Changes in rat testicular adenylate cyclase activities and gonadotrophin binding during unilateral experimental cryptorchidism

Unilaterally cryptorchid rats were examined at 3, 8, 15, 22 and 28 days after operation. There was a selective decrease in the adenylate cyclase (ATP pyrophosphate--lyase (cyclizing), EC 4.6.1.1) responses to gonadotrophin stimulation in the abdominal testis. This was associated with a parallel decrease in specific FSH and LH binding. There was no reduction in the response of testicular adenylate cyclases to prostaglandin (PG) E-1 or fluoride stimulation, indicating that both the GTP binding protein (N-component) and the catalytic subunit of the adenylate cyclase complexes were intact. The reduction in FSH-responsive adenylate cyclase activity in the abdominal testis was not due to a change in the Km for adenylate cyclase activation, but was due to a reduction in maximal velocities. Unilateral cryptorchidism was also associated with a rapid decline in soluble Mn2+-dependent adenylate cyclase activity in germ cells (spermatids). By 3 days after operation there was an 82% decrease in germ cell adenylate cyclase activity. The loss of soluble Mn2+-dependent adenylate cyclase activity was associated with a parallel decrease in Sertoli cell secretion of androgen binding protein, indicating that Sertoli cell factors may be important for the maintenance of germ cell adenylate cyclase activity. The desensitization of the gonadotrophin--responsive adenylate cyclases and the loss of gonadotrophin receptors in Leydig and Sertoli cells were not due to changes in plasma gonadotrophin values because LH concentrations were within normal limits and plasma FSH was only marginally elevated in the cryptorchid rats. No significant alterations of any of these parameters were seen in the scrotal testis of unilaterally cryptorchid rats when compared to values for intact controls.     

Lipolysis and beta-adrenergic receptor binding on adipocytes of spontaneously hypertensive rats

Adipocytes from spontaneously hypertensive rats demonstrated a blunted lipolytic response to isoproterenol and dibutyryl cyclic AMP. (-)-[3H]Dihydroalprenolol binding was examined in adipocytes from normotensive and spontaneously hypertensive rats. Increasing concentrations of isoproterenol decreased total (-)-[3H]dihydroalprenolol binding to intact cells from normotensive rats, and the efficacy of competition was decreased in adipocytes from spontaneously hypertensive rats. Scatchard analysis indicated that the number of (-)-[3H]dihydroalprenolol binding sites and the affinity of dihydroalprenolol binding were comparable between normotensive and spontaneously hypertensive rats. Isoproterenol- and Gpp(NH)p-stimulated adenylate cyclase activity was consistently depressed in adipocyte membranes from spontaneously hypertensive rats as compared to normotensive rats. No difference in fluoride-stimulated adenylate cyclase activity was observed. The blunted lipolytic and cyclic AMP response to isoproterenol in these cells suggest a postreceptor lesion of the lipolytic pathway (possibly the guanine nucleotide regulatory protein) in adipocytes from spontaneously hypertensive rats. The blunted lipolytic response to dibutyryl cyclic AMP suggests defective regulation of lipolytic enzymes at the protein kinase-hormone-sensitive lipase level.     

Evidence for a defect in the number of β-adrenergic receptors and in the adenylate cyclase responsiveness to guanine nucleotides in fat cells after adrenalectomy

Receptor binding studies (?)-[³H]dihydroalprenolol as the ligand revealed, in adrenalectomized rat fat cells, a 50% decrease in the number of β-adrenergic receptors. er cell with no change in the receptor affinity for this ligand. Adrenalectomy caused no change in the binding affinity for isoproterenol of both high affinity and low affinity populations of the β-adrenergic receptors. Guanine nucleotide sensitivity of the agonist binding to β-receptors was also unaltered by adrenalectomy. Adrenalectomy caused a 30–40% decrease in the maximal response of adenylate cyclase to (?)-isoproterenol only when guanine nucleotides were present in the assay, without altering the (?)-isoproterenol concentration giving half-maximal adenylate cyclase stimulation (K_act values). The maximal response of adenylate cyclase to Gpp(NH)p also was lower in adrenalectomized membranes, indicating a defect at the guanine nucleotide regulatory site. Removal of adenosine by addition of adenosine deaminase failed to reverse the decreased adenylate cyclase response to isoproterenol in adrenalectomized rats. However, in intact fat cells, in which cyclic AMP accumulation in response to isoproterenol was decreased by adrenalectomy, removal of adenosine almost completely corrected this defect. These results indicate that the observed changes in the number of β-adrenergic receptors and in the ability of guanine nucleotides to stimulate adenylate cyclase, though explaining the decreased adenylate cyclase responsiveness to catecholamines, do probably not contribute significantly to the mechanism by which adrenalectomy decreases the lipolytic responsiveness of adipocyte to catecholamines. In addition, this study also suggests that the increased sensitivity to adenosine of lipolysis reported in adipocytes from adrenalectomized rats may result from an action of adenosine at a post-adenylate cyclase step, possibly on the cyclic AMP phosphodiesterase.     

Ontogenetic development of isoproterenol subsensitivity of myocardial adenylate cyclase and beta-adrenergic receptors in spontaneously hypertensive rats

[3H]Dihydroalprenolol binding and adenylate cyclase activity in the myocardial membranes of Kyoto Wistar normotensive rats and spontaneously hypertensive rats were compared at various stages of postnatal development ranging from 2 to 36 weeks. Basal as well as agonist-stimulated myocardial adenylate cyclase activity was consistently decreased in spontaneously hypertensive rats as compared to normotensive rats as early as 2 weeks of age with significant differences (P < 0.05) observed after 6 weeks of age. When results were expressed as percent stimulation over the basal activity, only isoproterenol plus GTP-stimulated enzyme activity was reduced by 25--30% in spontaneously hypertensive rats, suggesting a specific loss of stimulation by isoproterenol in hypertensive animals. The number of [3H]dihydroalprenolol binding sites of KD for dihydroalprenolol binding were comparable between spontaneously hypertensive and normotensive rats at 3, 6 and 12 weeks of age. The competition of isoproterenol with [3H]dihydroalprenolol for the specific binding sites showed that the affinity of isoproterenol binding was decreased 3--4-fold in spontaneously hypertensive compared with normotensive rats. With postnatal development in age, basal as well as agonist-stimulated activities decreased progressively in both spontaneously hypertensive and normotensive rats. Similarly, the number of [3H]dihydroalprenolol binding sites decreased with the development in age, whereas affinity of dihydroalprenolol binding increased up to 12 weeks of age. These results therefore suggest that adenylate cyclase activity and the number of beta-adrenergic receptors in rat heart, decrease with age and that in hypertension, specific decrease in isoproterenol stimulation of cyclase appears at all stages of development.     

Differences in the accessibility of the beta-adrenergic receptor in isolated hepatocytes from foetal and adult rats.

Beta-receptor number (measured by [3H]-CGP 12 177 binding) and beta-adrenergic response (measured by isoproterenol stimulated glucose liberation and isoproterenol stimulated adenylate cyclase activity) were compared in hepatocytes isolated from foetal (on day 22 of gestation), adult female and adult male rats. Beta-receptor numbers in crude membrane preparations of hepatocytes from adult female and adult male rats were found to be nearly equal (15.5 and 15.1 fmol/mg), but in crude membrane preparations of foetal rats beta-adrenergic receptor number was significantly higher (34.3 fmol/mg). Determination of number of beta-adrenergic surface receptors of intact hepatocytes showed relative high values in foetal rats (about 22,000/cell) and adult female rats (about 20,000/cell), but in male rats the number was less (about 6500/cell). Glucose liberation was stimulated by isoproterenol to the same extent in hepatocytes isolated from adult female and foetal rats (about 150% over basal), whereas no effect was found in hepatocytes isolated from adult male rats. Dose-response curves showed that in foetal rat hepatocytes glucose release was already increased by 10(-8) M isoproterenol, whereas in female rat hepatocytes at least 10(-6) M isoproterenol was required. Adenylate cyclase was stimulated by isoproterenol in lysates of hepatocytes from adult female rats by about 180% and from foetal rats by about 250%. No effects were observed using lysates of hepatocytes from adult male rats. We interpret the observed differences of beta-adrenergic responses between adult female and male rats as being primarily caused by different accessibility of the beta-receptor to the beta-agonist isoproterenol in intact hepatocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Increased adenylate cyclase catalytic activity explains how estrogens "in vivo" promote lipolytic activity in rat white fat cells

Estradiol administration (5 micrograms per day x 4 days) to ovariectomized rats resulted in a 60-70% increase in the maximal lipolytic response of their white adipocytes to isoproterenol, epinephrine, IBMX and forskolin. These altered lipolytic responses were accompanied by parallel changes in the intracellular cyclic AMP levels found in response to 1 mM IBMX alone (+ 106%) or combined with submaximal concentrations of isoproterenol (+205%), epinephrine (+190%) and forskolin (235%). Studies of the adenylate cyclase activity revealed an overall increase in the stimulatory responsiveness of the enzyme (+150 to +200%) after the estradiol-treatment, regardless of the stimulatory agents tested (GTP, GppNHp, fluoride, isoproterenol, ACTH, forskolin). Finally, the finding of a 2-fold enhancement of the Mn2+ (+/- GDP beta S)-stimulated adenylate cyclase activity after the estradiol-treatment strongly suggests that increased activity of the catalytic subunit of this enzyme is the likely mechanism whereby estrogens promote lipolysis in rat fat cells.     

Heart receptors for VIP, PHI and secretin are able to activate adenylate cyclase and to mediate inotropic and chronotropic effects. Species variations and physiopathology

We have assessed the presence of VIP/PHI/secretin receptors in heart by: (1) testing the ability of the corresponding peptides to activate adenylate cyclase in cardiac membranes from rat, dog, Cynomolgus monkey and man, and (2) examining the ability of the same peptides to exert inotropic and chronotropic effects on heart preparations from rat and Cynomolgus monkey in vitro. Based on their affinity for natural peptides and synthetic analogs, two types of VIP/PHI/secretin receptors were characterized: the relatively nonspecific "secretin/VIP receptor" of rat heart (that is "secretin-preferring" only in that secretin was more efficient than VIP in stimulating adenylate cyclase), and the "VIP/PHI-preferring" receptor of man, monkey and dog heart. Four physiopathological situations affecting secretin/VIP receptors in rat heart were explored: In male rats from the Okamoto strain and the Lyon strain, two strains presenting spontaneous hypertension, heart membranes exhibited a markedly decreased response of adenylate cyclase to secretin/VIP, with lesser alterations in the responses to isoproterenol and glucagon. This impairment developed in parallel with the occurrence of hypertension and was reproduced in normotensive rats submitted to chronic isoproterenol treatment (but not in Goldblatt hypertensive rats). These findings are consistent with a hyperactivity of norepinephrine pathways in spontaneously hypertensive rats, leading to a reduced number of cardiac post-junctional secretin/VIP receptors bound to adenylate cyclase. Heart membranes from genetically obese (fa/fa) Zucker rats also exhibited severely decreased responses to secretin/VIP with lesser alterations in the responses to glucagon and isoproterenol. These anomalies were specific for the heart, and developed in concomitance with obesity. The first anomaly could not be corrected by severe food restriction. Secretin stimulation of heart adenylate cyclase was also selectively altered in streptozotocin-diabetic rats. Thus, two types of diabetic cardiomyopathy were characterized by a severe local alteration of secretin/VIP receptors coupled to adenylate cyclase. Hypothyroidism, provoked in rat by thyroidectomy or propylthiouracil treatment, again induced a marked decrease in secretin-stimulated cardiac adenylate cyclase activity. In rat papillary muscle electrically stimulated in vitro, secretin exerted a positive inotropic effect. This effect was reduced in obese (fa/fa) Zucker rats. In rat right atrium, secretin also exerted a positive chronotropic effects.(ABSTRACT TRUNCATED AT 400 WORDS)     

Testosterone response of cryptorchid and hypophysectomized rats to human chorionic gonadotrophin (hCG) stimulation

The testosterone responses to a single injection of hCG (100 i.u.) in hypophysectomized (hypox.), cryptorchid or sham-operated rats were followed over a 5-day period. In sham-operated rats, hCG induced a biphasic rise in serum testosterone, peaks being observed at 2 and 72 h. Reduced testis weights, elevated FSH and LH levels and reduced serum testosterone levels were found after 4 weeks of cryptorchidism, but hCG stimulation resulted in a normal 2 h peak in serum testosterone. However, the secondary rise at 72 h in cryptorchid rats was significantly lower than sham-operated rats. Reduced testis weight and undetectable serum FSH and LH levels together with decreased testosterone levels were found 4 weeks after hypophysectomy. Serum testosterone levels rose 2 h after hCG in comparison to hypox. controls but this peak was significantly reduced compared with sham-operated rats. The second rise in serum testosterone began on day 2, peaking on day 4 at levels comparable to that seen in sham-operated rats after hCG. The in vitro basal and hCG stimulated secretion of testosterone by cryptorchid testes was greater than that secreted by normal rat testes (518.0 +/- 45.9 and 3337.6 +/- 304.1 pmol per testis per 4 h compared with 223.6 +/- 24.9 and 1312.9 +/- 141.4 pmol per testis per 4 h for normal rat testes). In cryptorchid animals a single injection of 100 i.u. hCG resulted in a pattern of in vitro refractoriness similar to normal rats, lasting from 12 h to 2 days, during which testosterone secretion was reduced to near basal levels. The in vitro basal and hCG-stimulated secretion of testosterone by hypox. rat testes was severely diminished compared with normal rat testes. The temporal pattern of in vitro secretion of testosterone from hypox. rat testes mimicked the in vivo serum testosterone pattern seen in these animals. This study demonstrates important differences in the in vivo and in vitro testosterone response to hCG after testicular damage.     

The effect of chronic gamma irradiation in small doses on the adenylate cyclase activity of lung tissue plasma membranes in rats]

A study was made of the response of adenylate cyclase of cytoplasmic membranes of pulmonary tissue to chronic gamma irradiation of rats at a low-dose rate (0.036 cGy/day). The adenylate cyclase complex receptors were shown to respond to such exposure by activation of the enzyme they control and by being more sensitive to their specific effector, isoproterenol. The role of receptors in the manifestation of radiation hormesis is discussed.     

Functional state of adenylate cyclase signaling system in testis and ovary of fasted rats

Sensitivity of the adenylate cyclase signaling system (ACSS) to polypeptide hormones and biogenic amines is studied in testis and ovary of rats after the 2- and 4-day fasting as compared with control animals. In tissues of the fasted rats there is shown a decrease in the basal activity of adenylate cyclase (AC) and of the basal level of the GTP binding of heterotrimeric G protein. An increase of duration of fasting from 2 to 4 days led to intensification of these changes. In the fasted rats, the stimulating effects of chorionic gonadotropin, PACAP-38. and isoproterenol on the AC activity realized via G protein of the stimulatory type are enhanced, whereas the inhibitory effects of somatostatin on the AC activity realized via G protein of the inhibitory type are reduced. In testis of the fasted rats the stimulating effect of serotonin acting on AC via both types of G proteins are increased, while the inhibitory effects of the hormone decrease. Thus, under conditions of fasting, in rat testis and ovary the ACSS sensitivity to regulatory effects of hormones is changing: its stimulatory effects are increased, while its inhibitory effects, on the contrary, are decreased. We suggest these changes is one of the key mechanisms of adaptation of organism to deficiency of nutritional resources to be aimed at intensifying the tissues catabolic processes, preferably, lypolysis.     

Loss of adenylate cyclase hormonal sensitivity in chemically transformed epithelial cells.

The hormonal sensitivity of adenylate cyclase from a normal rat liver epithelial cell line (K16) and its chemically transformed derivative (W8) were compared. Intact normal rat liver cells had markedly increased cAMP levels after brief exposure to epinephrine, isoproterenol, norepinephrine or prostaglandin E₁. In contrast, the cAMP levels of chemically transformed cells were relatively unaffected by these same compounds even after prolonged incubation. A comparison of broken cell adenylate cyclase activities revealed a decreased basal activity in the chemically transformed cells; the response to NaF was similar in the two cell lines, while the response to catecholamines and prostaglandins paralleled the intact cell studies. These data suggest that one reason for loss of adenylate cyclase hormonal responsiveness in chemically transformed rat liver epithelial cells may be a dysfunction or loss of hormone binding sites.     

Desensitization of beta-adrenoreceptors and its influence on manifestations of the antiradiation effect of isoproterenol

A study was made of the function of beta-adrenoreceptors and Chinese hamster fibroblast cAMP during their desensitization to isoproterenol. Desensitization of adenylate cyclase was demonstrated to lead to the reduction of both the cAMP response to isoproterenol and isoproterenol capacity to protect the cells from ionizing radiation. This did not entail any changes in the number of beta-adrenoreceptors or in the dissociation constant of the beta-antagonist. It is assumed that desensitization provokes functional dissociation of the beta-receptor and adenylate cyclase. Intact adenylate cyclase is an absolute must for realization of the antiradiation potency of isoproterenol.     

Studies on structure-function relationships of human choriogonadotropins with C-terminally shortened alpha-subunits. II. Stimulation of adenylate cyclase activity and depletion of cytochrome P-450 in the rat testis

Human choriogonadotropin (hCG) analogues, containing the native beta-subunit and alpha-subunits enzymatically shortened by 2-3 amino acid residues, were used for studying the influence of hCG on the content of microsomal progesterone-binding cytochrome P-450 in rat testis. When 2-3 residues have been removed from the alpha-subunit, the ability of the hormone analogue to stimulate adenylate cyclase of isolated rat Leydig cells was diminished by 55%. When the hCG analogue containing a des-(88-92)-alpha chain was applied, the residual activity of the adenylate cyclase was negligible. 18 h after administration to rats in vivo, the hormone species containing des-(Lys-91-Ser-92)-alpha or des-(90-92)-alpha, respectively, were found to have induced a decrease in microsomal cytochrome P-450 content with an effectiveness corresponding to their ability of stimulating the adenylate cyclase in vitro. However, when assayed 48 h after application, the desensitization of the microsomal cytochrome P-450 system had persisted in case of the hCG species containing a des-(90-92)-alpha chain but not in case of hCG consisting of des-(Lys-91-Ser-92)-alpha and a native beta-subunit. From these results, it is concluded that short-term effects of hCG on the microsomal content of progesterone-binding cytochrome P-450 are mediated by the stimulation of adenylate cyclase. In contrast, the long-lasting action of hCG on this system seems not to be exclusively mediated by the increase in intracellular cAMP.     

Temporal changes in rat Leydig cell function after the induction of bilateral cryptorchidism

Adult rats were made bilaterally cryptorchid and studied at intervals of 3, 7, 14 or 21 days to study temporal changes in Leydig cell function. Serum FSH and LH levels were measured and the cross-sectional area of the Leydig cells assessed by morphometry. The function of the Leydig cells was judged by the binding of 125I-labelled hCG to testicular tissue in vitro and the testosterone response of the testis to hCG stimulation in vitro. By 3 days after cryptorchidism, the binding of labelled hCG to testicular tissue was significantly decreased compared to that of controls, but the testes were able to respond to hCG stimulation in vitro. At 7, 14 and 21 days after cryptorchidism, an enhanced testosterone response was observed and the size of the Leydig cells was significantly greater than that of the controls, which indicated increased secretory activity by the cryptorchid testis. Although serum FSH levels were significantly elevated after 3 days of cryptorchidism, serum LH levels did not rise until 7 days, thereby suggesting that the loss of receptors is unlikely to result from down-regulation by LH. The reduced testosterone response of the cryptorchid testis in vivo to low doses of hCG and the enhanced response at high doses are probably related to the reduced blood flow to the cryptorchid testis and the decreased sensitivity of the Leydig cells induced by LH/hCG receptor loss.     

Phorbol esters and beta-adrenergic agonists mediate desensitization of adenylate cyclase in rat glioma C6 cells by distinct mechanisms

Exposure of rat glioma C6 cells to either isoproterenol or 12-O-tetradecanoylphorbol 13-acetate (TPA) resulted in desensitization of isoproterenol-stimulated adenylate cyclase activity. After either treatment, the affinity of beta-receptors for isoproterenol was reduced. Thus, desensitization by TPA or isoproterenol appeared to involve an "uncoupling" of the beta-receptor from the stimulatory regulatory component (Ns) of adenylate cyclase. The activity of Ns, assayed by reconstitution of S49 cyc- adenylate cyclase activity, was found to be unchanged after desensitization. The activity of beta-receptors was measured by inactivating Ns and the catalytic component of adenylate cyclase in C6 membranes and fusing them with membranes lacking beta-receptors. Receptors from isoproterenol-treated C6 cells were less active in "coupling" to the foreign adenylate cyclase than receptors from untreated cells, whereas receptors from TPA-treated cells were fully active. This unexpected latter result was explored further. Lysates from C6 cells were centrifuged on linear sucrose density gradients and the gradient fractions assayed for beta-receptor binding activity. Most of the receptors were recovered in a "heavy" plasma membrane peak but some receptors also appeared in a "light" membrane peak. After treatment of the cells with isoproterenol or TPA, the proportion of receptors in the light peak increased. Prior treatment of the cells with concanavalin A prevented the increase in light receptors caused by isoproterenol or TPA. In addition, the concanavalin A treatment prevented the desensitization of adenylate cyclase caused by TPA but not that caused by isoproterenol. Finally, desensitization of adenylate cyclase was reversed by polyethylene glycol-induced fusion of membranes from cells treated with TPA but not isoproterenol. We conclude that beta-agonists and phorbol esters desensitize adenylate cyclase by distinct mechanisms. Agonists cause a reduction in the functional activity of the beta-receptors followed by a segregation of the receptors into a light membrane fraction devoid of Ns. Phorbol esters do not alter the activity of the receptors but do cause their segregation.     

Effect of long-term and short-term diabetes on the parathyroid hormone sensitive rat renal adenylate cyclase: correlation with vitamin D metabolism

In short-term experiments, male Wistar rats were made diabetic for 10 days with a single injection of streptozotocin (65 mg/kg body weight). One group of diabetic rats was treated with insulin for 3 days prior to sacrifice. In long-term experiments, vitamin D replete or vitamin D depleted rats were made diabetic for 6 weeks. Criteria for diabetes were loss of weight, glycosuria (Tes-Tape), and hyperglycemia. In long-term diabetic rats the activity of renal mitochondrial 25-hydroxyvitamin D3 (25-(OH)D3) 1 alpha-hydroxylase was significantly decreased and that of 25-(OH)D3 24-hydroxylase increased. However, the parathyroid hormone (PTH) sensitive renal adenylate cyclase activity of diabetic rats was not different from that of the nondiabetic rats in either the vitamin D replete group or the vitamin D depleted group. On the other hand, the PTH-sensitive renal adenylate cyclase activity was significantly higher in short-term diabetic rats than in control and insulin-treated rats. These differences were observed at doses of 10(-8) to 10(-5) M of PTH. This study has demonstrated for the first time that there are differences in the PTH-sensitive adenylate cyclase response between long-term and short-term diabetic rats. The hypersensitivity to PTH of the renal adenylate cyclase observed in short-term diabetic rats probably represents a response to insulin deficiency during the early development of diabetes mellitus in the rats.     

Tumor necrosis factor and interleukin 1 inhibit parathyroid hormone-responsive adenylate cyclase in clonal osteoblast-like cells by down-regulating parathyroid hormone receptors.

The effects of the monokines tumor necrosis factor alpha (TNF) and interleukin 1 (IL 1) on parathyroid hormone (PTH)-responsive adenylate cyclase were examined in clonal rat osteosarcoma cells (UMR-106) with the osteoblast phenotype. Recombinant TNF and IL 1 incubated with UMR-106 cells for 48 hr each produced concentration-dependent inhibition of PTH-sensitive adenylate cyclase, with maximal inhibition of PTH response (40% for TNF, 24% for IL 1) occurring at 10(-8) M of either monokine. Both monokines also decreased adenylate cyclase stimulation by the tumor-derived PTH-related protein (PTHrP). In contrast, TNF and IL 1 had little or no inhibitory effect on receptor-mediated stimulation of adenylate cyclase by isoproterenol and nonreceptor-mediated enzyme activation by cholera toxin and forskolin; both monokines increased prostaglandin E2 stimulation of adenylate cyclase. Binding of the radioiodinated agonist mono-[125I]-[Nle8,18, Tyr34]bPTH-(1-34)NH2 to UMR-106 cells in the presence of increasing concentrations of unlabeled [Nle8,18, Tyr34]bPTH-(1-34)NH2 revealed a decline in PTH receptor density (Bmax) without change in receptor binding affinity (dissociation constant, Kd) after treatment with TNF or IL 1. Pertussis toxin increased PTH-sensitive adenylate cyclase activity but did not attenuate monokine-induced inhibition of PTH response. In time course studies, brief (1 hr) exposure of cells to TNF or IL 1 during early culture was sufficient to decrease PTH response but only after exposed cells were subsequently allowed to grow for prolonged periods. Inhibition of PTH response by monokines was blocked by cycloheximide. The results indicate that TNF and IL 1 impair responsiveness to PTH (and PTHrP) by a time- and protein synthesis-dependent down-regulation of PTH receptors linked to adenylate cyclase.     

Evidence that metabolically active synaptosomes lack functional cyclic AMP-dependent protein kinase.

Cyclic adenosine monophosphate (cAMP)-mediated signal transduction was evaluated in synaptosomes prepared from rat brain cortex. Adenylate cyclase was responsive to known adenylate cyclase stimulators including peptides (CRH and VIP), catecholamines (norepinephrine and isoproterenol) and ligands that directly stimulate adenylate cyclase (forskolin). Cyclic AMP accumulation also increased approximately 2 to 3-fold, but none of the agonists was able significantly to activate cyclic AMP-dependent protein kinase (A-kinase) in cortical synaptosomes. However, in parallel studies with slices prepared from rat brain cortex, adenylate cyclase activity, cAMP accumulation and A-kinase activity were all stimulated by CRH, VIP, norepinephrine, isoproterenol and forskolin. These data suggest that, in intact synaptosomes, either the cellular machinery which facilitates binding of cAMP to the regulatory subunit of A-kinase is missing or the cAMP produced by adenylate cyclase is not accessible to A-kinase.     

Desensitization of β-Adrenergic Receptor-Coupled Adenylate Cyclase in Cerebral Cortex After In Vivo Treatment of Rats with Desipramine

Continuous treatment (1-10 days) of rats with desipramine (10 mg/kg, twice per day) caused desensitization of the beta-adrenergic receptor-coupled adenylate cyclase system of cerebral cortical membranes. The decrease in the isoproterenol-stimulated adenylate cyclase activity was more rapid and greater than the decrease in the number of beta-adrenergic receptors in membranes during treatment of the membrane donor rats with desipramine, indicating that the desensitization occurring at an early stage of the treatment was not accounted for solely by the decrease in the receptor number. Neither the guanine nucleotide regulatory protein (N) nor the adenylate cyclase catalyst was impaired by the drug treatment, since there was no decrease in the cyclase activity measured in the presence or absence of GTP, guanyl-5'-yl-beta-gamma-imidodiphosphate [Gpp(NH)p], NaF, or forskolin. Gpp(NH)p-induced activation of membrane adenylate cyclase developed with a lag time of a few minutes in membranes from control or drug-treated rats. The lag was shortened by the addition of isoproterenol, indicating that beta-receptors were coupled to N in such a manner as to facilitate the exchange of added Gpp(NH)p with endogenous GDP on N. This effect of isoproterenol rapidly decreased during the drug treatment of rats. Thus, functional uncoupling of the N protein from receptors was responsible for early development of desensitization of beta-adrenergic receptor-mediated adenylate cyclase in the cerebral cortex during desipramine therapy.     

Inhibition of parathyroid hormone-responsive adenylate cyclase in clonal osteoblast-like cells by transforming growth factor alpha and epidermal growth factor

The effects of transforming growth factor alpha (TGF-alpha) and epidermal growth factor (EGF) on parathyroid hormone (PTH)-responsive adenylate cyclase were examined in clonal rat osteosarcoma cells (UMR-106) with the osteoblast phenotype. Recombinant TFG-alpha and EGF incubated with UMR-106 cells for 48 h each produced concentration-dependent inhibition of PTH-responsive adenylate cyclase, with maximal inhibition of 38-44% at 1-3 ng/ml of either growth factor. TGF-alpha and EGF also inhibited beta-adrenergic agonist (isoproterenol)-stimulated adenylate cyclase by 32%, but neither growth factor affected enzyme response to prostaglandin or basal (unstimulated) activity. Nonreceptor-mediated activation of adenylate cyclase by forskolin and cholera toxin was inhibited 18-20% by TGF-alpha and EGF. Pertussis toxin augmented PTH-stimulated adenylate cyclase, suggesting modulation of PTH response by a functional inhibitory guanine nucleotide-binding regulatory component of the enzyme. However, pertussis toxin had no effect on TGF-alpha inhibition of PTH response. Growth factor inhibition of PTH response was time-dependent, with maximal inhibition by 4-12 h of TGF-alpha exposure, and was reduced by prior treatment of UMR-106 cells with cycloheximide. TGF-alpha was not mitogenic for UMR-106 cells. The results indicate that TGF-alpha and EGF selectively impair PTH- and beta-adrenergic agonist-responsive adenylate cyclase of osteoblast-like cells. Growth factor inhibition of adenylate cyclase may be exerted at the receptor for stimulatory agonist and at nonreceptor components excluding pertussis toxin-sensitive guanine nucleotide-binding regulatory proteins. The inhibitory action of growth factors may also require protein synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)