Cyclic Adenine Nucleotide Research Articles

Testing sensomotoric capacity by a computer-aided tracking system

In different brain structures (telencephalon, tectum opticum, rhombencephalon, cerebellum and medulla oblongata) of the teleost Serranus scriba Cuv., the content of glycogen was observed during a 24-h period. A circadian rhythm of brain glycogen concentration was found. The results are discussed with particular reference to the possible relation between catecholamines, cyclic adenine nucleotide and glycogen metabolism in the brain.

Role of cyclic adenosine monophosphate in the induction of endothelial barrier properties

Cyclic adenosine monophosphate (AMP) has numerous important effects on cell structure and function, but its role in endothelial cells is unclear. Since cyclic AMP has been shown to affect transmembrane transport, cell growth and morphology, cellular adhesion, and cytoskeletal organization, it may be an important determinant of endothelial barrier properties. To test this we exposed bovine pulmonary artery endothelial cell monolayers to substances known to increase cyclic AMP and measured their effect on endothelial permeability to albumin and endothelial cell cyclic AMP concentrations. Cholera toxin (CT), a stimulant of the guanine nucleotide binding subunit of adenylate cyclase, led to a concentration-dependent 2-6-fold increase in cyclic AMP which was associated with a 3-10-fold reduction in albumin transfer across endothelial monolayers. The effect was not specific to albumin as similar barrier-enhancing effects were also noted with an unrelated macromolecule, fluorescein isothiocyanate (FITC)-dextran (MW 70,000). Barrier enhancement with cyclic AMP elevation was also observed with forskolin, a stimulant of the catalytic subunit of adenylate cyclase. The temporal pattern of barrier enhancement seen with these agents paralleled their effects on increasing cyclic AMP, and the barrier enhancement could be reproduced by incubation with either dibutyryl cyclic AMP or Sp-cAMPS, cyclic AMP-dependent protein kinase agonists. Furthermore, the forskolin effect on barrier enhancement was partially reversed with Rp-cAMPS, an antagonist of cyclic AMP-dependent protein kinase. Since endothelial actin polymerization may be an important determinant of endothelial barrier function, we sought to determine whether the cyclic AMP-induced effects were associated with increases in the polymerized actin pool (F-actin). Both cholera toxin and forskolin led to apparent endothelial cell spreading and quantitative increases in endothelial cell F-actin fluorescence. In conclusion, increased endothelial cell cyclic adenine nucleotide activity was an important determinant of endothelial barrier function in vitro. The barrier enhancement was associated with increased endothelial apposition and increases in F-actin, suggesting that influences on cytoskeletal assembly may be involved in this process.

Insulin inhibition of hepatic cAMP-dependent protein kinase: decreased affinity of protein kinase for cAMP and possible differential regulation of intrachain sites 1 and 2.

In hepatocytes stimulated with 8-bromo-cAMP, insulin decreases the affinity of the cAMP-dependent protein kinase for cAMP, shifting the Ka without affecting the Vmax activity. This occurs under conditions where cyclic adenine nucleotide concentrations are unchanged. We report here that glycogenolysis stimulated by 8-(4-chlorophenylthio)-cAMP, an analog with 100 times tighter affinity than cAMP for the protein kinase regulatory subunit, was only slightly antagonized by insulin. The tight binding of this analog appears to overcome the protein kinase affinity change induced by insulin. The relative importance of the two intrachain cAMP binding sites of the cAMP-dependent protein kinase regulatory subunit was investigated by using analogs with relative selectivity for each site. Analogs exhibiting preferential binding to site 2 were far less sensitive to insulin antagonism than were analogs binding preferentially at site 1 and less well at site 2. No other property of these analogs, including the rate of hydrolysis by phosphodiesterase, the IC50 for phosphodiesterase, the Ka for protein kinase, or the type I versus type II kinase specificity, could account for the ability of insulin to antagonize glycogenolysis stimulated by these analogs. These data indicate that insulin may act to decrease the affinity of protein kinases for cAMP through a possible regulation of intrachain site 2 binding.

Hormonally induced elevations of α- and β-casein mRNA levels are blocked by cyclic adenine nucleotide and prostaglandins

Normal mammary gland development during pregnancy follows a coordinated program of morphological development (formation of lobuloalveoli) and biochemical differentiation (casein production). In culture, whole mammary glands of Balb/c mice can be similarly induced by application of a mixture of insulin, prolactin, aldosterone and hydrocortisone (IPAH) for 7 days. Our previous reports have shown that lobuloalveolar development, induced by IPAH, is competitively inhibited by the simultaneous presence of dibutyryl cyclic AMP (Bt 2cAMP), prostaglandins (PGs) E 1, E 2, and B 1, and papaverine (pap). However, if this mixture is not added until day 4, lobuloalveolar development is relatively unaffected but casein synthesis is repressed. This report explores the mechanism by which cyclic adenine nucleotides and prostaglandins interfere with the normal developmental pathway. The accumulation of α- and β-casein mRNAs induced by prolactin, hydrocortisone and aldosterone is blocked by the combination of Bt 2cAMP, PGs E 1, E 2, and B 1, and pap added to the medium for the final 3 days (days 4–7). Under these conditions the glands retain their lobuloalveoli, and little squamous metaplasia can be discerned. Furthermore, de novo synthesis of both caseins is selectively inhibited, despite the continued presence of casein mRNAs in the glands and normal protein synthesis. In contrast, the synthesis of keratin is stimulated. Incomplete mixtures of Bt 2cAMP and pap or the combination of PGs E 1, E 2, and B 1, are only partly effective in preventing the accumulation of casein mRNAs. All three mixtures bring about similar effects on both α-and β-casein mRNAs. These findings suggest that the mixture of cyclic adenine nucleotide and prostaglandins may uncouple the normal link between lobuloalveolar development and normal biochemical differentiation in the mammary gland (casein synthesis). Furthermore, inhibition of the synthesis of both caseins in the continued presence of α- and β-casein mRNAs is suggestive that casein production is subject to multilevel regulation in the developing mammary gland. Finally, the parallel inhibition of both α- and β-casein mRNA accumulation further emphasizes the close coordinate regulation of casein mRNAs.

Site of insulin inhibition of cAMP-stimulated glycogenolysis.

Insulin antagonized the glycogenolytic actions of cAMP in isolated hepatocytes even in the presence of the phosphodiesterase inhibitors, 1-methyl-3-isobutylxanthine or d-4-(3-butoxy-4-methoxybenzyl)-2- imidizolidione . Stimulation of glucose release by 8-bromo-cAMP, which is not appreciably hydrolyzed by phosphodiesterase, was also inhibited by insulin in the presence of d-4-(3-butoxy-4-methoxybenzyl)-2- imidizolidione . This demonstrates that insulin can antagonize cAMP stimulation of glycogenolysis independent of possible effects of insulin on adenylate cyclase or phosphodiesterase. Under conditions where changes in cyclic adenine nucleotide concentrations (cAMP and 8-bromo-cAMP) were prevented, insulin depressed the 8-bromo-cAMP-stimulated protein kinase activity ratio. We conclude that phosphodiesterase activation is not required for insulin antagonism of cAMP-stimulated glycogenolysis. The effect of insulin under these conditions can be explained by an action of insulin on cAMP-dependent protein kinase independent of changes in cAMP levels.

Cyclic adenine nucleotide levels in lymphocytes from patients with systemic sclerosis: efficiency of prostaglandin infusions.

Basal intracellular cAMP levels of lymphocytes from patients with systemic sclerosis, all suffering from Raynaud's phenomenon, were significantly lower than those obtained from normal controls. Moreover, the cAMP response to isoprenaline of these patients' lymphocytes was inhibited. Intravenous infusion of prostaglandins (E1 and I2) restored the basal levels and greatly improved the responsiveness of the cells to isoprenaline stimulation.

Enhancement of Squamous Cell Development in Cultured Skin by Cyclic Adenine Nucleotide and Prostaglandins

The present study tests the hypothesis that agents known to elevate the level of intracellular cyclic adenine nucleotide may direct different epithelial cells onto a pathway of epidermoid (squamous) development and differentiation. We report here that the mixture of dibutyryl cyclic AMP (dbcAMP), prostaglandins E1, E2 and B1 (PG E1, E2, B1), and papaverine (pap) enhances the rate of normal squamous cell development in organ-cultured skin of chick embryos. The three components may act synergistically to elevate the level of intracellular cyclic adenine nucleotide. We recently reported that the same group of agents induces abnormal development (squamous metaplasia) and aberrant differentiation (keratin production) in the normally cuboidal epithelium of cultured whole mammary glands of mice [1]. Thus, dbcAMP, PG E1, E2, B1, and pap are effective in enhancing normal squamous cell development and also in inducing squamous metaplasia de novo in the epithelial components of two different organs of embryonic and adult animals of two classes of vertebrates. The combined findings are suggestive that cyclic adenine nucleotide together with the prostaglandins may act generally on diverse types of epithelia to bring about squamous cell development and a differentiation marked by keratin production.

Expression of a Phenotype of Normal Differentiation in Cultured Mammary Glands is Promoted by Epidermal Growth Factor and Blocked by Cyclic Adenine Nucleotide and Prostaglandins

Chemical inducers of development have been found to alter the functional normal differentiation of mouse mammary gland in culture. Normal development, indicated by formation of lobuloalveolar structures, and normal differentiation, evidenced as casein synthesis, were induced in cultured whole mammary glands of virgin Balb/c mice using the hormones insulin, prolactin, aldosterone, and hydrocortisone (IPAH) in serum-free medium. Following removal of all added hormones except insulin, which is needed for general maintenance of mammary epithelial cells, the lobuloalveoli involuted (regressed) as a result of the death of their differentiated cells. A second round of normal development and differentiation was then elicited by the combination of epidermal growth factor (EGF) and IPAH. The cultured glands were found to synthesize seven molecular species of casein in both the first and second normal developmental cycles. Another series of experiments examined the effects of abnormal development and aberrant differentiation on the normal differentiation phenotype. Mammary glands undergoing the first round of normal development and differentiation were simultaneously induced to undergo an abnormal course of development (squamous metaplasia) and differentiation (excessive keratin production) by the additional use of agents known to elevate the level of intracellular cyclic AMP, namely dibutyryl cyclic AMP (dbcAMP), prostaglandins (PG) E1, E2 and B1, and papaverine (pap). The complete mixture of dbcAMP, pap, and the PGs blocked casein synthesis. DbcAMP and pap, in the absence of PG, markedly depressed the production, while the three PGs alone were almost totally inhibitory. Two indications emerge from the present findings: First, EGF appears to play a positive inductive role in the functional normal differentiation of the mammary gland. Second, cyclic adenine nucleotide and specific prostaglandins may mediate the negative de-inductive regulation of that process.

Induction of abnormal development and differentiation in cultured mammary glands by cyclic adenine nucleotide and prostaglandins.

Squamous (epidermoid) metaplasia is a common abnormality of development and differentiation, in which various epithelia are replaced by cells resembling those of the epidermis. The metaplastic cells differentiate into flattened cells that frequently form keratotic flakes and whorls. The cells may regress, remain benignly squamous, or progress to epidermoid carcinoma1. Squamous metaplasias have previously been brought about by mechanical injury2, vitamin A deficiency3,4 and chemical carcinogens5–7. In search of the physiological mediators of abnormal development and differentiation in oncogenesis, we have now discovered that the combination of dibutyryl cyclic AMP, prostaglandins (PGs) E1, E2 and B1, and papaverine (Pap) induces squamous metaplasia with keratin production in the epithelium of cultured mammary glands. The three inducers may act synergistically to elevate the level of intracellular cyclic adenine nucleotide. Removal of these inducers causes the regression of the metaplastic cells. Retinoid does not block the induction of this squamous metaplasia, even though retinoid has previously prevented and reversed the squamous metaplasias caused by vitamin A deficiency or chemical carcinogens in other organs4,8. The findings suggest that cyclic adenine nucleotide and the PGs may mediate the squamous metaplasias present in many abnormal, transformed and malignant tissues, and possibly also the normal development and differentiation of epithelia into keratin-producing, stratified squamous cells, as in skin.

In vitro behavior of human intestinal mucosa. The influence of acetyl choline on ion transport.

The possibility that the autonomic nervous system may influence the function of intestinal mucosa was investigated by assessing the effect of acetyl choline on ion transport in human intestine. Isolated pieces of stripped ileal mucosa were mounted in Perspex flux-chambers and bathed in isotonic glucose Ringer's solution. Acetyl choline caused a rise in mean potential difference (8.8-12.3 mV, P less than 0.002) and short circuit current (287.7-417.2 muA-cm-2, P less than 0.01) (n = 12), observable at a concentration of 0.01 mM and maximal at 0.1 mM. This effect was enhanced by neostigmine and blocked by atropine. Isotopic flux determinations revealed a change from a small mean net Cl absorption (58) to a net Cl secretion (-4.3mueq-cm-2-h-1P less than 0.001) due predominantly to an increase in the serosal to mucosal unidirectional flux of Cl (10.63-14.35 mueq-cm-2-h-1P less than 0.05) and a smaller reduction in the mucosal to serosal flux (11.22 to 10.02 mueq-cm-2-h-1P less than 0.05). Unidirectional and net Na transport was unaffected. A similar electrical and ion transport response was observed in a single study of two pieces of jejunal mucosa. In the absence of glucose net chloride secretion was produced and again an insignificant effect on net sodium transport was noted. Acetyl choline did not provoke a sustained effect on mucosal cyclic adenine nucleotide levels although a short-lived cyclic adenine nucleotide response was seen in some tissues 20-30 s after drug addition. These studies demonstrate that acetyl choline does influence human intestinal ion transport by stimulating chloride secretion and suggest a possible mechanism by which the parasympathetic nervous system could be concerned in the control of ion transport.

Nucleolar changes in liver before the onset of deoxyribonucleic acid replication

A mixture of chemicals was previously devised (3, 3', 5'-triiodo-L-thyronine, amino acids, a butyryl derivative of cyclic adenosine 3':5'-monophosphate, theophylline, and heparin) that induces nuclear DNA replication in the liver of the unoperated rat (Short, J., Tsukada, K., Rudert, W.A. 7 Lieberman, I. (1975) J. Biol. Chem. 250, 3602-3606). The stimulation of DNA synthesis with the complete solution is greater than the sum of the responses to the thyroid hormone alone and to a mixture of the remaining components of the inductive solution alone. The effects of the complete mixture and of parts of it on three parameters involving the hepatocyte nucleolus have now been examined in intact animals. The complete solution increases the level of RNA polymerase I (measured with isolated nuclei), the rate of ribosome synthesis, and the total volume of nucleoli per nucleus. Nucleolar hypertrophy is unique among the three changes in showing a requirement, just as for DNA synthesis, for all or almost all of the components of the complete mixture, including the thyroid hormone, for a maximal effect. Enlargement of nucleoli is detectable as early as 2 hours after the start of treatment with the complete mixture and a large proportion of the total hepatocyte population is involved. Hypertrophy is accompanied by an increase in nucleolar RNA content. N2-Monobutyryl cyclic guanosine 3':5'-monophosphate is not able to substitute for the cyclic adenine nucleotide.

Circadian rhythm of glycogen content in various brain structures of Serranus scriba Cuv.

In different brain structures (telencephalon, tectum opticum, rhombencephalon, cerebellum and medulla oblongata) of the teleost Serranus scriba Cuv., the content of glycogen was observed during a 24-h period. A circadian rhythm of brain glycogen concentration was found. The results are discussed with particular reference to the possible relation between catecholamines, cyclic adenine nucleotide and glycogen metabolism in the brain.