Cyclic AMP Regulates the Expression of Neurokinin1 Receptors by Neonatal Rat Spinal Neurons in Culture

Neurokinin1 (NK1) receptors are up-regulated in the spinal cord during peripheral inflammation, but the biochemical mediators regulating this change have not been resolved. The promoter region of the gene encoding the NK1 receptor contains a cyclic AMP (cAMP)-responsive element. Therefore, we used primary cultures of neonatal rat spinal cord to test whether increasing intracellular cAMP can increase expression of NK1 receptors. Treatment with dibutyryl-cAMP (dbcAMP) resulted in a time-dependent increase in 125I-Bolton-Hunter-substance P (BHSP) binding in the cultures; treatment with dibutyryl-cyclic GMP did not. Treatment with forskolin plus 3-isobutyl-1-methylxanthine mimicked the increase in binding, providing further evidence for the involvement of cAMP in this effect. Scatchard analyses indicated that the increase in BHSP binding was due to an increase in binding capacity. The cAMP-induced increase in BHSP binding was preceded by an increase in levels of mRNA for NK1 receptor and was attenuated by pretreatment with cycloheximide. These data indicate that the cAMP-induced increase in binding was due to increased synthesis of NK1 receptors. Comparison of substance P (SP)-induced production of inositol phosphates between cultures pretreated with dbcAMP and controls suggested that increased expression of NK1 receptors did not result in increased generation of second messenger by NK1 receptor activation. Together, these data indicate that a persistent increase in intracellular cAMP increases expression of NK1 receptors. Because NK1 receptor activation contributes to increased excitability of spinal neurons, the increased expression of NK1 receptors may be important in maintaining responsiveness of spinal neurons to SP in central mechanisms underlying hyperalgesia.     

Photoaffinity labeling of the cell surface adenosine 3':5'-monophosphate receptor of Dictyostelium discoideum and its modification in down-regulated cells

The cAMP cell surface receptor of Dictyostelium discoideum amoebae was identified by the use of the photoaffinity analogue 8-N3-[32P]cAMP. Labeling by intact cells of one component, identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography, could be specifically inhibited by the presence of nonradioactive cAMP. The component, P45 (apparent molecular weight of 45,000), was not identified on vegetative cells but was labeled with increasing intensity as cells differentiated and increased their levels of surface cAMP binding sites. Developmental mutants, starved under conditions where they do not express significant levels of cAMP binding sites, did not incorporate radioactivity into this protein. These mutants did label P45 when starved under differentiation-inducing conditions such that their levels of surface cAMP binding sites increased. P45 co-purified with the plasma membrane fraction isolated from cells to which 8-N3-[32p]cAMP had been covalently bound. Down-regulated amoebae, which displayed approximately 25% of the binding activity of untreated cells, did not label P45. These cells did, however, label a new component with an apparent molecular weight of 47,000 (P47).l The appearance of this component represented the only discernible difference in labeling profile under these conditions. As in the case of P45, radioactive incorporation into P47 did not occur if the photoactivation of 8-N3-[32P]cAMP was performed in the presence of nonradioactive cAMP.     

Second messenger systems in the action of LH and oxytocin on porcine endometrial cells in vitro

LH/hCG as well as oxytocin receptors are present in the porcine endometrium. Oxytocin increases phosphatidylinositol hydrolysis in this tissue, but its action on adenylate cyclase activity is disputed. The second messenger system responding to LH/hCG in endometrial cells has not been established. In this study, we investigated the involvement of protein kinase A and C signaling mechanisms in the action of LH on porcine endometrial cells in vitro. The possibility of cAMP accumulation after treatment of endometrial cells with oxytocin was also investigated. Endometrial tissue was obtained from gilts during Days 12-15 of the estrous cycle. To study the adenylate cyclase system, endometrial cells were cultured for 48 h and then incubated with different doses of LH or oxytocin for 15, 30, 60, and 180 min. To study the phospholipase C system, dispersed cells were first labeled with myo-[3H]inositol and then treated with increasing doses of LH or 100 nM of oxytocin for 30 min. Time- and dose-dependent effect of LH and oxytocin on cAMP concentration was observed. After 30 min of incubation only the highest dose of LH (100 ng/ml) was able to increase cAMP concentration in medium (P < 0.05). Longer periods (1 and 3 h) caused increased cAMP accumulation after treatment with 10 and 100 ng/ml of LH (P < 0.001). Oxytocin-stimulated cAMP concentration was observed after 1 h when only the highest dose (1000 nM) of hormone was used (P < 0.01) and after 3 h of incubation with doses of 10-1000 nM (P < 0.01). LH (10 and 100 ng/ml) increased inositol phosphates (IPs) accumulation in endometrial cells after 30 min of incubation (P < 0.01). Oxytocin involvement in IPs synthesis was more apparent than was LH (P < 0.001 versus P < 0.01). This is the first demonstration that LH receptor signaling leads to increased cAMP generation as well as IPs turnover in porcine endometrium. Oxytocin-dependent cAMP production in endometrial cells of swine was found after longer periods (3 h) of incubation. Our observations lead to the conclusion that both protein kinase A and C second messenger systems are involved in LH action and that oxytocin is able to stimulate adenylate cyclase activity in porcine endometrial cells.     

Regulation of prostaglandin E2 binding to a murine macrophage cell line, P388D1, by insulin.

Preincubation of murine macrophage-like P388D1 cells with physiological amounts of insulin resulted in an increase in prostaglandin E2 binding to these cells, by approximately 2-fold, when compared to untreated cells. Scatchard analysis of the binding of PGE2 to insulin-treated cells indicated that the enhanced binding was due to an increase in receptor number (from 0.30 +/- 0.02 to 0.63 +/- 0.03 fmol/10(6) cells for the high affinity receptor binding sites, and from 2.4 +/- 0.31 to 5.0 +/- 0.41 fmol/10(6) cells for the low affinity receptor binding sites) rather than to an increase in the affinity of the binding sites. The insulin-stimulation of PGE2 binding appeared to be associated with a lowering of the cAMP level in these cells; treatment of cells with insulin lowered the cAMP level by increasing the cAMP phosphodiesterase activity of both the membrane and cytosolic fractions. However, enhanced PGE2 binding to the cells resulted in an increase in cAMP level in the cells. This increase in cAMP level may help to enhance the immunosuppressive action of this prostanoid, as PGE2 is known to suppress many steps in the immune response, including interleukin-1 expression, by raising cAMP levels via activation of receptor-linked adenylate cyclase. Our data suggest that insulin at physiological concentrations may enhance the immunosuppressive action of PGE2.     

Signaling pathways in ascidian oocyte maturation: the roles of cAMP/Epac, intracellular calcium levels, and calmodulin kinase in regulating GVBD

Most mature ascidian oocytes undergo germinal vesicle breakdown (GVBD) when released by the ovary into sea water (SW). Acidic SW blocks this but they can be stimulated by raising the pH, increasing intracellular cAMP levels by cell permeant forms, inhibiting its breakdown or causing synthesis. Boltenia villosa oocytes undergo GVBD in response to these drugs. However, the cAMP receptor protein kinase A (PKA) does not appear to be involved, as oocytes are not affected by the kinase inhibitor H-89. Also, the PKA independent Epac agonist 8CPT-2Me-cAMP stimulates GVBD in acidic SW. GVBD is inhibited in calcium free sea water (CaFSW). The intracellular calcium chelator BAPTA-AM blocks GVBD at 10?μM. GVBD is also inhibited when the ryanodine receptors (RYR) are blocked by tetracaine or ruthenium red but not by the IP(3) inhibitor D-609. However, dimethylbenzanthracene (DMBA), a protein kinase activator, stimulates GVBD in BAPTA, tetracaine or ruthenium red blocked oocytes. The calmodulin kinase inhibitor KN-93 blocks GVBD at 10?μM. This and preceding papers support the hypothesis that the maturation inducing substance (MIS) produced by the follicle cells in response to increased pH causes activation of a G protein which triggers cAMP synthesis. The cAMP then activates an Epac molecule, which causes an increase in intracellular calcium from the endoplasmic reticulum ryanodine receptor. The increased intracellular calcium subsequently activates calmodulin kinase, which causes an increase in cdc25 phosphatase activity, activating MPF and the progression of the oocyte into meiosis.     

Effects of altered thyroid status on beta-adrenergic actions on skeletal muscle glycogen metabolism

The effects of hypothyroidism on glycogen metabolism in rat skeletal muscle were studied using the perfused rat hindlimb preparation. Three weeks after propylthiouracil treatment, serum thyroxine was undetectable and muscle glycogen and Glc-6-P were decreased. Basal and epinephrine-stimulated phosphorylase a and phosphorylase b kinase activities were also significantly reduced, as were epinephrine-stimulated cAMP accumulation and cAMP-dependent protein kinase activity. Conversely, basal and epinephrine-stimulated glycogen synthase I activities were significantly higher while the Ka of the enzyme for Glc-6-P was lower in hypothyroid animals. Propylthiouracil-treated rats also had increased phosphoprotein phosphatase activities towards phosphorylase and glycogen synthase and decreased activity of phosphatase inhibitor 1. beta-Adrenergic receptor binding and basal and epinephrine-stimulated adenylate cyclase activities were reduced in muscle particulate fractions from hypothyroid rats. Administration of triiodothyronine to rats for 3 days after 3 weeks of propylthiouracil treatment restored the altered metabolic parameters to normal. It is proposed that the decreased beta-adrenergic responsiveness of the enzymes of glycogen metabolism in hypothyroid rat skeletal muscle is due to increased activity of phosphoprotein phosphatases and to reduced beta-adrenergic receptors and adenylate cyclase activity.     

Purification and characterization of a membrane-associated cAMP-binding protein from developing Dictyostelium discoideum

Plasma membranes of 6-h differentiated Dictyostelium discoideum cells contain a cAMP-binding protein with the properties ascribed to the chemotaxis receptor present on these cells. We have purified this cAMP-binding protein using DEAE-Sephadex chromatography, hydrophobic chromatography on decylagarose and preparative polyacrylamide gel electrophoresis in nonionic detergent. Photoaffinity labeling of the DEAE-purified material with 8-azido-[32P] cAMP shows that only an Mr = 70,000 species on sodium dodecyl sulfate gels contains a cAMP-binding site. Two-dimensional polyacrylamide gel electrophoresis of material eluted from decyl-agarose and photoaffinity labeled indicates that the cAMP-binding protein is the most acidic of many Mr = 70,000 proteins present. This method is readily scaled up to process up to 10(11) cells which yield from 25 to 100 micrograms of cAMP-binding protein. Nucleotide specificity studies established that the cAMP-binding site of the protein is similar to that of the cAMP receptor assayed on intact cells and membranes. The rates of association and dissociation of the cAMP-binding protein are extremely rapid as found for the receptor, and its affinity for cAMP is comparable. The cAMP-binding protein is a concanavalin A binding glycoprotein, and is resistant to proteolysis by trypsin, but not chymotrypsin. Like the cAMP receptor in membranes and crude detergent extracts, this cAMP-binding protein is inhibited by phenylmethylsulfonyl fluoride. The purified binding protein exists in solution largely as a monomeric species, with some dimer being detected on gel filtration. Based on these criteria, we conclude that this cAMP binding protein represents the binding subunit of the cAMP chemotaxis receptor.     

Rolipram,a selective c-AMP phosphodiesterase inhibitor suppresses oro-facial dyskinetic movements in rats

Since striatal dopamine D2 receptor supersensitivity in the etiology of tardive dyskinesia has been suggested and dopamine D2 receptors are known to inhibit adenylate cyclase activity resulting in a decrease of cyclic adenosine 3′,5′-monophosphate (cAMP) levels, we hypothesized that an increase in cAMP levels ameliorates the condition. In the present study, 21-day haloperidol treatment (1.5 mg/kg I.P.) in rats resulted in an increase in striatal [³H]-spiperone (D2) binding whereas [³H] SCH23390 (D1) binding was unaltered. This haloperidol treatment also induced a significantly increase in the frequency of involuntary chewing movements and tongue protrusions, which are considered as a model of tardive dyskinesia. These dyskinetic movements were suppressed by administration of rolipram (0.5 and 1.0 mg/kg I.P.), an inhibitor of the cAMP phosphodiesterase type IV. The present results suggest that selective cAMP phosphodiesterase type IV inhibitors could be putative therapeutic drugs for tardive dyskinesia.     

Activation of protein kinase in thyroid slices by thyroid-stimulating hormone.

Protein kinase activity in homogenates of control thyroid slices and those incubated with thyroid-stimulating hormone (TSH) and prostaglandin EI was assayed and correlated with changes in cyclic adenosine 3':5'-monophosphate (cAMP) concentrations and binding of [3H]cAMP. Both TSH and prostaglandin E1 (25 mug/ml) increased protein kinase activity and the activity ratio (expressed as activity - cAMP to activity plus cAMP). It is unlikely that such activation reflects effects of the increased cAMP liberated at the time of homogenization. Hormone-induced activation of protein kinase persisted even after the homogenate had been diluted so that its cAMP concentration would be insufficient to achieve maximal activation of the enzyme. In contrast to the previous results of J. D. Corbin, T. R. Soderling, and C. R. Park ((1973 J. Biol. Chem. 248, 1813) using adipose tissue, homogenization of thyroid tissue in 0.5 M NaCl and chromatography using Sephadex G-100 did not seem to stabilize dissociation of protein kinase into its receptor and catalytic subunits. However, increasing amounts of NaCl in the homogenizing buffer were associated with an increase in the cAMP independence of enzyme activity. Dilution of the homogenate did not change the protein kinase activity ratio whether the homogenizing buffer contained NcCl or not. Increasing concentrations of NaF inhibited protein kinase activity. Within 1 to 3 min of incubation of thyroid slices with TSH, protein kinase activity and the activity ratio were increased significantly. This correlated quite well with increased cAMP concentrations in the slices and inhibition of [3H]cAMP binding to the homogenates. Maximal activation of the enzyme was achieved by 10 min which corresponds to the time of maximal effect on cAMP concentrations. Activation of protein kinase was achieved by 0.125 milliunit/ml of TSH and maximal effects with 0.5 to 1.25 milliunits/ml. These amounts agree well with those required for other effects of TSH. Although larger amounts of TSH produced even greater increases in cAMP concentrations this was not always associated with augmented inhibition of [3H]cAMP binding. These results are compatible with the concept that the TSH-mediated increase in cAMP is associated with activation of protein kinase in the intact cell. They also suggest that not all of the intracellular cAMP is available for activation of protein kinase.     

Divergent effects of cations on follicle-stimulating hormone- and forskolin-activated adenylyl cyclase in granulosa cells

The divalent cations magnesium, calcium and manganese, and the monovalent cation, potassium, but not sodium, enhance binding of [125I]iodo-porcine follicle-stimulating hormone to follicle-stimulating hormone (FSH) receptors in membranes of porcine granulosa cells via an increase in the apparent number of binding sites. The objective of the present studies was to determine if increased binding of FSH to its receptor causes increased adenylyl cyclase activity in response to FSH, or conversely, if enhancement of the cyclase or one of its components causes increased binding, or if the two processes are modulated independently. MgCl2 and CaCl2, which both enhance binding in intact cells and in cell-free membrane preparations, had opposite effects on cyclase-MgCl2 stimulatory, CaCl2 inhibitory. In isotonic NaCl, MgCl2 did not enhance binding, but it did increase FSH-stimulated production of cyclic adenosine 3',5'-monophosphate (cAMP). NaCl did not enhance FSH binding and it did not enhance cyclase in cell-free membranes, but it did increase FSH- and forskolin-stimulated cAMP production in intact cells. In intact cells, maximally effective concentrations of MgCl2 and KCl were additive in enhancing cAMP production whereas the effects of NaCl and KCl together were synergistic. The results indicate that although cationic effects on FSH binding are not causally related to effects on cyclase, the cationic microenvironment of the granulosa cell membrane is critical to both FSH receptor and adenylyl cyclase functions.     

Beta adrenergic receptors and cyclic AMP levels in intact human lymphocytes: Effects of age and gender

A saturable, stereospecific high affinity beta₂ adrenergic receptor was demonstrated on intact human peripheral blood lymphocytes using the ligand [¹²⁵I]-iodocyanopindolol ([¹²⁵I]ICYP). A method is described for parallel measurements of saturation binding isotherms and isoproterenol-cAMP responsiveness in split samples of intact lymphocytes isolated from 40 ml. of whole blood. A significant positive correlation between beta receptor density (Bmax) and the ratio of maximal isoproterenol-generated cAMP to basal levels was found in healthy subjects (r=0.65, p < 0.001). A significant positive correlation was found between age and the fold increase over basal cAMP levels induced by isoproterenol. Older females had a significantly higher fold increase in cAMP levels after isoproterenol than older males. These effects were largely accounted for by the lower basal levels of cAMP in older subjects. Beta receptor binding indices (Bmax and K_D) did not differ between males and females, or change with aging. The effects of age and sex upon cAMP levels appear to be at least partly mediated by mechanisms independent of the beta receptor. The method, which describes a convenient assay for parallel measurement of beta receptor binding and cAMP levels in small blood samples, represents a useful model for studying human beta receptor function.     

Modulation of mouse Leydig cell steroidogenesis through a specific arginine-vasopressin receptor

Characterization of specific vasopressin binding sites was investigated in purified mouse Leydig cells using tritiated arginine-vasopressin. Binding of radioligand was saturable, time- and temperature-dependent and reversible. (3H)-AVP was found to bind to a single class of sites with high affinity (Kd = 2.20 +/- 0.18 nM) and low capacity (Bmax = 17.4 +/- 1.8 fmol/10(6) Leydig cells). Binding displacements with specific selective analogs of AVP indicated the presence of V1 subtype receptors on Leydig cells. The ability of AVP to displace (3H)-AVP binding was greater than LVP and oxytocin. The unrelated peptides, somatostatin and substance P, were less potent, while neurotensin and LHRH did not displace (3H)-AVP binding. The time-course effects of AVP-pretreatment on basal and hCG-stimulated testosterone and cAMP accumulations were studied in primary culture of Leydig cells. Basal testosterone accumulation was significantly increased by a 24 h AVP-pretreatment of Leydig cells (P less than 0.001). This effect was potentiated by the phosphodiesterase inhibitor (MIX) and was concomitantly accompanied by a slight but significant increase in cAMP accumulation (P less than 0.01). AVP-pretreatment of the cells for 72 h had no effect on basal testosterone accumulation, but exerted a marked inhibitory effect on the hCG-stimulated testosterone accumulation (P less than 0.001). This reduction of testosterone accumulation occurred even in the presence of MIX and was not accompanied by any significant change of cAMP levels. We conclude from these data that AVP is capable of modulating steroidogenesis in Leydig cells through specific and functionally V1 receptor subtype and postulate that this effect may be part of an intratesticular paracrine/autocrine control mechanism.     

The cell density factor CMF regulates the chemoattractant receptor cAR1 in Dictyostelium

Starving Dictyostelium cells aggregate by chemotaxis to cAMP when a secreted protein called conditioned medium factor (CMF) reaches a threshold concentration. Cells expressing CMF antisense mRNA fail to aggregate and do not transduce signals from the cAMP receptor. Signal transduction and aggregation are restored by adding recombinant CMF. We show here that two other cAMP-induced events, the formation of a slow dissociating form of the cAMP receptor and the loss of ligand binding, which is the first step of ligand-induced receptor sequestration, also require CMF. Vegetative cells have very few CMF and cAMP receptors, while starved cells possess approximately 40,000 receptors for CMF and cAMP. Transformants overexpressing the cAMP receptor gene cAR1 show a 10-fold increase of [3H]cAMP binding and a similar increase of [125I]CMF binding; disruption of the cAR1 gene abolishes both cAMP and CMF binding. In wild-type cells, downregulation of cAR1 with high levels of cAMP also downregulates CMF binding, and CMF similarly downregulates cAMP and CMF binding. This suggests that the cAMP binding and CMF binding are closely linked. Binding of approximately 200 molecules of CMF to starved cells affects the affinity of the majority of the cAR1 cAMP receptors within 2 min, indicating that an amplifying mechanism allows one activated CMF receptor to regulate many cARs. In cells lacking the G-protein beta subunit, cAMP induces a loss of cAMP binding, but not CMF binding, while CMF induces a reduction of CMF binding without affecting cAMP binding, suggesting that the linkage of the cell density-sensing CMF receptor and the chemoattractant cAMP receptor is through a G-protein.     

A novel GIP receptor splice variant influences GIP sensitivity of pancreatic beta-cells in obese mice

Gastric inhibitory polypeptide (GIP) is an incretin that potentiates insulin secretion from pancreatic beta-cells by binding to GIP receptor (GIPR) and subsequently increasing the level of intracellular adenosine 3',5'-cyclic monophosphate (cAMP). We have identified a novel GIPR splice variant in mouse beta-cells that retains intron 8, resulting in a COOH-terminal truncated form (truncated GIPR). This isoform was coexpressed with full-length GIPR (wild-type GIPR) in normal GIPR-expressing tissues. In an experiment using cells transfected with both GIPRs, truncated GIPR did not lead to cAMP production induced by GIP but inhibited GIP-induced cAMP production through wild-type GIPR (n = 3-4, P < 0.05). Wild-type GIPR was normally located on the cell surface, but its expression was decreased in the presence of truncated GIPR, suggesting a dominant negative effect of truncated GIPR against wild-type GIPR. The functional relevance of truncated GIPR in vivo was investigated. In high-fat diet-fed obese mice (HFD mice), blood glucose levels were maintained by compensatory increased insulin secretion (n = 8, P < 0.05), and cAMP production (n = 6, P < 0.01) and insulin secretion (n = 10, P < 0.05) induced by GIP were significantly increased in isolated islets, suggesting hypersensitivity of the GIPR. Total GIPR mRNA expression was not increased in the islets of HFD mice, but the expression ratio of truncated GIPR to total GIPR was reduced by 32% compared with that of control mice (n = 6, P < 0.05). These results indicate that a relative reduction of truncated GIPR expression may be involved in hypersensitivity of GIPR and hyperinsulinemia in diet-induced obese mice.     

Regulatory factors of glucocorticoid binding in early and term rat placenta

We have measured by an exchange procedure the binding of [3H]dexamethasone in cytosol of early (10-13 days) and late (19-22 days) placentas from pregnant rats. Binding was 3-fold higher in late placentas both in the presence of Na2MoO4. We then studied some possible regulatory factors in order to explain differences in binding at both gestational ages. The activity of enzymes compromising the phosphorylation (acid and alkaline phosphatases) or stability (protease) of the receptor were normal or lower in early as opposed to late placenta, discarding these enzymes as leading regulatory factors. Cyclic nucleotides were also studied, in view that they regulate steroid binding in uterus and placenta. Both basal and epinephrine-stimulated production of cAMP were higher in early placenta. cAMP (but not cGMP) inhibited [3H]dexamethasone binding by reducing the number of sites without changing the Kd. Moreover, addition of epinephrine in concentrations that maximally stimulated cAMP, inhibited subsequent binding of [3H]dexamethasone in cytosol. We suggest that cAMP may be a modulator of glucocorticoid binding at the early stages of placental development. The significance of this mechanism may be understood in terms of the opposing effects of cAMP and glucocorticoids on placental progesterone production.     

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.     

Independent control of selected insulin-sensitive cell membrane and intracellular functions — the linkage of cell membrane and intracellular events controlled by insulin

Summary The effects of pH, oxidation reduction compounds and trypsin on insulin binding, hexose transport, and activation of glycogen synthase were studied utilizing rat adipocytes. In this paper the effect of pH is examined; while in the subsequent two papers the effects of glutathione and trypsin are examined. Increase in pH from 6 to 8.5 increased labelled glucose oxidation, 2-deoxyglucose transport as well as labelled insulin binding to the receptor. Enhanced insulin binding was due to an increased rate of association k₊₁ with no effect the rate of dissociation k₋₁ resulting in a decreased equilibrium dissociation constant K_D. Glycogen synthase activity was unaffected by increase in pH when adipocytes were incubated with or without glucose. Insulin in contrast to pH was effective in increasing the activity of glycogen synthase. With 2-deoxyglucose, % glycogen synthaseI activity was increased by an increase in pH. Glycogen synthase activity was thus stimulated by insulin by the direct mechanism, previously termed mechanism 1, involving the formation of a chemical mediator, and clearly distinguishable from the activation of hexose transport, previously termed mechanism 2(1). Increase in labelled glucose oxidation and in 2-deoxyglucose transport with increased pH, as well as insulin stimulation, was abolished by preincubation with trypsin, or cytochalasin B; suggesting that trypsin-sensitive and cytochalasin B-binding protein(s) presumably in the plasma membrane are involved in these effects of pH. Since increase in pH alone activates cell membrane-mediated hexose transport and insulin receptor binding under conditions where glycogen synthase is not activated, increase in pH acts presumably by a non-mediator mechanism. Insulin acts at the membrane to enhance further the effects of increased pH, via a mediator mechanism.     

Beta-adrenergic and somatostatin receptors regulate Na-H exchange independent of cAMP

Activation of beta-adrenergic and somatostatin receptors increases and attenuates, respectively, cAMP. We have determined, however, that in enteric endocrine cells beta-adrenergic and somatostatin receptors also regulate Na-H exchange activity, independent of their effects on cAMP. In cells loaded with a pH-sensitive dye, epinephrine, acting at a beta 2-adrenergic receptor induced an alkalinization while somatostatin caused an acidification of intracellular pH (pHi). These pHi changes were dependent on extracellular Na+ and inhibited by amiloride. Forskolin, dibutyryl-cAMP and 8-bromo-cAMP, however, had no effect on pHi. Cholera toxin, while decreasing the EC50 for epinephrine-stimulated increases in cAMP, had no effect on epinephrine-induced alkalinization, suggesting receptor coupling to Na-H exchange was not mediated by a cholera toxin-sensitive stimulatory GTP-binding protein (Gs). Additionally, epinephrine stimulated Na-H exchange in cyc- variants of S49 lymphoma cells, which lack a fundamental Gs. In the presence of pertussis toxin, somatostatin attenuation of cAMP was completely reversed; however, somatostatin inhibition of Na-H exchange was not affected. We suggest that beta-adrenergic and somatostatin receptors regulate Na-H exchange independent of changes in cAMP and possibly independent of GTP-binding proteins previously described as being coupled to these receptors.     

Influence of cAMP and calcium on epinephrine-mediated hepatic glycogenolysis in rainbow trout,Oncorhynchus mykiss

1. The role of cAMP and of calcium in mediating epinephrine-stimulated glycogenolysis was studied by incubating rainbow trout liver in vitro.2. Epinephrine significantly stimulates glucose release from liver pieces incubated in either calciumcontaining or calcium-free medium. However, the development of the glycogenolytic profile occurred more rapidly in the presence of calcium.3. The β-antagonist, propranolol, inhibited epinephrine-stimulated glucose release from liver pieces incubated in either calcium-containing or calcium-free medium.4. Calcium ionophore, A3187, stimulated glucose release from liver pieces incubated in calciumcontaining medium. Verapamil, a putative calcium channel blocker, had no effect on A23187-stimulated glycogenolysis. However, verapamil completely inhibited epinephrine-stimulated glycogenolysis.5. Dibutyryl cAMP and IBMX, singly or together, stimulated glucose release from liver pieces. cAMP-mediated glycogenolysis was more pronounced in liver pieces incubated in calcium-containing medium.6. These results indicate that epinephrine-stimulated hepatic glycogenolysis in rainbow trout proceeds through the activation of β-adrenergic receptors and that both cAMP and calcium are involved in the post-receptor signal transduction process.