Inhibition of glucagon-induced glycogenolysis in isolated rat hepatocytes by the Rp diastereomer of adenosine cyclic 3',5'-phosphorothioate

The ability of the Rp diastereomer of adenosine cyclic 3',5'-phosphorothioate (Rp cAMPS) to inhibit glucagon-induced glycogenolysis was studied in hepatocytes isolated from fed rats. Preincubation of the cells for 20 min with progressively higher concentrations of Rp cAMPS followed by a 1 X 10(-9) M glucagon challenge resulted in a 50% inhibition of glucose production over a 30-min period at 2-3 X 10(-6) M Rp cAMPS. A maximal inhibition of 50-74% was achieved, the actual value depending upon the length of preincubation with Rp cAMPS. The inhibitory effect did not increase when the concentration of Rp cAMPS was increased from 3 X 10(-6) to 3 X 10(-4) M. Addition of 1 X 10(-5) M Rp cAMPS to the cells followed by 10(-11) to 10(-6) M glucagon shifted the glucagon concentration required for half-maximal glucose production measured at 10 min to 6-fold higher glucagon concentrations and the concentration of glucagon required for apparent maximal glucose production measured at 10 min to greater than 10-fold higher glucagon concentrations. The cAMP-dependent protein kinase activation curve was similarly shifted to higher concentrations of glucagon. These data show that Rp cAMPS acts as a cAMP antagonist capable of opposing the glucagon-induced activation of cAMP-dependent protein kinase and the concomitant activation of the glycogenolytic cascade.     

A study of the mechanism of glucagon-induced protein phosphorylation in isolated rat hepatocytes using (Sp)-cAMPS and (Rp)-cAMPS, the stimulatory and inhibitory diastereomers of adenosine cyclic 3',5'-phosphorothioate

Maximal doses of glucagon increase the phosphorylation state of 12 cytosolic proteins in isolated hepatocytes from fasted rats (Garrison, J. C., and Wagner, J. D. (1982) J. Biol. Chem. 257, 13135-13143). Incubation of hepatocytes with lower concentrations of glucagon indicates that a hierarchy of substrates exists with the concentration of glucagon required for half-maximal increases in phosphorylation varying 5-15-fold. The proteins whose phosphorylation state is most sensitive to low concentrations of glucagon are pyruvate kinase and 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, both of which play key roles in the regulation of gluconeogenesis. Treatment of hepatocytes with (Sp)-cAMPS, the stimulatory diastereomer of adenosine cyclic 3',5'-phosphorothioate, mimics the response seen with glucagon. When hepatocytes are pretreated with the cAMP antagonist, (Rp)-cAMPS, the phosphorylation response is abolished at low concentrations of glucagon, and the dose of glucagon required for half-maximal stimulation of phosphorylation is increased 5-10-fold. The (Sp)-cAMPS-stimulated increases in phosphorylation state are also blunted by (Rp)-cAMPS. These results provide direct pharmacological evidence for the activation of the cAMP-dependent protein kinase in response to glucagon in the intact cell. Although low doses of glucagon appear to stimulate protein phosphorylation via the cAMP-dependent protein kinase, high doses of glucagon also cause a small increase in the concentration of free intracellular Ca2+ in hepatocytes. The glucagon-stimulated increases in the level of Ca2+ can be mimicked by (Sp)-cAMPS and inhibited by pretreatment with (Rp)-cAMPS. These results suggest that glucagon can elevate intracellular Ca2+ via cAMP and the cAMP-dependent protein kinase.     

Inhibition of glycogenolysis by 2,5-anhydro-D-mannitol in isolated rat hepatocytes

2,5-Anhydro-D-mannitol, an analog of D-fructofuranose, inhibited basal and glucagon-stimulated glycogenolysis and glucose production in hepatocytes isolated from fed rats. Glucose formation from galactose was unaffected by the inhibitor. 2,5-Anhydro-D-mannitol-1-phosphate inhibits phosphorylase alpha with a Ki value of 2.4 mM. This same phosphorylated metabolite accumulates to the extent of 9.2 mumol/g wet wt in treated hepatocytes suggesting that phosphorolysis is the locus of the inhibition of glucose production from glycogen. Our results suggest that 2,5-anhydro-D-mannitol can be used to produce a model of hereditary fructose intolerance and that it merits further study as a hypoglycemic agent.     

Inhibition of Escherichia coli growth by cyclic adenosine 3', 5'-monophosphate

Cyclic AMP inhibits growth rate of E. coli Hfr 3000. Doubling times in glucose minimal medium increased from 60 to about 90 minutes with the addition of 5 mM cAMP. This effect is specific since it was not observed when the cyclic nucleotide was replaced by 5′ AMP, ADP, ATP or adenosine. Half maximal inhibition was obtained with 1 to 3 mM cyclic AMP. This inhibition occurs only with those carbon sources which are known to decrease intracellular cyclic AMP levels, i.e. glucose and pyruvate. No inhibition was observed with succinate, malate or glycerol.     

Inhibition of choriogonadotropin-activated steroidogenesis in cultured Leydig tumor cells by the Rp diastereoisomer of adenosine 3',5'-cyclic phosphorothioate

The diastereoisomers of adenosine 3',5'-cyclic phosphorothioate, (Sp)-cAMPS and (Rp)-cAMPS, have been previously shown to act as agonists and antagonists, respectively, in the activation of several mammalian cAMP-dependent protein kinases. In an effort to characterize further the involvement of cAMP in the activation of Leydig cell steroidogenesis by lutropin/choriogonadotropin (LH/CG), we examined the effects of these cyclic nucleotide analogues on a clonal strain of cultured murine Leydig tumor cells (designated MA-10). Our results show that (i) (Sp)-cAMPS activates and (Rp)-cAMPS inhibits the isolated cAMP-dependent protein kinase of the MA-10 cells; (ii) both analogues inhibit the isolated cAMP phosphodiesterase(s); (iii) (Sp)-cAMPS activates steroid biosynthesis in intact cells, but (Rp)-cAMPS does not; and (iv) (Rp)-cAMPS is a competitive inhibitor of the activation of steroidogenesis by (Sp)-cAMPS, 8-bromo-cAMP, human CG, cholera toxin, and forskolin. However, (Rp)-cAMPS is a more effective inhibitor when steroidogenesis is activated by (Sp)-cAMPS or 8-bromo-cAMP than when it is activated by human CG, cholera toxin, or forskolin. This difference appears to be related to the combined effects of (Rp)-cAMPS on the cAMP-dependent protein kinases and cAMP phosphodiesterase(s). We conclude that cAMP is a quantitatively important mediator of the activation of steroidogenesis by LH/CG even at low concentrations of hormone where an increase in steroid biosynthesis cannot be easily correlated with increased cAMP accumulation. Thus, our data indicate that if other second messengers are involved in the activation of steroidogenesis by LH/CG, they must do so by acting together with, rather than independently of, cAMP.     

Effects of glucagon, phosphodiesterase inhibitors, and trypsin treatment on cyclic 3',5' adenosine monophosphate levels in isolated hepatocytes.

Cyclic 3',5' adenosine monophosphate (cyclic AMP) levels were measured in isolated hepatocytes under several conditions. Following the addition of glucagon cyclic AMP levels increased rapidly with peak values occurring at three minutes. The increase in cyclic AMP was dose dependent. Significant increases were found with 10(-10)M glucagon and a maximum increase of twenty fold was produced by 10(-8) M glucagon. This action of glucagon was augmented by the phosphodiesterase inhibitors, theophylline, SQ 20,009, and papaverine. Treatment of the hepatocytes with trypsin markedly reduced the response to glucagon.     

Fatty acid oxidation and esterification in isolated rat hepatocytes: regulation by dibutyryl adenosine 3',5'-cyclic monophosphate

Isolated rat hepatocytes rapidly utilized [(14)C]palmitate and, in particular, synthesized large amounts of neutral lipids from palmitate. Incorporation into cellular lipids occurred at a linear rate proportional to the medium concentration of fatty acids. Oxidation of [(14)C]palmitate to CO(2) increased with time and was much slower than palmitate esterification. Since [(14)C]acetate and [(14)C]glucose were oxidized to CO(2) at a linear rate, the lag in fatty acid oxidation to CO(2) did not involve enzymatic steps subsequent to acetate formation. The relative contribution of palmitate to esterification and to CO(2) formation depended upon the molar ratio of palmitate to albumin (v) and the length of incubation. Dibutyryl cyclic AMP (1 mM) reduced the oxidation of palmitate and acetate to CO(2) by about 50 and 90%, respectively, but did not alter palmitate esterification. However, equivalent concentrations of sodium butyrate produced similar decreases in CO(2) formation. Dibutyryl cyclic AMP (1 mM) also stimulated palmitate oxidation to water-soluble products, principally ketone bodies, by 50-100%. Sodium butyrate exerted no effect, while monobutyryl cyclic AMP and cyclic AMP both stimulated this pathway significantly. These results indicate that both v and dibutyryl cyclic AMP regulate the metabolism of fatty acids by isolated hepatocytes and suggest that hormonal stimulation of adenyl cyclase controls hepatic lipid metabolism.     

Endogenous inhibitors of cyclic adenosine 3',5'-monophosphate-phosphodiesterase in rat epididymis

Stabilization of cyclic adenosine 3',5'-monophosphate (cAMP)-phosphodiesterase (PDE) in 50% glycerol made possible the removal of endogenous inhibitors from tissue extracts by dialysis and the storage of the extracts at -20 degrees C without loss of PDE activity. Dialysates of heat-inactivated epididymal extracts were fractionated by liquid chromatography, and 4 fractions-F2, F5, F7, and F12-were found to contain endogenous inhibitors of PDE. The masses of the fractions required to inhibit low-Km PDE activity by ca. 50% in 430-microliter incubation mixtures were F2, 89 micrograms; F5, 23 micrograms; F7, 275 micrograms; and F12, 1.2 mg. The mechanisms of inhibition of low-Km PDE by the endogenous inhibitors were investigated by kinetic analysis of enzyme-inhibitor interaction. F2 and F12 inhibited PDE competitively; F5 and F7 decreased both apparent Km and Vmax, suggesting an uncompetitive mechanism of inhibition. The high potency of F5 in low concentration suggests that it may be a physiological modulator of low-Km cAMP-PDE activity.     

Inhibition of cGMP-dependent protein kinase by (Rp)-guanosine 3',5'-monophosphorothioates

The activation of the cGMP-dependent protein kinase and cAMP-dependent protein kinase by the diastereomers of guanosine 3',5'-monophosphorothioate, (Sp)-cGMPS and (Rp)-cGMPS, and 8-chloroguanosine 3',5'-monophosphorothioate, (Sp)-8-Cl-cGMPS and (Rp)-8-Cl-cGMPS, was investigated using the peptide Kemptide as substrate. The (Sp)-diastereomers, which have an axial exocyclic sulfur atom, bound to the cGMP-dependent protein kinase and stimulated its phosphotransferase activity. In contrast, the (Rp)-isomers, which have an equatorial exocyclic sulfur atom, bound to the enzyme without stimulation of its activity. (Rp)-cGMPS and (Rp)-8-Cl-cGMPS antagonized the activation of the cGMP-dependent protein kinase with a Ki of 20 microM and 1.5 microM, respectively. (Rp)-cGMPS also antagonized the activation of cAMP-dependent protein kinase with a Ki of 20 microM. In contrast, (Rp)-8-cGMPS ws a weak inhibitor of the cAMP-dependent protein kinase with a Ki of 100 microM. (Rp)-8-Cl-cGMPS appears to be a rather selective inhibitor of the cGMP-dependent protein kinase and may be a useful tool for studying the role of cGMP in broken and intact cell systems.     

Modulation of oocyte maturation by cyclic adenosine 3', 5'-pyrophosphate

The germinal vesicle (GV) of follicle-enclosed oocytes in mammals remains arrested at the dictyate state of meiosis. Upon releasing the oocytes from the follicles, the meiotic process resumes, leading to dissolution of the GV (GVBD), suggesting that factors in the follicular fluid sustain the meiotic arrest of oocytes. In the present study the spontaneous resumption of meiosis was blocked by the addition of cyclic adenosine 3', 5'-pyrophosphate (cAPP) plus dibutyryl cyclic adenosine 3',5'-monophosphate (dbcAMP), at final concentrations of 25 and 50 microM, respectively. These compounds were ineffective when added separately at these concentrations. None of the other related compounds tested with dbcAMP blocked GVBD. Bovine follicular fluid (BFF) was analyzed for inhibitors of GVBD. BFF was extracted with 70% ethanol and the ethanolic extract chromatographed on Dowex 1-X8 column. The fraction eluted with 0.1 N HCl markedly inhibited GVBD of isolated mouse oocytes in combination with dbcAMP. The active BFF substance and cAPP block spontaneous GVBD of mouse oocytes and may be related substances. The present study supports the thesis that meiotic arrest at the dictyate stage in oocytes is sustained by factors present in follicular fluid and may act in association with cAMP.     

An equilibrium study of the cooperative binding of adenosine cyclic 3',5'-monophosphate and guanosine cyclic 3',5'-monophosphate to the adenosine cyclic 3',5'-monophosphate receptor protein from Escherichia coli

The binding of adenosine cyclic 3',5'-monophosphate (cAMP) and guanosine cyclic 3',5'-monophosphate (cGMP) to the adenosine cyclic 3',5'-monophosphate receptor protein (CRP) from Escherichia coli was investigated by equilibrium dialysis at pH 8.0 and 20 degrees C at different ionic strengths (0.05--0.60 M). Both cAMP and cGMP bind to CRP with a negative cooperativity that is progressively changed to positive as the ionic strength is increased. The binding data were analyzed with an interactive model for two identical sites and site/site interactions with the interaction free energy--RT ln alpha, and the intrinsic binding constant K and cooperativity parameter alpha were computed. Double-label experiments showed that cGMP is strictly competitive with cAMP, and its binding parameters K and alpha are not very different from that for cAMP. Since two binding sites exist for each of the cyclic nucleotides in dimeric CRP and no change in the quaternary structure of the protein is observed on binding the ligands, it is proposed that the cooperativity originates in ligand/ligand interactions. When bound to double-stranded deoxyribonucleic acid (dsDNA), CRP binds cAMP more efficiently, and the cooperativity is positive even in conditions of low ionic strength where it is negative for the free protein. By contrast, cGMP binding properties remained unperturbed in dsDNA-bound CRP. Neither the intrinsic binding constant K nor the cooperativity parameter alpha was found to be very sensitive to changes of pH between 6.0 and 8.0 at 0.2 M ionic strength and 20 degrees C. For these conditions, the intrinsic free energy and entropy of binding of cAMP are delta H degree = -1.7 kcal . mol-1 and delta S degree = 15.6 eu, respectively.     

Regulation of prostaglandin endoperoxide synthase by cyclic adenosine 3',5'-monophosphate in the in vitro-perfused rat ovary

The preovulatory regulation of two enzymes in the prostaglandin biosynthetic pathway, prostaglandin endoperoxide synthase (PGS) and prostacyclin synthase (ISN), was examined in granulosa cells and residual tissue of rat ovaries perfused in vitro. Ovaries from rats primed with pregnant mare's serum gonadotropin (20 IU) were perfused for up to 20 h starting the morning of induced proestrus. The amounts of PGS and ISN present were analyzed with immunoblotting techniques. Soluble extracts from granulosa cells and residual ovarian tissues were obtained at different times (0 h, 3 h, 7 h, 12 h) after treatment in vitro with luteinizing hormone (LH, 0.1 microgram/ml) and 3-isobutyl-1-methylxanthine (IBMX; 0.2 mM) and at 7 h in untreated control ovaries or after treatment with forskolin (30 microM) or LH (0.1 microgram/ml). The levels in the perfusion medium of cyclic adenosine 3',5'-monophosphate (cAMP), progesterone, testosterone, and estradiol were measured and the number of ovulations were examined. The levels of PGS after treatment with LH + IBMX increased up to 7 h and remained high at 12 h, a time that is close to the time of ovulation. The increase was more pronounced in the granulosa cells than in the residual tissue. Treatment with forskolin induced synthesis of PGS in granulosa cells, and the levels at 7 h were similar to those after stimulation with LH + IBMX. The levels of PGS were lower in granulosa cells of the group stimulated with LH alone than in granulosa cells from ovaries stimulated with LH + IBMX or forskolin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Excretion and degradation of exogenous adenosine 3',5'-monophosphate by isolated perfused rat kidney

To determine the role played by the kidney in the metabolism and excretion of plasma adenosine 3′,5′-monophosphate (cAMP) we have studied the fate of this nucleotide (0.01–1.0mM) when it is perfused in a recirculating medium through the isolated rat kidney. cAMP was rapidly taken up and degraded by the kidney, the rate of its disappearance from the perfusate being at least twice its rate of excretion in the urine. Nevertheless, the cAMP excretory rate exceeded the filtration rate by 1.5 to 2 fold, and thus net secretion (transtubular transport) was demonstrated. The rates of filtration, perfusate clearance, and degradation of cAMP were proportional to its perfusate concentration. Methyl xanthines (caffeine and aminophylline) at 10mM, and probenecid at 0.9mM abolished transtubular transport of cAMP and greatly retarded disappearance of the nucleotide from the perfusate. It is concluded that there is a ready penetration of cAMP into renal cells from peritubular capillaries. Depending on the perfusate concentration of cAMP, transtubular transport may or may not exceed the simultaneous intra-renal breakdown of the compound. A low rate of cAMP excretion in the urine may accompany a considerably higher rate of cAMP clearance from the perfusate by the kidney.