Fluoride-stimulated Adenylate Cyclase Activity Research Articles

Glucagon and Insulin Binding to Liver Membranes in a Partially Nephrectomized Uremic Rat Model

To investigate the role of glucagon and insulin receptor binding in the glucagon hypersensitivity and insulin resistance which characterize the glucose intolerance of uremia, liver plasma membranes were prepared from control rats (blood urea nitrogen [BUN] 15+/-1 mg/100 ml, creatinine 0.7+/-0.2 mg/100 ml), and from 70% nephrectomized rats (BUN 30+/-2 mg/100 ml, creatinine 2.2+/-0.2 mg/100 ml), and from 90% nephrectomized rats (BUN 46+/-3 mg/100 ml, creatinine 4.20+/-0.7 mg/100 ml), 4 wk after surgery. As compared to controls, the 90% nephrectomized rats had significantly higher levels of plasma glucose (95+/-4 vs. 125+/-11 mg/100 ml), plasma insulin (28+/-9 vs. 52+/-11 muU/ml), and plasma glucagon (28+/-5 vs. 215+/-18 pg/ml). Similar, but less marked, elevations were observed in the 70% nephrectomized animals. In liver plasma membranes from nephrectomized rats, specific binding of (125)I-glucagon was increased by 80-120%. Furthermore, glucagon (2 muM)-stimulated adenylate cyclase activity in nephrectomized rats was twofold higher than in controls. In contrast, fluoridestimulated adenylate cyclase activity was similar in both groups of rats. In marked contrast to glucagon binding, specific binding of (125)I-insulin to liver membranes from nephrectomized rats was reduced by 40-50% as compared to controls. Data analysis suggested that the changes in both glucagon and insulin binding are a consequence of alterations in binding capacity rather than changes in affinity. Liver plasma membranes from nephrectomized rats degraded (125)I-glucagon and (125)I-insulin to the same extent as control rats. THESE RESULTS DEMONSTRATE THAT: (a) the 70 and 90% nephrectomized rats simulate the hyperglycemia, hyperinsulinemia, and hyperglucagonemia observed in clinical uremia; (b) in these animals specific binding of glucagon to liver membranes is increased and is accompanied by higher glucagon-stimulated adenylate cyclase activity; and (c) specific binding of insulin is markedly decreased. These findings thus provide evidence of oppositely directed, simultaneous changes in glucagon and insulin receptor binding in partially nephrectomized rats. Such changes may account for the hypersensitivity to glucagon and may contribute to resistance to insulin observed in the glucose intolerance of uremia.

Effect of physical training on control mechanisms of lipolysis in rat fat cell ghosts

Fat cell ghosts and homogenates of fat cells were used to study the influence of training on the regulatory system for lipolysis in adipose tissue of female rats. A training effect was identified from elevated succinate dehydrogenase activities in the soleus and plantaris muscles. Neither basal nor maximal (NaF-stimulated) adenylate cyclase activities per mg protein of fat cell ghosts were altered by training. Fluoride-stimulated adenylate cyclase activity per microgram DNA was lower in the trained than untrained group. Adenylate cyclase activities in response to norepinephrine expressed either on a per mg protein or per microgram DNA basis were lower (P less than 0.05) in fat cell ghosts from trained rats. Phosphodiesterase activity was higher (P less than 0.05) in fat cell ghosts from trained rats for cyclic AMP concentrations of 1--5.0 micrometer. The apparent Km's of phosphodiesterase were 1.19 and 2.0 micrometer of cyclic AMP for the untrained and trained groups, respectively (P less than 0.05). Protein kinase activity in the supernatant fraction of homogenates of fat cells was unchanged due to training. The overall effect of training was to blunt the system for cyclic AMP production in rat adipocytes. This may explain, at least partially, the lower plasma free fatty acid levels observed in trained compared to untrained persons during submaximal exercise.

Ontogeny of β-adrenergic receptors in rat cerebral cortex

The ontogeny of β-adrenergic receptors in rat cerebral cortex has been studied using [ 125I]iodohydroxyzylpindolol as a ligand in an in vitro binding assay. The concentration of β-adrenergic receptors was very low during the first week after birth. Between days 7 and 14 there was a rapid increase in the density of receptors. Adult levels were reached by the end of the second week. The affinities of 1-isoproterenol and iodohydroxybenzylpindolol for β-adrenergic receptors did not vary with the age of the animal. Fluoride stimulated adenylate cyclase activity in the cerebral cortex was 40% of the adult level at birth and gradually increased to maximal levels over the next two weeks. On the other hand, catecholamine stimulated cyclic-3′,5′-adenosine monophosphate accumulation was barely detectable during the first week after birth, but increased rapidly to adult levels bwetween days 7 and 14. The results suggest that is the development of β-adrenorgic receptors that permits the expression of catecholamine sensitive adenylate cyclase activity. Norepinephrine stores in the cerebral cortex developed slowly reaching adult levels approximately two months after birth. There is therefore little correlation between the ontogeny of presynaptic adrenergic nerve terminals and the postsynaptic development of β-adrenergic receptors.

Comparative studies on the effects of isoproterenol and glucagon on 3′,5′-cyclic monophosphate metabolism in embryonic and newborn chick liver

1. 1. The endogenous level of adenosine 3′,5′-cyclic monophosphate (cyclic-AMP) in developing chick liver was determined and found to be the highest at the earliest stage studied (6-day embryo) and to fall progressively to reach a lower plateau after about two weeks incubation. 2. 2. Isoproterenol or glucagon produce elevations in the cyclic-AMP content of 7–9-day embryonic liver in vitro but have little effect on the cyclic-AMP content of newborn chick liver slices in vitro. 3. 3. Kinetic plots of the phosphodiesterase activity in whole homogenates of embryonic or newborn chick liver are biphasic indicating the presence of 2 phosphodiestcrases. The activities of these 2 enzymes do not change markedly during development. 4. 4. The basal and fluoride stimulated adenylate cyclase activities of preparations of chick liver are lower than similar preparations of 7–9-day embryonic liver. Both types of preparations are stimulated by isoproterenol. 5. 5. The relationships between cyclic-AMP and the control of glycogen metabolism in the developing chick are discussed.

Electron microscopic cytochemical demonstration of adenyl cyclase activity in psoriatic epidermis.

Isoproterenol and sodium fluoride stimulated adenyl cyclase activity was detected in epidermal tissue from 2 patients with untreated psoriasis by an electron microscopic cytochemical technique. Adenyl cyclase activity was present on the outer surface of the cell membranes, predominantly in the basal cells and in the 4-5 lower Malpighian cell layers, while the superficial layers, stratum granulosum and stratum corneum showed no activity. The precipitates (lead-PPi complexes) after isoproterenol stimulation were larger and fewer in number than those seen after sodium fluoride stimulation. Isoproterenol stimulation was abolished by propranolol. Neither the uninvolved epidermis from the 2 patients with psoriasis nor the normal skin from 2 volunteer individuals showed any difference from the psoriatic epidermis.

Adenyl cyclase-cyclic AMP response during bowel obstruction

The massive intestinal secretion seen with cholera and other enterotoxins has been correlated with elevated mucosal adenyl cyclase and cyclic AMP levels. Bowel obstruction is associated with intestinal secretion, and it was of interest to investigate whether this secretion also involved an altered second messenger system. Sixteen rats were obstructed by tying a ligature around the mid-intestine. The bowel proximal to the obstruction showed decreased adenyl cyclase activity by 24 hr and decreased cyclic AMP levels by 48 hr. There was no change in sodium fluoride-stimulated adenyl cyclase activity, indicating normal functioning of the catalytic subunit of the enzyme. Thus, intestinal secretion seen during bowel obstruction is apparently not mediated in the same way as that of cholera, and the decreased adenyl cyclase activity may be ascribed to membrane alterations incurred by bowel distention.

Effect of exercise training on adenyl cyclase and phosphodiesterase in skeletal muscle, heart, and liver

Adaptive changes in the activities of adenyl cyclase and phosphodiesterase in response to exercise training were studied in muscle, heart, and liver. Basal adenyl cyclase activities were increased in muscle and decreased in heart as a result of training. Epinephrine-stimulated adenyl cyclase activity of heart was depressed by training. Fluoride-stimulated adenyl cyclase activity was not altered by training in any of the tissues studied. Phosphodiesterase activity was depressed in liver of trained rats but unaltered in muscle and heart. These changes in adenyl cyclase and phosphodiesterase activities suggest that the capacity to respond to hormones may be altered by training.

The effect of alkylating agents on adenosine 3′,5′-monophosphate metabolism in walker carcinoma

The effect of some alkylating agents on the activity of the enzymes adenylate cyclase and cyclic 3′,5′-nucleotide phosphodiesterase has been studied using Walker carcinoma cells in tissue culture. The monofunctional agent 5-aziridinyl-2,4-dinitrobenzamide (CB 1954), which has previously been shown to elevate the level of adenosine 3′,5′-monophosphate (cyclic AMP) in sensitive Walker cells, has been shown to have no effect on the activity of adenylate cyclase either in the presence or absence of the protecting agent 4-amino-2-phenylimidazole-5-carboxamide (2-phenyl-AlC). Chlorambucil ( p- N, N(di-2-chloroethylamino)phenylbutyric acid) (5 μg/ml) while having no effect on either the basal or fluoride-stimulated adenylate cyclase activity caused an inhibition of the high affinity form of the cyclic AMP phosphodiesterase which reached a maximum after 1 hr. This was accompanied by an increase in the intracellular level of cAMP which was proportional to the dose of chlorambucil up to a maximal 2-fold increase at 6.4 μg/ml, a dose which caused complete inhibition of cell growth. Further increases in the concentration of chlorambucil up to 100 μg/ml caused no further increase in cAMP level. Merophan ( dl- o -N,N( di-2- chloroethylamino) phenylalanine ) (0.5 μg/ml) similarly caused an inhibition of the low K m form of the phosphodiesterase, but the rate of inhibition was slower than that observed with chlorambucil. The molecular forms of the cAMP phosphodiesterase in Walker cells sensitive or resistant to chlorambucil have been resolved using Sepharose 6B gel chromatography. The resistant lines displayed a reduction in the specific activity of the high affinity form of the enzyme which was accompanied by a shift to lower molecular weight forms. This could explain the lack of effect of chlorambucil on cAMP levels in Walker cells with acquired resistance to this agent.

Activation by GTP of liver adenylate cyclase in the presence of high concentrations of ATP

Effect of GTP on adenylate cyclase of liver plasma membrane was examined using ATP which was extensively purified by DEAE-cellulose column chromatography. In the incubation containing 2mM purified ATP as substrate, GTP enhanced basal and glucagon- or fluoride-stimulated activities. When the unpurified ATP at 2mM was used, all the activities were high and the stimulatory effect of GTP was not detected. The substance(s) which was recovered from a small but significant peak on DEAE-cellulose column was equivalent to 10–100μM GTP in stimulating adenylate cyclase. These results indicate that, if highly purified ATP is used as substrate, GTP can enhance adenylate cyclase activity in the presence of millimolar concentration of ATP and that GTP enhances not only the glucagon-stimulated adenylate cyclase but also the basal as well as fluoride-stimulated adenylate cyclase activities.

Effect of Chilling on Platelet Cyclic Adenosine 3:5‐Monophosphate and Adenylate Cyclase Activity

Exposure of platelets to 1 C led to a transient increase in cyclic AMP levels (determined either by a protein binding method or by radioimmunoassay) within five to ten minutes reaching a maximum 10 to 15 minutes after chilling was begun and returning subsequently to baseline values. Addition of EDTA to the platelet suspension medium prevented this increase. Rewarming at 37 C produced a sudden reduction in platelet cyclic AMP. To determine whether the cold-induced increase in cyclic AMP was due to a transient stimulation of platelet adenylate cyclase or a rapid inhibition of phosphodiesterase, these enzymes were assayed in ruptured platelet suspensions. Platelet adenylate cyclase activity was found to possess certain characteristics similar to those of the enzyme derived from other sources but there was a marked potentiation of fluoride-stimulated adenylate cyclase activity by 0.001 M EDTA. This effect was limited to low EDTA concentrations. Exposure of platelets to 1 C for up to 60 minutes did not increase adenylate cyclase activity but lowered it substantially compared with controls kept at room temperature. Phosphodiesterase activity at 1 C was depressed sooner and to a greater extent than was adenylate cyclase. The transient rise in cyclic AMP levels in chilled platelets appears to be due to a disproportionate reduction of cyclic nucleotide phosphodiesterase activity.

Stimulation of rat liver adenylate cyclase by halothane

Rat liver membranes prepared by a modification of the procedure of Neville were exposed to clinical and toxic concentrations of the general anesthetic, halothane, for 10 minutes. Basal, glucagon (5 × 10 −5M) and sodium fluoride (20 mM) stimulated adenylate cyclase activity was assayed. Clinical and toxic concentrations of halothane augmented basal adenylate cyclase activity. Glucagon and sodium fluoride stimulated adenylate cyclase activity was enhanced at greater than clinically useful halothane concentrations only. The study provides direct evidence that halothane stimulates adenylate cyclase, the extent of augmentation of enzyme activity is halothane concentration dependent, and modified by other drugs.

Liver membrane adenylate cyclase. Synergistic effects of anions on fluoride, glucagon, and guanyl nucleotide stimulation

Some effects of salts on the adenylate cyclase of partially purified plasma membranes from rat liver have been studied. Under conditions where cyclic adenosine 3':5'-monophosphate formation was linear with respect to time and protein concentration, the enzyme was stimulated 3- to 6-fold by 10 mM NaF, 10- to 30-fold by 1 muM glucagon, 4- to 5-fold by 0.1 mM 5'-guanylylimidodiphosphate, and in the presence of 3 muM GTP, 2-fold by 10 mug/ml of prostaglandin E1. Various salts were found to stimulate basal activity slightly, but enhanced the response to NaF 3- to 4-fold, to glucagon 1.5- to 2-fold, to 5'-guanylylimidodiphosphate 2- to 3-fold, and to prostaglandin E1 1.5-fold. This enhancement was observed at maximally effective concentrations of each of the respective activators. Of the salts tested, NaN3 and the Na- or K-halides were most effective. Their action appeared to be due to the respective anions. Stimulation was detectable with 1.5 mM NaN3 or 3 mM NaCl and was maximal with 30 mM NaN3 or 60 mM NaCl. The stimulatory effect of NaN3 was not due to ATP-sparing, nor to an altered cyclic adenosine 3':5'-monophosphate recovery. It was independent of the chromatography and assay methods used, and was therefore not due to procedural artifact. Fluoride-stimulated cyclase activity was enhanced by salts to a greater degree than were 5'-guanylylimidodiphosphate-, glucagon-, or (prostaglandin E1 + GTP)-stimulated activities. The effects of NaN3 were not the result of significant changes in the enzyme's responses to GTP, which increased basal and glucagon-stimulated activities but inhibited F--stimulated activity. The effects of NaN3 were greater when cyclase was assayed with Mn2+ than with Mg2+. The facilitatory effect of NaN3 or NaCl on fluoride-stimulated adenylate cyclase activity was partially reversible as was the stimulatory effect of fluoride in the presence of NaN3. Enhancement of hormonal stimulation by NaN3 was also demonstrable with cardiac and adipose tissue adenylate cyclase. However, NaN3 did not stimulate detergent-dispersed adenylate cyclases from either liver plasma membranes or brain. The data suggest that stimulation of adenylate cyclase by salts may require the added presence of other stimulatory agents and an intact membrane structure.

Alterations in adenyl cyclase activity and glucose utilization of Bordetella pertussis-sensitized mouse spleen

The effects of Bordetella pertussis sensitization on the cyclic AMP content, adenyl cyclase activity and glucose uptake and utilization of mouse spleen were examined. Injection of B. pertussis vaccine into mice resulted in a decrease (36 per cent) in cyclic AMP content of the spleens 4 days after vaccination, while decrease in blood glucose (53 per cent) occurred within 6 hr. Experiments in vitro using membrane fractions of spleens have shown that epinephrine, norepinephrine, isoproterenol and sodium fluoride stimulated adenyl cyclase activity to a much less extent in those derived from pertussis-sensitized mice than in those from nonsensitized mice. When nonsensitized mice were injected with propranolol (5–40 mg/kg), a beta-adrenergic blocking agent, cyclic AMP content decreased (58 per cent) to a level equivalent to that obtained by pertussis sensitization. Whereas epinephrine and aminophylline enhanced spleen cyclic AMP content (62 per cent) and blood glucose levels (115 per cent), pretreatment with propranolol blocked this enhancement. These observations are consistent with the hypothesis that adenyl cyclase activity in the spleen is a function of a beta-adrenergic receptor which is subject to blockade by pertussis sensitization. Uptake and utilization of glucose is markedly enhanced in spleen slices of pertussis-sensitized animals compared with that in nonsensitized, reflecting a probable generalized increase in peripheral uptake which may, in part, explain the hypoglycemia and adjuvancy observed in sensitization.

Effects of glucagon on cyclic AMP and carbohydrate metabolism in livers from diabetic rats

Cyclic AMP metabolism in normal and diabetic livers was studied using the isolated perfused rat liver and plasma membrane and supernatat fractions from rat liver. Liver from alloxan- or streptozotocin-diabetic rats had increased tissue levels of cyclic AMP and showed increased release of cyclic AMP during perfusion. Addition of glucagon or cyclic AMP to the medium produced little or no increase in the high rates of glucose production and lactate gluconeogenesis in diabetic livers. Low concentrations of glucagon (2·10 −10 M or less) did not increase tissue accumulation or release of cyclic AMP in livers from diabetic rats but were effective in normal livers. Higher concentrations of the hormone produced normal responses in diabetic livers. Basal or fluoride-stimulated adenylate cyclase activity in plasma membranes isolated from diabetic livers was not altered, but the enzyme was subnormally normally responsive to concentrations of glucagon within the range 10 −10−10 −8 M. Insulin (10 −11−10 −6 M) added in vitro was without effect on basal or glucagon-stimulated plasma membrane adenylate cyclase. Neither modulation of the Mg 2+ concentration, nor addition of Ca 2+, GTP, theophylline or ouabain caused the emergence of an insulin effect. Rat liver plasma membranes contained phosphodiesterase activity with two apparent K m values of about 0.5 and 70 μM. The activity of the low K m enzyme was decreased in plasma membranes from diabetic rats and was increased by insulin treatment of the rats in vivo. Both low and high K m activities were also decreased in supernatant fractions from livers of diabetic rats. Insulin at 10 −9 or 10 −7 M concentration had no effect in vitro on plasma membrane phosphodiesterase activities.

Influence of pyrophosphate and diphosphonates on rat liver adenylate cyclase.

SUMMARY 1. Inorganic pyrophosphate (PPi) and diphosphonates inhibit glucagon-stimulated and fluoride-stimulated adenylate cyclase activity of rat liver. 2. Concentrations of diphosphonates required to produce inhibition are lower than those of PPi, and PPi inhibited fluoride-stimulated activity to a greater extent than glucagon-stimulated activity. 3. Diphosphonates have inhibitory activity in the presence and absence of an ATP regenerating system in the adenylate cyclase assay, and do not substantially affect the efficiency of the ATP regenerating system. No evidence was obtained that reversal of adenylate cyclase was responsible for PPi inhibition. 4. Fluoride virtually completely inhibits pyrophosphatase activity in crude membrane preparations from rat liver, whereas glucagon has no detectable effect.

The metabolism of subcutaneous adipose tissue in the immediate postnatal period of human neonates. 3. Role of fetal glycogen in lipolysis and fatty acid esterification in the first hours of life.

Extract: Total glycogen phosphorylase activity in neonatal adipose tissue is significantly increased immediately after birth (0–6 hr) and rapidly decreases to a relatively constant level. Fluoride-stimulated adenyl cyclase activity in homogenates of adipose tissue from neonates is directly correlated with the length of time from the onset of labor (y = 0.665 + 7.88x; P < 0.02). No significant differences are found between infants 1–10 hr of age and 2–4 days of age. The ATP content decreases rapidly in the first hours of life and is inversely correlated with the length of time since the onset of labor (log y = −0.657 - 0.0164x; P < 0.01). The ATP content starts to increase approximately 24 hr after birth. Changes in ATP content are directly correlated with glycogen content and glycerol release from neonatal adipose tissue.Speculation: The intensity of lipolysis in neonatal adipose tissue depends on various factors, one of the most important being an adequate energy supply. The increased glycogen content in adipose tissue immediately after birth and the rapid rate of utilization of glycogen-derived glucose appear to be major factors involved in the production of ATP required for glycogenolysis, lipolysis, and fatty acid activation during the early adaptation of the neonate to extrauterine life.

Demasquage presumé des récepteurs de la noradrénaline par enrichissement en acide linoléique des phospholipides membranaires de la cellule adipeuse

Membrane phospholipid composition influences the noradrenaline reactivity of adipose cell ghosts in vitro. Basal and fluoride-stimulated adenylate cyclase activities and the phosphodiesterase activity were unchanged whether or not structural phospholipids were enriched in linoleic acid. However, the adenylate cyclase activation increased with increased proportion of essential fatty acids, under in vitro hormonal stimulation.

Effects of pyrophosphate and diphosphonates on parathyroid hormone- and fluoride-stimulated adenylate cyclase activity

1. Introduction Parathyroid hormone (PTH) promotes adenylate cyclase activity in renal cortex [ 1,2] leading to an increased concentration of cyclic adenosine 3’S’-mono- phosphate (CAMP) in renal parenchymal cells [3,4]. This cyclic nucleotide is proposed as an intermediate in the action of PTH

Alterations in lipolysis, adenylate cyclase and adenosine 3', 5'-monophosphate levels in isolated fat cells following adrenalectomy.

Isolated fat cells prepared from the epididymal fat pads of adrenalectomized rats exhibited a reduced lipolytic response to epinephrine. Reduced responsiveness was first observed at 3 hr and was maximal at 24 hr following surgery. Plasma levels of corticosterone had fallen to near zero by 3 hr. The lipolytic responses to ACTH, dibutyryl cyclic AMP and theophylline were also reduced. Maximum lipolytic rates produced by a combination of epinephrine and theophylline were not altered. Loss of lipolytic response to epinephrine could be prevented by subcutaneous injection of hydrocortisone. Basal, epinephrinestimulated, and sodium fluoride—stimulated adenylate cyclase activity in fat cell homogenates were not altered by adrenalectomy; however, the response to ACTH was nearly abolished. Elevations in intracellular levels of cyclic AMP by ACTH were greatly reduced by adrenalectomy while the response to epinephrine was reduced to a lesser extent. With both hormones the effect of adrenalectomy was greater when the ...

Studies on plasma membranes XIV. Adenyl cyclase in plasma membranes isolated from rat and mouse livers and hepatomas, and its hormone sensitivity

1. 1. For a proper assay of the adenyl cyclase of isolated plasma membranes using [8- 14C]ATP as substrate complete separation of the cyclic adenosine 3′,5′-monophosphate (cyclic AMP) from labeled adenine, an ATP degradation product, is essential. Otherwise erroneously high basal activity and low hormone stimulation are recorded. This may explain certain discrepancies found in the literature on this liver enzyme activity. 2. 2. Rat-liver plasma membranes formed 2.3 ± 1.0 nmoles cyclic AMP from ATP per mg membrane protein per h. This activity was some 20-fold increased by 10 −5 M glucagon, 5-fold by 10 −4 M epinephrine and 10-fold by 10 −2 M fluoride under conditions in which ATP was not a limiting factor. 3. 3. The basal adenyl cyclase activity displayed by the plasma membranes isolated from the rather anaplastic rat hepatoma-484A was less than half that of the liver membranes, the relative stimulation by fluoride was about the same, but that of glucagon was lower than in the case of liver membranes, whereas epinephrine was virtually without effect. By contrast, plasma membranes isolated from mouse liver and the well differentiated mouse hepatoma-147042 did not differ much in basal, hormone- and fluoride-stimulated adenyl cyclase activity. 4. 4. The significance of the adenyl cyclase system for growth control and neoplasia is briefly discussed.