RESUMO
Protein kinase C (PKC) delta is dramatically upregulated in the corpus luteum in the second half of pregnancy in the rat. To gain insight into the hormonal regulation of PKC delta expression, studies were undertaken to analyze the regulation of PKC delta expression in a luteinized rat granulosa cell model. PKC delta protein expression was evaluated in luteinized granulosa cells, isolated from human (h)CG-treated immature female rats 7 h after the injection of an ovulatory dose of hCG and cultured up to 12 days. Cytochrome P450 cholesterol side chain cleavage enzyme expression was observed throughout the culture period, and a majority of the cells expressed steroidogenic acute regulatory protein and responded to rat placental lactogen (rPL)-1 by exhibiting hypertrophy, consistent with maintenance of the luteal phenotype. Both PKC delta protein and mRNA expression increased 3.5-4-fold with time of culture, and PKC delta mRNA expression could be eliminated by treatment of cells with the PKC inhibitor GF109203X. E(2) caused a specific dose- and time-dependent increase in expression of PKC delta protein of twofold, whereas PKC delta mRNA was unaffected by E(2) over a 12-day culture period. Treatment of cells with 500 ng/ml rPL-1 for the final 4 days of a 12-day culture in the absence of E(2) had no effect on PKC delta protein or mRNA expression, while treatment with 500 or 3000 ng/ml rPL-1 in the presence of E(2) significantly enhanced both PKC delta protein and mRNA expression (up to threefold). These results show that two of the major regulators of luteal function in the second half of pregnancy in the rat, E(2) and rPL-1, cooperate to regulate PKC delta expression in luteinized granulosa cells.
Assuntos
Estradiol/farmacologia , Células da Granulosa/efeitos dos fármacos , Células da Granulosa/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Lactogênio Placentário/farmacologia , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Animais , Enzima de Clivagem da Cadeia Lateral do Colesterol/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfoproteínas/metabolismo , Gravidez , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Transdução de SinaisRESUMO
Undifferentiated cells from preantral (PA) follicles respond to high levels of cAMP in a different manner than do differentiated cells from preovulatory (PO) follicles. We hypothesized that this differential response of PA and PO cells to cAMP could be due, in part, to either a difference in the profile of isoforms that comprise the cAMP-dependent protein kinase (PKA) holoenzymes and/or a difference in the interaction of PKA with A-kinase-anchoring proteins (AKAPs). To test these hypotheses, PKA activity, PKA holoenzymes, PKA subunits and AKAPs from PA and PO ovaries were compared. Soluble PKA holoenzymes and regulatory (R) subunits were separated by DEAE-cellulose chromatography and sucrose-density-gradient centrifugation. PKA R subunits were distinguished by photoaffinity labelling, autophosphorylation, size, isoelectric point and immunoreactivity. AKAPs were identified by RII subunit overlay assays and immunoreactivity. The results showed that extracts from PA and PO ovaries exhibited equivalent PKA holoenzyme profiles and activities, characterized by low levels of PKA type I (PKAI) holoenzyme and two distinct PKAII holoenzyme peaks, one containing only RIIbeta subunits (PKAIIbeta) and one containing both PKAIIbeta and PKAIIalpha holoenzymes. Both PA and PO ovarian extracts also contained PKA catalytic (C)-subunit-free RIalpha, while only PO ovaries exhibited C-subunit-free RIIbeta. Consistent with the elevated levels of C-subunit-free RIIbeta in PO cells, PKA activation in PO cells required higher concentrations of forskolin than that in PA cells. While extracts of PA and PO ovaries exhibited a number of similar AKAPs, including four prominent ones reactive with anti-AKAP-KL antisera (where AKAP-KL is an AKAP especially abundant in kidney and liver), cAMP-agarose affinity chromatography revealed two major differences in AKAP binding to purified R subunits. PO ovaries contained increased levels of AKAP80 (AKAP of 80 kDa) bound selectively to R subunits in DEAE-cellulose peak 2 (comprising PKAIIbeta and RIalpha), but not to R subunits in DEAE-cellulose peak 3 (comprising PKAIIalpha, PKAIIbeta and RIIbeta). PO ovaries also showed increased binding of R subunits to AKAPs reactive with anti-AKAP-KL antisera at 210, 175, 150 and 115 kDa. Thus in PO ovaries, unlike in PA ovaries, the majority of AKAPs are bound to R subunits. These results suggest that altered PKA-AKAP interactions may contribute to the distinct responses of PA and PO follicles to high levels of cAMP, and that higher cAMP levels are required to activate PKA in PO ovaries.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Folículo Ovariano/citologia , Animais , Proteínas de Transporte/isolamento & purificação , Diferenciação Celular , Cromatografia DEAE-Celulose , AMP Cíclico/análise , Proteínas Quinases Dependentes de AMP Cíclico/isolamento & purificação , Ativação Enzimática , Feminino , Células da Granulosa/enzimologia , Holoenzimas/isolamento & purificação , Isoenzimas/isolamento & purificação , Isoenzimas/metabolismo , Folículo Ovariano/química , Folículo Ovariano/enzimologia , Ligação Proteica , Ratos , Ratos Sprague-DawleyRESUMO
We have previously shown that estrogen up-regulates expression of protein kinase C (PKC) delta in the rat and rabbit corpus luteum as well as in luteinized rat granulosa primary cell cultures. To determine whether a similar regulation of the PKC delta isoform by estrogen occurred in another estrogen responsive system, we investigated the estrogen receptor positive MCF-7 human breast cancer cells. In a characterization of PKC isoforms in MCF-7 cells we determined that PKC delta was the predominant PKC isoform. However in contrast to the effect of estrogen on PKC delta expression in ovarian cells, estrogen treatment of MCF-7 cells resulted in a significant decrease in PKC delta protein and mRNA expression in a time and dose dependent manner. Treatment of MCF-7 cells with 10(-10)-10(-8) M estrogen for 7 days down-regulated specifically PKC delta mRNA and protein while expression of other PKC isoforms was unchanged. The opposite regulation of PKC delta expression in ovarian and breast cancer cells prompted us to evaluate the type of estrogen receptor present in both cell types. Results showed that luteinized rat granulosa cells expressed predominantly estrogen receptor beta while the MCF-7 cells expressed predominantly estrogen receptor alpha and barely detectable levels of estrogen receptor beta. These results suggest that the differential ability of estrogen to regulate PKC beta expression could potentially be a result of differential signaling through the two estrogen receptor subtypes.
Assuntos
Estrogênios/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Isoenzimas/genética , Proteína Quinase C/genética , Animais , Neoplasias da Mama , Corpo Lúteo/enzimologia , Estradiol/análogos & derivados , Estradiol/farmacologia , Antagonistas de Estrogênios/farmacologia , Feminino , Células da Granulosa/enzimologia , Humanos , Isoenzimas/metabolismo , Alcamidas Poli-Insaturadas , Protamina Quinase/metabolismo , Proteína Quinase C/metabolismo , Proteína Quinase C-delta , RNA Mensageiro/genética , Coelhos , Ratos , Receptores de Estrogênio/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
The Raf-1 serine/threonine kinase is a key protein involved in the transmission of many growth and developmental signals. In this report, we show that autoinhibition mediated by the noncatalytic, N-terminal regulatory region of Raf-1 is an important mechanism regulating Raf-1 function. The inhibition of the regulatory region occurs, at least in part, through binding interactions involving the cysteine-rich domain. Events that disrupt this autoinhibition, such as mutation of the cysteine-rich domain or a mutation mimicking an activating phosphorylation event (Y340D), alleviate the repression of the regulatory region and increase Raf-1 activity. Based on the striking similarites between the autoregulation of the serine/threonine kinases protein kinase C, Byr2, and Raf-1, we propose that relief of autorepression and activation at the plasma membrane is an evolutionarily conserved mechanism of kinase regulation.
Assuntos
Proteínas Proto-Oncogênicas c-raf/metabolismo , Animais , Catálise , Mutação , Proteínas Proto-Oncogênicas c-raf/química , Proteínas Proto-Oncogênicas c-raf/genética , Xenopus laevisRESUMO
We have identified mutations in Raf-1 that increase binding to Ras. The mutations were identified making use of three mutant forms of Ras that have reduced Raf-1 binding (Winkler, D. G., Johnson, J. C., Cooper, J. A., and Vojtek, A. B. (1997) J. Biol. Chem. 272, 24402-24409). One mutation in Raf-1, N64L, suppresses the Ras mutant R41Q but not other Ras mutants, suggesting that this mutation structurally complements the Ras R41Q mutation. Missense substitutions of residues 143 and 144 in the Raf-1 cysteine-rich domain were isolated multiple times. These Raf-1 mutants, R143Q, R143W, and K144E, were general suppressors of three different Ras mutants and had increased interaction with non-mutant Ras. Each was slightly activated relative to wild-type Raf-1 in a transformation assay. In addition, two mutants, R143W and K144E, were active when tested for induction of germinal vesicle breakdown in Xenopus oocytes. Interestingly, all three cysteine-rich domain mutations reduced the ability of the Raf-1 N-terminal regulatory region to inhibit Xenopus oocyte germinal vesicle breakdown induced by the C-terminal catalytic region of Raf-1. We propose that a direct or indirect regulatory interaction between the N- and C-terminal regions of Raf-1 is reduced by the R143W, R143Q, and K144E mutations, thereby increasing access to the Ras-binding regions of Raf-1 and increasing Raf-1 activity.
Assuntos
Cisteína/análise , Proteínas Proto-Oncogênicas c-raf/química , Proteínas Proto-Oncogênicas c-raf/metabolismo , Tirosina 3-Mono-Oxigenase , Proteínas ras/metabolismo , Proteínas 14-3-3 , Animais , Catálise , Códon , Ensaio de Imunoadsorção Enzimática , Humanos , Mutagênese Sítio-Dirigida , Oócitos/metabolismo , Ligação Proteica/genética , Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-raf/genética , Relação Estrutura-Atividade , Xenopus , Leveduras , Proteínas ras/genéticaRESUMO
Small heat-shock proteins (sHSPs) are widely expressed 25-28 kDa proteins whose functions are dynamically regulated by phosphorylation. While recent efforts have clearly delineated a stress-responsive p38 mitogen-activated protein-kinase (MAPK)-dependent kinase pathway culminating in activation of the heat-shock (HSP)-kinases, mitogen-activated protein-kinase-activated protein kinase-2 and -3, not all sHSP phosphorylation events can be explained by the p38 MAPK-dependent pathway. The contribution of protein kinase C (PKC) to sHSP phosphorylation was suggested by early studies but later questioned on the basis of the reported poor ability of purified PKC to phosphorylate sHSP in vitro. The current study re-evaluates the role of PKC in sHSP phosphorylation in the light of the isoform complexity of the PKC family. We evaluated the sHSP phosphorylation status in rat corpora lutea obtained from two stages of pregnancy, mid-pregnancy and late-pregnancy, which express different levels of the novel PKC isoform, PKC-delta. Two-dimensional Western blot analysis showed that HSP-27 was more highly phosphorylated in vivo in corpora lutea of late pregnancy, corresponding to the developmental stage in which PKC-delta is abundant and active. Late-pregnant luteal extracts contained a lipid-sensitive HSP-kinase activity which exactly co-purified with PKC-delta using hydroxyapatite and S-Sepharose column chromatography. To determine whether there might be preferential phosphorylation of sHSP by a particular PKC isoform, purified recombinant PKC isoforms corresponding to those PKC isoforms detected in rat corpora lutea were evaluated for HSP-kinase activity in vitro. Recombinant PKC-delta effectively catalysed the phosphorylation of sHSP in vitro, and PKC-alpha was 30-50% as effective as an HSP-kinase; other PKCs tested (beta1, beta2, epsilon and zeta) were poor HSP-kinases. These results show that select PKC family members can function as direct HSP-kinases in vitro. Moreover, the observation of enhanced luteal HSP-27 phosphorylation in vivo, in late pregnancy, when PKC-delta is abundant and active, suggests that select PKC family members contribute to sHSP phosphorylation events in vivo.
Assuntos
Corpo Lúteo/enzimologia , Proteínas de Choque Térmico/metabolismo , Isoenzimas/metabolismo , Proteínas Quinases Ativadas por Mitógeno , Proteína Quinase C/metabolismo , Animais , Western Blotting , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Cromatografia em Gel , Feminino , Peptídeos e Proteínas de Sinalização Intracelular , Fosforilação , Gravidez , Proteína Quinase C-delta , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/metabolismo , Fatores de Tempo , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
This study was undertaken to compare the effects of chronic angiotensin-converting enzyme (ACE) inhibition on blood pressure (BP) and renal hemodynamics in older black and nonblack hypertensive patients with chronic renal insufficiency. A multicenter, placebo lead-in double-blind, parallel group study was performed to compare the antihypertensive efficacy and renal hemodynamic response to the once-daily ACE inhibitor fosinopril (n = 14) and lisinopril (n = 13) over a 22-week period. The study goal was to lower diastolic blood pressure (DBP) to 90 mm Hg or less. Furosemide was added after 6 weeks if blood pressure goal was not achieved. At outpatient clinics at university medical centers, 27 older hypertensive patients (> or = 45 years; 12 blacks, 15 nonblacks; 19 male, eight female) with DBP of 95 mm Hg or higher and 4-hour creatinine clearance 20 to 70 mL/min/1.73 m2 were studied. Changes (delta) from baseline in BP, glomerular filtration rate (GFR), and renal plasma flow (RPF) were measured. Mean systolic blood pressure (SBP) and DBP decreased significantly and to a similar extent in randomized groups: fosinopril (mean +/- SEM) delta DBP at 6 weeks was -13 +/- 2 (P < 0.0001; 95% CI, -16 to -9) and at 22 weeks was -12 +/- 2 (P < 0.0001; 95% CI, -16 to -9); lisinopril delta DBP at 6 weeks was -14 +/- 6 (P < 0.0001; 95% CI, -10 to -18) and at 22 weeks was -16 +/- 2 (P < 0.0001; 95% CI, -12 to -21). GFR and RPF did not change significantly in either group. BP was significantly reduced and to a similar extent in blacks and nonblacks: for blacks, delta DBP at 6 weeks was -11 +/- 3 (P < 0.05; 95% CI, -0.01 to -9) and at 22 weeks was -16 +/- 2 (P < 0.0001; 95% CI, -11 to -20); for nonblacks, delta DBP at 6 weeks was -14 +/- 1 (P < 0.0001; 95% CI, -12 to -17) and at 22 weeks was -12 +/- 2 (P < 0.0001; 95% CI, -16 to -8). Eight patients (five blacks and three nonblacks) required an addition of furosemide after 6 weeks to reach the DBP goal of < or = 90 mm Hg at 22 weeks. GFR was not significantly altered for either racial group at 6 weeks; however, at 22 weeks; however, at 22 weeks, GFR decreased significantly in blacks (delta GFR, -16 +/- 5; P < 0.006; 95% CI, -26 to -5) and tended to increase in nonblacks (delta GFR, 7 +/- 6; P > 0.25). delta GFR correlated directly with the delta RPF (delta GFR = 0.0611* delta RPF -2.35 +; r = 0.68; P < 0.003). There was no correlation between delta MAP and delta GFR or delta RPF in blacks or nonblacks. We conclude that chronic ACE inhibition with fosinopril and lisinopril alone or in combination with furosemide lowers BP in older blacks and nonblacks with hypertension and chronic renal insufficiency. Racial differences in the renal hemodynamic response to chronic ACE inhibition were noted and appear to be independent of diuretic use and the magnitude of BP lowering.
Assuntos
Inibidores da Enzima Conversora de Angiotensina/farmacologia , Anti-Hipertensivos/farmacologia , População Negra , Hipertensão/tratamento farmacológico , Hipertensão/etnologia , Falência Renal Crônica/etnologia , Rim/efeitos dos fármacos , Idoso , Idoso de 80 Anos ou mais , Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Anti-Hipertensivos/uso terapêutico , Método Duplo-Cego , Feminino , Fosinopril/farmacologia , Taxa de Filtração Glomerular/efeitos dos fármacos , Humanos , Hipertensão/complicações , Hipertensão/fisiopatologia , Recém-Nascido , Rim/fisiopatologia , Falência Renal Crônica/complicações , Falência Renal Crônica/fisiopatologia , Lisinopril/farmacologia , Masculino , Pessoa de Meia-Idade , Estudos ProspectivosRESUMO
An interaction with the Ras proto-oncogene product is a requirement for Raf-1 activation in many signaling cascades. The significance of this interaction is demonstrated by the fact that a mutation preventing the Ras-Raf interaction severely impairs the function of both mammalian (Raf-1) and Drosophila (D-Raf) Raf proteins. In D-Raf, however, dominant intragenic mutations have been identified that suppress the effect of the Ras-binding site (RBS) mutation. To address the mechanism by which these mutations restore Raf signaling, we have introduced the suppressor mutations into the analogous residues of mammalian Raf-1. Here, we show that rather than compensating for the RBS mutation by restoring the Ras-Raf-1 interaction, the suppressor mutations increase the enzymatic and biological activity of Raf-1, allowing Raf-1 to signal in the absence of Ras binding. Surprisingly, we find that while one of the suppressor mutations (P181L) increases the basal kinase activity of Raf-1, it also abolishes the ability of wild-type Raf-1 to become activated by Ras. This mutation occurs in the cysteine-rich domain (CRD) of Raf-1 and demonstrates the importance of this region for a productive Ras-Raf interaction. Finally, we present evidence that the most activating suppressor mutation (G498S) increases Raf-1 activity by introducing a novel phosphorylation site into the L12 activation loop of the Raf-1 kinase domain.
Assuntos
Proteína Oncogênica p21(ras)/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais/fisiologia , Supressão Genética/fisiologia , Tirosina 3-Mono-Oxigenase , Proteínas 14-3-3 , Animais , Ácido Aspártico/metabolismo , Sítios de Ligação , Linhagem Celular , Drosophila/genética , Drosophila/fisiologia , Ativação Enzimática , Glicina/metabolismo , Humanos , Rim , Meiose , Proteína Oncogênica p21(ras)/genética , Oócitos , Fosfosserina/análise , Proteínas Serina-Treonina Quinases/genética , Proteínas/metabolismo , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-raf , XenopusRESUMO
The activation of the serine/threonine kinase Raf-1 is proving to be an intricate multistep process. Recent advances in elucidating how Raf-1 becomes activated in response to signaling events have emphasized the role of phosphorylation and protein interactions in Raf-1 regulation. The picture clearly emerging is that Raf-1 activity can be regulated by multiple mechanisms.
Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Ativação Enzimática , Humanos , Fosforilação , Proteínas Proto-Oncogênicas c-raf , Proteínas ras/metabolismoRESUMO
In response to many scientific discoveries linking cancer in certain families to inherited factors, the Vermont Cancer Center established the Familial Cancer Program (FCP) in December 1993. This multifaceted program combines the expertise of clinicians and researchers in many disciplines, including genetics, oncology, psychology, and molecular biology. The program's goals are identification of families in its region with excess cancer, provision of clinical services to such families, and use of research protocols when available and appropriate. This article describes the experience of setting up a familial cancer program in a rural area and discusses both successes and challenges in such an endeavor.
RESUMO
Flurbiprofen, an arylpropionic acid (APA) class nonsteroidal antiinflammatory drug (NSAID), is commercially available only as the racemic mixture, although its pharmacologic effect has been credited primarily to the S isomer. In humans, the bioavailability of racemic flurbiprofen absorbed from the oral cavity has been studied measuring the total concentration of S- and R-flurbiprofen, and the pharmacokinetics of S- and R-flurbiprofen have been studied after oral administration of racemic flurbiprofen. In this study, the plasma concentrations of S-flurbiprofen and to some extent R-flurbiprofen were studied after brushing with a toothpaste containing different mixtures of S- and R-flurbiprofen. The toothpaste formulations contained 1% racemic (50:50), eutectic (14:86), 1%, 0.5%, and 0.25% (5:95) R- to S-flurbiprofen. Both S- and R-flurbiprofen were rapidly absorbed, with a time to reach maximum concentration (tmax) of 1.2 to 1.4 hours. Based on the AUC, the amount of S-flurbiprofen absorbed increased proportionally when given as the 0.25% (5:95) preparation to the 0.5% (5:95) mixture but did not increase significantly above the 0.5% (5:95) mixture when given as 1% (5:95) R- to S-flurbiprofen. This suggests that dose-proportional absorption of S-flurbiprofen is not maintained at higher concentrations. The elimination of S-flurbiprofen appears to be variable and prolonged after this mode of administration, as observed from plasma concentrations. Further controlled and more prolonged studies of S- and R-flurbiprofen are needed to confirm these observations.
Assuntos
Anti-Inflamatórios não Esteroides/farmacocinética , Flurbiprofeno/farmacocinética , Boca/metabolismo , Adulto , Área Sob a Curva , Meia-Vida , Humanos , Masculino , Estereoisomerismo , Cremes DentaisRESUMO
The present studies were undertaken to examine the cellular distribution and regulation of delta protein kinase-C (PKC) at different stages of luteal differentiation in the rat. Results from in situ hybridization studies with a delta PKC-specific probe demonstrated that delta PKC was localized specifically in granulosa cells of healthy preantral, small antral, and preovulatory follicles and corpora lutea from the second half of pregnancy. Northern and Western blot analyses showed that levels of delta PKC protein and messenger RNA were elevated 25- and 35-fold, respectively, in the second half of pregnancy over levels in preovulatory follicle-enriched ovaries, peaking between days 18-20 of pregnancy. Levels of alpha PKC, beta PKC, and zeta PKC remained unchanged during luteal differentiation. In rats hypophysectomized and hysterectomized on day 12 of pregnancy and in immature rats containing corpora lutea induced with PMSG followed by hCG injections, exogenous estrogen caused 3- and 7-fold increases in delta PKC protein, respectively. In the immature rat model, delta PKC messenger RNA levels were not altered by exogenous estrogen. Although exogenous testosterone also elevated delta PKC protein levels, exogenous dihydrotestosterone and progesterone were ineffective. These results suggest that estrogen may participate, at a posttranscriptional level, in the dramatic induction of delta PKC seen at the end of pregnancy in rat corpora lutea.
Assuntos
Estrogênios/farmacologia , Isoenzimas/análise , Isoenzimas/fisiologia , Ovário/enzimologia , Proteína Quinase C/fisiologia , Animais , Northern Blotting , Western Blotting , Gonadotropina Coriônica/farmacologia , Corpo Lúteo/efeitos dos fármacos , Corpo Lúteo/fisiologia , Feminino , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Hibridização In Situ , Isoenzimas/genética , Fase Luteal/fisiologia , Ovário/fisiologia , Gravidez , Proteína Quinase C/genética , RNA Mensageiro/análise , RNA Mensageiro/genética , Radioimunoensaio , Ratos , Ratos Sprague-Dawley , Transcrição GênicaRESUMO
Studies were undertaken to classify protein kinase C (PKC) forms present in rat corpora lutea and to begin to evaluate their regulation during ovarian differentiation. Hydroxyapatite (HAP) column chromatography of rat luteal tissue revealed the presence of multiple forms of PKC (alpha, beta, delta, zeta). Identification of the PKC isoforms was based upon elution positions from HAP column chromatography and immunoreactivity. The delta PKC isoform was identified as the major Ca(2+)-independent form of PKC present in rat luteal tissue. The Ca(2+)-independent, lipid-dependent phosphorylation of the 80-kDa delta PKC was readily detectable in soluble luteal extracts and was shown to reflect autophosphorylation of delta PKC. To evaluate the regulation of PKC isoforms during ovarian differentiation, PKC protein levels were compared between preovulatory follicle-enriched ovaries and corpora lutea obtained on day 16 of pregnancy. Levels of delta PKC protein were greatly elevated in corpora lutea compared to levels in preovulatory follicles. In contrast, levels of alpha and beta PKC protein remained constant while levels of zeta PKC were slightly higher in the follicular than the luteal extract. Levels of delta PKC mRNA were also higher in corpora lutea than in preovulatory follicles. These results are the first to demonstrate the physiological regulation of delta PKC with follicular differentiation into corpora lutea and implicate a role for this prominent PKC form in the corpus luteum during pregnancy.
Assuntos
Isoenzimas/isolamento & purificação , Ovário/enzimologia , Proteína Quinase C/isolamento & purificação , Animais , Autorradiografia , Corpo Lúteo/enzimologia , Durapatita , Feminino , Regulação da Expressão Gênica , Isoenzimas/genética , Ovulação , Gravidez , Proteína Quinase C/genética , Proteína Quinase C-delta , RNA Mensageiro/análise , Ratos , Ratos Sprague-DawleyRESUMO
Rabbit corpora lutea were tested for the presence of phosphorylative responses sensitive to estrogen. Luteal Ca(2+)-independent lipid-stimulated kinase activity was detected by phosphorylation of the endogenous substrate, p76. Estrogen treatment, by way of estradiol-17 beta implant, increased levels of the lipid-stimulated phosphoprotein 2-3-fold throughout pseudopregnancy. Midpseudopregnant rabbit luteal extracts were further evaluated to determine the identity of the lipid-stimulated kinase. Results of low pH-activated phosphorylation were consistent with the identification of p76 as an autophosphorylated member of the protein kinase C (PKC) family. Partial purification of the luteal lipid-stimulated kinase was performed using sequential DEAE-cellulose/hydroxylapatite chromatographies and using gel filtration. Western immunoblot with type-specific anti-PKC delta antiserum showed coelution of kinase p76 activity with immunoreactive PKC delta. Immunoblot analysis confirmed that luteal levels of PKC delta were increased by estrogen treatment.
Assuntos
Cálcio/farmacologia , Corpo Lúteo/enzimologia , Diglicerídeos/farmacologia , Estradiol/farmacologia , Isoenzimas/metabolismo , Fosfatidilserinas/farmacologia , Proteína Quinase C/metabolismo , Proteínas Quinases/metabolismo , Pseudogravidez/enzimologia , Animais , Western Blotting , Gonadotropina Coriônica , Cromatografia , Cromatografia em Gel , Durapatita , Estradiol/administração & dosagem , Feminino , Hidroxiapatitas , Isoenzimas/isolamento & purificação , Cinética , Fosforilação , Protamina Quinase , Proteína Quinase C/isolamento & purificação , Proteínas Quinases/isolamento & purificação , Coelhos , Elastômeros de SiliconeRESUMO
Based upon recent reports that the rat testis exhibits mRNAs for cAMP-dependent protein kinase (A-kinase) regulatory (R) subunits RI alpha, RI beta, RII alpha, and RII beta, this study was designed to identify R proteins present in extracts of germ cell-rich testis from adult and Sertoli cell-enriched, germ cell-poor testis from 14-15-day-old rats. Following separation by DEAE-cellulose, R subunits were identified by Mr: (a) upon labeling with 8-N3[32P]cAMP and 32P in an RII phosphorylation reaction and; (b) by Western blot analysis using R-specific antibodies on one- and two-dimensional gel electrophoresis. Elution of R subunits as catalytic (C) subunit-free dimers or in association with C subunits to form holoenzyme was determined by their sedimentation characteristics on sucrose gradient centrifugation in conjunction with their cAMP-stimulated activation characteristics on Eadie-Scatchard analysis. Soluble extracts of testes, from both adult and 14-15 day-old rats, showed the presence of a prominent type I holoenzyme containing RI alpha subunits (47 kDa, peak 1), a minor type II holoenzyme, containing RII beta subunits (52 kDa, peak 2), and a second, more abundant, type II holoenzyme peak containing predominantly RII alpha and, to a lesser extent RII beta subunits (peak 3). The 53 kDa RI beta protein predicted by mRNA studies was only tentatively identified by Western blot analysis. Testes extracts of 14-15-day-old, but not adult, rats exhibited high levels of C subunit-free RI alpha, a result not predicted by mRNA studies. This latter result may be attributable to direct RI alpha regulation or to indirect RII beta regulation at a time during testis development prior to germ cell maturation.
Assuntos
Proteínas Quinases/metabolismo , Testículo/enzimologia , Animais , Western Blotting , Centrifugação com Gradiente de Concentração , Cromatografia DEAE-Celulose , Eletroforese em Gel Bidimensional , Masculino , Proteínas Quinases/química , Ratos , Ratos EndogâmicosRESUMO
The phosphinyl ester prodrug fosinopril, a new angiotensin converting enzyme (ACE) inhibitor, is fully hydrolysed after oral administration to the pharmacologically active diacid, fosinoprilat. This metabolite is cleared by both hepatic and renal routes, while most other ACE inhibitors are cleared exclusively by the kidney. In the present study, after administration of multiple fixed oral doses the accumulation of the active moieties of fosinopril, enalapril and lisinopril was compared in patients with renal insufficiency. 29 patients with creatinine clearances (CLCR) less than 30 ml/min received either fosinopril 10mg (n = 9), enalapril 2.5mg (n = 10) or lisinopril 5mg (n = 10) once daily for 10 days in a nonblind (open-label) parallel study. Pharmacokinetic parameters including area under the serum concentration-time curve (AUC), peak serum concentration (Cmax) and time to peak concentration (tmax), as well as renal function, blood pressure, and plasma renin activity (PRA) and aldosterone levels, were determined on the first and last days of the study. The percentage (+/- SEM) increases in AUC from day 1 to day 10 for fosinoprilat, enalaprilat and lisinopril were 26.8 +/- 9.9 (nonsignificant), 76.6 +/- 16.6 (p less than 0.001) and 161.7 +/- 31.8% (p less than 0.001), respectively. These results indicate that there was significantly less accumulation of fosinoprilat, based on accumulation indices, relative to either enalaprilat (p less than 0.05) or lisinopril (p less than 0.001) during the study. The Cmax of fosinopril increased significantly less than that of lisinopril (21.1 vs 123.6%; p less than 0.01). Renal function was not altered in any group, and blood pressure changed modestly.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Inibidores da Enzima Conversora de Angiotensina/farmacocinética , Enalapril/análogos & derivados , Enalapril/farmacocinética , Falência Renal Crônica/metabolismo , Prolina/análogos & derivados , Idoso , Inibidores da Enzima Conversora de Angiotensina/sangue , Enalapril/sangue , Feminino , Fosinopril , Humanos , Lisinopril , Masculino , Pessoa de Meia-Idade , Prolina/sangue , Prolina/farmacocinéticaRESUMO
Regulatory (R) subunits and their association with catalytic subunits to form cAMP-dependent protein kinase holoenzymes were investigated in corpora lutea of pregnant rats. Following separation by DEAE-cellulose chromatography, R subunits were identified by labeling with 8-N3[32P]cAMP and autophosphorylation on one and two-dimensional gel electrophoresis and by reactivity with antisera. DEAE-cellulose elution of R subunits with catalytic subunits as holoenzymes or without catalytic subunits was determined by sedimentation characteristics on sucrose density gradient centrifugation and by cAMP-stimulated kinase activation characteristics on Eadie-Scatchard analysis. We identified the presence of a type I holoenzyme containing RI alpha (Mr 47,000) subunits, a prominent type II holoenzyme containing RII beta (Mr 52,000) subunits, and a second more acidic type II holoenzyme peak containing both RII beta and RII alpha (Mr 54,000) subunits. However, the majority of total R subunit activity was associated with a catalytic subunit-free peak of RI alpha protein which on elution from DEAE-cellulose was associated with cAMP. This report establishes the more basic elution position from DEAE-cellulose of the prominent rat luteal RII beta holoenzyme in very close proximity to free RI alpha and presents one of the few reports of a normal tissue containing a large percentage of catalytic subunit-free RI alpha.
Assuntos
Corpo Lúteo/enzimologia , Isoenzimas/metabolismo , Proteínas Quinases/metabolismo , Animais , Centrifugação com Gradiente de Concentração , Cromatografia DEAE-Celulose , AMP Cíclico/metabolismo , Eletroforese em Gel de Poliacrilamida , Feminino , Isoenzimas/isolamento & purificação , Cinética , Substâncias Macromoleculares , Peso Molecular , Gravidez , Proteínas Quinases/isolamento & purificação , Ratos , Ratos EndogâmicosRESUMO
This study compares pharmacokinetic parameters and colonic tissue concentrations of cyclosporine administered by olive-oil or water-retention enemas with conventional intravenous (i.v.) and oral dosing. Five medical students were enrolled in a prospective crossover study. All subjects received a single dose of cyclosporine on four separate occasions, once orally, once as an olive-oil enema, once as a water enema, and once i.v. Cyclosporine concentration was measured in blood and in colonic tissue obtained by flexible sigmoidoscopy. Bioavailability was 18 +/- 7% (mean +/- SD) for the oral dose and was unmeasurable for the oil and water enemas. The concentration of cyclosporine in colon tissue was 32,443 +/- 17,251 ng/g (mean +/- SD) for the i.v. dose, 2797 +/- 1812 ng/g for the oral dose, 21,727 +/- 14,090 ng/g for the oil enema, and 25,318 +/- 30,408 ng/g for the water enema. The authors conclude that the bioavailability of cyclosporine, and thus the systemic absorption after administration by a retention enema, is negligible. The colonic tissue concentration of cyclosporine after i.v. or rectal administration via an enema is tenfold higher than that for oral dosing. These findings suggest that cyclosporine-retention enemas produce high distal colonic tissue concentrations with negligible systemic absorption after a single dose in healthy subjects and should be evaluated as treatment for patients with left-sided colitis. Because cyclosporine administered by the i.v. route provided sharply higher colonic tissue concentrations than those seen with oral therapy, pulse i.v. cyclosporine should be tried for patients with severe ileitis and colitis.
Assuntos
Ciclosporinas/farmacocinética , Administração Oral , Administração Retal , Colo/metabolismo , Ciclosporinas/administração & dosagem , Ciclosporinas/sangue , Feminino , Humanos , Injeções Intravenosas , Masculino , Músculo Liso/metabolismoRESUMO
Soluble ovarian extracts were incubated with protein kinase effectors in the presence of [gamma 32P]ATP and proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Autoradiograms revealed phosphorylation of an ovarian Mr = 80,000 substrate in the presence of EGTA ([ethylenebis(oxyethylenenitrilo)]tetraacetic acid), phosphatidylserine and 1,2-diolein. In contrast to a classical response pattern to C-kinase effectors, the ovarian Mr = 80,000 phosphorylation was inhibited by 2 x 10(-7) M or greater free Ca2+. The ovarian Mr = 80,000 substrate was distinguished from the myristoylated acidic Mr = 80,000 C-kinase substrate of brain tissue on the basis of heat stability and phosphorylative response to effectors. Phosphorylation of the exogenous substrate myelin basic protein by DEAE-resolved ovarian kinase showed the variant effector dependence, maximal in the presence of EGTA, phosphatidylserine and 1,2-diolein. Finally, the effect of Ca2+ on ovarian Mr = 80,000 [32P]phosphate content could not be accounted for by post-phosphorylation activities, or by DEAE-resolvable or hydroxylapatite-resolvable inhibitory activities.
Assuntos
Ovário/metabolismo , Fosfoproteínas/metabolismo , Proteínas Quinases/metabolismo , Animais , Encéfalo/enzimologia , Encéfalo/metabolismo , Cálcio/fisiologia , Diglicerídeos/fisiologia , Feminino , Peso Molecular , Proteína Básica da Mielina/metabolismo , Ovário/enzimologia , Fosfolipídeos/fisiologia , Proteínas Quinases/classificação , Proteínas Quinases/isolamento & purificação , Ratos , Ratos Endogâmicos , Especificidade por SubstratoRESUMO
To examine the renal effects of ibopamine HCl we evaluated 15 patients with New York Heart Association Class II-III congestive heart failure and mild renal insufficiency (creatinine clearance [CLcr] = 45-85 ml min-1). Diuretics and vasodilators were withheld and a sodium (Na+)-restricted diet was initiated. All patients exhibited positive Na+ balance at the time of evaluation. Hourly urine volumes, urine chemistries, serum chemistries, PAH and inulin/iothalamate clearances were determined 2 h pre and 4 h post a single 200 mg oral dose of ibopamine. Effective renal plasma flow, creatinine clearance, filtration fraction, and the fractional excretion of sodium and potassium were not significantly altered postdose. A significant increase in urine output and decrease in urine osmolality were seen at all time points postdose. A significant reduction in serum potassium (2 and 3 h) and blood urea nitrogen (1, 3 and 4 h) concentrations occurred. Measurements of glomerular filtration rate by inulin or [125I]-iothalamate produced differing results in the patient groups studied. We conclude that a single dose of ibopamine does not produce significant improvements in renal function in patients with congestive heart failure, mild renal insufficiency and positive sodium balance.