Mannose, glucosamine and inositol monophosphate inhibit the effects of insulin on lipogenesis. Further evidence for a role for inositol phosphate-oligosaccharides in insulin action.

The mechanism of insulin signalling is not yet understood in detail. Recently, a role for inositol phosphate (IP)-oligosaccharides as second messengers transmitting the insulin signal at the post-kinase level was proposed. To evaluate this hypothesis further, we studied whether IP-oligosaccharides isolated from 'haemodialysate' have insulin-like activity. We found that these compounds mimic, in a dose-dependent fashion, the following effects of insulin in adipocytes. (1) Lipogenesis. Incorporation of [3H]glucose into lipids (expressed in nmol/min per 10(6) cells): basal, 0.74 +/- 0.05; insulin (1 mu unit/ml), 4.43 +/- 0.21; IP-oligosaccharide (2 micrograms/ml), 4.07 +/- 0.19. (2) Inhibition of isoprenaline (isoproterenol) (1 microM)-stimulated cyclic AMP levels and lipolysis. Cyclic AMP (pmol/10(5) cells): basal 0.84 +/- 0.05; isoprenaline, 4.03 +/- 0.19; isoprenaline + insulin (200 mu units/ml), 2.06 +/- 0.7; isoprenaline + IP-oligosaccharides (2 micrograms/ml), 2.4 +/- 0.29. Inhibition of lipolysis (mumol of glycerol/mg of protein): isoprenaline (1 microM), 166 +/- 11; isoprenaline+insulin (150 mu units/ml), 53 +/- 3.5; isoprenaline+IP-oligosaccharides (2 micrograms/ml), 58 +/- 5. (3) Stimulation of 3-O-methylglucose transport; basal, 9 +/- 3%; insulin (1 mu unit/ml), 67 +/- 4%, IP-oligosaccharides (2 micrograms/ml), 54 +/- 2%. To identify the active molecules of the IP-oligosaccharide fraction, competition experiments were performed. IP-oligosaccharide effects on lipogenesis were blocked by inositol monophosphate, glucosamine and mannose. In contrast, these compounds did not inhibit IP-oligosaccharide effects on membrane-mediated functions (3-O-methylglucose transport, cyclic AMP levels, lipolysis). We also found that the effect of insulin on lipogenesis was blocked by mannose, glucosamine and inositol monophosphate, whereas the insulin effects on 3-O-methylglucose, cyclic AMP and lipolysis were unaffected. The following conclusions were reached. (1) IP-oligosaccharides mimic the major metabolic effects of insulin in adipocytes. This is consistent with the proposed role of IP-oligosaccharides as second messengers of certain insulin effects. (2) Mannose and glucosamine are functionally important sugar residues for the effect of IP-oligosaccharide on lipogenesis. (3) The observation that mannose, inositol monophosphate and glucosamine block the action of insulin of on lipogenesis supports a role of mannose- and glucosamine-containing IP-oligosaccharides as second messengers for this insulin effect.     

Insulin-dependent contractility of glomerular mesangial cells in response to angiotensin II, platelet-activating factor and endothelin is attenuated by prostaglandin E2.

Culture of glomerular mesangial cells in the absence of insulin decreased the degree of contraction of individual cells in response to vasoconstrictive agonists, angiotensin II, platelet-activating factor and endothelin 1, as compared with cells cultured in the presence of insulin (0.7 nM). This change was associated with a decreased sensitivity of the intracellular Ca2+ response to vasoactive agents in fura-2-loaded cells and with an increase in the basal level of prostanoid [prostaglandins (PG) E1 and E2] production estimated by radioimmunoassay. Addition of exogenous PGE2 to insulin-exposed cells decreased the contractile response to that observed in insulin-deficient cells. Inclusion of 8-bromo cyclic AMP had a similar effect. In 45Ca2(+)-release studies it was shown that, in saponin-permeabilized insulin-exposed cells, preincubation with exogenous PGE2 or 8-bromo cyclic AMP decreased the sensitivity of 45Ca2+ release in response to Ins(1,4,5)P3, as demonstrated by an increase in the EC50 (concn. giving half-maximal effect) to 0.182 +/- 0.024 microM and 0.457 +/- 0.031 microM respectively, as compared with untreated permeabilized cells (EC50 0.091 +/- 0.021 microM). A similar decrease in Ins(1,4,5)P3-sensitive 45Ca2+ release was seen in permeabilized cells from insulin-free conditions of culture (EC50 0.20 +/- 0.061 microM). As altered glomerular haemodynamics are found in insulinopaenic diabetic conditions, it is proposed that a decrease in intracellular Ca2+ availability in response to vasoactive agonists and consequent decrease in mesangial-cell contractility contributes to the hyperfiltration seen in this condition.     

Adenosine 3′:5′-cyclic monophosphate-dependent protein kinase(s) of rat ovarian cells. Gonadotropin regulation of adenosine 3′:5′-cyclic monophosphate-receptor activity

Regulation of cyclic AMP-dependent protein kinase, cyclic AMP-receptor activity and intracellular cyclic AMP concentrations by choriogonadotropin was studied in ovarian cells prepared from 26-day-old rats. A close correlation was observed between phospho-transferase activity and cyclic AMP-receptor activity in 12000g supernatant fractions from rat ovarian homogenate. The apparent activation constant (K(a)) and I(50) (concentration required to produce 50% inhibition) of different cyclic nucleotides for phosphotransferase and cyclic AMP receptor activities respectively were also determined. Cyclic AMP and 8-bromo cyclic AMP were most effective, giving K(a) values of 0.08 and 0.09mum and I(50) of 0.12 and 0.16mum respectively. Other nucleotides were also effective, but required higher concentrations to give a comparable effect. An increased concentration of cyclic AMP produced by choriogonadotropin (1mug/ml) treatment was accompanied by decreased cyclic AMP binding as early as 5min after hormone addition. Choriogonadotropin also stimulated the protein kinase activity ratio (-cyclic AMP/+cyclic AMP) under identical experimental conditions. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine potentiated the action of choriogonadotropin on the three parameters measured in a dose- and time-dependent manner. The maximal cyclic AMP-binding capacity, as determined by cyclic AMP-exchange assay, remained unchanged before and after hormone addition. The endogenously bound cyclic AMP was determined from the difference between the maximal binding capacity and the exogenously bound cyclic AMP. With different choriogonadotropin concentrations, a quantitative correlation was established between maximal binding capacity, exogenous binding and endogenous binding activities. Approx. 60% of total binding sites were endogenously occupied in untreated cells, and choriogonadotropin (1mug/ml) treatment fully saturated available binding sites with a parallel 10-fold increase in cellular cyclic AMP. The present results provide evidence for a probable intracellular compartmentalization of cyclic AMP in the ovarian cell, and suggest that in the unstimulated state all cyclic AMP present in the ovarian cell may not be available for protein kinase activation.     

Insulin modifies the properties of glucose transporters in rat brown adipose tissue.

The influence of cyclic AMP on cartilage degradation was investigated by using phosphodiesterase inhibitors [theophylline and 3-isobutyl-1-methylxanthine (IBMX)], forskolin (which activates the catalytic subunit of adenylate cyclase) and cyclic AMP analogues (dibutyryl and 8-bromo). Breakdown was assessed by quantification of proteoglycans released into the media of 8-day bovine nasal-septum cartilage cultures. Theophylline (1-20 mM), IBMX (0.01-2 mM) and dibutyryl cyclic AMP (0.1-2 mM) had little or no influence on the rate of proteoglycan release from unstimulated (no-endotoxin) cartilages. A small but detectable increase in breakdown was observed with 8-bromo cyclic AMP (0.5-2 mM) and forskolin (50-75 micrograms/ml). To examine potential inhibitory influences of these agents, the cyclic AMP modulators were added to cultures simultaneously treated with Salmonella typhosa endotoxin (12-25 micrograms/ml), a potent stimulator of cartilage degradation. The 3-4-fold stimulation of breakdown by endotoxin was strikingly inhibited by all three classes of cyclic AMP regulators. Optimal inhibition was found at 10-20 mM-theophylline, 1-2 mM-IBMX, 50-75 micrograms of forskolin/ml, 2 mM-dibutyryl cyclic AMP and 2 mM-8-bromo cyclic AMP. Inhibition was shown to be reversible, indicating that cartilages were viable after treatment. Sepharose CL-2B chromatography of proteoglycan products released from treated cartilages showed that the endotoxin-stimulated shift to lower average Mr was significantly prevented by cyclic AMP analogues and phosphodiesterase inhibitors. Together, these results show that agents which increase cyclic AMP inhibit both quantitative and qualitative aspects of endotoxin-mediated cartilage degradation.     

Down regulation of alpha 2 adrenergic receptor numbers in platelets by insulin.

Incubation of platelets from normal volunteers, who had not taken any medication at least for 2 weeks, with insulin (200 mu units/ml), resulted in the inhibition of the potentiation of ADP-induced platelet aggregation in the presence of (-)-epinephrine by 50-60% when compared with the control. The inhibitory effect of insulin was not related to the increased cyclic AMP level or decreased thromboxane A2 synthesis in these cells. It was found that the treatment of platelets with insulin decreased alpha 2 adrenergic receptors number of these cells from 413 +/- 92/platelet to 206 +/- 84/platelet as determined by Scatchard analysis of [3H]yohimbine binding. The affinity of the receptors (1.05 +/- 0.02 nM) remained essentially unchanged due to the treatment of platelets with the hormone (1.40 +/- 0.60 nM).     

The insulin-like effect of growth hormone on insulin-like growth factor II receptors is opposed by cyclic AMP. Evidence for a common post-receptor pathway for growth hormone and insulin action.

The counter-regulatory effects of beta-adrenergic stimulation and cyclic AMP on the insulin-like action of growth hormone (GH) on the subcellular distribution of insulin-like growth factor II (IGF-II) receptors were studied in fat cells from hypophysectomized (Hx) and sham-operated rats. For comparison, the effect of insulin on this process was also studied. Basal IGF-II binding was increased by approx. 2-fold in cells from Hx as compared with sham-operated animals. The stimulatory effect of insulin was decreased in Hx cells, mainly due to a basal redistribution but also to a reduced total number of receptors. GH exerted an acute insulin-like effect in cells from Hx rats and stimulated the translocation of IGF-II receptors from an intracellular pool to the plasma membrane. beta-Adrenergic stimulation with isoprenaline or addition of the non-metabolizable cyclic AMP-analogue N6-monobutyryl cyclic AMP induced a cellular resistance to both GH and insulin and also reduced the responsiveness to these hormones. Adenosine exerted a modulatory effect on both hormones. Binding of 125I-labelled GH to its receptors was not significantly changed by any of these factors. It is concluded that: (1) beta-adrenergic stimulation and cyclic AMP induce a cellular GH resistance at a level distal to the GH-binding site, and (2) the insulin-like effect of GH shares a common pathway with insulin which occurs at the post-binding level.     

Evidence for convertible forms of soluble uterine cyclic nucleotide phosphodiesterase

The cyclic nucleotide phosphodiesterase (3':5'-cyclic nucleotide 5'-nucleotidohydrolase, EC 3.1.4.17) systems of many tissues show multiple physical and kinetic forms. In contrast, the soluble rat uterine phosphodiesterase exists as a single enzyme form with non-linear Lineweaver-Burk kinetics for cyclic AMP (app. Km of approx. 3 and 20 microM) and linear kinetics for cyclic GMP (app. Km of approx. 3 microM) since the two hydrolytic activities are not separated by a variety of techniques. In uterine cytosolic fractions, cyclic AMP is a non-competitive inhibitor of cyclic GMP hydrolysis (Ki approx. 32 microM). Also, cyclic GMP is a non-competitive inhibitor of cyclic AMP hydrolysis (Ki approx 16 microM) at low cyclic GMP/cyclic AMP substrate ratios. However, cyclic GMP acts as a competitive inhibitor of cyclic AMP phosphodiesterase (Ki approx 34 microM) at high cyclic GMP/cyclic AMP substrate ratios. When a single hydrolytic form of uterine phosphodiesterase, separated initially by DEAE anion-exchange chromatography, is treated with trypsin (0.5 microgram/ml for 2 min) and rechromatographed on DEAE-Sephacel, two major forms of phosphodiesterase are revealed. One form elutes at 0.3 M NaOAc- and displays anomalous kinetics for cyclic AMP hydrolysis (app. Km of 2 and 20 microM) and linear kinetics for cyclic GMP (app. Km approx. 5 microM), kinetic profiles which are similar to those of the uterine cytosolic preparations. A second form of phosphodiesterase elutes at 0.6 M NaOAc- and displays a higher apparent affinity for cyclic AMP (app. Km approx. 1.5 mu) without appreciable cyclic GMP hydrolytic activity. These data provide kinetic and structural evidence that uterine phosphodiesterase contains distinct catalytic sites for cyclic AMP and cyclic GMP. Moreover, they provide further documentation that the multiple forms of cyclic nucleotide phosphodiesterase in mammalian tissues may be conversions from a single enzyme species.     

The effects of Ca2+ and guanylnucleotides on isoprenaline-stimulated cyclic AMP formation in rat reticulocyte ghosts

We have studied beta-adrenergic stimulation of cyclic AMP formation in fragmented membranes and in unsealed or resealed ghosts prepared from rat reticulocytes. The maximal rate of isoprenaline-stimulated cyclic AMP formation with saturating MgATP concentrations and in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine was 5-8 nmol/min per ml ghosts and remained constant for at least 15 min. Transition from resealed ghosts to fragmented membranes was associated with a shift of the activation constant (Ka) for (+/-)-isoprenaline from 0.1 to 0.6 microM. THe apparent dissociation constant for propranolol (0.01 microM) remained unchanged. The Ka values for isoprenaline in native reticulocytes and in resealed ghosts were identical. The stimulating effect of NaF on cyclic AMP formation in resealed ghosts reached 15% of maximal beta-adrenergic stimulation. Cyclic AMP formation, both in fragmented membranes and in ghosts, was half-maximally inhibited with Ca2+ concentrations ranging between 0.1 and 1 microM. GTP stimulated isoprenaline-dependent cyclic AMP formation in unsealed ghosts and in fragmented reticulocyte membranes by a factor of 3-5 but did not change the Ka value for isoprenaline. Ka values for the guanylnucleotides in different experiments varied between 0.3 and 2 microM. Ca2+ concentrations up to 4.6 microM reduced the maximal activation by GTP and Gpp(NH)p but did not affect their Ka values. Compared to GTP, maximal activation by Gpp(NH)p was higher in fragmented membranes, but much lower in ghosts. Our results suggest that the native beta-receptor adenylate cyclase system of reticulocytes is more closely approximated in the ghost model than in fragmented membrane preparations. Membrane properties seem to modulate the actions of guanylnucleotides on isoprenaline-dependent cyclic AMP formation in ghosts. Some of these effects are not observed in isolated membranes.     

Ontogenetic changes in adenylate cyclase, cyclic AMP phosphodiesterase and calmodulin in chick ventricular myocardium.

The activities of cyclic AMP phosphodiesterase (3',5'-cyclic nucleotide 5'-nucleotidohydrolase, EC 3.1.4.17) and adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] and calmodulin content during development of chick ventricular myocardium were determined. The specific activity of cyclic AMP phosphodiesterase was relatively low in early embryos, increased during embryogenesis by about 4-fold to reach highest values just before hatching, and then decreased by approx. 30% within 1 week after hatching. In contrast, adenylate cyclase did not change during embryonic development, but increased by approx. 50% within 1 week after hatching. Calmodulin content remained constant at 9 micrograms/g wet wt. during embryonic development and decreased to 6 micrograms/g wet wt. by 1 week after hatching. DEAE-Sephacel chromatography of chick ventricular supernatant revealed a single major form of cyclic nucleotide phosphodiesterase activity in early embryonic (9-day E) and hatched (6-day H) chicks. This enzyme form was eluted at approx. 0.27 M-sodium acetate, hydrolysed both cyclic AMP and cyclic GMP, and was sensitive to stimulation by Ca2+-calmodulin, with an apparent Km for calmodulin of approx. 1 nM. In contrast, ventricular supernatant from late-embryonic (18-day E) chicks contained two forms of phosphodiesterase separable on DEAE-Sephacel: the same form as that seen at other ages, plus a cyclic AMP-specific form which was eluted at approx. 0.65 M-sodium acetate and was insensitive to stimulation by Ca2+-calmodulin. The ontogenetic changes in cyclic AMP phosphodiesterase activity in chick ventricular myocardium are consistent with reported ontogenetic changes in the steady-state contents of cyclic AMP in this tissue and suggest that this enzyme may be responsible for the changes that occur in this nucleotide during development of chick myocardium.     

Cyclic nucleotide phosphodiesterase in silkworm. Separation and characterization of multiple forms of the enzyme.

The existence of two forms of cyclic AMP phosphodiesterase (3',5'-cyclic AMP 5'-nucleotidohydrolase, EC 3.1.4.17) was demonstrated in silkworm larvae by kinetic analysis and DEAE-cellulose column chromatography. The two forms of the enzyme (phosphodiesterase II and III) differ apparently in their characteristics from the previously reported cyclic nucleotide phosphodiesterase (phosphodiesterase I) of silkworm. The higher K-m form (phosphodiesterase II) has a molecular weight of approx. 50 000 and optimum pH of 7.8, and requires Mn-2-+ for maximum activity. The lower K-m form (phosphodiesterase III) has a molecular weight of approx. 97 000 and optimum pH of 7.2, and requires Mg-2-+ for maximum activity. Phosphodiesterase II and probably phosphodiesterase III are specific enzymes for the hydrolysis of cyclic AMP.     

Cyclic AMP mediates beta-adrenergic-induced increases in N-linked protein glycosylation in rat parotid acinar cells.

beta-Adrenergic stimulation of rat parotid cells by isoprenaline (isoproterenol) results in 2-3-fold increases in [3H]mannose incorporation into N-linked oligosaccharides. This occurs without perceptible lag and is linear with time for 60 min after agonist addition. Concomitantly, isoprenaline markedly increases cellular cyclic AMP. Examination of individual proteins by sodium dodecyl sulphate/polyacrylamide-gradient-gel electrophoresis reveals that glycosylation changes are primarily associated with four secretory proteins, of approx. Mr 17000, 32000, 38000 and 220000. Beta-Adrenoreceptor activation additionally elicits a slight increase in parotid protein synthesis. The greatest increase in [14C]leucine incorporation is that into another secretory protein (Mr approx. 24000). Exposure of cells to dibutyryl cyclic AMP yields results comparable with those after isoprenaline treatment. Forskolin, which increases parotid-cell cyclic AMP, also causes similar effects. Conversely, dibutyryl cyclic GMP shows no such response. The data are consistent with the notion that beta-adrenergic stimulation of N-linked protein glycosylation in rat parotid cells is mediated by cyclic AMP.     

Stimulatory effect of vasoactive intestinal peptide (VIP) on cyclic AMP production in rat peritoneal macrophages.

Vasoactive intestinal peptide (VIP) stimulated cyclic AMP production in rat peritoneal macrophages. The stimulatory effect of VIP was dependent on time, temperature and cell concentration, and was potentiated by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). At 15 degrees C, the response occurred in the 0.1-1000 nM range of VIP concentrations. Half maximal stimulation of cellular cyclic AMP (ED50) was obtained at 1.2 +/- 0.5 nM VIP, and maximal stimulation (about 3-fold basal level) was obtained between 100-1000 nM. The cyclic AMP system of rat peritoneal macrophages showed a high specificity for VIP. The order of potency observed in inducing cyclic AMP production was VIP greater than rGRF greater than hGRF greater than PHI greater than secretin. Glucagon, insulin, pancreastatin and octapeptide of cholecystokinin did not modify cyclic AMP levels at concentrations as high as 1 microM. The beta-adrenergic agonist isoproterenol increased the cyclic AMP production and show additive effect with VIP. Somatostatin inhibits the accumulation of cyclic AMP in the presence of both vasoactive intestinal peptide and isoproterenol. The finding of a VIP-stimulated cyclic AMP system in rat peritoneal macrophages, together with the previous characterization of high-affinity receptors for VIP in the same cell preparation, strongly suggest that VIP may be involved in the regulation of macrophage function.     

Receptor-mediated gonadotropin action in the ovary. Regulatory role of cyclic nucleotide phosphodiesterase(s) in intracellular adenosine 3′:5′-cyclic monophosphate turnover and gonadotropin-stimulated progesterone production by rat ovarian cells

The regulatory role of cyclic nucleotide phosphodiesterase(s) and cyclic AMP metabolism in relation to progesterone production by gonadotropins has been studied in isolated rat ovarian cells. Low concentrations of choriogonadotropin (0.4-5ng/ml) increased steroid production without any detectable increase in cyclic AMP, when experiments were carried out in the absence of phosphodiesterase inhibitors. The concentration of choriogonadotropin (10ng/ml) that stimulated progesterone synthesis maximally resulted in a minimal increase in cyclic AMP accumulation and choriogonadotropin binding. Choriogonadotropin at a concentration of 10ng/ml and higher, however, significantly stimulated protein kinase activity and reached a maximum between 250 and 1000ng of hormone/ml. Higher concentrations (50-2500ng/ml) of choriogonadotropin caused an increase in endogenous cyclic AMP, and this increase preceded the increase in steroid synthesis. Analysis of dose-response relationships of gonadotropin-stimulated cyclic AMP accumulation, progesterone production and protein kinase activity revealed a correlation between these responses over a wide concentration range when experiments were performed in the presence of 3-isobutyl-1-methylxanthine. The phosphodiesterase inhibitors papaverine, theophylline and 3-isobutyl-1-methylxanthine each stimulated steroid production in a dose-dependent manner. Incubation of ovarian cells with dibutyryl cyclic AMP or 8-bromo cyclic AMP mimicked the steroidogenic action of gonadotropins and this effect was dependent on both incubation time and nucleotide concentration. Maximum stimulation was obtained with 2mm-dibutyryl cyclic AMP and 8-bromo cyclic AMP, and this increase was close to that produced by a maximally stimulating dose of choriogonadotropin. Other 8-substituted derivatives such as 8-hydroxy cyclic AMP and 8-isopropylthio cyclic AMP, which were less susceptible to phosphodiesterase action, also effectively stimulated steroidogenesis. The uptake and metabolism of cyclic [(3)H]AMP in ovarian cells was also studied in relation to steroidogenesis. When ovarian cells were incubated for 2h in the presence of increasing concentrations of cyclic [(3)H]AMP, the radioactivity associated with the cells increased almost linearly up to 250mum-cyclic [(3)H]AMP concentration in the incubation medium. The (3)H label in the cellular extract was recovered mainly in the forms ATP, ADP, AMP, adenosine and inosine, with cyclic AMP accounting for less than 1% of the total tissue radioactivity. Incubation of cyclic AMP in vitro with ovarian cells resulted in a rapid breakdown of the nucleotide in the medium. The degradation products in the medium have been identified as AMP, adenosine and inosine. The rapid degradation of cyclic AMP by phosphodiesterase(s) makes it difficult to correlate changes in cyclic AMP concentrations with steroidogenesis. These observations thus provide an explanation for the previously observed lack of cyclic AMP accumulation under conditions in which low doses of choriogonadotropin stimulated steroidogenesis without any detectable changes in cyclic AMP accumulation.     

Further characterization of the inhibition of casein production in a primary mouse mammary epithelial cell culture by epidermal growth factor. Antagonism by cyclic AMP

Epidermal growth factor (EGF) inhibited casein production and the accumulation of casein mRNA activity induced by insulin (I), cortisol (F) and prolactin (P) in a primary culture of mammary epithelial cells from pregnant mice. The inhibitory effects of EGF were blocked by 8-bromo cyclic AMP (8-br-cAMP) in a dose-dependent manner. The effect of 8-br-cAMP was observed at a concentration as low as 20 microM and was maximal at 500 microM. Dibutyryl cyclic AMP (db-cAMP), cAMP, and 3-isobutylmethylxanthine (IBMX), an inhibitor of phosphodiesterase, also antagonized the inhibitory effect of EGF on casein production. 8-Br-cAMP had, however, no effect on the mitogenic activity of EGF in this system. These results suggest a possible modulatory role of cAMP in EGF-induced inhibition of casein production in cultured mammary epithelial cells.     

Cyclic AMP increases the concentration of insulin receptors in cultured fibroblasts and lymphocytes.

Incubation of SV40 transformed fibroblasts with dibutyryl cyclic AMP, 8-bromo-cyclic AMP, or 1-methyl-3-isobutylxanthine (MIX), a phosphodiesterase inhibitor, produced a two-fold increase in insulin receptor concentration without an effect on receptor affinity. The increase was dose-dependent, was observed after 8 hrs of treatment, and reached a maximum level by 12 to 24 hours. Upon removal of the nucleotide, receptor number decreased towards basal level.Incubation of cultured human lymphocytes (IM-9 line) with cyclic AMP derivatives or MIX also increased the number of insulin receptors without an alteration in receptor affinity. This effect was partially blocked by inhibition of protein synthesis and was independent of changes in cell cycle. The increase in insulin receptors was a specific response to cyclic AMP as the number of receptors for human growth hormone was unaltered. Incubation with 8-bromo-cyclic GMP did not alter the level of insulin binding.     

Resensitization of hepatocyte glucagon-stimulated adenylate cyclase can be inhibited when cyclic AMP phosphodiesterase inhibitors are used to elevate intracellular cyclic AMP concentrations to supraphysiological values.

Treatment of intact hepatocytes with glucagon led to the rapid desensitization of adenylate cyclase, which reached a maximum around 5 min after application of glucagon, after which resensitization ensued. Complete resensitization occurred some 20 min after the addition of glucagon. In hepatocytes which had been preincubated with the cyclic AMP phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), glucagon elicited a stable desensitized state where resensitization failed to occur even 20 min after exposure of hepatocytes to glucagon. Treatment with IBMX alone did not elicit desensitization. The action of IBMX in stabilizing the glucagon-mediated desensitized state was mimicked by the non-methylxanthine cyclic AMP phosphodiesterase inhibitor Ro-20-1724 [4-(3-butoxy-4-methoxylbenzyl)-2-imidazolidinone]. IBMX inhibited the resensitization process in a dose-dependent fashion with an EC50 (concn. giving 50% of maximal effect) of 26 +/- 5 microM, which was similar to the EC50 value of 22 +/- 6 microM observed for the ability of IBMX to augment the glucagon-stimulated rise in intracellular cyclic AMP concentrations. Pre-treatment of hepatocytes with IBMX did not alter the ability of either angiotensin or the glucagon analogue TH-glucagon, ligands which did not increase intracellular cyclic AMP concentrations, to cause the rapid desensitization and subsequent resensitization of adenylate cyclase. It is suggested that, although desensitization of glucagon-stimulated adenylate cyclase is elicited by a cyclic AMP-independent process, the resensitization of adenylate cyclase can be inhibited by a process which is dependent on elevated cyclic AMP concentrations. This action can be detected by attenuating the degradation of cyclic AMP by using inhibitors of cyclic AMP phosphodiesterase.     

The effect of trypsin on sugar uptake in rat thymocytes. Modulation of cellular cyclic AMP concentration and the sugar-transport system.

I have shown that cyclic AMP stimulates sugar uptake in rat thymocytes. However, trypsin treatment, which increases rat thymocyte cyclic AMP concentration, fails to increase sugar uptake. The purpose of the present study is to examine this seeming inconsistency, and to evaluate further the function of trypsin. Mild trypsin treatment of rat thymocytes produced a dose-related increase in cellular cyclic AMP concentration. Trypsin produced the same proportionate increase in cyclic AMP concentration in the presence or absence of optimal concentrations of the phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine, which suggests that trypsin acts to increase thymocyte cyclic AMP concentration by stimulating adenylate cyclase activity. Trypsin at concentrations of 0.3 mg/ml and less had no effect on the uptake of the glucose analogue 2-deoxy-D-glucose (2-DG), whereas at concentrations of 1 mg/ml and higher trypsin produced a small, dose-related, decrease in basal 2-DG uptake, becoming significantly lower than control values only at 5 mg/ml (-22.7%, P less than 0.05). Thymocyte sugar transporters, characterized by means of cytochalasin B binding, consist of a single class of sites with an apparent KD of 0.15 microM and maximum binding capacity of 2.73 pmol/20 x 10(6) cells (8.4 x 10(4) sites/thymocyte). Trypsin produced a dose-related decrease in the sugar-displaceable binding of cytochalasin B, so that at 5 mg of trypsin/ml the number of sugar transporters was decreased by approx. 50%. Thus trypsin treatment of rat thymocytes on the one hand increases cellular cyclic AMP concentration, which itself potentiates 2-DG uptake, and on the other hand decreases the number of sugar transporters, which itself decreases cellular sugar uptake, indicating that the apparent effect of trypsin on thymocyte 2-DG uptake is the result of the balance of its effects on these two systems.     

Prostaglandins and muscarinic agonists induce cyclic AMP attenuation by two distinct mechanisms in the pregnant-rat myometrium. Interaction between cyclic AMP and Ca2+ signals.

In pregnant-rat myometrium (day 21 of gestation), isoprenaline-induced cyclic AMP accumulation, resulting from receptor-mediated activation of adenylate cyclase, was negatively regulated by prostaglandins [PGE2, PGF2 alpha; EC50 (concn. giving 50% of maximal response) = 2 nM] and by the muscarinic agonist carbachol (EC50 = 2 microM). PG-induced inhibition was prevented by pertussis-toxin treatment, supporting the idea that it was mediated by the inhibitory G-protein Gi through the inhibitory pathway of the adenylate cyclase. Both isoprenaline-induced stimulation and PG-evoked inhibition of cyclic AMP were insensitive to Ca2+ depletion. By contrast, carbachol-evoked attenuation of cyclic AMP accumulation was dependent on Ca2+ and was insensitive to pertussis toxin. The inhibitory effect of carbachol was mimicked by ionomycin. Indirect evidence was thus provided for the enhancement of cyclic AMP degradation by a Ca2(+)-dependent phosphodiesterase activity in the muscarinic-mediated effect. The attenuation of cyclic AMP elicited by carbachol coincided with carbachol-stimulated inositol phosphate (InsP3, InsP2 and InsP) generation, which displayed an almost identical EC50 (3 microM) and was similarly unaffected by pertussis toxin. Both carbachol effects were reproduced by oxotremorine, whereas pilocarpine (a partial muscarinic agonist) failed to induce any decrease in cyclic AMP accumulation and concurrently was unable to stimulate the generation of inositol phosphates. These data support our proposal for a carbachol-mediated enhancement of a Ca2(+)-dependent phosphodiesterase activity, compatible with the rises in Ca2+ associated with muscarinic-induced increased generation of inositol phosphates. They further illustrate that a cross-talk between the two major transmembrane signalling systems contributed to an ultimate decrease in cyclic AMP in the pregnant-rat myometrium near term.     

Use of forskolin to study the relationship between cyclic AMP formation and bone resorption in vitro.

The effect of the adenylate cyclase activator forskolin on bone resorption and cyclic AMP accumulation was studied in an organ-culture system by using calvarial bones from 6-7-day-old mice. Forskolin caused a rapid and fully reversible increase of cyclic AMP, which was maximal after 20-30 min. The phosphodiesterase inhibitor rolipram (30 mumol/l), enhanced the cyclic AMP response to forskolin (50 mumol/l) from a net cyclic AMP response of 1234 +/- 154 pmol/bone to 2854 +/- 193 pmol/bone (mean +/- S.E.M., n = 4). The cyclic AMP level in bones treated with forskolin (30 mumol/l) was significantly increased after 24 h of culture. Forskolin, at and above 0.3 mumol/l, in the absence and the presence of rolipram (30 mumol/l), caused a dose-dependent cyclic AMP accumulation with an calculated EC50 (concentration producing half-maximal stimulation) value at 8.3 mumol/l. In 24 h cultures forskolin inhibited spontaneous and PTH (parathyroid hormone)-stimulated 45Ca release with calculated IC50 (concentration producing half-maximal inhibition) values at 1.6 and 0.6 mumol/l respectively. Forskolin significantly inhibited the release of 3H from [3H]proline-labelled bones stimulated by PTH (10 nmol/l). The inhibitory effect by forskolin on PTH-stimulated 45Ca release was significant already after 3 h of culture. In 24 h cultures forskolin (3 mumol/l) significantly inhibited 45Ca release also from bones stimulated by prostaglandin E2 (1 mumol/l) and 1 alpha-hydroxycholecalciferol (0.1 mumol/l). The inhibitory effect of forskolin on spontaneous and PTH-stimulated 45Ca release was transient. A dose-dependent stimulation of basal 45Ca release was seen in 120 h cultures, at and above 3 nmol of forskolin/l, with a calculated EC50 value at 16 nmol/l. The stimulatory effect of forskolin (1 mumol/l) could be inhibited by calcitonin (0.1 unit/ml), but was insensitive to indomethacin (1 mumol/l). Forskolin increased the release of 3H from [3H]proline-labelled bones cultured for 120 h and decreased the amount of hydroxyproline in bones after culture. Forskolin inhibited PTH-stimulated release of Ca2+, Pi, beta-glucuronidase and beta-N-acetylglucosaminidase in 24 h cultures. In 120 h cultures forskolin stimulated the basal release of minerals and lysosomal enzymes.(ABSTRACT TRUNCATED AT 400 WORDS)     

An assessment of the ability of insulin-stimulated cyclic AMP phosphodiesterase to decrease hepatocyte intracellular cyclic AMP concentrations.

Treatment of hepatocytes with either NH4Cl (10mM) or fructose (10mM) blocks insulin's activation of the 'dense-vesicle' cyclic AMP phosphodiesterase. The ability of insulin (10 nM) to decrease intracellular cyclic AMP concentrations raised by glucagon (10 nM) was unaffected by pre-treatment with either NH4Cl (10 mM) or fructose (10 mM). It is concluded that the 'dense-vesicle' enzyme does not play a significant role in this action of insulin and that as yet unidentified cyclic AMP phosphodiesterase(s) must be activated by insulin. Treatment of hepatocytes with either NH4Cl or fructose appeared to increase, reversibly, cyclic AMP phosphodiesterase activity. When N6-(phenylisopropyl)adenosine was used to prevent glucagon from blocking insulin's activation of the plasma-membrane cyclic AMP phosphodiesterase activity, insulin's ability to decrease intracellular cyclic AMP concentrations in glucagon-treated hepatocytes was increased markedly. Insulin's activation of the plasma-membrane cyclic AMP phosphodiesterase activity can exert a potent effect in decreasing intracellular cyclic AMP concentrations elevated by glucagon.