Gene silencing of human RAMP2 mediated by short-interfering RNA

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Abstract.Previous studies have shown that proteasome inhibitors promote the accumulation of steroidogenic acute regulatory protein (StAR) in cultured rat adrenocortical cells. Unexpectedly, this response was associated with a moderate lowering in the corticosterone secretion and proliferation rate of cultured cells. Hence, we studied the effects of proteasome inhibitors MG115 and MG132 on the secretion and proliferative activity of the regenerating adrenal cortex in rats 5 days after surgery. Animals were given two subcutaneous injections of 0.15 or 1.5 nmol/100 g of inhibitors 24 and 12 h before decapitation. Real-time PCR and Western blotting showed that StAR expression, both mRNA and protein, was markedly lower in regenerating adrenals than in the intact gland of sham-operated rats. Neither MG115 nor MG132 affected StAR expression in regenerating gland. Inhibitors induced a slight decrease in the plasma concentrations of aldosterone and corticosterone, but did not significantly alter metaphase index of the regenerating adrenal cortex. Our findings provide the first evidence that down-regulation of StAR occurs during the early stages of adrenal regeneration. Moreover, this suggests that the steroidogenic pathway is more sensitive to proteasome inhibitors than that regulating proliferative activity of regenerating adrenal cortex in the rat.

Section: Reverse Transcription (Rt)-polymerase Chain Reaction (Pcr)mentioning

confidence: 99%

Section: Reverse Transcription (Rt)-polymerase Chain Reaction (Pcr)mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Steroidogenic acute regulatory protein gene expression, steroid-hormone secretion and proliferative activity of adrenocortical cells in the presence of proteasome inhibitors: In vivo studies on the regenerating rat adrenal cortex

Ruciński¹,

Tortorella²,

Ziółkowska³

et al. 2008

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“…Total RNA extraction from frozen dispersed cells and stomach fragments, its RT to cDNA (13,14), and the amplification of the resulting cDNA (thermal cycler 489 DNA TC; Perkin-Elmer Life Sciences, Milan, Italy) were performed as previously described (15,16). To rule out the possibility of amplifying genomic DNA, in certain experiments, PCR was carried out without prior RT of the RNA.…”

Section: Rt-pcrmentioning

confidence: 99%

Galanin stimulates cortisol secretion from human adrenocortical cells through the activation of galanin receptor subtype 1 coupled to the adenylate cyclase-dependent signaling cascade

Belloni

Malendowicz²,

Ruciński³

et al. 2007

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Abstract. Previous studies showed that galanin receptors are expressed in the rat adrenal, and galanin modulates glucocorticoid secretion in this species. Hence, we investigated the expression of the various galanin receptor subtypes (GAL-R1, GAL-R2 and GAL-R3) in the human adrenocortical cells, and the possible involvement of galanin in the control of cortisol secretion. Reverse transcription-polymerase chain reaction detected the expression of GAL-R1 (but not GAL-R2 and GAL-R3) in the inner zones of the human adrenal cortex. The galanin concentration dependently enhanced basal, but not ACTH-stimulated secretion of cortisol from dispersed inner adrenocortical cells (maximal effective concentration, 10 -8 M). The cortisol response to 10 -8 M galanin was abrogated by GAL-R1 immunoneutralization, and unaffected by GAL-R2 or GAL-R3 immunoneutralization. Galanin (10 -8 M) and ACTH (10 -9 M) enhanced cyclic-AMP production from dispersed cells, and the response was suppressed by the adenylate cyclase inhibitor SQ-22536 (10 -4 M). Galanin did not affect inositol triphosphate release, which, in contrast, was raised by angiotensin-II (10 -8 M). SQ-22536 and the protein kinase (PK)A inhibitor H-89 (10 -5 M) abolished the cortisol response to 10 -8 M galanin, while the phospholipase C inhibitor U-73122 and the PKC inhibitor calphostin-C were ineffective. Preincubation with pertussis toxin (Ptx) (0.5 μg/ml) partially inhibited the cortisol response to galanin. We conclude that galanin stimulates cortisol secretion from human inner adrenocortical cells, acting through GAL-R1 coupled to the adenylate cyclase/PKA-dependent signaling cascade via a Ptx-sensitive Gα protein.

“…Total RNA was extracted from the frozen specimens, and reverse transcribed to cDNA (41)(42)(43)(44). PCR was carried out, as previously detailed (45)(46)(47), in a Roche LightCycler 2.0, using the following primers: NMUR1 sense (481-500), 5'-GCC-ATC-TGG-GTC-TTC-GCT-AT-3' and antisense (797-816) 5'-CAC-CTG-TCT-GCG-TTC-CCT-AT-3' (336 bp; accession number, AF-242873).…”

Section: Rt-pcrmentioning

confidence: 99%

Effects of neuromedin-U on immature rat adrenocortical cells: In vitro and in vivo studies

Ziółkowska¹,

Macchi²,

Trejter³

et al. 2008

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Abstract. Neuromedin U (NMU) is a brain-gut peptide, that in the peripheral organs and tissues acts via a G protein-coupled receptor, called NMUR1. Reverse transcription-polymerase chain reaction showed the expression of NMUR1 mRNA in either cortex and medulla or dispersed zona glomerulosa and zona fasciculata-reticularis cells of the immature rat adrenals. Accordingly, immunocytochemistry demonstrated the presence of NMUR1-like immunoreactivity in the cortex and medulla of immature adrenals. NMU8 administration to immature rats was found to raise aldosterone, but not corticosterone, plasma concentration, without altering adrenal growth. Conversely, the exposure to NMU8 markedly enhanced the proliferative activity of immature rat inner adrenocortical cells in primary in vitro culture, without significantly affecting their corticosterone secretion. Collectively, our findings suggest that adrenals of immature rats may be a target for circulating NMU. However, the physiological significance and relevance of the adrenal effects of NMU remain to be ascertained.