scholarly journals Role of actin in the responses of adrenal cells to ACTH and cyclic AMP: inhibition by DNase I.

1984 ◽  
Vol 99 (4) ◽  
pp. 1335-1342 ◽  
Author(s):  
S Osawa ◽  
G Betz ◽  
P F Hall
Keyword(s):  
Cyclic Amp ◽  
Adrenal Tumor ◽  
Dnase I ◽  
Erythrocyte Ghosts ◽  
Dibutyryl Cyclic Amp ◽  
Inner Mitochondrial Membrane ◽  
Steroid Synthesis ◽  
Adrenal Cells ◽  
Amp Inhibition

Erythrocyte ghosts were loaded with pancreatic DNase I and fused with Y-1 adrenal tumor cells to test the possibility that this enzyme might inhibit the steroidogenic responses of the cells to ACTH and cyclic AMP. Fusion of erythrocyte ghosts loaded with DNase I, but not those containing albumin, ovalbumin, boiled DNase I, or DNase I with excess G-actin, inhibited the increase in production of 20 alpha-dihydroprogesterone produced by ACTH and dibutyryl cyclic AMP; inhibition was concentration-dependent with 50% inhibition by 3 X 10(7) molecules of DNase I per cell. It was found that inhibition by DNase I was exerted at the step in the steroidogenic pathway at which cholesterol is transported to mitochondria where steroidogenesis begins. This was shown by measuring transport of cholesterol into the inner mitochondrial membrane, by measuring the production of pregnenolone by isolated mitochondria and by demonstrating that DNase I was without effect on the conversion of pregnenolone to 20 alpha-dihydroprogesterone (an end-product of steroid synthesis). The actin content of Y-1 cells was measured by two methods based upon inhibition of DNase I and by SDS gels following centrifugation. The cells were found to contain 2-3 X 10(7) molecules of actin per cell of which two-thirds is present as G-actin. Since DNase I is known to bind to G-actin to give a one to one complex, these and other findings suggest that at least some of the G-actin in the cells may be necessary for the steroidogenic responses to ACTH and cyclic AMP.

scholarly journals A role for calmodulin in the regulation of steroidogenesis

1981 ◽  
Vol 90 (2) ◽  
pp. 402-407 ◽  
Author(s):  
PF Hall ◽  
S Osawa ◽  
CL Thomasson
Keyword(s):  
Cyclic Amp ◽  
Adrenal Tumor ◽  
Side Chain ◽  
Dibutyryl Cyclic Amp ◽  
Steroid Production ◽  
Steroid Synthesis ◽  
Side Chain Cleavage ◽  
Cleavage Activity ◽  
Chain Cleavage

Two approaches were used to study the possible role of calmodulin in the regulation of steroid synthesis by mouse adrenal tumor cells: trifluoperazine was used as an inhibitor of calmodulin and liposomes were used to deliver calmodulin into the cells. Trifluoperazine inhibits three steroidogenic responses to both ACTH and dibutyryl cyclic AMP: (a) increase in steroid production, (b) increased transport of cholesterol to mitochondria, and (c) increased side-chain cleavage by mitochondria isolated from cells incubated with ACTH or dibutyryl cyclic AMP. When calmodulin is introduced into the cells via liposomes, steroid synthesis is slightly stimulated. When calmodulin extensively dialyzed against EGTA, this stimulation is abolished. Ca(2+) introduced via liposomes was also without effect. However, when both calmodulin and Ca(2+) are introduced via liposomes (either in separate liposomes or in the same liposomes), steroid synthesis is stimulated. This stimulation does not occur when either anticalmodulin antibodies or EGTA is also present in the liposomes or when trifluoperazine is present in the incubation medium. Calmodulin and Ca(2+) presented together in liposomes to the cells stimulate transport of cholesterol to mitochondria, and side-chain cleavage activity is greater in mitochondria isolated from cells previously fused with liposomes containing calmodulin and Ca(2+) than in mitochondria from cells fused with liposomes containing buffer only. These observations suggest that calmodulin may be involved in regulating the transport of cholesterol to mitochondria, a process which is stimulated by ACTH and dibutyryl cyclic AMP and which may account, at least in part, for the increase in steroid synthesis produced by these agents.

Roles of Cyclic Nucleotides in the Inhibition of Platelet Function by Physiolocic and Pharmacological Agents

1979 ◽  
Author(s):  
R.J. Haslam
Keyword(s):  
Platelet Aggregation ◽  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Platelet Function ◽  
Inhibitory Effects ◽  
Pharmacological Agents ◽  
Arterial Blood ◽  
Cyclic Cmp ◽  
Amp Inhibition

Cyclic AMP mediates the inhibitions of platelet aggregation caused by PCI2, PGE1 and PGD2. Thus, these compounds activate platelet adenylate cyclase and Increase platelet cyclic AMP; their inhibitory effects are blockod by inhibitor? of adenylate cyclase, are potentiated by inhibitors of cyclic AKP phosphodiesterase and are mimicked hy N6 ,2'-0-dibutyryl cyclic AMP. Inhibition of adenylate cyclase does not potentiate platelet aggregation in the absence of inhibitory prostaglandins, indicating that platelet cyclic AMP is too low to affect aggregation under these conditions. To determine whether platelets in the circulation are exposed to agents that increase platelet cyclic AMP, washed rabbi platelets labelled with [3H] adenine were incubated with rabbit arterial blood under various conditions; any increases in cyclic [3H]AMP were measured. These experiments showed that freshly taken rabbit arterial blood does not normally contain any factors that can increase platelet cyclic AMP sufficiently to affect platelet function; specifically, circulating PGI2 was less than 0.1 pmol/ml of blood. It follows that increases in cyclic AMP in circulating rabbit platelets must occur only locally or under special conditions. The role of the moderate increases in platelet cyclic CMP caused by aggregating agents remains uncertain, but the inhibition of aggregation by compounds such as sodium nitroprusside that increase cyclic CMP up to 100-fold suggests that cyclic CMP may, like cyclic AMP, be an inhibitory mediator.

scholarly journals Ultrastructural changes induced by ACTH in normal adrenocortical cells in culture.

1977 ◽  
Vol 72 (3) ◽  
pp. 757-763 ◽  
Author(s):  
A T Suyama ◽  
J A Long ◽  
J Ramachandran
Keyword(s):  
Cyclic Amp ◽  
Calcium Ions ◽  
Monolayer Culture ◽  
Ultrastructural Changes ◽  
Dibutyryl Cyclic Amp ◽  
Adrenocortical Cells ◽  
Adult Rats ◽  
Adrenal Cells ◽  
Camp Synthesis ◽  
High Concentrations

The effects of ACTH, its o-nitrophenyl sulfenyl derivative (NPS-ACTH) and dibutyryl cyclic AMP (dbc AMP) on the ultrastructural morphology of adrenocortical cells of adult rats in monolayer culture have been investigated. NPS-ACTH, which has previously been shown to stimulate steroidogenesis but not cAMP synthesis in adrenal cells, induced the same characteristic transformation of mitochondrial architecture as produced by ACTH or high concentrations of dbcAMP. All three agents caused the disappearance of electron-opaque granules present in the mitochondria of unstimulated cells. It was found that these granules could be extracted with EGTA (ethylene glycol-bis(beta-aminoethyl ether) N,N,N',N'-tetraacetate). These results are discussed in the light of the known importance of calcium ions in the actions of ACTH.

scholarly journals Receptor-mediated gonadotropin action in the ovary. Regulatory role of cyclic nucleotide phosphodiesterase(s) in intracellular adenosine 3′:5′-cyclic monophosphate turnover and gonadotropin-stimulated progesterone production by rat ovarian cells

1979 ◽  
Vol 180 (1) ◽  
pp. 201-211 ◽  
Author(s):  
Salman Azhar ◽  
K. M. Jairam Menon
Keyword(s):  
Cyclic Amp ◽  
Kinase Activity ◽  
Phosphodiesterase Inhibitors ◽  
Protein Kinase Activity ◽  
Dibutyryl Cyclic Amp ◽  
Steroid Production ◽  
Progesterone Production ◽  
Ovarian Cells ◽  
Cyclic Amp Accumulation

The regulatory role of cyclic nucleotide phosphodiesterase(s) and cyclic AMP metabolism in relation to progesterone production by gonadotropins has been studied in isolated rat ovarian cells. Low concentrations of choriogonadotropin (0.4–5ng/ml) increased steroid production without any detectable increase in cyclic AMP, when experiments were carried out in the absence of phosphodiesterase inhibitors. The concentration of choriogonadotropin (10ng/ml) that stimulated progesterone synthesis maximally resulted in a minimal increase in cyclic AMP accumulation and choriogonadotropin binding. Choriogonadotropin at a concentration of 10ng/ml and higher, however, significantly stimulated protein kinase activity and reached a maximum between 250 and 1000ng of hormone/ml. Higher concentrations (50–2500ng/ml) of choriogonadotropin caused an increase in endogenous cyclic AMP, and this increase preceded the increase in steroid synthesis. Analysis of dose–response relationships of gonadotropin-stimulated cyclic AMP accumulation, progesterone production and protein kinase activity revealed a correlation between these responses over a wide concentration range when experiments were performed in the presence of 3-isobutyl-1-methylxanthine. The phosphodiesterase inhibitors papaverine, theophylline and 3-isobutyl-1-methylxanthine each stimulated steroid production in a dose-dependent manner. Incubation of ovarian cells with dibutyryl cyclic AMP or 8-bromo cyclic AMP mimicked the steroidogenic action of gonadotropins and this effect was dependent on both incubation time and nucleotide concentration. Maximum stimulation was obtained with 2mm-dibutyryl cyclic AMP and 8-bromo cyclic AMP, and this increase was close to that produced by a maximally stimulating dose of choriogonadotropin. Other 8-substituted derivatives such as 8-hydroxy cyclic AMP and 8-isopropylthio cyclic AMP, which were less susceptible to phosphodiesterase action, also effectively stimulated steroidogenesis. The uptake and metabolism of cyclic [3H]AMP in ovarian cells was also studied in relation to steroidogenesis. When ovarian cells were incubated for 2h in the presence of increasing concentrations of cyclic [3H]AMP, the radioactivity associated with the cells increased almost linearly up to 250μm-cyclic [3H]AMP concentration in the incubation medium. The 3H label in the cellular extract was recovered mainly in the forms ATP, ADP, AMP, adenosine and inosine, with cyclic AMP accounting for less than 1% of the total tissue radioactivity. Incubation of cyclic AMP in vitro with ovarian cells resulted in a rapid breakdown of the nucleotide in the medium. The degradation products in the medium have been identified as AMP, adenosine and inosine. The rapid degradation of cyclic AMP by phosphodiesterase(s) makes it difficult to correlate changes in cyclic AMP concentrations with steroidogenesis. These observations thus provide an explanation for the previously observed lack of cyclic AMP accumulation under conditions in which low doses of choriogonadotropin stimulated steroidogenesis without any detectable changes in cyclic AMP accumulation.

CYCLIC AMP AND THE DIFFERENTIATION OF ADRENAL CORTICAL CELLS GROWN IN TISSUE CULTURE

1972 ◽  
Vol 55 (2) ◽  
pp. 405-413 ◽  
Author(s):  
A. JOSEPHINE MILNER
Keyword(s):  
Cyclic Amp ◽  
Smooth Endoplasmic Reticulum ◽  
Synthetic Activity ◽  
Cell Type ◽  
Cortical Cells ◽  
Steroid Synthesis ◽  
Adrenal Cells ◽  
Adrenal Cortical Cells ◽  
Primary Tissue ◽  
Fibroblastic Cells

SUMMARY Primary tissue cultures of adrenal cells were prepared from foetal rat adrenals. The effect of cyclic AMP (75 μmol/l) on the morphology and steroid synthetic activity of the cortical cells was examined in order to determine whether cyclic AMP mimics the effects induced by adrenocorticotrophin (ACTH); namely transformation to the differentiated cell type and increase in steroid synthetic activity. Cyclic AMP was found to induce some, but not all, of the changes normally induced by ACTH. In particular, the mitochondria in the cortical cells developed vesicular cristae and there was a proliferation of the smooth endoplasmic reticulum. These two organelles are the principle sites of enzymes involved in steroid synthesis and the ultrastructural transformation was accompanied by an increase in the steroidogenic activity of the cells. However, clear differences in the ultrastructure of the cyclic AMP- and ACTH-treated cells were noted. The effects of cyclic AMP on the ultrastructure of cortical and fibroblastic cells present in the adrenal cultures were found to be dependent upon the cell type.

Effects of Dibutyryl Cyclic AMP and Papaverine on Intrahepatocytic Bile Acid Transport Role of Vesicle Transport

1993 ◽  
Vol 28 (9) ◽  
pp. 833-838 ◽  
Author(s):  
M. Hoshino ◽  
T. Ohiwa ◽  
T. Hayakawa ◽  
Y. Kamiya ◽  
A. Tanaka ◽  
...  
Keyword(s):  
Bile Acid ◽  
Cyclic Amp ◽  
Vesicle Transport ◽  
Acid Transport ◽  
Dibutyryl Cyclic Amp ◽  
Bile Acid Transport

Regulation of Pancreatic Acinar Function: Effects of Cyclic AMP, Dibutyryl Cyclic AMP, and Theophylline in Vitro

1974 ◽  
Vol 52 (4) ◽  
pp. 780-785 ◽  
Author(s):  
T. H. Brian Haig
Keyword(s):  
Protein Synthesis ◽  
Cyclic Amp ◽  
Digestive Enzymes ◽  
Amylase Secretion ◽  
Dibutyryl Cyclic Amp ◽  
Rat Pancreas ◽  
Rule Out

The role of cyclic AMP in the regulation of pancreatic acinar function has been assessed by measuring the effects of exogenous cyclic AMP, dibutyryl cyclic AMP, and theophylline on protein synthesis and amylase secretion. The rate at which slices of rat pancreas incorporated leucine into protein did not change as a consequence of treatment with either cyclic AMP or dibutyryl cyclic AMP, nor did the slices alter their rate of amylase secretion. Moreover, theophylline did not enhance the ability of submaximal doses of pancreozymin to stimulate amylase secretion or to suppress protein synthesis. These results fail to demonstrate that cyclic AMP regulates either the synthesis or secretion of pancreatic digestive enzymes but they do not rule out the possibility.

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