Increase In Cellular Cyclic AMP Research Articles

Suppressing actions of butyrate on growth hormone (GH) secretion induced by GH-releasing hormone in rat anterior pituitary cells

We assessed the inhibitory effects of butyrate on the growth hormone (GH) secretion in order to investigate the cellular mechanisms in rat somatotrophs. Isolated anterior pituitary cells were cultured in DMEM for several hours, either in the presence (1, 3, or 10 mM) or absence of butyrate, and then stimulated with 10 −7 M GHRH for 30 min, in the presence of butyrate at the concentrations used for the previous culture. The increase in GHRH-induced GH release was significantly reduced in a time-dependent and concentration-dependent manner in the cells previously cultured with butyrate. GH content (the sum of GH released into the medium induced by GHRH stimulation and the GH remaining in the cells after stimulation) was reduced by the culture of cells in the presence of butyrate, which was also inversely dependent on the concentrations used for the culture. Simultaneous addition of an L-type Ca 2+ channel blocker, nifedipine (10 pM), to the medium during 10 −9 M GHRH stimulation significantly reduced the stimulated GH release, which was further significantly decreased by a simultaneous addition of 10 mM butyrate. Butyrate blunted the GHRH (10 −9 M)-induced increase in cellular cyclic AMP and calcium ion concentrations, the activity of protein kinases (A and C), and GHmRNA expression. The expression of mRNA for GPR 41 and 43, known as receptors for short-chain fatty acids, was confirmed in the anterior pituitary cells. These findings suggest that butyrate inhibits GHRH-induced GH release as well as GH production, and the cellular inhibitory actions of butyrate occur in diverse cellular signaling pathways of rat somatotrophs.

Beta-adrenergic stimulation of volume-sensitive chloride transport in lamprey erythrocytes.

We measured the effects of a beta-adrenergic agonist, isoproterenol, on chloride transport and volume regulation of lamprey (Lampetra fluviatilis) erythrocytes in isotonic (288 mosm L(-1)) and hypotonic (192 mosm L(-1)) medium. Isoproterenol at a high concentration (10(-5) M) did not influence chloride transport in isotonic medium but markedly increased chloride fluxes in hypotonic conditions: unidirectional flux increased from 100 mmol kg dcw(-1) h(-1) in the absence to 350 mmol kg dcw(-1) h(-1) (dcw=dry cell weight) in the presence of isoproterenol. Simultaneously, the half-time for volume recovery decreased from 27 to 9 min. Isoproterenol caused an increase in cellular cyclic AMP (cAMP) concentration. The stimulation of chloride transport in hypotonic conditions could be induced by application of the permeable cAMP analogue, 8-bromo-cyclic AMP, suggesting that the effect of beta-adrenergic stimulation on chloride transport occurs downstream of cAMP production. As isoproterenol did not affect unidirectional rubidium fluxes in hypotonic conditions, the transport pathway influenced by beta-adrenergic stimulation is most likely the swelling-activated chloride channel. Because the beta-adrenergic agonist only influenced the transport in hypotonic conditions despite the fact that cAMP concentration also increased in isotonic conditions, the activation may involve a volume-dependent conformational change in the chloride channel.

The roles of microfilaments and intermediate filaments in the regulation of steroid synthesis

Much of the cholesterol used in steroid synthesis is stored in lipid droplets in the cytoplasm of steroid-forming cells. The cholesterol ester in these droplets is transported to the inner mitochondrial membrane where it enters the pathway to steroid hormones as free cholesterol—the substrate for the first enzyme, namely P450 scc. It has been shown that this transport process governs the rate of steroid synthesis and is specifically stimulated by ACTH and its second messenger. The stimulating influence of ACTH on cholesterol transport is inhibited by cytochalasins, by monospecific anti-actin and by DNase I demonstrating that the steroidogenic cell must possess a pool of monomeric actin available for polymerization to F actin if it is to respond to ACTH and cyclic AMP. It has been shown that the two structures involved in cholesterol transport (droplets and mitochondria) are both bound to vimentin intermediate filaments in adrenal and Leydig cells. In addition these filaments are closely associated with the circumferential actomyosin ring in which they are crosslinked by actin microfilaments. In permeabilized adrenal cells Ca 2+ calmodulin phosphorylates vimentin and this change is known to disrupt intermediate filaments and to cause contraction of actomyosin by phosphorylating myosin light chain kinase. Ca 2+ calmodulin stimulated cholesterol transport and steroid synthesis and causes rounding of the responding cells by contraction of the actomyosin, if ATP is also added at the same time. Other agents that disrupt intermediate filaments include anti-vimentin plus ATP in permeabilized cells which also results in rounding of the cell. Acrylamide exerts a similar effect in intact adrenal cells and in addition causes rounding of the cells and increase in steroid synthesis without increase in cyclic AMP. It is also known that if adrenal cells are grown on surfaces treated with poly(HEMA), the cells grow in rounded form and steroid synthesis is increased in proportion to the degree of rounding ( r = 0.92). This response does not involve increase in cellular levels of cylic AMP. It is proposed that in vivo where the cell is always round and cannot show more than strictly limited change in shape, ACTH activates Ca 2+ calmodulin possibly by redistributing cellular Ca 2+. Ca 2+ calmodulin in turn promotes phosphorylation of vimentin and myosin light chain. The first of these phosphorylations shortens intermediate filaments and the second promotes contraction of the actomoyosin ring with internal shortening and approximation of lipid droplets and mitochondria. Details of the earlier events (activation of Ca 2+ calmodulin ) and later changes (transfer of cholesterol to the inner membrane) remain to be elucidated. It is clear however that the action of ACTH requires increase in cellular cyclic AMP. These experimental responses bypass this step in the response to ACTH.

Protein kinase C and protein kinase A regulate the expression of angiotensin II receptor mRNA in smooth muscle cells

Using reverse transcription polymerase chain reaction and fura-2 microfluorometry of intracellular Ca 2+ concentrations, we investigated the effects of angiotensin II and cyclic AMP on the expression of angiotensin II type 1 receptor mRNA and the relationship between angiotensin II receptor mRNA level and physiological responsiveness in rat aortic smooth muscle cells in primary culture. Angiotensin II (1 μM) induced a time- and dose-dependent transient decrease in the level of angiotensin II receptor mRNA. The maximal decrease (50 ± 13% of control) occurred at 6 h, followed by a gradual return to the control level within 24 h. H-7 (30 μM), a relatively specific protein kinase C inhibitor, inhibited decrease in the expression of angiotensin II receptor mRNA induced by 6 h angiotensin II treatment, while 6 h stimulation by 0.3 μM phorbol 12-myristate 13-acetate also induced a decrease (35 ± 8% of control) in the expression of angiotensin II receptor mRNA. An increase in cellular cyclic AMP induced by 10 μM forskolin plus 10 μM 3-isobutyl-1-methyl-xanthine, decreased the angiotensin II receptor mRNA level to 50 ± 13% of the control at 6 h and increased it to 219 ± 39% of the control at 48 h of treatment. Angiotensin II-induced decrease and cyclic AMP-induced increase in the expression of angiotensin II receptor mRNA were accompanied by a reduction and enhancement in [Ca 2+] i transients induced by angiotensin II, respectively. In contrast with angiotensin II receptor mRNA level (102 ± 8% of control), the [Ca 2+] i transients were markedly decreased in angiotensin II plus H-7 treated cells. Although the angiotensin II receptor mRNA level was reduced (50 ± 13% of control), the first phasic elevation of [Ca 2+] i was unchanged (104 ± 14% of control) in 6 h forskolin plus 3-isobutyl-1-methyl-xanthine treated cells. These results suggest that (1) angiotensin II, protein kinase C and protein kinase A regulate the expression of angiotensin II receptor mRNA, and that (2) the increase in receptor mRNA level is accompanied by an increase in physiological responsiveness, while the decrease in receptor mRNA is not always accompanied by a decrease in physiological responsiveness.

Cell density‐dependent decrease in cytoskeletal actin and myosin in cultured osteoblastic cells: Correlation with cyclic AMP changes

During bone development, osteoblasts form a contiguous layer along recently deposited osteoid and their morphology changes from fibroblast-like to cuboidal. In culture, similar changes occur with increased cell density. We examined the possible role of cyclic AMP in this process since cyclic AMP was reported to increase in fibroblasts with increased cell density and similar shape changes were seen in response to parathyroid hormone, which also increases cellular cyclic AMP in osteoblastic cells. Osteoblast-enriched rat calvaria cells were seeded at increasing density. The distribution between Triton X-100 extractable and nonextractable actin and myosin was estimated by polyacrylamide gel electrophoresis. Intracellular cyclic AMP was estimated by prelabeling the cellular ATP pool with 3H-adenine, followed by extraction and separation of 3H-cAMP by high-performance liquid chromatography. We found that osteoblastic cells contain about 40 pg actin and 5.3 pg myosin per cell. Around 60% of the actin and 70% of the myosin were in the nonextractable (crosslinked) form at cell densities of 10,000 to 50,000 cells per cm2. Above 50,000 cells/cm2, there was a cell density-dependent reduction in crosslinked actin and myosin and a concomitant increase in cellular cyclic AMP. A comparable rise in cyclic AMP, produced by incubation with phosphodiesterase inhibitors, and treatment with other agents that increase cyclic AMP produced a similar decrease in the level of cytoskeletal actin and myosin. Cytochalasin B treatment, through its effect on actin polymerization, produced similar changes in cell shape and cytoskeletal actin. The findings suggest that an elevation in intracellular cyclic AMP may play a role in the density-dependent changes in cell shape and microfilament organization observed in osteoblasts.

Pharmacological and biochemical effects of the cardiotonic agent Org10325 in isolated cardiac and vascular tissue preparations

1. The pharmacological and biochemical effects of a novel cardiotonic agent, Org10325 have been studied in isolated cardiac and vascular tissue preparations. 2. Org10325 produced concentration-dependent (0.15-4.8 mM) positive inotropic, positive chronotropic and vascular relaxant responses in rabbit isolated papillary, atrial and aortic preparations, respectively. The maximal chronotropic effect (45%) was significantly less than the isoprenaline maximum. The inotropic effects of Org10325 were not modified by alpha- or beta-adrenoceptor blockade or by pretreatment with reserpine. Org10325 was at least 23 times more potent at relaxing aortic strips pre-contracted with phenylephrine than with KCl. 3. Org10325 (74 microM) potentiated (10-14 fold) the positive inotropic effects of isoprenaline in rabbit isolated papillary muscles. Carbachol inhibited the positive inotropic effect of Org10325. Both the positive inotropic and vasorelaxant effects of Org10325 were accompanied by increases in cyclic AMP but not cyclic GMP. 4. In rat perfused heart preparation Org10325 increased phosphorylase a, cyclic AMP-dependent protein kinase activities and stimulated phosphorylation of contractile proteins (troponin-I and C-protein). 5. Org10325 selectively inhibited the cyclic AMP hydrolytic activity of cyclic AMP high affinity cyclic nucleotide phosphodiesterase (PDE) isoenzymes, PDE III (IC50 65 microM) and PDE IV (IC50 71 microM), from rabbit cardiac ventricle. Weak inhibition (IC50 greater than 250 microM) of PDE I and PDE II was observed. 6. The results show that the cardiac and vascular effects of Org10325 are mediated by an increase in cellular cyclic AMP due to inhibition of PDE III and PDE IV activities. However, in contrast to other PDE-inhibitors OrglO325 produced a marked increase in relaxation time of isolated papillary muscle suggesting the involvement of additional cyclic AMP-independent mechanisms of action.

12‐O‐Tetradecanoylphorbol 13‐Acetate and Forskolin Modify Muscarinic Receptor‐Linked Ca2+ Mobilization in SH‐SY5Y Neuroblastoma Cells Through Different Mechanisms

The phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), which causes differentiation of SH-SY5Y neuroblastoma cells, reduces carbachol binding and carbachol-stimulated Ca2+ mobilization in these cells. The decrease in responsiveness to carbachol is due partially to a reduction in the amount of Ca2+ released by the cells and partially to a decrease in the sensitivity of the cells to carbachol. These effects probably can be attributed to a reduction in muscarinic receptor number and a decrease in receptor affinity, respectively. Forskolin, an alkaloid known to cause an increase in cellular cyclic AMP, enhances Ca2+ influx into the cells without affecting the cytosolic free Ca2+ concentration. The alkaloid causes an apparent restoration of the reduced Ca2+ release, caused by TPA, but does not affect the sensitivity of the cells to carbachol. Forskolin increases the decay of carbachol-induced increase in cytosolic Ca2+. The effects of TPA appear to be linked directly to receptor function, whereas those of forskolin are due to the effect of cyclic AMP on cellular Ca2+ metabolism.

Effect of adenosine and adenosine analogs on [14C]aminopyrine accumulation by rabbit parietal cells

Adenosine receptors that modulate adenylate cyclase activity have been identified recently in a number of tissues. Adenosine A2 receptor is stimulatory to adenylate cyclase, whereas adenosine A1 receptor is inhibitory to adenylate cyclase. We investigated the effect of adenosine and its analogs on [14C]aminopyrine accumulation by rabbit parietal cells. Rabbit gastric mucosal cells were isolated by enzyme digestion. Parietal cells were enriched by nonlinear percoll gradients. [14C]Aminopyrine accumulation was used as an indicator of acid secretion. The effect of 2-chloroadenosine on histamine-stimulated [14C]aminopyrine accumulation was studied. The effects of N-ethylcarboxamideadenosine, 2-chloroadenosine, stable analogs of adenosine, and adenosine on [14C]aminopyrine accumulation were assessed. Cyclic AMP content of parietal cells was determined by radioimmunoassay. Histamine and carbachol, known secretagogues, stimulated [14C]aminopyrine accumulation. 2-Chloroadenosine did not suppress histamine-stimulated [14C]aminopyrine accumulation. 2-Chloroadenosine, N-ethylcarboxamideadenosine, and adenosine dose dependently increased [14C]aminopyrine accumulation. The order of potency was N-ethylcarboxamideadenosine greater than 2-chloroadenosine greater than adenosine. 8-Phenyltheophylline and theophylline, adenosine-receptor antagonists, or cimetidine did not have significant effects on the increase of AP uptake induced by 2-chloroadenosine. Coadministration of dipyridamole, and adenosine uptake inhibitor, augmented the effect of adenosine on [14C]aminopyrine accumulation. 2-Chloroadenosine, N-ethylcarboxamideadenosine, and adenosine each induced a significant increase in cellular cyclic AMP. We conclude that there may be adenosine A2 receptors on rabbit parietal cells which modulate gastric acid secretion.

Further evidence for a phospholipase C-coupled G protein in hamster fibroblasts. Induction of inositol phosphate formation by fluoroaluminate and vanadate and inhibition by pertussis toxin.

We have previously reported that alpha-thrombin induces in resting hamster fibroblasts (CCL39) the formation of inositol phosphates (IP) by activating a GTP-binding protein (G protein) sensitive to pertussis toxin (Paris, S., and Pouysségur, J. (1986) EMBO J. 5, 55-60). Here we show that IP formation in CCL39 cells can also be induced by NaF with AlCl3 and by vanadate. In the presence of Li+, IP accumulation is linear over 30 min with no detectable lag and is concentration-dependent. NaF alone is slightly stimulatory, but a marked potentiation is observed in the presence of AlCl3, by itself without effect. Maximal stimulation is obtained with 10 mM NaF and 3 microM AlCl3, and with vanadate half-maximal effect is achieved at 0.3 mM. Both stimulations are markedly inhibited (up to 80%) by pertussis toxin (half-maximal inhibition at 1-2 ng/ml). We therefore conclude that phospholipase C is stimulated by NaF plus AlCl3 (presumably acting as AlF-4) and by vanadate by direct activation of the regulatory G protein. In addition, NaF inhibits the inositol-1-phosphatase, but this effect is not potentiated by AlCl3. Similarly, vanadate inhibits inositol trisphosphate degradation. Maximal stimulations of phospholipase C by AlF-4 and vanadate are not additive, whereas they are both additive with thrombin effects. Pretreatment of cells for 15 min with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate nearly completely abolishes induction of IP formation by AlF-4 and vanadate, suggesting that protein kinase C exerts a feedback negative control either on the G protein or on phospholipase C itself. An increase in cellular cyclic AMP similarly results in a marked attenuation of AlF-4-induced IP formation, indicating that activation of phospholipase C can be controlled also by cyclic AMP. However, the stimulatory effect of AlF-4 on phospholipase C is clearly dissociated from its effect on the adenylate cyclase system.

Properties of a germination mutant of Phycomyces blakesleeanus

Spores of the Phycomyces blakesleeanus strain S440 germinated only for some 4 to 7% when activated with a heat treatment or with ammonium acetate. Contrary to wild type spores, they showed no increase in trehalase activity during or after the activating treatment. This was not due to a variant trehalase or a defective protein kinase but rather to the absence of an increase in cellular cyclic AMP which normally occurs in the wild type. Phosphodiesterase activity in the mutant was comparable to wild type activity and in both strains phosphodiesterase was inactivated by a heat treatment. The phosphodiesterase inhibitor 1-isobutyl, 3-methyl xanthine caused germination and trehalase activation in the wild type but not in the mutant. The results corroborate the importance of cyclic AMP in the breaking of dormancy and the activation of trehalase in this fungus.

Sodium-dependent phosphate transport inhibited by parathyroid hormone and cyclic AMP stimulation in an opossum kidney cell line.

In the present study we investigated the characteristics of the transport of inorganic phosphate (Pi) in an opossum kidney cell line endowed with parathyroid hormone (PTH) receptors. In confluent epithelial cell culture, a Na-dependent Pi transport (NaPiT) was identified. Preincubation for 1 h with bovine (b)PTH(1-34) at 10(-7) M inhibited the NaPiT from 2.76 +/- 0.11 to 1.08 +/- 0.10 nmol/mg protein X 2 min-1 (p less than 0.001). This inhibition was already expressed 5 min after exposure to 10(-7) M bPTH. It was associated with a 4-fold increase in cellular cyclic AMP. The NaPiT was significantly inhibited at 10(-9) M bPTH, a hormonal concentration which stimulated the cellular cyclic AMP by only 30%. Kinetic analysis of the NaPiT inhibition by 10(-7) M bPTH revealed a decrease in Vmax (from 4.14 +/- 0.32 to 2.41 +/- 0.14 nmol/mg protein X 2 min-1) with no change in Km (0.093 +/- 0.016 versus 0.094 +/- 0.012 mM). The effect of bPTH on NaPiT was not associated with a change in the Na-dependent glucose methylglucopyranoside transport also present in the opossum kidney cell line. The inhibitory influence of bPTH on NaPiT was not affected by blockage of new protein synthesis by cycloheximide. Stimulation of cyclic AMP production by 10(-5) M forskolin, 10 micrograms/ml cholera toxin, 10(-5) M prostaglandin E2 or addition of 10(-5) M dibutyryl cyclic AMP mimicked the PTH-induced reduction in NaPiT. In conclusion, the present study indicates that the opossum epithelial cell line is endowed with a Na-dependent Pi transport system which is selectively inhibited by PTH and agents which increase cyclic AMP production.

On the effect of glucagon on mitochondrial calcium retention in isolated hepatocytes.

Exposure of isolated hepatocytes to glucagon for 45 min caused a 2.5-fold increase in the time (Ca2+ retention time) for which mitochondria subsequently isolated from the cells retained a load of exogenous Ca2+ before its spontaneous release. Half maximal effect of glucagon was observed at a concentration of 0.6 nM. An increase in the Ca2+ retention time was observed after 30 but not 15 min exposure of cells to the hormone. Incubation of hepatocytes with dexamethasone, epinephrine, vasopressin, dibutyryl cyclic AMP or 8-bromo cyclic GMP also induced an increase in mitochondrial Ca2+ retention time. The effect of glucagon was associated with an increase in cellular cyclic AMP and was inhibited by puromycin, cycloheximide and cordycepin, but not by actinomycin D or chloramphenicol. Puromycin caused only a small inhibition of the stimulation by glucagon of mitochondrial pyruvate carboxylation. It is concluded that the effects of glucagon on mitochondrial Ca2+ retention require nuclear DNA-directed protein synthesis and differ, in this respect, from the rapid-onset effects of the hormone on other mitochondrial properties, including pyruvate carboxylation.

Experimental metastasis correlates with cyclic AMP accumulation in B16 melanoma clones.

Metastasis is a complex process whereby tumour cells from a primary neoplastic growth disseminate throughout the body and establish secondary tumour foci in distant organs. Biochemical traits associated with, or essential for, the expression of the metastatic phenotype have not yet been identified. In the course of examining stimulation of the B16 murine melanoma adenylate cyclase by melanocyte-stimulating hormone (MSH) and by the diterpene forskolin, we noted that tumour cell clones isolated from common parent cell populations differed widely in their responses to these agonists. We report here that the accumulation of cyclic AMP induced by MSH or forskolin shows a strong positive correlation with the ability of B16 melanoma clones to form pulmonary tumour colonies when injected intravenously (i.v.) into syngeneic mice ('experimental metastasis'). In parallel in vitro analyses of cyclic AMP metabolism and in vivo assays of experimental metastasis using replicate cell preparations, highly metastatic tumour cell clones consistently show greater than a 30-fold increase in cellular cyclic AMP when exposed to MSH or forskolin. By contrast, clones with limited metastatic abilities respond to the same agonists with only a two- to threefold increase in cellular cyclic AMP. These data suggest that cyclic AMP metabolism is linked with biochemical pathways that are responsible for the formation of experimental metastasis by the B16 melanoma.

Mechanical and hormonal stimulation of cell cultures derived from young rat mandible condyle

Collagenase digestion of young rat condyles released cells which were grown in culture during two weeks. Morphologically, two populations of cells were distinguished, one of which reacted to alkaline phosphatase and resembled chondroblast or osteoblast-like cells. Parathyroid hormone stimulated a 2-fold increase in cellular cyclic AMP, whereas calcitonin had no effect. Physical forces activated cellular cyclic AMP to a 2.5-fold of control levels and increased the incorporation of radioactive thymidine into DNA by 50 per cent. In contrast to cultured bone cells, the response to physical forces was not inhibited by indomethacin in cultured condyle cells. It seems, therefore, that condyle cells are specific in their response to bone-seeking hormones and physical forces.

Irreversible Activation of the Opiate Receptor of Neuroblastoma × Glioma Hybrid Cells by an Alkylating Benzomorphan Derivative

The benozomorphan derivative (-)-2-[2-(p-bromoacetamidophenyl)ethyl]-5,9 alpha-dimethyl-2'-hydroxy-6,7-benzomorphan (BAB), capable of reacting with nucleophilic groups, acts on neuroblastoma X glioma hybrid cells as a potent, irreversible opiate agonist. Its potency in inhibiting the increase in cellular cyclic AMP, evoked by prostaglandin E1, is comparable to that of Leu-enkephalin. This also applies to its capacity to compete with [3H]D-Ala2-Met-enkephalinamide ([3H]DAEA) in binding on cell membrane preparations. The comparatively lower potency of (-)-2-[2-(p-acetamidophenyl)-ethyl]-5,9 alpha-dimethly-2'-hydroxy-5,7-benzomorphan (AB), which differs from BAB in the substitution of the bromoacetamido group by an acetamido group, is of the same order of magnitude as that of morphine. The covalent interaction of BAB with the opiate receptors is deduced from the observations that (1) it is not possible to wash away this compound from the receptors, (2) the potency of BAB in inhibiting the specific binding of [3H]DAEA increases with prolonged preincubation time, and (3) AB behaves as a reversible agonist.

Action of cholera toxin on dispersed acini from rat pancreas: Post-receptor modulation involving cyclic AMP and calcium

In dispersed acini from rat pancreas, cholera toxin caused a significant increase in cellular cyclic AMP but little or no change in amylase secretion. The presence of a secretagogue that causes mobilization of cellular calcium (e.g., cholecystokinin, carbamylcholine, bombesin or ionophore A23187) caused a substantial increase in the effect of cholera toxin on enzyme secretion. Cholera toxin did not alter calcium transport or the changes in calcium transport caused by other secretagogues, and secretagogues that mobilize cellular calcium did not alter cellular cyclic AMP or the increase in cyclic AMP caused by cholera toxin. These results indicate that in dispersed acini from rat pancreas there is post-receptor modulation of the action of cholera toxin by secretagogues that mobilize cellular calcium and that this modulation is a major determinant of the effect of the toxin on enzyme secretion.

Direct action of somatostatin on dispersed mucosal cells from guinea-pig stomach

In dispersed mucosal cells from guinea-pig stomach, somatostatin inhibited in a noncompetitive fashion ( K i, 2 · 10 −8 M) the increase in cellular cyclic AMP caused by histamine but not by prostaglandin E 1 or phosphodiesterase inhibitors. Somatostatin also inhibted the increase in [ 14C]aminopyrine uptake caused by low concentrations of histamine probably by interfering with the synthesis of cellular cyclic AMP.

Unsaturated fatty acid effect on cyclic amp levels in human embryo lung fibroblasts

The addition of arachidonic acid at 250 μM to cultures of human embryo lung fibroblasts (IMR-90) increases cellular cyclic AMP levels within 5 minutes to approximately 15-fold over basal. Other unsaturated fatty acids, 11, 14, 17-eicosatrienoic, linoleic, 8, 11, 14-eicosatrienoic and oleic also cause similar rapid elevation of cellular cyclic AMP. During this time interval, no detectable conversion of the added linoleic or arachidonic acids to prostaglandin is observed. These cells produce prostaglandins at measurable concentrations in response to treatment with ascorbic acid or bradykinin. Saturated fatty acids have no influence on cyclic AMP levels in these cells. This effect of unsaturated fatty acids on cellular cyclic AMP levels varies with the cell type. For example, smooth muscle and endothelial cells obtained from the calf pulmonary artery show very little or no increase in cellular cyclic AMP upon exposure to arachidonic acid.

Action of cholera toxin on dispersed acini from guinea pig pancreas

In dispersed acini from guinea pig pancreas cholera toxin bound reversibly to specific membrane binding sites to increase cellular cyclic AMP and amylase secretion. Cholera toxin did not alter outflux of 45Ca or cellular cyclic AMP. Binding of 125I-labeled cholera toxin could be detected within 5 min; however, cholera toxin did not increase cyclic AMP or amylase release until after 40 min of incubation. There was a close correlation between the dose vs. response curve for inhibition of bindind of 125I-labeled cholera toxin by native toxin and the action of native toxin on cellular cyclic AMP. With different concentrations of cholera toxin, maximal stimulation of amylase release occurred when the increase in cellular cyclic AMP was approximately 35% of maximal. Cholera toxin did not alter the increase in 45Ca outflux or cellular cyclic GMP caused by cholecystokinin or carbachol but significantly augmented the increase in cellular cyclic AMP caused by secretion or vasoactive intestinal peptide. The increase in amylase secretion caused by cholera toxin plus secretin or vasoactive intestinal peptide was the same as that with cholera toxin alone. On the other hand, the increase in amylase secretion caused by cholera toxin plus cholecystokinin or carbachol was significantly greater than the sum of the increases caused by each agent alone.

The role of RNA and protein synthesis in mediating the action of MSH on mouse melanoma cells

Exposure of mouse melanoma cells in culture to MSH (melanocyte stimulating hormone) results in a marked increase in tyrosinase (O-diphenyl: O 2 oxidoreductase) activity following a lag period of 6–9 hours. Within 20 minutes after exposure of cells to MSH, the intracellular levels of cyclic AMP rise to levels which are ten times those of controls but fall to concentrations twice control values by 60 minutes. Transient increases in both protein and RNA synthetic rates also occur following MSH administration correlating in time with the dramatic but rapidly decaying increase in cellular cyclic AMP. The increase in tyrosinase activity observed in response to either MSH, dibutyryl cAMP, or theophylline, is completely suppressed by the addition of either cycloheximide (0.28 μg/ml) or actinomycin D (0.05 μg/ml) as is the basal activity of the enzyme. Results from 14C/ 3H leucine studies suggest that MSH may cause increased de novo synthesis of tyrosinase.