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1.
Horm Metab Res ; 40(12): 848-53, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18810711

RESUMO

Evidence indicates that dietary lipids influence adrenocortical function. In the present study, weanling rats were fed isocaloric synthetic diets for 6 and 12 months that contained 10% of one of the selected fatty acids as the predominant lipid: butter fat (high saturated, low polyunsaturated fat); olive oil (monounsaturated); corn oil (polyunsaturated); omega-3 ethyl ester mixture (long-chain polyunsaturates); elevated eicosapentaenoic acid; elevated docosahexaenoic acid. Adrenocortical cells derived from individual rats were evaluated for corticosterone and aldosterone responses to adrenocorticotropic hormone (ACTH). All comparisons were to the butter fat diet. Adrenocortical cell sensitivity to ACTH was not affected by the diets. However, there were differences in basal and maximal ACTH-induced corticosteroid production. Compared to the butter fat diet, the other diets variably decreased cellular corticosteroid production. Corticosterone and aldosterone production were affected similarly. The greatest decrease was most often seen with the omega-3 mixture diet (about -67%). At 6 months, the docosahexaenoic acid-elevated diet had selective suppressive actions on adrenocortical function whereas at 12 months, both docosahexaenoic and eicosahexaenoic acid-elevated diets had similar suppressive efficacies. The data indicate that a diet rich in high saturated, low polyunsaturated fat augments adrenocortical function and increasing the representation of long-chain unsaturated fatty acids suppresses adrenocortical function.


Assuntos
Córtex Suprarrenal/citologia , Córtex Suprarrenal/efeitos dos fármacos , Gorduras na Dieta/farmacologia , Testes de Função do Córtex Suprarrenal , Hormônio Adrenocorticotrópico/sangue , Animais , Dieta , Ácidos Docosa-Hexaenoicos/sangue , Ácido Eicosapentaenoico/sangue , Ácidos Graxos/farmacologia , Masculino , Ratos , Ratos Sprague-Dawley
2.
J Paediatr Child Health ; 36(6): 569-73, 2000 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-11115033

RESUMO

OBJECTIVE: Postnatal investigation of mild degrees of fetal hydronephrosis has allowed subsequent detection of infants with vesicoureteric reflux (VUR). This study was designed to provide short to medium term information on such infants who had primary VUR, the rates of renal damage and progression over time, the risk factors for such damage and to compare the characteristics of those who had mild dilatation of the fetal renal pelvis (4-9 mm) with those who had moderate-severe dilatation (> or = 10 mm). METHODOLOGY: Since June 1989, infants whose antenatal sonography had identified a fetal renal pelvis with an anteroposterior diameter of > 4 mm were investigated postnatally with renal ultrasonography and micturating cystourethrogram (MCU), and placed on antimicrobial prophylaxis. Those with VUR received 99mTc-dimercaptosuccinic acid (DMSA) scintigraphy. Infants were followed until discharge based on resolution of VUR, surgery, or low grade VUR. A 5.5 year cohort between June 1989 and December 1994 formed the study population. A review of notes and clinical review (if still under follow up) was undertaken. Vesicoureteric reflux on MCU was regraded according to the International Classification, and reflux nephropathy on DMSA scans was regraded according to criteria proposed by Goldraich. Regression analysis was used to assess risk factors for renal damage. RESULTS: There were 69 infants (37 girls, 32 boys) who were identified with primary VUR, with 37/69 having bilateral reflux. Eight had a urinary tract infection during the follow-up period. There was a broad distribution of grades of reflux detected (Grades I-3, Grades II-23, Grades III-19, Grades IV - 17, Grades V-7). 99m-Tc-dimercaptosuccinic acid scans on 57/69 (83%) demonstrated renal damage in eight infants (14%). This was predominantly global contraction of function. No progression of renal damage was seen over 2-7 years. Regression analysis showed a strong association between Grades IV, V reflux and the presence of renal damage (P < 0.001). Review of the degrees of fetal renal pelvic dilatation showed that 60/69 infants were detected because of mild (4-9 mm) dilatation. The majority (43/60) had lower grades of reflux (Grades I, II, 3), but there was no obvious cut-off between 4 and 9 mm that could predict high grade VUR (Grades IV, V). CONCLUSIONS: The use of 4 mm to define an abnormal fetal renal pelvis allows a much larger group of infants with high grade primary VUR to be detected than if a higher cut-off measurement is used. Although it also detects many more infants with low grade primary VUR, there is no obvious cut-off point at which this effect predominates. Progressive renal damage was not seen in follow up of up to 7 years of age. Renal damage on DMSA scanning in this group is almost exclusively a pattern of global contraction of function. The presence of high-grade VUR appears to be the only important factor in predicting the presence of renal damage.


Assuntos
Pelve Renal/diagnóstico por imagem , Ultrassonografia Pré-Natal , Refluxo Vesicoureteral , Criança , Pré-Escolar , Feminino , Seguimentos , Humanos , Lactente , Recém-Nascido , Nefropatias/diagnóstico , Nefropatias/diagnóstico por imagem , Nefropatias/etiologia , Masculino , Gravidez , Cintilografia , Compostos Radiofarmacêuticos , Ácido Dimercaptossuccínico Tecnécio Tc 99m , Refluxo Vesicoureteral/complicações , Refluxo Vesicoureteral/congênito , Refluxo Vesicoureteral/diagnóstico , Refluxo Vesicoureteral/diagnóstico por imagem
3.
Gen Comp Endocrinol ; 113(2): 255-66, 1999 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-10082628

RESUMO

Previous work with growing chickens (Gallus gallus domesticus) indicates that transient dietary protein restriction induces long-term enhancement of adrenal steroidogenic function in response to adrenocorticotropin (ACTH). The present study investigated two possible cellular functions mediating this enhanced response: (a) ACTH signal transduction and dissemination and (b) short-loop feedback inhibition of ACTH-induced corticosterone production by exogenous corticosterone. Cockerels (2 weeks old) were fed isocaloric synthetic diets containing either 20% (control) or 8% (restriction) soy protein for 4 weeks. Adrenal glands were processed for the isolation of adrenal steroidogenic cells nearly devoid of chromaffin cells ( approximately 90% adrenal steroidogenic cells). Results of experiments to assess signal transduction and dissemination indicated that protein restriction selectively enhanced ACTH-induced corticosterone production mediated by the cyclic AMP (cAMP)-dependent pathway. In addition, protein restriction substantially counteracted exogenous corticosterone-dependent inhibition of acute ACTH-induced corticosterone production (by 40.7% vs control). The proximal portion of the cAMP pathway seemed most affected by this stressor. Protein-restricted cells exhibited enhanced homologous sensitization to ACTH (136% greater than that of control cells) which appeared to be localized at a step(s) prior to or at the formation to cAMP. Also, maximal ACTH-induced cAMP production and sensitivity to ACTH in terms of cAMP production by protein-restricted cells were, respectively, 2.2 and 15.8 times those of control cells. However, variable results were obtained from other experiments designed to pinpoint the altered early steps in ACTH-transmembranous signaling. For example, with intact cells, cAMP responses to cholera toxin (CT) and forskolin (FSK) did not corroborate the results suggesting an augmentation of ACTH-signal transduction induced by protein restriction. Furthermore, basal and stimulatable (by ACTH, CT, FSK, and NaF) adenylyl cyclase activities from membranes from protein-restricted cells were, respectively, 47.2 and 40.2% less than those from control cells (normalized to 10(7) cell equivalents of crude membranes). Collectively, these findings suggest that protein restriction stress potentiates ACTH-induced corticosterone secretion by chicken adrenal steroidogenic cells in at least two ways: (1) on the proximal end, by modulating unknown factors which enhance cellular sensitivity to ACTH, ACTH receptor-adenylyl cyclase coupling, and adenylyl cyclase activity, and (2) on the distal end, by suppressing end-product corticosterone negative feedback, thus facilitating an increase in net corticosterone secretion.


Assuntos
Glândulas Suprarrenais/metabolismo , Hormônio Adrenocorticotrópico/farmacologia , Galinhas/fisiologia , Corticosterona/metabolismo , Dieta com Restrição de Proteínas/veterinária , Transdução de Sinais/fisiologia , Adenilil Ciclases/análise , Adjuvantes Imunológicos/farmacologia , Glândulas Suprarrenais/efeitos dos fármacos , Glândulas Suprarrenais/fisiologia , Animais , Galinhas/metabolismo , Toxina da Cólera/farmacologia , Cromatografia por Troca Iônica/veterinária , Colforsina/farmacologia , Corticosterona/análise , AMP Cíclico/análise , AMP Cíclico/metabolismo , Diglicerídeos/farmacologia , Inibidores Enzimáticos/farmacologia , Retroalimentação/fisiologia , Masculino , Radioimunoensaio/veterinária , Contagem de Cintilação/veterinária
4.
Gen Comp Endocrinol ; 109(1): 140-53, 1998 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-9446731

RESUMO

In the present study, we investigated the influence of dietary protein restriction stress on adrenal steroidogenic function of the domestic turkey. Immature male turkeys (2 weeks old) were fed isocaloric synthetic diets containing either 28% (control) or 8% (restriction) soy protein for 4 weeks. Trunk plasma was processed for the determination of adrenocorticotropin (ACTH), corticosterone, aldosterone, and total 3, 5, 3'-triiodothyronine (T3). In addition, adrenal glands were processed for the isolation of defined, density-separable, adrenal steroidogenic cell subpopulations: three low-density adrenal steroidogenic cell subpopulations [LDAC-1 (rho = 1.0350-1.0490 g/ml). LDAC-2 (rho = 1.0490-1.0570 g/ml), and LDAC 3 (rho = 1.0370-1.0585 g/ml)] and a high-density subpopulation [HDAC (rho = 1.0590-1.0720 g/ml)], and the steroidogenic function of these cell subpopulations was evaluated. Protein restriction did not influence plasma ACTH However, it increased relative adrenal weight (mg/100 g body wt) (+37.8%) and plasma corticosterone (+317%). By contrast, it depressed plasma aldosterone (-51.2%). In addition, it caused a modest depression in plasma T3 (-25.9%). At the cellular level, protein restriction induced panhypofunction. Basal corticosteroid (aldosterone and corticosterone) production values of LDAC-1, -2, and -3 and HDAC from protein-restricted birds were, respectively, 42.9, 47.9, 30.8, and 57.5% less than those of corresponding cell subpopulations from control birds. In addition, maximal corticosteroid production values of LDAC-1, -2, and -3 and HDAC from protein-restricted birds, in response to ACTH, angiotensin II (AngII), and 25-hydroxycholesterol support, were depressed by 56.8, 55.1, 22.7, and 42.9%, respectively. Interestingly, LDAC-3 was relatively refractory to the influence of this stressor. By contrast, there was the lack of a concentration-dependent aldosterone response of LDAC-1 and -2 to AngII with protein restriction. This was not due to a failure in cell function since aldosterone responses of these cell subpopulations to ACTH and to 25-hydroxycholesterol support were apparent. In addition, the concentration of AngII receptors of cell subpopulations from protein-restricted turkeys, if anything, was greater than that of cell subpopulations from control turkeys. Protein restriction also altered the cell subpopulation composition of the adrenal gland: compared to control, it decreased the proportion of LDAC-2 by 42.3% and increased the proportion of LDAC-3 and HDAC by 68.7 and 302%, respectively. Thus, dietary protein restriction induces adrenal steroidogenic hypofunction in turkeys. In addition, the present study suggests that this nutritional stressor induces marked remodeling of the steroidogenic tissue in the turkey adrenal gland.


Assuntos
Glândulas Suprarrenais/metabolismo , Aldosterona/biossíntese , Corticosterona/biossíntese , Dieta com Restrição de Proteínas/efeitos adversos , Doenças das Aves Domésticas/fisiopatologia , Estresse Fisiológico/veterinária , Perus/metabolismo , Glândulas Suprarrenais/citologia , Hormônio Adrenocorticotrópico/farmacologia , Angiotensina II/farmacologia , Animais , Centrifugação com Gradiente de Concentração , Estudos de Coortes , Relação Dose-Resposta a Droga , Hidroxicolesteróis/farmacologia , Masculino , Concentração Osmolar , Doenças das Aves Domésticas/etiologia , Doenças das Aves Domésticas/metabolismo , Receptores de Angiotensina/metabolismo , Estresse Fisiológico/metabolismo , Estresse Fisiológico/fisiopatologia , Perus/crescimento & desenvolvimento
5.
Pharmacol Toxicol ; 80(1): 24-9, 1997 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-9148278

RESUMO

In order to examine the role of cytoskeleton in modulating the cell surface receptors, AtT-20 cells (stably expressing thyrotropin-releasing hormone receptors) were incubated with drugs that are known to modify the tubulin-microtubule system. The binding of [3H]methyl thyrotropin-releasing hormone ([3H]mTRH) to intact cells increased as a function of time, and was linear from 1.25 x 10(6) to 6.25 x 10(6) cells/ml. Cells incubated with colchicine, vinblastine, and taxol for 16 hr were harvested and the cell concentration was determined using a haemocytometer. Because the drugs inhibited the cell proliferation at 100 nM, it was decided to examine the effect of 100 nM of each of the three drugs on the ability of [3H]mTRH to bind cell surface receptors. Cells were incubated with the drugs for 16 hr at 37 degrees. After the incubation, cells (5 x 10(6) cells/ml) from each group were assayed for [3H]mTRH binding. Colchicine, vinblastine, and taxol stimulated [3H]mTRH binding by up to 27, 27, and 21%, respectively, without altering the Ka of the ligand to the receptor. These results suggest that perturbation of cytosolic microtubules leads to a reorganization of the spatial location of hormone receptors.


Assuntos
Neoplasias Hipofisárias/metabolismo , Receptores do Hormônio Liberador da Tireotropina/metabolismo , Tubulina (Proteína)/biossíntese , Animais , Antineoplásicos Fitogênicos/farmacologia , Contagem de Células , Divisão Celular/efeitos dos fármacos , Colchicina/farmacologia , Cinética , Camundongos , Paclitaxel/farmacologia , Receptores do Hormônio Liberador da Tireotropina/genética , Hormônio Liberador de Tireotropina/metabolismo , Transfecção , Células Tumorais Cultivadas , Vimblastina/farmacologia
6.
Pharmacol Toxicol ; 81(6): 294-9, 1997 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-9444672

RESUMO

To understand the mechanism of ethanol action on G protein-mediated signal transduction pathway, the effect of ethanol on muscarinic receptor-G protein coupling in the rat cerebral cortex was examined. Acetylcholine (ACh)-stimulated G protein GTPase activity was used as an index of receptor-G protein coupling. ACh stimulation of G protein GTPase activity was time- and concentration-dependent, and atropine-sensitive. Rats injected with ethanol (3 g/kg body weight) were sacrificed after 4 hr, and the cerebral cortices removed. The ability of ACh to stimulate GTPase activity was similar in cortical cell membranes obtained from control and ethanol-treated rats; ACh maximally stimulated the enzymatic activity by 22% in membranes from both groups of rats. Next, in cortical cell membranes obtained from control rats (i.e., not injected with ethanol) the ability of ACh to stimulate GTPase activity in the presence of ethanol was examined. ACh stimulated GTPase activity in a concentration-dependent manner; the activity was 12.3 +/- 0.1, 14.5 +/- 0.64, 15.7 +/- 0.54, and 16.1 +/- 0.33 Pi pmol/min/mg protein, at 0, 0.01, 0.1, and 1 mM ACh, respectively (P < 0.05). In the presence of 100 mM ethanol ACh-stimulated GTPase activity was significantly inhibited. The IC50 value of ethanol inhibition of ACh-stimulated GTPase activity was approximately 50 mM. These results suggest that: 1) in vitro, ethanol impairs ACh-stimulated G protein GTPase activity in the rat cortical cell membranes, and 2) in vivo, the acute effects of alcohol on G protein function may be transient and reversible.


Assuntos
Depressores do Sistema Nervoso Central/toxicidade , Córtex Cerebral/efeitos dos fármacos , Etanol/toxicidade , Proteínas de Ligação ao GTP/metabolismo , Receptores Muscarínicos/efeitos dos fármacos , Acetilcolina/farmacologia , Animais , Sítios de Ligação , Membrana Celular/efeitos dos fármacos , Córtex Cerebral/metabolismo , Relação Dose-Resposta a Droga , GTP Fosfo-Hidrolases/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley , Receptores Muscarínicos/metabolismo
7.
Proc Soc Exp Biol Med ; 210(2): 180-90, 1995 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-7568289

RESUMO

Environmental lead (Pb2+) contributes a small but significant risk to human hypertension. It is postulated that the hypertensinogenic action of Pb2+ may be due, in part, to its direct action on vascular smooth muscle cells. To investigate this hypothesis, freshly isolated rat aortic smooth muscle (RASM) cells were propagated in defined media containing one of two Centers for Disease Control-based concentrations of Pb2+ (as lead citrate): 100 and 500 micrograms Pb2+/l (i.e., equivalent to 5.5 and 27.5 micrograms Pb2+/dl blood; designated 100-RASM and 500-RASM). Control (CON-RASM) cells received sodium citrate. 500-RASM cells exhibited suppressed propagation and fell out of propagation synchrony with CON-RASM cells: when CON-RASM cell approached confluence (approximately 90%), 500-RASM cell density was 6.4% that of CON-RASM cell density. By contrast, 100-RASM cells exhibited marked hyperplasia albeit this was not apparent until passage 3 (p3). Overall, when p3-p6 CON-RASM cells approached confluence, 100-RASM cell density was 107.6% greater than CON-RASM cell density. The protein content of CON-RASM and 100-RASM was not different, whereas that of 500-RASM cells was 29% greater than that of CON-RASM and 100-RASM cells. Phase-contrast microscopy revealed that 100 micrograms Pb2+/l converted normal spindle-shaped/ribbon-shaped RASM cells into less spread, cobblestone-shaped, neointimal-like cells. Immunocytochemical analysis revealed that 100-RASM cells lacked or had markedly fewer actin cables, characteristic of rapidly dividing cells. In addition, Pb(2+)-treated RASM cells exhibited altered membrane fatty acyl composition with a trend towards an increase (by as much as 50%) in membrane arachidonic acid. Interestingly, hyperplastic 100-RASM cells exhibited a 70.6% reduction in angiotensin II (Ang II) receptor concentration whereas the concentrations of alpha 1- and beta-adrenergic and atrial natriuretic peptide (ANP) receptors were not affected. In addition, in experiments designed to control for Pb(2+)-associated differences in RASM cell propagation, there was a concentration-dependent decrease in Ang II receptor concentration: for 100 and 500 micrograms Pb2+/l, Ang II receptor concentration was decreased 39.6% and 65.5%, respectively. Thus, although Pb2+, depending on its concentration, had contrasting effects on RASM cell propagation, it had a consistent, concentration-dependent inhibitory effect on Ang II receptor concentration. Recovery (r) from Pb2+ required at least two additional passages. At p71r the enhanced propagation (+54%) and reduced Ang II receptor concentration (-49%) of 100r-RASM cells persisted.(ABSTRACT TRUNCATED AT 400 WORDS)


Assuntos
Angiotensina II/metabolismo , Aorta/metabolismo , Chumbo/farmacologia , Músculo Liso Vascular/metabolismo , Receptores de Angiotensina/metabolismo , Actinas/metabolismo , Animais , Aorta/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Poluição Ambiental , Imunofluorescência , Humanos , Hipertensão/epidemiologia , Cinética , Masculino , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Receptores Adrenérgicos alfa 1/efeitos dos fármacos , Receptores Adrenérgicos alfa 1/metabolismo , Receptores Adrenérgicos beta/efeitos dos fármacos , Receptores Adrenérgicos beta/metabolismo , Receptores de Angiotensina/efeitos dos fármacos , Receptores do Fator Natriurético Atrial/efeitos dos fármacos , Receptores do Fator Natriurético Atrial/metabolismo , Valores de Referência , Fatores de Risco
8.
Gen Comp Endocrinol ; 99(3): 364-72, 1995 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-8536948

RESUMO

The inhibitory action of atrial natriuretic peptides (ANPs) on mammalian aldosterone synthesis is well documented. In addition, other work indicates that ANP and an analogue of its second messenger, 8-Br-cGMP, inhibit aldosterone production by chicken adrenal steroidogenic cells. However, the interaction between angiotensin II (AII) and ANP in the regulation of avian aldosterone production is poorly understood because chicken adrenal steroidogenic cells, the commonly used in vitro avian model, are comparatively unresponsive to AII. By contrast, turkey (Meleagris gallopavo) adrenal steroidogenic cells are sensitive to AII. Thus, in the present study, the action of ANPs and related peptides and their interaction with other stimulators of aldosterone production were investigated using freshly isolated and briefly cultured turkey adrenal steroidogenic cells. Surprisingly, several ANPs [rat (r), human (h), chicken (c)], and rat brain natriuretic peptide (rBNP) were as efficacious as [Ile5]AII for stimulating aldosterone production (2 hr) in freshly isolated cell suspensions but were less potent than [Ile5]AII (ED50 of ANPs approximately 5-10 nM; [Ile5]AII ED50 approximately 0.1 nM). In addition, chicken ANP enhanced maximal aldosterone production induced by [Ile5]AII (1 nM), K+ (25 mM), and hACTH-(1-39) (ACTH) (1 nM): maximal enhancement of the action of these secretagogues was +49%, +137% and +15%, respectively (P < 0.05; n = 3). Furthermore, other ANPs and related peptides [rBNP and bovine aldosterone secretion inhibiting factor (bASIF)] enhanced maximal [Ile5]AII-induced aldosterone production: the order of maximal enhancement was rBNP (+180%) > hANP/rANP (+50%) > bASIF (+25%) (P < 0.05; n = 3).(ABSTRACT TRUNCATED AT 250 WORDS)


Assuntos
Glândulas Suprarrenais/efeitos dos fármacos , Aldosterona/biossíntese , Fator Natriurético Atrial/farmacologia , Perus/metabolismo , Glândulas Suprarrenais/metabolismo , Animais , Bovinos , Galinhas , GMP Cíclico/biossíntese , Estudos de Avaliação como Assunto , Humanos , Masculino , Ratos , Receptores do Fator Natriurético Atrial/efeitos dos fármacos , Estimulação Química
9.
Endocrinology ; 136(4): 1626-34, 1995 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-7895673

RESUMO

In mammalian zona glomerulosa cells, angiotensin II (AII)-induced increases in intracellular Ca2+ ([Ca2+]i) and AII-induced aldosterone production seem to be inextricably linked. However, in avian adrenal steroidogenic (adrenocortical) cells studied thus far, inducible aldosterone production seems to be insensitive to alterations in the mobilization of cellular Ca2+. This raises the hypothesis that alternative signal transduction pathways are implemented to induce aldosterone production in avian adrenocortical cells. In the present study, this hypothesis was investigated by using isolated turkey (Meleagris gallopavo) adrenocortical cells that are known to be three times more sensitive to AII than to ACTH for aldosterone production. In isolated turkey adrenocortical cells, the mammalian AII receptor antagonist, [Sar1,Ile8]AII, was as efficacious as [Ile5]AII in stimulating aldosterone production, albeit it had about 1/150 the potency of [Ile5]AII. The actions of both analogs required extracellular K+, suggesting a voltage-sensitive event. However, a maximal aldosteronogenic concentration of [Sar1,Ile8]AII not only failed to increase [Ca2+]i but also completely blocked maximal (10(-8) M)[Ile5]AII-induced increases in [Ca2+]i when added before [Ile5]AII and partially dampened (approximately 50%) maximal [Ile5]AII-induced increases in [Ca2+]i when added after (3 min) [Ile5]AII. This blockade in [Ca2+]i elevation was surmounted by high concentrations of [Ile5]AII (> 10(-6) M). By contrast, [Sar1,Ile8]AII did not alter maximal aldosterone production induced by [Ile5]AII and vice versa, thus suggesting that the action of both analogs converged on the same aldosteronogenic pathway, and that AII-induced aldosterone production was not coupled to elevations in [Ca2+]i. Detailed homologous-heterologous ligand-binding analyses supported the presence of two AII-binding sites that were discriminated by [Sar1,Ile8]AII (dissociation constants, 4.2 +/- 1.4 and 21.9 +/- 2.2 nM; concentration distribution, approximately 40% and approximately 60%, respectively; mean +/- SE, n = 4) but not by [Ile5]AII (dissociation constant, 2.1 +/- 0.1 nM for both sites). In addition, [Sar1,Ile8]AII- and [Ile5]AII-binding sites exhibited different physicochemical and pharmacological properties. The sensitivity of [Sar1,Ile8]AII-binding sites was about twice that of [Ile5]AII-binding sites to dithiothreitol. In addition, whereas both the high- and low-affinity sites detected by [Sar1,Ile8] AII exhibited equivalent competitive sensitivities to the type-1 receptor, the nonpeptidic antagonist, losartan (DuP 753), the sensitivity of the low-affinity site was 2.7 times that of the high-affinity site to the type-2 receptor, nonpeptidic antagonist, PD123319. Taken collectively, the data suggest that in turkey adrenocortical cells, elevations in [Ca2+]i and aldosterone production are dissociable events regulated by distinct AII receptor subtypes or isomorphs.


Assuntos
Córtex Suprarrenal/efeitos dos fármacos , Aldosterona/biossíntese , Angiotensina II/farmacologia , Cálcio/metabolismo , Receptores de Angiotensina/fisiologia , Perus , 1-Sarcosina-8-Isoleucina Angiotensina II/metabolismo , 1-Sarcosina-8-Isoleucina Angiotensina II/farmacologia , Córtex Suprarrenal/metabolismo , Angiotensina II/metabolismo , Animais , Sítios de Ligação , Ligação Competitiva , Masculino , Zona Glomerulosa/efeitos dos fármacos , Zona Glomerulosa/metabolismo
10.
Gen Comp Endocrinol ; 98(1): 57-72, 1995 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-7781965

RESUMO

A body of histological and functional evidence supports the hypothesis that there are functionally distinct subpopulations of steroidogenic cells comprising the avian adrenal gland. In the present study, we tested this hypothesis by evaluating the steroidogenic responses of density-dependent subpopulations of adrenal steroidogenic cells isolated from domestic turkeys fed either a high-normal (control) sodium diet (0.4% Na+) or a Na(+)-restricted diet (0.04% Na+) for 8 days, the latter to stimulate the activity or appearance of possible zona glomerulosa-like cells. Subpopulations were visually yet reproducibly determined by their density-dependent separation on a continuous density gradient of Percoll (45%). The subpopulations were arbitrarily ascribed as being either low-density or high-density adrenal steroidogenic cells [LDAC (p = 1.0350-1.0585 g/ml) and HDAC (p = 1.0590-1.0720 g/ml), respectively]. LDAC and HDAC comprised 95.2 and 4.8%, respectively, of the total number of adrenal steroidogenic cells isolated. The LDAC was further subdivided into three visually distinct subpopulations. The functional differences between the LDAC subpopulations is discussed but was less dramatic than the functional distinction between the HDAC subpopulation and the pooled LDAC subpopulations. Basal aldosterone production values between control LDAC and HDAC were equivalent. In addition, there were no differences in maximal aldosterone production between control LDAC and HDAC in response to [Ile5]angiotensin II (AII), the avian equivalent, [Val5]AII, K+ (as KCl), and that supported by exogenous corticosterone. However, maximal aldosterone production in response to human ACTH-(1-39) (ACTH) of the LDAC was 32% greater than that of the HDAC. Na+ restriction enhanced basal aldosterone production of the LDAC by 84% over the control LDAC. In addition, it enhanced maximal aldosterone production of the LDAC in response to AII peptides, K+, ACTH and that supported by corticosterone by 54, 164, 83, and 74%, respectively, over that of the control LDAC. However, Na+ restriction disproportionately enhanced basal aldosterone production of the HDAC by 348% over that of the control HDAC. In addition, with Na+ restriction, maximal aldosterone production of the HDAC in response to AII, K+, and ACTH and that supported by exogenous corticosterone was consistently greater than that of the LDAC. Moreover, with Na+ restriction, maximal aldosterone production of the HDAC in response to AII peptides and K+ was increased over that of the control HDAC to a greater extent than was maximal aldosterone production in response to ACTH and that supported by corticosterone (% enhancement over control was as follows: AII peptides, 502%; K+, 668%; ACTH, 273%; corticosterone, 183%).(ABSTRACT TRUNCATED AT 400 WORDS)


Assuntos
Glândulas Suprarrenais/citologia , Esteroides/biossíntese , Perus , Glândulas Suprarrenais/efeitos dos fármacos , Glândulas Suprarrenais/metabolismo , Hormônio Adrenocorticotrópico/farmacologia , Aldosterona/biossíntese , Angiotensina II/farmacologia , Animais , Contagem de Células , Corticosterona/biossíntese , Corticosterona/farmacologia , Dieta Hipossódica , Masculino , Potássio/farmacologia
11.
Gen Comp Endocrinol ; 96(1): 108-21, 1994 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-7843558

RESUMO

The hormonal and cationic regulation of aldosterone production by freshly isolated turkey (Meleagris gallopavo) adrenal steroidogenic cells was investigated. Angiotensin II (AII), ACTH [human ACTH-(1-39)], and K+ stimulated aldosterone production in a concentration-dependent manner albeit these agents exhibited considerable differences in lag time for the significant stimulation of aldosterone production over basal production. By contrast, Ca2+ was without effect except at a high concentration (10 mM). Although ACTH was more efficacious than AII, it had about one-third the potency of AII for stimulating aldosterone production. However, ACTH potentiated the maximal aldosterone response to AII [maximal enhancement (+499%) at 3 x 10(-10) M ACTH]. Extracellular K+ was an absolute requirement for AII-induced aldosterone production (threshold concentration = 3 mM), and maximal enhancement (+200%) occurred with 5 mM (a physiological concentration). Although extracellular Ca2+ was not an absolute requirement for inducible aldosterone production, it enhanced AII-induced aldosterone production in a concentration-dependent manner [maximal enhancement (+727%) at 3 mM], albeit it did not alter the half-maximal steroidogenic concentration (EC50) of AII. Ca2+ also enhanced maximal ACTH-induced aldosterone production but to a lesser extent (+96% with 1 mM Ca2+). However, Ca2+ dramatically enhanced ACTH potency (ED50) (nearly 100 times at 1 mM Ca2+). The acute augmentation of AII-induced aldosterone production by ACTH, K+, and Ca2+ was not accompanied by increases in the cellular concentration and affinity of AII receptors, suggesting that the agents acted at intracellular loci distal to the AII receptor. Several aspects of the present study with isolated turkey adrenal steroidogenic cells differ markedly from those of studies with isolated chicken (Gallus gallus domesticus) adrenal steroidogenic cells and mammalian zona glomerulosa cells, thus suggesting interclass and intraclass differences in homeothermic vertebrate adrenal steroidogenic regulation.


Assuntos
Glândulas Suprarrenais/metabolismo , Aldosterona/biossíntese , Perus/metabolismo , Glândulas Suprarrenais/efeitos dos fármacos , Hormônio Adrenocorticotrópico/farmacologia , Angiotensina II/farmacologia , Animais , Cálcio/farmacologia , Sinergismo Farmacológico , Cinética , Masculino , Potássio/farmacologia
12.
Gen Comp Endocrinol ; 96(1): 92-107, 1994 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-7843572

RESUMO

In the present study, the properties of angiotensin II (AII) receptors of intact domestic turkey adrenal steroidogenic cells were characterized. AII (but not ACTH) induced an immediate and sustained increase in intracellular Ca2+. In addition, dithiothreitol inhibition of maximal AII-induced aldosterone production was closely correlated with its inhibition of binding suggesting that these receptors are type 1-like and operate through a non-"spare" receptor mode. Equilibrium-binding analysis revealed a single class of binding sites at a concentration of 63,500 sites/cell and having an apparent dissociation constant (Kd) of 1.21 nM. However, the Kd derived from kinetic analyses, 0.27 nM, was lower. Both empirically determined and model-based calculated distributions of bound hormone indicated that at equilibrium, about 30% of hormone-receptor complexes were internalized whereas 70% remained on the surface. This distribution contrasts sharply with that reported for mammalian (rat) adrenocortical cells. In keeping with recent cloning studies, these avian AII receptors of intact adrenal steroidogenic cells discriminated angiotensins and mammalian peptidic and nonpeptidic antagonists differently from mammalian adrenocortical and duck adrenal receptor preparations. Importantly, turkey adrenal steroidogenic cell AII receptors poorly discriminated the nonpeptide antagonists, losartan (DuP 753) (type-1 specific) and PD123177 (type-2 specific). Thus, AII receptors of freshly isolated, intact turkey adrenal steroidogenic cells are pharmacologically distinct from mammalian adrenocortical type-1 receptors.


Assuntos
Glândulas Suprarrenais/metabolismo , Receptores de Angiotensina/metabolismo , Perus , Glândulas Suprarrenais/efeitos dos fármacos , Hormônio Adrenocorticotrópico/farmacologia , Aldosterona/biossíntese , Angiotensina II/análogos & derivados , Angiotensina II/metabolismo , Angiotensina II/farmacologia , Animais , Cálcio/metabolismo , Ditiotreitol/farmacologia , Radioisótopos do Iodo , Cinética , Masculino
13.
Biochem Biophys Res Commun ; 191(3): 1073-80, 1993 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-7916599

RESUMO

A turkey adrenocortical cell AII receptor cDNA fragment (714 bp) was isolated by RT-PCR using oligonucleotide primers based on rat aortic smooth muscle (RASM) and bovine adrenal type-1 (AT1) receptor cDNA coding sequences as primers. Sequence analysis indicated 73% nucleotide identity and 78% amino acid identity to the RASM AT1 receptor. Notable differences were 1) two additional Cys at positions 92 and 99 (first extracellular loop), 2) deletion of amino acid 168, formation of a triplet Asn sequence (Asn 186, 187, 188) and substitution of Arg192 with Pro (second extracellular loop) and 3) two additional potential protein kinase C phosphorylation sites, Thr221 and Thr233 (third intracellular loop). Southern blot analysis indicated that the receptor is a product of a single-copy gene. Northern blot analysis indicated at least three mRNA transcripts (7.5, 3.5 and 2.0 kb) expressed predominantly in the adrenal gland.


Assuntos
Glândulas Suprarrenais/metabolismo , Receptores de Angiotensina/genética , Sequência de Aminoácidos , Angiotensina II/metabolismo , Animais , Sequência de Bases , Clonagem Molecular , DNA/genética , Expressão Gênica , Genes , Dados de Sequência Molecular , RNA Mensageiro/genética , Receptores de Angiotensina/metabolismo , Mapeamento por Restrição , Perus
14.
Biol Reprod ; 47(1): 97-104, 1992 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-1637954

RESUMO

Previous work has suggested that rat luteal cells have two populations of LH/hCG receptors that are located in different parts of the cell membrane. The possibility that these two receptor pools may have functional differences has been investigated through examination of the binding and action of native and deglycosylated hCG to different membrane fractions. Ovaries from eCG/hCG-primed immature female rats were separated into 1,000 x g (heavy) and 20,000 x g (light) particulate fractions. Increasing concentrations of NaCl had a biphasic effect on the binding of native and deglycosylated hCG to both membrane fractions, causing an increase in binding at low concentrations and a decrease in binding at higher concentrations. The binding of deglycosylated hCG to both membrane preparations and the binding of native hCG to light-membrane preparations was maximal at approximately the same NaCl concentration (50-65 mM). This was higher than the concentration of NaCl necessary for maximal binding of native hCG to the heavy-membrane preparation. In addition, maximal native hCG binding to this preparation occurred over a broader NaCl concentration range (15-65 mM). Equilibrium binding experiments showed differences in hCG binding to both fractions. In light membranes there were significantly more receptor sites for deglycosylated hCG (11.2 +/- 4.8 fmol/mg ovary) than for native hCG (4.8 +/- 0.7 fmol/mg ovary), with no significant different in affinity. In contrast, in heavy membranes the affinity for deglycosylated hCG (6.30 +/- 0.19.10(9) M-1), was significantly higher than that for native hCG (2.60 +/- 0.13.10(9) M-1), with no significant differences in receptor number.(ABSTRACT TRUNCATED AT 400 WORDS)


Assuntos
Gonadotropina Coriônica/metabolismo , Corpo Lúteo/química , Receptores da Gonadotropina/análise , Animais , Fracionamento Celular , Membrana Celular/química , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Corpo Lúteo/citologia , Corpo Lúteo/ultraestrutura , Relação Dose-Resposta a Droga , Feminino , Glicosilação , Ligantes , Hormônio Luteinizante/metabolismo , Ratos , Receptores da Gonadotropina/metabolismo , Receptores da Gonadotropina/fisiologia , Receptores do LH/análise , Receptores do LH/metabolismo , Cloreto de Sódio/farmacologia
15.
Biochem Biophys Res Commun ; 171(2): 525-30, 1990 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-1698360

RESUMO

Molecular analysis of the induction of the lutropin/choriogonadotropin receptor during the process of luteinization of the rat ovary was performed. The appearance of receptor binding activity and an immunological analysis of the receptor using Triton solubilized membrane proteins show little receptor present in luteal tissue through day 3 subsequent to hCG treatment, with some in day 4, and a marked increase by day 5. A similar pattern was found in the analysis of RNA hybridizing to several probes derived from the published cDNA sequence suggesting that receptor induction occurs primarily at the level of transcription.


Assuntos
Corpo Lúteo/fisiologia , Ovário/metabolismo , Receptores do LH/genética , Animais , Gonadotropina Coriônica/metabolismo , Feminino , Hibridização de Ácido Nucleico , Sondas de Oligonucleotídeos , RNA/genética , RNA/isolamento & purificação , Ratos , Ratos Endogâmicos , Receptores do LH/biossíntese , Receptores do LH/metabolismo , Transcrição Gênica
16.
Biochem Cell Biol ; 66(12): 1258-64, 1988 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-3245903

RESUMO

The effect of various salts on the binding of human choriogonadotropin to rat luteal membranes has been examined. Increasing salt concentrations had biphasic effects, initially increasing binding, then decreasing it. With NaCl, these effects were on both the affinity and the number of receptor sites. The affinity increased with increasing NaCl concentrations, to a maximum at 40 mM, and then decreased. Above 40 mM NaCl, the number of binding sites increased. NaCl also altered the effects of Mg2+ and guanyl nucleotides. At low ionic strength, Mg2+ was necessary to observe binding. Guanine nucleotides modulated this binding by decreasing the affinity. At 40 mM NaCl, Mg2+ increased receptor number without altering affinity. Guanyl nucleotides modulated this binding by reducing the number of sites to that observed in the absence of Mg2+. At 150 mM NaCl, Mg2+ and guanine nucleotides had no effect. The results suggest the presence of two pools of human choriogonadotropin receptor in rat corpus luteum, one coupled to the guanine nucleotide regulatory protein (Ns) and being Mg2+ dependent and guanine nucleotide sensitive, and the other not coupled to Ns and being Mg2+ independent and guanine nucleotide insensitive.


Assuntos
Gonadotropina Coriônica/metabolismo , Animais , Corpo Lúteo/metabolismo , Feminino , Humanos , Magnésio/farmacologia , Potássio/farmacologia , Ratos , Sódio/farmacologia , Trometamina
17.
Biol Reprod ; 38(5): 1012-8, 1988 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-3408769

RESUMO

Guanyl nucleotides are known to play a dual role in the activation of the adenylate cyclase system of the rat corpus luteum, being required for human choriogonadotropin (hCG) stimulation of the enzyme and modulating hCG binding to some hormone receptors. Current models of adenylate cyclase activation require that guanyl nucleotide binding be enhanced by hormones, and we have examined this binding in rat luteal membrane preparations known to contain guanyl nucleotide-modulated hCG receptors. [3H] Guanylyl-imidodiphosphate (GMPPnP), a nonhydrolyzable analog of guanosine triphosphate (GTP), was used to investigate binding to urea-washed, heavy rat luteal membranes. Binding was found to be linear, with respect to the amount of membranes added, in the range of 2-10 mg wet wt. tissue equivalents, and equilibrium was reached after a 30-min incubation at 30 degrees C. Analysis of equilibrium binding experiments gave a Ka of 1.2.10(7) +/- 0.9.10(7) M-1, with 460 +/- 430 fmol binding sites per mg tissue in the absence of hormone, Kinetic experiments showed an association rate constant of 2.6.10(5) +/- 0.5.10(5) M-1 min-1 and a dissociation rate constant of 1.8.10(-2) +/- 0.9.10(-2) min-1. In the presence of hCG, the Ka was unchanged; however, the number of binding sites increased by 50-120%. Competitive binding assays utilizing other nucleotides revealed that a hierarchy of GMPPnP = GTP greater than guanosine diphosphate (GDP) greater than inosine triphosphate (ITP) in displacing labeled GMPPnP. A similar hierarchy was also found for hCG-stimulated adenylate cyclase activity (GMPPnP = GTP greater than ITP) and for modulation of hCG binding (GMPPnP greater than GTP greater than ITP).(ABSTRACT TRUNCATED AT 250 WORDS)


Assuntos
Gonadotropina Coriônica/metabolismo , Corpo Lúteo/metabolismo , Nucleotídeos de Guanina/metabolismo , Adenilil Ciclases/análise , Animais , Ligação Competitiva , Corpo Lúteo/enzimologia , Técnicas de Cultura , Feminino , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Guanilil Imidodifosfato/metabolismo , Inosina Trifosfato/metabolismo , Cinética , Ratos , Ratos Endogâmicos , Receptores da Gonadotropina/metabolismo
18.
Ann Biomed Eng ; 16(2): 201-14, 1988.
Artigo em Inglês | MEDLINE | ID: mdl-3382067

RESUMO

A technique is described for exact estimation of kernels in functional expansions for nonlinear systems. The technique operates by orthogonalizing over the data record and in so doing permits a wide variety of input excitation. In particular, the excitation is not limited to inputs that are white, Gaussian, or lengthy. Diagonal kernel values can be estimated, without modification, as accurately as off-diagonal values. Simulations are provided to demonstrate that the technique is more accurate than the Lee-Schetzen method with a white Gaussian input of limited duration, retaining its superiority when the system output is corrupted by noise.


Assuntos
Engenharia Biomédica , Modelos Teóricos
19.
Endocrinology ; 119(4): 1805-9, 1986 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-3019644

RESUMO

Guanyl nucleotides are known to have a dual effect on most hormone-sensitive adenylate cyclase systems, regulating activation of the adenylate cyclase enzyme and binding of hormone to receptor. In the ovary, guanyl nucleotides have been shown to be required for hCG stimulation of luteal adenylate cyclase, but no effect on binding has been observed. Evidence has been obtained which suggests that guanyl nucleotide regulation of hCG binding is masked in most luteal membrane preparations by the presence of a large excess of unregulated receptor. A highly purified urea-washed membrane fraction has been prepared. Binding of hCG to this preparation was decreased (approximately 45%) in the presence of 5'-guanylylimidodiphosphate (GMPPnP). The maximal effect of GMPPnP occurred at 10 microM, with the half-maximal effect at 0.2 microM. These levels compare favorably with the GTP concentrations required for hCG stimulation of luteal adenylate cyclase. Analysis of equilibrium binding experiments showed that GMPPnP acted by reducing the number of binding sites by 45%. However, kinetic experiments suggested that this effect was due to a significant decrease in the affinity of a fraction of the hCG-binding sites. Association of hCG and its receptor was unaffected by the presence of GMPPnP (100 microM), whereas the dissociation of 40-50% of bound hormone was significantly accelerated (30-fold) by its presence. The guanyl nucleotide effect required the presence of MG+2; other divalent cations (Ca+2, Mn+2, and Co+2 could not be substituted. The ratio of beta-adrenergic to hCG-binding sites in the urea-washed heavy membrane preparation was elevated, suggesting that a sizable fraction of uncoupled hCG receptor had been removed. The results show that hCG binding to its luteal receptor is modulated by guanyl nucleotide and suggest that the modulation only occurs in those receptors that are directly coupled to the adenylate cyclase enzyme.


Assuntos
Gonadotropina Coriônica/metabolismo , Corpo Lúteo/metabolismo , Nucleotídeos de Guanina/farmacologia , Animais , Membrana Celular/metabolismo , Feminino , Guanilil Imidodifosfato/farmacologia , Cinética , Magnésio/farmacologia , Cloreto de Magnésio , Ratos , Ratos Endogâmicos , Receptores Adrenérgicos beta/metabolismo , Receptores do LH/efeitos dos fármacos , Receptores do LH/metabolismo
20.
Arch Biochem Biophys ; 233(2): 652-60, 1984 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-6435529

RESUMO

The ADP-ribosylation of rat luteal membrane proteins has been investigated. In the presence of cholera toxin two membrane proteins, Mr 115,000 and 46,000, incorporated [32P]ADP-ribose from [32P]NAD+. The larger protein also incorporated [32P]ADP-ribose in the absence of cholera toxin. The smaller protein was identified as the guanine nucleotide regulatory protein of adenylate cyclase. To facilitate studies concerning this species, a simple and convenient method of measuring ADP-ribose incorporation into the protein was developed. Levels of the protein were found to be approximately equal to those of the hCG receptor and 7- to 10-fold higher than those of the beta-adrenergic receptor. Luteinization of rat ovaries by injection of hCG indicated that G/F concentrations increased approximately 2-fold over a 5- to 11-day period, and correlated significantly with increased beta-adrenergic receptors, and beta-adrenergic and NaF-stimulated adenylate cyclase, but not with hCG receptors or hCG-stimulated adenylate cyclase. The distribution of the G/F protein in purified luteal membrane preparations mimicked the distribution of adenylate cyclase activity. No evidence could be found for hormonally induced alterations in ADP-ribose incorporation into this protein under either ribosylation or adenylate cyclase conditions. The exact role of the protein in the activation of rat luteal adenylate cyclase has yet to be determined.


Assuntos
Adenilil Ciclases/metabolismo , Corpo Lúteo/enzimologia , Proteínas de Ligação ao GTP/metabolismo , Guanosina Trifosfato/fisiologia , Proteínas de Membrana/metabolismo , Adenosina Difosfato Ribose/metabolismo , Animais , Centrifugação com Gradiente de Concentração , Toxina da Cólera/farmacologia , Gonadotropina Coriônica/farmacologia , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática/efeitos dos fármacos , Feminino , Ratos
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