Sucrase-isomaltase is an adenosine 3',5'-cyclic monophosphate-dependent epithelial chloride channel

BACKGROUND & AIMS: We previously isolated a monoclonal antibody against a Necturus gallbladder epitope that blocks native adenosine 3',5'-cyclic monophosphate (cAMP)-dependent chloride channels in intestine, gallbladder, urinary bladder, and airway epithelia in various animals. METHODS: Using this antibody, we purified a 200-kilodalton protein that, when reconstituted in lipid bilayers, forms 9-pS chloride channels that are blocked by the antibody. RESULTS: Amino acid sequencing of the purified protein showed strong homology to rabbit sucrase-isomaltase, an abundant intestinal enzyme. Western blot analysis of the in vitro-translated sucrase-isomaltase was indistinguishable from that of the protein used in the lipid bilayer studies. Expression of this protein in Chinese hamster ovary cells and in Xenopus laevis oocytes yielded cAMP-dependent chloride currents that in the latter system were blocked by the antibody. CONCLUSIONS: Because the monoclonal antibody blocks cAMP-dependent currents in epithelia as well as those produced both by the reconstituted and by the heterologously expressed protein, sucrase-isomaltase is a cAMP-dependent epithelial chloride channel. Thus an enzyme that can also function as an ion channel has been described for the first time.     

Hormonal regulation of phospholipid methyltransferase by 3',5'-cyclic adenosine monophosphate-dependent and independent mechanisms

Treatment of isolated rat adipocytes with epinephrine or isoproterenol caused a time- and concentration-dependent increase in phospholipid methyltransferase (PLMT) activity that was blocked by propranolol and unaffected by phentolamine. Forskolin mimicked the stimulatory effect on PLMT, and insulin inhibited this effect. In both the absence and presence of insulin, there was a linear relationship between PLMT activity and lipolysis. PLMT activity was also increased in response to oxytocin, which does not activate adenylate cyclase in adipocytes and does not stimulate lipolysis. The effects of oxytocin were inhibited by insulin and were additive with those of isoproterenol on PLMT. These data support the hypothesis that in adipocytes, PLMT is activated by a cAMP-dependent protein kinase and a cAMP-independent mechanism, both of which can be regulated independently, and both of which are sensitive to inhibition by insulin.     

Identification, characterization, and hormonal regulation of 3', 5'-cyclic adenosine monophosphate-dependent protein kinases in rat Sertoli cells.

Recent studies have disclosed multiple isoforms of regulatory (R) and catalytic (C) subunits of cAMP-dependent protein kinase (PKA) at the protein and messenger RNA (mRNA) levels. The purpose of the present study was to identify, characterize, and quantify individual R subunits in rat Sertoli cells both at the mRNA and protein levels. Unstimulated Sertoli cells contain high levels of R (approximately 9.2 +/- 0.8 pmol/mg protein) and C (approximately 7.3 +/- 0.7 pmol/mg protein). Stimulation with (Bt)2cAMP (0.1 mM) for 24 and 48 h revealed a time-dependent increase in [3H]cAMP-binding activity. During the same time period the catalytic activity remained relatively constant, resulting in an increase in the R/C ratio from approximately 1.3 to 3.0. Using diethylaminoethyl cellulose chromatography, 8-N3-[32P]cAMP photoaffinity labeling, autophosphorylation by gamma-[32P]ATP, and specific antibodies, we show that unstimulated Sertoli cells contain approximately 75% RI alpha, 25% RII alpha, and very low levels of RII beta. Stimulation of Sertoli cells with (Bt)2cAMP (0.1 mM, 48 h) was associated with a 2.1-fold increase in RI alpha (6.6-14 pmol/mg) and a 10- to 20-fold increase in RII beta (less than 0.1-1.1 pmol/mg), with little or no change in RII alpha (1.9-2.3 pmol/mg). Treatment with cAMP was associated with a slight increase in RI/RII ratio (3.3-4.1). mRNA levels for RII beta increased 30- to 50-fold after (Bt)2cAMP stimulation, whereas only minor changes in mRNA levels for RI alpha, RII alpha, and C alpha were observed (1.5- to 2.0-fold). mRNA levels for RI beta, C beta, and C gamma were not detected in either unstimulated or in cAMP-stimulated Sertoli cells. It is concluded that chronic treatment with cAMP changes the relative proportion of R subunits of PKA in a manner reflecting the changing levels in respective mRNAs. Furthermore, such treatment is associated with the appearance of a new PKA R subunit (RII beta), which is absent in untreated Sertoli cells.     

Hormonal effects on the immunocytochemical location of 3',5'-cyclic adenosine monophosphate-dependent protein kinase in rat tissues

Homogeneous preparations of type I and type II regulatory subunits (RI and RII, respectively) of cAMP-dependent protein kinase (cAMP kinase) were utilized as antigens to obtain isozyme specific antisera. Injections of pure catalytic subunit (C) from the type I isozyme resulted in antisera that reacted with C subunit obtained from either isozyme type. Cross-reactivity of the antisera raised against isolated subunits of the kinase was assessed by immunodiffusion analysis and by measuring the cAMP binding and phosphotransferase activities of the subunits after immunoprecipitation. These antisera were used to localize subunits of type I and type II cAMP kinases in rat skeletal muscle, liver, and adrenal by using indirect immunofluorescence and immunoperoxidase techniques. Specificity of the immunofluorescence was shown by absorption of the antisera with pure homologous antigens. In skeletal muscle, both R and C subunits of the type I and type II cAMP kinases were localized in the area of the sarcoplasmic reticulum and in periodic crossbands. Specific fluorescence for these components was observed in both isotropic and anisotropic band regions of the sarcomere. Densitometric determinations of immunoperoxidase staining revealed a larger amount of RI, RII, and C subunits in the isotropic band than in the anisotropic band regions. In liver, C, RI, and RII subunits were distributed both in cytoplasmic and nuclear areas and along plasma membranes of hepatocytes; however, there were qualitative differences observed among these various subcellular sites. With each antiserum, fluorescence was blocked by prior absorption with homologous antigen. After treatment of rats with glucagon, dramatic changes in the relative distribution patterns of C and RII were noted in the nucleus. In the adrenal gland, RI, RII, and C subunits were localized in both cytoplasmic and nuclear areas, and an apparent redistribution of these subunits occurred after treatment of (dexamethasone-suppressed) rats with ACTH. The application of this immunocytochemical approach provides a tool for examining and monitoring the subcellular distribution of these components of cAMP kinase in biological systems.     

Stimulatory effect of vanadate on 3',5'-cyclic guanosine monophosphate-inhibited low Michaelis-Menten constant 3',5'-cyclic adenosine monophosphate phosphodiesterase activity in isolated rat fat pads.

When isolated rat fat pads were incubated with vanadate, the low Michaelis-Menten constant (Km) cAMP phosphodiesterase (PDE) activity in the microsomal fraction was increased in a time- and dose-dependent manner with vanadate. 3',5'-Cyclic GMP inhibited the vanadate-stimulated PDE activity with similar profile to the insulin-stimulated one. The stimulatory effect of vanadate was inhibited by inhibitors of tyrosine kinases such as amiloride, biochanin A, and genistein to various extents. Vanadate and insulin both showed the full effect in the absence of either K+, N+, or Ca2+ in the medium, while preincubation of the fat pads with a chelator of intracellular Ca2+ inhibited the vanadate action in a dose-dependent manner. The insulin action was not inhibited by it at tested concentrations. These results suggest that the vanadate action, in contrast to the insulin one, is dependent on the intracellular level of Ca2+. Preincubation of the fat pads with inhibitors of protein kinase C such as 1-(5-isoquinoline sulfonyl)-2-methylpiperazine (H-7) and staurosporine inhibited, in part, the vanadate action but did not inhibit the insulin one. Furthermore, vanadate increased the protein kinase C activity in fat pads but insulin did not increase. H-7 and amiloride showed a significant inhibition of stimulation of protein kinase C activity by vanadate. These results suggest that vanadate stimulates, in part, the 3',5'-cyclic GMP-inhibited low Km cAMP PDE activity in the microsomal fraction of fat pads through the activation of tyrosine kinase and protein kinase C-mediated processes.     

A 3',5'-cyclic adenosine monophosphate-dependent pathway is responsible for a rapid increase in c-fos messenger ribonucleic acid by adrenocorticotropin.

ACTH rapidly and transiently increases c-fos mRNA in the rat adrenals in vivo. The present investigation was undertaken in order to determine what kind(s) of second messenger systems is involved in this increase. Rat adrenal cells were grown in monolayers in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. After 2 days of culture, cells were treated with ACTH and various agents alone or in combination. The amount of c-fos mRNA was determined by dot blot hybridization and corticosterone levels in the media were measured by RIA. ACTH (300 pg/ml) increased c-fos mRNA transiently with a peak level after 60 min. A similar increase was observed when (Bu)2cAMP (1 mM) was substituted for ACTH. Pretreatment with N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide (H-89), a selective inhibitor of cAMP-dependent protein kinase, suppressed both basal and ACTH-increased c-fos mRNA. H-89 also suppressed corticosterone production. On the other hand, neither 12-O-tetradecanoyl-phorbol-13-acetate (100 ng/ml) nor elevated potassium ion (50 mM) affected the amount of c-fos mRNA and corticosterone production. Furthermore, pretreatment with cycloheximide (5 micrograms/ml) increased both basal and ACTH-increased c-fos mRNA. These results indicate that ACTH increases c-fos mRNA by phosphorylation of preexisting trans-acting factor(s) via cAMP-dependent protein kinase in common with steroidogenesis.     

Up-regulation of 3'5'-cyclic guanosine monophosphate-specific phosphodiesterase in the porcine cumulus-oocyte complex affects steroidogenesis during in vitro maturation

The 3'5'-cyclic GMP (cGMP) pathway is known to influence ovarian functions, including steroidogenesis, ovulation, and granulosa cell proliferation. We show here that cGMP-phosphodiesterase (PDE) activity increased in a gonadotropin-dependent manner more than 3-fold in the cumulus-oocyte complex (COC) after 24 h in vitro maturation (IVM) and up to 5-fold after 48 h. Further characterization of this increase demonstrated that the activity was located primarily in cumulus cells, and was sensitive to sildenafil and zaprinast, two inhibitors specific to both type 5 and 6 PDEs. RT-PCR experiments showed that the mRNAs for cGMP-degrading PDEs 5A and 6C are present in the COC before and after 30 h IVM. Western blotting confirmed the presence of PDE 5A in the COC. Western blotting of PDE 6C revealed a significant up-regulation in the COC during IVM. Isolation and analysis of detergent-resistant membranes suggested that PDE 6C protein, along with half of the total sildenafil-sensitive cGMP-degradation activity, is associated with detergent-resistant membrane in the COC after 30 h IVM. Treatment of porcine COC with sildenafil during IVM caused a significant decrease in gonadotropin-stimulated progesterone secretion. Together, these results constitute the first report exploring the contribution of cGMP-PDE activity in mammalian COC, supporting a functional clustering of the enzyme, and providing the first evidence of its role in steroidogenesis.     

Characterization of a liver low Michaelis-Menten constant 3',5'-cyclic adenosine monophosphate phosphodiesterase activity sensitive to thyroid status

These studies were undertaken to assess the subcellular distribution and some biochemical properties of the hepatic cAMP phosphodiesterase(s) whose activity is modulated by the thyroid status in the rat. Thyroidectomy led to a 2-fold increase in low Michaelis-Menten constant (Km) cAMP phosphodiesterase activity in Golgi-endosomal fractions, but little affected this activity in crude particulate fractions. On analytical sucrose density gradients, an increase in cAMP phosphodiesterase activity in particulate elements which equilibrated at densities 1.17-1.22 was also observed. Acute insulin treatment did not further increase cAMP phosphodiesterase activity in Golgi-endosomal fractions of thyroidectomized rats. Up to 75% of the cAMP phosphodiesterase activity associated with Golgi-endosomal fraction of euthyroid and hypothyroid rats was inhibited by cGMP (IC50, 10 microM and 1 microM, respectively). Activity was also potently inhibited by griseolic acid, cilostamide, and cilostazole (IC50, less than 1 microM) but was much less sensitive to R0-20-1724 (IC50, 1 mM). Treatment of Golgi-endosomal fractions by a hypotonic extract of rat liver lysosomes led to the solubilization of 50% of low Km cAMP phosphodiesterase activity. On sucrose density gradients, the solubilized activity migrated as a slightly asymmetrical peak with a sedimentation coefficient of 6 S in euthyroid rats and 6.9 S in hypothyroid rats. On nondenaturing polyacrylamide gel electrophoresis, the activity migrated as two majors peaks with Rf values of 0.23 and 0.50; only the activity associated with the fast-moving peak was increased by thyroidectomy. On diethylaminoethyl-Sephacel chromatography, four peaks of cAMP phosphodiesterase activity, two of which were cGMP-inhibitable, were resolved. Thyroidectomy increased the activity associated with one of the cGMP-inhibitable peaks (eluted at 0.7-0.9 M sodium acetate) and led to the appearance of a new peak of activity (eluted at 0.4 M), which was not sensitive to cGMP. These results show that the low Km phosphodiesterase activity associated with liver Golgi-endosomal fractions, previously shown to be increased in hyperinsulinemic rats, is also increased in hypothyroid animals. They also suggest that, based on pharmacological and physical criteria, the enzyme species affected by the thyroid status belongs to the cGMP-and cilostamide-inhibited subclass of low Km cAMP phosphodiesterases.     

Interrelationship between adenylate cyclase activity, adenosine 3':5' cyclic monophosphate phosphodiesterase activity, adenosine 3':5' cyclic monophosphate levels, and growth of cells in culture

To investigate how cell population density influences the intracellular concentration of cyclic AMP we have measured adenylate cyclase and cyclic AMP phosphodiesterase activities and cyclic AMP levels at various stages of cell density in normal rat-kidney (NRK) cells, which exhibit contact-inhibition of growth, and in normal chick-embryo fibroblasts (CEF), which do not show contact inhibition of growth under our conditions. Until NRK cells reach confluency, both activities increase with increasing cell population and cyclic AMP levels are low. As NRK cells reach confluency, cyclic AMP phosphodiesterase activity decreases somewhat whereas adenylate cyclase activity continues to rise. This increase in synthetic ability is accompanied by the increase in cyclic AMP levels which occurs in these cells at confluency. In CEF grown in 5% serum where density-dependent inhibition of growth is not observed, both adenylate cyclase and cyclic AMP phosphodiesterase activities increase proportionately with increasing cell population density. No significant alteration occurs in the ratio between these two enzyme activities and no change is observed in cyclic AMP levels.The NaF-stimulated activity in NRK cells increases with increasing cell density until the cells reach confluency; thereafter the NaF-stimulated activity remains constant. In contrast, the NaF-stimulated activity observed in CEF does not vary appreciably between light and heavy density.The observed changes in the enzymes of cyclic AMP metabolism accurately reflect the changes in cyclic AMP concentration as a function of cell population density. The data indicate that these two enzyme activities respond to increasing cell density to elicit a rise in intracellular cyclic AMP levels. The elevated cyclic AMP levels are thought to be involved in the regulation of cellular growth rate and the mediation of contact inhibition of growth.     

Glucocorticoid enhancement of adrenocorticotropin-induced 3',5'-cyclic adenosine monophosphate production by cultured ovine adrenocortical cells

The present study examines the effect of chronic treatment of glucocorticoids on ACTH1-24- or forskolin-induced cAMP output of cultured sheep adrenocortical cells. Cells cultured for 2 days in the presence of 1 microM dexamethasone released more cAMP in response to ACTH1-24 than did untreated cells, both in the absence and presence of 0.5 mM 1-methyl-3-isobutylxanthine. Such an enhancing effect required greater than or equal to 21 h of treatment and was both concentration-dependent and steroid specific. The ED50 of dexamethasone was about 10 nM, while that of cortisol and corticosterone was about 1 microM; testosterone at concentrations less than or equal to 10(-5) M had no enhancing effect. Glucocorticoids enhanced the cAMP response to ACTH1-24 without altering its ED50. Treatment of cultures with aminoglutethimide or the antiglucocorticoid RU 38486 for 48 h resulted in a dose-dependent decrease in ACTH1-24-induced cAMP output. Moreover, RU 38486 antagonized the enhancing effect of dexamethasone. Glucocorticoids did not increase the cAMP response to forskolin. These results suggest that chronic exposure to glucocorticoids is necessary for the full expression of the cAMP response to ACTH1-24 of adrenocortical cells from adult sheep.     

Cyclic adenosine 3',5'-monophosphate-dependent activation of mitogen-activated protein kinase in cumulus cells is essential for germinal vesicle breakdown of porcine cumulus-enclosed oocytes

MAPK plays an important role during meiotic maturation in mammalian oocytes, whereas the necessity of MAPK during meiotic resumption in porcine oocytes is still controversial. Here, by applying the method of ultracentrifugation to move the opaque lipid droplets to the edge of the oocyte, therefore allowing clear visualization of porcine germinal vesicles, oocytes just before germinal vesicle breakdown (GVBD) and those that had just undergone GVBD were selected for the assay of MAPK activation. Our results showed that phosphorylation of MAPK in oocytes occurred after GVBD in all three different culture models: spontaneous maturation model, inhibition-induction maturation model, and normal maturation model. Moreover, we found that activation of MAPK in cumulus cells but not in oocytes was essential for GVBD in cumulus-enclosed oocytes. Then the cross-talk between cAMP and MAPK in cumulus cells was investigated by using cell-type-specific phosphodiesterase (PDE) isoenzyme inhibitors. Our results showed that PDE3 subtype existed in oocytes, whereas PDE4 subtype existed in cumulus cells. PDE3 inhibitor prevented meiotic resumption of oocytes, whereas PDE4 inhibitor enhanced the ability of FSH or forskolin to activate MAPK in cumulus cells. We propose that increased cAMP resulting from inhibition of PDE3 in oocytes blocks GVBD, whereas increased cAMP resulting from inhibition of PDE4 activates MAPK pathway in cumulus cells, which is essential for GVBD induction.     

Divalent cations modulate PTH-dependent 3',5'-cyclic adenosine monophosphate production in renal proximal tubular cells.

The kidney and parathyroid gland play key roles in calcium (Ca++) homeostasis. Recent data suggest that the kidney, in addition to being a primary target for PTH, also recognizes changes in the concentration of extracellular Ca++, thereby modulating hormone-dependent cAMP production, 1,25-dihydroxyvitamin D synthesis, and renin secretion. In this study, we examined: 1) the effects of varying concentration of divalent cations on PTH-dependent cAMP production in renal proximal tubular cells; and 2) the mechanisms by which extracellular Ca++ exerts its inhibitory effects on cAMP production. Single cell suspensions composed of 80-90% proximal tubular cells were prepared from cortical homogenates by collagenase digestion and sieving. In the presence of 1 mM isobutylmethylxanthine, cAMP content was measured by RIA in 5-15 min incubations and showed a 5- to 6-fold increase in response to PTH (10(-11) -10(-6) M). Increasing extracellular Ca++ and magnesium (Mg++) from 0 and 0.5 mM, respectively, to 5.0 mM inhibited PTH-dependent (3 x 10(-9) M) cAMP production by 54 +/- 4% and 47 +/- 6%, respectively. The half maximal inhibitory concentration for both Ca++ and Mg++ was 0.9 mM. In addition, increasing extracellular barium (Ba++) or strontium (Sr++) from 0-10 mM inhibited PTH-dependent (3 x 10(-9) M) production by 54 +/- 7% and 62 +/- 6% with half of the maximal observed inhibition at 2.2 and 2.7 mM, respectively. The inhibition of PTH-dependent cAMP production by 2.5 mM Ca++ was not reversed by the calcium channel blockers diltiazem or verapamil (10(-4) M). However, changes in intracellular calcium may play some role in the inhibitory effects of Ca++ on cAMP production, since ionomycin (10(-6)-10(-5) M) lowered PTH-dependent cAMP production by 25-36%. Our data suggest that the proximal tubular cell can sense physiologically relevant changes in Ca++, providing a potential mechanism for the modulation of 1,25-dihydroxyvitamin D production or other tubular functions relevant to fluid and mineral homeostasis.     

GATA4 reduction enhances 3',5'-cyclic adenosine 5'-monophosphate-stimulated steroidogenic acute regulatory protein messenger ribonucleic acid and progesterone production in luteinized porcine granulosa cells

Previous studies with cultured granulosa cells implicated GATA4 in gonadotropin regulation of the steroidogenic acute regulatory protein (STAR) gene. Caveats to these prior studies exist. First, GATA4 levels are reduced in granulosa-luteal cells after the LH surge when GATA6 expression is relatively high. Second, STAR mRNA expression is negligible in granulosa cells until after the LH surge. Both exogenous GATA4 and GATA6 can transactivate STAR gene promoter constructs. We used an RNA interference (RNAi) approach to determine the contributions of GATA4 and GATA6 to cAMP analog regulation of the endogenous STAR gene in luteinizing granulosa cells. STAR mRNA was stimulated by cAMP under control RNAi conditions. Surprisingly, GATA4 reduction by its respective RNAi approximately doubled the cAMP induction of STAR mRNA. At 24 h cAMP treatment, this augmentation was abolished by co-down-regulation of GATA4+GATA6. GATA6 down-regulation by itself did not alter STAR mRNA levels. GATA4+GATA6 co-down-regulation elevated basal CYP11A mRNA at 24 h treatment but did not affect its induction by cAMP. Basal levels of HSD3B mRNA were reduced by GATA4 RNAi conditions leading to a greater fold induction of its mRNA by cAMP. Fold cAMP-stimulated progesterone production was enhanced by GATA4 down-regulation but not by GATA4+GATA6 co-down-regulation. These data implicate GATA6 as the facilitator in cAMP-stimulated STAR mRNA and downstream progesterone accumulation under reduced GATA4 conditions. Data also demonstrate that basal levels of GATA4/6 are not required for cAMP induction of the STAR gene. The altered ratio of GATA4 to GATA6 after ovulation may allow GATA6 to enhance STAR mRNA accumulation.     

Bovine pituitary folliculo-stellate cells have beta-adrenergic receptors positively coupled to adenosine 3',5'-cyclic monophosphate production

The specific beta-adrenergic radioligand [125I]iodocyanopindolol (ICYP) was used to identify and characterize beta-adrenergic receptors in bovine pituitary folliculo-stellate cells (bFSC) grown in culture. Saturation analysis demonstrated the binding of ICYP to bFSC particulate fractions to be of high affinity (apparent Kd = 80 pM) and low capacity (Bmax = 37 fmol/mg protein). The specific beta-adrenergic radioligand [3H] dihydroalprenolol also bound to bFSC particulate preparations with parameters compatible with binding to the beta-adrenergic receptor (Kd = 3.0 nM; Bmax = 52 fmol/mg protein). No specific binding was observed with either the dopamine receptor radioligand [3H]spiperone or the alpha-adrenergic radioligand [3H]dihydro-alpha-ergocryptine. The bFSC beta-adrenergic receptors were further characterized by computer modeling of competition studies with a variety of agonists and antagonists selective for beta-adrenergic subtypes. The pharmacological profiles of ICYP binding obtained from these studies indicated that approximately equal proportions of both beta 1- and beta 2-adrenergic subtypes are expressed in cultured bFSC. Bovine FSC beta-adrenergic receptors are functionally coupled to activation of cAMP. The beta-adrenergic agonists isoproterenol, epinephrine, and norepinephrine provoked a rapid and marked stimulation of intracellular cAMP accumulation. The approximately equipotent effect of epinephrine and norepinephrine indicated that the beta-adrenergic effect on cAMP production is principally mediated via the beta 1-adrenergic receptor. The identification of beta-adrenergic receptors on bFSC positively coupled to adenylate cyclase provides a possible regulatory control pathway for the proposed role of pituitary FSC in the modulation of anterior pituitary hormone secretion.     

Effect of partially purified NSILA on adenylate cyclase, phosphodiesterase and 3',5'-cyclic AMP in fat cells.

Partially purified, non-suppressible, insulin-like material (NSILA-S) was studied with respect to its effect on the levels of 3',5'-cyclic adenosine monophosphate (cAMP) and its mechanism of action in the control of this nucleotide in rat fat cells. NSILA-S prevents the rise of cAMP in fat cells under the influence of isoproterenol with similar kinetics to insulin. A maximal effect is observed at about 70 ng/ml with a biological activity equivalent to 200 muU/ml of insulin. NSILA-S inhibits norepinephrine-stimulated adenylate cyclase activity in fat cell ghosts and partially purified plasma membrane preparations. At 10 mM Mg2+, the inhibition is characterized by an effect of Vmax without change in affinity towards ATP (apparent KM 30 muM). Similarly there is no observed change in affinity towards Mg2+. With respect to inhibition of norepinephrine-stimulated adenylate cyclase, the dose-response curve of NSILA-S is similar to that already found with intact cells. The effect of norepinephrine is inhibited throughout the dose-response range between 5 X 10(-7) and 5 X 10(-4) M. In contrast to previous observations with insulin in ghosts, NSILA-S inhibits the basal adenylate cyclase activity. Cyclic nucleotide phosphodiesterase activity in homogenates as measured at 1.0 muM substrate is increased by 90% after previous incubation of fat cells with NSILA-S. The study suggests that the anti-lipolytic effect of NSILA-S is mediated by a lowering of cAMP through inhibition of the adenylate cyclase and/or stimulation of the phosphodiesterase system.     

Inhibition of thyroxine 5'-deiodination type II in cultured human placental cells by cortisol, insulin, 3', 5'-cyclic adenosine monophosphate, and butyrate

The regulation of conversion of thyroxine (T4) to 3,5,3'-triiodothyronine (T3) by the type II iodothyronine deiodinating pathway was studied in normal human placental cells cultured from the chorionic membrane. T4 5'-deiodination was measured in cell sonicates after intact cells were incubated with test agents for 24 to 48 hours. Stimulation of T4 5'-deiodination occurred to a similar degree after depriving cells of thyroid hormone in serum-free medium and in medium containing 10% calf serum. Cortisol at 10 to 100 nmol/L in serum-free medium inhibited T4 5'-deiodination up to 36%, and 1 to 100 nmol/L of insulin inhibited deiodination up to 50%. Dibutyryl-cyclic AMP (dbcAMP) inhibited deiodination, but this appeared to result from the inhibitory effects of butyrate. Addition to the culture media of 8-bromo-cAMP, cholera toxin, and theophylline each caused partial inhibition of T4 5'-deiodination, strongly suggesting an inhibitory effect of raised intracellular cAMP. Neither alpha- nor beta-adrenergic agonists had any effect when added to the culture medium, nor did glucagon or cysteamine. These results demonstrate a complex, multihormonal control of human placental type II iodothyronine deiodination, and suggest that changes in the activity of this pathway may result in altered intracellular, and conceivably circulating, T3 concentrations in states of cortisol excess and marked hyperinsulinism. The factor that regulates type II deiodination via cAMP remains to be identified.     

Regulation of tetrahydrobiopterin biosynthesis in cultured adrenal cortical tumor cells by adrenocorticotropin and adenosine 3',5'-cyclic monophosphate

Y-1 adrenal cortical tumor cells in culture, which contain substantial amounts of tetrahydrobiopterin [6R-(L-erythro-1',2'-dihydroxypropyl)5,6,7,8-tetrahydropterin] (BH4) and GTP cyclohydrolase (GTP-CH), were used to study the regulation of BH4 biosynthesis by ACTH and cAMP. ACTH produced a dose-dependent increase in steroidogenesis, BH4 levels and GTP-CH activity. Maximal stimulation of BH4 biosynthesis occurred at the same concentration of ACTH that caused maximal stimulation of steroidogenesis. ACTH-(1-24) was more potent than ACTH-(1-39). The stimulation of BH4 biosynthesis by ACTH was dependent on cell density, being greater at lower cell densities, but was independent of time in culture. The lack of stimulation by ACTH at higher cell densities was due to an increase in the specific activity of GTP-CH in the control cells as density increased. This increase may be due in part to the increased release of steroids, since exogenous steroids added to low density cultures also resulted in an increase in the specific activity of the enzyme. Addition of steroids had no effect on ACTH-dependent stimulation of BH4 biosynthesis at low cell densities. (Bu)2cAMP, 8-bromo-cAMP, and forskolin all produced time- and dose-dependent increases in BH4 levels, GTP-CH activity, and steroidogenesis. Maximum increases in GTP-CH and BH4 occurred at concentrations similar to those required for maximal stimulation of steroidogenesis. In the Kin-8 mutant of Y-1 cells, which has a type 1 cAMP-dependent protein kinase with an altered regulatory subunit, ACTH was unable to increase BH4 levels or GTP-CH activity at a concentration that produced maximal stimulation of BH4 and steroid biosynthesis in the parent Y-1 line. These studies indicate that Y-1 cells in culture are useful for studying the regulation of BH4 biosynthesis in the adrenal cortex.