Adenylate Cyclase In Plasma Membranes Research Articles

The role of a guanine nucleotide-binding protein in the activation of rat liver plasma-membrane adenylate cyclase by forskolin.

The effects of the photoreactive GTP analogue GTP-gamma-azidoanilide on rat liver plasma-membrane adenylate cyclase are described. U.v. irradiation in the presence of the analogue abolished activation by any effector or combination of effectors that function via the activatory G protein. Partial protection against this inhibition was given by F- and guanosine 5'-[gamma-thio]triphosphate. It is concluded that GTP-gamma-azidoanilide acts by a light-induced covalent reaction with the G protein. In the dark the effects of the analogue were similar to those of GTP. Irradiation in the presence of GTP-gamma-azidoanilide was found to reduce but not to abolish activation of rat liver plasma membrane adenylate cyclase by forskolin. The activation by forskolin and GTP together were greater than the sum of the individual activations. Forskolin doubled adenylate cyclase activity in the presence of glucagon and guanosine 5'-[beta, gamma-imido]triphosphate, which might be expected to activate to the maximum possible extent via the G protein. It is concluded that there are two components to the forskolin activation, a guanine nucleotide-dependent and a guanine nucleotide-independent component.

Protamine inhibits adenylate cyclase activity: a possible reason for the toxicity of protamine.

Protamine is an effective inhibitor of the various activated forms of adenylate cyclase of liver plasma membranes. Inhibition of adenylate cyclase may account for its toxic but not its antitumor effects.

Decrease in adenylate cyclase activity in dog livers during endotoxic shock.

The effects of endotoxin administration on adenylate cyclase in dog liver plasma membranes were studied. The basal, fluoride-, guanine nucleotide-, isoproterenol-, and glucagon-stimulated adenylate cyclase activities were decreased by 61, 62, 69, 83, and 63%, respectively, 2 h after in vivo administration of endotoxin. Endotoxin (100 micrograms/ml) in vitro decreased the guanine nucleotide-, isoproterenol-, and glucagon-stimulated adenylate cyclase activities by 24, 25, and 21%, respectively. These data demonstrate that endotoxin administered in vivo or in vitro had an inhibitory effect on the adenylate cyclase enzyme system in dog liver plasma membranes. Because of the involvement of the adenylate cyclase-adenosine 3',5'-cyclic monophosphate (cAMP) system in the regulation of hepatic carbohydrate metabolism, the finding that endotoxin administration decreased adenylate cyclase activity in the liver should contribute to the understanding of the pathophysiology of altered hepatic glucose homeostasis during shock.

Changes in renal phospholipid fatty acids in diabetes mellitus: correlation with changes in adenylate cyclase activity.

Male Sprague-Dawley rats made diabetic with alloxan (37.5 mg/kg) or streptozotocin (65 mg/kg) were killed after 3-6 weeks of disease; renal tissues were studied for phospholipid content and for fatty acid composition of the phospholipids. No consistent change was noted in total phospholipid content nor in the proportion of various phospholipids in diabetics. However, diabetic animals showed a consistent reduction of arachidonic acid content in phosphatidylcholine (PC) and phosphatidylethanolamine in whole renal cortex, plasma membranes purified from renal cortex, and in isolated glomeruli. Associated with the fall in arachidonic acid was a rise in linoleic acid in the samples studied. Insulin therapy returned the fatty acid profiles to normal. These results are similar to patterns observed in other diabetic tissues and suggest that diabetes is associated with generalized changes in cell membranes. That these structural changes may have functional significance is suggested by demonstrated alterations in the temperature-dependence of adenylate cyclase in renal plasma membranes of diabetic animals. Adenylate cyclase is thought to be intimately associated with PC in plasma membranes, a phospholipid showing significant changes in fatty acid content in diabetes (unsaturation index 165 +/- 2 for normals, 147 +/- 5 for diabetics). Na+,K+-ATPase which is thought to be primarily associated in vivo with phosphatidylinositol (PI), shows no change in apparent energy of activation in diabetes. The fatty acid content of PI is minimally altered in diabetes, and the unsaturation index is unchanged.

Renal cortical adenylate cyclase: characterization of magnesium activation.

The kinetic characteristics of Mg2+ and substrate (Mg-ATP) utilization by renal cortical plasma membrane adenylate cyclase were investigated in the presence or absence of PTH and with either guanosine 5′-triphosphate (GTP) or guanyl-5′-yl-imidodiphosphate [Gpp(NH)p] included in the reaction mixture. Basal- and PTH-stimulated adenylate cyclase activity was found to be dependent on Mg2+. Gpp(NH)p, but not GTP, resulted in hysteretic activation of renal cortical adenylate clase which was abolished with PTH activation. Gpp(NH)p augmented basal adenylate cyclase activity approximately 5-fold greater than GTP. PTH caused a 2-fold increase in activity over basal in the presence of 0.01 mm Gpp(NH)p but a 10-fold increase was seen in the presence of 0.01 mm and 0.10 mm GTP. Maximum velocity (Vmax) under basal conditions and with PTH activation was similar regardless of which guanine nucleotide was included in the reaction mixture. Linear regression analysis of cAMP accumulation with varying Mg2+ concentrations (0....

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.

The biological activity of glucagon-phospholipid complexes.

Glucagon can form water-soluble complexes with phospholipids. The incorporation of glucagon into these lipoprotein particles reduces the biological activity of the hormone. The effect is observed only at temperatures below the phase transition temperature of the phospholipid and results in a decreased stimulation of the adenylate cyclase of rat liver plasma membranes by the lipoprotein complex as compared with the hormone in free solution. Two- to five-fold higher concentrations of glucagon are required for half-maximal stimulation of adenylate cyclase when the hormone is complexed with dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, or bovine brain sphingomyelin. A possible role of lipoprotein-associated hormones in the development of insulin resistance is discussed.

Adenylate cyclase is inhibited upon depletion of plasma-membrane cholesterol.

A procedure has been developed that allows for the depletion of rat liver plasma membrane cholesterol by incubation with liposomes at 4 degrees C. Upon cholesterol depletion, adenylate cyclase activity was inhibited and the membranes became more rigid, as determined by the flexibility of an incorporated fatty acid spin probe. Decreasing the cholesterol/phospholipid molar ratio elicited a pronounced drop in the net fold-stimulation of adenylate cyclase activity by glucagon. Two lipid phase separations were detected in cholesterol-depleted membranes at around 25 degrees C and 13 degrees C respectively. Breaks at these temperatures were observed in Arrhenius plots of both the mobility of the spin probe and the glucagon-stimulated adenylate cyclase activity for the range 2-40 degrees C, but only the one at the lower temperature for the fluoride-stimulated activity. It is proposed that the lipid phase separation occurring at 25 degrees C is localized in the external half of the bilayer, whereas that at 13 degrees C is due to lipids in the inner half of the bilayer. Similar structural and functional perturbations were manifest if the cholesterol-complexing polyene antibiotic amphotericin B was added to native membranes. The mechanism of adenylate cyclase inhibition achieved by cholesterol depletion and the domain structure of the plasma membrane in relation to cholesterol distribution are discussed. Native cholesterol/phospholipid ratios appear to optimize the functioning of adenylate cyclase in liver plasma membranes.

Circulating bovine lymphocytes contain receptors for parathyroid hormone.

No cell type practicably obtainable in vivo, such as blood cells, is known to contain parathyroid hormone (PTH) receptors; this deficiency has hampered investigation of receptor regulation. Second, PTH in vivo is among the potent stimulators of osteoclastic activity, although no direct hormonal effects on these cells have been identified. Several lines of evidence suggest that cells of the immune system may mediate PTH effects on osteoclasts. We, therefore, studied bovine blood cells for the presence of PTH receptors and PTH-stimulated adenylate cyclase. Using an analogue of bovine PTH, (125)I-labeled [Nle(8),Nle(18),Tyr(34)]bPTH-(1--34)amide, we found PTH-specific binding to intact, nonadherent mononuclear cells (lymphocytes) and PTH-stimulated adenylate cyclase in plasma membranes prepared from these cells, and not with cells or membranes from other blood cells. Lymphocytes may serve to study the effects of physiologic and pathologic perturbations on PTH-receptor function in vivo. Exploration of PTH-related lymphocyte responses may help define the relation between cells of the immune system and osteoclastic bone resorption.

Conformational and biological properties of di[delta-(5-nitro-2-pyrimidyl)ornithine 17,18]glucagon. Role of the arginine residues.

The reaction of nitromalondialdehyde with the arginine residues of glucagon results in the conversion of the 2 arginine residues in the peptide to delta-(5-nitro-2-pyrimidyl)ornithine to form di[delta-(5-nitro-2-pyrimidyl)ornithine 17,18]glucagon (NP-glucagon). The modified peptide does not exhibit any loss in ability to activate adenylate cyclase of rat liver plasma membranes or to stimulate glycogenolysis in cortisone-primed rabbits relative to the native hormone despite this marked alteration in structure. The CD of dilute solutions of NP-glucagon is similar to that of the native hormone. In the absence of salt, the CD of NP-glucagon is independent of peptide concentration, but structures of higher helical content are observed in concentrated peptide solutions in the presence of 0.1 M NaCl and in methanol. The extent of helix formation under these conditions is greater than that given by glucagon. Results from viscosity and proton magnetic resonance spectra confirm and extend previous studies to indicate that this fully active derivative is in a compact folded conformation.

Guanine nucleotide mediated desensitization of adenylate cyclase in cell free preparations from a Leydig cell tumour.

Cell free desensitization of a tumour Leydig cell plasma membrane adenylate cyclase has been demonstrated in the presence of guanine nucleotides. In experiments in which the membranes were pre-incubated with various nucleotides and LH, it was shown that this decreased adenylate cyclase activity was dependent on the presence of GTP and occurred both in the presence and absence of ATP. While pre-treatment with LH alone appeared to enhance subsequent adenylate cyclase activity, this hormone was able to potentiate the desensitizing effect of GTP. The desensitizing effect of GTP was not inhibited by sodium fluoride. In contrast, the GTP analogue p(NH)ppG (guanosine 5'beta, gamma-imido triphosphate) caused a persistent activation of the adenylate cyclase. GMP and guanosine also initially inhibited the adenylate cyclase activity, but this was entirely reversed by p(NH)ppG plus LH. GDP in addition to GTP caused desensitization but this was only partially reversed by p(NH)ppG plus LH. It is proposed that in similarity with the ovary (Bockaert et al. 1976; Ezra & Salomon 1982a) desensitization of Leydig tumour cell plasma membrane adenylate cyclase may involve a GTP-mediated phosphorylation step.

Differential acid stabilities of citraconylated amino groups of glucagon Preparation of Nα-Citraconyl glucagon and evaluation of its biological properties

Acylation of the α- and ϵ-amino groups of histidine-1 and lysine-12 in glucagon with citraconic anhydride resulted in the formation of amide bonds which displayed different stabilities to hydrolysis under mild acid conditions. Treatment of N α, ϵ -dicitraconyl glucagon at pH 4.0 and room temperature regenerated the free ϵ-amino group within 16 h, while the citraconyl-α-amino group was stable. N α -Citraconyl glucagon was purified by anion-exchange chromatography and was a weak partial agonist in stimulating adenylate cyclase in rat liver plasma membranes. The derivative exhibited 1% of the biological potency and 35–40% of the maximal stimulation of glucagon. Binding affinity to plasma membranes was also reduced, but not to as great an extent as adenylate cyclase activity. Removal of the α-citraconyl group by treatment with 10 mM HCl at 40°C restored full potency and stimulation to glucagon. These results suggest that the N-terminal histidine of glucagon is involved in both binding to plasma membranes and transduction of the signal to adenylate cyclase.

Multiple expressions of the activity of guanine nucleotide regulatory protein in human thyroid adenylate cyclase

Adenylate cyclase (ATP pyrophosphate-lyase, EC 4.6.1.1) in plasma membranes from human thyroid was highly responsive to thyrotropin. Pretreatment of thyroid plasma membranes with 5′-guanylylimidodiphosphate (Gpp(NH)p) in the presence of Mg 2+ led to a temperature-dependent activation, which was seen neither in the absence of Mg 2+ nor at 4 °C. By contrast, thyrotropin bound to its receptors regardless of the temperature and produced its maximal effect after 2 min of preincubation in the absence or presence of Mg 2+. Furthermore, activation was seen after treatment with thyrotropin and Gpp(NH)p even carried out in the absence of Mg 2+ or at 4 °C. However, the full activation by Gpp(NH)p required Mg 2+, hormone, and elevated temperature. These observations suggest that there appears to be two types of nucleotide interaction responsible for the Gpp(NH)p activation in human thyroid membrane; one type seen in the absence of hormone may represent the system uncoupled from hormone receptor, while the fully coupled hormone-sensitive adenylate cyclase accounts for the second type of interaction which requires the presence of hormone.

Mechanism of action of ATP on intestinal epithelial cells: Cyclic AMP-mediated stimulation of active ion transport

ATP, ADP and AMP but not adenosine increased cyclic AMP in dispersed enterocytes prepared from guinea pig small intestine. This action of ATP was augmented by IBMX and was reproduced by App(NH)p or App(CH 2)p. ATP also increased the formation of cyclic [ 14C]AMP in enterocytes that had been preincubated with [ 14C]adenine. Gpp(NH)p and NaF each caused persistent activation of adenylate cyclase in plasma membranes from enterocytes and ATP caused significant augmentation of this persistent activation. In addition to increasing cellular cyclic AMP and agumenting Gpp(NH)p and NaF-stimulated persistent activation of adenylate cyclase, ATP increased the I sc across mounted strips of small intestine and inhibited net absorption of fluid and electrolytes in segments of everted small intestine. These results indicate that intestinal epithelial cells possess a receptor that interacts with ATP and other adenine nucleotides and that receptor occupation by ATP causes activation of adenylate cyclase, increased cyclic AMP and changes in active ion transport across intestinal mucosa.

Inhibition of renal adenylate cyclase by plasma from uremic patients.

The native plasma of patients suffering from renal insufficiency inhibited the parathyroid hormone (PTH), the vasopressin (VP) and the NaF-activated adenylate cyclase (AC) in bovine renal plasma membranes. A crude immunoglobulin fraction, prepared by (NH4)2SO4 precipitation, decreased only the hormone, but not the NaF activated AC. Chromatography of the native plasma on a AcA 34 column revealed two peaks inhibiting the PTH and to a lesser extent the VP dependent AC. Only the second peak reduced the NaF stimulated cAMP production. None of the column samples inhibited the isoproterenol stimulated adenylate cyclase in turkey red blood cells. Further purification of the first peak on a Sepharose 6B column confirmed previous results that immunoglobulins may block the PTH receptor in the kidney. Repurification of the second peak of inhibition on a Bio-Gel P10 column suggested that small peptides crossreacting in the radioimmunoassay for PTH may desensitize the hormonal receptor.

Adenylate Cyclase in Rat Synaptosomal Plasma Membranes and Caudate Nucleus Homogenate: Effects of Dopamine, E Prostaglandins, Guanyl‐5′‐yl‐imidodiphosphate and Morphine

The adenylate cyclase in two particulate preparations from rat brain, a homogenate from caudate nucleus (CN-homogenate) and a synaptosomal plasma membrane fraction (SPM-fraction) from whole rat brain was investigated. Stimulation of the enzyme by dopamine and prostaglandins E1 and E2 was found in the CN-homogenate while only a weak prostaglandin E1 and E2 stimulation and no dopamine stimulation could be found in the SPM-fraction. Guanyl-5'-yl-imidophosphate (GppNHp) and NaF could stimulate the adenylate cyclase in both preparations. Morphine up to 10(-5) M altered neither the basal enzyme activity nor any of the stimulated enzyme activities.

Increased dose-response relationship of liver plasma membrane adenylate cyclase to glucagon stimulation in diabetic rats. A possible role of the guanyl nucleotide-binding regulatory protein.

In view of controversial findings regarding the mechanism for the increased intracellular hepatic cyclic 3':5' adenosine monophosphate levels in diabetic rats, we studied the dose-response relationship of the adenylate cyclase to glucagon stimulation in severely diabetic and in diabetic, insulin-treated rats. An enhanced response to glucagon and an additional augmenting effect of guanosine triphosphate on hormonal stimulation of the adenylate cyclase activity were found in diabetes which were reversible with insulin treatment. The results suggest a role of the regulatory guanyl nucleotide-binding protein in diabetes leading to an increased dose response relationship of the hepatic adenylate cyclase system to glucagon.

Photoaffinity labeling of the canine renal receptor for parathyroid hormone.

Studies were undertaken to identify and characterize components of the parathyroid hormone receptor. An analog of the bovine parathyroid hormone(1-34) sequence was derivatized at the 23-tryptophan position with the photoreactive reagent 2-nitro-5-azidophenylsulfenyl chloride. 2-Nitro-5-azidophenylsulfenyl-bovine parathyroid hormone (NAPS-bPTH) analog retained full biological activity with respect to receptor binding and activation of adenylate cyclase in canine renal cortical plasma membranes. 125I-bPTH(1-34) analog was also derivatized without loss of receptor-binding activity. When 125I-NAPS-bPTH(1-34) analog was incubated with canine renal plasma membranes and then photolyzed, at least two 125I-labeled membrane components were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The incorporation of 125I radioactivity into one of these components (Mr congruent to 60,000) was inhibited when incubation and photolysis were performed in the presence of excess, unlabeled bPTH(1-34). Furthermore, photolysis of membranes in the presence of NAPS-bPTH(1-34) analog led to activation of adenylate cyclase which persisted following washing to remove noncovalently bound peptide, suggesting a functional, covalent hormone-receptor complex had been formed. We conclude that the M r congruent to 60,000 membrane component may be a part of the renal receptor for parathyroid hormone.

Opiate receptor-mediated inhibition of adenylate cyclase in rat striatal plasma membranes.

Plasma membranes from rat striatum contain adenylate cyclase activity that is subject to dual regulation by GTP. Low concentrations (up to 30 nM) of the nucleotide increase activity whereas higher concentrations evoke a steady decline in activity; such behavior characterizes dually regulated adenylate cyclase systems. The opiates, morphine sulfate and D-Ala-Met-enkephalin, produce naloxone-reversible inhibition of the enzyme that is dependent on "inhibitory concentrations" of GTP (above 50 nM). In the absence of GTP no inhibition is observed. Sodium ions decrease the inhibition of activity promoted by GTP alone, but amplify the degree of inhibition seen in the presence of the opiates and GTP. The potencies of the opiates in mediating these effects mirror their affinities for delta opiate receptors in striatum. It is suggested that this action of the opiates may represent their primary action in striatum.

Developmental changes in the form of the Arrhenius plots of rat liver plasma membrane adenylate cyclase and 5′-nucleotidase activities

Summary Arrhenius plots of both adenylate cyclase and 5′nucleotidase activity in liver plasma membranes from rats 6 weeks of age, and older, exhibit a single well-defined break at around 28°C with an activation energy at temperatures above the break point that is lower than that found at temperatures below the break. In contrast, animals at 4 weeks of age, whilst exhibiting a similar break temperature, have activation energies for these enzymes which are higher at temperatures above the break than below.