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1.
J Endocrinol ; 207(3): 289-300, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20880984

RESUMO

The expression of type I GNRH receptor (GNRHR-I) and the direct role of GNRH-I on corpora lutea (CL) function were studied in the pseudopregnant rabbit model. Immunohistochemistry evidenced GNRHR-I and GNRH-I in luteal cells at early (day 4 pseudopregnancy)-, mid (day 9)-, and late (day 13)-luteal stages. Real-time RT-PCR and western blotting revealed GNRHR-I mRNA and protein at the three luteal stages. Buserelin in vivo treatment at days 9 and 13 decreased plasma progesterone levels for 48 and 24  h respectively. In in vitro cultured CL, buserelin reduced progesterone secretion, increased prostaglandin F(2α) (PGF(2α)) secretion and cyclo-oxygenase-2 (COX-2) and nitric oxide synthase (NOS) activities at days 9 and 13, and decreased PGE2 at day 13. Co-incubation with antagonists for GNRH-I (antide), inositol 1,4,5-trisphosphate (IP3, 2-amino-ethoxydiphenylborate), and diacylglycerol (DAG, 1-hexadecyl-2-acetyl glycerol) or inhibitors for phospholipase C (PLC, compound 48/80), and protein kinase C (PKC, staurosporine) counteracted the buserelin effects. Buserelin co-incubated with COX inhibitor (acetylsalicylic acid) increased progesterone and decreased PGF(2α) and NOS activity at days 9 and 13, whereas co-incubation with NOS inhibitor (N-nitro-l-arginine methyl ester) increased progesterone at the same luteal stages. These results suggest that GNRHR-I is constitutively expressed in rabbit CL independently of luteal stage, whereas GNRH-I down-regulates directly CL progesterone production via PGF(2α) at mid- and late-luteal stages of pseudopregnancy, utilizing its cognate type I receptor with a post-receptorial mechanism that involves PLC, IP3, DAG, PKC, COX-2, and NOS.


Assuntos
Corpo Lúteo/metabolismo , Hormônio Liberador de Gonadotropina/metabolismo , Pseudogravidez/metabolismo , Receptores LHRH/metabolismo , Animais , Aspirina/administração & dosagem , Busserrelina/administração & dosagem , Corpo Lúteo/efeitos dos fármacos , Ciclo-Oxigenase 2/metabolismo , Diglicerídeos/administração & dosagem , Dinoprosta/metabolismo , Inibidores Enzimáticos/administração & dosagem , Feminino , Inositol 1,4,5-Trifosfato/administração & dosagem , NG-Nitroarginina Metil Éster/administração & dosagem , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/metabolismo , Oligopeptídeos/administração & dosagem , Progesterona/sangue , Progesterona/metabolismo , Proteína Quinase C/antagonistas & inibidores , Coelhos , Receptores LHRH/antagonistas & inibidores , Estaurosporina/administração & dosagem , Fosfolipases Tipo C/antagonistas & inibidores , p-Metoxi-N-metilfenetilamina/administração & dosagem
2.
BMC Physiol ; 8: 10, 2008 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-18492257

RESUMO

BACKGROUND: Serotonin induces fluid secretion from Calliphora salivary glands by the parallel activation of the InsP3/Ca2+ and cAMP signaling pathways. We investigated whether cAMP affects 5-HT-induced Ca2+ signaling and InsP3-induced Ca2+ release from the endoplasmic reticulum (ER). RESULTS: Increasing intracellular cAMP level by bath application of forskolin, IBMX or cAMP in the continuous presence of threshold 5-HT concentrations converted oscillatory [Ca2+]i changes into a sustained increase. Intraluminal Ca2+ measurements in the ER of beta-escin-permeabilized glands with mag-fura-2 revealed that cAMP augmented InsP3-induced Ca2+ release in a concentration-dependent manner. This indicated that cAMP sensitized the InsP3 receptor Ca2+ channel for InsP3. By using cAMP analogs that activated either protein kinase A (PKA) or Epac and the application of PKA-inhibitors, we found that cAMP-induced augmentation of InsP3-induced Ca2+ release was mediated by PKA not by Epac. Recordings of the transepithelial potential of the glands suggested that cAMP sensitized the InsP3/Ca2+ signaling pathway for 5-HT, because IBMX potentiated Ca2+-dependent Cl- transport activated by a threshold 5-HT concentration. CONCLUSION: This report shows, for the first time for an insect system, that cAMP can potentiate InsP3-induced Ca2+ release from the ER in a PKA-dependent manner, and that this crosstalk between cAMP and InsP3/Ca2+ signaling pathways enhances transepithelial electrolyte transport.


Assuntos
Cálcio/metabolismo , AMP Cíclico/administração & dosagem , Dípteros/metabolismo , Retículo Endoplasmático/metabolismo , Inositol 1,4,5-Trifosfato/administração & dosagem , Glândulas Salivares/metabolismo , Serotonina/metabolismo , Animais , Células Cultivadas , Dípteros/efeitos dos fármacos , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Retículo Endoplasmático/efeitos dos fármacos , Glândulas Salivares/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia
3.
Eur Biophys J ; 36(3): 187-97, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17262224

RESUMO

Considering the ATP-driven (SERCA) pump flux as function of glucose concentration and the calcium flux from the endoplasmic reticulum (ER) through the IP(3)R channel, the calcium-based phantom bursting model (PBM) of beta-cells (Bertram and Sherman in Bull Math Biol 66:1313, 2004) is theoretically extended to discuss the effects of glucose and inositol 1,4,5-trisphosphate (IP(3)) concentration on the membrane potential activities. When IP(3) concentration is fixed, it is found that there is a critical glucose concentration at which electrical bursting oscillations transfer into spiking, and the critical concentration of glucose is increased with the increasing of IP(3) concentration. To get the bursting oscillations in beta-cells, our theoretical results show that the stimulatory glucose concentration should be more than 10 mM, which is consistent with the normal physiological IP(3) level. When the stochastic opening and closing of IP(3)R channels are considered, it is shown that the membrane potential oscillation transfers from spiking to bursting with the channel number decreasing, and the average cytosolic free Ca(2+) concentration is increased with the increase of glucose concentration.


Assuntos
Potenciais de Ação/fisiologia , Sinalização do Cálcio/fisiologia , Glucose/administração & dosagem , Receptores de Inositol 1,4,5-Trifosfato/fisiologia , Inositol 1,4,5-Trifosfato/administração & dosagem , Células Secretoras de Insulina/fisiologia , Modelos Biológicos , Potenciais de Ação/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Simulação por Computador , Relação Dose-Resposta a Droga , Interações Medicamentosas , Receptores de Inositol 1,4,5-Trifosfato/efeitos dos fármacos , Células Secretoras de Insulina/efeitos dos fármacos , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/fisiologia
5.
Methods Mol Biol ; 312: 213-28, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-21341102

RESUMO

Many receptor tyrosine kinases and seven-transmembrane receptors are directly coupled or coupled via G proteins, respectively, to the activation of phosphoinositidase Cs. These enzymes catalyze the hydrolysis of phosphatidylinositol 4,5-bisphosphate to produce the second messengers, myo-inositol(1,4,5)trisphosphate [Ins(1,4,5)P(3)] and diacylglycerol. Ins(1,4,5)P(3) interacts with a specific receptor that is a ligand-gated channel that allows mobilization of non-mitochondrial intracellular calcium (Ca(2+)) stores. Because Ins(1,4,5)P(3) is plasma membrane impermeant, this phenomenon was first demonstrated in permeabilized pancreatic acinar cells, and all subsequent studies in cells have involved introduction of Ins(1,4,5)P(3) by rendering a cell population permeable (3), using microinjection techniques, or by the presentation of chemically modified membrane-permeable Ins(1,4,5)P(3) analogs, such as photolabile "caged Ins(1,4,5)P(3)". An alternative approach involves disruption of the plasma membrane and preparation of microsomes from the intracellular vesicular Ca(2+) stores; however, microsomal preparations exhibit a loss of Ins(1,4,5)P(3) responsiveness compared to permeabilized and intact cells.


Assuntos
Cálcio/metabolismo , Fibroblastos/metabolismo , Inositol 1,4,5-Trifosfato/administração & dosagem , Microinjeções/métodos , Microscopia de Contraste de Fase/métodos , Osteoblastos/metabolismo , Animais , Adesão Celular , Linhagem Celular , Humanos , Inositol 1,4,5-Trifosfato/análogos & derivados , Inositol 1,4,5-Trifosfato/metabolismo , Células L , Camundongos , Polifosfatos/administração & dosagem , Polifosfatos/metabolismo , Espectrometria de Fluorescência
6.
Anim Reprod Sci ; 80(1-2): 101-12, 2004 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-15036519

RESUMO

Inositol 1,4,5-trisphosphate (IP3) is considered to be important for activation of mammalian oocytes at the time of fertilization, and activation induces a rise in intracellular Ca2+ concentration ([Ca2+]i) by release from the Ca2+ stores in the oocytes. Therefore, IP3 could act as an artificial activator of porcine oocytes. Activation and development, and rise in [Ca2+]i in matured oocytes injected with various concentrations of IP3 were investigated in this study. Porcine oocytes were recovered from the ovaries of prepubertal gilts, matured for 46-48 h and cultured in vitro for 7 days in following treatments as non-injected oocytes (NI), injected with carrier buffer, 2.5, 5 and 500 microM of IP3. The result showed that IP3 activated porcine oocytes matured in vitro (NI 3.8%, buffer 7.1%, 2.5 microM IP3 73.5%, 5 microM IP3 76.2%, 500 microM IP3 85.2%). There was a slight but not significant increase in the proportion of oocytes activated as the level of IP3 increased. The rate of development to the cleavage stage increased remarkably when the concentration of IP3 increased (NI 4.9%, buffer 5.7%, 2.5 microM IP3 30.3%, 5 microM IP3 47.1%, 500 microM IP3 78.1%). Blastocyst development was only observed in oocytes that had been injected with a higher concentration of IP3 (5 microM IP3 6.1% and 500 microM IP3 5.3%). Both the peak value and duration of [Ca2+]i rise also increased as the concentration of IP3 increased. Baseline values (ratio value, R) for [Ca2+]i ranged from 1.51 to 1.57 and was not affected by the buffer treatment. The peak value of [Ca2+]i rose significantly with increasing level of IP3 treatment (2.5 microM IP3, 3.54 +/- 0.32; 5 microM IP3, 7.50 +/- 0.37; 500 microM IP3, 8.54 +/- 0.33). Similarly, the duration of the [Ca2+]i rise increased as the level of IP3 increased (2.5 microM IP3, 43.7+/- 7.00 s; 5 microM IP3, 93.5 +/- 9.17 s; 500 microM IP3, 160.6 +/- 18.9 s). It was concluded that injected IP3 promotes the development of porcine matured oocytes and that their developmental ability is positively correlated with the rise in [Ca2+]i induced by IP3.


Assuntos
Inositol 1,4,5-Trifosfato/administração & dosagem , Oócitos/efeitos dos fármacos , Oócitos/fisiologia , Suínos/fisiologia , Animais , Blastocisto/fisiologia , Cálcio/metabolismo , Células Cultivadas , Feminino , Microinjeções
7.
Neurosci Lett ; 326(3): 167-70, 2002 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-12095648

RESUMO

In voltage-clamped guinea-pig chromaffin cells, muscarine (50 microM) or caffeine (30 mM) produced a transient intracellular Ca(2+) concentration ([Ca(2+)](i)) increase, catecholamine release and an outward K(+) current mediated through Ca(2+) released from internal Ca(2+) stores at a holding potential of -40 mV. Caffeine followed by muscarine failed to evoke these responses, while muscarine followed by caffeine was effective in producing about 30% of [Ca(2+)](i) increase and catecholamine secretion. In cells dialyzed with inositol 1,4,5-trisphosphate (IP(3)), caffeine failed to produce the [Ca(2+)](i) increase. Intracellular application of cyclic adenosine 5'-diphosphate-ribose (cADP-ribose) or 8-bromo cADP-ribose exerted no effect on the resting [Ca(2+)](i) and the caffeine-induced [Ca(2+)](i) increase. These results suggest that IP(3)-sensitive stores are functionally divided into two subpopulations, sensitive and insensitive to caffeine, and it is unlikely that cADP-ribose plays a role as a Ca(2+) releaser in guinea-pig adrenal chromaffin cells.


Assuntos
Cálcio/metabolismo , Células Cromafins/metabolismo , Glândulas Suprarrenais/efeitos dos fármacos , Glândulas Suprarrenais/fisiologia , Animais , Cafeína/farmacologia , Células Cultivadas , Estimulantes do Sistema Nervoso Central/farmacologia , Células Cromafins/efeitos dos fármacos , ADP-Ribose Cíclica/administração & dosagem , ADP-Ribose Cíclica/antagonistas & inibidores , ADP-Ribose Cíclica/farmacologia , Diálise , Cobaias , Inositol 1,4,5-Trifosfato/administração & dosagem , Inositol 1,4,5-Trifosfato/farmacologia , Masculino , Potenciais da Membrana/efeitos dos fármacos , Muscarina/farmacologia , Agonistas Muscarínicos/farmacologia , Técnicas de Patch-Clamp
8.
J Vet Sci ; 2(2): 131-7, 2001 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-14614284

RESUMO

For parthenogenetic activation as a model system of nuclear transfer, microinjection and electroporation as activation treatments in bovine metaphase II oocytes were administered to each of three groups as follows: control group (treatments with Ca2+, Mg2+ -free PBS+100 micro M EGTA), IP3 group (control+25 micro M IP3) and IP3+ ryanodine group (control+25 micro M IP3+10 mM ryanodine). In experiments using microinjection, no significant differences were observed between any of the developmental stages of the electroporation experiment. For electroporation, cleavage rates were significantly higher in the IP3+ryanodine group than in the IP3 or control group (85.6% vs 73.7% or 67.6%, respectively). In the subsequent stages of embryonic development, such as morula and blastocyst formation, the IP3 and ryanodine group exhibited significantly higher rates of morula fomation than the IP3 or control groups (40.6% vs 24.2% or 16.7%, respectively). Similarly, the rate of blastocyst formation in the IP3+ryanodine group was significantly higher than the control group (16.3% vs 6.9%) but did not differ significantly from the IP3 group (16.3% vs 9.5%). In nuclear transfer, activation was performed at 30 hpm by microinjection and elecroporation with 25 micro M IP3+ 10 mM ryanodine followed by 6-DMAP treatment. No significant differences were observed at any stage of embryonic development and none of the embryos activated by electroporation reached either the morula or blastocyst stage. However, 3.8% and 1.9% of embryos activated by microinjection sucessfully developed to the morula and blastocyst stages, respectively. In conclusion, activation treatments using IP3 and ryanodine are able to support the development of bovine parthenogenetic and reconstructed embryos.


Assuntos
Adenina/análogos & derivados , Bovinos/embriologia , Desenvolvimento Embrionário e Fetal/efeitos dos fármacos , Inositol 1,4,5-Trifosfato/farmacologia , Partenogênese/efeitos dos fármacos , Rianodina/farmacologia , Adenina/administração & dosagem , Adenina/farmacologia , Animais , Bovinos/fisiologia , Fusão Celular , Eletroporação/veterinária , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/farmacologia , Feminino , Inositol 1,4,5-Trifosfato/administração & dosagem , Microinjeções/veterinária , Técnicas de Transferência Nuclear , Oócitos/efeitos dos fármacos , Oócitos/crescimento & desenvolvimento , Inibidores de Proteínas Quinases , Rianodina/administração & dosagem , Pele/citologia
9.
Artigo em Inglês | WPRIM (Pacífico Ocidental) | ID: wpr-104743

RESUMO

For parthenogenetic activation as a model system of nuclear transfer, microinjection and electroporation as activation treatments in bovine metaphase II oocytes were administered to each of three groups as follows: control group (treatments with Ca2+, Mg2+ -free PBS+100 micro M EGTA), IP3 group (control+25 micro M IP3) and IP3+ ryanodine group (control+25 micro M IP3+10 mM ryanodine). In experiments using microinjection, no significant differences were observed between any of the developmental stages of the electroporation experiment. For electroporation, cleavage rates were significantly higher in the IP3+ryanodine group than in the IP3 or control group (85.6% vs 73.7% or 67.6%, respectively). In the subsequent stages of embryonic development, such as morula and blastocyst formation, the IP3 and ryanodine group exhibited significantly higher rates of morula fomation than the IP3 or control groups (40.6% vs 24.2% or 16.7%, respectively). Similarly, the rate of blastocyst formation in the IP3+ryanodine group was significantly higher than the control group (16.3% vs 6.9%) but did not differ significantly from the IP3 group (16.3% vs 9.5%). In nuclear transfer, activation was performed at 30 hpm by microinjection and elecroporation with 25 micro M IP3+ 10 mM ryanodine followed by 6-DMAP treatment. No significant differences were observed at any stage of embryonic development and none of the embryos activated by electroporation reached either the morula or blastocyst stage. However, 3.8% and 1.9% of embryos activated by microinjection sucessfully developed to the morula and blastocyst stages, respectively. In conclusion, activation treatments using IP3 and ryanodine are able to support the development of bovine parthenogenetic and reconstructed embryos.


Assuntos
Animais , Feminino , Adenina/administração & dosagem , Bovinos/embriologia , Fusão Celular , Eletroporação/veterinária , Desenvolvimento Embrionário e Fetal/efeitos dos fármacos , Inibidores Enzimáticos/administração & dosagem , Inositol 1,4,5-Trifosfato/administração & dosagem , Microinjeções/veterinária , Técnicas de Transferência Nuclear , Oócitos/efeitos dos fármacos , Partenogênese/efeitos dos fármacos , Inibidores de Proteínas Quinases , Rianodina/administração & dosagem , Pele/citologia
10.
EMBO J ; 19(14): 3608-17, 2000 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-10899115

RESUMO

Ca(2+) liberation by inositol 1,4,5-trisphosphate (IP(3)) is 'quantal', in that low [IP(3)] causes only partial Ca(2+) release, but further increasing [IP(3)] evokes more release. This characteristic allows cells to generate graded Ca(2+) signals, but is unexpected, given the regenerative nature of Ca(2+)-induced Ca(2+) release through IP(3) receptors. Two models have been proposed to resolve this paradox: (i) all-or-none Ca(2+) release from heterogeneous stores that empty at varying [IP(3)]; and (ii) phasic liberation from homogeneously sensitive stores. To discriminate between these hypotheses, we imaged subcellular Ca(2+) puffs evoked by IP(3) in Xenopus oocytes where release sites were functionally uncoupled using EGTA. Puffs were little changed by 300 microM intracellular EGTA, but sites operated autonomously and did not propagate waves. Photoreleased IP(3) generated flurries of puffs-different to the prolonged Ca(2+) elevation following waves in control cells-and individual sites responded repeatedly to successive increments of [IP(3)]. These data support the second hypothesis while refuting the first, and suggest that local Ca(2+) signals exhibit rapid adaptation, different to the slower inhibition following global Ca(2+) waves.


Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Cálcio/metabolismo , Inositol 1,4,5-Trifosfato/farmacologia , Animais , Soluções Tampão , Sinalização do Cálcio/efeitos da radiação , Quelantes/administração & dosagem , Quelantes/farmacologia , Ácido Egtázico/administração & dosagem , Ácido Egtázico/farmacologia , Processamento de Imagem Assistida por Computador , Inositol 1,4,5-Trifosfato/administração & dosagem , Inositol 1,4,5-Trifosfato/metabolismo , Cinética , Microinjeções , Microscopia Confocal , Modelos Biológicos , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Oócitos/efeitos da radiação , Fotólise/efeitos da radiação , Raios Ultravioleta , Xenopus laevis
11.
FEBS Lett ; 466(1): 112-4, 2000 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-10648823

RESUMO

Livers from connexin32 (Cx32) deficient mice have been shown to be defective in hormonally induced glucose mobilization. In order to determine whether this effect is due to decreased diffusion of the second messenger inositol 1,4,5-trisphosphate (IP(3)) between hepatocytes, we injected iontophoretically different amounts of IP(3) in Fura-2 loaded hepatocyte doublets (i.e. cell pairs) from wild type or Cx32 deficient mice. Whereas 84% of wild type hepatocytes showed an intercellular Ca(2+) wave spreading from the injected cell to the neighboring cell, only 25% of Cx32 deficient hepatocyte doublets did so. The amount of IP(3) necessary to induce an intercellular Ca(2+) wave in Cx32 deficient hepatocyte doublets was estimated to be about 25-fold higher than in wild type doublets. This confirms the notion that the low hormonally or electrically induced glucose mobilization found in Cx32 deficient livers relative to wild type livers is due to largely hindered diffusion of IP(3) between Cx32 deficient hepatocytes.


Assuntos
Conexinas/deficiência , Junções Comunicantes/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Fígado/metabolismo , Animais , Permeabilidade da Membrana Celular , Conexinas/genética , Conexinas/metabolismo , Feminino , Glucose/metabolismo , Técnicas In Vitro , Inositol 1,4,5-Trifosfato/administração & dosagem , Iontoforese , Fígado/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Sistemas do Segundo Mensageiro , Proteína beta-1 de Junções Comunicantes
12.
EMBO J ; 18(12): 3222-31, 1999 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-10369663

RESUMO

Intercellular Ca2+ signaling in intact salivary glands of the blowfly Calliphora erythrocephala was studied by fluorimetric digital imaging combined with microinjection of putative messenger molecules. Iontophoretic injection of D-myo-inositol 1,4, 5-trisphosphate (InsP3) into salivary gland cells evoked regenerative intercellular Ca2+ waves that spread through the impaled cell and several rows of surrounding cells. Ca2+ increases induced by microinjection of Ca2+ ions were confined to the injected cells and their nearest neighbors. Depletion of intracellular Ca2+ stores by thapsigargin pre-treatment did not alter the time course of the Ca2+ increase caused by Ca2+ injection. However, activation of Ca2+ release became clearly evident when Ca2+ was injected in the presence of serotonin (5-HT). Under these conditions, injection of Ca2+ triggered intercellular Ca2+ waves that consecutively passed through >10 cells. The phospholipase C inhibitor U73122 blocked 5-HT-induced Ca2+ increases but did not affect InsP3-dependent Ca2+ spiking and intercellular Ca2+ wave propagation. The results demonstrate that propagation of agonist-evoked Ca2+ waves in the blowfly salivary gland requires supra-basal [InsP3] but does not depend on feedback activation of phospholipase C. We conclude that the intra- and intercellular transmission of these Ca2+ waves is mediated by diffusion of Ca2+ and Ca2+-induced Ca2+ release via the InsP3 receptor channel.


Assuntos
Sinalização do Cálcio , Dípteros/metabolismo , Animais , Cálcio/administração & dosagem , Cálcio/metabolismo , Cálcio/farmacologia , Canais de Cálcio/fisiologia , Sinalização do Cálcio/efeitos dos fármacos , Difusão , Ativação Enzimática , Estrenos/farmacologia , Retroalimentação , Técnicas In Vitro , Inositol 1,4,5-Trifosfato/administração & dosagem , Inositol 1,4,5-Trifosfato/farmacologia , Receptores de Inositol 1,4,5-Trifosfato , Microinjeções , Pirrolidinonas/farmacologia , Receptores Citoplasmáticos e Nucleares/fisiologia , Glândulas Salivares/citologia , Glândulas Salivares/efeitos dos fármacos , Glândulas Salivares/enzimologia , Glândulas Salivares/metabolismo , Serotonina/farmacologia , Antagonistas da Serotonina/farmacologia , Tapsigargina/farmacologia , Fatores de Tempo , Fosfolipases Tipo C/antagonistas & inibidores , Fosfolipases Tipo C/metabolismo
13.
Biochem J ; 318 ( Pt 3): 849-55, 1996 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-8836128

RESUMO

The effect of cGMP on noradrenaline-induced intracellular Ca2+ mobilization was investigated in whole-cell voltage-clamped guinea-pig hepatocytes. Treatment of the cells with 8-Br-cGMP (1-500 microM) resulted in an increase in the sensitivity of the cells to noradrenaline and to inositol 1,4,5-trisphosphate (InsP3) photo-released from caged InsP3. The positive effect of 8-Br-cGMP on the Ca2+ release evoked by Ca(2+)-mobilizing agonists or InsP3 was blocked by a protein kinase G (PKG; cGMP-dependent protein kinase) inhibitor, the RP-8-(4-chlorophenylthio)guanosine 3':5'-monophosphorothioate. 8-Br-cGMP affected neither the basal InsP3 concentration nor the noradrenaline-induced production of InsP3. In permeabilized hepatocytes, the dose-response curve for InsP3-induced Ca2+ release was shifted to the left in the presence of 8-Br-cGMP. Furthermore, the treatment with 8-Br-cGMP did not affect the Ca2+ content of the InsP3-sensitive Ca2+ stores. These results indicate that intracellular cGMP potentiates the noradrenaline-induced Ca2+ response by enhancing Ca2+ release from the intracellular Ca2+ stores. We suggest that cGMP increases the apparent affinity of InsP3 receptors for InsP3 in guinea-pig hepatocytes probably by phosphorylation via the activation of PKG.


Assuntos
Cálcio/metabolismo , GMP Cíclico/análogos & derivados , Inositol 1,4,5-Trifosfato/farmacologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Animais , Cálcio/farmacologia , Canais de Cálcio/metabolismo , GMP Cíclico/administração & dosagem , GMP Cíclico/metabolismo , GMP Cíclico/farmacologia , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Sinergismo Farmacológico , Cobaias , Técnicas In Vitro , Inositol 1,4,5-Trifosfato/administração & dosagem , Receptores de Inositol 1,4,5-Trifosfato , Líquido Intracelular/metabolismo , Cinética , Masculino , Norepinefrina/administração & dosagem , Norepinefrina/farmacologia , Fotólise , Receptores Citoplasmáticos e Nucleares/metabolismo
14.
Cell Calcium ; 20(3): 243-55, 1996 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-8894271

RESUMO

Continuous perfusion of immobilized electropermeabilized SH-SY5Y neuroblastoma cells was utilised as a novel approach to the assessment of incremental activation and inactivation of myo-inositol 1,4,5-trisphosphate (IP3)-induced calcium (Ca2+) mobilisation (IICM). SH-SY5Y cells when stimulated with sub-optimal IP3 exhibited a rapid concentration dependent activation of Ca2+ mobilization followed by a partial inactivation. Although this partial inactivation allowed net Ca2+ mobilized to be stringently returned to basal levels, a concentration-dependent depletion of the store was maintained while ever perfusion with the stimulating IP3 concentration was sustained. This partial inactivation of IP3-induced quantal Ca2+ release (QCR) was only compromised if cells, with replete Ca2+ stores, were perfused with supra-maximally effective concentrations of IP3 (5-10 microM). Thus, at supra-optimal IP3 concentrations, a reproducible plateau of Ca2+ release lying 50-150 nM above the basal Ca2+ concentration was observed. Feedback on IP3R sensitivity by gross cytosolic Ca2+ levels could be eliminated as the sustained and exclusive mediator of incremental activation/inactivation cycle of IICM in SH-SY5Y cells, since released Ca2+ was perfused away from the immobilized cells. Thus, while ever the cells were continuously perfused with IP3, impressive incremental inactivation was apparent. Additionally, IP3R partial agonists were found to exhibit lower intrinsic activity for both activation and inactivation of QCR, suggesting that ligand-induced inactivation of the IP3R was more important than inactivation mechanisms reliant on either Ca2+ flux through the channel and/or calcium store depletion. Therefore, we suggest that, in perfused SH-SY5Y cells, the most parsimonious explanation of our data is that IP3 binding probably activates and then partially inactivates its receptor in a concentration-dependent fashion to produce the QCR phenomenon.


Assuntos
Cálcio/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Neuroblastoma/metabolismo , Linhagem Celular , Humanos , Inositol 1,4,5-Trifosfato/administração & dosagem , Perfusão
15.
Arch Int Pharmacodyn Ther ; 331(3): 221-31, 1996.
Artigo em Inglês | MEDLINE | ID: mdl-9124995

RESUMO

The effects of the antimalarial drug, mefloquine, on the uptake and release of Ca2+ by crude microsomes from dog brain were investigated using a spectrophotometric method. Mefloquine inhibited the inositol-1,4,5-phosphate (IP3)-induced Ca2+ release with an IC50 of 42 microM, but was a weaker inhibitor of the uptake of Ca2+ into the vesicles (IC50: 272 microM). These effects of mefloquine are in contrast to its actions on Ca2+ uptake and release by skeletal muscle microsomes, where its predominant effect was seen to be the inhibition of Ca2+ uptake into the vesicles. Mefloquine was found to be more potent than quinine as a specific inhibitor of Ca2+ release from IP3-sensitive stores in dog brain microsomes. The possibility of the drug affecting cellular IP3-linked signal transduction processes should be considered.


Assuntos
Antimaláricos/farmacologia , Encéfalo/efeitos dos fármacos , Cálcio/metabolismo , Mefloquina/farmacologia , Animais , Encéfalo/metabolismo , Cães , Inositol 1,4,5-Trifosfato/administração & dosagem , Inositol 1,4,5-Trifosfato/antagonistas & inibidores , Microssomos/efeitos dos fármacos , Quinina/farmacologia
16.
Mol Pharmacol ; 49(2): 360-4, 1996 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-8632770

RESUMO

Microinjection, but not extracellular application, of cytidine-5'-diphosphate-D-glucose (CDPG) has been shown to elicit Ca(2+)-dependent currents in Xenopus laevis oocytes. These responses were comparable to those of inositol-1,4,5-trisphosphate (InsP3) in being both rapid and dose dependent. For example, maximal amplitudes of CDPG-induced current were similar (approximately 365 +/- 75 nA at 1 microM CDPG) to those of InsP3. The CDPG currents were insensitive to removal of extracellular Ca2+, indicating the dependence on Ca2+ release from intracellular Ca2+ stores but not on Ca2+ entry through plasma membrane. CDPG-induced currents were reduced or abolished by pretreatment with thapsigargin, by injection of the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, or by extracellular perfusion of the Cl- channel blocker niflumic acid but were insensitive to injection of the InsP3 antagonist heparin. These results suggest that CDPG induces Ca2+ discharge from intracellular Ca2+ stores via a mechanism distinct from that of InsP3 in Xenopus oocytes. Another pyrimidine nucleotide-glucose derivative, uridine-5'-diphosphate-alpha-D-glucose, also induced Ca(2+)-dependent currents, but the activity was lower than that of CDPG (maximal amplitude, 272 +/- 62 nA). Other nucleotide-glucose compounds (adenosine-5'-diphosphate-D-glucose, guanosine-5'-diphosphate-D-glucose, and thymidine-5'-diphosphate-D-glucose) had no current responses when injected into oocytes. After injection of CDPG, CDPG-induced Ca2+ release appeared to couple to a Ca2+ entry pathway similar to that coupled to InsP3. These results indicate that pyrimidine nucleotide-glucose conjugates may provide novel pharmacological tools for the study of Ca2+ signaling in oocytes.


Assuntos
Cálcio/farmacologia , Membrana Celular/fisiologia , Inositol 1,4,5-Trifosfato/farmacologia , Açúcares de Nucleosídeo Difosfato/farmacologia , Oócitos/fisiologia , Adenosina Difosfato Glucose/farmacologia , Animais , Membrana Celular/efeitos dos fármacos , Quelantes/farmacologia , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Feminino , Glucose/análogos & derivados , Glucose/farmacologia , Glicina/análogos & derivados , Glicina/farmacologia , Açúcares de Guanosina Difosfato/farmacologia , Inositol 1,4,5-Trifosfato/administração & dosagem , Cinética , Potenciais da Membrana/efeitos dos fármacos , Microinjeções , Oócitos/efeitos dos fármacos , Sulfetos , Tiofenos , Uridina Difosfato Glucose/farmacologia , Xenopus
17.
Biophys J ; 69(6): 2378-91, 1995 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-8599644

RESUMO

To study the initial kinetics of Ins(1,4,5)P3-induced [Ca2+]i elevations with a high time resolution and to avoid the problem of cell-to-cell heterogeneity, we have used the combined patch-clamp/microfluorimetry technique. The mathematical description of the microperfusion of Ins(1,4,5)P3 and the subsequent Ca2+ release consists of a monoexponential decay (cytosolic Ins(1,4,5)P3 concentration) and a Hill equation (Ins(1,4,5)P3 dose-response curve). Two additional Hill equations and an integration were necessary to include a putative dependence of Ins(1,4,5)P3-induced Ca2+ release on [Ca2+]i. Best-fitting analysis assuming [Ca2+]i-independent Ca2+ release yielded Hill coefficients between 4 and 12. The high cooperativity was also observed with the poorly metabolizable analog Ins(2,4,5)P3 and was independent of extracellular [Ca2+]. Best-fitting analysis including a positive [Ca2+]i feedback suggested a cooperativity on the level of Ins(1,4,5)P3-induced channel opening (n = 2) and an enhancement of Ins(1,4,5)P3-induced Ca2+ release by [Ca2+]i. In summary, the onset kinetics of Ins(1,4,5)P3-induced [Ca2+]i elevations in single HL-60 granulocytes showed a very high cooperativity, presumably because of a cooperativity on the level of channel opening and a positive Ca2+ feedback, but not because of Ca2+ influx or Ins(1,4,5)P3 metabolism. This high cooperativity, acting in concert with negative feedback mechanisms, might play an important role in the fine-tuning of the cellular Ca2+ signal.


Assuntos
Cálcio/metabolismo , Inositol 1,4,5-Trifosfato/farmacologia , Radioisótopos de Cálcio , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular , Corantes Fluorescentes , Fura-2 , Células HL-60 , Humanos , Inositol 1,4,5-Trifosfato/administração & dosagem , Inositol 1,4,5-Trifosfato/metabolismo , Cinética , Matemática , Microquímica , Modelos Teóricos , Técnicas de Patch-Clamp , Perfusão/métodos , Frações Subcelulares/metabolismo , Fatores de Tempo
18.
Cardiovasc Res ; 30(6): 905-14, 1995 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-8746205

RESUMO

OBJECTIVE: We studied the effect of calcium on inositol 1,4,5-trisphosphate (InsP3)-induced contractions in saponin-permeabilized chick atrial muscle (80-100 microns in diameter). Calcium has been proposed to modulate InsP3-induced response and InsP3 binding in other tissue. METHODS: A transient increase in tension was used to detect the release of calcium in this multicellular preparation. Pulsed (2-3 s) superfusion of either calcium or InsP3 produced a transient contraction. RESULTS: Pulsed coapplication of both InsP3 and calcium resulted in a contraction greater than that predicted by an additive effect of both intracellular messengers. The release of calcium by InsP3 is positively and negatively modulated by myoplasmic calcium (EC50 congruent to 0.17 microM and IC50 of approximately 1.5 microM). The peak potentiation occurred at approximately pCa = 6.3 (0.51 microM free calcium). CONCLUSIONS: These observations indicate that an increase of InsP3 in the heart, as produced by cardiac neurotransmitters and hormones, may regulate the force of contraction by virtue of its synergistic action with calcium. While calcium remains the primary trigger for calcium release during excitation-contraction (E-C) coupling, we propose that changes in cytoplasmic calcium can modulate InsP3-induced calcium release on a beat-to-beat basis. Thus, InsP3 may regulate force generation during E-C coupling by activating calcium release during a heart beat.


Assuntos
Cálcio/farmacologia , Inositol 1,4,5-Trifosfato/farmacologia , Contração Miocárdica/efeitos dos fármacos , Animais , Cálcio/administração & dosagem , Permeabilidade da Membrana Celular , Galinhas , Citoplasma/metabolismo , Interações Medicamentosas , Eletrofisiologia , Átrios do Coração , Técnicas In Vitro , Inositol 1,4,5-Trifosfato/administração & dosagem , Miocárdio/metabolismo , Estimulação Química
19.
Biochem J ; 312 ( Pt 3): 789-94, 1995 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-8554521

RESUMO

Submaximal concentrations of inositol 1,4,5-trisphosphate (InsP3) rapidly release only a fraction of the InsP3-sensitive intracellular Ca2+ stores, despite the ability of further increases in InsP3 concentration to evoke further Ca2+ release. The mechanisms underlying such quantal Ca2+ mobilization are not understood, but have been proposed to involve regulatory effects of cytosolic Ca2+ on InsP3 receptors. By examining complete concentration-effect relationships for InsP3-stimulated 45Ca2+ efflux from the intracellular stores of permeabilized hepatocytes, we demonstrate that, at 37 degrees C, responses to InsP3 are quantal in Ca(2+)-free media heavily buffered with either EGTA or BAPTA [1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid]. Lower concentrations of InsP3 were used to examine the kinetics of Ca2+ mobilization at 2 degrees C, because at the lower temperature the stores were more sensitive to InsP3: the concentration of InsP3 causing half-maximal Ca2+ release (EC50) after a 30 s incubation decreased from 281 +/- 37 nM at 37 degrees C to 68 +/- 3 nM at 2 degrees C. At 2 degrees C, the EC50 for InsP3-stimulated Ca2+ mobilization decreased as the duration of exposure to InsP3 was increased: the EC50 was 68 +/- 3 nM after 30 s, and 29 +/- 2 nM after 420 s. InsP3-stimulated Ca2+ mobilization is therefore non-quantal at 2 degrees C: InsP3 concentration determines the rate, but not the extent, of Ca2+ release. By initiating quantal responses to InsP3 at 37 degrees C and then simultaneously diluting and chilling cells to 2 degrees C, we demonstrated that the changes that underlie quantal responses do not rapidly reverse at 2 degrees C. At both 37 degrees C and 2 degrees C, modest increases in cytosolic Ca2+ increased the sensitivity of the stores to InsP3, whereas further increases were inhibitory; both Ca2+ effects persisted after prior removal of ATP. We conclude that the effects of Ca2+ on InsP3 receptors are unlikely either to be enzyme-mediated or to underlie the quantal pattern of Ca2+ release evoked by InsP3.


Assuntos
Canais de Cálcio/fisiologia , Cálcio/metabolismo , Inositol 1,4,5-Trifosfato/farmacologia , Fígado/metabolismo , Receptores Citoplasmáticos e Nucleares/fisiologia , Trifosfato de Adenosina/farmacologia , Animais , Quelantes/farmacologia , Relação Dose-Resposta a Droga , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Inositol 1,4,5-Trifosfato/administração & dosagem , Receptores de Inositol 1,4,5-Trifosfato , Cinética , Fígado/efeitos dos fármacos , Masculino , Ratos , Ratos Wistar , Temperatura
20.
Neuroreport ; 6(10): 1434-6, 1995 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-7488742

RESUMO

The C57BL/6J-bgJ/bgJ (beige-J) mutation imparts a blunted response to intracerebroventricular (i.c.v.) morphine in the tail-flick test, without altered micro-opioid receptor number or morphine affinity. We now report that co-administration of IP3 (36.1 nmol) restored morphine responsiveness of beige-J mice to essentially that of normal littermates (bg+/bg-; ED50 = 3.9 and 3.5 nmol, respectively). IP3 had no effect on morphine-induced antinociception in control animals. Neither myoinositol at 36.1 nmol nor IP6 at the highest testable dose (4.5 nmol) had a similar effect. Myo-inositol at 5.5 mumol restored beige-J responsiveness to that of littermates. These findings implicate some component of the phosphoinositide cycle in the antinociceptive defect of beige-J mice.


Assuntos
Analgésicos/farmacologia , Inositol 1,4,5-Trifosfato/farmacologia , Inositol/farmacologia , Dor/genética , Animais , Relação Dose-Resposta a Droga , Injeções Intraventriculares , Inositol/administração & dosagem , Inositol 1,4,5-Trifosfato/administração & dosagem , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Morfina/farmacologia , Dor/psicologia , Medição da Dor/efeitos dos fármacos , Tempo de Reação/efeitos dos fármacos , Sistemas do Segundo Mensageiro/fisiologia
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