Daesety Vishnuvardhan

Selective roles for the PC2 processing enzyme in the regulation of peptide neurotransmitter levels in brain and peripheral neuroendocrine tissues of PC2 deficient mice.

The prohormone convertase 2 (PC2) is hypothesized to convert multiple pro-neuropeptides into active peptides that function as neurotransmitters. To examine the in vivo role of PC2 in neuropeptide production, the tissue contents of six different neuropeptides in brain and peripheral nervous tissues were examined in PC2 deficient mice. Specific neuropeptide radioimmunoassays and RP-HPLC (reverse-phase HPLC) provided evaluation of processed, active neuropeptides in brain and neuroendocrine tissues of PC2 deficient mice. Results demonstrated three features with regard to the selective roles of PC2 in determining the production of NPY, somatostatin-28, enkephalin, VIP, galanin, and CRF in neuroendocrine tissues. Firstly, PC2 deficient mice showed changes in several neuropeptides, but not all neuropeptides examined. The absence of active PC2 resulted in altered cellular levels of NPY, somatostatin-28, and (Met)enkephalin; few changes in VIP or galanin occurred in the tissues examined. CRF content was not altered in brains of PC2 deficient mice. Secondly, comparison of a single neuropeptide among different tissues of PC2 deficient mice demonstrated tissue-selective roles for PC2 in production of the neuropeptide. For example, NPY levels were decreased in ileum of PC2 deficient mice, but NPY content was not altered in hypothalamus that is abundant in NPY. In addition, (Met)enkephalin levels in hypothalamus and cortex were decreased in PC2 deficient mice, but no changes were observed in adrenal or intestine. Thirdly, a single tissue region often showed selective alterations among different neuropeptides. For example, the neuropeptide-rich hypothalamus region showed decreased (Met)enkephalin in PC2 deficient mice, but NPY, VIP, galanin, and CRF were not altered. These results demonstrate the selective role of PC2 in neuropeptide production that provides active peptide neurotransmitter or hormones for biological functions in brain and neuroendocrine systems.

Obliteration of α‐melanocyte‐stimulating hormone derived from POMC in pituitary and brains of PC2‐deficient mice

Alpha‐melanocyte‐stimulating hormone (α‐MSH) is a neuropeptide expressed in pituitary and brain that is known to regulate energy balance, appetite control, and neuroimmune functions. The biosynthesis of α‐MSH requires proteolytic processing of the proopiomelanocortin (POMC) precursor. Therefore, this study investigated the in vivo role of the prohormone convertase 2 (PC2) processing enzyme for production of α‐MSH in PC2‐deficient mice. Specific detection of α‐MSH utilized radioimmunoassay (RIA) that does not crossreact with the POMC precursor, and which does not crossreact with other adrenocorticotropin hormone (ACTH) and β‐endorphin peptide products derived from POMC. α‐MSH in PC2‐deficient mice was essentially obliterated in pituitary, hypothalamus, cortex, and other brain regions (collectively), compared to wild‐type controls. These results demonstrate the critical requirement of PC2 for the production of α‐MSH. The absence of α‐MSH was accompanied by accumulation of ACTH, ACTH‐containing imtermediates, and POMC precursor. ACTH was increased in pituitary and hypothalamus of PC2‐deficient mice, evaluated by RIA and reversed‐phase high pressure liquid chromatography (RP‐HPLC). Accumulation of ACTH demonstrates its role as a PC2 substrate that can be converted for α‐MSH production. Further analyses of POMC‐derived intermediates in pituitary, conducted by denaturing western blot conditions, showed accumulation of ACTH‐containing intermediates in pituitaries of PC2‐deficient mice, which implicate participation of such intermediates as PC2 substrates. Moreover, accumulation of POMC was observed in PC2‐deficient mice by western blots with anti‐ACTH and anti‐β‐endorphin. In addition, increased β‐endorphin1−31 was observed in pituitary and hypothalamus of PC2‐deficient mice, suggesting β‐endorphin1−31 as a substrate for PC2 in these tissues. Overall, these studies demonstrated that the PC2 processing enzyme is critical for the in vivo production of α‐MSH in pituitary and brain.

Effect of bupropion on CYP2B6 and CYP3A4 catalytic activity, immunoreactive protein and mRNA levels in primary human hepatocytes: comparison with rifampicin

Animals treated with multiple doses of bupropion have had increased bupropion clearance or increased liver weight, suggesting induction of drug‐metabolizing activity. The possibility of cytochrome p450 (CYP) induction by bupropion (10 μM) was evaluated in‐vitro by comparing catalytic activity, immunoreactive protein and CYP mRNA levels from human hepatocytes in primary culture versus cells treated with vehicle (0.5% methanol) and with rifampicin (rifampin) as a positive control. mRNA levels were analysed using a branched DNA luminescent assay. CYP2B6 activity, protein and mRNA levels were increased by 2.5, 1.5 and 2.1 fold, respectively, by 20 μM rifampicin. However, 10 μM bupropion minimally altered CYP2B6 (1.4, 1.1, 0.8 fold). Although CYP3A4 activity, protein, and mRNA levels were increased by 4.0, 2.3, and 14.0 fold, respectively, by 20 μM rifampicin, 10 μM bupropion minimally altered CYP3A4 (1.4, 1.0, 0.8 fold). Rifampicin (20 μM) increased CYP2E1 protein by 2.1 fold, while 10 μM bupropion minimally altered CYP2E1 protein (1.2 fold). Overall, results of this study suggest that multiple doses of bupropion are not likely to induce CYP2B6, 3A4 or 2E1 in‐vivo in man.

Neuronal cell lines expressing PC5, but not PC1 or PC2, process Pro-CCK into glycine-extended CCK 12 and 22.

Endocrine tumor cells in culture and in vitro cleavage assays have shown that PC1 and PC2 are capable of processing pro-CCK into smaller, intermediate and final, bioactive forms. Similar studies have shown that PC5 has the ability to process a number of propeptides. Here, we use GT1-7 (mouse hypothalamic) and SK-N-MC and SK-N-SH (human neuroblastoma) tumor cell lines to study the ability of PC5 to process pro-CCK. RT-PCR and Western blot analysis showed that the cells express PC5 mRNA and protein, but not PC1 or PC2. They were engineered to stably overexpress CCK and cell media was analyzed for pro-CCK expression and cleavage of the prohormone. Radioimmunoassays showed that pro-CCK was expressed, but no amidated CCK was detected. Lack of production of amidated CCK may be due to the lack of the appropriate carboxypeptidase and amidating enzymes. Production of glycine-extended CCK processing products was evaluated by treatment of media with carboxypeptidase B followed by analysis with a CCK Gly RIA. Glycine-extended forms of the peptide were found in the media. The predominant forms co-eluted with CCK 12 Gly and CCK 22 Gly on gel filtration chromatography. The results demonstrate that these cell lines which express PC5 and not PC1 or PC2 have the ability to process pro-CCK into intermediate, glycine-extended forms more closely resembling pro-CCK products in intestine than in brain.

Cholecystokinin Levels in Prohormone Convertase 2 Knock-out Mouse Brain Regions Reveal a Complex Phenotype of Region-specific Alterations *

Prohormone convertase 2 is widely co-localized with cholecystokinin in rodent brain. To examine its role in cholecystokinin processing, cholecystokinin levels were measured in dissected brain regions from prohormone convertase 2 knock-out mice. Cholecystokinin levels were lower in hippocampus, septum, thalamus, mesencephalon, and pons in knock-out mice than wild-type mice. In cerebral cortex, cortex-related structures and olfactory bulb, cholecystokinin levels were higher than wild type. Female mice were more affected by the loss of prohormone convertase 2 than male mice. The decrease in cholecystokinin levels in these brain regions shows that prohormone convertase 2 is important for cholecystokinin processing. Quantitative polymerase chain reaction measurements were performed to examine the relationship between peptide levels and cholecystokinin and enzyme expression. They revealed that cholecystokinin and prohormone convertase 1 mRNA levels in cerebral cortex and olfactory bulb were actually lower in knock-out than wild type, whereas their expression in other brain regions of knock-out mouse brain was the same as wild type. Female mice frequently had higher expression of cholecystokinin and prohormone convertase 1, 2, and 5 mRNA than male mice. The loss of prohormone convertase 2 alters CCK processing in specific brain regions. This loss also appears to trigger compensatory mechanisms in cerebral cortex and olfactory bulb that produce elevated levels of cholecystokinin but do not involve increased expression of cholecystokinin, prohormone convertase 1 or 5 mRNA.

Lovastatin is a potent inhibitor of cholecystokinin secretion in endocrine tumor cells in culture

Lovastatin prevents isoprene synthesis thereby affecting the structural organization of proteins involved in protein transport and secretion. Lovastatin at 1 microM decreases CCK 8 secretion by over 50% in WE cells and in CCK 8 expressing AtT20 cells. At 10 microM CCK 8 secretion was inhibited by two thirds and at 100 microM, cytotoxic effects were observed in both cell types. Addition of mevalonate does not restore CCK secretion and stimulation of secretion by forskolin is also partially inhibited. Cellular content of CCK 8 and pro-CCK were not altered in either of these cell lines except at 100 microM lovastatin. Our results clearly demonstrate that lovastatin at 1 microM strongly inhibits CCK 8 secretion at multiple levels while having little or no effect on its synthesis. This effect on secretion may be partly responsible for the adverse gastrointestinal side effects of lovastatin in patients.

Inhibition of prohormone convertase 1 (PC1) expression in cholecystokinin (CCK) expressing At-T20 cells decreased cellular content and secretion of CCK and caused a shift in molecular forms of CCK secreted

Two different RNAi methods were used to inhibit the expression of prohormone convertase 1 (PC1) in At-T20 cells. Transient transfection of double stranded RNA and stable expression of a vector expressing hairpin-loop RNA targeting PC1 reduced cholecystokinin (CCK) secretion from At-T20 cells. PC1 mRNA and protein were also decreased in the vector transfected cells. This treatment caused a shift in the forms of cholecystokinin (CCK) secreted, decreasing CCK 22 and increasing CCK 8. Stable expression of RNAi effectively decreased PC1 expression. The observed decrease in CCK seen with these RNAi treatments further supports a role for PC1 in CCK processing in these cells.

Production, purification, and characterization of rat pro-CCK from serum-free adapted Drosophila cells.

The precursor of cholecystokinin (pro-CCK) was expressed and purified from media of stably transfected D.Mel-2 cell as an V5-His tagged fusion protein. Its identity was confirmed using SDS-PAGE, immunoblotting, gel filtration chromatography, HPLC, and Mass Spectroscopy. Two major forms of pro-CCK were found with a molecular weight of about 14.4 and 11.3 kDa. The smaller form represents the V5-His tagged pro-CCK after cleavage at a single arginine residue at CCK-58. This cleavage is probably being performed by endogenous proteases in these cells. Purification of the desired larger form of pro-CCK is possible using a nickel column with a recovery of about 20%, yielding 500 microg/L media. The purified protein is stable for several months and can be used for further functional studies of pro-CCK.

Use of expression of antisense mRNA for proprotein convertases 1 and 2 in prohormone processing.

Publisher Summary Studies using antisense inhibition have been criticized because of the possible nonspecific cellular effects complicating interpretation of the results. In light of these concerns, several controls have been performed to ensure that the effects of antisense expression on the processing of pro-cholecystokinin (CCK) are specific. The two most important controls performed were to measure the effects of antisense expression on target messenger ribonucleic acid (mRNA) and target protein. Another control that was performed was to measure the effect of polycystin 2 (PC2) message and protein level in antisense PC1-transfected cells and vice versa. Although PC1 and PC2 are homologous genes within the same family of endoproteases, the effect of antisense is specific for each. These observations suggest that the effect of antisense on the RNA level is complex but specific and may occur at multiple levels to produce inhibition of the protein product. Knowledge of the actual enzymes responsible for prohormone cleavages in specific tissues lags behind progress in other areas of the peptide hormone field owing to the technical difficulty of identification, isolation, characterization, and inhibition of these enzymes. As important as the PC enzymes appear to be, it is possible that proteases other than the PCs are involved in prohormone processing.