Increased cyclic AMP response to forskolin in Epstein-Barr virus-transformed human B-lymphocytes derived from schizophrenics

Phorbol 12-myristate-13-acetate (PMA), a protein kinase C (PKC) activator, elevated basal cyclic AMP levels and enhanced isoproterenol-, prostaglandin E₁- (PGE₁), forskolin- and cholera toxin-stimulated cyclic AMP accumulation in Epstein-Barr virus (EBV)-transformed human B-lymphocytes. Staurosporine, a PKC inhibitor, significantly antagonized the increase in cyclic AMP accumulation produced by PMA, whereas the inactive phorbol ester, 4α-phorbol 12,13-didecanoate (4αPDD), had no effect. Basal levels of cyclic AMP and the accumulation of cyclic AMP produced by PMA, isoproterenol, PGE₁, cholera toxin and the combination of these compounds with PMA were not significantly different between schizophrenics and controls. The cyclic AMP response to forskolin in the presence and absence of PMA was significantly greater in EBV-transformed human B-lymphocytes from schizophrenics. These results suggest that activation of adenylyl cyclase by forskolin is elevated in EBV-transformed B-lymphocytes derived from schizophrenics and that this elevation is further enhanced through a PKC-dependent phosphorylation mechanism. Received: 20 May 1996/Final version: 6 November 1996     

Inotropic responses of the frog ventricle to dibutyryl cyclic AMP and 8-bromo cyclic GMP and related changes in endogenous cyclic nucleotide levels

Isolated frog ventricles were superfused with solutions containing either N⁶, O²-dibutyryl adenosine 3',5'-cyclic monophosphate (dibutyryl 3',5'-cyclic AMP) or 8-bromo guanosine 3',5'-cyclic monophosphate (8-br 3',5'-cyclic GMP) and changes in isometric twitch tension and in the levels of endogenous 3',5'-cyclic nucleotides were measured. The results show that dibutyryl 3',5'-cyclic AMP potentiates the twitch, whereas 8-br 3',5'-cyclic GMP depresses it. Both effects are dose-related. The magnitude of the decrease in contractile force produced by 8-br 3',5'-cyclic GMP, and of the increase caused by dibutyryl 3',5'-cyclic AMP, are each paralleled by quantitatively-equivalent changes in the ratio 3',5'-cyclic AMP: 3',5'-cyclic GMP. The effect of 8-br 3',5'-cyclic GMP on the twitch is associated with a marked reduction in endogenous 3',5'-cyclic AMP levels, which is linearly related to the increment in intracellular 3',5'-cyclic GMP (slope of regression line ± S.E.: ?10.4 ± 0.6 pmoles 3',5'-cyclic AMP·pmole^?1 increase in 3',5'-cyclic GMP). The entry of dibutyryl 3',5'-cyclic AMP into the fibres is accompanied by a relatively small change in intracellular 3',5'-cyclic GMP, in this case, an increase (slope of regression line ± S.E.: +0.018 ± 0.002 pmoles 3',5'-cyclic GMP·pmole^?1 increase in 3',5'-cyclic AMP). These results suggest that both 3',5'-cyclic nucleotides are normally involved in regulating contractility, 3',5'-cyclic AMP potentiating the twitch and 3',5'-cyclic GMP exerting a counter-action; and that 3',5'-cyclic GMP may constitute part of a feedback mechanism which serves to regulate 3',5'-cyclic AMP levels.     

Reduced responsiveness of adenylate cyclase to forskolin in human lymphoma cells.

The beta 2-adrenergic transmembrane signal transduction was investigated in malignant B-cells from 15 patients with low grade non-Hodgkin's lymphoma as compared with normal lymphocytes of seven healthy adults. The number of beta 2-adrenoceptors and the response of adenylate cyclase (AC) to isoproterenol were slightly decreased in lymphoma cells. The responsiveness of AC to forskolin was 8-fold lower in lymphoma cells, whereas the response to cholera toxin showed no difference. These findings demonstrate an impairment of the beta 2-adrenergic signal transduction in low grade lymphoma cells that particularly affects the function of AC. The comparison with forskolin resistant mutants of an adrenocortical tumor cell line, Y1 (Schimmer et al., J Biol Chem 262: 15521-15526, 1987), suggests that the availability of functional active alpha subunits of stimulatory G proteins (Gs) might be reduced in human B-cell lymphoma, although other mechanisms known to inhibit the AC activity might be involved.     

Effects of forskolin and isoproterenol on cyclic AMP and tension in the myometrium

The effects of forskolin and isoproterenol on total cyclic AMP accumulation and relaxation were compared in uterine segments from estrogen-treated rabbits and term-pregnant rats. In the rabbit, forskolin (0.2-5.0 microM) inhibited spontaneous and acetylcholine-induced contractions and increased cyclic AMP levels. At lower doses forskolin significantly inhibited contractions but had little effect on cyclic AMP (cAMP) levels. Isoproterenol (0.005-0.5 microM), doses equi-effective to those of forskolin for inhibition of contractions, did not alter cyclic AMP levels. In the rat, forskolin (0.5 microM) and isoproterenol (0.05 microM) inhibited spontaneous and potassium-induced contractions by 80%. This reduction was accompanied by an increase in cyclic AMP levels, but the increase was significantly greater with forskolin than with isoproterenol. We conclude that: (1) cAMP may mediate relaxation under certain conditions but it is not an obligatory mediator, (2) some form of compartmentalization of cyclic AMP might exist in the myometrium.     

Effects of a water-soluble forskolin derivative (NKH477) and a membrane-permeable cyclic AMP analogue on noradrenaline-induced Ca2+ mobilization in smooth muscle of rabbit mesenteric artery.

1. Effects were studied of 6-(3-dimethylaminopropionyl) forskolin (NKH477), a water-soluble forskolin derivative and of dibutyryl-cyclic AMP, a membrane-permeable cyclic AMP analogue on noradrenaline (NA)-induced Ca2+ mobilization in smooth muscle strips of the rabbit mesenteric artery. The intracellular concentration of Ca2+ ([Ca2+]i), isometric force and cellular concentration of inositol 1,4,5-trisphosphate (InsP3) were measured. 2. NA (10 microM) produced a phasic, followed by a tonic increase in both [Ca2+]i and force in a solution containing 2.6 mM Ca2+. NKH477 (0.01-0.3 microM) attenuated the phasic and the tonic increases in both [Ca2+]i and force induced by 10 microM NA, in a concentration-dependent manner. 3. In Ca(2+)-free solution containing 2 mM EGTA with 5.9 mM K+, NA (10 microM) produced only phasic increases in [Ca2+]i and force. NKH477 (0.01 microM) and dibutyryl-cyclic AMP (0.1 mM) each greatly inhibited these increases. 4. NA (10 microM) led to the production of InsP3 in intact smooth muscle strips and InsP3 (10 microM) increased Ca2+ in Ca(2+)-free solution after a brief application of Ca2+ in beta-escin-skinned smooth muscle strips. NKH477 (0.01 microM) or dibutyryl-cyclic AMP (0.1 mM) modified neither the NA-induced synthesis of InsP3 in intact muscle strips nor the InsP3-induced Ca2+ release in skinned strips. 5. In Ca(2+)-free solution, high K+ (40 and 128 mM) itself failed to increase [Ca2+]i but concentration-dependently enhanced the amplitude of the increase in [Ca2+]i induced by 10 microM NA with a parallel enhancement of the maximum rate of rise.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of forskolin on bone. Stimulation of cyclic AMP accumulation and calcium efflux from chick embryo tibiae in organ culture

Forskolin (FSK), an activator of adenylate cyclase in many tissues, was investigated to determine its potential for studying cyclic AMP-mediated, physiological events in bone. Tibiae from 14-day chick embryos were cultured in modified Eagle's medium for up to 8 hr, and the cyclic AMP and Ca2+ efflux responses to parathyroid hormone (PTH) and FSK were observed. FSK caused a concentration-related increase in tissue cyclic AMP, with 10(-6) M FSK producing a cyclic AMP response of similar magnitude to that elicited by 10(-6) M PTH (1-34). However, while 10(-6) M PTH was the maximally-activating PTH concentration, a further elevation was produced by 10(-5) M FSK. Chick embryo tibiae cultured for 8 hr in the presence of PTH exhibited a concentration-related stimulation of Ca2+ efflux up to 10(-6) M PTH. When this physiological effect was examined in bones cultured in the presence of FSK, a biphasic response to increasing FSK concentrations was found; Ca2+ efflux was stimulated by 10(-6) M and 3 X 10(-6) M FSK, but not by 10(-7) M or 10(-5) M FSK. In summary, concentrations of FSK, which produced tissue cyclic AMP accumulation similar to that produced by physiologically effective PTH concentrations, also resulted in net Ca2+ efflux from bone. These results indicate that FSK may be a useful agent for studying the role of cyclic AMP in the response of bone to hormones.     

Histamine dependent cyclic AMP generating system in rabbit CNS: interaction with neuroregulators and forskolin

Histamine (HI) potently stimulated, through H2-receptors, cAMP accumulation in rabbit cerebral cortical slices but not in retinal pieces. The action of HI in the cerebral cortex was not significantly affected by dopamine, serotonin, melatonin and GABA-ergic drugs. Forskolin markedly stimulated cAMP accumulation both in the cerebral cortex and retina. There was a synergistic interaction between HI and forskolin in the cerebral cortex, whereas in the retina HI decreased the forskolin-activated cAMP accumulation.     

Interaction between cyclic GMP protein kinase and cyclic AMP may be diminished in stunned cardiac myocytes

We tested the hypothesis that the importance of the negative functional effects of the cyclic GMP protein kinase would be reduced in stunned (simulated ischemia/reperfusion) cardiac myocytes. Ventricular cardiac myocytes were isolated from New Zealand white rabbits (N=7). Myocytes were studied at baseline and after simulated ischemia (15 min of 95% N(2)-5% CO(2) at 37 degrees C) followed by simulated reperfusion (reoxygenation). Cell shortening was studied with a video edge detector; O(2) consumption was measured using O(2) electrodes. Protein phosphorylation was measured autoradiographically after gel electrophoresis. Functional and metabolic data were acquired after: (1) 8-(4-chlorophenylthio)guanosine-3',5'-monophosphate (PCPT, cGMP protein kinase agonist) 10(-7) or 10(-5) M; (2) 8-Br-cAMP 10(-5) M followed by PCPT 10(-7) or 10(-5) M; (3) beta-phenyl-1, N(2)-etheno-8-bromoguanosine-3',5'-monophosphorothioate, SP-isomer (SP, cGMP protein kinase agonist) 10(-7) or 10(-5) M; (2) 8-Br-cAMP 10(-5) M followed by SP 10(-7) or 10(-5) M. At baseline, percent of shortening (Pcs) and maximal rate of shortening (Rs) were significantly lower in the stunned myocytes (Pcs: 5.0+/-0.2% control vs. 3.8+/-0.3 stunned; Rs: 64.8+/-5.9 microm/s control vs. 46.9+/-4.8 stunned). In both groups, PCPT and SP dose-dependently decreased Pcs and Rs. The effects were slightly, but not significantly, less in stunned myocytes. 8-Br-cyclic AMP significantly increased function in control, but not stunned myocytes (Pcs, 4.5+/-0.5 to 6.2+/-0.8 control vs. 3.1+/-0.2 to 3.6+/-0.2 stunned). The negative functional effects of PCPT and SP were diminished after 8-Br-cyclic AMP in control (from -39% to-29%) and diminished significantly more in the stunned myocytes (-19%). PCPT and cyclic AMP phosphorylated similar protein bands. In stunned myocytes, three (22, 31 and 53 kDa) bands were enhanced less by PCPT.     

A method to assess histamine-induced cyclic AMP production in isolated gastric mucosal cells from human biopsies.

Human gastric mucosal cells were isolated by digestion of fundic biopsies with pronase and collagenase. The mean value of gastric cells per milligram biopsy specimen +/- SEM was 59,000 +/- 4,300 (n = 31) with a viability of 90 +/- 5%. With the cell yield of 1 patient a series of approximately 70-80 cyclic AMP measurements was possible. Histamine stimulated intracellular cyclic AMP production with an EC50 value of 35 +/- 25 mumol/l (SEM; n = 4). In the presence of 100 mumol/l histamine the Ki values (mumol/l) for the histamine H2 receptor antagonists averaged 1.45 (cimetidine), 0.10 (ranitidine), and 0.02 (famotidine). No significant inhibition of histamine-induced cyclic AMP production was obtained with the histamine H1 receptor antagonist triprolidine. With the new method histamine-induced cyclic AMP production can be measured in intact human gastric mucosal cells from fundic biopsy samples.     

Dibutyryl cyclic AMP and forskolin inhibit phosphatidylinositol hydrolysis, Ca2+ influx and contraction in vascular smooth muscle.

Dibutyryl cyclic AMP and forskolin inhibited the contraction induced by norepinephrine (NE) more strongly than the high K(+)-induced contraction in isolated rat aorta. These inhibitors inhibited the 45Ca2+ influx stimulated by NE but not that by high K+, and they inhibited NE-induced inositol monophosphate accumulation. These results suggest that cAMP inhibits NE-induced contraction, at least partly, by inhibiting the alpha-adrenoceptor-mediated signal transduction and high K(+)-induced contraction by decreasing Ca2+ sensitivity but not Ca2+ influx.     

Opposing functional effects of cyclic GMP and cyclic AMP may act through protein phosphorylation in rabbit cardiac myocytes

1. We tested the hypothesis that the negative functional effects of cyclic GMP (cGMP) oppose the positive effects of cyclic AMP (cAMP) in cardiac myocytes through interaction at the level of their respective protein kinases. 2. Cell shortening was studied using a video-edge detector. The O2 consumption of a suspension of rabbit ventricular myocytes was measured using O2 electrodes. Protein phosphorylation was measured autoradiographically following SDS-PAGE. Data were collected with: (1) 8-bromo-cGMP (8-Br-cGMP) 10(-7) or 10(-5) M; (2) 8-bromo-cAMP (8-Br-cAMP) 10(-7) or 10(-5) M; (3) 8-Br-cAMP 10(-5) M followed by 8-Br-cGMP 10(-7) or 10(-5) M; (4) 8-Br-cGMP 10(-5) M followed by 8-Br-cAMP 10(-7) or 10(-5) M; (5) 8-Br-cGMP 10(-7) or 10(-5) M followed by KT 5720 (cAMP-dependent protein kinase inhibitor) or KT 5823 (cGMP-dependent protein kinase inhibitor) 10(-6) M; and (6) 8-Br-cAMP 10(-7) or 10(-5) M followed by KT 5720 or KT 5823 10(-6) M. 3. 8-Br-cGMP 10(-5) M decreased percent shortening (Pcs) from 6.3+/-0.6 to 3.6+/-0.4% and rate of shortening (Rs) from 66.7+/-4.4 to 41.8+/-4.2 microm s(-1). 8-Br-cAMP 10(-5) M increased Pcs (from 3.7+/-0.2 to 4.8+/-0.2) and Rs (from 50.0+/-3.0 to 60.0+/-3.1). With 8-Br-cAMP 10(-5) M, 8-Br-cGMP 10(-5) M decreased Pcs and Rs less. The positive functional effects of 8-Br-cAMP 10(-7) or 10(-5) M were also diminished with 8-Br-cGMP 10(-5) M. Following 8-Br-cGMP 10(-7) or 10(-5) M, KT 5720 10(-6) M further decreased Pcs to 2.5+/-0.3 and Rs to 30.0+/-4.1. KT 5823 10(-6) M returned Pcs to 4.7+/-0.4 and Rs to 61.3+/-5.3. Following 8-Br-cAMP 10(-7) or 10(-5) M, KT 5720 decreased the elevated Pcs and Rs significantly and KT 5823 10(-6) M further increased these parameters. 4. cGMP and cAMP phosphorylated the same five protein bands. With KT 5720 or KT 5823, all of the bands were lighter at the same concentration of 8-Br-cAMP and 8-Br-cGMP. 5. We conclude that, in rabbit ventricular myocytes, the opposing functional effects of cGMP and cAMP are related to the interaction at the level of their respective protein kinases.     

Direct and indirect stimulations of cyclic AMP formation in human brain.

1. Adenosine 3':5'-cyclic monophosphate (cyclic AMP) formation, by use of an [3H]-adenine prelabelling assay, was measured in fragments of human cerebral cortex, taken in the course of various neurosurgical procedures. 2. Large accumulations of [3H]-cyclic AMP due to isoprenaline, noradrenaline and adenosine and small effects due to forskolin were observed. Histamine, 5-hydroxytryptamine (5-HT) and the excitatory amino acids glutamate and quisqualate were ineffective. 3. The response to noradrenaline consisted of two components; a direct beta-adrenoceptor response and an enhancement mediated by an alpha-adrenoceptor which appears to be similar to that in rat cerebral cortex. 4. The response to isoprenaline was also potentiated by histamine H1 receptor stimulation but the direct effect of 2-chloroadenosine was not altered by histamine, 5-HT or quisqualate. 5. It is concluded that some, but not all, of the indirect modulations of cyclic AMP formation previously observed in experimental animal brain exist in human cerebral cortex.     

ACTH, prolactin, corticosterone and pituitary cyclic AMP responses to repeated stress

The present experiment was conducted to determine whether the plasma hormonal and pituitary cyclic AMP responses observed following a single exposure to an acute stressor would diminish following reexposures to the same stressor. Fifteen-min stress exposures (forced running) were separated by 45-min recovery periods. Separate groups of control and stressed animals were sacrificed before and after each of four 15-min stress periods and after each recovery period. The first exposure to 15 min of forced running raised plasma ACTH, corticosterone and pituitary cyclic AMP levels approximately 6-fold and more than tripled levels of plasma prolactin. Plasma ACTH and pituitary cyclic AMP responses to the second, third and fourth stress exposures were very similar to the responses to the first stress exposure, and levels of these substances returned to prestress levels during each 45-min recovery period. Plasma prolactin responses to the four stress sessions were somewhat variable but no significant trend among the responses was seen. Plasma prolactin levels also returned to prestress levels between stress exposures. Corticosterone levels were similar following each of the four stress sessions but levels remained elevated compared to prestress levels between stress exposures. These data suggest that pituitary responses to acute stress are rapid, that return to prestress levels is also rapid, with the exception of corticosterone, and that repeated responses of the same magnitude may be evoked when stressors are separated by short recovery periods.     

Contraction of cardiac myocytes from noradrenaline-treated rats in response to isoprenaline, forskolin and dibutyryl cAMP

Rats were treated with noradrenaline at either 300 micrograms/kg per h for 1 week (Group 1) or 600 micrograms/kg per h for 2 weeks (Group 2) using subcutaneously implanted osmotic minipumps. Control rats were sham-operated. Ventricular myocytes were isolated and contraction amplitude and rats of shortening and relaxation measured using a video and edge-detection device. Maximum contraction amplitude achieved in high calcium was similar for all groups, indicating that there was little change in the basic contractility of cells from noradrenaline-treated animals. Cells from Group 1 treated rats showed a depressed concentration-response curve to isoprenaline but maximum contraction amplitudes in forskolin and dbcAMP were unchanged. In cells from Group 2 treated rats the concentration for half-maximal effect (EC50) of isoprenaline was increased and the maximum contraction amplitude was depressed (P less than 0.001). The ratio between maximum response to calcium and that to isoprenaline in the same cell was also significantly reduced (P less than 0.001). There were significant decreases in response to forskolin, with both the maximum contraction amplitude (P less than 0.01) and forskolin/calcium ratio (P less than 0.001) being attenuated in myocytes from noradrenaline-treated rats. The dbcAMP/calcium ratio was also depressed after noradrenaline treatment (P less than 0.02). The extent of the reduction in response was greatest for isoprenaline and least for dbcAMP. These results suggest that desensitisation progresses from a homologous to a heterologous form with increased dose and time of exposure to circulating catecholamines and that, in the latter, lesions occur at several stages of the adenylate cyclase cascade.     

A method to simultaneously investigate histamine-induced cyclic AMP and aminopyrine accumulation in isolated gastric mucosal cells from human biopsies.

A method has been developed to simultaneously measure two parameters of histamine-induced gastric secretion, cyclic AMP and aminopyrine accumulation, in gastric mucosal cells from human biopsies. Histamine stimulated cyclic AMP and aminopyrine accumulation with different time courses. Cyclic AMP production reached a maximum at 30 min, whereas maximal aminopyrine accumulation was obtained after the cells had been incubated for 90 min in presence of 100 mcM histamine. Histamine was more potent in stimulating aminopyrine than cyclic AMP accumulation. The ED50 values for histamine-induced aminopyrine accumulation were estimated to be 0.88 +/- 0.02 and 25.53 +/- 8.75 mcM for cyclic AMP production, respectively. The aminopyrine accumulation was more than half-maximally increased at 1 mcM histamine without significant effects on the cyclic AMP basal level. It remains to be elucidated whether this finding indicates, besides cyclic AMP, the involvement of calcium in histamine stimulus. The histamine H2-receptor antagonists cimetidine and ranitidine inhibited both in vitro parameters, whereas the gastric proton pump inhibitor omeprazole only affected aminopyrine accumulation. Our method might be a valuable tool for in vitro pharmacological and clinical investigations on histamine-induced gastric secretion in human biopsies.     

Effects of beta-adrenergic blockade on plasma cyclic AMP and blood sugar responses to glucagon and isoproterenol in man.

In healthy humans glucagon infusion resulted in a significant increase in blood sugar and in plasma cyclic AMP. No discernible hemodynamic effects were found. Isoproterenol infusion on a mole per mole basis in the same subjects induced a significant, although less pronounced rise in plasma cyclic AMP, heart rate, and a fall in diastolic blood pressure but had no effect on blood sugar. Propranolol administration abolished the hemodynamic effects of isoproterenol and significantly decreased the response of plasma cyclic AMP; the same blocking dosage had little effect on plasma cyclic AMP changes induced by glucagon wheras the response in blood sugar was significantly reduced. These data in vivo are compatible with the in vitro demonstration of separate receptors for glucagon and isoproterenol.     

Dibutyryl cyclic AMP and adrenaline increase contractile force and 45Ca uptake in mammalian cardiac muscle

Summary The effects of dibutyryl cyclic AMP (DB-AMP; 10^–3M) and adrenaline (2.2×10^–6 M) on contractile force, ⁴⁵Ca uptake, and total myocardial Ca concentration were investigated in electrically driven left auricles isolated from rat hearts. The experiments were performed at an extracellular Ca concentration of 0.45 mM and at low frequency of stimulation (15 beats/min). ⁴⁵Ca exposure was 5 min. Under the conditions used, both drugs increased contractile force and enhanced ⁴⁵Ca uptake (expressed as relative specific activity) by about 30% (DB-AMP) and 40% (adrenaline), respectively. Thus, the results provide evidence that the effects of adrenaline on ⁴⁵Ca uptake in mammalian cardiac muscle can be mimicked by DB-AMP.     

The role of calcium and cyclic AMP in the contractile action of angiotensin II upon rat descending colon.

The effect of procedures which affect calcium availability and tissue levels of cyclic AMP were investigated upon the contractions of rat colon to angiotensin II, PGE2 and KCl. Calcium free Tyrode containing EDTA (25 microM) reduced the response to angiotensin more than to PGE2 and KCl. An increase in calcium concentration to 3.6 mM potentiated responses to angiotensin and KCl. A further increase to either 7.2 mM or 10.8 mM still potentiated responses to angiotensin but those to KCl wre reduced. SKF 525A (2.6 x 10(-5) M) and verapamil (3.5 x 10(-6) M) inhibited the responses to KCl but the responses to PGE2 and angiotensin were less affected. Isoprenaline, Theophylline and dibutyryl cyclic AMP preferentially reduced the responses to angiotensin and PGE2. It is suggested that part of the response to angiotensin involves an influx of calcium, independent of depolarisation.