Dibutyryl cAMP Stimulation Research Articles

Prohibitin-1 is an ACTH-Regulated Protein in Human and Mouse Adrenocortical Cells and Plays a Role in Corticosteroid Production

Cell-intrinsic early events involved in different trophic hormone-induced steroidogenesis in their respective steroidogenic cell type are very similar. For example, the activation of the cAMP-PKA signaling pathway in response to trophic hormone stimulation and, subsequently, cholesterol transport to the mitochondria to initiate steroidogenesis is common to them. Recently, we have found that an evolutionarily conserved protein, prohibitin-1 (PHB1), is regulated by Luteinizing Hormone (LH) in murine Leydig cells and plays a role in interconnected cell signaling and mitochondrial steps pertaining to testosterone production. Among the primary steroidogenic tissues, PHB1 expression levels are highest in the adrenal cortex (The Human Protein Atlas); however, its regulation and role in adrenocortical cells are virtually unknown. We investigated the regulation and the role of PHB1 in adrenocortical cells in vitro using human HAC15 and mouse Y-1 cell culture models. It was found that Adrenocorticotrophic Hormone (ACTH) stimulation upregulates PHB1 levels in adrenocortical cells in a time-dependent manner. A similar effect on PHB1 levels was also observed in response to dibutyryl-cAMP stimulation, a cell-permeable analogue of cAMP (the second messenger for ACTH action). Moreover, manipulating PHB1 levels in adrenocortical cells affected mitochondria, lysosomes, and lipid droplet characteristics, modulated phospho-PKA and phospho-ERK1/2 levels, and altered corticosteroid production. This finding suggests that ACTH regulates PHB1 in adrenocortical cells and plays a role in corticosteroid production, which was previously unknown.

Differential regulation of two 3' end variants of P450 aromatase transcripts and of a new truncated aromatase protein in rabbit preovulatory granulosa cells.

In rabbit granulosa cells, two cytochrome P450 aromatase (P450 arom) mRNAs issued from promoter II were described: a full-length and a truncated transcript. Western blot analysis showed two P450 arom proteins with apparent molecular masses of 53 and 46 kDa, which are consistent with the predicted theoretical sizes of proteins encoded by these two transcripts. To examine the involvement of the truncated transcript in the regulation of P450 arom gene expression, the level of each transcript was specifically quantified in cultured granulosa cells by competitive quantitative RT-PCR. FSH induced a dose-dependent increase in both estradiol production and P450 arom mRNAs levels with a much more enhancement in the full-length mRNA. The half-life of the transcripts could not explain this differential regulation. Upon dibutyryl cAMP stimulation, the full-length mRNA was less abundant than the truncated one. In contrast, Western blot analysis revealed a stimulation of the 53-kDa protein content, whereas the 46-kDa protein amount was apparently unaffected. TGF beta in FSH-stimulated conditions decreased both estradiol production and P450 arom transcripts levels. TGF beta did not modify estradiol production and aromatase protein amounts induced by dibutyryl cAMP, whereas the two P450 arom mRNAs levels were increased. In conclusion, we report for the first time that a protein encoded by a truncated P450 arom mRNA could be involved in the regulation of estrogen production. Moreover, we show that the two P450 arom mRNAs are regulated in a differential manner, probably through hormonal control of the alternative splicing.

Effects of troglitazone on intracellular cholesterol distribution and cholesterol-dependent cell functions in MA-10 Leydig tumor cells

Troglitazone treatment of MA-10 Leydig tumor cells resulted in cellular cholesteryl esters decreasing and cell free cholesterol increasing. This was not an effect unique to this chemical entity; rosiglitazone and pioglitazone caused these changes also. The excess free cholesterol was recovered largely in the cholesterol oxidase susceptible, plasma membrane cholesterol pool. This effect of troglitazone probably is not mediated by activation of peroxisome proliferator activated receptors since it immediately reversed with washing and did not occur at all in cells treated with the peroxisome proliferator activated receptor agonist, 15-deoxy Δ 12,14 prostaglandin J-2. Plasma membrane cholesterol esterification was inhibited by troglitazone in a dose-dependent manner. Plasma membrane cholesterol esterification was inhibited half-maximally by 14 μM troglitazone and by more than 90% by 40 μM troglitazone. This effect was not unique for MA-10 cells. Similar results were found using fibroblasts. Troglitazone was not simply inhibiting internalization of plasma membrane cholesterol. Dibutyryl-cAMP stimulation of troglitazone-treated cells resulted in more progesterone synthesis than in stimulated control cells; moreover, radioactive plasma membrane cholesterol was readily converted into progesterone in troglitazone-treated cells. Studies of LDL uptake in troglitazone-treated cells indicated that intracellular membranes were cholesterol replete. Troglitazone inhibited plasma membrane cholesterol esterification with kinetics similar to 58-035, a known inhibitor of the acyl coenzyme A: cholesterol acyltranserase (ACAT) enzyme. It is not likely an ACAT inhibitor since troglitazone did not block incorporation of exogenous free fatty acids into cholesteryl esters. Thus, it appears that troglitazone prevented presentation of free fatty acid to the ACAT enzyme.

An investigation into the effects of stimulators of fluid production on Locusta Malpighian tubule intracellular elemental composition

The intracellular elemental concentrations of Na, K, P, S, Cl and Mg in the type 1 cells of Malpighian tubules of Locusta migratoria L. have been measured using electron probe X-ray microanalysis. The effects of in vitro stimulation with 1 mM cAMP and corpora cardiaca extract (CC-extract) on the elemental concentrations have been quantified. The distribution of elements, particularly Na, K and Cl is not homogeneous in control cells, and concentration gradients exist within the cytoplasm. Dibutyryl-cAMP (DB-cAMP) caused a decrease in [K]i without disrupting the gradient which increased from the basal to the apical surface, the apical [Na]i was increased as was the [Cl]i. In contrast, in vitro application of CC-extract did not cause changes to the intracellular elemental composition as compared with control cells These data are consistent with the interpretation that exogenous cAMP only partially activated the full stimulatory response of Malpighian tubule cells observed with CC-extract. The changes observed in the density and elemental composition of the `dark bodies' in response to DB-cAMP and CC-extract stimulation suggest that these structures have a role in the ionic economy of Malpighian tubule cells.

Dibutyryl Cyclic AMP-Enhanced Germ Tube Formation in Exponentially GrowingCandida albicansCells

Competence in germ tube formation for exponential phase cells of severalCandida albicanswas low (less than 14%) in 0.1Mphosphate buffer (pH 6.8) containing 1 mMproline. Addition of 5 mMdibutyryl cyclic AMP (cAMP), a lipophilic cAMP analog, to the induction medium increased germ tube formation up to 50%. This enhancement occurred only when the cells were grown in medium lacking glucose or fructose as a carbon source.N-Acetylglucosamine-induced germ tube formation was enhanced by dibutyryl cAMP under similar conditions, but the enhancement was only slight. Cyclic GMP (cGMP) or AMP did not enhance germ tube formation, while dibutyryl cGMP, theophylline, and butyric acid were inhibitory. These results show that dibutyryl cAMP stimulation of the germ tube formation depends on the growth phase and the nutritional status of the cell.

<b>STUDIES ON THE PHOTOSENSITIVITY OF TIN-PROTOPORPHYRINTREATED RATS </b>

We have demonstrated previously by cDNA vinalysis that VIP is derived together with PHI-like peptide (PHM) from a common precursor (preproVIP) in a humain neuroblastoma cell line called NB-OK-1. The humain neuroblastosma cell line produces and releases the large variants (8K and 18K dalton) of VIP-like immunoreactivity (IR-VIP) in response to dibutyryl cAMP stimulation. The purpose of the present study is to purify the 8Kd vind 18Kd forms of VIP-relaited peptides from the human neuroblastoma culture media and to characterize their structure. IR-VIP in the culture media was fractionated with 40% to 60% of saturated ammonium sulfate (SAS)

Substituted thieno[3,4-d]imidazoles, a novel group of H +/K +-ATPase inhibitors. Differentiation of their inhibition characteristics from those of omeprazole

The action of the H +/K +-ATPase inhibitors, Hoe 731 and S 4216, both thieno-imidazole derivatives, was compared with that of the benzimidazole derivative, omeprazole. In intact, gastric membrane vesicles under conditions shown to result in acidification of the vesicle interior, Hoe 731 and S 4216 inhibited H +/K +-ATPase activity with an IC 50 value of about 1.0 μM. In the absence of a generated pH gradient the respective IC 50 values were 5.5 and 2.1 μM. In contrast, omeprazole inhibited the enzyme only in the presence of proton accumulation (IC 50: 0.7 μM). The inhibitory action of omeprazole on H +/K +-ATPase-mediated proton transport was prevented by the membrane permeable mercaptane, dithioerythritol, but not by the membrane impermeable, mercaptane glutathione, whereas both mercaptanes were able to prevent the effect of Hoe 731 and S 4216. These results indicate that the thienoimidazoles react with intravesicular (luminal) and extravesicular (cytosolic) SH groups of the H +/K +-ATPase, whereas omeprazole interacts uniquely with luminal SH groups of the enzyme. In isolated parietal cells all drugs caused a concentration-dependent inhibition of HCl production, as measured by [ 14C]aminopyrine uptake, during histamine and dibutyryl-cAMP stimulation. The IC 50 value was 0.1 μM for Hoe 731 and omeprazole and 0.4 μM for S 4216 after 30-min incubation. The inhibitory action of Hoe 731 and S 4216 faded with increasing incubation time, whereas omeprazole caused an unchanged inhibition over the entire 120-min incubation period. We suggest that several factors, e.g. weaker chemical stability of the drugs or perturbation of cellular glutathione levels, may be responsible for the fading inhibitory action of thienoimidazoles in the parietal cell.

Adenosine receptor-dependent modulation of inhibin secretion in cultured immature rat Sertoli cells

Inhibitory (A1) adenosine receptors that attenuate adenylate cyclase activity are present in cultured Sertoli cells. To investigate the possible effect of activating these receptors on the secretion of inhibin by the Sertoli cell, immature rat Sertoli cells were incubated for 24 h with follicle-stimulating hormone (FSH) in the absence or presence of the non-metabolizable, adenosine agonist phenyl-isopropyl-adenosine (PIA), and the accumulation of α-inhibin immunoreactivity was measured in the medium. Although devoid of effects when added alone, PIA inhibited the FSH-dependent secretion of α-inhibin in a concentration-dependent manner (ED n50 = 1–1.5 nM). PIA treatment of the Sertoli cells also rendered the cells less sensitive to FSH in terms of α-inhibin secretion. The concentration-response curve to FSH was shifted to the right when cells were incubated in the presence of 100–1000 nM PIA. In contrast, dibutyryl cAMP stimulation of α-inhibin accumulation was unaffected by treatment with PIA, indicating that the site of PIA action is at the level of cAMP synthesis. These data provide experimental evidence of adenosine modulation of inhibin secretion by the Sertoli cell and suggest that adenosine may act as a local modulator within the pituitary-testicular axis.

Functional and structural differences between cultured outer and inner layer cells of bovine adrenal cortex.

Outer and inner layer cells of bovine adrenal cortex were cultured separately to compare cellular structural characteristics and functional differences. Outer layer cells were polygonal in shape with radially distributed lipid droplets in the cytoplasm, and produced mainly aldosterone and cortisol. The aldosterone production increased upon stimulation with angiotensin II or dibutyryl-cAMP. In contrast, inner layer cells were spindle-shaped and had fine diffused lipid droplets. They produced four times as much cortisol as outer layer cells but no aldosterone. Cortisol production increased with ACTH or dibutyryl-cAMP stimulation. When stimulated by ACTH or by dibutyryl-cAMP, both types of adrenocortical cells showed "cellular retraction" whereby the number of cytoplasmic lipid droplets decreased and microvilli on the cellular surface increased. At the same time, the transverse distribution of actin fibers disappeared and the microtubules changed their distribution pattern from circular to radial. Stimulation by angiotensin II, on the other hand, brought no marked structural changes. These results indicate that, in functional terms, the outer layer cells and the inner layer cells in this culture system reflect zona glomerulosa and zona fasciculata-reticularis, respectively.

Comparative effects of cytochalasin D on the protein discharge induced by α- and β-adrenergic or cholinergic agonists in rat exorbital lacrimal glands

Cytochalasin D altered the kinetics of peroxidase and radiolabeled protein discharge from rat exorbital lacrimal glands in vitro, in response to various secretagogues. The changes were different with each inducer. The discharge due to isoproterenol was immediately inhibited by 95%; the discharge evoked by noradrenaline via α-adrenergic receptors was progressively reduced and was inhibited by 50% after 30 min, whereas that evoked by carbachol was not influenced during the initial discharge period and was diminished by only 30% after 30 min. When calcium was removed from the incubation medium, the secretory responses were lowered and the inhibitory effect of cytochalasin D was still observed. The rate of protein discharge inhibition was related to the dose and was maximal with 2·10 −6 M cytochalasin D when the discharge resulted from cholinergic, α- or β-adrenergic or dibutyryl cAMP stimulation. Cytochalasin D did not impair cellular energetics nor other stimulations induced through muscarinic or adrenergic receptors. Cytochalasin D effects could be related to interaction with actin, leading to the inhibition of the release of proteins into the incubation medium following the activation of the adrenergic system.

Role of protein phosphorylation in regulating rat submandibular mucin secretion.

The possible involvement of protein phosphorylation during beta-adrenergic receptor stimulation in rat submandibular gland was investigated in vitro using a dispersed cell preparation. (-)-Isoproterenol, a beta-adrenergic agonist, or dibutyryl cAMP stimulation was associated with an enhanced phosphorylation of three protein bands having apparent molecular weights of 34,000, 26,000, and 21,000, respectively. (-)-Propranolol, a beta-adrenergic antagonist, inhibited the phosphorylation of the three proteins during beta-adrenergic stimulation but not during dibutyryl cAMP stimulation. The three proteins were not fragments of a higher-molecular-weight protein. Subcellular fractionation using differential centrifugation, fractionation in an aqueous two-phase polymer system, and discontinuous sucrose gradient ultracentrifugation coupled with marker enzyme analysis indicated that all three proteins were enriched in the same subfractions: a heavy plasma membrane fraction and a fraction containing plasma membrane and Golgi membrane material. The extent of protein phosphorylation for all three proteins increased as a function of time and dose after beta-adrenergic stimulation. After 20 min of maximal beta-adrenergic stimulation, the addition of a beta-adrenergic blocker caused a time-dependent decrease in the 32P content of all three proteins. Pure cholinergic or pure alpha-adrenergic receptor stimulation had no effect on the 32P content of the three proteins. These data are consistent with a role for cAMP-mediated protein phosphorylation during mucin secretion from rat submandibular cells.

ACTH stimulation of adrenal steroidogenesis is sensitive to trypsin inhibitors.

Various trypsin inhibitors (lima bean, soybean, and p-tosyl-L-lysine chloromethyl ketone (TLCK)) decrease the sensitivity of rat adrenocortical cells isolated from the inner zones to ACTH-stimulated corticosterone production. Basal steroidogenesis is not affected. The inhibitors produce dose-dependent effects at concentrations greater than 10 μ M. Dibutyryl cAMP stimulation of this system also is antagonized by TLCK, though not by lima bean trypsin inhibitor. When various steroid intermediates are added to the incubations as sources of substrate, the inhibitory effect of TLCK on corticosterone production decreases in parallel with the number of hydroxylation reactions required to generate corticosterone. These results suggest that trypsin inhibitors may block specific ACTH-linked proteolytic events in the adrenal cortex. Alternatively, they may interfere non-specifically with ACTH binding and/or adrenocortical metabolism. In either case, the common practice of including a trypsin inhibitor in adrenal cell...

<b>Dibutyryl cAMP stimulation of production and release of VIP-like immunoreactivity in a human neuroblastoma cell </b><b>line </b>

<b>Dibutyryl cAMP stimulation of production and release of VIP-like immunoreactivity in a human neuroblastoma cell </b><b>line </b>