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Different roles of protein kinase C isozymes in differentiation of PC12h cells
1376 die dm-aaged ~pocytochrome P450 by cytosolic proteases may be one mechanism by which DDEP-mediated loss of susceptible P450 isozymes occurs "in vivo". Supported by NIH grants DX 26506 and GM 44037. I P.we.243 1
Rat liver cytochrome P-450 and ageing Horbach, G.J.M.J., Van Asten, J.G., Rietjens *, I.C.M. and Van Bezooijen, C.F.A. TNO Institute for Experimental Gerontology, P.O. Box 5815, 2280 HV Rijswijk and * Agricultural University Wageningen, The Netherlands
Age-related changes in the cytochrome P-450 syst~,n in the ra~ are often described and generally a decrease in both content and activity are reported, although increases have been described as well. The age-re~ated changes are more pronounced in male rats than in female ones, dl:,, to changes in gender-specific, constitutive forms of cvtochrome P-450, which in male rats are dependent on testosterone levels. As far as the inducible types of cytochrome P-45~ ~ e concerned, little 6ata are available on the influence of age. In this study, data will be presented on the levels of r;:;~ssenger RNA and enzymes for the cytochromes P-450 IA1, IA2, IIBl. and lIB2 in male rats either untreated or maximally induc~:~ ~,ith phenobarbital, 3-methylcholanthrene or isosafrole. Also the specific activities of P-450 enzymes were m~asurea using the highly specific substrates, pentoxyresorufin and ethoxyresorufin. The results of this study show that after maximal ~,~duction there are considerable changes with age in the levels of mRNAs for F-4501A1, IA2, liB1 and liB2. The inducibility of the mRNAs for P-45011B1 and lIB2 by both phenobarbital and isosafrole shows a marked decrease between 12 and 36 months of age. A decrease in the inducibility of the mRNAs for P-4501A1 and IA2 is only observed when using isosafrole as the inducer and not when using 3-methylcholanthrene. From these observations, it can be concluded that changes with age in the mRNA levels are different for the different P-450 enzymes and are dependent on the inducer used. However, after maximal induction no age-related changes were observed in the amount and activity of the cytochrome P-4501A1, IA2, IIB1 and lIB2. Therefore, the lower mRNA levels with age are not reflected in lower enzyme levels, but rather result in a decreased synthesis rate of these enzymes. I P.we.244 ]
Different roles of protein kinase C isozymes in differentiation of PCI2h cells Kunugi, Y.-U., Tamura, H., Shimohama *, S., Saitoh * *, T. Taniguchi, T. and K i m u r a *, J. Department of Neurobiology, Kyoto Pharmaceutical University, Kyoto, 607, • Department of Neurology, Kyoto University School of Medicine, Kyoto, 606, Japan and * * Department of Neurosciences, School of Medicine, University o.f California, San Diego, U.S.A.
Protein kinase C has been implicated in the regulation of various cellular processes including growth, differentiation, hormone and neurotransmitter release, gene expression and cellular n'Letabolism. However, the role of each protein kinase C isozyme (a, ill, flII or v type) in the regulation of different cellular functions remains unknown. Rat pheochromocytoma PC12 cells can be induced to differentiate into cells with typical morphology by a number of substrates including nerve growth factor (NGF), cAMP derivatives and TPA, an activator of protein kinase C. Although this differentiation process is well known to be accompanied by various physiological responses, the molecular mechanisms of their signal transductions are not clear. In order to clarify the contribution of protein kinase C-related signal transduction pathway, and moreover, to corelate the presence of a protein kinase C isozyme with
1377 specialized physiological response charateristic to the differntiation process of stimulated PC12 cells, we examined the different expression of four types of protein kinase C isozyme in PC12h cells when stimulated by dibutyryl cAMP (diBtcAMP), which is a stable derivative of cAMP and can enter the living cells. PC12h cells were subcultured in serum-free medium, Dulbecco's modified Eagle's medium/Ham'sF12 (50:50) supplemented with 5 p g / m l insulin, 5/~g/ml transferrin and 20 nM progesterone. Cells were passaged routinely prior to confluence and plated at a density of 1.0 × 105 cells/cm 2 onto poly-L-lysine-precoated plastic dishes (Nune). After 1 day, diBtcAMP were added at a final concentration of 1 mM into the chemically defined medium to stimulate the neurite outgrowth of PC12h cells. For immunoblotting analyses, cells were homogenized in a sucrose buffer solution by a Teflon-glass homogenizer arid stored at - 8 0 ° C until use. By immunoblotting analyses using four specific antibodies against protein kinase C isozymes, we detected two isozymes of protein ldnase C, a and fill types in homogenates of PC12h cells. Both isozymes showed two immunoreactive protein band, 81 kDa native enzyme and its presumed proteolytie fragment 72 kDa species. When exposed to I mM diBtcAMP, PC12h cells clearly began to exhibite the neurite outgrowth at around 30 rain after the addition of diBtcAMP. In this stage, neurites looked narrow and unstable. After 24 hours, however, these neurites became much stable and wide ribbon-like shapes. When immunoblotting was carried out for the homogenates of diBtcAMP-stimulated PC12h cells, two protein kinase C isozymes mentioned above showed the different profiles time-dependently and quantitatively. As for the a type, 72 kDa band protein increased within 5 min after the addition of diBteAMP, and then began to decrease rapidly with increase of the smaller immunoreactive component, 35 kDa band protein after about 1 hour- treatment by diBtcAMP. On the other hand, fill type isozyme showed the sharp increase of 72 kDa species within 5 min, and this prompt accumulation of f l l type enzyme protein was observed to succeed until nearly 24 hours after the addition of diBtcAMP, where the slight decrease of this 72 kDa band was observed. These results suggest that a and fill types of protein kinase C isozyme may be involved in the different cellular processes and regulate the specialized physiological responses in the differentiation of PC12h cells. Furthermore, it may be estimated that the concerted action or cross-talk between protein kinase C and cAMP-dependent signal tansduction pathway works on the differentiation process of PC12h cells.
iP.wo.245] Carboxylesterases: comparisons between cloned rat and human forms Long *, R.M. and Pohl * *, L.R, • Dept. of Pharmacology and Toxicology, University of Maryland. Baltimore. MD. 21201 and * * Lab. of Chemical Pharmacology. NHLBI. NIH, Bethesda, MD. 20892. U.S.A.
Carboxylesterases comprise a family of isoenzymes that hydrolyze a wide range of substrates, including therapeutic drugs, chemical toxicants, and endogenous lipids. Lambda gt11 libraries prepared from rat and human livers were screened with antibodies raised to a purified rat liver carboxylesterase (Satoh et al., 1989 an~ ~everal clones were isolated and sequenced. The longest eDNA isolated from a rat library (Long et al., 1988) contained an open reading frame of 531 amino acids that represented 100% of the sequence of a mature carboxylesterase protein, and the longest cDNA isolated from a human library contained an open reading frame of 521 amino acids that had 98% of the sequence of a processed carboxylesterase protein. Evidence was found for a signal peptide that presumably facilitates passage of the carboxylesterase pre-protein ~cross the endoplasmic reticulum membrane. The sequences possessed many structural features that are highly conserved among these clones and a purified rabbit !iver carboxylesterase protein (Korza and Ozols, 1988), including Ser, His, and Asp residues that form the active site, two pairs of Cys residues that can participate in disulfide bond formation, and one Asn-Xxx-Thr site for N-linked carbohydrate addition. When the clones were used to probe rat and human liver genomic DNA that had been digested with restriction enzymes, several bands of varying intensity were observed. The results suggested that carboxylesterases are
Rat liver cytochrome P-450 and ageing Horbach, G.J.M.J., Van Asten, J.G., Rietjens *, I.C.M. and Van Bezooijen, C.F.A. TNO Institute for Experimental Gerontology, P.O. Box 5815, 2280 HV Rijswijk and * Agricultural University Wageningen, The Netherlands
Age-related changes in the cytochrome P-450 syst~,n in the ra~ are often described and generally a decrease in both content and activity are reported, although increases have been described as well. The age-re~ated changes are more pronounced in male rats than in female ones, dl:,, to changes in gender-specific, constitutive forms of cvtochrome P-450, which in male rats are dependent on testosterone levels. As far as the inducible types of cytochrome P-45~ ~ e concerned, little 6ata are available on the influence of age. In this study, data will be presented on the levels of r;:;~ssenger RNA and enzymes for the cytochromes P-450 IA1, IA2, IIBl. and lIB2 in male rats either untreated or maximally induc~:~ ~,ith phenobarbital, 3-methylcholanthrene or isosafrole. Also the specific activities of P-450 enzymes were m~asurea using the highly specific substrates, pentoxyresorufin and ethoxyresorufin. The results of this study show that after maximal ~,~duction there are considerable changes with age in the levels of mRNAs for F-4501A1, IA2, liB1 and liB2. The inducibility of the mRNAs for P-45011B1 and lIB2 by both phenobarbital and isosafrole shows a marked decrease between 12 and 36 months of age. A decrease in the inducibility of the mRNAs for P-4501A1 and IA2 is only observed when using isosafrole as the inducer and not when using 3-methylcholanthrene. From these observations, it can be concluded that changes with age in the mRNA levels are different for the different P-450 enzymes and are dependent on the inducer used. However, after maximal induction no age-related changes were observed in the amount and activity of the cytochrome P-4501A1, IA2, IIB1 and lIB2. Therefore, the lower mRNA levels with age are not reflected in lower enzyme levels, but rather result in a decreased synthesis rate of these enzymes. I P.we.244 ]
Different roles of protein kinase C isozymes in differentiation of PCI2h cells Kunugi, Y.-U., Tamura, H., Shimohama *, S., Saitoh * *, T. Taniguchi, T. and K i m u r a *, J. Department of Neurobiology, Kyoto Pharmaceutical University, Kyoto, 607, • Department of Neurology, Kyoto University School of Medicine, Kyoto, 606, Japan and * * Department of Neurosciences, School of Medicine, University o.f California, San Diego, U.S.A.
Protein kinase C has been implicated in the regulation of various cellular processes including growth, differentiation, hormone and neurotransmitter release, gene expression and cellular n'Letabolism. However, the role of each protein kinase C isozyme (a, ill, flII or v type) in the regulation of different cellular functions remains unknown. Rat pheochromocytoma PC12 cells can be induced to differentiate into cells with typical morphology by a number of substrates including nerve growth factor (NGF), cAMP derivatives and TPA, an activator of protein kinase C. Although this differentiation process is well known to be accompanied by various physiological responses, the molecular mechanisms of their signal transductions are not clear. In order to clarify the contribution of protein kinase C-related signal transduction pathway, and moreover, to corelate the presence of a protein kinase C isozyme with
1377 specialized physiological response charateristic to the differntiation process of stimulated PC12 cells, we examined the different expression of four types of protein kinase C isozyme in PC12h cells when stimulated by dibutyryl cAMP (diBtcAMP), which is a stable derivative of cAMP and can enter the living cells. PC12h cells were subcultured in serum-free medium, Dulbecco's modified Eagle's medium/Ham'sF12 (50:50) supplemented with 5 p g / m l insulin, 5/~g/ml transferrin and 20 nM progesterone. Cells were passaged routinely prior to confluence and plated at a density of 1.0 × 105 cells/cm 2 onto poly-L-lysine-precoated plastic dishes (Nune). After 1 day, diBtcAMP were added at a final concentration of 1 mM into the chemically defined medium to stimulate the neurite outgrowth of PC12h cells. For immunoblotting analyses, cells were homogenized in a sucrose buffer solution by a Teflon-glass homogenizer arid stored at - 8 0 ° C until use. By immunoblotting analyses using four specific antibodies against protein kinase C isozymes, we detected two isozymes of protein ldnase C, a and fill types in homogenates of PC12h cells. Both isozymes showed two immunoreactive protein band, 81 kDa native enzyme and its presumed proteolytie fragment 72 kDa species. When exposed to I mM diBtcAMP, PC12h cells clearly began to exhibite the neurite outgrowth at around 30 rain after the addition of diBtcAMP. In this stage, neurites looked narrow and unstable. After 24 hours, however, these neurites became much stable and wide ribbon-like shapes. When immunoblotting was carried out for the homogenates of diBtcAMP-stimulated PC12h cells, two protein kinase C isozymes mentioned above showed the different profiles time-dependently and quantitatively. As for the a type, 72 kDa band protein increased within 5 min after the addition of diBteAMP, and then began to decrease rapidly with increase of the smaller immunoreactive component, 35 kDa band protein after about 1 hour- treatment by diBtcAMP. On the other hand, fill type isozyme showed the sharp increase of 72 kDa species within 5 min, and this prompt accumulation of f l l type enzyme protein was observed to succeed until nearly 24 hours after the addition of diBtcAMP, where the slight decrease of this 72 kDa band was observed. These results suggest that a and fill types of protein kinase C isozyme may be involved in the different cellular processes and regulate the specialized physiological responses in the differentiation of PC12h cells. Furthermore, it may be estimated that the concerted action or cross-talk between protein kinase C and cAMP-dependent signal tansduction pathway works on the differentiation process of PC12h cells.
iP.wo.245] Carboxylesterases: comparisons between cloned rat and human forms Long *, R.M. and Pohl * *, L.R, • Dept. of Pharmacology and Toxicology, University of Maryland. Baltimore. MD. 21201 and * * Lab. of Chemical Pharmacology. NHLBI. NIH, Bethesda, MD. 20892. U.S.A.
Carboxylesterases comprise a family of isoenzymes that hydrolyze a wide range of substrates, including therapeutic drugs, chemical toxicants, and endogenous lipids. Lambda gt11 libraries prepared from rat and human livers were screened with antibodies raised to a purified rat liver carboxylesterase (Satoh et al., 1989 an~ ~everal clones were isolated and sequenced. The longest eDNA isolated from a rat library (Long et al., 1988) contained an open reading frame of 531 amino acids that represented 100% of the sequence of a mature carboxylesterase protein, and the longest cDNA isolated from a human library contained an open reading frame of 521 amino acids that had 98% of the sequence of a processed carboxylesterase protein. Evidence was found for a signal peptide that presumably facilitates passage of the carboxylesterase pre-protein ~cross the endoplasmic reticulum membrane. The sequences possessed many structural features that are highly conserved among these clones and a purified rabbit !iver carboxylesterase protein (Korza and Ozols, 1988), including Ser, His, and Asp residues that form the active site, two pairs of Cys residues that can participate in disulfide bond formation, and one Asn-Xxx-Thr site for N-linked carbohydrate addition. When the clones were used to probe rat and human liver genomic DNA that had been digested with restriction enzymes, several bands of varying intensity were observed. The results suggested that carboxylesterases are