N6-monobutyryl cAMP Research Articles

Pivotal role of cAMP in the activation of liver glycogen breakdown in high-fat diet fed mice

AimsLiver glycogen catabolism was evaluated in male Swiss mice fed a high-fat diet rich in saturated fatty acids (HFD) or normal fat diet (NFD) during one week. Main methodsLiver glycogenolysis (LG) and liver glucose production (LGP) were measured either under basal or stimulated conditions (infusion of glycogenolytic agents). Thus, isolated perfused livers from HFD and NFD mice were infused with glycogenolytic agents, i.e., glucagon, epinephrine, phenylephrine, isoproterenol, adenosine-3′-5′-cyclic monophosphate (cAMP), N6,2′-O-dibutyryl-cAMP (DB-cAMP), 8-bromoadenosine-cAMP (8-Br-cAMP) or N6-monobutyryl-cAMP (N6-MB-cAMP). Moreover, glycemia and liver glycogen content were measured. Key findingsGlycemia, liver glycogen content and basal rate of LGP and LG were not influenced by the HFD. However, LGP and LG were lower (p<0.05) in HFD mice during the infusions of glucagon (1nM), epinephrine (20μM) or phenylephrine (20μM). In contrast, the activation of LGP and LG during the infusion of isoproterenol (20μM) was not different (HFD vs. NFD). Because glucagon showed the most prominent response, the effect of cAMP, its intracellular mediator, on LGP and LG was investigated. cAMP (150μM) showed lower activation of LGP and LG in the HFD group. However, the activation of LGP and LG was not influenced by HFD whether DB-cAMP (3μM), 8-Br-cAMP (3μM) or N6-MB-cAMP (3μM) were used. SignificanceThe activation of LGP and LG depends on the intracellular availability of cAMP. It can be concluded that cAMP played a pivotal role on the activation of LG in high-fat diet fed mice.

Decreased response to cAMP in the glucose and glycogen catabolism in perfused livers of Walker-256 tumor-bearing rats

The hepatic response to cyclic adenosine monophosphate (cAMP) and N6-monobutyryl-cAMP (N6-MB-cAMP) in the glucose and glycogen catabolism and hepatic glycogen levels were evaluated in Walker-256 tumor-bearing rats, on days 5 (WK5), 8 (WK8), and 11 (WK11) after the implantation of tumor. Rats without tumor fed ad libitum (fed control rats) or that received the same daily amount of food ingested by anorexics tumor-bearing rats (pair-fed control rats) or 24 h fasted (fasted control rats) were used as controls. Glucose and glycogen catabolism were measured in perfused liver. Hepatic glycogen levels were lower (p < 0.05) in WK5, WK8, and WK11 rats in comparison with fed control rats, but not in relation to the pair-fed control rats. However, the stimulatory effect of cAMP (3 and 9 μM) in the glycogen catabolism was lower (p < 0.05), respectively, in WK5 and WK8 rats compared to the pair-fed and fed control rats. Accordingly, the suppressive effect of cAMP (6 μM) in the glucose catabolism, under condition of depletion of hepatic glycogen (24 h fast), was lower (p < 0.05) in WK5 and WK11 rats than in fasted control rats. Similarly, the suppressive effect of N6-MB-cAMP (1 μM), a synthetic analogue of cAMP that it is not degraded by phosphodiesterase 3B (PDE3B), in the glucose catabolism was lower (p < 0.05) in WK5 rats compared to fasted control rats. In conclusion, livers of Walker-256 tumor-bearing rats showed lower response to cAMP in the glucose and glycogen catabolism in various stages of tumor development (days 5, 8 and 11), which was probably not due to the lower hepatic glycogen levels nor due to the increased activity of PDE3B.

Vacuolar H+-ATPase apical accumulation in kidney intercalated cells is regulated by PKA and AMP-activated protein kinase

The vacuolar H(+)-ATPase (V-ATPase) in type A kidney intercalated cells is a major contributor to acid excretion in the collecting duct. The mechanisms of V-ATPase-trafficking regulation in kidney intercalated cells have not been well-characterized. In developmentally related epididymal clear cells, we showed previously that PKA, acting downstream of soluble adenylyl cyclase (sAC), induces V-ATPase apical membrane accumulation. These PKA-mediated effects were inhibited by activators of the metabolic sensor AMP-activated kinase (AMPK) in clear cells. Here, we examined the regulation of V-ATPase subcellular localization in intercalated cells by PKA and AMPK in rat kidney tissue slices ex vivo. Immunofluorescence labeling of kidney slices revealed that the PKA activator N(6)-monobutyryl cAMP (6-MB-cAMP) induced V-ATPase apical membrane accumulation in collecting duct intercalated cells, whereas the V-ATPase had a more cytosolic distribution when incubated in Ringer buffer alone for 30 min. V-ATPase accumulated at the apical membrane in intercalated cells in kidney slices incubated in Ringer buffer for 75 min, an effect that was prevented by treatment with PKA inhibitor (mPKI). The V-ATPase distribution was cytosolic in intercalated cells treated with the carbonic anhydrase inhibitor acetazolamide or the sAC inhibitor KH7, effects that were overridden by 6-MB-cAMP. Preincubation of kidney slices with an AMPK activator blocked V-ATPase apical membrane accumulation induced by 6-MB-cAMP, suggesting that AMPK antagonizes cAMP/PKA effects on V-ATPase distribution. Taken together, our results suggest that in intercalated cells V-ATPase subcellular localization and therefore its activity may be coupled to acid-base status via PKA, and metabolic status via AMPK.

Cyclic AMP regulates the expression and nuclear translocation of RFC40 in MCF7 cells

We have previously shown that the regulatory subunit of PKA, RIα, functions as a nuclear transport protein for the second subunit of the replication factor C complex, RFC40, and that this transport appears to be crucial for cell cycle progression from G1 to S phase. In this study, we found that N6-monobutyryl cAMP significantly up-regulates the expression of RFC40 mRNA by 1.8-fold and its endogenous protein by 2.3-fold with a subsequent increase in the RIα-RFC40 complex formation by 3.2-fold. Additionally, the nuclear to cytoplasmic ratio of RFC40 increased by 26% followed by a parallel increase in the percentage of S phase cells by 33%. However, there was reduction in the percentage of G1 cells by 16% and G2/M cells by 43% with a concurrent accumulation of cells in S phase. Interestingly, the higher percentage of S phase cells did not correlate with a parallel increase in DNA replication. Moreover, although cAMP did not affect the expression of the other RFC subunits, there was a significant decrease in the RFC40-37 complex formation by 81.3%, substantiating the decrease in DNA replication rate. Taken together, these findings suggest that cAMP functions as an upstream modulator that regulates the expression and nuclear translocation of RFC40.

Cyclic AMP-mediated induction of the glial fibrillary acidic protein is independent of protein kinase A activation in rat C6 glioma

N6-O'2-dibutyryl cAMP (dbcAMP), N6-monobutyryl cAMP (N6-mbcAMP), 8-Chloro cAMP (ClcAMP), and O'2-monobutyryl cAMP (O'2-mbcAMP) were used to study glial fibrillary acidic protein (GFAP) induction in rat C6 glioma. With the exception of O'2-mbcAMP, these cAMP analogs induced GFAP after stimulation of cells with a concentration of 0.5-1 mM. Only dbcAMP and N6-mbcAMP increased the intracellular concentration of cAMP. Protein kinase A (PKA) activation is often proposed to be involved in GFAP expression in astrocytes. Ion-exchange chromatography indicated that protein kinase activity is associated with PKA type II in C6. dbcAMP, N6-mbcAMP, and ClcAMP upregulated the amount of cAMP-binding proteins approximately twofold. RI was upregulated in the cytosol and particulate fraction, whereas RII was not affected after stimulation with dbcAMP. Concomitant, the PKA activity decreased approximately 60% and 40% in the cytosol and particulate fraction, respectively. CREB is constitutively expressed in C6 and is downregulated after stimulation with dbcAMP. The membrane-permeable PKA inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline sulfonamide (H89) did not suppress the induction of GFAP-mRNA and its translation into GFAP. On the contrary, depending on the time difference between H89 and dbcAMP addition to C6, GFAP synthesis could even be potentiated more than twofold. Experiments in the presence of cycloheximide showed that protein synthesis is necessary for GFAP transcription. Although all components of the PKA signal transduction pathway are present in C6, GFAP synthesis is not dependent on PKA activation but required the synthesis of an unidentified factor.

The effect of cyclic AMP analogues on cholesteryl ester synthesis and hydrolysis in cultured rat peritoneal macrophages.

The accumulation of large amounts of cholesteryl ester in macrophages which have invaded the artery wall leads to the formation of foam cells, and is one of the early events in the development of atherosclerosis. The activities of both acyl Coenzyme A: cholesterol acyltransferase (ACAT), which is responsible for cholesterol esterification, and cholesteryl ester hydrolase (CEH) have been shown to be increased in foam cells ( 1 ), so that the cholesteryl ester undergoes a continual cycle of formation and hydrolysis ( l,2). Cyclic AMP (CAMP) has been reported to both stimulate (3) and inhibit (4) ACAT activity in the murine macrophage cell line 5774, while in a recent study we were unable to demonstrate any effect (5). CEH activity, on the other hand, has been shown to be increased by dibutyryl cAMP (Bt2cAMP) in two other murine cell lines, P388DI and WEHl (6.7). In the present work, we have investigated the role of cAMP in the regulation of cholesteryl ester metabolism and foam cell formation in cultured rat peritoneal macrophages using cellpermeable cAMP analogues. Macrophages were collected from male Wistar rats (250300g) by peritoneal washing (8) 2 days after intraperitoneal injection of 3ml thioglycollate (3%). Cells from 3-4 rats were pooled for each preparation. After washing, the cells were cultured as monolayers in supplemented Dulbecco's modified Eagle's medium at 37OC in 95% air l 5% CO?. Rat lipoprotein of density < 1.050glml was isolated from rat serum by ultracentrifugation and acetylated by incubation with acetic anhydride (9). Cholesterol esterification was assayed by the incorporation of radioactivity from potassium ('HI =!ca:c in:o cholesteryl esters during a 5h incubation period. Lipids were extracted with chloroform: methanol (2: 1, v:v) and separated by thin layer chromatography. For the determination of CEH activity, the cells were removed from the plates with a plastic scraper, washed with phosphate-buffered saline and disrupted by sonication. The release of I I-'%]oleate from cholesterol I I '%)oleate was then measured in the resulting homogenate (10). The cAMP analogues used were N6 benzoyl cAMP (BEAMP), B-(4-chlorophenylthio) cAMP (CPTcAMP), 8 chloro cAMP (ClcAMP), N6 monobutyryl cAMP (MBcAMP), 8-(6-amino hexyl) amino cAMP (AHA CAMP), 8-piperidino cAMP (PIP CAMP) and dibutyryl cAMP (BtZcAMP). Significance limits were calculated using Student's t test. Cholesterol esterification in the cultured macrophages was unaffected by a range of cAMP analogues at a concentration of

Insulin promotes and cyclic adenosine 3',5'-monophosphate impairs functional insertion of insulin receptors in the plasma membrane of rat adipocytes: evidence for opposing effects of tyrosine and serine/threonine phosphorylation.

The aim of the present study was to elucidate events in the plasma membrane (PM) associated with the previously described effect of insulin to rapidly enhance the number of cell surface insulin binding sites in rat adipocytes. [125I]insulin was cross-linked to cell surface insulin receptors of intact cells that had been preincubated with or without insulin. Subsequently prepared PM displayed a approximately 3-fold increase in bound [125I]insulin when cells had been pretreated with 6 nM insulin for 20 min compared to membranes from control cells, and SDS-PAGE with autoradiography showed that this occurred at the insulin receptor alpha-subunit. The magnitude of the effect was similar to that found for insulin binding to intact cells that had been preincubated with insulin. In contrast, the insulin binding capacity in the PM was not affected by prior treatment of cells with insulin when assessed with the addition of [125I]insulin directly to solubilized PM; this suggests an unchanged total number of PM receptors. Thus, the enhancement of cell surface insulin binding capacity produced by insulin is not due to the translocation of receptors, but instead appears to be confined to receptors already present in the PM. The addition of phospholipase C (from Clostridium perfringens), which cleaves PM phospholipids, mimicked the effect of insulin to enhance cell surface binding in adipocytes, and this suggests a pool of cryptic PM receptors. Both the nonmetabolizable cAMP analog N6-monobutyryl cAMP (N6-mbcAMP) and the serine/threonine phosphatase inhibitor okadaic acid abolished the effect of concomitant insulin treatment to increase binding capacity. In contrast, the tyrosine phosphatase inhibitor vanadate increased insulin binding even in the presence of okadaic acid or N6-mbcAMP. The effect of N6-mbcAMP to impair cell surface insulin binding was also evident in the presence of a peptide derived from the major histocompatibility complex type I that effectively impairs receptor internalization, but the amount of PM receptors assessed by immunoblot was unaltered. Taken together, the data suggest that insulin exposure leads to the uncovering of cryptic receptors associated with the PM. It is also suggested that tyrosine phosphorylation promotes this process, whereas enhanced serine phosphorylation, e.g. produced by cAMP, impairs the functional insertion of the receptors, rendering them unable to bind insulin.

Insulin Promotes and Cyclic Adenosine 3',5'-Monophosphate Impairs Functional Insertion of Insulin Receptors in the Plasma Membrane of Rat Adipocytes: Evidence for Opposing Effects of Tyrosine and Serine/Threonine Phosphorylation

The aim of the present study was to elucidate events in the plasma membrane (PM) associated with the previously described effect of insulin to rapidly enhance the number of cell surface insulin binding sites in rat adipocytes. [125I]insulin was cross-linked to cell surface insulin receptors of intact cells that had been preincubated with or without insulin. Subsequently prepared PM displayed a ∼3-fold increase in bound [125I]insulin when cells had been pretreated with 6 nm insulin for 20 min compared to membranes from control cells, and SDS-PAGE with autoradiography showed that this occurred at the insulin receptorα -subunit. The magnitude of the effect was similar to that found for insulin binding to intact cells that had been preincubated with insulin. In contrast, the insulin binding capacity in the PM was not affected by prior treatment of cells with insulin when assessed with the addition of [125I]insulin directly to solubilized PM; this suggests an unchanged total number of PM receptors. Thus, the enhancement of cell surface insulin binding capacity produced by insulin is not due to the translocation of receptors, but instead appears to be confined to receptors already present in the PM. The addition of phospholipase C (from Clostridium perfringens), which cleaves PM phospholipids, mimicked the effect of insulin to enhance cell surface binding in adipocytes, and this suggests a pool of cryptic PM receptors. Both the nonmetabolizable cAMP analog N6-monobutyryl cAMP (N6-mbcAMP) and the serine/threonine phosphatase inhibitor okadaic acid abolished the effect of concomitant insulin treatment to increase binding capacity. In contrast, the tyrosine phosphatase inhibitor vanadate increased insulin binding even in the presence of okadaic acid or N6-mbcAMP. The effect of N6-mbcAMP to impair cell surface insulin binding was also evident in the presence of a peptide derived from the major histocompatibility complex type I that effectively impairs receptor internalization, but the amount of PM receptors assessed by immunoblot was unaltered. Taken together, the data suggest that insulin exposure leads to the uncovering of cryptic receptors associated with the PM. It is also suggested that tyrosine phosphorylation promotes this process, whereas enhanced serine phosphorylation, e.g. produced by cAMP, impairs the functional insertion of the receptors, rendering them unable to bind insulin.

Interactions between angiotensin II and adenosine 3´:5´-cyclic monophosphate in the regulation of amino acid transport by vascular smooth muscle cells

The regulation of amino acid transport by angiotensin II (AII) and cyclic AMP (cAMP) was assessed in cultured vascular smooth muscle cells, using a nonmetabolizable amino acid, alpha-[3H]aminoisobutyric acid (AIB). An exposure time in excess of 2 h was required for AII to elicit a stimulatory response, the magnitude of which increased in a time-dependent manner for 12 h. AII-induced transport was blocked by [1-sarcosine, 8-isoleucine]AII, a competitive inhibitor of AII binding. The effect of AII was not abolished by downregulation protein kinase C with phorbol 12,13-dibutyrate or by use of a protein kinase C inhibitor, suggesting that transport in response to AII can be mediated by a protein kinase C independent pathway. In contrast, the elimination of calcium from the incubation medium reduced AII-stimulated AIB uptake. The calmodulin inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide partially inhibited AIB uptake in response to AII, suggesting that calmodulin may be involved in the modulation of AII-stimulated amino acid transport. AIB transport was also increased by elevating intracellular cAMP levels via beta-adrenergic receptor stimulation, the use of a cAMP analog (N6-monobutyryl cAMP), or a phosphodiesterase inhibitor (3-isobutyl-1-methylxanthine) or by direct stimulation of adenylate cyclase with forskolin. cAMP-induced AIB transport was evident within 10 min and peaked within 1 h. At 1 h AII enhanced cAMP-stimulated AIB transport. A possible mechanism for this effect is suggested by the observation that AII potentiated cAMP production in response to isoproterenol and 3-isobutyl-1-methylxanthine.

A stable peroxovanadium compound with insulin-like action in human fat cells

Aqueous solutions of peroxovanadium (pV) compounds are potent insulin-mimics in various types of cell. Since chemical instability is a problem with these agents, we studied the insulin-like action in human fat cells of a stable pV complex, bpV(pic). It enhanced 14C-U-glucose uptake in a dose-dependent manner by approximately twofold which was slightly less than the effect of insulin (approximately threefold). The pV complex did not alter cell-surface insulin binding and submaximal concentrations did not influence cellular sensitivity to insulin action on glucose uptake. The bpV(pic) inhibited the lipolytic effect of isoprenaline to the same extent as insulin; however, when the cGMP-inhibitable low-K(m) phosphodiesterase (cGI-PDE) was blocked with the specific inhibitor OPC 3911, the antilipolytic effect of insulin, but not that of bpV(pic), was completely prevented. Moreover, when lipolysis was stimulated by the non-hydrolysable cAMP analogue N6-monobutyryl cAMP, bpV(pic), in contrast to insulin, maintained an antilipolytic effect. These findings indicate that bpV(pic) exerts its antilipolytic effect not only through cGI-PDE activation, similar to the effect of insulin, but also by means of other mechanisms. The tyrosine kinase activity of insulin receptors from human placenta was not altered by the pV compound itself, whereas bpV(pic) clearly enhanced insulin-stimulated activity. In contrast, in situ tyrosine phosphorylation of the insulin receptor beta-subunit as well as that of several other proteins was clearly increased in cells which were treated with bpV(pic), whereas vanadate only amplified insulin-stimulated tyrosine phosphorylation. In conclusion, bpV(pic) exerts powerful insulin-like effects in human fat cells and may be a new and potentially useful agent in the management of insulin-resistant states.

Interactions between angiotensin II and adenosine 3´:5´-cyclic monophosphate in the regulation of amino acid transport by vascular smooth muscle cells

The regulation of amino acid transport by angiotensin II (AII) and cyclic AMP (cAMP) was assessed in cultured vascular smooth muscle cells, using a nonmetabolizable amino acid, alpha-[3H]aminoisobutyric acid (AIB). An exposure time in excess of 2 h was required for AII to elicit a stimulatory response, the magnitude of which increased in a time-dependent manner for 12 h. AII-induced transport was blocked by [1-sarcosine, 8-isoleucine]AII, a competitive inhibitor of AII binding. The effect of AII was not abolished by downregulation protein kinase C with phorbol 12,13-dibutyrate or by use of a protein kinase C inhibitor, suggesting that transport in response to AII can be mediated by a protein kinase C independent pathway. In contrast, the elimination of calcium from the incubation medium reduced AII-stimulated AIB uptake. The calmodulin inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide partially inhibited AIB uptake in response to AII, suggesting that calmodulin may be involved in the modulation of AII-stimulated amino acid transport. AIB transport was also increased by elevating intracellular cAMP levels via beta-adrenergic receptor stimulation, the use of a cAMP analog (N6-monobutyryl cAMP), or a phosphodiesterase inhibitor (3-isobutyl-1-methylxanthine) or by direct stimulation of adenylate cyclase with forskolin. cAMP-induced AIB transport was evident within 10 min and peaked within 1 h. At 1 h AII enhanced cAMP-stimulated AIB transport. A possible mechanism for this effect is suggested by the observation that AII potentiated cAMP production in response to isoproterenol and 3-isobutyl-1-methylxanthine.

Differential Regulation of Oocyte Maturation and Cumulus Expansion in the Mouse Oocyte– Cumulus Cell Complex by Site-Selective Analogs of Cyclic Adenosine Monophosphate

In the present study, we have examined how differential distribution of cyclic adenosine 5′-monophosphate (cAMP)-dependent protein kinase isozymes within the mouse oocyte–cumulus cell complex might influence the physiological response of the complex to cAMP, by determining the actions of site-selective cAMP analogs on oocyte maturation and cumulus expansion. Five different analogs of cAMP were utilized: 8-thiomethyl-cAMP and 8-bromo-cAMP, which bind to site 1 on the type II regulatory subunit (RII) of cAMP-dependent protein kinase A (PKA); 8-aminohexylamino-cAMP, which binds to site 1 on the type I regulatory subunit (RI) of PKA; N6-monobutyryl cAMP, which binds to site 2 on either RI or RII; and 8-piperidino-cAMP, which binds to either site 1 on RII or site 2 on RI. These analogs were tested alone or in paired combinations that synergistically activate either the type I or type II PKA isozyme. When tested alone, analogs that can bind to, and presumably activate, type I PKA were the most potent inhibitors of germinal vesicle breakdown (GVB) in both cumulus cell-enclosed and denuded oocytes. Consistent with this result was the finding that paired combinations of analogs that selectively activate type I PKA were also most effective in preventing GVB. On the other hand, pulsing meiotically arrested cumulus cell-enclosed oocytes with high concentrations of analogs that bind to PKA II, or with paired combinations of analogs that selectively activate type II PKA, led to induction of GVB; stimulation with analogs or combinations thereof that presumably stimulate type I PKA was less effective. Cumulus expansion in response to PKA stimulation showed similar selectivity in that type II PKA-stimulating treatments were considerably more effective in provoking expansion than type I PKA-stimulating treatments. 8-N3-[32P]cAMP photoaffinity labeling of PKA regulatory subunits revealed that only RI was present in oocyte extracts, while extracts from oocyte–cumulus cell complexes contained both RI and RII. These results support the hypothesis that type II PKA mediates cAMP-stimulated cumulus expansion and resumption of meiotic maturation, while direct elevation of type I PKA within the oocyte is instrumental in maintaining meiotic arrest.

CAMP Regulates Infection Structure Formation in the Plant Pathogenic Fungus Magnaporthe grisea.

Magnaporthe grisea, the causal agent of rice blast, is one of the most destructive fungal pathogens of rice throughout the world. Infection of rice by M. grisea requires the formation of an appressorium, a darkly pigmented, dome-shaped structure. The germ tube tip differentiates into an appressorium following germination of conidia on a leaf surface. When conidia germinate on growth medium or other noninductive surfaces, the emerging germ tube does not differentiate and continues to grow vegetatively. Little is known about the endogenous or exogenous signals controlling the developmental process of infection structure formation. We show here that a hydrophobic surface was sufficient for the induction of the appressorium. Furthermore, we demonstrate that the addition of cAMP, its analogs (8-bromo cAMP and N6-monobutyryl cAMP), or 3-isobutyl-1-methylxanthine (an inhibitor of phosphodiesterase) to germinating conidia or to vegetative hyphae induced appressorium formation on noninductive surfaces. The identification of cAMP as a mediator of infection structure formation provides a clue to the regulation of this developmental process. Elucidation of the mechanism involved is not only of biological interest but may also provide the basis for new disease control strategies.

Vanadate increases cell surface insulin binding and improves insulin sensitivity in both normal and insulin-resistant rat adipocytes.

The aim of this study was to elucidate the acute effects of vanadate on cell surface insulin binding and insulin sensitivity in rat adipocytes. The cells were preincubated at 37 degrees for 20 min followed by energy depletion with potassium cyanide, extensive washing and 125I-insulin binding. The presence of vanadate or insulin during the preincubation period dose-dependently enhanced 125I-insulin binding to normal adipocytes (maximally 4-5-fold) through an increased number of binding sites without any change in receptor affinity. Submaximal concentrations of vanadate added together with insulin enhanced the cellular sensitivity to the effect of insulin to stimulate 3-O-methylglucose transport. Vanadate, but not insulin, was also capable of increasing insulin binding as well as insulin sensitivity in insulin-resistant cells (treatment with N6-monobutyryl cAMP or amiloride and adipocytes from obese, aging rats). There was a correlation between the effect of vanadate to augment insulin binding and its ability to enhance cellular insulin sensitivity. Thus, the data suggest that short-term vanadate treatment improves insulin sensitivity through enhanced receptor binding and that this occurs in both normal and insulin-resistant cells.

Regulation of 11β- and 17α-Hydroxylases in Cultured Bovine Adrenocortical Cells: 3′,5′-Cyclic Adenosine Monophosphate, Insulin-Like Growth Factor-I, and Activators of Protein Kinase C*

The induction of steroid 11 beta-hydroxylase and 17 alpha-hydroxylase was studied in bovine adrenocortical cell cultures in serum-free medium. In the absence of insulin-like growth factor (IGF)-I or insulin, cholera toxin failed to increase 11 beta-hydroxylase enzyme activity or messenger RNA (mRNA) levels; cholera toxin increased 11 beta-hydroxylase activity and mRNA only in the presence of 10 nM IGF-I or of higher concentrations of insulin. 17 alpha-Hydroxylase enzyme activity and mRNA, in contrast, were increased maximally by cholera toxin in the absence of insulin or IGF. We also compared the induction of 11 beta-hydroxylase and 17 alpha-hydroxylase by intracellular second messengers. When cultures were incubated with cholera toxin, cAMP analogs, forskolin, ACTH, or prostaglandin E1 in defined medium with insulin, all agents increased the mRNA levels for 11 beta-hydroxylase and 17 alpha-hydroxylase. 11 beta-Hydroxylase enzyme activity was detectable in control (insulin only) cultures and was increased to varying extents by the different agents. 17 alpha-Hydroxylase enzyme activity was undetectable in control cultures and was increased more than 50-fold by all agents. We compared the sensitivity of induction of 11 beta-hydroxylase and 17 alpha-hydroxylase enzyme activities by cAMP using serial dilutions of an equimolar mixture of N6-monobutyryl cAMP and 8-bromo cAMP. For both enzymes, the response curve was biphasic, with a maximal response in the range of 20 to 100 microM each analog, but the decline in response at higher cAMP concentrations was much more marked for 11 beta-hydroxylase than for 17 alpha-hydroxylase. The effects of activation of protein kinase C were studied in cultures incubated with 12-O-tetradecanoylphorbol-13-acetate (TPA) together with a cAMP analog mixture. TPA decreased cAMP-induced 11 beta-hydroxylase mRNA; TPA also decreased the induction of 17 alpha-hydroxylase mRNA, as previously reported. TPA caused a dose-dependent decrease in cAMP-induced 11 beta-hydroxylase enzyme activity. Angiotensin II at 0.1 to 10 microM also decreased induction of 11 beta-hydroxylase. Induction of 11 beta-hydroxylase and 17 alpha-hydroxylase is coordinately regulated by cAMP, protein kinase C, and IGF-I/insulin, but responses to these regulators differ in various respects between these two cytochrome P450 enzymes.

Cyclic adenosine monophosphate acts synergistically with dexamethasone to inhibit the entrance of cultured adult rat hepatocytes into S‐phase: With a note on the use of nucleolar and extranucleolar [3H]‐thymidine labelling patterns to determine rapid changes in the rate of onset of DNA replication

Analogs of cyclic adenosine monophosphate (cAMP) (N6benzoyl cAMP and N6monobutyryl cAMP) as well as agents that increased the intracellular level of cAMP (glucagon and isobutylmethylxanthine) inhibited the EGF-stimulated DNA replication of adult rat hepatocytes in primary culture independently of cell density. This inhibition was strongly potentiated by the glucocorticoid dexamethasone. The effect of cAMP (and dexamethasone) was not due to toxicity, because the inhibition was reversible and the cell ultrastructure preserved. cAMP acted by decreasing the rate of transition from G1- to S-phase, the duration of G2- and S-phase of the hepatocyte cell cycle being unaffected. DNA replication started in the extranucleolar compartment of the nucleus and ended in the nucleolar compartment as described earlier for cells grown in the absence of cAMP (O.K. Vintermyr and S.O. Døskeland, J. Cell. Physiol., 1987, 132:12-21). The action of cAMP was very rapid: significant inhibition of the transition was noted 2 hr after the addition of glucagon/IBMX and half-maximal inhibition after 4 hours. The determination of extranucleolarly labelled nuclei in cells pulse-labelled with [3H]thymidine allowed precise analysis of rapid changes in the probability of transition from G1- to S-phase. The extranucleolar labelling index could also be determined in cells continuously exposed to [3H]thymidine.

Abnormal Adenosine 3'5'-Monophosphate Stimulation of Renal 1,25-Dihydroxyvitamin D Production in Hyp Mice: Evidence that 25-Hydroxyvitamin D-lα- Hydroxylase Dysfunction Results from Aberrant Intracellular Function*

Previously we have established that abnormal regulation of renal 25-hydroxyvitamin D (25OHD)-1 alpha-hydroxylase in Hyp mice involves the PTH-adenylate cyclase component of enzyme activation. However, it remains unknown if the muted effects of PTH result from 1) abnormal second messenger production or 2) an intracellular defect limiting enzyme activation. To distinguish between these possibilities, we compared cAMP stimulation of renal 25OHD-1 alpha-hydroxylase in normal, phosphate-depleted normal, and Hyp mice. Administration of N6-monobutyryl cAMP iv (200 mg/kg/day) increased enzyme activity in normal (4.1 +/- 1.7 vs. 40.7 +/- 7.0 fmol/mg kidney.min) and phosphate-depleted mice (13.3 +/- 1.8 vs. 78.2 +/- 10.4) to a level significantly greater than that achieved in Hyp mice (7.4 +/- 1.1 vs. 22.7 +/- 3.6). Moreover, similar to our observations after PTH stimulation, the apparent abnormal cAMP effect did not result from an altered time course of enzyme activation or a rightward shift in the dose response. Collectively, these data indicate that abnormal regulation of 1,25-dihydroxyvitamin D production in Hyp mice results from aberrant intracellular regulation of 25OHD-1 alpha-hydroxylase, a defect probably related to deranged phosphate transport in the renal tubule.

A point mutation abolishes binding of cAMP to site A in the regulatory subunit of cAMP-dependent protein kinase.

Each regulatory subunit of cAMP-dependent protein kinase has two tandem cAMP-binding sites, A and B, at the carboxyl terminus. Based on sequence homologies with the cAMP-binding domain of the Escherichia coli catabolite gene activator protein, a model has been constructed for each cAMP-binding domain. Two of the conserved features of each cAMP-binding site are an arginine and a glutamic acid which interact with the negatively charged phosphate and with the 2'-OH on the ribose ring, respectively. In the type I regulatory subunit, this arginine in cAMP binding site A is Arg-209. Recombinant DNA techniques have been used to change this arginine to a lysine. The resulting protein binds cAMP with a high affinity and associates with the catalytic subunit to form holoenzyme. The mutant holoenzyme also is activated by cAMP. However, the mutant R-subunit binds only 1 mol of cAMP/R-monomer. Photoaffinity labeling confirmed that the mutant R-subunit has only one functional cAMP-binding site. In contrast to the native R-subunit which is labeled at Trp-260 and Tyr-371 by 8-N3cAMP, the mutant R-subunit is convalently modified at a single site, Tyr-371, which correlates with a functional cAMP-binding site B. The lack of functional cAMP-binding site A also was confirmed by activating the mutant holoenzyme with analogs of cAMP which have a high specificity for either site A or site B. 8-NH2-methyl cAMP which preferentially binds to site B was similar to cAMP in its ability to activate both mutant and wild type holoenzyme whereas N6-monobutyryl cAMP, a site A-specific analog, was a very poor activator of the mutant holoenzyme. The results support the conclusions that 1) Arg-209 is essential for cAMP binding to site A and 2) cAMP binding to domain A is not essential for dissociation of the mutant holoenzyme.

Hormonal stimulation of avian embryonic cartilage growth in vitro: histologic and ultrastructural features.

We studied the histologic and ultrastructural features of embryonic chick cartilage after the cartilage had been incubated in serum-free medium that contained hormones and growth factors known to stimulate in vitro cartilage growth. Pelvic cartilages from 9 d chick embryos were incubated in BGJb ( Fitton -Jackson modification) medium alone (control) or medium containing one of the following: N6 monobutyryl cyclic AMP 0.5 mM, forskolin 100 microM, triiodothyronine (T3) 10 nM, insulin 45 nM, or somatomedin C 0.67 nM. At the end of 3 d of incubation the cartilages were fixed in buffered formalin. Significant growth (increases in size, wet and dry weight) was seen with each treatment group. N6-Monobutyryl cAMP treated cartilage had an increased number of flattened immature chondrocytes with large nuclei and prominent nucleoli. The histologic and ultrastructural features of forskolin treated cartilage were indistinguishable from N6-monobutyryl cAMP treatment. The T3 treated cartilage contained large hypertrophic chondrocytes with prominent lacunar typical of mature cartilage. T3 treated cartilage had considerable vacuole formation and dilated endoplasmic reticulum. Insulin and somatomedin treated cartilage had histologic appearance similar to control cartilage. Thus, the effects of various hormones on embryonic cartilage growth in vitro can be separated as to whether growth is the result of chondrocytic hyperplasia (cyclic AMP mediated), chondrocytic hypertrophy with maturation (T3), or a combination of both hyperplasia and hypertrophy (insulin and somatomedin-C).

Cyclic nucleotides and somatomedin action in cartilage.

The role of cyclic nucleotides was evaluated in the stimulation of cartilage metabolism by somatomedin-C (Sm-C). The effects of cAMP and cyclic guanosine monophosphate (cGMP) analogs on matrix synthesis were evaluated. The effects of Sm-C on tissue concentrations of these cyclic nucleotides were investigated. Likewise, the direct effects of Sm-C on the activities of cartilage adenylate cyclase, guanylate cyclase, and phosphodiesterase were determined. We found that tissue concentrations of cAMP in cartilage declined rapidly during organ culture, despite the presence of serum or Sm-C, cGMP concentrations in cartilage declined rapidly during control incubations but were augmented significantly at 30 and 60 min of incubation with the addition of serum or Sm-C. Thereafter, cGMP concentrations declined toward the levels of incubated control cartilages. Sm-C had no effect on phosphodiesterase activity. N6-Monobutyryl cAMP stimulated sulfate uptake, but dibutyryl cGMP did not. Sm-C did not stimulate adenylate cyclase in purified plasma membranes from chondrocytes, whereas it stimulated both plasma membrane and cytosol guanylate cyclase at concentrations of Sm-C as low as 10(-12) M. These data would indicate that cAMP is not the intracellular second messenger for Sm-C in cartilage. The data for cGMP are provocative and suggest it as a candidate for a second messenger mediating a portion of Sm's stimulation of cartilage metabolism.