induced decrease in cyclic GMP measured in these experiments are comparable to values obtained previously (Woodruff et al. 1977. J. Gen. Physiol. 69:677-679; Woodruff and Bownds. 1979. J. Gen. Physiol. 73:629-653). The data are consistent with a role for cyclic GMP in visual transduction irrespective of the calcium concentration.     

Mechanism of cyclic GMP inhibition of inositol phosphate formation in rat aorta segments and cultured bovine aortic smooth muscle cells

In order to clarify the mechanism(s) by which cyclic GMP inhibits the generation of inositol phosphates in rat aorta segments and cultured bovine aortic smooth muscle cells, we studied phosphoinositide hydrolysis and GTPase activity in homogenates and membrane preparations of cultured bovine aortic smooth muscle cells. Pretreatment of homogenate preparations with cyclic GMP plus ATP did not inhibit [8-arginine, 3H] vasopressin (AVP) binding, but resulted in a total suppression of the AVP-induced GTPase activation. The pretreatment with cyclic GMP and ATP also inhibited the formation of inositol phosphates induced by AVP in the presence of low concentrations of guanosine 5'-(gamma-thio)triphosphate (GTP gamma S), or by high concentrations of GTP gamma S alone. However, the formation of inositol phosphates by high concentrations of Ca2+ alone was not blocked. These results suggest that the ability of cyclic GMP to inhibit phosphoinositide hydrolysis results from an inhibition of a guanine nucleotide regulatory protein activation, and the interaction between guanine nucleotide regulatory protein and phospholipase C. While the precise site of this inhibition is not presently known, the inhibition by cyclic GMP is dependent upon the addition of ATP and probably entails a phosphorylation event since adenylylimidodiphosphate can not substitute for the ATP requirement.     

Inhibition of estrogen-induced increases in uterine guanosine 3',5'-cyclic monophosphate levels by inhibitors of protein and RNA synthesis.

Uterine guanosine 3',5'-cyclic monophosphate (cyclic GMP) levels are elevated significantly from 2 to 12 h after a single injection of estradiol-17 beta or diethylstilbestrol to mature, ovariectomized, or immature rats. The accumulation of cyclic GMP is greater in endometrial- than myometrial-enriched uterine tissue. The estrogen-induced increase in cyclic GMP can be prevented by administration of the protein synthesis inhibitors, cycloheximide and puromycin, or by relatively large doses of the RNA synthesis inhibitor, actinomycin D, but not by the muscarinic antagonist, atropine. The requirement for a protein with a relatively rapid rate of turnover is suggested by the demonstration that cycloheximide, when administered after estrogen, can within a 3-h period restore the estrogen-elevated levels of cyclic GMP to those of the non-estrogen-treated tissue.     

Role of Intercellular and Intracellular Communication by Nitric Oxide in Coupling of Muscarinic Receptors to Activation of Guanylate Cyclase in Neuronal Cells

Abstract: Muscarinic receptor-mediated cyclic GMP formation and release of nitric oxide (NO) (or a precursor thereof) were compared in mouse neuroblastoma N1E-115 cells. [³H]Cyclic GMP was assayed in cells prelabeled with [³H]guanine. Release of NO upon the addition of muscarinic agonists to unlabeled neuroblastoma cells (NO donor cells) was quantitated indirectly by its ability to increase the [³H]cyclic GMP level in labeled cells whose muscarinic receptors were inactivated by irreversible alkylation (NO detector cells). Carbachol increased NO release in a concentration-dependent manner, with half-maximal stimulation at 173 μ M (compared to 96 μ M for direct activation of cyclic GMP formation). The maximal effect of carbachol in stimulating release of NO when measured indirectly was lower than that in elevating [³H]cyclic GMP directly in donor cells. Hemoglobin was more effective in blocking the actions of released NO than in attenuating direct stimulation of [³H]cyclic GMP synthesis. There was a good correlation between the ability of a series of muscarinic agonists to release NO or to activate [³H]cyclic GMP formation directly, and the potency of pirenzepine in inhibiting the two responses. Furthermore, there was a similar magnitude of desensitization of both responses by prolonged receptor activation or stimulation of protein kinase C. NO release was also regulated in relation to the cellular growth phase. A model is proposed in which a fraction of NO generated upon receptor activation does not diffuse extracellularly and stimulates cyclic GMP synthesis within the same cell where it is formed (locally acting NO). The remainder of NO that is extruded extracellularly might travel to neighboring cells (neurotransmitter NO) or might be taken back into the cells of origin (homing NO).     

A cyclic GMP-dependent signalling pathway regulates bacterial phytopathogenesis

Cyclic guanosine 3′,5′‐monophosphate (cyclic GMP) is a second messenger whose role in bacterial signalling is poorly understood. A genetic screen in the plant pathogen Xanthomonas campestris (Xcc) identified that XC_0250, which encodes a protein with a class III nucleotidyl cyclase domain, is required for cyclic GMP synthesis. Purified XC_0250 was active in cyclic GMP synthesis in vitro. The linked gene XC_0249 encodes a protein with a cyclic mononucleotide‐binding (cNMP) domain and a GGDEF diguanylate cyclase domain. The activity of XC_0249 in cyclic di‐GMP synthesis was enhanced by addition of cyclic GMP. The isolated cNMP domain of XC_0249 bound cyclic GMP and a structure–function analysis, directed by determination of the crystal structure of the holo‐complex, demonstrated the site of cyclic GMP binding that modulates cyclic di‐GMP synthesis. Mutation of either XC_0250 or XC_0249 led to a reduced virulence to plants and reduced biofilm formation in vitro. These findings describe a regulatory pathway in which cyclic GMP regulates virulence and biofilm formation through interaction with a novel effector that directly links cyclic GMP and cyclic di‐GMP signalling.     

Hepatic cyclic GMP formation is regulated by similar factors that modulate activation of purified hepatic soluble guanylate cyclase

The same factors that regulate the activation of purified hepatic soluble guanylate cyclase by diverse agents possessing distinct requirements for enzyme activation were found to modulate cyclic GMP formation in intact viable hepatic cells. A comparison was made between activation of heme-deficient or heme-reconstituted guanylate cyclase and stimulation of cyclic GMP formation in mouse hepatic slices that were 95% viable and showed no active efflux of cyclic GMP. Heme-dependent activators of guanylate cyclase elicited a greater -fold increase in hepatic cyclic GMP levels in slices from phenobarbital-pretreated than control mice. Brilliant cresyl blue and KCN inhibited both enzyme activation and hepatic cyclic GMP accumulation caused by agents that generate nitric oxide. Hepatic slices from 3,5-diethoxycarbonyl-1,4-dihydrocollidine-treated mice, which are known to develop sharp increases in hepatic protoporphyrin IX/heme concentration ratios, showed elevated resting cyclic GMP levels whereas phenobarbital pretreatment produced decreased resting cyclic GMP levels compared to controls. Guanylate cyclase activation by azide required added catalase, and both enzyme activation and hepatic cyclic GMP formation were inhibited by aminotriazole. Enzyme activation by glyceryl trinitrate and NaNO2 required added thiols. Hepatic slices from acetaminophen-pretreated mice showed marked depletion of sulfhydryls and decreased cyclic GMP formation in response to these enzyme activators. Both effects were completely restored by treatment of thiol-depleted mice with N-acetylcysteine. These observations lend support to the general view that information gained from studies on the regulatory properties of purified soluble guanylate cyclase bears a close relationship to studies on regulatory mechanisms that modulate cyclic GMP formation in intact cells.     

Regulation of Neuronal Nitric Oxide and Cyclic GMP Formation by Ca2+

Nitric oxide (NO) acts as a messenger molecule in the CNS by activating soluble guanylyl cyclase. Rat brain synaptosomal NO synthase was stimulated by Ca2+ in a concentration-dependent manner with half-maximal effects observed at 0.3 microM and 0.2 microM when its activity was assayed as formation of NO and L-citrulline, respectively. Cyclic GMP formation was apparently inhibited, however, at Ca2+ concentrations required for the activation of NO synthase, indicating a down-regulation of the signal in NO-producing cells. Purified synaptosomal guanylyl cyclase was not inhibited directly by Ca2+, and the effect was not mediated by a protein binding to guanylyl cyclase at low or high Ca2+ concentrations. In cytosolic fractions, the breakdown of cyclic GMP, but not that of cyclic AMP, was highly stimulated by Ca2+, and 3-isobutyl-1-methylxanthine did not block this reaction effectively. The effects of Ca2+ on cyclic GMP hydrolysis and on apparent guanylyl cyclase activities were abolished almost completely in the presence of the calmodulin antagonist calmidazolium, whose effect was attenuated by added calmodulin. Thus, a Ca2+/calmodulin-dependent cyclic GMP phosphodiesterase is highly active in synaptic areas of the brain and may prevent elevations of intracellular cyclic GMP levels in activated, NO-producing neurons.     

Prostaglandin Induces Ca2+ Influx and Cyclic GMP Formation in Mouse Neuroblastoma × Rat Glioma Hybrid NG108–15 Cells in Culture

Various prostaglandins (PGs) (10 nM-30 microM) were added to NG108-15 cells in culture, and changes in the levels of intracellular cyclic GMP and Ca2+ were investigated. Exposure of the cells to PGF2 alpha, PGD2, and PGE2 (10 microM) transiently increased the cyclic GMP content 7.5-, 3.9-, and 3.1-fold, respectively. Furthermore, the increased levels of cyclic GMP correlated well with the rise in cytosolic free Ca2+ concentrations induced by the PGs. Other PGs (10 microM), including metabolites and synthetic analogs, which had no effect on intracellular Ca2+, failed to increase the cyclic GMP content in the cells. When extracellular Ca2+ was depleted from the culture medium, the PG-induced increase in cyclic GMP level was almost completely abolished. In addition, treatment of the cells with quin 2 tetraacetoxymethyl ester dose-dependently inhibited the PG-induced cyclic GMP formation. The increase in cyclic GMP content caused by treatment of the cells with a high K+ level (50 mM) was completely blocked by voltage-dependent Ca2+ entry blockers, such as verapamil (10 microM), nifedipine (1 microM), and diltiazem (100 microM); however, the PG (10 microM)-induced increase in cyclic GMP content was not affected by such Ca2+ entry blockers. These findings indicate that PG-induced cyclic GMP formation may require the rise in intracellular Ca2+ level and that the voltage-dependent Ca2+ channels may not be involved in the PG-induced rise in Ca2+ content.     

A possible role for guanosine 3′,5′-monophosphate in the stimulus-secretion coupling in exocrine pancreas

Carbamylcholine, caerulein and cholecystokinin octapeptide rapidly increased the cyclic GMP concentration and amylase secretion in isolated guinea pig pancreatic slices. The cyclic GMP concentration was increased eight-fold over the basal concentration in 30 s, with concomitant increase in the rate of amylase secretion. The tissue concentration of cyclic GMP then rapidly declined to a plateau value of approx. 16% of the peak level within 10 min and was maintained at that concentration for the duration of the experiment. We have shown earlier (Kapoor, C.L. and Krishna, G. (1977) Science 196, 1003–1005) that the decrease of tissue cyclic GMP was due mainly to the secretion of cyclic GMP into the medium. The cyclic AMP concentration in the tissue was not changed, nor was it secreted into the medium.There was a correlation between the concentration response to various agents for the increase in cyclic GMP concentration and amylase secretion in pancreatic slices. Carbamylcholine increased both the cyclic GMP concentration and amylase secretion; the half-maximal effect was achieved at 1.5 μM concentration. Caerulein and cholecystokinin octapeptide were 5000 times more potent than carbamylcholine in increasing cyclic GMP concentration and amylase secretion; the half-maximal effect was achieved at 0.3 nM concentration. Atropine, which completely inhibited the increase in cyclic GMP and amylase secretion induced by carbamylcholine, did not block the effects of caerulein or cholecystokinin octapeptide. These results suggest that various secretagogues induced amylase secretion by increasing the cyclic GMP concentration, but the mechanism by which cyclic GMP caused amylase secretion remains to be elucidated.     

A possible role for guanosine 3',5'-monophosphate in the stimulus-secretion coupling in exocrine pancreas.

Carbamylcholine, caerulein and cholecystokinin octapeptide rapidly increased the cyclic GMP concentration and amylase secretion in isolated guinea pig pancreatic slices. The cyclic GMP concentration was increased eight-fold over the basal concentration in 30 s, with concomitant increase in the rate of amylase secretion. The tissue concentration of cyclic GMP then rapidly declined to a plateau value of approx. 16% of the peak level within 10 min and was maintained at that concentration for the duration of the experiment. We have shown earlier (Kapoor, CL. and Krishna, G. (1977) Science 196, 1003--1005) that the decrease of tissue cyclic GMP was due mainly to the secretion of cyclic GMP into the medium. The cyclic AMP concentration in the tissue was not changed, nor was it secreted into the medium. There was a correlation between the concentration response to various agents for the increase in cyclic GMP concentration and amylase secretion in pancreatic slices. Carbamylcholine increased both the cyclic GMP concentration and amylase secretion; the half-maximal effect was achieved at 1.5 micrometer concentration. Caerulein and cholecystokinin octapeptide were 5000 times more potent than carbamylcholine in increasing cyclic GMP concentration and amylase secretion; the half-maximal effect was achieved at 0.3 nM concentration. Atropine, which completely inhibited the increase in cyclic GMP and amylase secretion induced by carbamylcholine, did not block the effects of caerulein or cholecystokinin octapeptide. These results suggest that various secretagogues induced amylase secretion by increasing the cyclic GMP concentration, but the mechanism by which cyclic GMP caused amylase secretion remains to be elucidated.