AMP Production In Response Research Articles

Chapter 6 Regulation of renal salt and water transporters during vasopressin escape

Hyponatremia, defined as a serum sodium < 135 mmol/l, is one of the most commonly encountered and serious electrolyte disorders of clinical medicine. The predominant cause of hyponatremia is an inappropriate elevation of circulating vasopressin levels relative to serum osmolality or the 'syndrome of inappropriate antidiuretic hormone secretion' (SIADH). Fortunately, the degree of the hyponatremia is limited by a process that counters the water-retaining action of vasopressin, namely 'vasopressin escape'. Vasopressin escape is characterized by a sudden increase in urine volume with a decrease in urine osmolality independent of circulating vasopressin levels. Until recently, little was known about the molecular mechanisms underlying escape. In the 1980s, we developed an animal model for vasopressin escape in which male Sprague-Dawley rats were infused with dDAVP, a V2-receptor-selective agonist of vasopressin, while being fed a liquid diet. Rats drank a lot of water in order to get the calories they desired. Using this model, we demonstrated that the onset of vasopressin escape (increased urine volume coupled to decreased urine osmolality) coincided temporally with a marked decrease in renal aquaporin-2 (water channel) protein and mRNA expression in renal collecting ducts. This protein reduction was reversible and correlated to decreased water permeability of the collecting duct. Studies examining the mechanisms underlying AQP2 decrease revealed a decrease in V2-receptor mRNA expression and binding, as well as a decrease in cyclic AMP production in response to acute-dDAVP challenge in collecting duct suspensions from these escape animals. Additional studies showed an increase in sodium transporters of the distal tubule. These changes, hypothetically, might help to attenuate the hyponatremia. Future studies are needed to fully elucidate systemic, intra-organ, and cellular signaling responsible for the physiological phenomenon of vasopressin escape.

Cloning and Characterization of a Novel WD-40 Repeat Protein That Dramatically Accelerates Osteoblastic Differentiation

The bone morphogenetic proteins (BMPs) play a pivotal role in endochondral bone formation. Using differential display polymerase chain reaction, we have identified a novel gene, named BIG-3 (BMP-2-induced gene 3 kb), that is induced as a murine prechondroblastic cell line, MLB13MYC clone 17, acquires osteoblastic features in response to BMP-2 treatment. The 3-kilobase mRNA encodes a 34-kDa protein containing seven WD-40 repeats. Northern and Western analyses demonstrated that BIG-3 mRNA and protein were induced after 24 h of BMP-2 treatment. BIG-3 mRNA was expressed in conditionally immortalized murine bone marrow stromal cells, osteoblasts, osteocytes, and growth plate chondrocytes, as well as in primary calvarial osteoblasts. Immunohistochemistry demonstrated that BIG-3 was expressed in the osteoblasts of calvariae isolated from mouse embryos. To identify a role for BIG-3 in osteoblast differentiation, MC3T3-E1 cells were stably transfected with the full-length coding region of BIG-3 (MC3T3E1-BIG-3) cloned downstream of a cytomegalovirus promoter in pcDNA3.1. Pooled MC3T3E1-BIG-3 clones expressed alkaline phosphatase activity earlier and achieved a peak level of activity 10-fold higher than cells transfected with the empty vector (MC3T3E1-EV) at 14 days. Cyclic AMP production in response to parathyroid hormone was increased 10- and 14-fold at 7 and 14 days, respectively, in MC3T3E1-BIG-3 clones, relative to MC3T3E1-EV clones. This increase in cAMP production was associated with an increase in PTH binding. Expression of BIG-3 increased mRNA levels encoding Cbfa1, type I collagen, and osteocalcin and accelerated formation of mineralized nodules. In conclusion, we have identified a novel WD-40 protein, induced by BMP-2 treatment, that dramatically accelerates the program of osteoblastic differentiation in stably transfected MC3T3E1 cells.

Relaxation and modulation of cyclic AMP production in response to atrial natriuretic peptides in guinea pig tracheal smooth muscle

Relaxation and modulation of cyclic AMP production in response to atrial natriuretic peptides were investigated in epithelium-denuded guinea pig tracheal rings, treated with indomethacin (5 μM) and phosphoramidon (1 μM) and contracted with histamine (3 μM). Atrial natriuretic peptide (ANP) was a more potent relaxant than C-type natriuretic peptide whereas ANP-(4–23) was inactive suggesting the involvement of ANP A receptors in the relaxant effect of ANP. ODQ (1 H-[1,2,4]oxadiazolo[4,3- A]quinoxalin-1-one, 10 μM), a selective inhibitor of soluble guanylyl cyclase, markedly inhibited the relaxant response to sodium nitroprusside. The relaxant response to ANP was not altered by ODQ demonstrating the involvement of particulate guanylyl cyclase. ANP-induced relaxations, as well as sodium nitroprusside-induced relaxations, were similarly potentiated by rolipram (4-(3-(cyclopentyloxy)-4-methoxyphenyl)pyrrolidin-2-one, 3 μM), a type IV phosphodiesterase inhibitor, and by zaprinast (2-(2-propyloxyphenyl)-8-azapurin-6-one, 10 μM), a type V phosphodiesterase inhibitor. ANP-mediated response was unaffected by glibenclamide (10 μM), a selective blocker of ATP-sensitive K + channels, and by apamin (1 μM), a selective blocker of small-conductance Ca 2+-activated K + channels. Iberiotoxin (100 nM) extensively prevented the relaxant effect of ANP suggesting the activation of large-conductance Ca 2+-activated K + channels. In addition, ANP (10 nM) and ANP-(4–23) (100 nM) significantly reduced forskolin (1 μM)-stimulated cAMP accumulation suggesting, for the first time, the presence of functional ANP C receptors in guinea pig airway smooth muscle. However, relaxations to forskolin and to isoproterenol were not altered in the presence of ANP-(4–23) or ANP demonstrating that the inhibitory effect of ANP-(4–23) and ANP on adenylyl cyclase was not sufficient to alter the functional response induced by these two activators of adenylyl cyclase.

Identification of multiple human calcitonin receptor isoforms: heterologous expression and pharmacological characterization.

The human breast carcinoma cell line T47D is known to express high-affinity calcitonin receptors (CTRs). PCR amplification of the CTR cDNA from T47D mRNA resulted in the identification of two different cDNAs that encode distinct receptor isoforms, h alpha CTR and h beta CTR. The two cDNAs are identical except that the h alpha CTR cDNA contains a 48 bp insert sequence that encodes a 16 amino acid domain in the first cytosolic loop of the receptor. Stable transfection of each receptor cDNA into murine erythroleukaemia (MEL) cells resulted in the expression of receptors with high affinity for radiolabelled salmon calcitonin (h alpha CTR Kd 0.09 nM, h beta CTR Kd 0.12 nM). Ligand competition binding studies did not reveal any significant pharmacological difference between the receptor isoforms. In transfected MEL cells and COS-1 cells the h beta CTR isoform was expressed at tenfold higher levels than the h alpha CTR. A reporter gene assay that monitored the coupling of CTR to adenylate cyclase by increases in beta-galactosidase activity indicated that both receptors were able to stimulate cyclic AMP production in response to ligand binding.

The effects of β‐adrenoceptor stimulation on adhesion of human natural killer cells to cultured endothelium

1. The circulation of natural killer (NK) cells in vivo is influenced by physical exercise, mental stress, and infusion of beta-adrenoceptor agonists. We have previously presented in vitro data, showing that beta 2-adrenoceptor agonists induce detachment of NK cells from endothelial cells (EC), supporting the hypothesis that NK cells can be recruited from the marginating pool in blood vessels. 2. Because NK cells as well as EC express beta 2-adrenoceptors, the present study was conducted to investigate whether stimulation of the beta-adrenoceptors on NK cells, EC or both cell types is required to induce detachment from EC. 3. Cells were pretreated (15 min) with a selective beta 2-adrenoceptor antagonist, GR81706, at various concentrations. The duration of beta-adrenoceptor blockade was tested by determining the adenosine 3',5'-cyclic monophosphate (cyclic AMP) production induced by terbutaline (a beta 2-adrenoceptor specific agonist). This receptor-mediated response was effectively inhibited for at least 4 h, whereas the cyclic AMP production in response to forskolin (a direct activator of adenylate-cyclase) was not affected. 4. Functional adhesion assays were then performed to determine the role of beta-adrenoceptors on the different cell types involved (NK and EC) in catecholamine-induced detachment. Peripheral blood mononuclear cells were allowed to adhere for 1 h to monolayers of unstimulated EC in the presence or absence of cyclic AMP inducing agents, and the percentage of NK cells in the adhering lymphocyte fraction was determined by flow cytometry. 5. Both adrenaline (10(-5) M) and forskolin (10(-5) M) caused detachment of NK cells from EC. After blockade of the P2-adrenoceptors on NK cells by pretreatment with GR81706 (10-6 M), the effect of adrenaline on NK cells adhesion was pretented; after blockade of the beta2-adrenoceptors on EC, NK cell adhesion was still significantly reduced by adrenaline. In all cases, forskolin caused detachment of NKcells.6. To establish further that stimulation of beta-adrenoceptors on NK cells is sufficient to cause detachment,we showed that adrenaline also reduced adhesion of NK cells to monolayers of Chinese hamster ovary cells, which do not express beta-adrenoceptors.7. Together, these results show that stimulation of beta2-adrenoceptors on NK cells negatively influences their capacity to adhere to EC, and that beta2-adrenoceptors on EC play a negligible role in this phenomenon.

Effects of porcine relaxin on contraction, membrane response and cyclic AMP content in rat myometrium in comparison with the effects of isoprenaline and forskolin

1. The longitudinal muscle from the uterus of oestrogen-treated rats was quiescent in Mg-free Krebs solution. Electrical stimulation generated phasic contraction, which was depressed to 35% and 18% by 50 mu and 150 mu porcine relaxin, respectively. 2. The phasic contractions were more strongly depressed to 26% by 50 mu relaxin in solution containing 0.6 mM Mg, and the depression lasted for more than 4 h after the removal of relaxin. During the persisting depression, raising the external Ca to 7.5 mM did not restore the contraction, but the contraction was restored by removal of Mg. 3. The depression of the phasic contraction by relaxin, examined in Mg-free solution, was enhanced and reduced by pretreatment of the tissue with 0.6 mM Mg and 0.6 mM Mn, respectively, for about 15 min. In contrast, the depression of contraction by isoprenaline or forskolin was enhanced by pretreatment with either Mg or Mn. 4. The cellular content of cyclic AMP was measured in Krebs solution containing 0.6 mM Mg. The values were 1.24 (pmol mg-1 protein) in control solution, and 2.31 and 1.56 when the tissues were treated with 150 mu relaxin and 10(-9) M isoprenaline, respectively. 5. The cyclic AMP production in response to 10(-7) M forskolin measured in Mg-free solution was enhanced when the tissue was pretreated with either 0.6 mM Mg or Mn for 15 min. The cyclic AMP production in response to 100 mu relaxin was increased when the tissue was pretreated with 0.6 mM Mg, and was unchanged by pretreatment with Mn. The cyclic AMP production in response to 10(-9) M isoprenaline was unchanged by pretreatment with the divalent cations. 6. The membrane potential of the muscle was -60.8 mV in Krebs solution containing 0.3 mM Mg, and electrical stimulation induced an action potential which consisted of spike and plateau components. Application of 150 mu relaxin reduced the duration of the plateau; the contractions were progressively depressed. The resting membrane potential and membrane resistance were unchanged by application of 150 mu relaxin. The membrane was hyperpolarized by 2.8 mV, accompanied by a decrease in membrane resistance, when 10(-9) M isoprenaline was applied. 7. Although there were several differences between the effects of relaxin and isoprenaline, it is probable that some process, which is cyclic AMP-dependent, accelerated by Mg and depressed by Mn, is involved in the depressant action of relaxin on contraction.

Stimulatory and inhibitory cyclic AMP responses in rabbit ciliary processes after cervical ganglionectomy

Cyclic AMP production in response to agonists which act at a variety of receptors to either stimulate or inhibit cyclic AMP production has been studied in intact, dissected ciliary processes from rabbit eyes after unilateral surgical removal of the cervical ganglion. Cyclic AMP responses to stimulatory ligands vasoactive intestinal peptide (VIP), isoproterenol, and forskolin and inhibitory agonists neuropeptide Y (NPY), the synthetic somatostatin analogue SMS 201-995, and alpha-adrenergic agents were investigated in tissues from normal eyes and compared to the same responses in tissues from sympathetically denervated eyes. Neither stimulated cyclic AMP production nor inhibition of stimulated cyclic AMP production was significantly different in tissues from denervated vs. normal eyes. Inhibition of VIP-stimulated cyclic AMP production by epinephrine and paraaminoclonidine in tissues from both normal and denervated eyes was blocked by the alpha 2-adrenergic antagonist yohimbine but not by the alpha 1-adrenergic antagonist prazosin. These data indicate that the VIP, NPY, somatostatin, and alpha 2- and beta 2-adrenergic receptors which regulate cyclic AMP production in rabbit ciliary processes are postjunctional and suggest that ligands known to modulate cyclic AMP levels in this tissue may exert effects on aqueous humor formation independently of adrenergic innervation.

Regulation of interleukin-6 release by human thyrocytes

Autoimmune thyroiditis is characterized by lymphocytic accumulation within the thyroid which may be the result, in part, of immunomodulatory cytokine secretion by thyrocytes. We have tested human thyroid cell cultures (n = 9) for interleukin-6 (IL-6) release using two bioassays. IL-6 was detected in all culture supernatants under basal conditions and was increased by gamma-interferon, tumour necrosis factor and TSH in a dose-dependent manner. The bioactivity was confirmed as IL-6 by immunoblotting experiments and could not be accounted for by contamination of the thyroid cell cultures with fibroblasts, lymphocytes or monocytes. Circulating IL-6 levels were not raised in patients with Graves' hyperthyroidism. Exogenous recombinant IL-6 reduced cyclic AMP production in response to TSH when added to thyroid cell cultures. Since IL-6 plays a major role in B cell differentiation and T cell activation, release of IL-6 by thyrocytes may increase the intrathyroidal autoimmune response in Graves' disease and Hashimoto's thyroditis. Our results also suggest that IL-6 may modulate thyroid cell function.

Comparison of the effects of amino-terminal synthetic parathyroid hormone-related peptide (PTHrP) of malignancy and parathyroid hormone on resorption of cultured fetal rat long bones

We have compared the effects of of various synthetic amino-terminal forms of human parathyroid hormone-related peptide (PTHrP) of malignancy with synthetic parathyroid hormone (PTH) on the resorptive responses of fetal rat long bones in organ culture. PTH and PTHrP increased 45Ca release at concentrations of 0.1-25 nM. PTHrP (1-40) and bovine PTH (1-34) were more potent than human PTH (1-34) and PTHrP (1-34). However, the slopes of the dose-response curves and the maximal resorptive effects were similar. There was a marked decrease in the potency of amino-terminal PTHrP peptides as the length was decreased. PTHrP (1-29) and PTHrP (1-25) were inactive at 120 nM. Further comparison of bPTH (1-34) and PTHrP (1-34) showed that both could induce bone resorption after a brief (6 hours) exposure and that the response to PTHrP (1-34) was qualitatively similar to that of bPTH (1-34) with respect to enhancement by ACTH and inhibition by calcitonin and glucocorticoids. Hydroxyurea and indomethacin did not block the resorptive response to either agonist. Cyclic AMP production in response to PTHrP (1-34) and (1-40) was similar to that for bPTH (1-34) in ROS 17/2.8 cells. The cyclic AMP (cAMP) response was much smaller in fetal rat long bones and calvariae, and bPTH was more potent than PTHrP. These studies confirm that PTHrP is quantitatively similar in its effects on bone resorption to PTH and are consistent with the two agents acting on the same receptor.

Variable Stimulation of Adenylate Cyclase Activity by Vasoactive Intestinal-Like Peptides and β-Adrenergic Agonists in Murine T Cell Lymphomas of Immature, Helper, and Cytotoxic Types

We examined several cultured murine T cell lymphomas, induced by a radiation leukemia virus MuRadLV, including cell lines derived from immature T cells (5 clones of the BL/VL3 cell line), antigen-specific T helper cells (5 lines of the TL2 series), and one T cytotoxic cell line (NS8).With one exception (the TL2-9 cell line), these cells showed common characteristics: 1) an efficient adenylate cyclase system; 2) increased cyclic AMP production in response to at least one type of neurotransmitter, i.e., to the catecholamine isoproterenol and/or the neuropeptide VIP; 3) on the basis of adenylate cyclase stimulation, beta- adrenoceptors were of the beta 2 subtype and VIP receptors were of a «helodermin-preferring» subtype previously encountered in a human T lymphoblast cell line. Although we analyzed only a limited number of cell lines, it appeared that the immature T BL/VL3 clones responded to peptides of the VIP family with higher potency and efficacy than T helper and T cytotoxic cells.The membranes from the specific TL2-9 helper cell line were without adenylate cyclase activity in the presence of Gpp[NH]p, NaF, and GTP alone or GTP in the presence of isoproterenol or VIP.They produced cyclic AMP in the presence of Mn 2+ and forskolin only, suggesting a defect in Gs as in S49 cyc mouse lymphosarcoma cells.This was further demonstrated by the absence of cholera toxin-stimulated ADP-ribosylation in TL2-9 membranes.

Characterization of primar cell cultures derived from rat renal proximal tubules

Proximal tubules were prepared from rat kidney cortex by collagenase digestion and purified by Percoll gradient centrifugation. Their enrichment was estimated by comparing the specific activities of various cell-specific enzymes in homogenates of renal cortex and of the isolated tubules. The tubules were cultured in a 50:50 mixture of Dulbecco's modified Eagle's and Ham's F12 media supplemented with insulin, transferrin, epidermal growth factor, hydrocortisone, and prostaglandin E1. After 2 to 3 d an extensive outgrowth of epithelial cells developed from the attached tubules. After 5 to 7 d near confluent monolayers were obtained. Hormonal responsiveness, marker enzyme activities, and transport properties were determined to further characterize the primary cultures. The cultured cells exhibited increased cyclic AMP production in response to parathyroid hormone but not calcitonin or vasopressin, consistent with the absence of cells derived from distal and collecting tubules. The cells also retained significant levels of 25-hydroxyvitamin D3-1 alpha-hydroxylase, alkaline phosphatase, and gamma-glytamyl-transpeptidase, three enzymes that are primarily associated with the proximal tubule. The cultured epithelial cells also exhibit a Na+-dependent phosphate and glucose transport systems. Therefore, the cells retain many functional properties that are characteristic of proximal tubules. Thus, the primary cultures should be suitable for the study of processes that occur specifically within this segment of the rat nephron.

Effect of ouabain on cellular free calcium and cellular cyclic AMP production in response to arginine vasopressin in rat renal papillary collecting tubule cells in culture

The effect of extracellular calcium (Ca2+) on the cellular action of arginine vasopressin (AVP) was examined using an Na+, K+-ATPase inhibitor in rat renal papillary collecting tubule cells in culture. The pretreatment of cells with ouabain enhanced basal and AVP-induced cAMP production in a dose-dependent manner. The augmentation by ouabain of cellular cAMP production in response to AVP was totally abolished by co-treatment with cobalt, lanthanum, verapamil or Ca2+-free medium containing 1 mmol EGTA/l, each blocking cellular Ca2+ uptake by different mechanisms. Two other findings indicated that ouabain directly stimulated cellular Ca2+ mobilization; namely, that ouabain significantly increased 45Ca2+ influx and cellular free Ca2+ concentration [( Ca2+]i) determined by Fura-2 fluorescence. The ouabain-induced increase in [Ca2+]i was completely blocked by either cobalt or Ca2+-free medium containing 1 mmol EGTA/l. AVP at 0.1 mumol/l increased [Ca2+]i to 177.1 +/- 26.2 nmol/l from 92.2 +/- 8.0 nmol/l (P less than 0.01) in renal papillary collecting tubule cells, and ouabain significantly enhanced the AVP-induced increase in [Ca2+]i. The increase of cellular free Ca2+ induced by ouabain probably binds to calmodulin to form an active complex of Ca2+-calmodulin in the cell, since two chemically dissimilar antagonists of calmodulin attenuated the enhancement by ouabain of cAMP production in response to AVP. These results therefore indicate that ouabain increases cellular Ca2+ uptake and enhances AVP-induced cellular free Ca2+ mobilization and its own second messenger cAMP production in renal papillary collecting tubule cells, and that extracellular Ca2+ is an important source for ouabain-mobilized cellular Ca2+.

Calmodulin regulation of cellular cyclic AMP production in response to arginine vasopressin, prostaglandin E2 and forskolin in rat renal papillary collecting tubule cells in culture.

The effect of calmodulin on the stimulation of cyclic AMP production by arginine vasopressin (AVP), prostaglandin E2 (PGE2) and forskolin was examined in cultured renal papillary collecting tubule cells of the rat. In the presence of the phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine submaximal concentrations of AVP (1 nmol/l), PGE2 (20 nmol/l) and forskolin (240 nmol/l) significantly increased cellular cyclic AMP accumulation by 2.3-, 6.0- and 8.4-fold respectively. Two chemically dissimilar inhibitors of calmodulin, namely trifluoperazine and N-(6-amino-hexyl)-5-chloro-1-naphthalenesulphonamide (W-7), attenuated the AVP-, PGE2- and forskolin-stimulated cellular production of cyclic AMP in a dose-related manner. Cellular production of cyclic AMP was inhibited by 50% (ID50) by doses ranging from 16 to 28 mumol trifluoperazine/l and 35 to 44 mumol W-7/1. Basal accumulation of cellular cyclic AMP was also decreased by treatment with either trifluoperazine or W-7, but the effective dose was higher than that which inhibited cellular cyclic AMP production stimulated by AVP, PGE2 and forskolin. Since forskolin directly activates adenylate cyclase at a site of the catalytic unit and the cellular action of AVP to activate adenylate cyclase is mediated through receptor-guanine nucleotide regulatory-catalytic units, the present study indicates calmodulin regulation of basal, AVP-, PGE2- and forskolin-activated adenylate cyclase in the papillary collecting tubule cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of cortisol on cyclic AMP production stimulated by 1–24 ACTH in adrenal cortex membranes

The effect of cortisol on cyclic AMP production by crude bovine adrenal cortex membrane preparations has been investigated. Results demonstrate that in the presence of cortisol, cyclic AMP production in response to 1–24 ACTH was enhanced up to a concentration of about 74 μM cortisol. At higher concentrations the effect was reversed. Cortisol had no effect on cyclic AMP production in the absence of 1–24 ACTH, and cyclic AMP production was completely inhibited when 5 mM EDTA was added to the incubation tubes with the cortisol.

The functional activity of adult mouse Leydig cells in monolayer culture: Effect of lutropin and foetal calf serum

Purified Leydig cells were obtained from adult mouse testes by mechanical dispersion followed by Percoll density-gradient centrifugation as described by Schumacher et al. (1978). The cells were then established in monolayer culture by maintaining them in medium and 10% serum at 32°C in 95% O 2, 5% CO 2. The cells rapidly attached to the culture dishes, gradually flattened and became epitheloid in appearance. Testosterone production by the cells in response to maximum stimulating levels of LH (100 ng/ml) and dibutyryl cyclic AMP (1 mM) was maintained for at least 2 days (∼ μg/10 6 cells/ 2 h) and then declined to lower levels by days 3–4. Cyclic AMP production in response to LH was higher on day 1 than day 0 and then declined to lower levels by days 3—4. Binding of [ 125I]hCG was similar on day 0 and day 1 (∼20 fmoles/10 6 cells) and then declined to lower levels by days 3–4. The functional activity of the cells cultured in 0, 1 and 10% foetal calf serum was also examined; no significant effect of the serum on LH-stimulated testosterone or cyclic AMP production was found; however, a decrease of up to 50% in the binding of [ 125I]hCG to the Leydig cells occurred in the presence of serum. These results demonstrate that the function of differentiated adult Leydig cells can be maintained for at least 2 days in culture.

Receptor mediated gonadotropin action in the ovary

Abstract. The role of macromolecular synthesis in gonadotrophin, cholera enterotoxin and cAMP stimulated progesterone production by rat ovarian cells has been investigated. Both progesterone and cAMP accumulation were increased by cholera enterotoxin in a concentration dependent manner. Incubation of cells with cholera enterotoxin resulted in a 10–20-fold increase in cAMP levels. The effect was observed 15–20 min after addition of toxin. In contrast, stimulation of cAMP levels by hCG was immediate. Cyclic AMP production in response to trophic hormone reached a maximum at 60 min, while maximum stimulation in response to toxin was attained after 2–3 h of incubation. Activation of steroidogenesis in response to both cholera enterotoxin and gonadotrophin showed a lag period. hCG and cholera enterotoxin stimulated a maximum amount of progesterone production after 2 and 3 h of incubation, respectively. With the maximum effective concentration of hCG, addition of choleragen did not result in any further increase in steroidogenesis. Similarly 8 Br-cAMP and Bt2-cAMP also stimulated steroidogenesis. Progesterone response to cholera enterotoxin, hCG, LH, 8 Br-cAMP and Bt2-cAMP was completely blocked by incubation of cells with cordycepin, actinomycin D, emetine and cycloheximide. That the specific effect of these inhibitors was on macromolecular synthesis rather than a general non-specific toxic effect was demonstrated by inhibition of [3H]proline incorporation and the lack of effect of these inhibitors on cAMP production in response to hCG, LH and cholera enterotoxin. Emetine (10 μm) and cycloheximide (50 μm) inhibited cholera enterotoxin and hCG stimulated progesterone production when added at different time points during the incubation. Cordycepin (250 μm) also blocked toxin and hCG induced steroidogenesis in a similar manner. The concentrations of cordycepin, cycloheximide and emetine required to completely block hCG stimulated progesterone production were 250, 50, and 5 μm, respectively. Similar concentrations of these inhibitors also maximally inhibited 8 Br-cAMP and cholera enterotoxin stimulated steroidogenesis. No effect of these inhibitors on basal production of progesterone was observed. These results suggest that gonadotrophin induced progesterone synthesis is dependent upon the continued synthesis of short lived mRNA and protein(s).

Calcium-dependent hormonal regulation of amino acid transport and cyclic AMP accumulation in rat hepatocyte monolayer cultures.

The effect of glucagon, epinephrine, norepinephrine, dexamethasone, insulin, and dexamethasone plus glucagon on the transport of 2-aminoisobutyric acid (AIB) and that of glucagon on the production of cyclic AMP were examined in rat hepatocyte monolayer cultures under three different culture conditions involving calcium. The hepatocytes were studied in calcium-contaning medium after treatment with or without 0.033% dimethyl sulfoxide, the solvent for the calcium ionophore A23187 (calcium controls); calcium-free medium after treatment with A23187 (calcium-depleted); and calcium-containing medium after treatment with ionophore (calcium-restored). The basal and hormonally regulated rates of AIB transport for hepatocytes in calcium control and calcium-depleted cultures were comparable. The restoration of calcium in calcium-restored cultures increased the basal and the hormonally stimulated transport of AIB when compared to the other conditions. Calcium markedly enhanced the stimulation of AIB transport in cultures treated with glucagon, catecholamines, and dexamethasone plus glucagon. The level of cyclic AMP production in response to glucagon in calcium control and calcium-depleted cultures was the same and it was conspicuously higher than the level in calcium-restored cultures. Varying the concentration of calcium in the medium used to maintain the hepatocytes in calcium control cultures did not affect the stimulation of AIB transport or cyclic AMP production by glucagon. However, in calcium-restored cultures, increasing the calcium concentration of the medium resulted in increased stimulation of AIB transport and decreased production of cyclic AMP by glucagon. In the calcium-restored cultures, calcium in the absence of glucagon enhanced AIB transport but had no effect on cyclic AMP production. Cultures maintained for 6 hr in calcium-free medium after the depletion of calcium showed a 6- to 7-fold increase in the production of cyclic AMP in response to glucagon, but no stimulation of AIB transport. We suggest that mobilization of cellular calcium by glucagon either directly or through cyclic AMP mediates its stimulation of amino acid transport.

Mepacrine blocks beta-adrenergic agonist-induced desensitization in astrocytoma cells.

C6 astrocytoma cells contain beta-adrenergic receptors coupled to adenylate cyclase. A 2-hr exposure to l-isoproterenol results in an 80% decrease in cyclic AMP production in response to a subsequent challenge by l-isoproterenol (desensitization). This loss in responsiveness is paralleled by a 20-30% decrease in the apparent number of beta-adrenergic receptors and by increased release of arachidonic aciid into the medium. The increased release of arachidonic acid is caused by the action of phospholipase A2 (phosphatide 2-acylhydrolase, EC 3.1.1.4) and corresponds to increased turnover of methylated phospholipids. Mepacrine and tetracaine, both inhibitors of this phospholipase A2, are able to block l-isoproterenol-induced desensitization of cyclic AMP production and the decrease in beta-adrenergic receptors. Mellitin and phorbol ester, two activators of phospholipase A2, when preincubated with the cells cause a decreased cyclic AMP response of the cells to l-isoproterenol. These results suggest that the activation of phospholipase A2 in the local domain of the beta-adrenergic receptor may be involved in desensitization.

Cyclic AMP metabolism in adipose tissue of exercise-trained rats.

Cyclic AMP metabolism in epididymal adipose tissue of exercise-trained rats was examined to determine if training induced changes in cyclic AMP production or inactivation. Beginning at 7 weeks of age, male rats were physically trained by 12 weeks of treadmill running. Pair-fed control rats remained sedentary in their cages for the duration of the experiment. Tissue levels of cyclic AMP were measured in epididymal adipose tissue slices incubated with norepinephrine. Adenyl cyclase was assayed in adipocyte ghost cell prepartions and low-Km phosphodiesterase was assayed in homogenates of adipose tissue. In response to norepinephrine stimulation, tissue cyclic AMP levels were reduced in trained compared to untrained rats. Training increased the ratio of activity of phosphodiesterase relative to adenyl cyclase. The results of this study indicate that cyclic AMP production in response to norepinephrine stimulation is not increased by training and may even be reduced, implying that adipose tissue cyclic AMP levels may be under a greater degree of control in trained rats. Modulation of adipose tissue cyclic AMP levels may function to regulate more closely the duration of lipolysis in exercise-trained rats.

Epinephrine-induced cyclic AMP production and vasoconstriction in the noncyclically perfused rat hindlimb: A possible role for insulin

The effects of epinephrine alone and in combination with insulin and adrenergic blocking agents on cyclic AMP production and vascular tone are studied in noncyclically perfused rat hindlimbs. Epinephrine increases cyclic AMP production 11-fold and vascular resistance 2-fold. Insulin has no effect on either parameter but blunts cyclic AMP production in response to epinephrine by 50% without altering the epinephrine induced change in resistance. Alpha and beta adrenergic blocking agents have divergent and contrasting effects when infused with epinephrine. Phentolamine, but not propranolol, blocks the increase in vascular resistance observed with epinephrine; propranolol, but not phentolamine, inhibits epinephrine stimulated cyclic AMP release. These studies suggest that cyclic AMP may not play a significant role in alpha adrenergic mediated vasoconstriction.