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Chronic opioids regulate KATP channel subunit Kir6.2 and carbonic anhydrase I and II expression in rat adrenal chromaffin cells via HIF-2? and protein kinase A.

ABSTRACT: At birth, asphyxial stressors such as hypoxia and hypercapnia are important physiological stimuli for adrenal catecholamine release that is critical for the proper transition to extrauterine life. We recently showed that chronic opioids blunt chemosensitivity of neonatal rat adrenomedullary chromaffin cells (AMCs) to hypoxia and hypercapnia. This blunting was attributable to increased ATP-sensitive K(+) (KATP) channel and decreased carbonic anhydrase (CA) I and II expression, respectively, and involved ?- and ?-opioid receptor signaling pathways. To address underlying molecular mechanisms, we first exposed an O2- and CO2-sensitive, immortalized rat chromaffin cell line (MAH cells) to combined ? {[d-Arg(2),Ly(4)]dermorphin-(1-4)-amide}- and ? ([d-Pen(2),5,P-Cl-Phe(4)]enkephalin)-opioid agonists (2 ?M) for ?7 days. Western blot and quantitative real-time PCR analysis revealed that chronic opioids increased KATP channel subunit Kir6.2 and decreased CAII expression; both effects were blocked by naloxone and were absent in hypoxia-inducible factor (HIF)-2?-deficient MAH cells. Chronic opioids also stimulated HIF-2? accumulation along a time course similar to Kir6.2. Chromatin immunoprecipitation assays on opioid-treated cells revealed the binding of HIF-2? to a hypoxia response element in the promoter region of the Kir6.2 gene. The opioid-induced regulation of Kir6.2 and CAII was dependent on protein kinase A, but not protein kinase C or calmodulin kinase, activity. Interestingly, a similar pattern of HIF-2?, Kir6.2, and CAII regulation (including downregulation of CAI) was replicated in chromaffin tissue obtained from rat pups born to dams exposed to morphine throughout gestation. Collectively, these data reveal novel mechanisms by which chronic opioids blunt asphyxial chemosensitivity in AMCs, thereby contributing to abnormal arousal responses in the offspring of opiate-addicted mothers.

SUBMITTER: Salman S

PROVIDER: S-EPMC4121586 | biostudies-literature | 2014 Aug

REPOSITORIES: biostudies-literature

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Chronic opioids regulate KATP channel subunit Kir6.2 and carbonic anhydrase I and II expression in rat adrenal chromaffin cells via HIF-2α and protein kinase A.

Salman Shaima S Holloway Alison C AC Nurse Colin A CA

American journal of physiology. Cell physiology 20140604 3

At birth, asphyxial stressors such as hypoxia and hypercapnia are important physiological stimuli for adrenal catecholamine release that is critical for the proper transition to extrauterine life. We recently showed that chronic opioids blunt chemosensitivity of neonatal rat adrenomedullary chromaffin cells (AMCs) to hypoxia and hypercapnia. This blunting was attributable to increased ATP-sensitive K(+) (KATP) channel and decreased carbonic anhydrase (CA) I and II expression, respectively, and i ...[more]

PMID: 24898587

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Project description:Overexpression of carbonic anhydrase (CA IX) is associated with poor survival in several adult-type cancers but its expression is undocumented in Wilms tumour (WT), the most common tumour of the paediatric kidney.CA9 expression was measured using polymerase chain reaction (PCR) in 13 WTs and matched-paired non-neoplastic kidneys (NKs). CA IX and hypoxia-inducible factor-1 ?-subunit (HIF-1?) protein were quantified in 15 matched-paired WTs and NKs using enzyme-linked immunosorbent assays. CA IX and HIF-1? were localised by immunostaining tissue sections of 70 WTs (untreated WTs, n = 22; chemotherapy-treated WTs, n = 40; relapsed/metastatic WTs, n = 8). CA IX-positive untreated WTs (n = 14) were immunostained for vascular endothelial growth factor (VEGF), glucose transporter-1 (GLUT1) and CD31. Double staining for CA IX and CD31 was performed in WTs (n = 14).CA9 full length (FL) was significantly up-regulated in WTs compared to NKs (p = 0.009) by real-time PCR. Conventional PCR showed expression of alternative splice variant in all NKs and WTs but FL in WTs only. WTs showed a 2-fold increase in CA IX protein over NKs (p = 0.01). HIF-1? levels were up-regulated in WTs compared to NKs, although the difference was not statistically significant (p = 0.09). CA IX and HIF-1? immunolocalisation were observed in 63% and 93% of WTs, respectively. The median fraction of cells staining positively for CA IX and HIF-1? was 5% and 22%, respectively. There was no significant association between the expression of either CA IX or HIF-1? and clinicopathological variables in WTs resected following chemotherapy. VEGF and GLUT1 immunoreactivity was seen in 94% and 100% with the median fraction of 10% and 60% respectively. Co-expression and co-localisation of all four hypoxia markers was seen in 7/14 and 6/14 cases respectively. CA IX was seen in well vascularised areas as well as in the peri-necrotic areas.Carbonic anhydrase 9 (mRNA and protein), and HIF-1? protein are overexpressed in a significant portion of WTs. No significant association was detected between the expression of either CA IX or HIF-1? and clinicopathological variables in WTs resected following chemotherapy. Cellular localisation studies in untreated WTs suggest that CA IX and HIF-1? are regulated by hypoxia and non-hypoxia mechanisms.

Dataset Information

Chronic opioids regulate KATP channel subunit Kir6.2 and carbonic anhydrase I and II expression in rat adrenal chromaffin cells via HIF-2? and protein kinase A.

Publications

Chronic opioids regulate KATP channel subunit Kir6.2 and carbonic anhydrase I and II expression in rat adrenal chromaffin cells via HIF-2α and protein kinase A.

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