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1.
J Physiol Pharmacol ; 62(4): 461-8, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22100847

RESUMO

Endogenous opioid peptides are involved in the regulation of the HPA-axis function and stress response mechanism. However, there is a scarcity of data on opioid involvement in the regulation of the adrenocortical endocrine function. This study was performed to: 1) establish the expression of proenkephalin, POMC and prodynorphin genes in the porcine adrenal cortex and test in vitro the influence of ACTH, angiotensin II, CRH and epinephrine on this expression, and 2) determine the effects of opioid receptor agonists on basal and ACTH- or angiotensin II-affected secretion of cortisol, aldosterone and progesterone by the cultured adrenocortical cells. Our experiment has demonstrated the presence of mRNAs for opioid precursors in cells isolated from the adrenal cortex and the significant effects of ACTH and angiotensin II, but not CRH or epinephrine, on adrenocortical transcription of the analyzed genes. Angiotensin II reduced the expression of the POMC gene but stimulated that of prodynorphin. In turn, ACTH decreased the transcription of prodynorphin. The study has also demonstrated the effects of selective opioid receptor agonists - DPLPE (delta), FK33-824 (mu) and U50,488 (kappa) - on adrenal steroidogenesis in pigs. Basal secretion of cortisol was enhanced after the activation of mu or kappa receptors, whereas ACTH-stimulated cortisol output was increased only by the mu receptor agonist. Angiotensin II-treated cells significantly decreased aldosterone secretion in the presence of the kappa receptor agonist. The present results suggest that opioid peptides are synthesized in the porcine adrenal cortex, indicating their involvement in the regulation of adrenal steroidogenesis through autocrine and/or paracrine interactions.


Assuntos
Córtex Suprarrenal/efeitos dos fármacos , Expressão Gênica , Peptídeos Opioides/genética , Receptores Opioides/agonistas , Esteroides/biossíntese , (trans)-Isômero de 3,4-dicloro-N-metil-N-(2-(1-pirrolidinil)-ciclo-hexil)-benzenoacetamida/farmacologia , Córtex Suprarrenal/citologia , Córtex Suprarrenal/metabolismo , Aldosterona/biossíntese , Aldosterona/metabolismo , Animais , Técnicas de Cultura de Células , Células Cultivadas , D-Ala(2),MePhe(4),Met(0)-ol-encefalina/farmacologia , D-Penicilina (2,5)-Encefalina/farmacologia , Expressão Gênica/efeitos dos fármacos , Hidrocortisona/biossíntese , Hidrocortisona/metabolismo , Sistema Hipotálamo-Hipofisário/efeitos dos fármacos , Sistema Hipotálamo-Hipofisário/metabolismo , Sistema Hipófise-Suprarrenal/efeitos dos fármacos , Sistema Hipófise-Suprarrenal/metabolismo , Progesterona/biossíntese , Progesterona/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Suínos
2.
Anim Reprod Sci ; 91(1-2): 107-22, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16310101

RESUMO

It is known that acute action of mu opioid receptor agonist, FK 33-824, results in an inhibition of oestradiol (E2) secretion by porcine granulosa cells from large follicles, but the opioid mode of action is unknown. In the present study, the involvement of two signal transduction pathways, phospholipase C/protein kinase C and adenylyl cyclase/protein kinase A, in mechanism of the opioid action was investigated. Treatment of pig granulosa cells with FK 33-824 at the dose 1 nM suppressed E2 secretion. Protein kinase C (PKC) inhibitors - staurosporine (1-100 nM), d-sphingosine (10-500 nM) and PKCi (100-2000 nM) - both alone and in combination with FK 33-824 reduced E2 release from the cells in relation to the control group. The inhibitory effect of the opioid on E2 output was also observed in PKC-deficient granulosa cells. PKC activator, PMA (10 and 100 nM) significantly attenuated the inhibitory effect of the opioid agonist. FK 33-824 also inhibited 3[H]phorbol 12,13 dibutyrate (3[H]PDBu) specific binding by granulosa cells. Adenylyl cyclase (AC) engagement in opioid signal transduction was assayed after 2-h and 4-h incubations of granulosa cells. During 2-h incubation, FK 33-824 at the dose 1 nM decreased cAMP secretion. Prolongation of the incubation up to 4 h caused disappearance of the opioid action. The addition of protein kinase A (PKC) inhibitor, PKAi (100-2000 nM), alone or together with FK 33-824, was followed by an inhibition of E2 secretion. FK 33-824 with the highest dose of PKAi (2000 nM) significantly inhibited E2 secretion by the cells in comparison to these agents tested separately. The opioid added in combination with PKA activator, 8BrcAMP (1000 microM), caused attenuation of stimulatory effect of 8BrcAMP. Collectively, these results suggest that acute action of mu opioid agonist on porcine granulosa cells leads to decrease of enzymatic activity of PKC and AC/PKA. It is not ruled out that other signal transduction pathways - not considered in this study - may also be engaged in the opioid mechanism of action in these cells.


Assuntos
D-Ala(2),MePhe(4),Met(0)-ol-encefalina/farmacologia , Estradiol/metabolismo , Células da Granulosa/metabolismo , Proteínas Quinases/metabolismo , Receptores Opioides mu/agonistas , Transdução de Sinais , Adenilil Ciclases/metabolismo , Animais , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Feminino , Células da Granulosa/efeitos dos fármacos , Células da Granulosa/enzimologia , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Suínos , Fosfolipases Tipo C/metabolismo
3.
Domest Anim Endocrinol ; 27(4): 379-96, 2004 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-15519041

RESUMO

Opioids were found as factors affecting porcine ovarian steroidogenesis. The mechanism of opioid action, however, on porcine theca interna cells is completely unknown. Therefore, the present study was designed to investigate the possible involvement of two intracellular pathways, phospholipase C/protein kinase C and adenylyl cyclase/protein kinase A, in opioid signal transduction in porcine theca cells treated with mu opioid receptor agonist, FK 33-824. Incubation of the cells for 4 h with FK 33-824 at the dose 1 nM resulted in decreases in inositol phosphate accumulation as well as androstenedione (A(4)), testosterone (T), and estradiol (E(2)) secretions. Protein kinase C (PKC) inhibitors, staurosporine (1-100 nM), D-sphingosine (10-500 nM), and PKCi (100-2000 nM), both added alone and together with the opioid agonist, depressed release of the steroid hormones. PKC activator, phorbol ester (PMA, 1-100 nM), used alone was without effect on theca cell steroidogenesis, but added in combination with FK 33-824 abolished inhibitory influence of the opioid on A(4), T, and E(2) output. The steroid hormone secretion by PKC-deficient theca cells was inhibited by the opioid agonist. FK 33-824 also suppressed PKC activity reducing [(3)H]PDBu specific binding to theca cells, whereas ionomycin (a positive control) increased labeled phorbol ester binding to the cells. In the next experiment, cAMP release from theca cells during 2 and 4 h incubations with FK 33-824 (1-100 nM), naloxone (10 microM; opioid receptor antagonist), and LH (100 ng/mL; a positive control) was examined. FK 33-824 at the dose 1 nM inhibited cAMP secretion during 2 h incubation, but had no effect during longer incubation. LH in a manner independent on incubation time multiplied cAMP release. Protein kinase A inhibitor, PKAi (100-2000 nM), alone and in combination with FK 33-824 (1 nM), inhibited A(4), T, and E(2) secretions by theca cells. PKA activator, 8BrcAMP (10-1000 microM), stimulated the steroid hormone release, but this stimulatory effect was diminished in the presence of FK 33-824. The results allow to suggest that opioid peptides affect porcine theca cell steroidogenesis and their acute action on the cells is connected with the inhibition of phospholipase C/protein kinase C and adenylyl cyclase/protein kinase A signal transduction systems.


Assuntos
D-Ala(2),MePhe(4),Met(0)-ol-encefalina/farmacologia , Enzimas/efeitos dos fármacos , Hormônios Esteroides Gonadais/metabolismo , Receptores Opioides mu/agonistas , Transdução de Sinais/efeitos dos fármacos , Células Tecais/enzimologia , Adenilil Ciclases/efeitos dos fármacos , Adenilil Ciclases/metabolismo , Análise de Variância , Androstenodiona/metabolismo , Animais , Proteínas Quinases Dependentes de AMP Cíclico/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Inibidores Enzimáticos/farmacologia , Enzimas/metabolismo , Estradiol/metabolismo , Feminino , Hormônio Luteinizante/fisiologia , Naloxona/farmacologia , Antagonistas de Entorpecentes/farmacologia , Proteína Quinase C/efeitos dos fármacos , Proteína Quinase C/metabolismo , Suínos , Testosterona/metabolismo , Células Tecais/citologia , Células Tecais/efeitos dos fármacos , Células Tecais/metabolismo , Fosfolipases Tipo C/efeitos dos fármacos , Fosfolipases Tipo C/metabolismo
4.
Reprod Domest Anim ; 39(1): 25-32, 2004 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-15129917

RESUMO

The present studies were undertaken to examine the influence of mu (beta-endorphin, DAMGO, FK 33-824), delta (met-enkephalin, leu-enkephalin, DPLPE) and kappa opioid receptor agonists (dynorphin A, dynorphin B, U 50488) used at different doses (1-1000 nM) alone and in combination with LH (100 ng/ml) on steroidogenesis in porcine granulosa cells derived from large follicles. The effects of mu, delta and kappa receptor agonists on both basal and LH-induced progesterone (P4) secretion were negligible. Agonists of mu opioid receptors reduced basal androstenedione (A4), testosterone (T) and oestradiol (E2) release. Co-treatment with LH entirely abolished the inhibitory effect of these agonists on A4 and E2 secretion and resulted in an increase in T release. The addition of delta receptor agonists was followed by a decrease in basal A4, T and E2 secretion. The cells incubated in the presence of LH increased the androgen production and abrogated the inhibitory effect of delta agonists on E2 output. Basal A4, T and E2 release was also suppressed by kappa receptor agonists. The presence of LH in culture media extended the inhibitory effect of these opioids on E2 output and caused either abolition of the inhibitory influence of kappa agonists or even augmentation of both androgen release in response to the opioids. In conclusion, these data support the involvement of three major types of opioid receptors in the regulation of porcine granulosa cell steroidogenesis.


Assuntos
Peptídeos Opioides/farmacologia , Folículo Ovariano/efeitos dos fármacos , (trans)-Isômero de 3,4-dicloro-N-metil-N-(2-(1-pirrolidinil)-ciclo-hexil)-benzenoacetamida/farmacologia , Animais , D-Ala(2),MePhe(4),Met(0)-ol-encefalina/farmacologia , Relação Dose-Resposta a Droga , Dinorfinas/farmacologia , Endorfinas/farmacologia , Ala(2)-MePhe(4)-Gly(5)-Encefalina/farmacologia , D-Penicilina (2,5)-Encefalina/farmacologia , Encefalinas/farmacologia , Feminino , Células da Granulosa/efeitos dos fármacos , Hormônio Luteinizante/farmacologia , Peptídeos Opioides/administração & dosagem , Folículo Ovariano/citologia , Suínos , beta-Endorfina/farmacologia
5.
J Pharmacol Exp Ther ; 310(3): 1020-6, 2004 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-15121763

RESUMO

We have previously documented that chronic alcohol consumption or alcohol withdrawal affects mu-opioid receptor density and receptor-mediated G protein coupling in Fawn-Hooded (FH) rat brain, especially in mesolimbic regions. FH rats demonstrate comorbid depression and high voluntary alcohol consumption; treatment with standard antidepressants improves both facets of this phenotype. Accordingly, we sought to examine whether mu-opioid receptor binding and the receptor-mediated functional coupling to G protein is affected by this drug treatment. Using quantitative autoradiography, binding of mu-opioid receptors labeled by [125I]FK33,824 (D-Ala2,N-Me-Phe4,Met(O)5-ol enkephalin) and the coupling between receptors and G proteins determined by agonist-stimulated guanosine 5'-O -(3-[35S]thio)triphosphate ([35S]GTPgammaS) binding was mapped throughout brain sections of FH rats after 10-day treatment with vehicle, desipramine, or sertraline. Both desipramine and sertraline produced significant decreases of [125I]FK33,824 binding in many brain regions; 13 of 20 measured regions for desipramine and 16 of 20 measured regions for sertraline. The coupling efficiency of mu-opioid receptors to G proteins was determined by an increase of [35S]GTPgammaS binding induced by stimulation with the mu-opioid receptor agonist [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (10 microM). In contrast to the receptor binding profile, functional coupling of receptors to G proteins was only significantly reduced in the amygdala, whereas it remained unchanged in other regions compared with control. The present findings suggest that antidepressants regulate opioid systems; however, this occurs differentially, and region-specific alteration of functional coupling of mu-opioid receptors to G proteins in the amygdala suggests that opioid function within the amygdala may be modulated by antidepressants.


Assuntos
Tonsila do Cerebelo/efeitos dos fármacos , Antidepressivos/farmacologia , Proteínas de Ligação ao GTP/metabolismo , Receptores Opioides mu/metabolismo , Tonsila do Cerebelo/metabolismo , Animais , D-Ala(2),MePhe(4),Met(0)-ol-encefalina/farmacologia , Desipramina/farmacologia , Interações Medicamentosas , Ala(2)-MePhe(4)-Gly(5)-Encefalina/farmacologia , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Masculino , Ratos , Sertralina/farmacologia , Radioisótopos de Enxofre
6.
Anim Reprod Sci ; 78(1-2): 71-84, 2003 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-12753784

RESUMO

The present study was designed to investigate basal and LH-induced steroidogenesis in porcine theca cells from large follicles in response to various concentrations (1-1000 nM) of mu opioid receptor agonists (beta-endorphin, DAMGO, FK 33-824), delta receptor agonists (met-enkephalin, leu-enkephalin, DPLPE) and kappa receptor agonists (dynorphin A, dynorphin B, U 50488). Agonists of mu opioid receptors suppressed basal androstenedione (A4), testosterone (T) and oestradiol-17beta (E2) secretion and enhanced LH-induced A4 and T release by theca cells. The inhibitory effect of the agonists on E2 secretion was abolished in the presence of LH. All delta receptor agonists depressed basal progesterone (P4) output. However, the influence of these agents on LH-treated cells was negligible. Among delta receptor agonist used only leu-enkephalin and DPLPE at the lowest concentrations inhibited basal A4 release. The presence of LH in culture media changed the influence of these opioids from inhibitory to stimulatory. Similarly, DPLPE reduced T secretion by non-stimulated theca cells and enhanced T secretion of stimulated cells. All of delta agonists inhibited basal E2 secretion and unaffected its release from LH-treated theca cells. Agonists of kappa receptors inhibited basal, non-stimulated, P4 secretion and two of them (dynorphin B, U 50488) potentiated LH-induced P4 output. Basal A4 and T release remained unaffected by kappa agonist treatment, but the cells cultured in the presence of LH generally increased both androgen production in response to these opioids. Basal secretion of E2 was also suppressed by kappa agonists. This inhibitory effect was not observed when the cells were additionally treated with LH. In view of these findings we suggest that opioid peptides derived from three major opioid precursors may directly participate in the regulation of porcine theca cell steroidogenesis.


Assuntos
Peptídeos Opioides/farmacologia , Esteroides/biossíntese , Suínos/metabolismo , Células Tecais/efeitos dos fármacos , Células Tecais/metabolismo , (trans)-Isômero de 3,4-dicloro-N-metil-N-(2-(1-pirrolidinil)-ciclo-hexil)-benzenoacetamida/farmacologia , Animais , Células Cultivadas , D-Ala(2),MePhe(4),Met(0)-ol-encefalina/farmacologia , Dinorfinas/farmacologia , Endorfinas/farmacologia , Ala(2)-MePhe(4)-Gly(5)-Encefalina/farmacologia , D-Penicilina (2,5)-Encefalina/farmacologia , Encefalina Leucina/farmacologia , Encefalina Metionina/farmacologia , Estradiol/biossíntese , Estradiol/metabolismo , Feminino , Progesterona/biossíntese , Progesterona/metabolismo , Receptores Opioides delta/agonistas , Receptores Opioides delta/efeitos dos fármacos , Receptores Opioides kappa/agonistas , Receptores Opioides kappa/efeitos dos fármacos , Receptores Opioides mu/agonistas , Receptores Opioides mu/efeitos dos fármacos , Testosterona/biossíntese , Testosterona/metabolismo , beta-Endorfina/farmacologia
7.
Acta Vet Hung ; 49(3): 319-29, 2001.
Artigo em Inglês | MEDLINE | ID: mdl-11702344

RESUMO

In earlier in vitro experiments opioids affected steroidogenesis in porcine luteal and granulosa cells. The present studies were undertaken to examine the effects of FK 33-824 (opioid agonist) alone or in combination with LH, PRL or naloxone (NAL, opioid antagonist) on steroidogenesis in cultured porcine theca cells. Moreover, we have tested beta-endorphin-like immunoreactivity (beta-END-LI) concentrations in culture media under control conditions and following treatments of theca cells with LH, PRL, progesterone (P4), oestradiol (E2) or testosterone (T). FK 33-824 and NAL significantly increased P4 release by theca cells and inhibited stimulatory effect of LH on this steroid output. PRL-induced P4 secretion from the cells was blunted only by FK 33-824. Secretion of androstenedione (A4) and T was essentially elevated in the presence of FK 33-824 and this potentiation of both androgen release was completely abolished by PRL. NAL blocked stimulatory effect of the opioid agonist only in case of T. Secretion of oestradiol and oestrone was completely free from the influence of both the opioid agonist and antagonist. Pig theca cells were able to produce beta-END-LI but none of tested hormones (LH, PRL, P4, E2 and T alone or in combination) significantly affected this production. In conclusion, these data indicate that porcine theca cells may produce beta-END-LI and change their steroidogenesis in response to opioid peptides.


Assuntos
D-Ala(2),MePhe(4),Met(0)-ol-encefalina/farmacologia , Entorpecentes/agonistas , Suínos/fisiologia , Células Tecais/metabolismo , beta-Endorfina/biossíntese , Animais , D-Ala(2),MePhe(4),Met(0)-ol-encefalina/antagonistas & inibidores , Feminino , Hormônios Esteroides Gonadais/metabolismo , Hormônios Esteroides Gonadais/farmacologia , Gonadotropinas Hipofisárias/farmacologia , Naloxona/farmacologia , Antagonistas de Entorpecentes/farmacologia , Células Tecais/efeitos dos fármacos , beta-Endorfina/metabolismo
8.
Z Naturforsch C J Biosci ; 56(7-8): 629-32, 2001.
Artigo em Inglês | MEDLINE | ID: mdl-11531100

RESUMO

The possible involvement of opioid system in the regulation of hemolymph glucose level in the fresh water crab Oziotelphusa senex senex Fabricius, was investigated. Opioid agonist and antagonist was also used in addition to methionine-enkephalin itself. Injection of the opioid, methionine-enkephalin and FK 33-824 significantly elevated hemolymph glucose level. In contrast, injection of naloxone in to crab resulted in decrease in hemolymph glucose level. Injection of naloxone prior to injection of methionine-enkephalin blocked the hyperglycemic action of methionine-enkephalin. Injection of methionine-enkephalin, FK 33824 and naloxone produced no significant effect on hemolymph glucose level in eyestalk-less crab. The alterations in the intact crab hemolymph glucose level hypothesised to be due to stimulation of release of hyperglycemic hormone during methionine-enkephalin and FK 33824 treatment and blocking of release of hyperglycemic hormone during naloxone treatment from the eyestalks of crab Oziotelphusa senex senex.


Assuntos
Braquiúros/fisiologia , D-Ala(2),MePhe(4),Met(0)-ol-encefalina/farmacologia , Encefalina Metionina/farmacologia , Glucose/metabolismo , Hemolinfa/fisiologia , Naloxona/farmacologia , Animais , Água Doce , Hemolinfa/efeitos dos fármacos , Homeostase
9.
J Biol Chem ; 276(41): 37779-86, 2001 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-11500514

RESUMO

The most convincing evidence demonstrating constitutive activation of mu-opioid receptors is the observation that putative inverse agonists decrease basal G-protein activity in membrane preparations. However, it is not clear whether constitutively active receptors in isolated membranes have any physiological relevance in intact cells. GH3 cells expressing mu-opioid receptors (GH3MOR) exhibit higher basal G-protein activity and lower basal cAMP levels than wild-type GH3 cells, indicative of constitutively active receptors. This study determined whether alkylation of mu-opioid receptors by the irreversible antagonist beta-funaltrexamine would decrease spontaneous receptor activity in intact cells, revealing constitutive activity. GH3MOR cells were pretreated with increasing concentrations of beta-funaltrexamine followed by functional testing after removal of unbound drug. beta-Funaltrexamine pretreatment produced a concentration-dependent decrease in mu-opioid receptor binding with an IC50 of 0.98 nm and an Emax of 77%. Similar concentrations of beta-funaltrexamine pretreatment produced a half-maximal reduction in basal [35S]GTPgammaS binding, a decrease in basal photolabeling of G-proteins with azidoanilido-[alpha-32P]GTP, and an increase in basal adenylyl cyclase activity in intact cells. Therefore, mu-opioid receptors are constitutively active in intact cells, producing stimulation of G-proteins and inhibition of adenylyl cyclase. Importantly, photolabeling of Galpha-subunits with azidoanilido-[alpha-32P]GTP demonstrated that constitutively active mu-opioid receptors activate individual G-proteins differently than the agonist [d-Ala2,N-MePhe4,Gly-ol5]enkephalin.


Assuntos
Inibidores de Adenilil Ciclases , D-Ala(2),MePhe(4),Met(0)-ol-encefalina/farmacologia , Proteínas de Ligação ao GTP/metabolismo , Naltrexona/análogos & derivados , Receptores Opioides mu/fisiologia , Toxina Adenilato Ciclase , Linhagem Celular , Ala(2)-MePhe(4)-Gly(5)-Encefalina/farmacologia , Naltrexona/farmacologia , Cloreto de Potássio/farmacologia , Ligação Proteica , Receptores Opioides mu/agonistas , Receptores Opioides mu/metabolismo , Fatores de Virulência de Bordetella/farmacologia
10.
J Physiol Pharmacol ; 51(2): 359-68, 2000 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-10898106

RESUMO

The aim of the present studies was to investigate (1) the presence of LH receptor (LHR) in porcine separated small (SLCs) and large (LLCs) luteal cells taken from mid-luteal corpora lutea and (2) the influence of opioid agonist, FK 33-824 (FK) on LHR gene expression in these cells. Immunocytochemistry revealed intense staining for LHR in both SLCs and LLCs. Reverse transcription-polymerase chain reaction (RT-PCR) and Southern hybridization were used to check the effect of FK and hCG on LHR gene expression. The LHR gene expression was observed in non-stimulated LLCs and in both types of cells after treatment with FK or hCG. FK stimulated LHR gene expression in SLCs and inhibited the gene expression in LLCs. Moreover, FK inhibited and potentiated stimulatory influence of hCG on the gene expression in LLCs and SLCs, respectively. These results suggest that LHR gene expression in porcine luteal cells can be regulated by opioid peptides.


Assuntos
Corpo Lúteo/metabolismo , Endorfinas/fisiologia , Expressão Gênica , Receptores do LH/genética , Receptores do LH/metabolismo , Animais , Tamanho Celular , Células Cultivadas , Gonadotropina Coriônica/farmacologia , Corpo Lúteo/citologia , Corpo Lúteo/efeitos dos fármacos , D-Ala(2),MePhe(4),Met(0)-ol-encefalina/farmacologia , Endorfinas/antagonistas & inibidores , Feminino , Expressão Gênica/efeitos dos fármacos , Suínos , Distribuição Tecidual
11.
Reprod Nutr Dev ; 40(1): 63-75, 2000.
Artigo em Inglês | MEDLINE | ID: mdl-10737551

RESUMO

Beta-endorphin-like immunoreactivity (beta-END-LI) was measured by radioimmunoassay in porcine ovarian follicular fluid (FF) from small, medium and large follicles throughout the oestrous cycle. The concentration of beta-END-LI in FF from small follicles collected on days 1-5 of the cycle was at least tenfold higher than in the fluid from any other follicles independently from their size and the period of the cycle. The level of beta-END-LI in small follicles on days 6-10 was drastically decreased. Subsequently, on days 11-16 its concentration was enhanced and reduced again in pre-ovulatory period of the cycle. Concentrations of beta-END-LI in FF from medium follicles were relatively equal throughout the cycle (days 6-21). No significant differences in beta-END-LI levels were found between small, medium and large follicles from days 17-21. However, beta-END-LI concentrations in medium follicles on days 11-13 and 14-16 were statistically lower than those in small follicles. Moreover, the effects of FSH, prolactin (PRL), progesterone (P4), testosterone (T) and 17 beta-oestradiol (E2) on beta-END-LI release by granulosa cells (GCs) from large follicles and, on the other hand, the effects of the opioid agonist FK 33-824 alone or in combination with FSH, PRL or naloxone (NAL) on follicular steroidogenesis were studied. FSH drastically increased beta-END-LI output in a dose-dependent fashion. This stimulatory effect of the gonadotrophin was inhibited by the highest dose of P4 (10(-5) M). The effect of PRL and the steroids added to the cultures on beta-END-LI release was negligible. FSH- or PRL-induced P4 secretion by GCs was essentially abolished by both FK 33-824 and NAL. However, androstenedione (A4) and testosterone output by the cells was greatly potentiated by FK 33-824. In the presence of NAL, FSH or PRL, A4 release stimulated by FK 33-824 was suppressed to the basal level. Secretion of E2 was completely free from the influence of FK 33-824 or NAL; only oestrone (E1) output was modulated by them in cultures where FSH or PRL was present. In conclusion, FSH appears to be the key regulator of beta-END-LI secretion by porcine granulosa cells. Moreover, steroidogenesis in pig granulosa cells is modulated by opioid peptides acting both alone and by way of interaction with FSH or PRL.


Assuntos
Folículo Ovariano/fisiologia , Suínos/fisiologia , beta-Endorfina/fisiologia , Animais , D-Ala(2),MePhe(4),Met(0)-ol-encefalina/farmacologia , Sinergismo Farmacológico , Feminino , Hormônio Foliculoestimulante/farmacologia , Líquido Folicular/química , Células da Granulosa/efeitos dos fármacos , Células da Granulosa/metabolismo , Naloxona/farmacologia , Antagonistas de Entorpecentes/farmacologia , Prolactina/farmacologia , Esteroides/farmacologia , beta-Endorfina/análise , beta-Endorfina/metabolismo
12.
Anim Reprod Sci ; 56(3-4): 245-57, 1999 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-10497920

RESUMO

The present study was designed to investigate the effect of the opioid agonist FK 33-824 on basal and hCG-induced progesterone (P4), cAMP and cGMP secretion and on the phosphoinositide-specific phospholipase C signalling system in separated porcine small (SLCs) and large luteal cells (LLCs). Unit gravity sedimentation was used to produce cultures of small and large luteal cells from corpora lutea (CL) on days 8-10 of the oestrous cycle. In order to examine the effect of FK 33-824 on P4 and cyclic nucleotide release, SLCs and LLCs were incubated in M199 medium at 37 degrees C in 5% CO2:95% air, for 12 h. Small and large luteal cells were treated with hCG (100 ng/ml) alone, FK 33-824 (10(-9) M) alone or were co-treated with FK 33-824 and hCG and with the opioid antagonist, naloxone (NAL, 10(-5) M). FK 33-824 alone did not influence P4 secretion by LLCs and SLCs. However, FK 33-824 completely abolished the stimulatory effect of hCG on P4 secretion by SLCs. The addition of FK 33-824 was followed by a significant increase in cAMP release (p<0.01) by LLCs and a decrease in cGMP secretion by SLCs (p<0.05). The effect of FK 33-824 was blocked by NAL, which strongly suggests that the observed influence of this opioid agonist was achieved through its binding to opioid receptors in luteal membranes. In the presence of hCG, cAMP secretion by both SLCs and LLCs was many-fold higher than in the control group. As regards cGMP output, only LLCs showed elevated secretion of this cyclic nucleotide under the influence of hCG. With the aim of examining the influence of FK 33-824 on phosphatidylinositol hydrolysis, LLCs, SLCs and mixed small and large cells were labelled with [3H]-myo-inositol (100 microCi/ml) for 3 h at 37 degrees C. The cells were then incubated in M199 medium supplemented with 10 mM LiCl, 1% BSA, and antibiotics in the presence and absence of FK 33-824 (10(-9) M) at 37 degrees C for 30 min. Liberated labelled inositol mono-, bis-, and trisphosphates (IPs) were isolated and quantified by affinity chromatography on columns of AG 1-X8 resin, followed by liquid scintillation spectroscopy. Inositol phosphate accumulation in LLCs, SLCs, and mixed small and large cells was not altered by treatment with FK 33-824 at the dose used. In view of these findings we suggest that opioid peptides affect pig corpus luteum steroid secretion, and the response is probably mediated through cyclic nucleotides, but not IPs.


Assuntos
Corpo Lúteo/efeitos dos fármacos , D-Ala(2),MePhe(4),Met(0)-ol-encefalina/farmacologia , Fase Luteal/fisiologia , Suínos/fisiologia , Animais , Gonadotropina Coriônica/farmacologia , Cromatografia de Afinidade/veterinária , Corpo Lúteo/fisiologia , AMP Cíclico/análise , AMP Cíclico/metabolismo , GMP Cíclico/análise , GMP Cíclico/metabolismo , Feminino , Fosfatos de Inositol/análise , Fosfatos de Inositol/metabolismo , Naloxona/farmacologia , Antagonistas de Entorpecentes/farmacologia , Ovário/fisiologia , Progesterona/análise , Progesterona/metabolismo , Radioimunoensaio/veterinária
13.
Alcohol Clin Exp Res ; 23(6): 1008-14, 1999 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-10397284

RESUMO

BACKGROUND: Relapse after abstinence can be modelled in rats using an alcohol deprivation effect (ADE) of enhanced ethanol consumption after a period of enforced abstinence from ethanol; however, not all rat strains display such an effect. We wanted to examine the effect of naltrexone on ethanol consumption by ethanol-preferring Fawn-Hooded (FH) rats using such a model. METHODS: FH rats were given continual free-choice access to a 5% ethanol solution or water (4 weeks) followed by 2 weeks of water alone. At the end of this abstinence period, osmotic minipumps were implanted subcutaneously to deliver saline (n = 4) or naltrexone (n = 4; 8.4 mg/kg/day for 4 weeks). After recovery from surgery, the rats were again given access to 5% ethanol under the same free-choice conditions (4 weeks). A third group of age-matched controls drank only water during the behavioral trial. At the end of the behavioral trial, the rats were decapitated and an autoradiographic examination was made of micro-opioid receptor density through the forebrain using the ligand [125I]FK-33824. RESULTS: First, a period of enforced abstinence from ethanol consumption caused a significant (p < 0.05) and prolonged increase in ethanol preference (+18%) and decrease in water consumption (-53%), although the volume of ethanol consumed (ml/day) did not vary, indicating an atypical ADE in this rat strain. Second, naltrexone significantly (p < 0.05) decreased ethanol consumption by the FH rats in terms of absolute amount of ethanol consumed and preference for ethanol solution, but this effect of naltrexone diminished over time, concurrent with a robust and significant elevation in micro-opioid receptor density in all brain regions examined (p < 0.05). Finally, ethanol consumption alone also upregulated micro-opioid receptor density relative to nondrinking controls in a number of brain regions, which included the nucleus accumbens (+29%) and caudate-putamen (+15%,p < 0.05), but decreased micro-opioid receptor density in other regions including the substantia nigra pars reticulata, which was suggestive of an indirect effect on micro-opioid receptors. CONCLUSIONS: The data suggest that continual long-term naltrexone treatment may not be effective in the treatment of alcoholism, possibly because of the induced increase in micro-opioid receptor density.


Assuntos
Consumo de Bebidas Alcoólicas/tratamento farmacológico , Depressores do Sistema Nervoso Central/administração & dosagem , Etanol/administração & dosagem , Naltrexona/farmacologia , Antagonistas de Entorpecentes/farmacologia , Receptores Opioides mu/efeitos dos fármacos , Temperança , Animais , Sítios de Ligação , D-Ala(2),MePhe(4),Met(0)-ol-encefalina/análise , D-Ala(2),MePhe(4),Met(0)-ol-encefalina/farmacologia , Masculino , Prosencéfalo/química , Prosencéfalo/efeitos dos fármacos , Ratos , Receptores Opioides mu/análise
14.
Eur J Neurosci ; 9(1): 128-39, 1997 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-9042577

RESUMO

The distribution of mu opioid receptors was examined by light and electron microscopic autoradiography in the locus coeruleus of the rat following in vitro labelling with the iodinated agonist [125I]FK-33824. At the light microscopic level, specific mu opioid binding sites were concentrated over the perikarya and dendrites of neurons that were tyrosine hydroxylase-immunopositive in adjacent sections. Accordingly, both the number of tyrosine hydroxylase-immunoreactive neurons and the density of labelled mu receptors decreased markedly throughout the rostrocaudal extent of the nucleus following treatment with the catecholaminergic neurotoxin 6-hydroxydopamine. By electron microscopy, specifically labelled receptors were detected both inside and on the surface of locus coeruleus neurons. Intracellular sites were found by resolution circle analysis to be highly concentrated within the endoplasmic reticulum and Golgi apparatus, suggesting that the ligand recognizes both glycosylated and preglycosylated forms of receptor. The remainder were found mainly over the cytoplasmic matrix or intracytoplasmic vesicles, and were attributed to newly synthesized or recycled receptors in transit. Cell surface receptors were present over both dendritic and perikaryal membranes of noradrenergic cells. These were most highly concentrated opposite abutting axon terminals, suggesting the existence of receptor 'hot spots' at sites of putative endogenous ligand release. However, only a small proportion of these sites was associated with synaptic specializations. Furthermore, an important contingent was detected opposite non-axonal elements, such as dendrites and glial cells, suggesting that mu opioid ligands act mainly parasynaptically on locus coeruleus neurons. Finally, approximately 5% of labelled receptors were associated with axoglial interfaces, indicating that a minor action of mu opioids in the locus may be presynaptic and/or glial.


Assuntos
Locus Cerúleo/metabolismo , Locus Cerúleo/ultraestrutura , Neurônios/metabolismo , Neurônios/ultraestrutura , Receptores Opioides mu/metabolismo , Receptores Opioides mu/ultraestrutura , Sistema Nervoso Simpático/metabolismo , Sistema Nervoso Simpático/ultraestrutura , Animais , Autorradiografia , Grânulos Citoplasmáticos/efeitos dos fármacos , Grânulos Citoplasmáticos/fisiologia , Grânulos Citoplasmáticos/ultraestrutura , D-Ala(2),MePhe(4),Met(0)-ol-encefalina , Imuno-Histoquímica , Radioisótopos do Iodo , Locus Cerúleo/fisiologia , Masculino , Microscopia Eletrônica , Neurônios/fisiologia , Norepinefrina/fisiologia , Oxidopamina , Ensaio Radioligante , Ratos , Ratos Sprague-Dawley , Sistema Nervoso Simpático/fisiologia , Simpatolíticos
15.
J Neuroendocrinol ; 8(11): 883-7, 1996 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-8933366

RESUMO

The distribution and quantification of opioid receptor types in post-mortem human pituitary cryostat sections was determined by quantitative in vitro receptor autoradiography. Highly specific radioligands were used for each opioid receptor type i.e. [125l]-FK-33-824 for mu-opioid sites, [125l][D.Ala2]-Deltorphin-l for delta-opioid sites and 3H-U69,593 for kappa-opioid sites. None of the five specimens tested exhibited any labeling in the anterior lobe of the pituitary for the three radioligands. As for the posterior pituitary, all 5 specimens contained both mu and kappa-opioid binding sites whereas none of them showed delta-binding sites. The presence of both mu- and kappa-opioid binding sites in the human posterior pituitary contrasts with previous findings in the rat where only kappa-sites have been found. The present study could contribute to understanding of the functional action of opioids in the human pituitary.


Assuntos
Benzenoacetamidas , Neuro-Hipófise/metabolismo , Receptores Opioides/metabolismo , (trans)-Isômero de 3,4-dicloro-N-metil-N-(2-(1-pirrolidinil)-ciclo-hexil)-benzenoacetamida , Adulto , Sítios de Ligação , Ligação Competitiva , Cadáver , D-Ala(2),MePhe(4),Met(0)-ol-encefalina/metabolismo , Ala(2)-MePhe(4)-Gly(5)-Encefalina , Encefalinas/farmacologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Entorpecentes/metabolismo , Oligopeptídeos/farmacologia , Pirrolidinas/metabolismo , Distribuição Tecidual
16.
J Endocrinol ; 151(2): 195-201, 1996 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-8958779

RESUMO

To study possible age-related differences in the role of neuronal histaminergic pathways in the control of GH secretion, the effects of alpha-fluoromethylhistidine (alpha-FMH), an irreversible inhibitor of histamine (HA) synthesis, were examined on basal and opioid-induced GH release in neonatal and adult rats. The mechanisms involved in such effects were evaluated by measuring pituitary GH mRNA levels and hypothalamic levels of GH-releasing hormone (GHRH) and somatostatin (SRIF) mRNAs. Daily injection of alpha-FMH (20 mg/kg, s.c.) in pups of either sex, from birth until 10 days of age, caused a significant increase in baseline plasma GH and potentiated the GH response to the [Met5]-enkephalin analog FK 33-824 (1 mg/kg, s.c.) administered 3 h after the last alpha-FMH injection. GH and SRIF mRNA levels were significantly higher in alpha-FMH-treated pups than in controls, whereas no difference was observed in GHRH mRNA levels. In young adult male rats, acute administration of alpha-FMH (100 mg/kg, s.c., 3 h before) did not change significantly basal GH levels but potentiated FK 33-824 (0.3 mg/kg, intracarotid)-induced stimulation of GH secretion. Repeated administration of alpha-FMH (200 micrograms/rat, i.c.v., for 3 days) failed to modify basal and FK 33-824-induced GH secretion, caused a significant reduction in hypothalamic GHRH mRNA levels and left SRIF and GH mRNAs unchanged. These findings indicate that HA exerts an inhibitory effect on GH secretion in both neonatal and adult rats. The different effects of short-term HA depletion on hypothalamic and pituitary indices of somatotropic function observed at the two age periods may be ascribed to the immaturity of the HA system in early postnatal life and to a different functional role of GH-regulatory factors during ontogeny.


Assuntos
Envelhecimento/fisiologia , Hormônio do Crescimento/metabolismo , Antagonistas dos Receptores Histamínicos/farmacologia , Histidina Descarboxilase/antagonistas & inibidores , Hipotálamo/metabolismo , Metilistidinas/farmacologia , Análise de Variância , Animais , Animais Recém-Nascidos , D-Ala(2),MePhe(4),Met(0)-ol-encefalina/farmacologia , Feminino , Hormônio do Crescimento/sangue , Hormônio Liberador de Hormônio do Crescimento/genética , Hipotálamo/efeitos dos fármacos , Masculino , Hipófise/química , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Somatostatina/genética , Ganho de Peso/efeitos dos fármacos
17.
Hepatology ; 24(4): 895-901, 1996 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-8855194

RESUMO

Portacaval anastomosis (PCA) in the rat results in a broad spectrum of neurological and neurobehavioral changes, including alterations of circadian rhythms, impaired locomotor activity, and reflexes, as well as decreased threshold to noxious stimuli. In addition, following portacaval shunting, rats drink significantly more ethanol in a free-choice drinking paradigm. Available evidence suggests that many of these behavioral changes may be modulated by the endogenous opioid system of the brain. To evaluate this possibility, the effects of PCA on circulating beta-endorphin (beta-EP), as well as beta-EP content in the pituitary and specific brain nuclei, was evaluated using a sensitive radioimmunoassay. Furthermore, the characteristics and regional densities of mu and delta opioid receptors in the brains of PCA and sham-operated control rats were studied using an in vitro technique, as well as quantitative receptor autoradiography and the specific ligands 125I [D-Ala2, MePhe4, Met(o)ol5]enkephalin (FK 33-824) and 125I [2-D-penicillamine, 5-D-penicillamine]-enkephalin (DPDPE) for micro and delta sites, respectively. PCA resulted in region-selective modifications of beta-EP in brain, but not in pituitary or blood. Autoradiographic studies revealed a generalized decrease in mu binding sites (up to 70% decreases compared with sham-operated controls) and region-selective alterations of delta receptor densities following PCA. Portacaval-shunted rats drank significantly more ethanol in a free-choice drinking paradigm, an effect that was significantly attenuated by the administration of the opiate antagonist naloxone. Increased ethanol preference thus appeared to result from modifications of the endogenous opioid system in nucleus accumbens of rats following PCA.


Assuntos
Química Encefálica , Encéfalo/metabolismo , Naloxona/farmacologia , Antagonistas de Entorpecentes/farmacologia , Derivação Portocava Cirúrgica , beta-Endorfina/metabolismo , Consumo de Bebidas Alcoólicas , Análise de Variância , Animais , D-Ala(2),MePhe(4),Met(0)-ol-encefalina/metabolismo , D-Penicilina (2,5)-Encefalina , Encefalinas/metabolismo , Masculino , Hipófise/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores Opioides delta/metabolismo , Receptores Opioides mu/metabolismo , beta-Endorfina/análise
18.
J Neurophysiol ; 75(5): 2017-28, 1996 May.
Artigo em Inglês | MEDLINE | ID: mdl-8734600

RESUMO

1. Presynaptic inhibition of synaptic transmission in the hippocampus was investigated by comparing the effects of several agonists on miniature excitatory and inhibitory postsynaptic currents (mEPSCs and mIPSCs). 2. The Ca2+ ionophore ionomycin increased the frequency of mEPSCs and mIPSCs but did not affect their amplitude. Ionomycin-induced release required extracellular Ca2+ and was prevented by pretreatment with botulinum neurotoxin serotype F, like evoked synaptic transmission. Unlike evoked transmission, however, this increase did not involve activation of voltage-dependent Ca2+ channels because it was insensitive to Cd2+. 3. Both the lanthanide gadolinium and alpha-latrotoxin produced increases in the frequency of mEPSCs and mIPSCs, but their actions were independent of extracellular Ca2+. 4. Adenosine, the gamma-aminobutyric acid-B (GABAB) receptor agonist baclofen, and a mu-opioid receptor agonist strongly reduced the frequency of synaptic currents triggered by all three secretagogues. 5. We conclude that activation of these presynaptic receptors can reduce high frequencies of vesicular glutamate and GABA release by directly impairing transmitter exocytosis. Presynaptic inhibition of gadolinium- and alpha-latrotoxin-induced release indicates that this impairment occurs without changes in intraterminal Ca2+ homeostasis and when vesicle fusion is rendered Ca2+ independent, respectively. 6. The inhibition of ionomycin-induced release provides additional evidence for a direct, neurotransmitter receptor-mediated modulation of the proteins underlying vesicular docking or fusion as an important component of presynaptic inhibition of evoked synaptic transmission.


Assuntos
Cálcio/fisiologia , Hipocampo/fisiologia , Neurotransmissores/metabolismo , Receptores Pré-Sinápticos/fisiologia , Transmissão Sináptica/fisiologia , Adenosina/farmacologia , Animais , Baclofeno/farmacologia , Toxinas Botulínicas/farmacologia , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/metabolismo , Fármacos Cardiovasculares/farmacologia , D-Ala(2),MePhe(4),Met(0)-ol-encefalina/farmacologia , Depressão Química , Espaço Extracelular/efeitos dos fármacos , Espaço Extracelular/metabolismo , Agonistas GABAérgicos/farmacologia , Gadolínio/farmacologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Técnicas In Vitro , Ionomicina/farmacologia , Ionóforos/farmacologia , Técnicas de Patch-Clamp , Ratos , Receptores Pré-Sinápticos/agonistas , Receptores Pré-Sinápticos/antagonistas & inibidores , Venenos de Aranha/farmacologia , Transmissão Sináptica/efeitos dos fármacos
19.
Eur J Endocrinol ; 134(1): 73-6, 1996 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-8590960

RESUMO

Exogenous growth hormone (hGH) administration in humans attenuates the endogenous growth hormone (GH) response to some pharmacological stimuli; in particular, pretreatment with hGH completely blocks the serum GH response to growth hormone-releasing hormone. In order to evaluate the mechanism(s) whereby opiods induce GH secretion in man, we gave the following treatments to six healthy male volunteers: (a) IV saline; (b) a met-enkephalin analog G-DAMME 250 micrograms IV as a bolus at time 0'; (c) hGH 2 IU as an IV bolus at time -180'; (d) G-DAMME as above, preceded by hGH as above. In our study, G-DAMME stimulated GH secretion both basally (peak 17.9 +/- 6.0 ng/ml) and, to a lesser extent, after hGH pretreatment (6.0 +/- 2.7 ng/ml). Since in our study G-DAMME was able to partially overcome the inhibitory effect of hGH administration, it is suggested that opioids act through an inhibition of somatostatin release and not through a GHRH-dependent pathway. However, an additional direct effect of hGH on pituitary somatotrophes cannot be excluded.


Assuntos
D-Ala(2),MePhe(4),Met(0)-ol-encefalina/farmacologia , Hormônio do Crescimento/metabolismo , Hormônio do Crescimento/farmacologia , Adulto , Humanos , Masculino , Concentração Osmolar , Valores de Referência , Método Simples-Cego , Fatores de Tempo
20.
Peptides ; 17(6): 957-64, 1996.
Artigo em Inglês | MEDLINE | ID: mdl-8899814

RESUMO

The effects of a synthetic Met-enkephalin analogue D-Ala2, MePhe4Met(O)5-ol]enkephalin (DAMME) (1 mg/kg. IP) on gastric damage produced by necrotizing agents (0.6 N HCl, ethanol 1 ml/rat, PO) were evaluated, and the correlation between histamine and opioids in stomach was investigated, Rats pretreated with DAMME bad significantly less severe lesions and lower histamine content in gastric tissue. The histamine level, expressed in mg/g of gastric tissue, changed from 0.41 +/- 0.10 of control animals to 1.33 +/- 0.12 for HCl and 1.51 +/- 0.20 for ethanol treatment, whereas in animals pretreated with DAMME the values were significantly reduced to 0.55 +/- 0.13 and 0.65 +/- 0.15. These results confirm a link between the gastroprotection produced by opioids and the modulation of histaminergic activity in the rat stomach.


Assuntos
D-Ala(2),MePhe(4),Met(0)-ol-encefalina/farmacologia , Mucosa Gástrica/efeitos dos fármacos , Histamina/metabolismo , Entorpecentes/farmacologia , Animais , Células Enterocromafins/efeitos dos fármacos , Masculino , Muco/fisiologia , Células Parietais Gástricas/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Úlcera Gástrica/induzido quimicamente , Úlcera Gástrica/prevenção & controle
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