Alterations in adenylate cyclase activity due to streptozotocin-induced diabetic cardiomyopathy

The effect of chronic experimental diabetes on the adenylate cyclase system (AC) in the rat heart was investigated. Rats were made diabetic by an intravenous injection of streptozotocin (65 mg/kg), hearts were removed 8 weeks later and washed cell particles were isolated. AC activity was measured in the absence and presence of different concentrations of forskolin, NaF, GTP analogue [Gpp(NH)p] or epinephrine. A significant depression in the epinephrine stimulated AC activity was observed in diabetic hearts. Basal AC activity and stimulation of AC with forskolin, NaF and Gpp(NH)p were not significantly different between control and diabetic preparations. These results indicate no apparent alterations in the regulatory or catalytic properties of AC in hearts from chronic diabetic rats. The observed depression in epinephrine stimulated AC activity may account for the depressed inotropic action of catecholamines in the diabetic cardiomyopathy.     

Possible mechanism of regulating adenylate cyclase activity in adipocyte membranes from exercise-trained male rats

(-)-Isoproterenol-stimulated adenylate cyclase activities were significantly greater in membranes from exercise-trained male rats than in sedentary male rats. GTP-inhibition of forskolin (10 microM)-stimulated cyclase activities were observed in sedentary membranes, whereas the inhibitory actions of GTP were significantly reduced in membranes from trained rat adipocytes. Treatment of membranes with islet-activating protein, a pertusis toxin, completely abolished the differences in GTP-inhibition of forskolin-stimulated cyclase activities between the two groups. The amounts of the inhibitory regulatory protein (41kDa/40kDa polypeptides) were about 40% less in membranes from trained rats than in sedentary membranes, whereas that of the stimulatory regulatory protein (a 45kDa polypeptide) was equivalent. It is concluded that the enhanced cyclase activities of adipocyte membranes from trained male rats appear to result from, in part, an attenuation of the inhibitory pathway due to a specific decrease in the amount of the inhibitory regulatory proteins.     

cAMP and adenylate cyclase activity in Streptomyces granaticolor

Abstract Intracellular and extracellular cyclic adenosine 3',5'-monophosphate (cAMP) levels were determined during the growth of Streptomyces granaticolor . The intracellular level of cAMP represents not more than 10% of the total amount. cAMP synthesis varies in cultures growing on different carbon sources. The activity of adenylate cyclase in intact cells is strictly dependent on the presence of a metabolizable carbon source.     

Altered expression and function of regulator of G-protein signaling-17 (RGS17) in hepatocellular carcinoma

Guanine nucleotide regulatory proteins (G-proteins) are central to normal hepatocyte function and are implicated in hepatic disease initiation and progression. Regulators of G-protein signaling (RGS) are critical to defining G-protein-dependent signal fidelity, yet the role of RGS proteins in the liver is poorly defined. The aims of this study were to determine RGS17 expression in normal and transformed hepatic tissue and cells, and address the function of RGS17 in hepatic tumorgenicity. RGS17 expression was determined in human and rat HCC tissue and cell lines. Molecular approaches were used to alter RGS17 expression in HCC cells, effects on cell function measured, and RGS17 association with specific Gα-subunits determined. Using these approaches RGS17 mRNA, but not protein, was detectable in human and rat HCC tissue and cells. Conversely, RGS17 mRNA was not detected in normal tissue, isolated hepatocytes, or non-tumorigenic hepatic cells. Subsequent studies using transfected cells demonstrated that RGS17 proteins were not post-translationally modified in HCC cells, and RGS17 expression is governed by protein degradation and not via miRNAs. Notwithstanding inherently low RGS17 protein levels, altering RGS17 expression profoundly affected HCC cell mitogenesis and migration. Analysis of RGS17-G-protein interaction demonstrated RGS17 associates with both Giα- and Gqα-subunits in HCC cells of human and rat origin. In conclusion, these data demonstrate that, despite difficulties in measuring endogenous RGS protein expression, RGS17 is differentially expressed in HCC and plays a central role in regulating transformed hepatocyte tumorgenicity.     

Isolation and expression of the Bradyrhizobium japonicum adenylate cyclase gene (cya) in Escherichia coli

A 5.0-kilobase-pair HindIII fragment of Bradyrhizobium japonicum DNA containing the cya gene which encodes adenylate cyclase was isolated as an insert in pBR322, using marker rescue of the maltose-negative phenotype of an Escherichia coli cya mutant for identification. The isolated B. japonicum DNA fragment was capable of reversing the pleiotropic phenotype of cya mutations when inserted in either orientation in the HindIII site of pBR322. The complemented E. coli strains produced high levels of cyclic AMP. No sequence homology between the B. japonicum cya gene and that of E. coli was detected by hybridization analysis.     

Stimulation of diamondback moth (Plutella xylostella) adenylate cyclase activity by a carbodiimide

1. DFCD or 3-(2,6-diisopropyl-4-phenoxyphenyl)-1-tert-butylcarbodiimide, a toxic metabolite of the thiourea acaricide/insecticide diafenthiuron, stimulated adenylate cyclase activity in preparations from heads of adult diamondback moths, Ptutella xylostella (L.).2. Depending upon assay conditions, DFCD gave a biphasic response with K_a, values of 0.025 and 1.25 μM for high and low affinity components, respectively, or a monophasic response with a K_a of 0.4 μM.3. Studies with potential agonists and antagonists suggested that octopamine-sensitive (K_a 7.5 μM) and dopamine-sensitive (K_a, 1.0μM) adenylate cyclases were present.4. At maximally effective concentrations, the activity of DFCD was nonadditive to that of octopamine or dopamine.5. It appeared that DFCD was binding to octopamine- and dopamine-sensitive adenylate cyclases and that affinity of the carbodiimide was higher for the former than for the latter.6. These actions on biogenic amine-sensitive adenylate cyclases likely are involved in the toxicity of diafenthiuron to diamondback moths.     

Mouse neuroblastoma adenylate cyclase. Adenosine and adenosine analogues as potent effectors of adenylate cyclase activity.

1. Intact mouse neuroblastoma NS20 cells, in the presence of cyclic adenosine 3':5'-monophosphate (cAMP) phosphodiesterase inhibitor, responded to adenosine (200 muM) and 2-chloroadenosine (200 muM) with a 20-fold increase in intracellular cAMP levels. AMP (200 muM) additions caused only a 3.5-fold cAMP level elevation. ATP, ADP, guanosine, cytidine, uridine, and guanine, all at 200 muM, had no effect on the cAMP level of these cells. 2. Homogenate NS20 adenylate cyclase activity was increased 2.5- to 4-fold by addition of 200 muM adenosine, 2-chloroadenosine, 2-hydroxyadenosine, or 8-methylaminoadenosine. Prostaglandin E1 additions (1.4 muM) produced about an 8-fold stimulation of homogenate cyclase activity. The Km of homogenate cyclase activation by adenosine and 2-chloroadenosine was 67.6 and 6.7 muM, respectively. Addition of 7-deazaadenosine, tolazoline, yohimbine, guanosine, cytosine, guanine, 2-deoxy-AMP, and adenine 9-beta-D-xylopyranoside, all at 200 muM were found to be without effect on homogenate NS20 adenylate cyclase. Two classes of inhibitors of homogenate NS20 adenylate cyclase activity were observed. One class, which included AMP, adenine, and theophylline, blocked 2-chloroadenosine but not prostaglandin E1 stimulation of cyclase. Theophylline was shown to be a competitive inhibitor of 2-chloroadenosine, with a Ki of 35 muM. The second class of inhibitors, which included 2'- and 5'-deoxyadenosine, inhibited unstimulated, 2-chloroadenosine and prostaglandin E1-stimulated homogenate cyclase activity to about the same degree. 3. Activation of NS20 homogenate adenylate cyclase by adenosine appears to be noncooperative. 4. The inhibitory action of putative "purinergic" neurotransmitters is postulated to be due to their effects on adenylate cyclase activity.     

Altered expression of G-protein mRNA in spontaneously hypertensive rats.

We have recently demonstrated that the decreased ability of hormones, forskolin and GTP to stimulate adenylate cyclase in heart and aorta from spontaneously hypertensive rats (SHR), as compared to their age-matched Wistar-Kyoto control rats (WKY), was associated with enhanced levels of Gi- and not with Gs-regulatory proteins. In the present studies we have investigated the expression of Gi-regulatory proteins at the mRNA level by Northern blotting. Total RNA of heart ventricle and aorta from WKY and SHR was probed with radiolabeled cDNA inserts encoding Gi alpha-2 and Gi alpha-3. The Gi alpha-2 and Gi alpha-3 probes detected a message of 2-3 and 3-5 kb, respectively, in both WKY and SHR, however, the message was significantly enhanced in SHR, as compared by WKY. On the other hand the cDNA probe encoding Gs alpha detected a message of 1.8 kb in heart and aorta from both WKY and SHR, however, no difference in the levels of Gs alpha mRNA was detected in SHR and WKY tissues. These results indicate that the mRNA levels of Gi alpha-2 and Gi alpha-3 and not of Gs are overexpressed in heart and aorta from SHR, which may be responsible for the increased levels of Gi as shown earlier by immunoblotting techniques. It may be suggested that the enhanced vascular tone and impaired cardiac contractility in hypertension may partly be the consequences of increased levels of Gi in heart and aorta.     

Elevated plasma levels of pancreastatin (PST) in patients with non-insulin-dependent diabetes mellitus (NIDDM)

Pancreastatin (PST) is known as the peptide which inhibits first phase of glucose-stimulated insulin secretion. Fasting plasma PST levels and responses of PST after oral glucose ingestion in patients with non-insulin-dependent diabetes mellitus (NIDDM) were studied with human PST-specific radioimmunoassay. Fasting plasma PST in NIDDM patients was not different from healthy controls, although a slightly higher level of PST was observed in patients treated with sulfonylurea among NIDDM patients. No significant increase in plasma PST was observed after a glucose ingestion in healthy controls. In contrast, plasma PST levels in NIDDM patients rose significantly after glucose ingestion. These results suggest a possible pathophysiological role for PST in NIDDM.     

Ultrastructural localization of adenylate cyclase activity in chicken osteoclasts

Using lead citrate as a capture reagent and adenylate-(beta, gamma-methylene) diphosphate (AMP-PCP) as a substrate, we localized adenylate cyclase activity on the non-ruffled border plasma membrane of approximately half of the osteoclasts on trabecular bone surfaces in the tibial metaphyses of chickens fed a low (0.3%)-calcium diet. The enzyme was not detectable in osteoclasts when chickens were fed a normal calcium diet. Activity was observed on the entire plasma membrane of detached osteoclasts that were situated between osteoblasts on the bone surface and blood vessels in the marrow cavity. Detection of activity on detached osteoclasts required the presence of an activator, implying lower levels in these cells than in those with ruffled borders. Staining was greater on the lateral sides of osteoblasts and osteoclasts when they were in contact with each other. Reaction specificity was indicated by the demonstration of stimulation by forskolin, guanylate-(beta, gamma-methylene) diphosphate (GMP-PCP), dimethylsulfoxide, and NaF, inhibition by alloxan and 2',5'-dideoxyadenosine, and absence of activity when sections were incubated in substrate-free medium or when GMP-PCP replaced AMP-PCP as a substrate. The finding of adenylate cyclase in osteoclast plasma membrane provides structural evidence that the adenylate cyclase-cyclic AMP system has a role in regulation of osteoclast cell function. The low-calcium diet appears to have resulted in increased amounts of adenylate cyclase in osteoclasts.     

Particulate and soluble adenylate cyclase activities of mouse male germ cells

Germ cells from the mouse testis possess both a particulate and a soluble form of adenylate cyclase (EC 4.6.1.1). Germ cell adenylate cyclase activity is Mn⁺⁺ dependent and is not stimulable with either NaF or 5′guanylylimidodiphosphate. Both particulate and soluble adenylate cyclase specific activities increase as germ cells progress through their differentiative stages, but epididymal spermatozoa seem to lack a significant amount of soluble activity. Somatic cells of the seminiferous tubule possess only a membrane bound activity, which is Mg⁺⁺ and Mn⁺⁺ dependent, NaF and 5′guanylylimidodiphosphate stimulable. It is suggested that germ cell adenylate cyclases represent incomplete forms of the enzyme, devoid of regulative subunits.     

Uncoupling by proteolysis of alpha-adrenergic receptor-mediated inhibition of adenylate cyclase in human platelets

Ethyl 2{5(4-chlorophenyl)pentyl}oxiran-2-carboxylate (POCA) is a new hypoglycaemic compound. The POCA-CoA ester strongly inhibits β-oxidation at carnitine palmitoyltransferase I. Chronic administration of POCA to rats decreases plasma concentrations of cholesterol and triacylglycerol and increases the number of hepatic peroxisomes similarly to hypolipidaemic drugs related to clofibrate. Peroxisomal fractions from rats fed a diet containing 0.2% of POCA for 4 weeks were prepared on self-generated Percoll gradients. POCA induced a 4-fold increase in catalase activity and peroxisomal β-oxidation, agreeing with the morphological data. The increase in peroxisomal β-oxidation caused by POCA feeding does not prevent accumulation of lipid following the inhibition of mitochondrial β-oxidation.     

Ultracytochemical localization of adenylate cyclase activity in rat thymocytes

Summary Ultrastructural localization of adenylate cyclase (AC) activity was investigated in suspensions of unfixed isolated rat thymocytes using a medium containing 0.6 mM 5-adenylylimidodiphosphate (AMP-PNP) as a substrate, 10 mM MgSO₄ as an activator, 5 mM theophylline as an inhibitor of 3,5-AMP-phosphodiesterase and 2 mM lead nitrate as a capturing agent. AC activity was demonstrated in plasma membrane, perinuclear space, endoplasmic reticulum, Golgi complex, centriole microtubules and mitochondria. AC was activated with 10^–4 M adrenalin in the presence of 5-guanylylimido-diphosphate (GMP-PNP) as well as with 10^–2 M NaF. In the cells incubated in a medium devoid of theophylline and containing 5-AMP instead of AMP-PNP, 5-nucleotidase activity was observed in the same cell structures as AC activity. Hydrolysis of 5-AMP in the nucleus was much stronger than that of AMP-PNP. 10 mM NaF markedly inhibited hydrolysis of 5-AMP in all cell structures. No staining was observed with 2 mM -glycerophosphate as a substrate. Incubation of unfixed thymocytes in media containing AMP-PNP, 5-AMP or p-nitrophenyl phosphate, but not -glycerophosphate, induced both in the nucleus and in the cytoplasm in some cells an appearance of a transitory reticular formation consisting of about 30 nm thick strands which could penetrate the nuclear envelope and plasma membrane and form connections with adjacent cells. The transitory reticular formation seems to belong to the cytoskeleton and to be involved in cell aggregation.     

Adenyl cyclase in human platelets: activity and responsiveness

A clinical study was conducted whereby the activity of adenyl cyclase in the human platelet was demonstrated. The study showed that this activity can be stimulated and inhibited in vitro. Platelets were isolated from normal donors. The laboratory procedures involved in the study are described in detail. It seems that many of the biologic processes which occur in the human platelet are dependent on the breakdown of ATP (adenosine-tri-phosphate) to, among other substances, AMP (adenosine-3',5' monophosphate). Activity of the adenyl cyclase was stimulated by fluoride, prostaglandin E1, and glucagon; it was inhibited by thrombin, epinephrine, norepinephrine, and serotonin. PG (prostaglandin) E1 at concentrations of 20 ng/ml and above increased adenyl cyclase in 7 experiments by 3-5 times. Even at concentrations as low as 2 ng/ml., PGE2 caused perceptable stimulation. The PGE, while stimulating adenyl cyclase activity, also inhibited aggregation of platelets by a variety of substances. Results of the study suggest that adenosine-3',5' monophosphate may be important in the regulation of platelet adhesiveness.     

Membrane fluidity and adenylate cyclase activity in genetically obese mice

Adipocyte plasma membranes of genetically obese $\text{ob}\text{ob}$ mice are more fluid than their lean littermates but the fluidity was normalised in mice maintained at high environmental temperatures. The defective response of adenylate cyclase to isoproterenol was improved after normalisation of membrane fluidity. No major changes in the phospholipid composition of $\text{ob}\text{ob}$ membranes were detected.