Augmentation by chlorpropamide of 1-deamino-8-D-arginine vasopressin-induced antidiuresis and stimulation of renal medullary adenylate cyclase and accumulation of adenosine 3',5'-monophosphate

The effect of chlorpropamide was determined in Brattleboro diabetes insipidus (DI) rats that were injected with 1-deamino-8-D-arginine vasopressin (dDAVP). Chlorpropamide augmented the antidiuretic responses to 0.78 and 1.56 ng dDAVP but not to larger doses. In an effort to explain this observation we investigated the effect of chlorpropamide on renal medullary adenylate cyclase activation by dDAVP and on phosphodiesterase activity. We found that the injection of chlorpropamide increased adenylate cyclase activation by dDAVP added in vitro to renal medullary cell membrane preparations from Brattleboro DI rats but had no effect on phosphodiesterase activity. When kidneys from Brattleboro DI rats, treated and not treated with chlorpropamide, were perfused in vitro, we found that 10(-4) M dDAVP increased the concentration of cAMP in comparison to untreated and chlorpropamide-treated groups, and that chlorpropamide plus dDAVP resulted in a greater concentration of renal cAMP than was found with dDAVP alone. We believe that treatment with chlorpropamide increases dDAVP-stimulated renal medullary adenylate cyclase activity without altering phosphodiesterase activity and that this leads to increased renal cAMP concentrations. This, in turn, causes an augmented antidiuresis in response to dDAVP.     

Subcellular distribution of the enzymes related to the cellular action of vasopressin in renal medulla.

Subcellular distribution of the enzymes related to the cellular action of antidiuretic hormone was studied in bovine renal medulla. The highest activity of vasopressin-stimulated adenylate cyclase was found in plasma membranes. The basal activity increased two times above homogenate while vasopressin-stimulated and NaF-stimulated activities both increased five times. Adenylate cyclase activity was present also in other particulate fractions, but it was not significantly stimulated by vasopressin. Cyclic AMP phosphodiesterase was predominantly located in the cytosol when assayed with 0.5 mM cyclic AMP or with 5 muM cyclic AMP. However, with the latter concentration of cyclic AMP more activity remained associated with the particulate fractions and was more inhibited by theophylline. The highest cyclic AMP-stimulated protein kinase activity occurred in the cytosol. Protein kinase activity present in other subcellular fractions was not markedly stimulated by cyclic AMP. Protein phosphatase activity was highest in cytosol when assayed using 32P-histones, 32P-plasma membrane proteins, and 32P-cytoslic proteins. The activity was unaffected by 10-6M to 10-4M cyclic AMP or cyclic GMP. The activity was completely inhibited by 10mM ZnSO4 and 10mM CuSO4; 10mM NaF inhibited the activity by approximately 14%. The enzymes related to the cellular action of vasopressin are predominatly localized in the cytosol except for the vasopressin-sensitive adenylate cyclase which is plasma membrane bound. To mediate the effect of antidiuretic hormone and act on the luminal plasma membrane these soluble enzymes and their substrates should be compartmentalized, possibly by a system of cytoplasmic microtubules.     

Age-related postreceptor mechanisms: Changes in adenylate cyclase but not phosphodiesterase in isolated mouse renal medullary collecting ducts

Urine concentrating ability declines with increasing age, partly due to an impaired response of kidney medullary collecting ducts to the antidiuretic hormone, vasopressin. We investigated this change in isolated mouse medullary collecting ducts by measuring the activity of adenylate cyclase and phosphodiesterase, which catalyse the formation and hydrolysis of cAMP, respectively. Adenylate cyclase activity was measured in the presence of vasopressin (which stimulates adenylate cyclase via the receptor) or forskolin (which directly stimulates the catalytic subunit). We showed an age-related decrease in the catalytic subunit of adenylate cyclase, and no difference in the activity of phosphodiesterase, indicating that a reduction in the catalytic subunit of adenylate cyclase contributes towards the age-related decrease in cyclic AMP response of kidney to vasopressin.     

Decreased glucagon-stimulated cyclic AMP production by isolated liver cells of rats with type 2 diabetes

This study was undertaken to investigate the effect of experimental type 2 diabetes in the rat on the insulin and glucagon receptors and on the early steps of glucagon action. The binding of insulin and glucagon and the glucagon-stimulated cyclic AMP accumulation in the presence of a phosphodiesterase inhibitor (IBMX, 0.1 mmoles/l) were studied in liver cells isolated from 7-9-month-old rats with chronic type 2 diabetes and from control rats. No significant change was observed in [125I] insulin binding and [125I]glucagon binding of diabetic liver cells as compared to controls. Scatchard analysis of the competition experiments indicated that affinity and number of insulin and glucagon receptors were not significantly changed in the liver cells of diabetic rats. The basal cyclic AMP level was significantly lower in the diabetic hepatocytes (2.3 +/- 0.9 pmoles/10(6) cells) than in the controls (4.0 +/- 0.6 pmoles/10(6) cells). Cyclic AMP response to physiological concentrations of glucagon (0.1-1 nmoles/l) was about 2 times lower in the diabetic hepatocytes than in the controls. Furthermore, the basal liver membrane adenylate cyclase activity and the fluoride-activatable adenylate cyclase activity were about 2 times lower in the diabetics as compared to control rats, while the liver cyclic AMP and cyclic GMP phosphodiesterase activities were unchanged. The ability of glucagon to stimulate liver membrane adenylate cyclase over a 10(-12)-10(-6) M concentration range was decreased in diabetic rats. Taken together, these data are consistent with the thesis that the impairment of the liver cyclic AMP response to glucagon in rats with type 2 diabetes is caused by a decrease in the amount of adenylate cyclase in the liver plasma membranes.     

DDAVP (1-desamino-8-D-arginine-vasopressin) treatment of central diabetes insipidus--mechanism of prolonged antidiuresis.

DDAVP, 1-desamino-8-D-arginine-vasopressin, is a synthetic analog of arginine vasopressin which produces prolonged antidiuresis after intranasal administration to patients with complete central diabetes insipidus. We have studied the mechanism of the prolonged antidiuretic effect by specific radioimmunossay of DDAVP in plasma of patients and by in vitro studies on the adenylate cyclase-cylic AMP system of the rat outer renal medulla. When DDAVP was administredd to patients, all responded, but the duration of response among patients varied from 5-21 h. The peak level of DDAVP in plasma was achieved up to 4 h after administration indicating a slow absorption from the nasal mucosa. The disappearance time of DDAVP from plasma correlated significantly with the duration of antidiuresis, P less than 0.001. On a molar basis DDAVP was 3-fold greater than AVP in its stimulation of outer medullary adenylate cyclase activity and 10-fold greater than AVP in its stimulation of cyclic AMP content. The prolonged antidiuresis of intranasally administered DDAVP is due to slow absorption, presistence in plasma, and enchanced effect on the kidney.     

Effects of hormone treatment and age on the stimulation of rat anterior pituitary adenylate cyclase and cyclic adenosine 3',5' monophosphate by hypothalamic extract

Crude ovine hypothalamic extract in vitro significantly stimulated adenylate cyclase activity and LH release by pituitary gland obtained from newborn rats (0.25–0.5 days old) and increased cyclic adenosine 3′,5′-monophosphate (cyclic AMP) in 2-day-old rats. In adult rats, the stimulation of adenylate cyclase activity and cyclic AMP formation, but not LH release, induced by hypothalamic extract was inhibited by adrenalectomy. In vivo administration of pharmacologic doses of 1 mg dexamethasone asd 100 μg thyroxine for 1 day markedly reduced the effect of 0.2 mg/ml but not 1 mg/ml hypothalamic extract on cyclic AMP concentrations. The action of the latter dose of hypothalamic extract on cyclic AMP was inhibited by injection of dexamethasone and thyroxine for 3 days, while the stimulation of LH release was preserved. Such dissociation between activation of the adenylate cyclase-cyclic AMP system and LH release suggests that cyclic AMP formation may not be essential for increased release of LH. Adrenalectomy did not inhibit stimulation of adenylate cyclase activity or cyclic AMP formation caused by prostaglandin E₁ (PGE₁). Hypothalamic extract or PGE₁ added to intact pituitary in vitro increased protein kinase activity and decreased the protein binding of exogenous cyclic AMP, reflecting the increased generation of endogenous cyclic AMP.     

Effect of hypophysectomy on cyclic 3',5'-nucleotidemetabolizing enzymes in the rat thyroid gland

Adenylate cyclase and cyclic AMP phosphodiesterase activities in the thyroid gland were significantly reduced after hypophysectomy, followed by a gradual restoration of the enzyme activities to the levels seen in sham-operated rats whereas a slight and persistent reduction was evident in guanylate cyclase and cyclic GMP phosphodiesterase activities in the same tissue. These changes in enzyme activities were restored by TSH administration but not by ACTH. The recovery of activity produced by TSH administration was inhibited by cycloheximide. Hypophysectomy, or TSH and cycloheximide administration, did not produce any significant changes in the concentrations of calmodulin, suggesting that the alteration of these enzyme activities is not induced by a decrease in the concentration of calmodulin. Since forskolin activation of adenylate cyclase did not restore the reduced activity in the hypophysectomized rat thyroid to the level found in the sham-operated control rat thyroid, we conclude that there is a reduction of the amount of enzyme after hypophysectomy rather than a change of the active site on adenylate cyclase. The spontaneous restoration of adenylate cyclase and cyclic AMP phosphodiesterase activities after hypophysectomy implies that cyclic AMP-metabolizing enzymes are responsive to an autoregulatory mechanism in thyroid follicular cells.     

Neurohypophyseal peptides in hypothyroid rats: plasma levels and kidney response.

A degree of impairment of water excretion may be associated with hypothyroidism. The involvement of vasopressin has been suggested, but its role continues to be debated because of lack of studies where vasopressin was directly assayed. In this study, water excretion was assessed and arginine vasopressin was directly measured in unanesthetized and nonstressed normal and thyroidectomized rats at a basal state and after water loading. Following water load, both groups decreased their plasma osmolality. Plasma vasopressin demonstrated an elevated level in hypothyroid rats compared to control (2.04 and 1.04 μU/ml), respectively, at baseline and (1.32 and 0.68 μU/ml), respectively, after water loading. There was a significant and similar correlation between plasma vasopressin and plasma osmolality in both groups. The regression lines for urine osmolality and plasma vasopressin of the two groups were parallel but with a significantly greater vasopressin level for the hypothyroid rats at any given urine osmolality. This suggested that circulating plasma vasopressin was less active in hypothyroid than control rats; a hypothesis that was tested by measuring the responsiveness of renal medullary adenylate cyclase to vasopressin. Both basal and vasopressin stimulated cyclic AMP levels were less in hypothyroid than control rats. Thus, these studies demonstrate that hypothyroidism in the rat was associated with an elevated plasma vasopressin that did not appear to be fully effective in inducing an antidiuresis. Factors other than vasopressin may be more important in the water imbalance of hypothyroidism.     

Adenosine-mediated stimulation of bone cell adenylate cyclase activity.

Adenosine rapidly stimulated adenylate cyclase activity but did not modify cyclic AMP degradation when added to a particulate fraction prepared from isolated bone cells. The effect of adenosine was one-half maximal at 5-10 micronM, and was not mimicked by 5' AMP, inosine, guanosine, uridine, adenine, or ribose. Basal and adenosine-stimulated adenylate cyclase activites were directly proportional to the concentration of particulate protein in the assay system. Theophylline decreased the degree to which adenosine stimulated adenylate cyclase activity, whereas another phosphodiesterase inhibitor, RO-20-1724, failed to iiinfluence the effect of adenosine. Adenosine itself, and not a metabolite of adenosine is the stimulator of adenylate cyclase, since it was neither phosphorylated nor deaminated appreciably by the particulate fraction. The particulate fraction did not convert substrate ATP to adenosine in amounts sufficient to enhance adenylate cyclase. The stimulatory effect of adenosine was maximal at 1.2 mM Mg2+, declined with increases in the Mg2+ concentration, and was replaced by inhibition at 20 mM Mg2+. At 2.4 mM Mg2+, basal adenylate cyclase activity peaked at 1.1 mM ATP, and was inhibited by higher ATP concentrations. The magnitude of adenosine stimulation was greater at inhibitory concentrations of ATP than at concentrations which yielded maximum activity. The results suggest that the previously demonstrated ability of adenosine to increase cyclic 3'5' AMP levels in intact bone cells stems from its effect on adenylate cyclase. Adenosine may act by modifying the regulatory nfluence of free Mg2+, uncomplexed ATP, (or both), on adenylate cyclase. Theophylline appears to interfere with the action of adenosine by a mechanism which is distinct from its capacity to inhibit cyclic AMP phosphodiesterase activity. (Endocrinology 99:901,1976)     

Glucagon-sensitive adenylate cyclase in human renal medulla.

In cell-free preparations (washed 600 x g pellets) of human renal medulla, glucagon produced a dose-dependent stimulation of adenylate cyclase. The stimulation of renal medullary adenylate cyclase by saturating concentrations of glucagon was additive to the saturating doses of vasopressin. Furthermore, L-isoproterenol stimulated renal medullary adenylate cyclase in a dose-dependent manner, and this stimulation was blocked by DL-propranolol. Stimulation of the renal medullary adenylate cyclase by maximal doses of glucagon and L-isoproterenol was additive. DL-Propranolol did not inhibit stimulation of glucagon. Thus, the results indicate the existence of a specific adenylate cyclase that is responsive to glucagon--distinct from the isoproterenol-sensitive adenylate cyclase and the previously described vasopressin-sensitive adenylate cyclase in human renal medulla. We suggest that the renal tubular effect of glucagon may be mediated by glucagon-dependent cyclic-AMP production in renal tissue.     

Calmodulin regulation of cellular cyclic AMP production in response to arginine vasopressin, prostaglandin E2 and forskolin in rat renal papillary collecting tubule cells in culture

The effect of calmodulin on the stimulation of cyclic AMP production by arginine vasopressin (AVP), prostaglandin E2 (PGE2) and forskolin was examined in cultured renal papillary collecting tubule cells of the rat. In the presence of the phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine submaximal concentrations of AVP (1 nmol/l), PGE2 (20 nmol/l) and forskolin (240 nmol/l) significantly increased cellular cyclic AMP accumulation by 2.3-, 6.0- and 8.4-fold respectively. Two chemically dissimilar inhibitors of calmodulin, namely trifluoperazine and N-(6-amino-hexyl)-5-chloro-1-naphthalenesulphonamide (W-7), attenuated the AVP-, PGE2- and forskolin-stimulated cellular production of cyclic AMP in a dose-related manner. Cellular production of cyclic AMP was inhibited by 50% (ID50) by doses ranging from 16 to 28 mumol trifluoperazine/l and 35 to 44 mumol W-7/1. Basal accumulation of cellular cyclic AMP was also decreased by treatment with either trifluoperazine or W-7, but the effective dose was higher than that which inhibited cellular cyclic AMP production stimulated by AVP, PGE2 and forskolin. Since forskolin directly activates adenylate cyclase at a site of the catalytic unit and the cellular action of AVP to activate adenylate cyclase is mediated through receptor-guanine nucleotide regulatory-catalytic units, the present study indicates calmodulin regulation of basal, AVP-, PGE2- and forskolin-activated adenylate cyclase in the papillary collecting tubule cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interrelationship between adenylate cyclase activity, adenosine 3':5' cyclic monophosphate phosphodiesterase activity, adenosine 3':5' cyclic monophosphate levels, and growth of cells in culture

To investigate how cell population density influences the intracellular concentration of cyclic AMP we have measured adenylate cyclase and cyclic AMP phosphodiesterase activities and cyclic AMP levels at various stages of cell density in normal rat-kidney (NRK) cells, which exhibit contact-inhibition of growth, and in normal chick-embryo fibroblasts (CEF), which do not show contact inhibition of growth under our conditions. Until NRK cells reach confluency, both activities increase with increasing cell population and cyclic AMP levels are low. As NRK cells reach confluency, cyclic AMP phosphodiesterase activity decreases somewhat whereas adenylate cyclase activity continues to rise. This increase in synthetic ability is accompanied by the increase in cyclic AMP levels which occurs in these cells at confluency. In CEF grown in 5% serum where density-dependent inhibition of growth is not observed, both adenylate cyclase and cyclic AMP phosphodiesterase activities increase proportionately with increasing cell population density. No significant alteration occurs in the ratio between these two enzyme activities and no change is observed in cyclic AMP levels.The NaF-stimulated activity in NRK cells increases with increasing cell density until the cells reach confluency; thereafter the NaF-stimulated activity remains constant. In contrast, the NaF-stimulated activity observed in CEF does not vary appreciably between light and heavy density.The observed changes in the enzymes of cyclic AMP metabolism accurately reflect the changes in cyclic AMP concentration as a function of cell population density. The data indicate that these two enzyme activities respond to increasing cell density to elicit a rise in intracellular cyclic AMP levels. The elevated cyclic AMP levels are thought to be involved in the regulation of cellular growth rate and the mediation of contact inhibition of growth.     

Antilipolytic effects of insulin and adenylate cyclase inhibitors on isolated human fat cells

Antilipolysis induced by insulin by adenylate cyclase inhibitors was compared in isolated human fat cells when lipolysis was activated at well-defined steps in the cyclic AMP system. The latter was achieved with isoprenaline (beta-adrenoreceptor agonist), cholera toxin and pertussis toxin (acting on the GTP-sensitive coupling proteins), forskolin (stimulating the catalytic component of adenylate cyclase), enprofylline (selective phosphodiesterase inhibitor) and N6-monobutyryl-cyclic-AMP or 8-bromo cyclic-AMP (cyclic AMP analogues which are resistant or sensitive to phosphodiesterase, respectively). Clonidine (alpha 2-adrenoreceptor agonist), prostaglandin E2 and N6-(phenylisopropyl) adenosine (adenosine analogue) failed to inhibit lipolysis stimulated by cholera toxin or pertussis toxin, but were effective under all other conditions. Insulin failed to inhibit lipolysis stimulated by enprofylline or N6-monobutyryl cyclic AMP, but was effective under all other circumstances. In conclusion, insulin and adenylate cyclase inhibitors are antilipolytic in human fat cells through different mechanisms. Adenylate cyclase inhibitors act predominantly on the GTP-sensitive coupling proteins and, to a minor extent, at some yet unidentified distal step in the lipolytic machinery. As regards insulin, the major site of the antilipolytic action is phosphodiesterase.     

Effect of irradiation on enzyme activities of cyclic AMP system in the neuro- and adenohypophyses.

The enzyme activities of cyclic AMP system in the neuro- and adenohypophyses were studied, immediately after an irradiation by a single whole body exposure of 1600 R, in an attempt to find whether this intervention causes the changes in the responsiveness of the cyclic AMP regulatory system. In the irradiated rats the neurohypophyses revealed a reduced activity of adenylate cyclase, moderately increased activity of phosphodiesterase and slightly decreased activity of protein kinase, including the value stimulated by cyclic AMP. In the adenohypophyses the irradiation did not cause any significant changes in the enzyme activities of the cyclic AMP system, except of slightly decreased adenylate cyclase activity. The possible relationship of the plasma level of antidiuretic hormone immediately after irradiation and the enzyme activities of cyclic AMP system is discussed.     

Glucose Inhibition of Adenylate Cyclase in Intact Cells of Escherichia coli B

Previous studies in E. coli B have demonstrated an inverse correlation between the presence of glucose in the medium and the accumulation of cyclic AMP in the medium. This observation could not be explained by the action of glucose as a repressor of adenylate cyclase (EC 4.6.1.1) synthesis, as a stabilizer of cyclic AMP phosphodiesterase (EC 3.1.4.17) activity, or as a direct inhibitor of adenylate cyclase activity in cell-free preparations. The recent development of an in vivo assay for adenylate cyclase has provided a basis for further exploring the inhibitory action of glucose in intact cells. With this assay it has been possible to show that, while glucose does not affect adenylate cyclase in vitro, it rapidly inhibits the enzyme activity in intact cells. Extensive metabolism of glucose is not required, since alpha-methylglucoside also inhibits adenylate cyclase in vivo. When cells are grown on glucose as carbon source, some sugars (mannose, glucosamine) substitute for glucose as adenylate cyclase inhibitors while others (e.g., fructose) do not. Dose-response studies indicate that low concentrations of glucose lead to essentially complete inhibition of adenylate cyclase activity while only moderately decreasing intracellular cyclic AMP concentrations. The evidence presented suggests that the decreased cellular cyclic AMP levels resulting from glucose addition can be accounted for by inhibition of adenylate cyclase without any significant effect on cyclic AMP phosphodiesterase or the transport of cyclic AMP from the cells to the medium.     

Cooling enhances adenosine 3':5' monophosphate accumulation in thyrotropin stimulated dog thyroid slices

Dog thyroid slices have been stimulated in vitro by thyrotrophin (TSH) at 37 degrees C and 25 degrees C. Adenosine 3':5'-monophosphate (cyclic AMP) accumulation was enhanced by cooling to 25 degrees C. This observation has been extended to kidney cortex slices stimulated by parathyroid hormone (PTH). However, this phenomenon is not general: it does not apply to thyroid slices stimulated by prostaglandin E1 (PGE1) or adrenal cortex slices stimulated by adrenocorticotrophic hormone (ACTH). Slight cooling provides a useful tool to influence biochemical mechanisms in intact cells and therefore the mechanism of action of cooling on cyclic AMP was investigated in dog thyroid slices stimulated by TSH. Adenylate cyclase and cyclic nucleotide phosphodiesterase activities as measured in acellular preparations decreased in parallel with the temperature. A decrease of the cyclic AMP efflux from the cell at 25 degrees C, although not measurable in this preparation, did not seem to be responsible for the phenomenon. computer simulation of the kinetics of the cyclic AP accumulation curve is compatible with the hypothesis of decrease in the desensitization rate of adenylate cyclase at 25 degrees C. This was demonstrated using an experimental protocol in intact slices and by measurements of adenylate cyclase activities in particulate preparations of slices pretreated or not by the hormone. This decreased adenylate cyclase desensitization can explain the higher cyclic AMP levels in TSH stimulated thyroid slices incubated at 25 degrees C. However, this does not exclude complementary mechanisms.     

The adenylate cyclase-cyclic AMP-phosphodiesterase system in pathological human thyroid.

The adenylate cyclase-cyclic AMP-phosphodiesterase system of human thyroid tissues adjacent to cold nodules (control), two follicular adenomas, one hyperplastic thyroid and one hyperfunctioning follicular carcinoma have been compared. In the hyperfunctional follicular carcinoma the basal adenylate cyclase is much higher than in control tissue, carcinoma adenylate cyclase does not respond to TSH and prostaglandin E1, whereas it responds normally to fluoride. In the hyperplastic, but hypofunctional thyroid the basal adenylate cyclase is higher than in normal tissue whereas the response to TSH, PGE1, and fluoride is normal. No difference between the follicular adenomas and normal thyroid stimulated and unstimulated adenylate cyclase was observed. Furthermore in various thyroid tissues no changes in the level of cyclic AMP phosphodiesterase was found. Our data indicate a greater change in the synthesis rather than in degradation of cyclic AMP in the human pathological thyroids studied.     

Role of the calcium-calmodulin system in the adenylate cyclase activity of vascular smooth muscle

In order to evaluate the relationship between the calcium-calmodulin system and the adenylate cyclase activity of vascular smooth muscle, we examined the effects of several calcium effectors on the basal and stimulated adenylate cyclase activity. Thoracic aortae were removed from Wistar rats and the tissues were homogenated with cold homogenizing buffer containing 1 nM EDTA. Membrane protein fraction of the smooth muscle was prepared by centrifugation at 37,000 g. In this procedure, endogenous guanine nucleotides and contractile proteins remained. The protein fraction was incubated with 2 mM EGTA, 50 microM trifluoperazine, 0.1 microM A23187 or 25 microM calmodulin under basal and stimulated (50 microM isoproterenol, 100 microM GTP and 50 microM forskolin) conditions. The adenylate cyclase activity was determined by a method modified in our laboratory using double isotope counting. Trifluoperazine reduced the basal adenylate cyclase activity significantly (p less than 0.01) as well as the stimulated enzyme activities. A23187 did not affect the basal enzyme activity, but elevated the isoproterenol stimulated enzyme activity significantly (p less than 0.05). EGTA did not affect the basal and stimulated adenylate cyclase activities. Calmodulin elevated the basal enzyme activity significantly (p less than 0.02), but did not affect the stimulated enzyme activities. These results suggest that the calcium-calmodulin system is necessary for maintenance of the adenylate cyclase activity of vascular smooth muscle cells. The calmodulin acting site is considered to be the catalytic subunit, and stimulation of the enzyme is accelerated by calcium ion.     

Adenylate cyclase permanently uncoupled from hormone receptors in a novel variant of S49 mouse lymphoma cells.

A novel variant of the S49 mouse lymphoma has been selected from wild-type cells by growth in medium containing the beta-adrenergic agonist terbutaline and inhibitors of cyclic nucleotide phosphodiesterase. In contrast to the situation in the wild-type clone, synthesis of adenosine 3':5'-monophosphate (cyclic AMP) is not stimulated by beta-adrenergic agonists or by prostaglandin E1 either in intact variant cells or in membrane preparations of such clones. However, basal and NaF-stimulated activities of adenylate cyclase [ATP pyrophosphate-lyase (cyclizine), EC 4.6.1.1] are normal, enzyme activity is stimulated by guanyl-5'-yl imidodiphosphate [Gpp(NH)p], and intact cells accumulate cyclic AMP when exposed to cholera toxin. Furthermore, variant cell membranes possess ligand-binding activity consistent with the conclusion that a normal or an excessive number of beta-adrenergic receptors is present. Thus, interaction between the hormone-binding and the catalytic moieties of the adenylate cyclase system is lost. This variant phenotype, designated as uncoupled (UNC), has been stable for more than 100 generations without exposure to the drugs used for selection. Such cells should be useful for the elucidation of methanisms of transmission of information from hormone receptors to adenylate cyclase.     

Vascular adenylate cyclase: role of age and guanine nucleotide activation.

Particulate adenylate cyclase activity was examined in broken cell preparations of rat aorta and mesenteric artery from 3- to 5- and 9- to 13-week-old rats. While basal adenylate cyclase activity of the mesenteric artery was 4-fold greater than aortic enzyme activity, there was no difference in enzyme activity with age. GTP and the GTP analogue, 5'-guanylylimidodiphosphate [Gpp(NH)p] stimulated adenylate cyclase activity. Stimulation by Gpp(NH)p did not differ with age for either tissue and occurred without a detectable lag. The vasodilators, isoproterenol, 2-chloroadenosine and prostaglandin E1, were ineffective in increasing adenylate cyclase activity, although marked stimulation was demonstrated with both sodium fluoride and Gpp(NH)p. Even in combination with Gpp(NH)p, isoproterenol did not increase particulate adenylate cyclase activity of these blood vessels; however, with intact arteries, isoproterenol (10(-7)M) did increase aortic and mesenteric cyclic AMP levels. Isoproterenol increased cyclic AMP levels in rats of both ages, at a time when isoproterenol was less effective in maximally relaxing aortic strips from 9- to 13-week-old rats. These data indicate that diminished aortic relaxation with age is not associated with a reduced ability of vascular relaxants to increase aortic cyclic AMP levels. Furthermore, as a first step in establishing that guanine nucleotides are regulators of vascular adenylate cyclase, both GTP and Gpp(NH)p were found to be potent activators of adenylate cyclase from blood vessels.