RESUMO
T-type Ca(2+) channel family includes three subunits Ca(V)3.1, Ca(V)3.2 and Ca(V)3.3 and have been shown to control burst firing and intracellular Ca(2+) concentration ([Ca(2+)](i)) in neurons. Here, we investigated whether Ca(V)3.1 channels could generate a pacemaker current and contribute to cell excitability. Ca(V)3.1 clones were over-expressed in the neuronal cell line NG108-15. Ca(V)3.1 channel expression induced repetitive action potentials, generating spontaneous membrane potential oscillations (MPOs) and concomitant [Ca(2+)](i) oscillations. These oscillations were inhibited by T-type channels antagonists and were present only if the membrane potential was around -61mV. [Ca(2+)](i) oscillations were critically dependent on Ca(2+) influx through Ca(V)3.1 channels and did not involve Ca(2+) release from the endoplasmic reticulum. The waveform and frequency of the MPOs are constrained by electrophysiological properties of the Ca(V)3.1 channels. The trigger of the oscillations was the Ca(V)3.1 window current. This current induced continuous [Ca(2+)](i) increase at -60mV that depolarized the cells and triggered MPOs. Shifting the Ca(V)3.1 window current potential range by increasing the external Ca(2+) concentration resulted in a corresponding shift of the MPOs threshold. The hyperpolarization-activated cation current (I(h)) was not required to induce MPOs, but when expressed together with Ca(V)3.1 channels, it broadened the membrane potential range over which MPOs were observed. Overall, the data demonstrate that the Ca(V)3.1 window current is critical in triggering intrinsic electrical and [Ca(2+)](i) oscillations.
Assuntos
Canais de Cálcio Tipo T/metabolismo , Sinalização do Cálcio , Espaço Intracelular/metabolismo , Animais , Linhagem Celular , Ativação do Canal Iônico , Cinética , Potenciais da Membrana , Camundongos , Ratos , TransfecçãoRESUMO
Local Ca2+ rises and propagated Ca2+ signals represent different patterns that are differentially decoded for fine tuning cellular signalling. This Ca2+ concentration plasticity is absolutely required to allow adaptation to different needs of the cells ranging from contraction or increased learning to proliferation and cell death. A wide diversity of molecular structures and specific location of Ca2+ signalling molecules confer spatial and temporal versatility to the Ca2+ changes allowing specific cellular responses to be elicited. Various types of local Ca2+ signals have been described. Ca2+ spikes correspond to Ca2+ signals spanning several micrometers but displaying limited propagation into a cell leading to regulation of cellular functions in one particular zone of this cell. This is of particular relevance in cells presenting distinct morphological specializations, i.e. apical versus basal sites or dendritic versus somatic/axonal sites. More stereotyped elementary Ca2+ events (denominated Ca2+ sparks or Ca2+ puffs depending on the type of endoplasmic reticulum Ca2+ release channel involved) are highly confined and non-propagated Ca2+ rises which are observed in the close neighbouring of the Ca2+ channels. These elementary Ca2+ events play a major role in controlling cellular excitability. Elementary Ca2+ events involve Ca2+ release channels such as the ryanodine receptors (RyRs) and the inositol 1,4,5-trisphosphate receptors (InsP3Rs). The molecular bases underlying the various local Ca2+ release events will be discussed by reviewing the channels and particularly the different isoforms of RyRs and InsP3Rs and their role in inducing localized Ca2+ responses. These calcium release events are controlled by various second messengers and are regulated by Ca2+ channel-associated proteins, intra-luminal Ca2+ content of the endoplasmic reticulum (ER) and other Ca2+ organelles. We will discuss on how the control of local cellular Ca2+ content may account for cellular functions in physiological and physiopathological conditions.
Assuntos
Sinalização do Cálcio , Animais , HumanosRESUMO
Subtype 3 of the ryanodine receptor (RYR3) is a ubiquitous Ca2+ release channel which is predominantly expressed in smooth muscle tissues and certain regions of the brain. We show by reverse transcription-polymerase chain reaction (RT-PCR) that non-pregnant mouse myometrial cells expressed only RYR3 and therefore could be a good model for studying the role of endogenous RYR3. Expression of RYR3 was confirmed by Western blotting and immunostaining. Confocal Ca2+ measurements revealed that in 1.7 mM extracellular Ca2+, neither caffeine nor photolysis of caged Ca2+ were able to trigger any Ca2+ responses, whereas in the same cells oxytocin activated propagated Ca2+ waves. However, under conditions of increased sarcoplasmic reticulum (SR) Ca2+ loading, brought about by superfusing myometrial cells in 10 mM extracellular Ca2+, all the myometrial cells responded to caffeine and photolysis of caged Ca2+, indicating that it was possible to activate RYR3. The caffeine-induced Ca2+ responses were inhibited by intracellular application of an anti-RYR3-specific antibody. Immunodetection of RYR3 with the same antibody revealed a rather homogeneous distribution of fluorescence in confocal cell sections. In agreement with these observations, spontaneous or triggered Ca2+ sparks were not detected. In conclusion, our results suggest that under conditions of increased SR Ca2+ loading, endogenous RYR3 may contribute to the Ca2+ responses of myometrial cells.
Assuntos
Canal de Liberação de Cálcio do Receptor de Rianodina/fisiologia , Animais , Anticorpos/farmacologia , Cafeína/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Células Cultivadas , Ácido Edético/análogos & derivados , Ácido Edético/farmacologia , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Miométrio/citologia , Miométrio/fisiologia , Ocitocina/farmacologia , Fotólise , Canal de Liberação de Cálcio do Receptor de Rianodina/imunologiaRESUMO
Heterodimeric class I phosphoinositide 3-kinase (PI3K) has been shown to be involved in the stimulation of voltage-gated Ca(2+) channels by various mediators. In this study, we bring evidences that vascular L-type Ca(2+) channels can be modulated by both tyrosine kinase-regulated class Ia and G protein-regulated class Ib PI3Ks. Purified recombinant PI3Ks increased the peak Ca(2+) channel current density when applied intracellularly. Furthermore, PI3Kalpha-, beta-, and delta-mediated stimulations of Ca(2+) channel currents were increased by preactivation by a phosphotyrosyl peptide, whereas PI3Kgamma- and beta-mediated effects were increased by Gbetagamma. In freshly isolated and cultured vascular myocytes, angiotensin II and Gbetagamma stimulated L-type Ca(2+) channel current. In contrast, platelet-derived growth factor (PDGF)-BB and the phosphotyrosyl peptide did not stimulate Ca(2+) channel current in freshly isolated cells despite the presence of endogenous PDGF receptors and PI3Kalpha and PI3Kgamma. Interestingly, when endogenous PI3Kbeta expression arose in cultured myocytes, both PDGF and phosphotyrosyl peptide stimulated Ca(2+) channels through PI3Kbeta, as revealed by the inhibitory effect of an anti-PI3Kbeta antibody. These results suggest that endogenous PI3Kbeta but not PI3Kalpha is specifically involved in PDGF receptor-induced stimulation of Ca(2+) channels and that different isoforms of PI3K regulate physiological increases of Ca(2+) influx in vascular myocytes stimulated by vasoconstrictor or growth factor.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Músculo Liso Vascular/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Angiotensina II/farmacologia , Animais , Bário/metabolismo , Bário/farmacologia , Becaplermina , Cálcio/metabolismo , Canais de Cálcio Tipo L/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Produtos do Gene env/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Transporte de Íons/efeitos dos fármacos , Iontoforese , Isoenzimas/genética , Isoenzimas/metabolismo , Isoenzimas/farmacologia , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Técnicas de Patch-Clamp , Fragmentos de Peptídeos/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/farmacologia , Fator de Crescimento Derivado de Plaquetas/farmacologia , Proteínas Proto-Oncogênicas c-sis , Ratos , Ratos Wistar , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Transfecção , Vasoconstritores/farmacologiaRESUMO
Previous results have shown that in rat portal vein myocytes the betagamma dimer of the G(13) protein transduces the angiotensin II-induced stimulation of calcium channels and increase in intracellular Ca(2+) concentration through activation of phosphoinositide 3-kinase (PI3K). In the present work we determined which class I PI3K isoforms were involved in this regulation. Western blot analysis indicated that rat portal vein myocytes expressed only PI3Kalpha and PI3Kgamma and no other class I PI3K isoforms. In the intracellular presence of an anti-p110gamma antibody infused by the patch clamp pipette, both angiotensin II- and Gbetagamma-mediated stimulation of Ca(2+) channel current were inhibited, whereas intracellular application of an anti-p110alpha antibody had no effect. The anti-PI3Kgamma antibody also inhibited the angiotensin II- and Gbetagamma-induced production of phosphatidylinositol 3,4,5-trisphosphate. In Indo-1 loaded cells, the angiotensin II-induced increase in [Ca(2+)](i) was inhibited by intracellular application of the anti-PI3Kgamma antibody, whereas the anti-PI3Kalpha antibody had no effect. The specificity of the anti-PI3Kgamma antibody used in functional experiments was ascertained by showing that this antibody did not recognize recombinant PI3Kalpha in Western blot experiments. Moreover, anti-PI3Kgamma antibody inhibited the stimulatory effect of intracellularly infused recombinant PI3Kgamma on Ca(2+) channel current without altering the effect of recombinant PI3Kalpha. Our results show that, although both PI3Kgamma and PI3Kalpha are expressed in vascular myocytes, the angiotensin II-induced stimulation of vascular L-type calcium channel and increase of [Ca(2+)](i) involves only the PI3Kgamma isoform.
Assuntos
Angiotensina II/farmacologia , Canais de Cálcio Tipo L/fisiologia , Isoenzimas/metabolismo , Músculo Liso Vascular/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Animais , Anticorpos/farmacologia , Bário/farmacologia , Western Blotting , Cálcio/metabolismo , Canais de Cálcio Tipo L/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Membrana Celular/fisiologia , Classe Ib de Fosfatidilinositol 3-Quinase , Técnicas In Vitro , Isoenzimas/isolamento & purificação , Cinética , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Microssomos/enzimologia , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Técnicas de Patch-Clamp , Dibutirato de 12,13-Forbol/farmacologia , Fosfatidilinositol 3-Quinases/isolamento & purificação , Veia Porta/fisiologia , Subunidades Proteicas , Ratos , Proteínas Recombinantes/metabolismoRESUMO
1. Previous data have shown that activation of beta(3)-adrenoceptors stimulates vascular L-type Ca(2+) channels through a G alphas-induced stimulation of the cyclic AMP/PKA pathway. The present study investigated whether beta-adrenergic stimulation also uses the G beta gamma/PI3K/PKC pathway to modulate L-type Ca(2+) channels in rat portal vein myocytes. 2. Peak Ba(2+) current (I(Ba)) measured using the whole-cell patch clamp method was maximally increased by application of 10 microm isoprenaline after blockade of beta(3)-adrenoceptors by 1 microM SR59230A. Under these conditions, the isoprenaline-induced stimulation of I(Ba) was reversed by ICI-118551 (a specific beta(2)-adrenoceptor antagonist) but not by atenolol (a specific beta(1)-adrenoceptor antagonist). The beta(2)-adrenoceptor agonist salbutamol increased I(Ba), an effect which was reversed by ICI-118551 whereas the beta(1)-adrenoceptor agonist dobutamine had no effect on I(Ba). 3. Application of PKA inhibitors (H-89 and Rp 8-Br-cyclic AMPs) or a PKC inhibitor (calphostin C) alone did not affect the beta(2)-adrenergic stimulation of I(Ba) whereas simultaneous application of both PKA and PKC inhibitors completely blocked this stimulation. 4. The beta(2)-adrenergic stimulation of L-type Ca(2+) channels was blocked by a pre-treatment with cholera toxin and by intracellular application of an anti-G alphas antibody (directed against the carboxyl terminus of G alphas). In the presence of H-89, intracellular infusion of an anti-Gss(com) antibody or a beta ARK(1) peptide as well as a pre-treatment with wortmannin (a PI3K inhibitor) blocked the beta(2)-adrenergic stimulation of I(Ba). 5. These results suggest that the beta(2)-adrenergic stimulation of vascular L-type Ca(2+) channels involves both G alphas and G beta gamma subunits which exert their stimulatory effects through PKA and PI3K/PKC pathways, respectively.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Músculo Liso Vascular/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Animais , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Técnicas In Vitro , Músculo Liso Vascular/enzimologia , Veia Porta/citologia , Veia Porta/metabolismo , Proteína Quinase C/metabolismo , Ratos , Transdução de SinaisRESUMO
Using an antisense strategy, we have previously shown that in vascular myocytes, subtypes 1 and 2 of ryanodine receptors (RYRs) are required for Ca(2+) release during Ca(2+) sparks and global Ca(2+) responses, evoked by activation of voltage-gated Ca(2+) channels, whereas RYR subtype 3 (RYR3) has no contribution. Here, we investigated the effects of increased Ca(2+) loading of the sarcoplasmic reticulum (SR) on the RYR-mediated Ca(2+) responses and the role of the RYR3 by injecting antisense oligonucleotides targeting the RYR subtypes. RYR3 expression was demonstrated by immunodetection in both freshly dissociated and cultured rat portal vein myocytes. Confocal Ca(2+) measurements revealed that the number of cells showing spontaneous Ca(2+) sparks was strongly increased by superfusing the vascular myocytes in 10 mm Ca(2+)-containing solution. These Ca(2+) sparks were blocked after inhibition of RYR1 or RYR2 by treatment with antisense oligolucleotides but not after inhibition of RYR3. In contrast, inhibition of RYR3 reduced the global Ca(2+) responses induced by caffeine and phenylephrine, indicating that RYR3 participated together with RYR1 and RYR2 to these Ca(2+) responses in Ca(2+)-overloaded myocytes. Ca(2+) transients evoked by photolysis of caged Ca(2+) with increasing flash intensities were also reduced after inhibition of RYR3 and revealed that the [Ca(2+)](i) sensitivity of RYR3 would be similar to that of RYR1 and RYR2. Our results show that, under conditions of increased SR Ca(2+) loading, the RYR3 becomes activable by caffeine and local increases in [Ca(2+)](i).
Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Músculo Liso Vascular/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Animais , Oligonucleotídeos Antissenso , Veia Porta , Ratos , Transdução de SinaisRESUMO
Localized Ca(2+)-release signals (puffs) and propagated Ca(2+) waves were characterized in rat ureteric myocytes by confocal microscopy. Ca(2+) puffs were evoked by photorelease of low concentrations of Ins(1,4,5)P(3) from a caged precursor and by low concentrations of acetylcholine; they were also observed spontaneously in Ca(2+)-overloaded myocytes. Ca(2+) puffs showed some variability in amplitude, time course and spatial spread, suggesting that Ins(1,4,5)P(3)-gated channels exist in clusters containing variable numbers of channels and that within these clusters a variable number of channels can be recruited. Immunodetection of Ins(1,4,5)P(3) receptors revealed the existence of several spots of fluorescence in the confocal cell sections, supporting the existence of clusters of Ins(1,4,5)P(3) receptors. Strong Ins(1,4,5)P(3) photorelease and high concentrations of acetylcholine induced Ca(2+) waves that originated from an initiation site and propagated in the whole cell by spatial recruitment of neighbouring Ca(2+)-release sites. Both Ca(2+) puffs and Ca(2+) waves were blocked selectively by intracellular applications of heparin and an anti-Ins(1,4,5)P(3)-receptor antibody, but were unaffected by ryanodine and intracellular application of an anti-ryanodine receptor antibody. mRNAs encoding for the three subtypes of Ins(1,4,5)P(3) receptor and subtype 3 of ryanodine receptor were detected in these myocytes, and the maximal binding capacity of [(3)H]Ins(1,4,5)P(3) was 10- to 12-fold higher than that of [(3)H]ryanodine. These results suggest that Ins(1,4,5)P(3)-gated channels mediate a continuum of Ca(2+) signalling in smooth-muscle cells expressing a high level of Ins(1,4,5)P(3) receptors and no subtypes 1 and 2 of ryanodine receptors.
Assuntos
Cálcio/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Monócitos/enzimologia , Monócitos/metabolismo , Uretra/citologia , Acetilcolina/farmacologia , Animais , Membrana Celular/metabolismo , Cerebelo/citologia , Relação Dose-Resposta a Droga , Immunoblotting , Imuno-Histoquímica , Cinética , Luz , Microscopia Confocal , Microscopia de Fluorescência , Microssomos/metabolismo , Ligação Proteica , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rianodina/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Transdução de Sinais , Uretra/enzimologia , Uretra/metabolismoRESUMO
In this study, Gbeta specificity in the regulation of Gbetagamma-sensitive phosphoinositide 3-kinases (PI3Ks) and phospholipase Cbeta (PLCbeta) isozymes was examined. Recombinant mammalian Gbeta(1-3)gamma(2) complexes purified from Sf9 membranes stimulated PI3Kgamma lipid kinase activity with similar potency (10-30 nm) and efficacy, whereas transducin Gbetagamma was less potent. Functionally active Gbeta(5)gamma(2) dimers were purified from Sf9 cell membranes following coexpression of Gbeta(5) and Ggamma(2-His). This preparation as well as Gbeta(1)gamma(2-His) supported pertussis toxin-mediated ADP-ribosylation of Galpha(i1). Gbeta(1)gamma(2-His) stimulated PI3Kgamma lipid and protein kinase activities at nanomolar concentrations, whereas Gbeta(5)gamma(2-His) had no effect. Accordingly, Gbeta(1)gamma(2-His), but not Gbeta(5)gamma(2-His), significantly stimulated the lipid kinase activity of PI3Kbeta in the presence or absence of tyrosine-phosphorylated peptides derived from the p85-binding domain of the platelet derived-growth factor receptor. Conversely, both preparations were able to stimulate PLCbeta(2) and PLCbeta(1). However, Gbeta(1)gamma(2-His), but not Gbeta(5)gamma(2-His), activated PLCbeta(3). Experimental evidence suggests that the mechanism of Gbeta(5)-dependent effector selectivity may differ between PI3K and PLCbeta. In conclusion, these data indicate that Gbeta subunits are able to discriminate among effectors independently of Galpha due to selective protein-protein interaction.
Assuntos
Subunidades beta da Proteína de Ligação ao GTP , Subunidades gama da Proteína de Ligação ao GTP , Proteínas de Ligação ao GTP/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP , Isoenzimas/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosfolipídeos/metabolismo , Fosfolipases Tipo C/metabolismo , Ativação Enzimática , Humanos , Fosfolipase C beta , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
1. The effects of beta(3)-adrenergic stimulation were studied on the L-type Ca(2+) channel in single myocytes from rat portal vein using the whole-cell mode of the patch-clamp technique. 2. Reverse transcription-polymerase chain reaction showed that beta(1)-, beta(2)- and beta(3)-adrenoceptor subtypes were expressed in rat portal vein myocytes. Application of both propranolol (a non-selective beta(1)- and beta(2)-adrenoceptor antagonist) and SR59230A (a beta(3)-adrenoceptor antagonist) were needed to inhibit the isoprenaline-induced increase in L-type Ca(2+) channel current. 3. L-type Ca(2+) channels were stimulated by CGP12177A (a beta(3)-adrenoceptor agonist with potent beta(1)- and beta(2)-adrenoceptor antagonist property) in a manner similar to that of isoprenaline. The CGP12177A-induced stimulation of Ca(2+) channel current was blocked by SR59230A, cyclic AMP-dependent protein kinase inhibitors, H-89 and Rp 8-Br-cyclic AMPs, but was unaffected by protein kinase C inhibitors, GF109203X and 19-31 peptide. This stimulation was mimicked by forskolin and 8-Br-cyclic AMP. In the presence of okadaic acid (a phosphatase inhibitor), the beta(3)-adrenoceptor-induced stimulation was maintained after withdrawal of the agonist. 4. The beta(3)-adrenoceptor stimulation of L-type Ca(2+) channels was blocked by a pretreatment with cholera toxin and by the intracellular application of an anti-Galpha(s) antibody. This stimulation was unaffected by intracellular infusion of an anti-Gbeta(com) antibody and a betaARK(1) peptide. 5. These results show that activation of beta(3)-adrenoceptors stimulates L-type Ca(2+) channels in vascular myocytes through a Galpha(s)-induced stimulation of the cyclic AMP/protein kinase A pathway and the subsequent phosphorylation of the channels.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Músculo Liso Vascular/fisiologia , Veia Porta/fisiologia , Receptores Adrenérgicos beta/fisiologia , Sulfonamidas , 1-Propanol/farmacologia , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Agonistas Adrenérgicos beta/farmacologia , Antagonistas Adrenérgicos beta/farmacologia , Animais , Bário/farmacologia , Canais de Cálcio Tipo L/efeitos dos fármacos , Células Cultivadas , Toxina da Cólera/farmacologia , Colforsina/farmacologia , Relação Dose-Resposta a Droga , Estimulação Elétrica , Isoproterenol/farmacologia , Isoquinolinas/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Dibutirato de 12,13-Forbol/farmacologia , Veia Porta/citologia , Veia Porta/efeitos dos fármacos , Propanolaminas/farmacologia , RNA Mensageiro/genética , Ratos , Receptores Adrenérgicos beta/efeitos dos fármacos , Receptores Adrenérgicos beta/genética , Receptores Adrenérgicos beta 3 , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de SinaisRESUMO
While the roles of subtypes 1 and 2 of the ryanodine receptors (RYRs) have been studied in cellular systems expressing specifically one or the other of these subtypes (i.e. skeletal and cardiac muscle), the function of these receptors has not been evaluated in smooth muscles. We have previously reported RYR-mediated elementary (Ca(2+) sparks) and global Ca(2+) responses in rat portal vein myocytes. Here, we investigated the respective roles of all three RYR subtypes expressed in these cells as revealed by reverse transcriptase-polymerase chain reaction. Antisense oligonucleotides targeting each one of the three RYR subtypes were shown to specifically inhibit the expression of the corresponding mRNA and protein without affecting the other RYR subtypes. Confocal Ca(2+) measurements revealed that depolarization-induced Ca(2+) sparks and global Ca(2+) responses were blocked when either RYR1 or RYR2 expression was suppressed. Caffeine-induced Ca(2+) responses were partly inhibited by the same antisense oligonucleotides. Neither the corresponding scrambled oligonucleotides nor the antisense oligonucleotides targeting RYR3 affected depolarization- or caffeine-induced Ca(2+) responses. Our results show that, in vascular myocytes, the two RYR1 and RYR2 subtypes are required for Ca(2+) release during Ca(2+) sparks and global Ca(2+) responses, evoked by activation of voltage-gated Ca(2+) channels.
Assuntos
Cálcio/metabolismo , Músculo Liso Vascular/metabolismo , Isoformas de Proteínas/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Animais , Sequência de Bases , Cafeína/farmacologia , Primers do DNA , Técnicas In Vitro , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Oligonucleotídeos Antissenso/genética , Isoformas de Proteínas/genética , Ratos , Canal de Liberação de Cálcio do Receptor de Rianodina/genéticaRESUMO
In rat portal vein myocytes, Ca(2+) signals can be generated by inositol 1,4,5-trisphosphate (InsP(3))- and ryanodine-sensitive Ca(2+) release channels, which are located on the same intracellular store. Using a laser scanning confocal microscope associated with the patch-clamp technique, we showed that propagated Ca(2+) waves evoked by norepinephrine (in the continuous presence of oxodipine) were completely blocked after internal application of an anti-InsP(3) receptor antibody. These propagated Ca(2+) waves were also reduced by approximately 50% and transformed in homogenous Ca(2+) responses after application of an anti-ryanodine receptor antibody or ryanodine. All-or-none Ca(2+) waves obtained with increasing concentrations of norepinephrine were transformed in a dose-response relationship with a Hill coefficient close to unity after ryanodine receptor inhibition. Similar effects of the ryanodine receptor inhibition were observed on the norepinephrine- and ACh-induced Ca(2+) responses in non-voltage-clamped portal vein and duodenal myocytes and on the norepinephrine-induced contraction. Taken together, these results show that ryanodine-sensitive Ca(2+) release channels are responsible for the fast propagation of Ca(2+) responses evoked by various neurotransmitters producing InsP(3) in vascular and visceral myocytes.
Assuntos
Cálcio/metabolismo , Inositol 1,4,5-Trifosfato/fisiologia , Músculo Liso Vascular/metabolismo , Norepinefrina/farmacologia , Canal de Liberação de Cálcio do Receptor de Rianodina/fisiologia , Animais , Cafeína/farmacologia , Inositol 1,4,5-Trifosfato/metabolismo , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Neurotransmissores/farmacologia , Técnicas de Patch-Clamp , Veia Porta/efeitos dos fármacos , Veia Porta/metabolismo , Ratos , Ratos Wistar , Rianodina/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Vasoconstrição/fisiologiaRESUMO
In this study, we identified the receptor subtype activated by endothelin-1 (ET-1) and the subunit composition of the G protein coupling this receptor to increase in cytosolic Ca2+ concentration in rat portal vein myocytes. We used intranuclear antisense oligonucleotide injection to selectively inhibit the expression of G protein subunits. We show here that the endothelin receptor subtype A (ETA)-mediated increase in cytosolic Ca2+ concentration was mainly dependent on Ca2+ release from the intracellular store. ETA receptor-mediated Ca2+ release was selectively inhibited by antisense oligonucleotides that inhibited the expression of alpha11, beta3, and gamma5 subunits, as checked by immunocytochemistry. Intracellular dialysis of a carboxyl terminal anti-betacom antibody and a peptide corresponding to the Gbetagamma binding region of the beta-adrenergic receptor kinase-1 had no effect on the ETA receptor-mediated Ca2+ release. In contrast, a synthetic peptide corresponding to the carboxyl terminus of the alphaq/alpha11 subunit, heparin (an inhibitor of inositol 1,4,5-trisphosphate receptors), and U73122 (an inhibitor of phosphatidylinositol-phospholipase C) inhibited, in a concentration-dependent manner, the ETA receptor-mediated Ca2+ responses. Accumulation of [3H]inositol trisphosphate evoked by norepinephrine peaked at approximately 15 s, whereas that evoked by ET-1 progressively increased within 2 min. In myocytes injected with anti-alphaq antisense oligonucleotides, both amplitude and time course of the norepinephrine-induced Ca2+ release became similar to those of the ET-1-induced Ca2+ response. We conclude that the ETA receptor-mediated Ca2+ release is selectively transduced by the heterotrimeric G11 protein composed of alpha11, beta3, and gamma5 subunits, and that a delayed stimulation of phospholipase C occurs via the alpha11 subunit.
Assuntos
Cálcio/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Músculo Liso Vascular/metabolismo , Fosfatidilinositóis/metabolismo , Receptores de Endotelina/metabolismo , Animais , Endotelina-1/metabolismo , Heparina/farmacologia , Hidrólise , Fosfatos de Inositol/metabolismo , Norepinefrina/metabolismo , Veia Porta/citologia , Veia Porta/metabolismo , Ratos , Receptores de Endotelina/efeitos dos fármacos , Fatores de Tempo , Fosfolipases Tipo C/antagonistas & inibidoresRESUMO
We have previously reported that, in venous myocytes, Gbetagamma scavengers inhibit angiotensin AT1A receptor-induced stimulation of L-type Ca2+ channels (1). Here, we demonstrate that intracellular infusion of purified Gbetagamma complexes stimulates the L-type Ca2+ channel current in a concentration-dependent manner. Additional intracellular dialysis of GDP-bound inactive Galphao or of a peptide corresponding to the Gbetagamma binding region of the beta-adrenergic receptor kinase completely inhibited the Gbetagamma-induced stimulation of Ca2+ channel currents. The gating properties of the channel were not affected by intracellular application of Gbetagamma, suggesting that Gbetagamma increased the whole-cell calcium conductance. In addition, both the angiotensin AT1A receptor- and the Gbetagamma-induced stimulation of L-type Ca2+ channels were blocked by pretreatment of the cells with wortmannin, at nanomolar concentrations. Correspondingly, intracellular infusion of an enzymatically active purified recombinant Gbetagamma-sensitive phosphoinositide 3-kinase, PI3Kgamma, mimicked Gbetagamma-induced stimulation of Ca2+ channels. Both Gbetagamma- and PI3Kgamma-induced stimulations of Ca2+ channel currents were reduced by protein kinase C inhibitors suggesting that the Gbetagamma/PI3Kgamma-activated transduction pathway involves a protein kinase C. These results indicate for the first time that Gbetagamma dimers stimulate the vascular L-type Ca2+ channels through a Gbetagamma-sensitive PI3K.
Assuntos
Canais de Cálcio/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Ativação do Canal Iônico , Músculo Liso Vascular/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Animais , Canais de Cálcio Tipo L , Músculo Liso Vascular/citologia , Proteína Quinase C/antagonistas & inibidores , RatosRESUMO
Ca2+ signalling events were analyzed in single myocytes from rat portal vein by using a laser confocal microscope combined with the patch-clamp technique. Increase in inositol 1,4,5-trisphosphate (InsP3) concentration was obtained by photorelease from a caged precursor or intracellular dialysis of 3F-InsP3. Low InsP3 concentrations activated either small elevations of [Ca2+]i or localized Ca2+ transients whereas high InsP3 concentrations activated either homogeneous Ca2+ responses or propagated Ca2+ waves. The InsP3-evoked localized Ca2+ transients had spatio-temporal properties characteristic of Ca2+ sparks. In addition, compounds that blocked Ca2+ sparks and Ca2+ responses activated by Ca2+ jumps reduced the global InsP3-activated Ca2+ responses and suppressed the Ca2+ transients. In contrast, Ca2+ responses evoked by flash-photolytic Ca2+ jumps or caffeine were not affected by heparin (an InsP3 receptor antagonist). These results suggest that the absence of elementary Ca2+ events evoked by InsP3 may be related to the lack of clustered InsP3 receptor units in these cells, as confirmed by immunocytochemistry. Cooperativity between InsP3- and ryanodine-sensitive Ca2+ channels may represent a novel mechanism to amplify Ca2+ release from the same intracellular store and give rise to propagated Ca2+ waves.
Assuntos
Canais de Cálcio/efeitos dos fármacos , Inositol 1,4,5-Trifosfato/farmacologia , Veia Porta/efeitos dos fármacos , Receptores Citoplasmáticos e Nucleares/efeitos dos fármacos , Canal de Liberação de Cálcio do Receptor de Rianodina/efeitos dos fármacos , Rianodina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Acetatos/efeitos da radiação , Animais , Canais de Cálcio/metabolismo , Células Cultivadas , Etilenodiaminas/efeitos da radiação , Inositol 1,4,5-Trifosfato/análogos & derivados , Inositol 1,4,5-Trifosfato/efeitos da radiação , Receptores de Inositol 1,4,5-Trifosfato , Ativação do Canal Iônico/efeitos dos fármacos , Microinjeções , Microscopia Confocal , Técnicas de Patch-Clamp , Fotólise , Veia Porta/citologia , Veia Porta/metabolismo , Ratos , Ratos Wistar , Receptores Citoplasmáticos e Nucleares/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Transdução de Sinais/fisiologiaRESUMO
The molecular diversity of receptors and the capability of these receptors to activate multiple types of G proteins theoretically allow the transmission of signals through multiple effector pathways. In functional experiments, however, the number of possibilities may be strongly reduced. We have recently reported that in vascular myocytes, α(1)-adrenoceptors activate two G proteins composed of α(q)/ß(1)/γ(3) and α(11)/ß(3)/γ(2) subunits, leading to increase in cytoplasmic [Ca(2+)](i) concentration. Only the α(q) subunit transduces the signal to a phospholipase C-ß, which hydrolyzes phosphatidylinositol 4,5-bisphosphate to generate inositol 1,4,5-trisphosphate and the subsequent release of Ca(2+) from the intracellular store. In contrast, the α(11) subunit activates Ca(2+) entry through a nonspecific cation channel in the presence of increased [Ca(2+)](i) level. These coupling mechanisms reveal the distinct participation of G(q) and G(11) in the regulation of vascular contractility. Specific G(q)- or G(11)-activated pathways should be taken into account to understand the various contraction profiles induced by different vasoconstrictors.
RESUMO
A G protein composed of alpha13, beta1, and gamma3 subunits selectively couples the angiotensin AT1A receptors to increase cytoplasmic Ca2+ concentration ([Ca2+]i) in rat portal vein myocytes (Macrez-Leprêtre, N., Kalkbrenner, F., Morel, J. L., Schultz, G., and Mironneau, J. (1997) J. Biol. Chem. 272, 10095-10102). We show here that Gbetagamma transduces the signal leading to stimulation of L-type Ca2+ channels. Intracellular dialysis through the patch pipette of a carboxyl-terminal anti-betacom antibody and a peptide corresponding to the Gbetagamma binding region of the beta-adrenergic receptor kinase 1 inhibited the stimulation of Ca2+ channels and the increase in [Ca2+]i evoked by angiotensin II. The Gbetagamma binding peptide did not prevent the dissociation of the heterotrimeric G protein into its subunits, as it did not block activation of phospholipase C-beta by Galphaq in response to stimulation of alpha1-adrenoreceptors. Transient overexpression of the beta-adrenergic receptor kinase 1 fragment and of Galpha subunits also inhibited the angiotensin II-induced increase in [Ca2+]i. Both anti-alpha13 antibody and carboxyl-terminal alpha13 peptide abrogated the angiotensin II-induced stimulation of Ca2+ channels. We conclude that activation of angiotensin AT1 receptors requires all three alpha, beta, and gamma subunits of G13 for receptor-G protein interaction, whereas the transduction of the signal to L-type Ca2+ channels is mediated by Gbetagamma.
Assuntos
Canais de Cálcio/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Músculo Liso Vascular/metabolismo , Veia Porta/metabolismo , Receptores de Angiotensina/metabolismo , Sequência de Aminoácidos , Angiotensina II/farmacologia , Animais , Cálcio/metabolismo , Canais de Cálcio Tipo L , Separação Celular , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Dimerização , Condutividade Elétrica , Ativação Enzimática , Dados de Sequência Molecular , Proteínas Musculares/metabolismo , Veia Porta/citologia , Ratos , Receptor Tipo 1 de Angiotensina , Receptor Tipo 2 de Angiotensina , Receptores Adrenérgicos alfa 1/metabolismo , Transdução de Sinais , Fosfolipases Tipo C/metabolismo , Quinases de Receptores Adrenérgicos betaRESUMO
1. Cytosolic Ca2+ concentration ([Ca2+]i) during exposure to acetylcholine or caffeine was measured in mouse duodenal myocytes loaded with fura-2. Acetylcholine evoked a transient increase in [Ca2+]i followed by a sustained rise which was rapidly terminated after drug removal. Although L-type Ca2+ currents participated in the global Ca2+ response induced by acetylcholine, the initial peak in [Ca2+]i was mainly due to release of Ca2+ from intracellular stores. 2. Atropine, 4-diphenylacetoxy-N-methylpiperidine (4-DAMP, a muscarinic M3 antagonist), pirenzepine (a muscarinic M1 antagonist), methoctramine and gallamine (muscarinic M2 antagonists) inhibited the acetylcholine-induced Ca2+ release, with a high affinity for 4-DAMP and atropine and a low affinity for the other antagonists. Selective protection of muscarinic M2 receptors with methoctramine during 4-DAMP mustard alkylation of muscarinic M3 receptors provided no evidence for muscarinic M2 receptor-activated [Ca2+]i increase. 3. Acetylcholine-induced Ca2+ release was blocked by intracellular dialysis with a patch pipette containing either heparin or an anti-phosphatidylinositol antibody and by external application of U73122 (a phospholipase C inhibitor). 4. Acetylcholine-induced Ca2+ release was insensitive to external pretreatment with pertussis toxin, but concentration-dependently inhibited by intracellular dialysis with a patch pipette solution containing an anti-alpha q/alpha 11 antibody. An antisense oligonucleotide approach revealed that only the Gq protein was involved in acetylcholine-induced Ca2+ release. 5. Intracellular applications of either an anti-beta com antibody or a peptide corresponding to the G beta gamma binding domain of the beta-adrenoceptor kinase 1 had no effect on acetylcholine-induced Ca2+ release. 6. Our results show that, in mouse duodenal myocytes, acetylcholine-induced release of Ca2+ from intracellular stores is mediated through activation of muscarinic M3 receptors which couple with a Gq protein to activate a phosphatidylinositol-specific phospholipase C.
Assuntos
Cálcio/metabolismo , Duodeno/metabolismo , Proteínas de Ligação ao GTP/fisiologia , Músculo Liso/metabolismo , Fosfatidilinositóis/metabolismo , Receptores Muscarínicos/fisiologia , Acetilcolina/farmacologia , Animais , Cafeína/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Heparina/farmacologia , Hidrólise , Camundongos , Toxina Pertussis , Receptor Muscarínico M3 , Fosfolipases Tipo C/fisiologia , Fatores de Virulência de Bordetella/farmacologia , Quinases de Receptores Adrenérgicos betaRESUMO
Ca2+ signalling events and whole-cell Ca2+ currents were analyzed in single myocytes from rat portal vein by using a laser scanning confocal microscope combined with the patch-clamp technique. In myocytes in which the intracellular Ca2+ store was depleted or Ca2+ release channels were blocked by 10 microM ryanodine, inward Ca2+ currents induced slow and sustained elevations of [Ca2+]i. These Ca2+ responses were suppressed by 1 microM oxodipine and by depolarizations to +120 mV, a potential close to the reversal potential for Ca2+ ions, suggesting that they reflected Ca2+ influx through L-type Ca2+ channels. With functioning intracellular Ca2+ stores, flash photolysis of caged Ca2+ gave rise to a small increase in [Ca2+]i with superimposed Ca2+ sparks, reflecting the opening of clustered Ca2+ release channels. Brief Ca2+ currents in the voltage range from -30 to +10 mV triggered Ca2+ sparks or macrosparks that did not propagate in the entire line-scan image. Increasing the duration of Ca2+ current for 100 ms or more allowed the trigger of propagating Ca2+ waves which originated from the same initiation sites as the caffeine-activated response. Both Ca2+ sparks and initiation sites of Ca2+ waves activated by Ca2+ currents were observed in the vicinity of areas that excluded the Ca2+ probes, reflecting infoldings of the plasma membrane close to the sarcoplasmic reticulum, as revealed by fluorescent markers. The hierarchy of Ca2+ signalling events, from submicroscopic fundamental events to elementary events (sparks) and propagated waves, provides an integrated mechanism to regulate vascular tone.