RESUMO
Ca(v)2.1 channels (P/Q-type) play a prominent role in controlling neurotransmitter release. Transgenic mice in which the α1A pore-forming subunit of Ca(v)2.1 channels is ablated (KO) provide a powerful tool to study Ca(v)2.1 function in synaptic transmission in vivo. Whole-cell patch clamp was used to measure inhibitory glycinergic postsynaptic currents (IPSCs) from the lateral superior olive (LSO). Comparing wild-type (WT) and KO mice, we investigated the relevance of P/Q-type calcium channels at a glycinergic synapse mediated by multiple types of Ca(2+) channels, in opposition to synapses where only this type of Ca(2+) channels are in charge of transmitter release. We found that in KO mice, N-type and L-type Ca(2+) channels control synaptic transmission, resulting in a functional but reduced glycinergic transmitter release. Pair pulse facilitation of synaptic currents is retained in KO mice, even when synaptic transmission is driven by either N or L-type calcium channels alone, in contrast with lack of this phenomenon in other synapses which are exclusively mediated by P/Q-type channels. Thus, pointing a difference between P/Q- and N-type channels present in single or multiple types of calcium channels driven synapses. Significant alterations in short-term synaptic plasticity were observed. KO mice exhibited a stronger short term depression (STD) of IPSCs during repetitive stimulation at high frequency and recovered with a larger time constant compared to WT mice. Finally, transmitter release at the LSO synapse from KO mice was strongly modulated by presynaptic GTP-binding protein-coupled receptor γ-aminobutyric acid type B (GABA(B)).
Assuntos
Canais de Cálcio Tipo P/metabolismo , Plasticidade Neuronal/fisiologia , Neurotransmissores/metabolismo , Sinapses/metabolismo , Transmissão Sináptica/fisiologia , Animais , Tronco Encefálico/metabolismo , Canais de Cálcio/metabolismo , Canais de Cálcio Tipo Q/metabolismo , Potenciais Pós-Sinápticos Excitadores/fisiologia , Glicina/metabolismo , Potenciais Pós-Sinápticos Inibidores/fisiologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Neurônios/metabolismo , Técnicas de Cultura de Órgãos , Técnicas de Patch-ClampRESUMO
Spinal nucleus of bulbocavernosus and its target musculature, the bulbocavernosus and levator ani muscles, are sexually dimorphic, and their sexual differentiation depends on plasmatic levels of testosterone. Electrophysiological and immunocytochemical studies have demonstrated that at mammalian adult neuromuscular junctions only P/Q-type Ca2+ channels (Ca(v2.1)), mediate evoked transmitter release. Here we report that N-type Ca2+ channel (Ca(v2.2)) blocker omega-Conotoxin GVIA, as well as Ca(v2.1) blocker omega-Agatoxin IVA, significantly reduced quantal content of transmitter release by approximately 80% and approximately 70% respectively at levator ani muscle of the adult rats, indicating that neuromuscular transmission is jointly mediated by both types of channels. In these synapses, we also observed that castration and restitution of plasmatic testosterone in rats resulted in changes in the sensitivity to omega-Conotoxin GVIA. Castration induced, whereas testosterone treatment avoided, functional loss of Ca(v2.2), as mediators of transmitter release in these synapses. Strikingly, the expression and localization of alpha1B subunits, which form the pore of the Ca(v2.2) channel, were similar at control, gonadectomized and gonadectomized testosterone-treated rats, suggesting that testosterone may regulate the coupling mechanisms between Ca(v2.2) and transmitter release at the neuromuscular junctions of these sexually dimorphic motoneurons.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Junção Neuromuscular/efeitos dos fármacos , Testosterona/farmacologia , Animais , Animais Recém-Nascidos , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo N , Diafragma/citologia , Diafragma/efeitos dos fármacos , Interações Medicamentosas , Potenciais Evocados/efeitos dos fármacos , Potenciais Evocados/fisiologia , Potenciais Evocados/efeitos da radiação , Imuno-Histoquímica/métodos , Masculino , Orquiectomia/métodos , Diafragma da Pelve , Radioimunoensaio/métodos , Ratos , Ratos Sprague-Dawley , Receptores Colinérgicos/metabolismo , ômega-Agatoxina IVA/farmacologia , ômega-Conotoxina GVIA/farmacologiaRESUMO
Previously, we have presented evidence for the presence of L-type voltage-dependent Ca2+ channels (VDCC) in 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, (acetoxymethyl)ester (BAPTA-AM)-incubated motor nerve terminals (MNTs) of the levator auris muscle of mature mice. The aim of the present work was to study the coupling of these L-type VDCC to neurotransmitter release by inhibiting protein phosphatases. We thus studied the effects of the protein phosphatase inhibitors okadaic acid (OA) and pervanadate on quantal content (QC) of transmitter release with the P/Q-type channels fully blocked. The QC was not significantly different under the three experimental conditions tested: incubation with dimethylsulphoxide (DMSO), ethylene-glycol-bis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid, (acetoxymethyl)ester (EGTA-AM) and BAPTA-AM. After preincubation with OA (1 microM), but not with pervanadate, QC increased substantially in the BAPTA-AM-incubated (up to 400%) MNT, but not in those incubated with DMSO or EGTA-AM. The OA-induced increment of QC was attenuated greatly (approximately 95% reduction) by preincubation with either nitrendipine (10 microM) or calciseptine (300 nM). The effect of OA (1 microM) and pervanadate (0.1 mM) on spontaneous neurotransmitter release was also studied. After preincubation with OA, but not per-vanadate, miniature end-plate potential (MEPP) frequency increased only in the BAPTA-AM-incubated MNT (up to 700% increment). This response was attenuated (by approximately 80%) by nitrendipine (10 microM) or calciseptine (300 nM). In contrast, neither omega-agatoxin IVA (120 nM) nor omega-conotoxin GVIA (1 microM) affected this OA-induced increment significantly. We also evaluated the relationship between QC and extracellular [Ca2+] ([Ca2+]o) in BAPTA-AM-incubated MNT. Under conditions in which only P/Q-type VDCC were available to participate in neurotransmitter release, QC increased as [Ca2+]o was raised from 0.5 to 2 mM. However, when only L-type VDCC were available, QC increased when [Ca2+]o increased from 0.5 to 1 mM, but decreased significantly at 2 mM. The mean latency for P/Q-type VDCC-mediated EPP was 1.7-1.9 ms; for L-type VDCC-mediated EPP, 1.9-2.5 ms. The rise time of the L-type VDCC mediated EPP was significantly slower than that mediated by P/Q-type VDCC. Preincubation with H-7 (100 microM), a potent inhibitor of protein kinase C (PKC) and adenosine 3',5'cyclic monophosphate (cAMP)-dependent protein kinase (PKA), attenuated the OA-induced increment of both QC and MEPP frequency (50% and 70% decrement, respectively), suggesting the participation of at least these two protein kinases in the coupling of L-type VDCC. In summary, our results show coupling of L-type VDCC to neurotransmitter release when protein phosphatases are inhibited and intracellular [Ca2+] is buffered by the fast chelator BAPTA.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Placa Motora/metabolismo , Neurônios Motores/metabolismo , Neurotransmissores/metabolismo , Animais , Soluções Tampão , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Quelantes/farmacologia , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Venenos Elapídicos/farmacologia , Inibidores Enzimáticos/farmacologia , Potenciais Evocados/efeitos dos fármacos , Potenciais Evocados/fisiologia , Ionóforos/farmacologia , Camundongos , Placa Motora/efeitos dos fármacos , Músculo Esquelético/inervação , Músculo Esquelético/fisiologia , Nitrendipino/farmacologia , Ácido Okadáico/farmacologia , Terminações Pré-Sinápticas/efeitos dos fármacos , Terminações Pré-Sinápticas/metabolismo , Transmissão Sináptica/efeitos dos fármacos , Vanadatos/farmacologia , ômega-Agatoxina IVA/farmacologia , ômega-Conotoxina GVIA/farmacologiaRESUMO
The involvement of the different types of voltage-dependent calcium channels (VDCC) in both DM-BAPTA-AM-incubated and EGTA-AM-incubated mature mice levator auris neuromuscular junctions (NMJ) was studied. We evaluated the effects of omega-agatoxin IVA (omega-Aga IVA), nitrendipine and omega-conotoxin GVIA (omega-CgTX) (P/Q-, L- and N-type VDCC blockers, respectively) on perineurial calcium currents (ICa) and nerve-evoked transmitter release. The application of omega-Aga IVA (100 nM) drastically reduced perineurial ICa (>90%) and nerve-evoked transmitter release (>90% of reduction in quantal content, m) at both DM-BAPTA-AM-incubated and EGTA-AM-incubated NMJ. The L-type VDCC antagonist nitrendipine (10 microM) caused a significant reduction (23+/-9%, n=5) of perineurial ICa at DM-BAPTA-AM-incubated NMJ. In addition, after the block of P/Q-type VDCC with omega-Aga IVA (100 nM), nitrendipine reduced (>90%, n=2) the remaining perineurial ICa. Such reduction was not observed at EGTA-AM-incubated NMJ, before or after the total block of P/Q-type VDCC. Moreover, nitrendipine did not significantly reduce the quantal content of DM-BAPTA-AM-incubated NMJ. Finally, the application of omega-CgTX (5 microM) did not significantly affect perineurial ICa or nerve-evoked transmitter release at either DM-BAPTA-AM-incubated or EGTA-AM-incubated NMJ. These results show the existence of a nitrendipine-sensitive, L-type component of perineurial ICa in DM-BAPTA-AM-incubated NMJ of mature mice.
Assuntos
Canais de Cálcio/metabolismo , Cálcio/farmacologia , Placa Motora/metabolismo , Animais , Soluções Tampão , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L , Quelantes/farmacologia , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Condutividade Elétrica , Potenciais Evocados , Masculino , Camundongos , Nitrendipino/farmacologia , Peptídeos/farmacologia , Venenos de Aranha/farmacologia , ômega-Agatoxina IVA , ômega-Conotoxina GVIARESUMO
The effects of the membrane permeant Ca2+ chelator BAPTA-AM on voltage-gated Na+, Ca2+, K+ (I(Na), I(Ca) I(K), respectively) and Ca2+-activated K+ (I(KCa)) currents in cultured bovine chromaffin cells were investigated using the whole-cell patch-clamp technique. Superfusion with BAPTA-AM (50 microM) induced a rapid (< 60 s) and reversible block of both I(KCa) and I(K) (approximately 50%), without affecting either I(Ca) or I(Na). Preincubation with BAPTA-AM (50 microM, 30 min) or cell loading with the nonpermeable active form of BAPTA (10 mM in the pipette solution) permanently blocked I(KCa). BAPTA-AM superfusion (50 microM) also blocked I(K) (approximately 53%) after BAPTA-loading or BAPTA-AM preincubation. In conclusion, we show a fast and reversible block of I(KCa) and I(K) by BAPTA-AM, acting directly on K+ channels before it operates as a Ca2+ chelator, in cultured bovine chromaffin cells.