Thyroid-stimulating hormone and cyclic adenosine 3',5'-monophosphate in the regulation of thyroid gland function.

The initial step in TSH action reflects binding of the hormone to specific receptor sites on the plasma membrane. Such binding has been studied using plasma membranes, homogenates, isolated thyroid cells grown in culture, and thyroid slices. 3-H- and iodinated TSH preparations have been used; the latter have been prepared using both chloramine-T and lactoperoxidase. Some of the discrepancies reported in the literature might reflect the different thyroid and hormone preparations and the variable incubation conditions which have been used. In general, good correlation exists between binding of TSH and activation of adenylate cyclase in thyroid plasma membranes. Data is reviewed related to activation of protein kinase in intact thyroid cells by TSH. Although there is impressive evidence for cyclic AMP mediation of effects of TSH on the thyroid, some data that are inconsistent with this concept are considered, especially in relationship to 32-P incorporation into phospholipid. The role of cyclic GMP in thyroid function is discussed.     

Plasma cyclic adenosine 3'5'-monophosphate (AMP) levels in acute myocardial infarction

Plasma cyclic adenosine 3′5′-monophosphate (AMP) levels were measured in 44 patients with acute myocardial infarction, 33 patients with other cardiac and noncardiac diseases and 20 normal volunteers. The normal range of cyclic AMP was 4 to 16 picomoles/ml. The 35 surviving patients with acute myocardial infarction tended to have a slightly increased level of plasma cyclic AMP during the first 24 hours with a subsequent return to normal; the 9 nonsurvivors had abnormally high levels of cyclic AMP. An inverse correlation was found between cyclic AMP levels and stroke work index, and plasma cyclic AMP levels were of equal or better prognostic value than stroke work index. Plasma cyclic AMP levels were in the normal range in patients without acute myocardial infarction. Thus, very high levels of plasma cyclic AMP, found in patients with fatal myocardial infarction, appear to have clinical significance.     

The role of cyclic adenosine 3',5'-monophosphate in the ovulatory process of the in vitro perfused rabbit ovary

LH alters ovarian steroidogenesis via adenylate cyclase (AC) activation and cAMP production. Although LH also initiates ovarian follicle rupture, evidence is lacking for involvement of cAMP in this process. This work explores the involvement of cAMP in the ovulation of in vitro perfused rabbit ovaries by comparing LH stimulation of ovaries with that of LH plus 3-isobutyl-1-methyl-xanthine (IBMX), (an inhibitor of phosphodiesterase) and of forskolin (a nonreceptor-specific activator of AC). Venous perfusates were analyzed for cAMP, progesterone, 17 beta-estradiol, and testosterone, ovaries were analyzed for cAMP, and ovulations were noted. LH, LH plus IBMX, and forskolin all increased tissue cAMP levels significantly after 0.5 h, the perfusate levels increasing rapidly thereafter reaching plateau levels, while tissue levels returned to control levels after 2.4 h. LH plus IBMX and forskolin significantly increased cAMP release over LH controls, LH plus IBMX increasing and forskolin decreasing the number of ovulations. Forskolin significantly increased progesterone release over LH controls and, although no other significant steroid differences were seen, strong tendencies existed. Although forskolin could induce ovulations and could induce significantly higher release of cAMP than LH, it resulted in a lower ovulation rate than receptor-specific LH. LH plus IBMX also induced significantly higher cAMP release than LH, as did forskolin, and resulted in a higher ovulation rate than both LH and forskolin. These findings suggest, not only that cAMP production alone is sufficient for ovulation, but also that the receptor specificity of the cAMP production is important for the number of ovulations. Since tissue levels of cAMP peak several hours before ovulation, the cAMP is probably inducing a metabolic pathway leading to ovulation.     

Calcium-dependent regulation of guanosine 3',5'-monophosphate in renal cortex: effects of ionophore A23187 and tetracaine and evidence for independent control of adenosine 3',5'-monophosphate.

The effects of carbamylcholine (Cch), the divalent cation ionophore A23187 and Ca2+ on the cyclic 3',5'-guanosine monophosphate (cGMP) and cyclic 3',5'-adenosine monophosphate (cAMP) content of rat renal cortical slices were examined. In both the presence and absence of 10 mM theophylline, Cch detectably increased cGMP within 15 sec, with peak responses noted by 2 min. The maximal cGMP response to Cch alone (0.05 mM) was an increase of two- to three-fold over control. Theophylline, which was routinely present in the incubations and which alone increased cGMP of the slices two-fold over basal during 20 min incubations, potentiated the response to Cch (maximal increase, five- to sixfold over theophylline alone). The action of Cch to increase renal cortical cGMP was blocked by prior addition of atropine and was dependent upon the presence of Ca2+ in the incubation media. Exclusion of Ca2+ lowered basal cGMP and abolished increases mediated by Cch, while exclusion of Mg2+ was without detectable effect on cGMP. In slices incubated initially without Ca2+, reexposure to Ca2+ for 1min partially restored the cGMP response to Cch, and reexposure for 3 min completely restored this response. Since prior incubation of tissue in Ca2+-free buffer for only 2 min was sufficient to block the cGMP responses to Cch, depletion of tissue Ca2+ did not appear to be involved. A23187 also increased renal cortical cGMP fivefold in the presence of Ca2+. Its effects were not additive with those of Cch and were not additive with those of Cch and were not expressed by Mg2+ in Ca2+-free media. By contrast, tetracaine, which blocks Ca2+ transport across or binding to biologic membranes, reduced basal cGMP and inhibited the actions of Cch and A23187 to increase cGMP in cortical slices incubated with Ca2+. The action of 1 mM tetracaine to block Cch-mediated increases in cGMP was partially reversed by increasing media Ca2+ from 1.5 to 5 mM, but not by increasing media Mg2+ to 5 mM. In contrast to their effects on cGMP, Cch, A23187, Ca2+ exclusion, and tetracaine did not detectably alter basal renal cortical cAMP or cAMP responses to parathyroid hormone (PTH). Conversely, concentrations of PTH, glucagon, and isoproterenol which maximally increased renal cortical cAMP did not alter cGMP. Furthermore, prior incubation of slices with Cch did not alter their subsequent cAMP response to PTH at a time when cGMP levels were still elevated, while prior incubation with PTH did not affect the subsequent cGMP response to Cch at a time when cAMP was increased. These studies demonstrate modulation of renal cortical cGMP by cholinergic stimuli and Ca2+. They also indicate that cGMP and cAMP in renal cortex can be regulated independently.     

Effect of glucagon and cyclic adenosine 3',5'-monophosphate on protein phosphorylation in rat pancreatic islets

Isolated rat pancreatic islets, incubated in the presence of extracellular 32Pi to a state of steady 32P incorporation into cellular phosphopeptides, were exposed to glucagon, (Bu)2cAMP, or somatostatin for 10 min. In other experiments, homogenates of rat islets were phosphorylated using [gamma-32P]ATP with or without cAMP. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and phosphorylation of proteins was measured by liquid scintillation counting of gel slices. Glucagon (2.9 X 10(-7) M) stimulated the phosphorylation of 15 polypeptides (by approximately 20-50%) with major phosphorylation of proteins with mol wts of 138,000, 93,000, 53,000, 49,000, 35,000, 27,000 and 15,000 in intact rat islets and also stimulated insulin release by 202%. Somatostatin (6.6 X 10(-7) M) inhibited all the glucagon-stimulated phosphorylation by approximately 15-30% and also inhibited the glucagon-stimulated insulin release by 46%. (Bu)2cAMP (10(-3) M) stimulated 32P incorporation (by approximately 20-50%) into the same 15 peptides as did glucagon and also stimulated insulin release by 169%. When homogenates of rat islets were used. cAMP (10(-6) M) stimulated the phosphorylation of proteins (by approximately 25-60%) to an extent similar to that seen in the presence of glucagon or (Bu)2cAMP in intact islets. These findings indicate that the glucagon-stimulated phosphorylation of rat islet proteins may be mediated by cAMP-dependent protein kinase and that protein phosphorylation may be important in mediating the glucagon-stimulated insulin release.     

Atypical synergistic alpha 1- and beta-adrenergic regulation of adenosine 3',5'-monophosphate and guanosine 3',5'-monophosphate in rat pinealocytes

The adrenergic control of cAMP and 3',5'-cyclic GMP (cGMP) in dispersed adult rat pinealocytes was investigated. Norepinephrine treatment increased cAMP and cGMP content 60- and 400-fold, respectively; both alpha- and beta-adrenoceptors had to be activated for these responses to occur. Beta-Adrenergic stimulation alone produced only about 6- and 2-fold increase in cAMP and cGMP content, respectively. Alpha-Adrenergic stimulation, which alone had no effect on either cyclic nucleotide concentration, markedly amplified the beta-adrenergic stimulation of both cAMP and cGMP. The relative potency of alpha-adrenergic agonists and antagonists indicates the alpha 1-subclass of adrenoceptors is involved. A role of alpha 1-adrenoceptors in the control of pineal cAMP is consistent with published evidence of the presence of alpha 1-adrenoceptors on pinealocytes and their role in the regulation of N-acetyltransferase activity and melatonin production.     

Estrogen receptor-mediated down-regulation of corticotropin-releasing hormone gene expression is dependent on a cyclic adenosine 3',5'-monophosphate regulatory element in human placental syncytiotrophoblast cells

Placental CRH plays a major role in the mechanisms controlling human pregnancy and parturition. Understanding how placental CRH production is regulated is therefore of importance. Previously we have shown that placental expression of CRH peptide and mRNA are inhibited by estrogens, in contrast to the stimulatory effects of estrogen on hypothalamic CRH production. Our current study found that in placental cells cotransfected with a CRH promoter construct and an estrogen receptor-alpha expression vector results in a differential regulation whereby 17beta-estradiol (E2) decreased and the putative pure estrogen antagonist, ICI 182780, increased CRH promoter activity. Sequential deletion of the CRH promoter indicated that the region between -248 and -213 bp was essential for the effect of both E2 and ICI 182780. This region contains a consensus cAMP regulatory element (CRE) that is a requirement for E2- and ICI 182780-mediated activity because the CRE motif can confer E2 inhibition on a heterologous promoter such as rabbit beta-globin. Mutation of the CRE resulted in a complete reversal of E2 and ICI 182780 regulatory effects. In summary, our results demonstrate that a consensus CRE is required for the action of estrogen receptor ligands in human placental syncytiotrophoblast cells.     

Spontaneous diabetic BB rat: studies of cyclic adenosine 3',5'-monophosphate phosphodiesterase and calmodulin

Uncontrolled diabetes in man is associated with increased plasma and tissue levels of cAMP and decreased cAMP phosphodiesterase (PDE) activity. Spontaneously diabetic BB rats (SDR) were used in these experiments. Specific tissues (i.e. liver and epididymal fat) were studied without therapeutic insulin. Another group of normal animals were rendered diabetic by streptozotocin (STZ) and killed without benefit of insulin therapy. Calmodulin (CM), a small molecular weight protein essential for activation of specific cAMP PDE was assayed. STZ diabetes is associated with a decrease (58%) in CM biological activity and in immunoreactive CM in fat (69%) and liver (13%) tissues. Similarly, SDR rats and the nondiabetic genetic controls (NDR) demonstrate decreased CM bioactivity in fat (76% and 56%, respectively) and decreased CM immunoreactivity in liver (68% and 74%, respectively) compared to normal control rats. In addition, maximum velocity (Vmax) of the low Michaelis-Menten constant (Km) cAMP PDE is decreased in SDR animals, as compared to controls in both fat (42%) and liver (39%) tissues. Similar data are presented for NDR animals. STZ diabetes is also associated with a reduction in Vmax of the low Km cAMP PDE in both liver (70%) and fat (70%) tissues. These changes found in the NDR animals suggests that the diabetic defect may be under dual regulation: genetic and environmental.     

Urinary and plasma cyclic adenosine 3',5'-monophosphate in patients with idiopathic edema.

Urinary excretion and plasma concentrations of adenosine 3',5'-monophosphate were determined in idiopathic edema patients both at rest and after assumption of the upright position. Patients and normal subjects responded similarly to upright posture with decreases in urinary volume and creatine excretion and their urinary excretion of cyclic AMP was not significantly different. The plasma concentration of cyclic AMP was high in patients, both in the recumbent and upright positions, and its renal clearance (unlike that of creatinine) was low. Plasma cyclic AMP increased in response to upright posture both in patients and normal subjects. Even if the significance of elevated plasma cyclic AMP is unknown, its presence without an increase in urinary cyclic AMP suggests that the renal handling of cyclic AMP is abnormal is idiopathic edema; this may be related to an excessive beta-adrenergic tone.     

Catecholamine effect on cyclic adenosine 3':5'-monophosphate level in isolated catfish hepatocytes

The effect of catecholamines (epinephrine, norepinephrine, isoproterenol, and phenylephrine) on cyclic adenosine 3':5'-monophosphate (cAMP) level in isolated catfish (Ictalurus melas) liver cells was studied in the presence or absence of alpha (phentolamine) and beta (propranolol)-receptor antagonists. All catecholamines increased the hepatocyte cAMP level: the rank of their potency was epinephrine = isoproterenol greater than norepinephrine greater than phenylephrine. Propranolol completely blocked the catecholamine effect; phentolamine was ineffective. Results confirm previous findings (L. Brighenti, A. C. Puviani, M. E. Gavioli, and C. Ottolenghi, 1987, Gen. Comp. Endocrinol. 66, 306-313) that epinephrine and norepinephrine act via beta-receptor activation. However, the comparison of the effects of isoproterenol and phenylephrine on cAMP with those on phosphorylase alpha and on glycogen breakdown suggests that a more complex mechanism is possibly involved in the catecholamine effect on catfish glycogenolysis.     

Urinary excretion of cyclic adenosine 3',5'-monophosphate in children of different ages.

The urinary excretion of cyclic AMP was investigated in 97 healthy children, 3 months to 16 years old. When the excretion was expressed as mumol/24 h an increase with age (r = 0.693, P less than 0.001) and an increase with body weight (r = 0.700, P less than 0.001) were found to be quite similar. In relation to surface area, the average excretion for children up to 91/2 years old was 4.45 +/- 1.71 mumol/m2 in constrast with 2.22 +/- 0.66 mumol/m2 in older children (P less than 0.001). The decline appears to be associated with approaching puberty. When cAMP excretion was related to urinary creatinine, an inverse correlation with age was found (r = -0.772, P less than 0.001). In the youngest category, 3 months to 4 years old, the ratio was 9.26 +/- 1.49 mumol/g creatinine vs 4.67 +/- 1.05 mumol/g creatinine in the age group 12 to 16 years old (P less than 0.001), which compares closely with the normal adult average of 4.34 +/- 1.25 mumol/g creatinine found in our previous study. Throughout there was no evidence of sex differentiation.     

Dopamine inhibits prolactin release when cyclic adenosine 3',5'-monophosphate levels are elevated

We purified lactotrophs from pituitary tumors induced by estrogen in ovariectomized female Fischer 344 rats from 80% of the population before to more than 90% after purification through a continuous Percoll density gradient. The percentage of lactotrophs was evaluated by immunofluorescence. The patterns of PRL release stimulated by 100 nM TRH, 20 microM A23187 (a Ca++ ionophore), 50 nM 12-O-tetradecanoyl-phorbol-13-acetate (a C-kinase activator), or combinations of these agents, or inhibited by 10 microM dopamine were similar in perifused primary cultures of tumor lactotrophs to patterns in cultures of anterior pituitary cells from female retired breeders used previously. In particular, dopamine completely inhibited the release stimulated by forskolin. Intracellular cAMP concentrations and PRL accumulation in the medium were measured in monolayer cultures of purified tumor lactotrophs. In 9 separate experiments, forskolin (10 microM) increased intracellular cAMP concentrations more than 60-fold above control after 30 min of incubation. Preincubation (30 min) with dopamine (10 microM) reduced the cAMP accumulation caused by forskolin, but levels were still at least 20-fold above basal levels in most experiments. PRL release was stimulated 2-fold with forskolin alone, but there was no stimulation of PRL release by forskolin in the presence of dopamine even though cAMP levels were elevated above basal. Therefore, a decrease in cAMP levels is not necessary to inhibit PRL release, and dopamine must have a mechanism for inhibiting PRL release in addition to inhibiting adenylate cyclase.