Adenylate Cyclase In Plasma Membranes Research Articles

CAMP imaging of cells treated with pertussis toxin, cholera toxin, and anthrax edema toxin

The enzymatic activity of the three most studied bacterial toxins that increase the cytosolic cAMP level: pertussis toxin (PT), cholera toxin (CT), and anthrax edema toxin (ET), was imaged by fluorescence videomicroscopy. Three different cell lines were transfected with a fluorescence resonance energy transfer biosensor based on the PKA regulatory and catalytic subunits fused to CFP and YFP, respectively. Real-time imaging of cells expressing this cAMP biosensor provided time and space resolved pictures of the toxins action. The time course of the PT-induced cAMP increase suggests that its active subunit enters the cytosol more rapidly than that deduced by biochemical experiments. ET generated cAMP concentration gradients decreasing from the nucleus to the cell periphery. On the contrary, CT, which acts on the plasma membrane adenylate cyclase, did not. The potential of imaging methods in studying the mode of entry and the intracellular action of bacterial toxins is discussed.

Differential expression of vasoactive intestinal peptide and its functional receptors in human osteoarthritic and rheumatoid synovial fibroblasts

Vasoactive intestinal peptide (VIP) has shown potent antiinflammatory effects in murine arthritis and ex vivo in human rheumatoid arthritis (RA) synovial cells. To investigate the potential endogenous participation of this system in the pathogenesis of RA, we analyzed the expression and regulation of VIP and its functional receptors in human fibroblast-like synoviocytes (FLS) from patients with osteoarthritis (OA) and patients with RA. The expression of VIP was studied by reverse transcription-polymerase chain reaction (RT-PCR), enzyme immunoassay, and immunofluorescence in cultured FLS, and by immunohistochemical analysis in synovial tissue. The expression and function of the potential VIP receptors in FLS were studied by RT-PCR, determination of intracellular cAMP production, cell membrane adenylate cyclase (AC) activity, and interleukin-6, CCL2, and CXCL8 production in response to VIP or specific agonists and antagonists. VIP expression was detected in human FLS at the messenger RNA and protein levels, and it was significantly decreased in RA FLS compared with OA FLS. VIP receptor type 1 (VPAC1) was the dominant AC-coupled receptor in OA FLS, in contrast with RA FLS, in which VPAC2 was dominant. Tumor necrosis factor alpha-treated OA FLS reproduced the VIP and VPAC receptor expression pattern of RA FLS. The antagonistic effects of VIP on FLS proinflammatory factor production were reproduced by VPAC1- and VPAC2-specific agonists in OA FLS and RA FLS, respectively. VIP expression is down-regulated in RA and in tumor necrosis factor alpha-treated FLS, suggesting that down-regulation of this endogenous antiinflammatory factor may contribute to the pathogenesis of RA. In RA FLS, VPAC2 mediates the antiinflammatory effects of VIP, suggesting that VPAC2 agonists may be an alternative to VIP as antiinflammatory agents.

Role of beta3-adrenergic receptors in the action of a tumour lipid mobilizing factor.

Induction of lipolysis in murine white adipocytes, and stimulation of adenylate cyclase in adipocyte plasma membranes, by a tumour-produced lipid mobilizing factor, was attenuated by low concentrations (10−7–10−5 M) of the specific β3-adrenoceptor antagonist SR59230A. Lipid mobilizing factor (250 nM) produced comparable increases in intracellular cyclic AMP in CHOK1 cells transfected with the human β3-adrenoceptor to that obtained with isoprenaline (1 nM). In both cases cyclic AMP production was attenuated by SR59230A confirming that the effect is mediated through a β3-adrenoceptor. A non-linear regression analysis of binding of lipid mobilizing factor to the β3-adrenoceptor showed a high affinity binding site with a Kd value 78±45 nM and a Bmax value (282±1 fmole mg protein−1) comparable with that of other β3-adrenoceptor agonists. These results suggest that lipid mobilizing factor induces lipolysis through binding to a β3-adrenoceptor.British Journal of Cancer (2002) 86, 424–428. DOI: 10.1038/sj/bjc/6600086 www.bjcancer.com© 2002 The Cancer Research Campaign

Role of the cyclic AMP-dependent pathway in free radical-induced cholesterol accumulation in vascular smooth muscle cells

We have previously reported that free radical-treated vascular smooth muscle cells (SMC) lead to cholesterol accumulation in vitro. In the current study, we investigated the effects of oxidative stress on cyclic AMP concentration and cAMP-dependent enzymes involved in cholesterol homeostasis in A7r5 cells. Under our conditions of a mild oxidative stress, namely with no change in cell viability, we found that free radicals, initiated using azobis-amidinopropane dihydrochloride (AAPH), resulted in a dose-dependent decrease in cellular cAMP which was opposed by vitamin E preincubation. Although the addition of adenylate cyclase activators (carbacyclin and forskolin) increased cAMP levels it did not succeed in restoring the AAPH-induced decrease. The oxidative stress-induced increase in activities of 3-hydroxy-3-methylglutaryl coenzyme A reductase and of acyl coenzyme A: cholesterol acyltransferase and the decrease in neutral cholesteryl ester hydrolase activity were suppressed by addition of dibutyryl cAMP. Taken together, these results strongly suggest that free radicals reduce cAMP concentrations by altering cell membrane adenylate cyclase activity. The changes of cAMP-dependent enzymes induced by oxidative stress resulting in cholesterol accumulation might be one of the processes leading to SMC-derived foam cells depicted in atheroma plaque. Moreover, if extrapolated to in vivo, these data may explain in part the beneficial effects of antioxidants in the reduction of cardiovascular diseases.

Alterations of G-protein and adenylate cyclase signaling in rat liver during the progression of sepsis.

Changes in the protein level of various subunits of G-protein and the activity of adenylate cyclase in rat liver plasma membranes during different metabolic phases of sepsis were studied. Sepsis was induced by cecal ligation and puncture (CLP). The results show that the protein levels of Galphai-2 and Galphai-3 were unchanged during the early hypermetabolic (hyperglycemic) phase (9 h after CLP), whereas Galphaai-2 and Galphaai-3 were increased by 32.4 and 59.1%, respectively, during the late hypometabolic (hypoglycemic) phase (18 h after CLP) of sepsis. The protein levels of Galphas and Gbeta remained unaltered during both the early and the late phases of sepsis. The activity of adenylate cyclase remained unchanged during the early phase, whereas it was decreased by 26% (p < .05) during the late phase of sepsis. Since the G-protein/adenylate cyclase signaling system mediates hormonal control of hepatic glucose metabolism, the observed increases in the Galphai-2 and Galphai-3 protein levels coupled with a decrease in the activity of adenylate cyclase may contribute to the development of the hypoglycemia during the late stage of sepsis.

Significance of adenylate cyclase system of the liver in its chronic diseases

The activity of adenylate cyclase in the hepatocyte plasma membranes, content of cAMP, and cAMP/cGMP ratio in the liver and blood plasma are decreased in patients with chronic liver diseases (fatty dystrophy, chronic hepatitis, and cirrhosis). This decrease depends on the disease severity and is most pronounced in cirrhosis. The sensitivity of liver adenylate cyclase to insulin and glucagon is changed. It is concluded that disorders in the adenylate cyclase system are an important molecular mechanism in the pathogenesis of chronic liver diseases.

Nitric Oxide Increases Tumor Necrosis Factor Production in Differentiated U937 Cells by Decreasing Cyclic AMP

Nitric oxide (NO) increases tumor necrosis factor (TNF) synthesis in human peripheral blood mononuclear cells by a cGMP-independent mechanism. NO has been shown to inhibit adenylate cyclase in cell membranes. Since cAMP down-regulates TNF transcription, we examined the possibility that NO enhances TNF synthesis by decreasing cAMP. U937 cells were induced to differentiate using phorbol myristate acetate (100 nM for 48 h) and then were incubated for 24 h with sodium nitroprusside (SNP) or S-nitroso-N-acetylpenicillamine (SNAP). These NO donors increased TNF production (7.0- and 15.6-fold, respectively, at 500 microM) in a dose-dependent manner (p = 0.002). However, SNP and SNAP did not elevate cGMP levels in U937 cell cultures, and the cGMP analog, 8-bromo-cGMP, had no effect on TNF production. In contrast, SNP (p = 0.001) and SNAP (p = 0.009) decreased intracellular cAMP levels by up to 51.5% over 24 h and, in the presence of a phosphodiesterase inhibitor, blunted isoproterenol-stimulated increases in cAMP by 21.8% (p = 0.004) and 27.6% (p = 0.008), respectively. H89, an inhibitor of cAMP-dependent protein kinase, dose dependently increased TNF production in phorbol myristate acetate-differentiated U937 cells in the absence (6.5-fold at 30 microM; p = 0.035), but not in the presence (p = 0.77) of SNAP. Conversely, the cAMP analog dibutyryl cAMP (Bt2cAMP) blocked SNAP-induced TNF production (p = 0.001). SNP and SNAP (500 microM) increased relative TNF mRNA levels by 57.5% (p = 0.045) and 66.2% (p = 0.001), respectively. This effect was prevented by Bt2cAMP. These results indicate that NO up-regulates TNF production by decreasing intracellular cAMP.

β-Adrenergic responsiveness of adenylate cyclase in human adipocyte plasma membranes in obesity and after massive weight reduction

The aim of this study was to find out how β-adrenergic responsiveness of adipocytes is altered in obesity and by weight loss and to investigate what mechanisms lead to potential alterations in responsiveness. Crude plasma membranes were prepared from adipocytes of massively obese and normal-weight individuals, as well as previously obese patients that had lost an average of 38% of their initial weight after bariatric surgery. Stimulation of adenylate cyclase by isoproterenol varied considerably in fat cell plasma membranes from different individuals. Crude fat cell plasma membranes from obese patients were less responsive to isoproterenol than those from normal-weight subjects, whereas those from postgastroplasty patients were hyperresponsive. The response was correlated negatively with cell size and positively with β-adrenergic receptor density and with the ratio of β-receptors and stimulatory G-proteins (Gs). There was no correlation with Gs content. However, differences in receptor density between small and large cells or normal-weight, obese, and post-bypass patients could not explain the observed differences in responsiveness to isoproterenol between the different groups.

Glucagon-like peptide-1 binding to rat hepatic membranes.

We have found [125I]glucagon-like peptide (GLP)-1(7-36)amide specific binding activity in rat liver and isolated hepatocyte plasma membranes, with an M(r) of approximately 63,000, estimated by cross-linking and SDS-PAGE. The specific binding was time- and membrane protein concentration-dependent, and equally displaced by unlabelled GLP-1(7-36)amide and by GLP-1(1-36)amide, achieving its ID50 at 3 x 10(-9) M of the peptides. GLP-1(7-36)amide did not modify the basal or the glucagon (10(-8) M)-stimulated adenylate cyclase in the hepatocyte plasma membranes. These data, together with our previous findings of a potent glycogenic effect of GLP-1(7-36)amide in isolated rat hepatocytes, led us to postulate that the insulin-like effects of this peptide on glucose liver metabolism could be mediated by a type of receptor probably different from that described for GLP-1 in pancreatic B-cells or, alternatively, by the same receptor which, in this tissue as well as in muscle, uses a different transduction system.

Expression of beta 1- and beta 3-adrenergic-receptor messages and adenylate cyclase beta-adrenergic response in bovine perirenal adipose tissue during its transformation from brown into white fat.

Possible modifications of the beta-adrenergic effector system during the development of bovine perirenal brown adipose tissue (BAT) in utero and its transformation into white-like adipose tissue after birth were studied. The parameters assessed were the level of expression of beta 1-, beta 2- and beta 3-adrenergic receptor (AR) mRNAs and the response of the plasma-membrane adenylate cyclase to (-)-isoprenaline and to the beta 3-agonist BRL 37344. The beta 3-AR mRNA was found to be expressed very early in utero, i.e. before the third month of foetal life. Then it increased dramatically (9-fold) between month 6 of foetal life and birth. A high beta 3-AR mRNA level was maintained after birth up to an age of 3 months. After conversion of BAT into white-like adipose tissue, i.e. in the adult bovine, the beta 3-AR mRNA expression became small or not detectable, and the beta 1-AR mRNA, which was expressed much less than the beta 3-AR mRNA in foetal life, became predominant. A response of the adenylate cyclase to (-)-isoprenaline was observed in foetal life (3.1-fold stimulation). It decreased after birth (1.8-fold stimulation) and then remained constant until adulthood. A response to BRL 37344 was also observed in foetal life (1.8-fold stimulation). It was maintained after birth, but disappeared in the adult. A possible relationship between the beta-AR expression and the adenylate cyclase response to (-)-isoprenaline on the one hand and the uncoupling-protein expression on the other is discussed. The bovine might represent a good model to understand the transition from brown to white fat in the human.

Distribution of adenylate cyclase and GTP-binding proteins in hepatic plasma membranes.

Hepatic membrane subfractions prepared from control rats demonstrated forskolin (FSK)-stimulated adenylate cyclase activity in the basolateral (sinusoidal) but not apical (canalicular) plasma membrane. After bile duct ligation (BDL) for 12 or 24 h, there was an increase in FSK-stimulated adenylate cyclase activity in the apical membrane (54.2 +/- 3.9 pmol.mg-1 x min-1). The mechanism for this increase was explored further. ATP hydrolysis was found to be much higher in the apical than the basolateral membrane. Increasing the ATP levels in the assay enhanced apical membrane adenylate cyclase activity (10.5 +/- 0.2 pmol.mg-l.min-1); however, total adenosinetriphosphatase (ATPase) activity was not altered after BDL. Extraction of the apical membrane with bile acids or other detergents resulted in a two- to threefold increase in adenylate cyclase activity (30.6 +/- 3.6 pmol.mg-1 x min-1; detergent C12E8) This suggested that bile duct ligation was acting via the detergent-like action of bile acids to uncover latent adenylate cyclase activity on apical membranes. Further studies demonstrated that both BDL and detergent extraction also enhanced toxin-directed ADP-ribosylation of Gs alpha (cholera toxin) and Gi alpha (pertussis toxin) in the apical but not the basolateral membrane. After BDL, Gi alpha was found to be twofold greater in the apical membrane than the basolateral membrane. Immunoblotting using specific G protein antibodies further confirmed that apical membranes from control rats had a higher concentration of Gi1, 2 alpha and beta and slightly elevated levels of Gi3 alpha and Gs alpha compared with the basolateral membrane. The results demonstrate that adenylate cyclase and heterotrimeric GTP-binding proteins are present on the apical membrane, but measurement of their functional activity requires detergent permeabilization of apical membrane vesicles and is limited by the presence of high ATPase activity.

Regulation of adenylate cyclase in plasma membranes of human intraabdominal and abdominal subcutaneous adipocytes

Fat cells were isolated from human subcutaneous, omental, and mesenteric adipose tissue. Omental fat cells were the smallest (438 pL), subcutaneous cells were intermediate (494 pL), and mesenteric cells were the largest (600 pL). There was no difference in the stimulation of adenylate cyclase by isoproterenol in plasma membranes of adipocytes prepared from the three sites. N 6-(phenylisopropyl)adenosine inhibited 7-deacetyl-6-( N-acetylglycyl)forskolin-stimulated adenylate cyclase activity more potently in subcutaneous than in intraabdominal (especially omental) fat cell membranes. K d values of the adenosine A 1 receptors for 1,3-[ 3H]dipropyl-8-cyclopentylxanthine were similar in the three fat depots, but the receptor number as calculated per milligram protein was lower in omental than in abdominal subcutaneous adipocytes. Differences in adipocyte size cannot explain regional differences in the regulation of adenylate cyclase.

Involvement of the Golgi region in the intracellular trafficking of cholera toxin.

The intracellular pathway following receptor-mediated endocytosis of cholera toxin was studied using brefeldin A (BFA), which inhibited protein secretion and induced dramatic morphological changes in the Golgi region. In both mouse Y1 adrenal cells and CHO cells, BFA at 1 micrograms/ml caused a 80-90% inhibition of the cholera toxin (CT)-induced elevation of intracellular cAMP. The inhibition of the cytotoxicity of CT by BFA was also observed in a rounding assay of Y1 adrenal cells. The inhibition of CT cytotoxicity by BFA was dose dependent, with the ID50 value similar to the LD50 of BFA in Y1 adrenal cells. Binding and internalization of [125I]-labeled cholera toxin in Y1 adrenal cells was not affected by BFA. Unlike the BFA-sensitive cell lines such as Y1 adrenal and CHO cells, BFA at 1 micrograms/ml did not inhibit the cytotoxicity of CT in PtK1 cells, of which the Golgi structure was BFA-resistant. These results strongly suggest that a BFA-sensitive Golgi is required for the protection of CT cytotoxicity by BFA. In contrast, elevation of the intracellular cAMP by forskolin, which acts directly on the plasma membrane adenylate cyclase, was not affected by BFA. These observations indicate that the intoxication of target cells by CT requires an intact Golgi region for its intracellular trafficking and/or processing. In this respect, CT shares a common intracellular pathway with ricin, Pseudomonas toxin, and modeccin, even though their structures and modes of action are very different.

Attenuated adenosine-sensitivity and decreased adenosine-receptor number in adipocyte plasma membranes in human obesity.

Fat-cells were isolated from patients of body-mass indices (BMIs) ranging from 17.9 to 83.9 kg/m2. Isoprenaline-stimulated cyclic AMP accumulation in cells prepared from obese subjects as compared with normal-weight subjects, was less sensitive to inhibition by the adenosine agonist N6-(phenylisopropyl)adenosine (PIA) (P = 0.047). The inhibition of 7 beta-desacetyl-7 beta-[gamma-(N-methylpiperazino) butyryl]-forskolin-stimulated adenylate cyclase by PIA in the presence of adenosine deaminase was also much attenuated in crude plasma membranes of adipocytes prepared from massively obese patients as compared with lean controls (P = 0.0143). This difference was probably not due to different cell size, because adenylate cyclase of crude plasma membranes of large adipocytes was actually more sensitive to PIA than was adenylate cyclase of membranes of smaller fat-cells co-isolated from the same individual. The stimulatory effect of PIA on glucose uptake in the presence of adenosine deaminase was depressed in adipocytes prepared from obese subjects and correlated with BMI at r = -0.626 (P = 0.007) at 100 nM-PIA. The adenosine receptors were studied by using the adenosine antagonist 1,3-[3H]dipropyl-8-cyclopentylxanthine. The binding was rapid and proportional to protein concentration. There was no difference in the affinities of receptors in membranes of obese and normal-weight subjects; Kd values of all patients averaged 3.3 nM. Bmax values were 54 and 130 fmol/mg of protein in membranes prepared from seven obese and five control patients respectively. The Bmax values calculated per mg of protein correlated with BMI at r = -0.539 (P = 0.047). The adenosine content of adipose tissue was higher in obese than in control subjects. These results demonstrate an attenuated response of cyclic AMP accumulation, adenylate cyclase and glucose uptake to adenosine in fat-cells prepared from obese subjects, and suggest that this change is at least partly due to changes in the amount of adenosine receptors, but not their affinity. The decreased receptor number could be due to higher adenosine content. A higher adenosine concentration in adipose tissue could explain why lipolysis is inhibited in situ in obesity, and the desensitization could explain the diminished response to adenosine analogues in isolated fat-cells.

Effects of a membrane-bound trypsin-like proteinase and seminal proteinase inhibitors on the bicarbonate-sensitive adenylate cyclase in porcine sperm plasma membranes

Plasma membranes were purified from flagella of porcine cauda epididymal sperm and proteolytic regulation of bicarbonate-sensitive adenylate cyclase was studied. It was found that the epididymal sperm plasma membrane contained a trypsin-like proteinase which inactivated adenylate cyclase. Bicarbonate activates adenylate cyclase as reported previously, but, at the same time, the anions enhance the inactivation of the enzyme by the membrane-bound trypsin-like proteinase. This phenomenon is not due to the direct activation of the proteinase, but closely related to the activation of adenylate cyclase by bicarbonate. It was also found that seminal proteinase inhibitors blocked the inactivation of adenylate cylcase and maintained the bicarbonate activation of the enzyme at high level. Actually, bicarbonate keeps adenylate cyclase fully active in ejaculated sperm, because membrane-bound proteinase is completely inhibited by the seminal proteinase inhibitors. These results suggest that the interactions between membrane-bound proteinase and seminal proteinase inhibitor are involved in the regulation of the bicarbonate-sensitive adenylate cyclase system.

Vasoactive intestinal peptide stimulation of human renal adenylate cyclase in vitro.

1. A direct action of vasoactive intestinal peptide (VIP) upon human kidney was sought by measurement of renal adenylate cyclase in tissue homogenates and plasma membranes isolated from tissue samples excised for therapeutic reasons. 2. VIP (1 microM) produced a mean stimulation of adenylate cyclase activity of 3.5-fold compared to basal values in cortical plasma membranes; comparative stimulations of 2.8-fold and 27.3-fold were obtained with 1 microM-glucagon and 1 microM-h(1-34) parathyroid hormone respectively. 3. Half-maximal stimulation of human renal cortical plasma membrane adenylate cyclase was observed with a mean value of 35 nM-VIP. 4. The stimulation of renal adenylate cyclase by VIP appeared to be specific because stimulation by glucagon was additive to that obtained with VIP, and the VIP receptor antagonist (4 Cl-D-Phe6, Leu17)-VIP inhibited the VIP-dependent stimulation of adenylate cyclase activity.

Potent and cooperative feedback inhibition of adenylate cyclase activity by calcium in pituitary-derived GH 3 cells

Calcium (Ca 2+) ion concentrations that are achieved intracellularly upon membrane depolarization or activation of phospholipase C stimulate adenylate cyclase via calmodulin (CaM) in brain tissue. In the present study, this range of Ca 2+ concentrations produced unanticipated inhibitory effects on the plasma membrane adenylate cyclase activity of GH 3 cells. Ca 2+ concentrations ranging from 0.1 to 0.8 μM exerted an increasing inhibition on enzyme activity, which reached a plateau (35–45% inhibition) at around 1 μM. This inhibitory effect was highly cooperative for Ca 2+ ions, but was neither enhanced nor dependent upon the addition of CaM (1 μM) to EGTA-washed membranes. The inhibition was greatly enhanced upon stimulation of the enzyme by vasoactive intestinal peptide (VIP) and/or GTP. Prior exposure of cultured cells to pertussis toxin did not affect the inhibition of plasma membrane adenylate cyclase activity by Ca 2+, although in these membranes, hormonal (somatostatin) inhibition was significantly attenuated. Maximally effective concentrations of Ca 2+ and somatostatin produced additive inhibitory effects on adenylate cyclase. The addition of phosphodiesterase inhibitors demonstrated that inhibitory effects of Ca 2+ were not mediated by Ca 2+-dependent stimulation of a phosphodiesterase activity. These observations provide a mechanism for the feedback inhibition by elevated intracellular Ca 2+ levels on cAMP-facilitated Ca 2+ entry into GH 3 cells, as well as inhibitory crosstalk between Ca 2+-mobilizing signals and adenylate cyclase activity.

Induction of prostacyclin receptor expression in human erythroleukemia cells

We have identified both high-affinity (K D = 36±3 nM) and low-affinity (K D = 2.1±0.8 μM) prostacyclin (PGI 2)-receptor sites on human erythroleukemia (HEL) cells using the radiolabelled prostacyclin analogue, [ 3H]iloprost. The addition of the phorbol ester, TPA, to the culture medium caused a 5–10-fold increase in the number of both the low- and the high-affinity sites, without any change in their affinity constants. Iloprost stimulated HEL cell membrane adenylate cyclase activity 5-fold. This stimulation was potentiated in the presence of GTP, indicating a conventional PGI 2 receptor-G s-adenylate cyclase system. HEL cells represent a source of prostacyclin receptor mRNA which may be of value in expression cloning of this receptor.

Lipid Peroxidation and Cancer: A Critical Reconsideration

The author reviews the problem of the pattern of lipid peroxidation in cancer cells with special reference to a comparison between normal liver cells and hepatomas both transplanted and induced by diethylnitrosamine. It is stated that the loss of lipid peroxidation is proportional to the degree of de-differentiation of hepatoma cells. During carcinogenesis, however, the loss is already evident at the stage of preneoplastic nodules. A common feature of all tumors, independently of the extent of the loss of peroxidation in basal conditions, is the lack of further stimulation by ADP/iron or by ascorbate/iron. As regards the reasons for the decline in lipid peroxidation, they are certainly not unique. An important cause is the low activity of the enzymes of the monooxygenase microsomal chain. Another very important one is the change in lipid composition of membranes, with a marked decrease in polyunsaturated fatty acids, which are the main substrate for lipid peroxidation. It has been shown that enrichment of membranes of hepatomas with arachidonic acid results in restoration of stimulation of peroxidation by ascorbate/iron, but not with ADP/iron. The last type of stimulation mostly reflects the behaviour of the monooxygenase chain, whereas ascorbate/iron-induced stimulation does not require the presence of an efficient cytochrome P450-chain. Another cause for decreased lipid peroxidation in tumors is the increased rigidity of membranes, due to the large increase in cholesterol content: this prevents to some extent the influx of oxygen inside the membranes. Yet another cause is the presence of increased amounts of antioxidants in both cytosol and membranes. The main toxic product of lipid peroxidation, 4-hydroxynonenal, has been found to elicit several actions at extremely low concentrations. In fact, 4-hydroxynonenal stimulates chemotaxis of polymorphonuclear leukocytes, stimulates plasma membrane adenylate cyclase, stimulates plasma membrane guanylate cyclase, and stimulates phospholipase C. The last three enzymes involve the action of G-proteins. The effect of the aldehyde is present at less than micromolar concentrations, which may occur inside the cells in certain conditions. Moreover, at concentrations from 10(-6) to 10(-7) M, the aldehyde is able to block oncogene c-myc expression in the human erythroleukemic K562 cell line, which at the same time becomes able to express the gamma-globin gene. These facts are discussed with reference to a possible biological meaning of the loss of lipid peroxidation in tumors.

Guanine nucleotide binding regulatory proteins and adenylate cyclase in livers of streptozotocin- and BB/Wor-diabetic rats. Immunodetection of Gs and Gi with antisera prepared against synthetic peptides.

Adenylate cyclase in liver plasma membranes from streptozotocin-diabetic (STZ) or BB/Wor spontaneously diabetic rats showed increased responsiveness to GTP, glucagon, fluoroaluminate, and cholera toxin. Basal or forskolin-stimulated activity was unchanged in STZ rats, but increased in BB/Wor rats. No change in the alpha-subunit of Gi (alpha i) was observed in STZ or BB/Wor rats using pertussis toxin-stimulated [32P]ADP-ribosylation. Immunodetection using antibodies against the COOH-terminal decapeptides of alpha T and alpha i-3 showed no change in alpha i in STZ rats and a slight decrease in BB/Wor rats. Angiotensin II inhibition of hepatic adenylate cyclase was not altered in either diabetic rat. In both models of diabetes, Gs alpha-subunits were increased as measured by cholera toxin-stimulated [32P]-ADP-ribosylation of 43-47.5-kD peptides, reconstitution with membranes from S49 cyc- cells or immunoreactivity using antibodies against the COOH-terminal decapeptide of alpha s. These data indicate that STZ-diabetes increases hepatic Gs but does not change Gi or adenylate cyclase catalytic activity. In contrast, BB/Wor rats show increased hepatic Gs and adenylate cyclase. These changes could explain the increase in hepatic cAMP and related dysfunctions observed in diabetes.