Increased somatostatin release from pancreases of alloxan diabetic rats perfused in vitro.

Insulin, glucagon, and somatostatin release $\text{in vitro}$ from perfused pancreases of normal and alloxan-diabetic rats were compared. Insulin and glucagon responses to arginine were decreased in the diabetic group whereas both basal and arginine-stimulated somatostatin release was increased. These results suggest that alterations in pancreatic D cell $\text{function}$ as well as in D cell $\text{mass}$ may contribute to the abnormal insulin and glucagon secretion found in alloxan diabetes.     

Potentiation of arginine-induced glucagon secretion by adenosine

Adenosine and the synthetic adenosine agonists 2-chloroadenosine and N⁶-(L-2-phenylisopropyl)-adenosine were tested for effects on hormone secretion from the rat isolated perfused pancreas. These nucleosides, at concentrations of 5 μM, markedly potentiated both phases of arginine-induced glucagon release; the two synthetic agonists were more effective than adenosine. In the absence of arginine, each of the nucleosides induced a transient burst of glucagon. In contrast, adenosine and both synthetic agonists inhibited arginine-induced insulin secretion to varying degrees and caused only negligible insulin release when perfused without arginine. The adenosine antagonist 8-($\text{p}$-sulfophenyl)-theophylline prevented the actions of adenosine on hormone release from the pancreas. Our data suggest that adenosine potentiation of arginine-induced glucagon release may be mediated via adenosine receptors on alpha cell membranes; such a mechanism could provide an important endogenous control over glucagon secretion.     

Comparison of circulating and peritoneal leukocyte cationic proteins in release of histamine from rat mast cells

Lysosomal cationic proteins which release histamine from rat peritoneal mast cells were prepared from circulating as well as peritoneal leukocytes of rabbits. The release of histamine by cationic proteins and by compound $\text{48}\text{80}$ was compared as a function of temperature, pH and concentration. Cationic protein-mediated histamine release appears to be a non-cytotoxic energy requiring process similar to compound $\text{48}\text{80}\text{-}\text{mediated release}$. It was inhibited by iodoacetate, n-ethylmaleimide, 2,4-dinitrophenol, malonate, oxamate, glutamate and slightly inhibited by 2-deoxyglucose. Pharmacologic inhibition of release by isoproterenol, aminophylline, dibutyryl cyclic AMP and prednisone was also demonstrated.     

Hormonal regulation of amino acid accumulation in human fetal liver explants Effects of dibutyryl cyclic AMP,glucagon and insulin

α-Aminoisobutyrate accumulation by human fetal liver explants in organ culture is stimulated by dibutyryl cyclic AMP ($\text{N}{}^6\text{,}{}^2\text{′O-}\text{dibutyryl}$ adenosine 3′–5′: cyclic monophosphate), glucagon or insulin. Theophylline increased the effect of submaximal concentrations of dibutyryl cyclic AMP or glucagon. Maximal concentrations of glucagon and dibutyryl cyclic AMP yielded the same results as either agent alone. A period of about 4–6 h was required to observe the stimulatory effect of dibutyryl cyclic AMP or insulin, which could be completely prevented by simultaneous incubation with cycloheximide. Maximal effects of either dibutyryl cyclic AMP or glucagon plus insulin produced additive results. These data support the hypothesis that insulin acts via a mechanism independent of the glucagon—cyclic AMP pathway in liver tissue.In addition, the pharmacologic receptor for glucagon was detected in liver explants from a 30-mm (crown - rump) specimen (6 weeks gestation). The liver had the competence to respond to dibutyryl cyclic AMP by the 36-mm stage. Tissue from a 36-mm specimen did not respond to insulin, but a clear response was elicited from a specimen at the 48-mm stage. These data demonstrate the ability of human fetal liver to respond to hormones at a very early stage in gestation.     

Antagonism of prostaglandin-promoted pituitary cyclic amp accumulation and growth hormone secretion in vitro by 7-oxa-13-prostynoic acid

The studies reported here confirm the previously observed potent stimulus to growth hormone (GH) secretion by prostaglandin E₁ (PGE₁). Proportional increments in GH secretion were observed following $\text{in vitro}$ addition of PGE₁ over a concentration range of 10^?7 to 10^?5 M. Growth hormone secretion could not be further stimulated by higher concentrations of prostaglandin. Prostaglandin E₁ also increased cyclic AMP concentration in the pituitary explants in a proportional fashion, which correlated closely with its potency as a growth hormone secretogogue. In order to define more precisely the mechanism by which prostaglandin acts, the effects of prostaglandin antagonist, 7-oxa-13-prostynoic acid, on GH secretion and cyclic AMP accumulation were investigated. Addition of the antagonist alone had no consistent effects on GH secretion or cyclic AMP levels in the pituitary. However, the antagonist significantly reduced the stimulation of hormone release and cyclic AMP accumulation found following addition of PGE₁. Increasing the concentration of antagonist further diminished prostaglandin stimulated hormone release and nucleotide accumulation. The antagonist failed to block the stimulatory effects of theophylline and dibutyryl cyclic AMP on GH release, indicating that the inhibition observed occurred prior to intracellular accumulation of the cyclic nucleotide. These results are consistent with the hypothesis that a prostaglandin receptor on the pituitary somatotrope is linked to the adenyl cyclase-cyclic AMP system.     

Glucagon or cyclic AMP-stimulated synthesis of 5-phosphoribosyl 1-pyrophosphate in isolated hepatocytes and inhibition by antimicrotubular drugs.

Glucagon increased the level of 5-phosphoribosyl 1-pyrophosphate ($\text{PP}$Rib$\text{P}$) in isolated rat hepatocytes; a relatively high concentration of cyclic AMP could replace glucagon. In the presence of glucagon, the rate of incorporation of respective radioactive precursors into purine, pyrimidine, and oxidized pyridine nucleotides was accelerated, indicating that glucagon stimulates the synthesis of $\text{PP}$Rib$\text{P}$. Addition of 10^?6 M colchicine, vinblastin, or podophyllotoxin abolished the glucagon or cyclic AMP-induced increase in the $\text{PP}$Rib$\text{P}$ level. Colchicine did not affect accumulation of cyclic AMP induced by glucagon. These results suggest the involvement of tubulin or microtubules in the signal transfer from cyclic AMP to stimulated synthesis of $\text{PP}$Rib$\text{P}$.     

Opposite effects of acute and repeated administration of desmethylimipramine on adrenergic responsiveness in rat pineal gland.

The effect of acute and repeated desmethylimipramine (DMI) treatment on catecholamine-stimulated production of adenosine 3', 5'-monophosphate (cyclic AMP) in rat pineal gland was studied $\text{in}$$\text{vivo}$. In rats exposed to continuous illumination, the administration of isoproterenol (2μmol/kg) to control animals produced a marked increase in the concentration of cyclic AMP in pineal gland. In contrast, norepinephrine (2μmol/kg) failed to increase the levels of cyclic AMP. After acute treatment with DMI (single injection, 38μmol/kg, i. p.), the isoproterenol-induced rise in cyclic AMP was not significantly different from that measured in control animals. However, acute DMI treatment did allow a significant elevation in the concentration of cyclic AMP in pineal gland in response to norepinephrine. In rats given nine injections of DMI (38μmol/kg, i.p., twice daily) neither isoproterenol nor norepinephrine caused a significant increase in the concentration of cyclic AMP in pineal glands. Although acute treatment with DMI had no significant effect on [³H] dihydroalprenolol binding, chronic treatment with DMI significantly reduced [³H] dihydroalprenolol binding in the pineal gland. The results of this study suggest that while a single administration of DMI can enhance adrenergic responses elicited by norepinephrine, chronic administration of DMI leads to compensatory decreases in receptor density and adrenergic responsiveness.     

Adenosine 3′, 5′-cyclic monophosphate in Schistosoma mansoni

Homogenates of adult $\text{Schistosoma mansoni}$ (blood flukes), isolated from the porto-mesenteric veins of infected mice, contain substantial activities of adenylyl cyclase, cyclic AMP phosphodiesterase, and a cyclic AMP stimulated protein kinase. The adenylyl cyclase, which is largely sedimentable at 10,000xg, is stimulated 20-fold by 10mM sodium fluoride and 1.4 to 2-fold by serotonin, glucagon, prostaglandins E₁, E₂ or B₁. The phosphodiesterase, which is largely sedimentable at 10,000xg, is inhibited by both aminophylline and papaverine but is not influenced by 10mM sodium fluoride. The protein kinase, which is present in the 10,000xg supernatant is stimulated 4 to 8-fold by either cyclic AMP or cyclic GMP. There is a preference for cyclic AMP ($\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1.1×10}{}^{\mathrm{?7}}\text{M}$) over cyclic GMP ($\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 4.5×10}{}^{\mathrm{?6}}\text{M}$). If intact worms are incubated in a glucose free medium there is a mobilization of glycogen stores which is preceded by a rise in cyclic AMP concentration. In a medium with 5mM glucose there is neither a rise in cyclic AMP nor mobilization of glycogen.     

Effects of choleragen on hormonal responsiveness of adenylate cyclase in human fibroblasts and rat fat cells

Choleragen increases cyclic AMP content of confluent human fibroblasts. Maximally effective concentrations of isoproterenol and prostaglandin E₁ also induce large increases in cyclic AMP content of human fibroblasts and in confluent cultures the effect of prostaglandin E₁ is much greater than that of isoproterenol. After incubation with choleragen, the increment in cyclic AMP produced by 2 μM isoproterenol is increased and approaches that produced by 5.6 μM prostaglandin E₁. Although the concentration of isoproterenol which produces a maximal increase in cyclic AMP is similar in both control and choleragen-treated cells, lower concentrations of isoproterenol are more effective in the choleragen-treated cells. In choleragen-treated cells, although the response to 5.6 μM prostaglandin E₁ is reduced by as much as 50%, the concentration of prostaglandin E₁ required to induce a maximal increase in cyclic AMP is $\text{1}\text{10}$ that required in control cells. Thus the capacities of intact human fibroblasts to respond to isoproterenol and prostaglandin E₁ can be altered independently during incubation of intact cells with choleragen. Differences in responsiveness to the two agonists were not demonstrable in adenylate cyclase preparations from control or choleragen-treated cells.In rat fat cells, the effects of choleragen on cyclic AMP content were much smaller than those in fibroblasts. In contrast to its effect on intact fibroblasts, choleragen treatment of rat fat cells did not alter the accumulation of cyclic AMP in response to a maximally effective concentration of isoproterenol. The responsiveness of adenylate cyclase preparations to isoproterenol was also not altered by exposure of fat cells to choleragen.     

Cyclic AMP and cyclic GMP as the respective mediators of the intracycle stimulation of DNA synthesis and mitosis induced by glucagon and insulin in primary neonatal rat hepatocytes

Within a wide range of concentrations ($\text{i.e.}$, from 10^?15 to 10^?8 mole/1), equimolar mixtures of dibutyryl-cyclic AMP and dibutyryl-cyclic GMP or of glucagon and dibutyryl-cyclic GMP or of insulin and dibutyryl-cyclic AMP faithfully mimicked the stimulation of DNA-synthetic and mitotic activities elicited by equimolar associations of glucagon and insulin in 4-to-5-day-old neonatal rat hepatocytes in primary tissue culture. These observations strongly suggest that the intracycle, growth-promoting effects of the two pancreatic hormones are mediated via both purine cyclic nucleotides in the neonatal rat hepatocytes.     

The effect of various secretagogues on accumulation of cyclic AMP and secretion of α-amylase from rat exocrine pancreas

The relationship between accumulation of cyclic AMP and the secretion of α-amylase was investigated in the rat pancreas $\text{in vitro}$. Theophylline and secretin induced an increase in tissue cyclic AMP levels, however, only secretin stimulated secretion of α-amylase. Pancreozymin caused a release of α-amylase and had a biphasic effect on nucleotide levels — stimulation followed by inhibition. Carbachol, which induced a secretory response in the rat pancreas, reduced tissue levels of the cyclic nucleotide.     

Cyclic AMP stimulated phosphorylation of liver pyruvate kinase in hepatocytes.

The addition of glucagon (10^?6 M) to an incubation mixture containing ³²Pi and hepatocytes isolated from livers of rats fed $\text{ad libitum}$ results in both a 3-fold increased incorporation of ³²P into L-type pyruvate kinase and a decreased catalytic activity. The ³²P incorporated into pyruvate kinase was covalently bound to the enzyme as evidenced by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. In addition, exogenous cyclic AMP (10^?3 M) stimulated the phosphorylation and the suppression of catalytic activity to a similar extent. On the other hand, insulin (10^?7 M) had essentially no effect on the incorporation of ³²P into pyruvate kinase or on its catalytic activity under the conditions used in this study. These results suggest that phosphorylation of pyruvate kinase $\text{invivo}$ is stimulated by glucagon via cyclic AMP and cyclic AMP-dependent protein kinase and that the activity of the enzyme is, at least in part, regulated by a phosphorylation-dephosphorylation mechanism.     

A bicyclo-somatostatin analog,highly specific for the inhibition of growth hormone release

The tetradecapeptide somatostatin was cyclized by a combination of conventional and solid phase peptide synthesis methods, to a homodetic cyclic disulfide tetradecapeptide, Wy-40,391:

The analog inhibits the release of growth hormone (GH) $\text{in vivo}$ without affecting either insulin or glucagon secretion. A correlation between binding affinity to the receptors and specificity is suggested.     

The effect of cyclic AMP on glycoprotein secretion in isolated rat hepatocytes

The effects of dibutyryl cyclic AMP on glycoprotein biosynthesis, intracellular mobilization, and secretion in isolated rat hepatocytes are described. Dibutyryl cyclic AMP (2.5 mm) initially suppresses [³H]glucosamine or [³H]fucose incorporation into cellular macromolecular material; however, after $\text{3}\text{1}\text{2}$ h, the incorporation of these radiolabeled carbohydrates into macromolecular material was stimulated relative to control cells. The stimulation in accumulation of cellular glycoprotein occurred in membrane-associated fractions, with most of this accumulation occurring in the Golgi elements. The glycoprotein produced in the presence of dibutyryl cyclic AMP was quantitatively precipitated by antibodies directed against rat serum, suggesting that the accumulated cellular material is normally destined for secretion from the cell. Dibutyryl cyclic AMP also produced a drastic inhibition of glycoprotein secretion which persisted during the cellular accumulation of glycosylated material. Exposure of the hepatocytes to colchicine (10 μm) produced a similar increase in accumulation of [³H]glucosamine-containing immunoprecipitable material in the cellular fraction and a similar inhibition in secretion. The initial dibutyryl cyclic AMP-mediated suppression of synthesis of intracellular glycosylated material occurred entirely in non-membrane-associated intracellular fractions. Also, the initial accumulation of [³H]glucosamine-containing immunoprecipitable material was not suppressed during the first $\text{3}\text{1}\text{2}$ h after exposure to dibutyryl cyclic AMP, suggesting the initial suppression represents a metabolic process unrelated to secretion. The incorporation of [³H]leucine into macromolecular material was inhibited in both cellular and secreted fractions after exposure to dibutyryl cyclic AMP; however, the accumulation into the extracellular environment was inhibited to a greater extent. The patterns of [³H]glucosamine-containing lipid biosynthesis were unaffected by dibutyryl cyclic AMP.     

Malaria enhances cyclic AMP production by immature erythrocytes in vitro.

Infection with a chloroquine-susceptible line of $\text{Plasmodium}$$\text{berghei}$ NYU-2 enhanced the production of cyclic AMP in response to isoproterenol only in immature mouse erythrocytes, which possess a functional hormone-receptor-adenylate cyclase complex. With 10^?7 M isoproterenol the increases in cyclic AMP concentrations (picomoles per 10⁸ erythrocytes) were 19 for a preparation of mature erythrocytes, 81 for a preparation enriched with immature erythrocytes, 25 for a preparation of infected mature erythrocytes, and 900 for a preparation enriched with infected immature erythrocytes. Beta-adrenergic receptor blockade with propranolol prevented this response to isoproterenol but had no effect on the stimulation produced by prostaglandin E₁. These findings indicate that the malaria parasite enhances the responsiveness of a fundamental regulatory system that is intrinsic to immature erythrocytes.     

Studies on the mechanism of insulin-stimulated protein phosphorylation in adipocytes

Insulin exerts two types of effects on protein phosphorylation in adipocytes. First, insulin stimulates phosphorylation of a 123,000 dalton peptide (ATP citrate lyase); second, insulin inhibits the epinephrine-stimulated phosphorylation of a 69,000 dalton peptide.Propranolol, nicotinic acid and concanavalin A, agents which, like insulin, inhibit epinephrine-stimulated cAMP accumulation, also inhibit epinephrine-stimulated phosphorylation of the 69,000 dalton peptide. These agents do not, however, stimulate the phosphorylation of the 123,000 dalton peptide. Carbamylcholine and a variety of cyclic nucleotides (other than cyclic AMP and dibutyryl cAMP) do not alter protein phosphorylation in intact adipocytes. Finally, under conditions wherein insulin fails to inhibit dibutyryl cAMP-stimulated phosphorylation of the 69,000 dalton peptide, insulin-stimulated phosphorylation persists.Thus, while insulin $\text{inhibition}$ of epinephrine-stimulated phosphorylation may be mediated by insulin-induced alterations in cAMP accumulation or action, insulin-$\text{stimulated}$ phosphorylation is $\text{not}$ due to alterations in cyclic nucleotide accumulation or action.     

Pentagastrin and adenosine 3',5'-cyclic monophosphate stimulated secretion of somatostatin from perfused rat gastric mucosa.

Immunoreactive somatostatin is secreted by rat gastric mucosa perifused $\text{in vitro}$. Somatostatin release is stimulated by pentagastrin and cyclic AMP with theophylline. These results suggest that gastric mucosal somatostatin may have a paracrine action as feedback inhibitor of gastrin secretion.     

Somatostatin analogs which inhibit glucagon and growth hormone more than insulin release

Three analogs of somatostatin, [$\text{D}$-Cys¹⁴] -, [Ala², $\text{D}$-Cys¹⁴] - and [$\text{D}$-Trp⁸, $\text{D}$-Cys¹⁴] - somatostatin, were synthesized by the solid phase method, characterized by several means, and tested for their effects on the release of insulin, glucagon, and growth hormone. The peptides sharply suppressed the release of growth hormone $\text{in vitro}$ and glucagon $\text{in vivo}$, but had less effect on insulin secretion $\text{in vivo}$. These analogs, particularly [$\text{D}$-Trp⁸, $\text{D}$-Cys¹⁴] - somatostatin, could possibly be useful for the treatment of diabetes mellitus.     

Studies on submaxillary gland immunoreactive glucagon.

Biologically active immunoreactive glucagon is present in submaxillary gland of rat, mouse, guinea pig and human and can be extracted by saline adjusted to pH 2.8 with HCl. Chromatography on Sephadex G-150 indicates its molecular weight to be 29,000. It has similar immunologic characteristics as pancreatic glucagon. It is biologically active and elevates plasma glucose and insulin when injected intraperitoneally into rats. Compared to pancreatic glucagon, the hyperglycemic effect persists much longer. It competes with pancreatic glucagon for binding to specific glucagon receptors of rat liver plasma membranes. It is stable to pH changes, however, urea dissociates it into several smaller molecular weight fragments including that of 3500. It appears to be an aggregate of smaller glucagon molecules and is not responsible for immunoreactive glucagon in totally eviscerated rats. $\text{In vitro}$, the submaxillary gland does not release immunoreactive glucagon in response to arginine or glucose.