Cyclic AMP Generation In Response Research Articles

Urine osmolality, cyclic AMP and aquaporin-2 in urine of patients under lithium treatment in response to water loading followed by vasopressin administration

Lithium is the drug that is most frequently associated with acquired nephrogenic diabetes insipidus (NDI). The exact mechanism of lithium-induced NDI in man is unknown. The aim of the present study was to investigate the kidney response to minimal and maximal stimulation of the kidney urine concentrating mechanism by measuring urine osmolality, and urine levels of cAMP and AQP-2 in urine of patients under long-term lithium treatment. Twenty patients under long-term lithium treatment were included. The kidney urinary 3′,5′-cyclic adenosine monophosphate (cyclic AMP), aquaporin-2 levels and urine osmolality were determined during a situation of minimal kidney urine concentrating activity (induced by water loading) and during a situation following maximal stimulation of kidney urine concentrating activity (induced by 1-desamino-8- d-arginine-vasopressin). Patients were classified as NDI, partial NDI and non-NDI based on maximal reached urine osmolality. The partial correlation ( r) between urinary cyclic AMP levels (mol/l) and urine osmolality was 0.94 ( P < 0.001). No significant correlation was observed between urinary aquaporin-2 levels (mol/mol creatinine) and osmolality nor between urinary cyclic AMP and aquaporin-2 levels. The rise in urinary cyclic AMP but not aquaporin-2 levels upon 1-desamino-8- d-arginine-vasopressin administration after water loading significantly differed between the three categories, decreasing with increasing NDI category. In conclusion we found that in lithium-induced kidney urine concentrating deficit in man, the cyclic AMP generation in response to 1-desamino-8- d-arginine-vasopressin administration after water loading, is impaired. It remains to be elucidated whether principal cells, G-proteins or adenylate cyclase e.g. are the major targets for the mechanism underlying lithium-induced NDI in man.

Metabolic acidosis up-regulates PTH/PTHrP receptors in UMR 106-01 osteoblast-like cells

Metabolic acidosis results in skeletal demineralization by multiple mechanisms. One of these involves the inorganic phase of bone by which hydrogen ion is buffered by bone carbonate. In addition, the cellular components of bone participate by the induction and repression of several skeletal genes. Previous studies have suggested that the action of parathyroid hormone (PTH), a major regulator of bone turnover, might be altered by acidosis. The present studies were designed to test directly, in vitro, whether acidosis altered the effects of PTH in UMR 106-01 osteoblast-like cells. Studies were conducted in confluent cultures of UMR 106-01 cells in modified Eagle's medium (MEM) with 5% fetal bovine serum (FBS) at pH values varying from 7.4 to 7.1 by addition of HCl. After time periods of 4 to 48 hours, cells were tested for cyclic AMP generation in response to PTH. PTH binding and PTH/PTHrP receptor mRNA levels were determined by radioligand binding assay and Northern analysis respectively. After 48 hours, decreases in pH from 7.4 to 7.1 resulted in a progressive increase in PTH-stimulated cyclic-AMP generation from 1978 +/- 294 to 4968 +/- 929 pmol/culture/5 min (P < 0.05). Basal cyclic AMP concentrations were unchanged. PTH binding increased 1.5- to twofold. Competitive inhibition binding revealed an increase in receptor number supported by up-regulation of PTH/PTHrP receptor mRNA up to twofold from control levels. These findings demonstrate that metabolic acidosis stimulates the response to PTH in UMR 106-01 osteoblast-like cells by a mechanism that involves an increase in the levels of PTH/PTHrP receptor mRNA. Thus, the skeletal response to acidosis that includes an increase in bone resorption may result, at least in part, from an increase in PTH/PTHrP receptors leading to an enhanced effect of PTH on bone.

Role of cyclo-oxygenase-2 induction in interleukin-1beta induced attenuation of cultured human airway smooth muscle cell cyclic AMP generation in response to isoprenaline.

Airway smooth muscle (ASM) in human asthma shows reduced relaxation and cyclic AMP generation in response to beta-adrenoceptor agonists. IL-beta attenuates cyclic AMP generation but the underlying mechanism is unclear. We have reported that IL-1beta induces cyclo-oxygenase-2 (COX-2) in human ASM cells and results in a marked increase in prostanoid generation with PGE2 and PGI2 as the major products. We investigated the role of COX-2 induction and prostanoid release (measured as PGE2) in IL-1beta induced attenuation of cyclic AMP generation in response to the beta-adrenoceptor agonist isoprenaline (ISO). Pre-treatment of human ASM cells with IL-1beta significantly attenuated cyclic AMP generation in response to high concentrations of ISO (1.0-10.0 microM) in a time- and concentration-dependent manner. The effect was accompanied by a high concentration of PGE2 release. The non-selective COX inhibitor indomethacin (Ind), the selective COX-2 inhibitor NS-398, the protein synthesis inhibitors cycloheximide (CHX) and actinomycin D and the steroid dexamethasone (Dex) all abolished the PGE2 release and prevented the attenuated cyclic AMP generation. COX substrate arachidonic acid time- and concentration-dependently mimicked IL-1beta induced attenuation and the effect was prevented by the non-selective COX inhibitors Ind and flurbiprofen, but not by NS-398, CHX and Dex. In contrast to IL-1beta, TNFalpha and IFNgamma, which are ineffective in inducing COX-2 and releasing PGE2 from human ASM cells, did not affect the cyclic AMP formation. Our study demonstrates that COX-2 induction and the consequent release of prostanoids plays a crucial role in IL-1beta induced attenuation of human ASM cell cyclic AMP response to ISO.

Decreased transcript expression coincident with impaired glycosylation in the β2-adrenergic receptor gene does not result from differences in the primary sequence

Variants of the S49 mouse lymphoma cell line exhibit multiple lesions along the pathway of cyclic AMP generation in response to β 2-adrenergic stimulation. Two such variants, β p and β d, are characterized by decreased receptor binding and mRNA expression, 50% and 25% of wild-type receptor expression, respectively. The rate of β 2-adrenergic receptor synthesis was measured and found to be decreased in the βd cells vis-à-vis the rate in wild type cells. The molecular mass of the β 2-adrenergic receptor in the S49 wild-type, β p and β d variant cells was estimated by labeling the receptor with the photoaffinity probe [ 125I]iodocyanopindololdiazirine. Receptor size was found to be 67 000 and 47 000 Da in the wild-type and 60 000 and 42 000 in the two variant cells. This 6 kDa discrepancy in mass was abolished upon treatment of labeled cell extracts with N-glycosidase F, suggesting the possibility of either N-terminal truncation or altered glycosylation of the receptor in the variant cells. To distinguish between these possibilities, we sequenced the β 2-adrenergic receptor gene and two kilobases of the 5′-non-coding region. No differences were found in the coding region of the gene from wild-type, β p and β d S49 cells suggesting that both the diminished expression and the decreased size of β 2-adrenergic receptor in the β p and β d S49 variants are related to impaired glycosylation of the receptor. This hypothesis was substantiated by the reduced retention of the variant cells' β 2-adrenergic receptor on immobilized WGA. Furthermore, growth of the S49 cells in the presence of the α-mannosidase II inhibitor, swainsonine, preferentially impaired the ability of the receptors derived from the variant cells to bind to WGA. These results imply that altered expression and glycosylation of G-protein-linked receptors occur as a consequence of one or more mutations outside the receptor's open reading frame. ©1997 Elsevier Science B.V. All rights reserved.

Molecular Cloning and Functional Expression of a Human Pancreatic Secretin Receptor

Through binding to high affinity receptors, secretin is the major hormonal stimulant of ductular bicarbonate secretion in the pancreas, contributes toward enzyme secretion from pancreatic acinar cells, and has been implicated as a pancreatic growth factor. In this work, we utilized hybridization screening of a human pancreatic cDNA library to clone a 1.8-kb cDNA encoding a 440 amino acid protein 81% identical to the rat pancreatic secretin receptor. COS-7 cells transfected with this cDNA bound secretin with a high affinity, and vasoactive intestinal peptide with a lower affinity, appropriate for a secretin receptor. Receptor-bearing COS-7 cells also exhibited an increase in cyclic AMP generation in response to secretin stimulation. Studies analyzing the role of this receptor in human pancreatic physiology and pathophysiology are currently underway in our laboratory.

In vitro and in vivo activated T cells display increased sensitivity to PGE 2

In this study we report that in vitro activation of T cells increased the cyclic AMP response to subsequent prostaglandin E 2 (PGE 2) stimulation severalfold per cell. This sensitization of T cells to PGE 2-induced cyclic AMP generation was observed when the T cells had been stimulated in vitro for 5 days with either the CD3 monoclonal antibody OKT3, phytohemagglutinin, or the combination phytohemagglutinin plus the phorbol ester PMA. Enhanced cyclic AMP generation following mitogenic activation was seen in response to both PGE 2 and forskolin, a direct activator of the adenylate cyclase, indicating that the amount of adenylate cyclase had increased during the in vitro activation course. In order to investigate whether various T cell subsets in general and in vivo activated T cells in particular would differ in their susceptibility to PGE 2, we isolated CD4 +, CD8 +, CD4 −CD8 −, CD4 +CD45RO + (“memory”), and CD4 +CD45RA + (“virgin”) T cells and studied PGE 2-mediated inhibition of CD3-induced proliferation, as well as cyclic AMP generation in response to PGE 2, respectively. We found that CD8 + T cells are more susceptible to PGE 2 inhibition and produce more cyclic AMP than CD4 + T cells. Double-negative T cells (enriched for γδ T cell receptor positive cells) were found to be sensitive to PGE 2 as well. Within the CD4 + T cell population, CD45RO + (“memory”) T cells were significantly more sensitive to PGE 2-mediated suppression than CD45RA + (“virgin”) T cells. CD45RO + cells required a 10-fold lower dose of PGE 2 for half-maximum suppression of proliferation. However, no difference in cyclic AMP production could be demonstrated between these two subsets. We propose that substantial heterogeneity exists among peripheral blood T lymphocyte subsets regarding their sensitivity to the immunosuppressive action of PGE 2 and that the sensitivity of individual cells changes in the course of an immune response.

An inhibitory role for the protein kinase C pathway in ovarian steroidogenesis. Studies with cultured swine granulosa cells.

We have used primary cultures of swine granulosa cells to investigate the regulatory role of the protein kinase C pathway in the ovary. In this system, we observed the following. Swine granulosa cells bound [3H]phorbol 12,13-dibutyrate [( 3H]PDB) specifically with high affinity [apparent Ki for 12-O-tetradecanoylphorbol 13-acetate (TPA) = 3.1 (2.1-4.7) nM] and low capacity [0.68 (0.34-0.99) pmol/10(7) cells]. The cytosol of granulosa cells contained functionally active protein kinase C capable of phosphorylating distinct proteins in response to stimulation with active phorbol ester. TPA and PDB induced dose-dependent inhibition (greater than 85%) of follicle-stimulating-hormone (FSH)-stimulated progesterone production. Half-maximally inhibitory concentrations were 0.10 and 0.75 nM for TPA and PDB respectively, whereas phorbol analogues that do not activate protein kinase C were not inhibitory. TPA did not impede cyclic AMP generation in response to FSH, cholera toxin or forskolin acutely (within 48 h), but did inhibit the stimulatory effects of 8-bromo cyclic AMP, insulin and oestradiol on progesterone biosynthesis. In the presence of maximally effective concentrations of 25-hydroxy-, 20 alpha-hydroxy- or 22R-hydroxy-cholesterol as exogenous sterol substrates for cholesterol side-chain cleavage, treatment with TPA suppressed pregnenolone, progesterone and 20 alpha-hydroxypregn-4-en-3-one biosynthesis by more than 80%. The inhibitory effects of phorbol esters were not attributable to non-specific cytotoxicity, since prostaglandin F2 alpha production increased in the same cultures and aromatization of exogenously supplied testosterone to oestradiol was not suppressed. In intact granulosa cells, the effects of phorbol esters were mimicked by a synthetic non-diterpene diacylglycerol, 1-octanoyl-2-acetylglycerol, and the tumour promoter, mezerein, which specifically activates protein kinase C. We conclude that swine granulosa cells contain specific high-affinity receptors for phorbol esters that are functionally coupled to protein phosphorylation. Moreover, treatment with phorbol esters or non-phorbol activators of protein kinase C results in selective inhibition of cholesterol side-chain cleavage activity without impairing cyclic AMP generation or oestrogen biosynthesis.

Vasoactive intestinal peptide: vasodilatation and cyclic AMP generation.

Vasoactive intestinal peptide (VIP) is a putative neurotransmitter that causes vasodilation when injected intravenously. To learn more about the vasodilator actions of VIP, studies were performed on the rat mesenteric arterial bed and on cultured smooth muscle cells derived from both the rat mesenteric artery and aorta. In isolated perfused rat mesenteric artery beds, VIP caused relaxation at the threshold concentration of less than 1 nM and a maximal fall in perfusion pressure similar to that obtained with isoproterenol. VIP stimulated adenylate cyclase in cultured vascular smooth muscle cells in a concentration range of 10(-10)-10(-6) M; maximum activity was increased 2.7 +/- 0.6- and 3.4 +/- 0.4-fold above basal in mesenteric and aortic smooth muscle cells, respectively. The threshold and the half-maximal concentrations of VIP for adenylate cyclase stimulation were more than 50-fold lower than those for both prostaglandin E1 and isoproterenol in cultured mesenteric and aortic cells. Similar effects on cyclic AMP generation in response to VIP were observed in isolated rat mesenteric artery rings. Immunocytochemical examination of the rat mesenteric artery bed in situ demonstrated dense innervation of small- and medium-size vessels with nerve fibers containing VIP-like immunoreactivity. Thus VIP is present in the rat mesenteric artery, a peripheral arterial bed, and is a potent vasodilator that can activate vascular smooth muscle adenylate cyclase and thus potentially contribute to the regulation of vascular tone.

Properties of cardiac contractions in zero sodium solutions: Intracellular free calcium controls slow channel conductance

Guinea-pig, rat and frog atria bathed in zero sodium, contract in response to high intensity electrical stimuli. A transient positive inotropic response after sodium removal is followed by a decline and then leveling off of twitch tension. Abolition of sodium-calcium exchange in zero sodium medium results in a species-dependent decline in the rate of relaxation and an increase in diastolic tension. The characteristic positive inotropic effect of isoproterenol is reduced in rat and frog atria and abolished in guinea-pig atria bathed in zero sodium. Cyclic AMP generation in response to isoproterenol is reduced but not abolished in zero sodium, and time to peak tension is still shortened in response to the catecholamine. Addition of strontium ion to guinea-pig atria in zero sodium restores the ability of isoproterenol to increase twitch tension and to enhance slow channel conductance now carried by strontium. Chelation of calcium ion in the presence of strontium ion by the calcium chelator ethyleneglycol bis-(β-amino ethylether)- n,n,n′,n′ tetraacetic acid (EGTA) K A Ca K A Sr > 100 greatly increases the duration of slow action potentials carried by strontium. These data suggest that intracellular calcium accumulation, but not strontium accumulation, decreases slow channel conductance and the ability of isoproterenol to enhance slow channel conductance. It is concluded that sodium reduction tends to increase intracellular free calcium by decreasing the process of sodium-calcium exchange which normally contributes to relaxation. A large accumulation of intracellular calcium diminishes the ability of the tissue to respond to isoproterenol both by reducing the ability of the tissue to generate cyclic AMP and by directly inhibiting slow channel conductance.

Effect of fasting on tubular phosphorus reabsorption

In the thyroparathyroidectomized (TPTX) rat, fasting increased urinary phosphorus excretion by decreasing the tubular reabsorption of P1 (TRP) and resulted in hypophosphatemia. The administration of either sucrose or NaHCO3 prevented the metabolic acidosis associated with fasting and decreased the phosphaturia, indicating that the phosphaturia in fasting is in part due to metabolic acidosis. In rats on partial reduction of P1 intake selectively, the phosphaturic response to parathyroid hormone (PTH) was completely suppressed. On the other hand, the fasting rat partially retained the phosphaturic response to PTH, although dietary P1 intake was totally absent. These findings suggest that the renal P1 wasting in fasting may take place by dual mechanisms: a) the PTH-independent decrease in TRP, and b) an inability to totally suppress the response to PTH. Cyclic AMP generation in response to PTH, determined both in vivo and in vitro, was not measurably altered in fasting. However, the phosphaturic response to cyclic AMP was decreased in fasting, suggesting that the mechanism of partial resistance to PTH is probably not at but after cyclic AMP generation.

Alpha-Adrenergic reduction of cyclic adenosine monophosphate concentrations in rat myocardium.

We determined the effect of alpha-adrenergic receptor stimulation on cyclic adenosine monophosphate (cyclic AMP) concentrations in isolated myocytes derived from adult rat hearts and in isolated perfused rat hearts. Activation of alpha-adrenergic receptors with either phenylephrine (10(-8) M to 10(-6) M) or epinephrine (10(-8) M to 10(-6) M) plus propranolol (10(-6) M) resulted in a reduction in cyclic AMP levels in isolated myocytes. The action of phenylephrine was antagonized by phentolamine (10(-6) M). Phenylephrine (10(-5)M attenuated cyclic AMP generation in response to isoproterenol (10(-8) M and 10(-5) M). However, this effect of phenylephrine was not antagonized by phentolamine. Elevation of cyclic AMP concentrations produced by glucagon and by theophylline in isolated myocytes was attenuated by phenylephrine and by epinephrine plus propranolol and the attenuation was antagonized by phentolamine. In isolated perfused rat hearts epinephrine (10(-6) M), when given with propranolol, diminished the rate of development of tension and also reduced tissue levels of cyclic AMP. Epinephrine alone, as well as isoproterenol, increased contractility and myocardial cyclic AMP concentrations as expected. These results indicate that catecholamines may increase or decrease cyclic AMP levels in rat myocardium, depending on the intensity of stimulation of receptor types. Increases are mediated by beta-adrenergic receptors, whereas decreases appear to by mediated by alpha-adrenergic receptors.