NaF-stimulated Adenylate Cyclase Activity Research Articles

Effects of fluorotelomer alcohol 8:2 FTOH on steroidogenesis in H295R cells: Targeting the cAMP signalling cascade

Previous studies have demonstrated that perfluorinated chemicals (PFCs) can affect reproduction by disruption of steroidogenesis in experimental animals. However, the underlying mechanism(s) of this disruption remain unknown. Here we investigated the effects and mechanisms of action of 1H, 1H, 2H, 2H-perfluoro-decan-1-ol (8:2 FTOH) on steroidogenesis using a human adrenocortical carcinoma cell line (H295R) as a model. H295R cells were exposed to 0, 7.4, 22.2 or 66.6 μM 8:2 FTOH for 24 h and productions of progesterone, 17α-OH-progesterone, androstenedione, testosterone, deoxycorticosterone, corticosterone and cortisol were quantified by HPLC-MS/MS. With the exception of progesterone, 8:2 FTOH treatment significantly decreased production of all hormones in the high dose group. Exposure to 8:2 FTOH significantly down-regulated cAMP-dependent mRNA expression and protein abundance of several key steroidogenic enzymes, including StAR, CYP11A, CYP11B1, CYP11B2, CYP17 and CYP21. Furthermore, a dose-dependent decrease of cellular cAMP levels was observed in H295R cells exposed to 8:2 FTOH. The observed responses are consistent with reduced cellular cAMP levels. Exposure to 8:2 FTOH resulted in significantly less basal (+ GTP) and isoproterenol-stimulated adenylate cyclase activities, but affected neither total cellular ATP level nor basal (−GTP) or NaF-stimulated adenylate cyclase activities, suggesting that inhibition of steroidogenesis may be due to an alteration in membrane properties. Metabolites of 8:2 FTOH were not detected by HPLC-MS/MS, suggesting that 8:2 FTOH was not metabolized by H295R cells. Overall, the results show that 8:2 FTOH may inhibit steroidogenesis by disrupting the cAMP signalling cascade.

Functional desensitisation of beta-adrenergic receptors of avian erythrocytes by catecholamines and adenosine 3',5'-phosphate.

Prolonged exposure to beta-adrenergic agonists of pigeon erythrocytes causes a reversible loss (70%) of catecholamine-stimulated adenylate cyclase activity without reduction in the number of beta-adrenergic receptors. In addition a less pronounced decrease in non-stimulated and NaF-stimulated adenylate cyclase activity (15-22%) is observed, appearing at different agonist concentrations and at a different rate. Dibutyryladenosine 3',5'-phosphate and the phosphodiesterase inhibitor methylisobutylxanthine partially mimick the action of the beta-adrenergic agonist, thus pointing to a possible role of adenosine 3',5'-phosphate in establishing desensitization. When adenylate cyclase from desensitized cells is stimulated with 5'-guanylyl-imidodiphosphate in the presence or absence of catecholamines the lag period preceding the attainment of maximal activity is extended. Likewise the rate of reversal by GTP or GTP of persistent activation of adenylate cyclase is slowed down. This is therefore interpreted to mean that the loss in hormonal stimulation on treatment of pigeon red blood cells with beta-adrenergic agonists is due to a delayed exchange of GDP against GTP on the regulatory GTP-binding protein. Furthermore, we conclude that events causing the refractory state in avian erythrocytes should occur at a site distal to the beta-adrenergic receptor.

Effect of phosphatidylcholine structure on the adenylate cyclase activity of a murine fibroblast cell line

To determine which structural characteristics of membrane phospholipids influence adenylate cyclase activity, we measured basal and sodium fluoride-or forskolin-stimulated activity in a murine fibroblast cell line, i.e., Balb/c3T3 cells grown in media supplemented with fetal calf serum (FCS), lipid-depleted FCS (LD-FCS) or LD-FCS complexed with different phosphatidylcholine (PC) molecular species. Cells grown in the presence of LD-FCS showed a substantial decrease in their basal and NaF-stimulated adenylate cyclase activities; however, their forskolin-stimulated activity was not altered, suggesting that the enzyme's catalytic site is not affected by changes in membrane lipids. Media supplemented with different LD-FCS/PC complexes were shown to prevent the LD-FCS-mediated reduction of basal and NaF-stimulated adenylate cyclase activity to different extents. Addition of cis-9-16:1, cis-9-18:1/cis-9-18:1 or cis-9-18:1/cis-9,12-18:2 sn-glycerophosphocholine (GPC) completely restored adenylate cyclase activity, while cis-11-18:1/cis-11-18:1 GPC was not effective and only a partial recovery was observed with 16:0/16:0, 16:0/cis-9-18:1 and trans-9-18:1 GPC. Considering the structural features of these seven PC molecular species, the findings suggest that an optimal lipid environment is conferred to the enzyme by the presence of two cis double bonds, each located in delta 9 position of the PC acyl chains. The limited effect of cis-9-16:1/cis-9-18:1 GPC and cis-9-18:1/cis-9-16:1 GPC suggests that an equal length of the terminal hydrocarbon chains extending beyond the delta 9 double bonds is also important.(ABSTRACT TRUNCATED AT 250 WORDS)

Are cardiac G-proteins altered in rat models of hypertension?

To determine whether alterations in cardiac G-protein number or function, or both, are involved in the desensitization of adenylate cyclase responsiveness in the hypertensive state. Quantitation of G-protein subunits in myocardial membranes from four different rat models of hypertension in comparison with respective normotensive rats. We compared male and female adult spontaneously hypertensive rats (SHR) with age- and sex-matched Wistar-Kyoto (WKY) rats, rats from the highest-pressure quartile from an F2 generation of WKY x SHR hybrids with those from the lowest-pressure quartile, and one-kidney, one clip renal hypertensive with sham-operated rats. The function of Gs alpha was quantitated by reconstitution of cardiac cholate extracts into cyc- cell membranes with subsequent measurement of NaF-stimulated adenylate cyclase activity. The amounts of immunodetectable Gs alpha, Gi alpha, Gq alpha and G beta were determined from quantitative Western blotting experiments with [125I]-protein A detection. None of the parameters investigated differed significantly between hypertensive and normotensive rats in any of the models investigated. We conclude that major quantitative alterations in cardiac Gs, Gi or Gq are not a general feature of the hypertensive state.

?-Adrenergic receptor activity of cerebral microvessels is reduced in aged rats

The effect of age on beta- (beta) adrenergic receptor number (Bmax) and adenylate cyclase (AC) activity was determined in microvessels isolated from male F-344 rats at 3, 18, and 24 months of age. Scatchard analysis of [125I]iodocyanopindolol (ICYP) binding indicated reduced Bmax (fmol/mg) of microvessels isolated from 24 month old rats (27.2 +/- 4.9) compared with 3 month old (50.4 +/- 5.2) and 18 month old rats (p less than 0.01) (61.4 +/- 7.6). The basal AC activity (pmol cAMP/mg) in 24 month old rats (32.0 +/- 6.7) and in 18 month old rats (30.4 +/- 2.1) were significantly reduced compared to the basal activity in the young (50.1 +/- 4.2). The net isoproterenol or NaF stimulated AC activity in 24 month old rats (zero and 15.6 +/- 8.5 respectively) was also reduced compared to young rats (10.1 +/- 3.9 and 166.0 +/- 21.2 respectively). It is concluded that aging is associated with reduced isoproterenol stimulated AC activity of cerebral microvessels. This reduction is the product of reduced beta-adrenergic receptor number and reduced activity of AC in aged rat cerebral microvessels.

Effect of temperature on the adenylate cyclase activity of Mytilus galloprovincialis mantle tissue

1. 1. The basal and NaF-stimulated adenylate cyclase activities of Mytilus galloprovincialis mantle tissue were studied at different temperatures. 2. 2. There are no significant differences in the K m for ATP at 13°C and 20°C in both basal and NaF-stimulated conditions. 3. 3. NaF increases the V max of the enzyme (5-fold) and decreases about 50% the K m for ATP at both temperatures assayed. 4. 4. Activation energy of the enzyme reaction is 33.4 kJ/mol. K in basal conditions and 29.4 kJ/mol. K when NaF is present. The Q 10, at saturating substrate concentrations, is approximately 1.5 and this value is constant in the temperature range studied, 10–30°C. 5. 5. The adenylate cyclase starts being inactivated from 30°C. The enzyme shows greater sensitivity to denaturalization by temperature in NaF-stimulated than in basal conditions.

Beta-carotene induces morphological differentiation and decreases adenylate cyclase activity in melanoma cells in culture.

Several studies suggest that beta-carotene reduces the risk of some cancers. Except for its function as an antioxidant, the effect of this vitamin on mammalian cells remains poorly defined. This study was performed to show whether beta-carotene treatment of murine B-16 melanoma cells in culture induces differentiation and alters the adenylate cyclase (AC) system. The AC system mediates the action of agents which regulate cell differentiation and transformation. Results showed that beta-carotene treatment for a period of 24 hours or more caused morphological differentiation without changing the level of melanin, and reduced basal and melanocyte-stimulated hormone (MSH)-, sodium fluoride (NaF)-, and forskolin-stimulated AC activity in vitro. Retinol, a metabolite of beta-carotene, inhibited growth without morphological differentiation and reduced basal and MSH- and NaF-stimulated AC activity. However, butylated hydroxyanisole, a lipid-soluble antioxidant, also reduced growth without morphological differentiation, but it failed to alter basal or MSH-stimulated AC activity. The present and previous studies show that the AC system represents a common site where some antitumor-promoting vitamins (beta-carotene, retinol, retinoic acid, and alpha-tocopheryl succinate) act.

Deficient Activity of Nucleotide Binding Regulatory Protein Coupled With PGE2 Receptor in Renal Medulla of Spontaneously Hypertensive Rats

Prostaglandin (PG) E2 receptor-adenylate cyclase system was studied in the kidney of 12-week-old spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) to evaluate the role of this system in hypertension. PGE2 receptors were determined by a radioligand binding method using [3H]-PGE2. Adenylate cyclase responses to PGE2, sodium fluoride (NaF) and forskolin were also measured. The concentration of PGE2 receptor was increased in SHR compared with WKY. PGE2- and NaF-stimulated adenylate cyclase activities were significantly lower in SHR than WKY. There was no significant difference in forskolin-stimulated adenylate cyclase activity between SHR and WKY. NaF activates the nucleotide binding regulatory protein (G-protein) and forskolin directly activates the catalytic unit. These results indicate that the activity of G-protein coupled with renal PGE2 receptors is deficient in SHR. This defect may contribute to the elevation of blood pressure, through sodium retention.

Viral Pneumonia Attenuates Adenylate Cyclase But Not Beta-Adrenergic Receptors in Murine Lung

Respiratory infections provoke increased airway reactivity in both asthmatic and otherwise healthy subjects in part through impaired beta-adrenergic relaxation of bronchial and tracheal muscle. The precise mechanism remains obscure, but some studies report a decrease in the number of beta-adrenergic receptors. The present study was designed to assess the effect of viral respiratory infection with influenza A on lung beta-adrenergic receptors and adenylate cyclase activation in mice under two separate protocols. First, to determine whether changes are due to a local or systemic effect, we compared mice with influenza infections limited to the upper respiratory tract to mice with infection of the total respiratory tract. Four days after upper respiratory tract infection there were no changes in either isoproterenol- or NaF-stimulated adenylate cyclase activity. In contrast, there was an 82% decrease in isoproterenol- and a 25% decrease in NaF-stimulated adenylate cyclase activity on the fourth day after total respiratory tract infection. There were no changes in beta-adrenergic receptors or receptor coupling to adenylate cyclase with either type of infection. Our second protocol compared acutely infected mice to postrecovery mice. Twelve days after infection the virus was no longer present in the lungs, and adenylate cyclase activity was restored to normal. These data suggest that viral respiratory infection may impair airway function through attenuation of receptor and postreceptor activation of adenylate cyclase activity.

The novel thermogenic β-adrenergic agonist Ro 16-8714 increases the interscapular brown-fat β-receptor-adenylate cyclase and the uncoupling-protein mRNA level in obese (fa/fa) Zucker rats

In obese (fa/fa) rats both the total (-)-isoprenaline- and NaF-stimulated adenylate cyclase activities of interscapular brown-adipose-tissue (IBAT) plasma membranes were decreased as compared with lean rats by 40 and 38% respectively. An acute treatment with Ro 16-8714 increased (-)-isoprenaline- and NaF-stimulated adenylate cyclase activities by 2.5- and 2.0-fold respectively, and beta-adrenoceptor number 2.8-fold in obese rat IBAT, but it had no effect on these parameters in lean rats. It increased mRNA for mitochondrial uncoupling protein in both lean and obese rats to the same extent.

Inhibition of formation of cyclic AMP and cyclic GMP by vasopressin in smooth-muscle cells is insensitive to pertussis toxin.

Pretreatment of A-10 cells with pertussis toxin had no effect on [arginine]vasopressin-mediated inhibition of cyclic nucleotide accumulation. Pretreatment of the cells with the same concentration of pertussis toxin produced 90-95% inhibition of [32P]ADP ribosylation in membranes, suggesting that these cells possess pertussis-toxin substrate and that the toxin enters the cells to reach its site of action. The functional integrity of the pertussis-toxin substrate in these cells is confirmed by the observation that in these cell membranes increasing concentrations of GTP inhibited basal, forskolin- and NaF-stimulated adenylate cyclase activities, and this inhibition was abolished when the cells were pretreated with pertussis toxin. In addition, thrombin-mediated inhibition of isoprenaline-stimulated cyclic AMP accumulation was also inhibited by pertussis-toxin pretreatment of the cells. These data suggest that, unlike thrombin, [arginine]vasopressin-induced inhibitory effects on cyclic nucleotide accumulation in smooth-muscle cells are not mediated by pertussis-toxin substrate.

Modulation of receptor-mediated gonadotropin action in rat testes by dietary fat.

The effect of feeding diets enriched with 18:2 omega 6, 18:3 omega 3, or saturated fatty acids on lipid composition and receptor-mediated action of luteinizing hormone/human chorionic gonadotropin (LH/hCG) in rat testicular plasma membranes was investigated. Linoleic and alpha-linolenic acid treatments reduced total phospholipid and cholesterol content of the testicular plasma membrane and altered membrane phospholipid composition. Change in phospholipid and cholesterol content after feeding the polyunsaturated fats decreased cholesterol to phospholipid ratios and binding capacity of the LH/hCG receptor in the testicular plasma membrane. LH-stimulated adenylate cyclase activity was decreased in animals fed the linolenic acid-rich diet. NaF-stimulated adenylate cyclase activity was decreased in animals fed diets high in either polyunsaturated fatty acid. Decreased plasma membrane LH/hCG receptor content was associated with decreased testosterone production in Leydig cells in response to LH in the linolenic acid-fed group. It is suggested that change in cholesterol-to-phospholipid ratios alters the physical properties of testicular plasma membranes in a manner that influences accessibility of LH/hCG receptors in testicular tissue.

Age-associated decrease in beta-adrenergic receptors and adenylate cyclase activity in rat brown adipose tissue.

The ability to regulate body temperature diminishes with age. In the rat, nonshivering thermogenesis in brown adipose tissue (BAT) serves as the regulator of body temperature. This process is stimulated by catecholamine activation of adenylate cyclase through the beta-adrenergic receptor. However, beta-adrenergic responsiveness also diminishes with age. To investigate the age-related alterations in beta-adrenergic function in BAT, beta-adrenergic receptor number and cytochrome c oxidase activity were assessed in 3-, 12-, and 24-month-old rats, and adenylate cyclase activity was assessed in 3-, 12-, 18-, and 24-month-old rats. The amount of brown adipose tissue recovered from senescent rats was 30% less than that from either the 3- or 12-month-old rats. There was a corresponding decrease in the total amount of cytochrome c oxidase activity in the 24-month-old rats. In the senescent rats, the density of beta-adrenergic receptors was twofold less than in the 3- and 12-month-old rats. The decrease in density was predominately due to a decrease in the beta 1-adrenergic subtype. Isoproterenol- and NaF-stimulated adenylate cyclase activity were threefold greater and forskolin-stimulated activity was fourfold greater in the 3-month than in the older rats. There was no change in the Kact for isoproterenol with age. These biochemical alterations with age may contribute to the inability of older animals to thermoregulate when exposed to cold.

Effects of chronic ethanol exposure on adenylate cyclase activities in the rat.

The effects of chronic ethanol administration on striatal and cerebellar adenylate cyclase systems were investigated in the rat. The chronic ethanol treatment resulted in behavioral tolerance, but no difference in the sensitivity of adenylate cyclase to in vitro ethanol was observed. In one set of experiments using 173-195 g rats, GTP-, dopamine- and NaF-stimulated adenylate cyclase activities in the striatum were higher in rats chronically treated with ethanol when compared to animals pair-fed the liquid diet. However, no difference in adenylate cyclase activity was observed in cerebellar or striatal tissues when larger rats (280-385 g) were used. In conclusion, an adaptive change in activation of adenylate cyclase by in vitro ethanol does not occur after chronic ethanol treatment. The observed changes in enzyme activity measured in the absence of in vitro ethanol do not appear to be a simple, direct effect of chronic ethanol treatment.

Elevation of adenylate cyclase activity during leukemic cell differentiation

Exposure of the various human myeloid leukemic cell lines (HL60 and RDFD) to various compounds results in marked differentiation of the cells. This differentiation is associated with a marked increase in both basal and NaF-stimulated adenylate cyclase (AC) activity. The increase in AC activity occurs regardless of the differentiation inducer one has utilized (retinoic acid (RA), dimethyl formamide (DMF), hypoxanthine (HPX) or actinomycin D (actD) and is correlated with this process, as a variant of the HL60 cell (HL60-Blast) that does not differentiate upon exposure to the various inducers does not demonstrate this increase in AC activity. In addition, the differentiation process is associated with a rapid increase in intracellular cAMP within hours of adding the inducer, followed by a gradual decrease.

Deafferentation elicits increased dopamine-sensitive adenylate cyclase and receptor binding in the olfactory tubercle

Removal of a major non-catecholaminergic output from the olfactory bulb elicits sprouting of dopaminergic axons in the olfactory tubercle. The functional consequences of this increased dopaminergic innervation are presently not known. This study examined the question of whether lesion-induced sprouting of dopaminergic axons is associated with changes in dopamine-sensitive adenylate cyclase and dopamine receptor density in the partially denervated olfactory tubercle. The results indicate that dopamine- and NaF-stimulated adenylate cyclase activity increased as early as 7 d, while forskolin-sensitive activity increased at 3 d and persisted up to 20 d after lesioning; higher levels of GTP- and NaF-stimulated enzyme activity were found in detergent extracts of olfactory tubercle membranes from 20 d lesioned rats; higher levels of 3H-forskolin binding were found in membranes from 14 and 20 d lesioned rats; and there was an increase in dopamine receptor density, but not affinity, in olfactory tubercle membranes from lesioned rats. The data indicate that lesion-induced dopaminergic sprouting in the olfactory tubercle is temporally coordinated with the increased formation of dopamine receptors, both D1 and D2, the stimulatory guanine nucleotide regulatory protein (Ns) and the catalytic subunit (C) of adenylate cyclase in the postsynaptic membrane.

Calcium and calmodulin in the regulation of human thyroid adenylate cyclase activity.

TSH (thyrotropin)-stimulated human thyroid adenylate cyclase has a biphasic response to Ca2+, being activated by submicromolar Ca2+ (optimum 22nM), with inhibition at higher concentrations. Calmodulin antagonists caused an inhibition of TSH-stimulated adenylate cyclase in a dose-dependent manner. Inhibition of TSH-and TSIg-(thyroid-stimulating immunoglobulins)-stimulated activity was more marked than that of basal, NaF- or forskolin-stimulated activity. This inhibition was not due to a decreased binding of TSH to its receptor. Addition of pure calmodulin to particulate preparations of human non-toxic goitre which had not been calmodulin-depleted had no effect on adenylate cyclase activity. EGTA was ineffective in removing calmodulin from particulate preparations, but treatment with the tervalent metal ion La3+ resulted in a loss of up to 98% of calmodulin activity from these preparations. Addition of La3+ directly to the adenylate cyclase assay resulted in a partial inhibition of TSH- and NaF-stimulated activity, with 50% inhibition produced by 5.1 microM and 4.0 microM-La3+ respectively. Particulate preparations with La3+ showed a decrease of TSH- and NaF-stimulated adenylate cyclase activity (approx. 40-60%). In La3+-treated preparations there was a decrease in sensitivity of TSH-stimulated adenylate cyclase to Ca2+ over a wide range of Ca2+ concentrations, but most markedly in the region of the optimal stimulatory Ca2+ concentration. In particulate preparations from which endogenous calmodulin had been removed by La3+ treatment, the addition of pure calmodulin caused an increase (73 +/- 22%; mean +/- S.E.M., n = 8) in TSH-stimulated thyroid adenylate cyclase activity. This was seen in 8 out of 13 experiments.

Adenylate Cyclase Activity in Fetal Rabbit Myocardium

Adenylate cyclase activity [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] was determined in vitro in fetal rabbit myocardial membranes from individual fetal pups at 21 to 31 days gestation (term, 31 days). Basal and NaF-stimulated adenylate cyclase activities did not change during 21-31 days gestation. Significant stimulation of the enzyme by l-isoproterenol was observed only in the presence of guanosine triphosphate (100 microM). Under these conditions, maximal adenylate cyclase stimulation by l-isoproterenol (100 microM) was significantly higher at 25-31 than at 21 days gestation. Moreover, EC50 (Kact) for l-isoproterenol at 25-31 days was significantly lower than at 21 days gestation. We conclude that, in fetal rabbit myocardial membranes, there is an increase in the sensitivity of adenylate cyclase stimulation by l-isoproterenol from 21 to 25-31 days gestation.

Alterations in cerebral β-adrenergic receptor-adenylate cyclase system induced by halothane, ketamine and ethanol

The effect of halothane, ketamine and ethanol on ?-adrenergic receptor adenylate cyclase system was studied in the brain of rats. An anesthetic concentration of halothane and ketamine added in vitro decreased the stimulatory effect of norepinephrine on cyclic AMP formation in slices from the cerebral cortex. On the other hand, ethanol increased the basal activity of cerebral adenylate cyclase without affecting on the norepinephrine-stimulated activity. The increase of the basal activity induced by ethanol was not antagonized by propranolol, a ?-adrenergic antagonist. In the crude synaptosomal (P(2)) fraction, these drugs had no significant effect on the basal adenylate cyclase activity, binding of [(3)H]dihydroalprenolol to ?-receptor, and binding of [(3)H]guanylylimido diphosphate ([(3)H]Gpp(NH)p) to guanyl nucleotide binding site. In contrast, the adenylate cyclase activity stimulated by Gpp(NH)p or NaF was significantly inhibited by an anesthetic concentration of these drugs. An anesthetic concentration of these drugs increased the membrane fluidity of P(2) fraction monitored by the fluorescence polarization technique. The addition of linoleic acid (more than 500 ?M) also induced not only the increase of fluidity, but also the decrease of Gpp(NH)p- or NaF-stimulated adenylate cyclase activity in the cerebral P(2) fraction. The present results suggest that general anesthetics may interfere with the guanyl nucleotide binding regulatory protein-mediated activation of cerebral adenylate cyclase by disturbing the lipid region of synaptic membrane.

Synergistic effects of chymotrypsin and human chorionic gonadotrophin on rat luteal adenylate cyclase.

The effects of chymotrypsin on rat luteal adenylate cyclase have been investigated. Maximal stimulation occurred at a concentration of 10 micrograms/ml. This dose of chymotrypsin stimulated basal, GTP-,5'-(beta, gamma-imido)triphosphate-, and NaF-stimulated adenylate cyclase activity 2- to 3-fold. hCG-stimulated activities were stimulated 5- to 6-fold by the proteinase and showed a synergistic effect. The proteinase did not alter the hCG response curve, and the effect could not be mimicked by trypsin. Proteolytic activity was required, since chymotrypsinogen and inactivated chymotrypsin were not effective. The presence of chymotrypsin could not restore the hormonal sensitivity of a proteinase inhibitor-inactivated system. The results suggest that chymotrypsin stimulated rat luteal adenylate cyclase activity by two or more mechanisms: 1) a direct action on the catalytic subunit or a closely associated protein, and 2) an action somewhere in the pathway of hormonal stimulation.