RESUMO
BACKGROUND AND PURPOSE: Evidence suggests that phosphorylation of TRPV1 is an important component underlying its aberrant activation in pathological pain states. To date, the detailed pharmacology of diverse TRPV1 receptor agonists and antagonists has yet to be reported for native TRPV1 under phosphorylating conditions. Our goal was to optimize a relatively high-throughput methodology to allow pharmacological characterization of the native TRPV1 receptor using a spinal cord neuropeptide release assay under naive and phosphorylating states. EXPERIMENTAL APPROACH: Herein, we describe characterization of rodent TRPV1 by measurement of CGRP release from acutely isolated lumbar (L1-L6) spinal cord using a 96-well technique that combines use of native, adult tissue with quantitation of CGRP release by ELISA. KEY RESULTS: We have studied a diverse panel of TRPV1 agonists and antagonists under basal and phosphorylating conditions. We show that TRPV1-mediated CGRP release is evoked, in a temperature-dependent manner, by a PKC activator, phorbol 12,13-dibutyrate (PDBu); and that treatment with PDBu increases the potency and efficacy of known TRPV1 chemical agonists, in an agonist-specific manner. We also show that the pharmacological profile of diverse TRPV1 antagonists is dependent on whether the stimulus is PDBu or capsaicin. Of note, HPPB was identified as an antagonist of capsaicin-evoked, but a potentiator of PDBu-evoked, CGRP release. CONCLUSIONS AND IMPLICATIONS: Our findings indicate that both TRPV1 agonist and antagonist profiles can be differentially altered by PKC activation. These findings may offer new insights for targeting TRPV1 in pain states.
Assuntos
Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Medula Espinal/efeitos dos fármacos , Canais de Cátion TRPV , Animais , Cálcio/metabolismo , Capsaicina/farmacologia , Relação Dose-Resposta a Droga , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Proteína Quinase C/metabolismo , Pirazinas/farmacologia , Piridinas/farmacologia , Ratos , Ratos Endogâmicos , Medula Espinal/metabolismo , Canais de Cátion TRPV/agonistas , Canais de Cátion TRPV/antagonistas & inibidores , Canais de Cátion TRPV/genéticaRESUMO
OBJECTIVE: The present study examined whether local administration of the cannabinoid-2 (CB(2)) receptor agonist GW405833 could modulate joint nociception in control rat knee joints and in an animal model of osteoarthritis (OA). METHOD: OA was induced in male Wistar rats by intra-articular injection of sodium monoiodo-acetate with a recovery period of 14 days. Immunohistochemistry was used to evaluate the expression of CB(2) and transient receptor potential vanilloid channel-1 (TRPV1) receptors in the dorsal root ganglion (DRG) and synovial membrane of sham- and sodium mono-iodoacetate (MIA)-treated animals. Electrophysiological recordings were made from knee joint primary afferents in response to rotation of the joint both before and following close intra-arterial injection of different doses of GW405833. The effect of intra-articular GW405833 on joint pain perception was determined by hindlimb incapacitance. An in vitro neuronal release assay was used to see if GW405833 caused release of an inflammatory neuropeptide (calcitonin gene-related peptide - CGRP). RESULTS: CB(2) and TRPV1 receptors were co-localized in DRG neurons and synoviocytes in both sham- and MIA-treated animals. Local application of the GW405833 significantly reduced joint afferent firing rate by up to 31% in control knees. In OA knee joints, however, GW405833 had a pronounced sensitising effect on joint mechanoreceptors. Co-administration of GW405833 with the CB(2) receptor antagonist AM630 or pre-administration of the TRPV1 ion channel antagonist SB366791 attenuated the sensitising effect of GW405833. In the pain studies, intra-articular injection of GW405833 into OA knees augmented hindlimb incapacitance, but had no effect on pain behaviour in saline-injected control joints. GW405833 evoked increased CGRP release via a TRPV1 channel-dependent mechanism. CONCLUSION: These data indicate that GW405833 reduces the mechanosensitivity of afferent nerve fibres in control joints but causes nociceptive responses in OA joints. The observed pro-nociceptive effect of GW405833 appears to involve TRPV1 receptors.
Assuntos
Indóis/farmacologia , Articulação do Joelho/efeitos dos fármacos , Morfolinas/farmacologia , Osteoartrite do Joelho/complicações , Dor/tratamento farmacológico , Receptor CB2 de Canabinoide/agonistas , Animais , Canabinoides/análise , Modelos Animais de Doenças , Eletrofisiologia , Gânglios Espinais/metabolismo , Imuno-Histoquímica , Injeções Intra-Articulares , Articulação do Joelho/fisiologia , Masculino , Fibras Nervosas/efeitos dos fármacos , Fibras Nervosas/fisiologia , Osteoartrite do Joelho/metabolismo , Dor/etiologia , Ratos , Ratos Wistar , Membrana Sinovial/metabolismo , Canais de Cátion TRPV/análiseRESUMO
Nicotinic acetylcholine receptors (nAChRs) are widely expressed in the mammalian central nervous system (CNS). Despite this, very little was known, until recently, about their physiological role. In the periphery, nicotinic receptors mediate vital excitatory fast synaptic cholinergic transmission at both the neuromuscular junction and ganglia. In the brain, this role has been mainly "delegated" to glutamate receptors. The very broad cholinergic innervations of most brain areas, including the cortex, have implicated this system, and brain nicotinic receptors in particular, in a unique "modulatory" role of other transmitters systems. Recent evidence confirms, on one hand, that brain nicotinic receptors have a dominant "presynaptic" modulatory function, controlling the release of both acetylcholine (auto-receptors) and other neurotransmitters (hetero-receptors). On the other hand, more experimental data support the idea that a variable component of fast synaptic transmission in the brain can also be mediated by "postynaptic" nicotinic receptors, which, in turn, can control cell excitability. A challenging goal is to identify which one of the plethora of nicotinic receptor subtypes is mediating each effect in different brain areas, and which of these receptors and functions are lost or affected in different human neuro-psychiatric disorders. Needless to say, a better understanding of the physiological role of brain nicotinic receptors will drive our quest for more selective and efficacious nicotinic receptor targeted therapeutic agents.
Assuntos
Plasticidade Neuronal/fisiologia , Neurônios/metabolismo , Neurotransmissores/metabolismo , Receptores Nicotínicos/fisiologia , Transmissão Sináptica/fisiologia , Animais , Humanos , Receptores Nicotínicos/classificação , Receptores Nicotínicos/metabolismoRESUMO
The effects of 5-hydroxyindole (5-HI) have been investigated on human alpha 7 nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus oocytes and GH4 cells, on native alpha 7 nAChRs expressed by IMR-32 cells and on alpha 7 nAChR-mediated events in mossy fibre-granule cell synapses in rat cerebellar slices. In oocytes expressing alpha 7 nAChRs, 5-HI potentiated sub-maximal, 60 micro M ACh-induced ion currents in a concentration-dependent manner, the threshold effective concentration being 30 micro M. 5-HI itself did not act as an agonist on alpha 7 nAChRs. A maximum potentiation of 12 times the control was observed at 20 mM 5-HI. The effect of 1 mM 5-HI on the concentration-response curve for ACh revealed that 5-HI increased the potency as well as the efficacy of ACh on alpha 7 nAChRs. 5-HI also potentiated alpha 7-mediated increases in intracellular free calcium levels in both mammalian cells heterologously expressing human alpha 7 nAChRs and in human IMR-32 neuroblastoma cells expressing native alpha 7 nAChRs. At mossy fibre-granule cell synapses, application of 1 mM ACh induced glutamate-evoked excitatory post-synaptic currents (EPSCs). Co-application of 1 mM 5-HI with 1 mM ACh further increased the frequency of the EPSCs. The ACh-induced release, as well as the 5-HI-induced enhancement of release, were blocked by 1-10 nM methyllycaconitine or 200 nM alpha-bungarotoxin, demonstrating that both effects were mediated by presynaptic alpha 7 nAChRs. The results demonstrate that responses mediated by alpha 7 nAChRs are strongly potentiated by 5-HI.
Assuntos
Acetilcolina/farmacologia , Cerebelo/metabolismo , Ácido Glutâmico/metabolismo , Indóis/farmacologia , Receptores Nicotínicos/efeitos dos fármacos , Animais , Cálcio/metabolismo , Células Cultivadas , Cerebelo/efeitos dos fármacos , Relação Dose-Resposta a Droga , Eletrofisiologia , Feminino , Processamento de Imagem Assistida por Computador , Técnicas In Vitro , Potenciais da Membrana/fisiologia , Microeletrodos , Oócitos/efeitos dos fármacos , Técnicas de Patch-Clamp , Ratos , Transmissão Sináptica/efeitos dos fármacos , Xenopus laevis , Receptor Nicotínico de Acetilcolina alfa7RESUMO
Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand gated ion channels, which are found at the neuromuscular junction and in the central and peripheral nervous systems. The channels can be assembled from fourteen known subunits. The exact combination and function of all the channels are still not determined but in the CNS certain combinations have been identified which appear to modulate the release of specific neurotransmitters. Non-specific nAChR agonists like nicotine and epibatidine, have been shown to have interesting pharmacology but their clinical value is limited by their undesirable side effects. Selective ligands for different receptor subtypes have been reported and these compounds are probably the best tools for determining the function of the subtypes. The expectation is that some receptor subtype selective nAChR ligands will be clinically useful for the treatment of a broad range of CNS disorders. The development of stable cell lines functionally expressing specific combinations of subunits has greatly improved our understanding of ligand specificity. There have also been advances in the modelling of the ligand binding site, thanks to the discovery of a homologous snail ACh binding protein the X-ray structure of which was determined in 2001. These techniques should lead to rapid advances in the development of truly subtype selective ligands. In this review we describe recent progress in the area and describe the first 1000 fold selective low molecular weight ligands from the AstraZeneca group. We also comment on the first subtype specific channel modulators.
Assuntos
Receptores Nicotínicos/efeitos dos fármacos , Alcaloides/farmacologia , Animais , Azocinas , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Humanos , Ligantes , Agonistas Nicotínicos/farmacologia , Antagonistas Nicotínicos/farmacologia , Piridinas/farmacologia , Quinolizinas , Receptores Nicotínicos/químicaRESUMO
Capacitative Ca(2+) entry involves the regulation of plasma membrane Ca(2+) channels by the filling state of intracellular Ca(2+) stores in the endoplasmic reticulum (ER). Several theories have been advanced regarding the mechanism by which the stores communicate with the plasma membrane. One such mechanism, supported by recent findings, is conformational coupling: inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)) receptors in the ER may sense the fall in Ca(2+) levels through Ca(2+)-binding sites on their lumenal domains, and convey this conformational information directly by physically interacting with Ca(2+) channels in the plasma membrane. In support of this idea, in some cell types, store-operated channels in excised membrane patches appear to depend on the presence of both Ins(1,4,5)P(3) and Ins(1,4,5)P(3) receptors for activity; in addition, inhibitors of Ins(1,4,5)P(3) production that either block phospholipase C or inhibit phosphatidylinositol 4-kinase can block capacitative Ca(2+) entry. However, the electrophysiological current underlying capacitative Ca(2+) entry is not blocked by an Ins(1,4,5)P(3) receptor antagonist, and the blocking effects of a phospholipase C inhibitor are not reversed by the intracellular application of Ins(1,4,5)P(3). Furthermore, cells whose Ins(1,4,5)P(3) receptor genes have been disrupted can nevertheless maintain their capability to activate capacitative Ca(2+) entry channels in response to store depletion. A tentative conclusion is that multiple mechanisms for signaling capacitative Ca(2+) entry may exist, and involve conformational coupling in some cell types and perhaps a diffusible signal in others.
Assuntos
Canais de Cálcio/metabolismo , Sinalização do Cálcio , Cálcio/metabolismo , Ativação do Canal Iônico , Animais , Condutividade Elétrica , Retículo Endoplasmático/metabolismo , Exocitose , Humanos , Inositol 1,4,5-Trifosfato/metabolismo , Receptores de Inositol 1,4,5-Trifosfato , Modelos Biológicos , Receptores Citoplasmáticos e Nucleares/metabolismoRESUMO
In nonexcitable cells, the predominant mechanism for regulated entry of Ca(2+) is capacitative calcium entry, whereby depletion of intracellular Ca(2+) stores signals the activation of plasma membrane calcium channels. A number of other regulated Ca(2+) entry pathways occur in specific cell types, however, and it is not know to what degree the different pathways interact when present in the same cell. In this study, we have examined the interaction between capacitative calcium entry and arachidonic acid-activated calcium entry, which co-exist in HEK293 cells. These two pathways exhibit mutual antagonism. That is, capacitative calcium entry is potently inhibited by arachidonic acid, and arachidonic acid-activated entry is inhibited by the pre-activation of capacitative calcium entry with thapsigargin. In the latter case, the inhibition does not seem to result from a direct action of thapsigargin, inhibition of endoplasmic reticulum Ca(2+) pumps, depletion of Ca(2+) stores, or entry of Ca(2+) through capacitative calcium entry channels. Rather, it seems that a discrete step in the pathway signaling capacitative calcium entry interacts with and inhibits the arachidonic acid pathway. The findings reveal a novel process of mutual antagonism between two distinct calcium entry pathways. This mutual antagonism may provide an important protective mechanism for the cell, guarding against toxic Ca(2+) overload.
Assuntos
Ácido Araquidônico/metabolismo , Cálcio/antagonistas & inibidores , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Linhagem Celular , Humanos , Tapsigargina/farmacologiaRESUMO
We investigated the putative roles of phospholipase C, polyphosphoinositides, and inositol 1,4,5-trisphosphate (IP(3)) in capacitative calcium entry and calcium release-activated calcium current (I(crac)) in lacrimal acinar cells, rat basophilic leukemia cells, and DT40 B-lymphocytes. Inhibition of phospholipase C with blocked calcium entry and I(crac) activation whether in response to a phospholipase C-coupled agonist or to calcium store depletion with thapsigargin. Run-down of cellular polyphosphoinositides by concentrations of wortmannin that block phosphatidylinositol 4-kinase completely blocked calcium entry and I(crac). The membrane-permeant IP(3) receptor inhibitor, 2-aminoethoxydiphenyl borane, blocked both capacitative calcium entry and I(crac). However, it is likely that 2-aminoethoxydiphenyl borane does not inhibit through an action on the IP(3) receptor because the drug was equally effective in wild-type DT40 B-cells and in DT40 B-cells whose genes for all three IP(3) receptors had been disrupted. Intracellular application of another potent IP(3) receptor antagonist, heparin, failed to inhibit activation of I(crac). Finally, the inhibition of I(crac) activation by or wortmannin was not reversed or prevented by direct intracellular application of IP(3). These findings indicate a requirement for phospholipase C and for polyphosphoinositides for activation of capacitative calcium entry. However, the results call into question the previously suggested roles of IP(3) and IP(3) receptor in this mechanism, at least in these particular cell types.
Assuntos
Cálcio/fisiologia , Inositol 1,4,5-Trifosfato/metabolismo , Aparelho Lacrimal/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfolipases Tipo C/metabolismo , Androstadienos/farmacologia , Animais , Linfócitos B , Compostos de Boro/farmacologia , Cálcio/metabolismo , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/genética , Canais de Cálcio/fisiologia , Linhagem Celular Transformada , Células Cultivadas , Galinhas , Inibidores Enzimáticos/farmacologia , Estrenos/farmacologia , Heparina/farmacologia , Receptores de Inositol 1,4,5-Trifosfato , Cinética , Aparelho Lacrimal/citologia , Aparelho Lacrimal/efeitos dos fármacos , Leucemia Basofílica Aguda , Camundongos , Pirrolidinonas/farmacologia , Ratos , Receptores Citoplasmáticos e Nucleares/efeitos dos fármacos , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/fisiologia , Tapsigargina/farmacologia , Células Tumorais Cultivadas , WortmaninaRESUMO
The regulation of store-operated, calcium-selective channels in the plasma membrane of rat basophilic leukemia cells (RBL-2H3 m1), an immortalized mucosal mast cell line, was studied at the single-channel level with the patch clamp technique by removing divalent cations from both sides of the membrane. The activity of the single channels in excised patches could be modulated by Ca(2+), Mg(2+), and pH. The maximal activation of these channels by divalent cation-free conditions occurred independently of depletion of intracellular Ca(2+) stores, whether in excised patches or in whole cell mode. Yet, a number of points of evidence establish these single-channel openings as amplified store-operated channel events. Specifically, (i) the single channels are exquisitely sensitive to inhibition by intracellular Ca(2+), and (ii) both the store-operated current and the single-channel openings are completely blocked by the capacitative calcium entry blocker, 2-aminoethoxydiphenyl borane. In addition, in Jurkat T cells single-channel openings with lower open probability have been observed in the whole cell mode with intracellular Mg(2+) present (Kerschbaum, H. H., and Cahalan, M. D. (1999) Science 283, 836-839), and in RBL-2H3 m1 cells a current with similar properties is activated by store depletion.
Assuntos
Cálcio/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/fisiologia , Animais , Cálcio/farmacologia , Membrana Celular/fisiologia , Quelantes/farmacologia , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Concentração de Íons de Hidrogênio , Leucemia Basofílica Aguda , Magnésio/farmacologia , Técnicas de Patch-Clamp , Ratos , Canal de Liberação de Cálcio do Receptor de Rianodina/efeitos dos fármacos , Soluções , Células Tumorais CultivadasRESUMO
We have investigated the signaling pathways underlying muscarinic receptor-induced calcium oscillations in human embryonic kidney (HEK293) cells. Activation of muscarinic receptors with a maximal concentration of carbachol (100 microm) induced a biphasic rise in cytoplasmic calcium ([Ca2+]i) comprised of release of Ca2+ from intracellular stores and influx of Ca2+ from the extracellular space. A lower concentration of carbachol (5 microm) induced repetitive [Ca2+]i spikes or oscillations, the continuation of which was dependent on extracellular Ca2+. The entry of Ca2+ with 100 microm carbachol and with the sarcoplasmic-endoplasmic reticulum calcium ATPase inhibitor, thapsigargin, was completely blocked by 1 microm Gd3+, as well as 30-100 microm concentrations of the membrane-permeant inositol 1,4,5-trisphosphate receptor inhibitor, 2-aminoethyoxydiphenyl borane (2-APB). Sensitivity to these inhibitors is indicative of capacitative calcium entry. Arachidonic acid, a candidate signal for Ca2+ entry associated with [Ca2+]i oscillations in HEK293 cells, induced entry that was inhibited only by much higher concentrations of Gd3+ and was unaffected by 100 microm 2-APB. Like arachidonic acid-induced entry, the entry associated with [Ca2)]i oscillations was insensitive to inhibition by Gd3+ but was completely blocked by 100 microm 2-APB. These findings indicate that the signaling pathway responsible for the Ca2+) entry driving [Ca2+]i oscillations in HEK293 cells is more complex than originally thought, and may involve neither capacitative calcium entry nor a role for PLA2 and arachidonic acid.
Assuntos
Sinalização do Cálcio , Receptores Muscarínicos/metabolismo , Ácido Araquidônico/farmacologia , Transporte Biológico , Compostos de Boro/farmacologia , Carbacol/farmacologia , Interações Medicamentosas , Gadolínio/farmacologia , Humanos , Tapsigargina/farmacologiaRESUMO
We examined the activation and regulation of calcium release-activated calcium current (I(crac)) in RBL-1 cells in response to various Ca(2+) store-depleting agents. With [Ca(2+)](i) strongly buffered to 100 nM, I(crac) was activated by ionomycin, thapsigargin, inositol 1,4,5-trisphosphate (IP(3)), and two metabolically stable IP(3) receptor agonists, adenophostin A and L-alpha-glycerophospho-D-myoinositol-4,5-bisphosphate (GPIP(2)). With minimal [Ca(2+)](i) buffering, with [Ca(2+)](i) free to fluctuate I(crac) was activated by ionomycin, thapsigargin, and by the potent IP(3) receptor agonist, adenophostin A, but not by GPIP(2) or IP(3) itself. Likewise, when [Ca(2+)](i) was strongly buffered to 500 nM, ionomycin, thapsigargin, and adenophostin A did and GPIP(2) and IP(3) did not activate detectable I(crac). However, with minimal [Ca(2+)](i) buffering, or with [Ca(2+)](i) buffered to 500 nM, GPIP(2) was able to fully activate detectable I(crac) if uptake of Ca(2+) intracellular stores was first inhibited. Our findings suggest that when IP(3) activates the IP(3) receptor, the resulting influx of Ca(2+) quickly inactivates the receptor, and Ca(2+) is re-accumulated at sites that regulate I(crac). Adenophostin A, by virtue of its high receptor affinity, is resistant to this inactivation. Comparison of thapsigargin-releasable Ca(2+) pools following activation by different IP(3) receptor agonists indicates that the critical regulatory pool of Ca(2+) may be very small in comparison to the total IP(3)-sensitive component of the endoplasmic reticulum. These findings reveal new and important roles for IP(3) receptors located on discrete IP(3)-sensitive Ca(2+) pools in calcium feedback regulation of I(crac) and capacitative calcium entry.
Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Adenosina/análogos & derivados , Adenosina/farmacologia , Animais , Inibidores Enzimáticos/farmacologia , Retroalimentação , Fluorescência , Fura-2/química , Inositol 1,4,5-Trifosfato/farmacologia , Receptores de Inositol 1,4,5-Trifosfato , Fosfatos de Inositol/farmacologia , Ionomicina/farmacologia , Ionóforos/farmacologia , Técnicas de Patch-Clamp , Ratos , Tapsigargina/farmacologia , Células Tumorais CultivadasRESUMO
In A7r5 cells with empty intracellular Ca(2+) stores in which the cytosolic free Ca(2+) concentration ([Ca(2+)](i)) had been increased by capacitative Ca(2+) entry, stimulation of receptors linked to phospholipase C (PLC), including those for Arg(8)-vasopressin (AVP) and platelet-derived growth factor (PDGF), caused a decrease in [Ca(2+)](i.) This effect was further examined in a stable variant of the A7r5 cell line in which the usual ability of hormones to stimulate non-capacitative Ca(2+) entry is not expresssed. In thapsigargin-treated cells, neither AVP nor PDGF affected capacitative Mn(2+) or Ba(2+) entry, but both stimulated the rate of Ca(2+) extrusion, and their abilities to decrease [Ca(2+)](i) were only partially inhibited by removal of extracellular Na(+). These results suggest that receptors linked to PLC also stimulate plasma membrane Ca(2+) pumps. Activation of protein kinase C by phorbol 12, 13-dibutyrate (PDBu, 1 microM) also caused a decrease in [Ca(2+)](i) by accelerating Ca(2+) removal from the cytosol; the effect was again only partially inhibited by removal of extracellular Na(+). An inhibitor of PKC, Ro31-8220 (10 microM), abolished the ability of PDBu to decrease [Ca(2+)](i), without affecting the response to maximal or submaximal concentrations of AVP. Similar experiments with PDGF were impracticable because Ro31-8220, presumably by inhibiting the tyrosine kinase activity of the PDGF receptor, abolished all responses to PDGF. U73122 (10 microM), an inhibitor of PLC, completely inhibited PDGF- or AVP-evoked Ca(2+) mobilization, without preventing either stimulus from causing a decrease in [Ca(2+)](i). We conclude that receptors coupled to PLC, whether via G-proteins or protein tyrosine kinase activity, also share an ability to stimulate the plasma membrane Ca(2+) pump via a mechanism that does not require PLC activity.
Assuntos
Cálcio/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Receptores de Superfície Celular/metabolismo , Fosfolipases Tipo C/metabolismo , Animais , Arginina Vasopressina/antagonistas & inibidores , Arginina Vasopressina/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , ATPases Transportadoras de Cálcio/metabolismo , Cátions Bivalentes/metabolismo , Linhagem Celular , Citosol/efeitos dos fármacos , Citosol/enzimologia , Citosol/metabolismo , Ativação Enzimática/efeitos dos fármacos , Hidrólise/efeitos dos fármacos , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/enzimologia , Músculo Liso Vascular/metabolismo , Dibutirato de 12,13-Forbol/farmacologia , Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Fator de Crescimento Derivado de Plaquetas/farmacologia , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Ratos , Sódio/metabolismo , Tapsigargina/farmacologia , Fosfolipases Tipo C/antagonistas & inibidoresRESUMO
1. Depletion of the Ca2+ stores of A7r5 cells stimulated Ca2+, though not Sr2+, entry. Vasopressin (AVP) or platelet-derived growth factor (PDGF) stimulated Sr2+ entry. The cells therefore express a capacitative pathway activated by empty stores and a non-capacitative pathway stimulated by receptors; only the former is permeable to Mn2+ and only the latter to Sr2+. 2. Neither empty stores nor inositol 1,4,5-trisphosphate (InsP3) binding to its receptors are required for activation of the non-capacitative pathway, because microinjection of cells with heparin prevented PDGF-evoked Ca2+ mobilization but not Sr2+ entry. 3. Low concentrations of Gd3+ irreversibly blocked capacitative Ca2+ entry without affecting AVP-evoked Sr2+ entry. After inhibition of the capacitative pathway with Gd3+, AVP evoked a substantial increase in cytosolic [Ca2+], confirming that the non-capacitative pathway can evoke a significant increase in cytosolic [Ca2+]. 4. Arachidonic acid mimicked the effect of AVP on Sr2+ entry without stimulating Mn2+ entry; the Sr2+ entry was inhibited by 100 microM Gd3+, but not by 1 microM Gd3+ which completely inhibited capacitative Ca2+ entry. The effects of arachidonic acid did not require its metabolism. 5. AVP-evoked Sr2+ entry was unaffected by isotetrandrine, an inhibitor of G protein-coupled phospholipase A2. U73122, an inhibitor of phosphoinositidase C, inhibited AVP-evoked formation of inositol phosphates and Sr2+ entry. The effects of phorbol esters and Ro31-8220 (a protein kinase C inhibitor) established that protein kinase C did not mediate the effects of AVP on the non-capacitative pathway. An inhibitor of diacylglycerol lipase, RHC-80267, inhibited AVP-evoked Sr2+ entry without affecting capacitative Ca2+ entry or release of Ca2+ stores. 6. Selective inhibition of capacitative Ca2+ entry with Gd3+ revealed that the non-capacitative pathway is the major route for the Ca2+ entry evoked by low AVP concentrations. 7. We conclude that in A7r5 cells, the Ca2+ entry evoked by low concentrations of AVP is mediated largely by a non-capacitative pathway directly regulated by arachidonic acid produced by the sequential activities of phosphoinositidase C and diacylglycerol lipase.
Assuntos
Ácido Araquidônico/metabolismo , Arginina Vasopressina/farmacologia , Cálcio/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Animais , Linhagem Celular , Gadolínio/farmacologia , Transporte de Íons/efeitos dos fármacos , Manganês/metabolismo , Músculo Liso Vascular/citologia , Diester Fosfórico Hidrolases/metabolismo , Ratos , Estrôncio/metabolismoRESUMO
The complex changes in intracellular Ca2+ concentration that follow cell stimulation reflect the concerted activities of Ca2+ channels in the plasma membrane and in the membranes of intracellular stores, and the opposing actions of the mechanisms that extrude Ca2+ from the cytosol. Disentangling the roles of each of these processes is hampered by the lack of adequately selective pharmacological tools. In this review, Colin Taylor and Lisa Broad summarize the more serious problems associated with some of the commonly used drugs, and describe specific situations in which the multiple effects of drugs on Ca2(+)-signalling pathways have confused analysis of these pathways.
Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Cálcio/metabolismo , Membranas Intracelulares/efeitos dos fármacos , Animais , Canais de Cálcio/química , Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Humanos , Receptores de Inositol 1,4,5-Trifosfato , Membranas Intracelulares/metabolismo , Receptores Citoplasmáticos e Nucleares/química , Transdução de Sinais/efeitos dos fármacos , Fosfolipases Tipo C/antagonistas & inibidores , Vasodilatadores/farmacologiaRESUMO
Differentiation of BC3H1 cells leads to expression of a variety of proteins characteristic of smooth muscle and to changes in the behaviour of intracellular Ca2+ stores. Treatment of both differentiated and undifferentiated cells with thapsigargin (2 microM) emptied their intracellular Ca2+ stores, and in the presence of extracellular Ca2+ caused an increase in cytosolic [Ca2+] that rapidly reversed after its removal. The amplitudes of these capacitative Ca2+ entry signals were 101 +/- 8 nM (n = 42) in differentiated cells and 188 +/- 16 nM (n = 35) in undifferentiated cells. Mn2+ entry in thapsigargin-treated cells, measured by recording the quenching of cytosolic fura 2 fluorescence, was 374 +/- 26% (n = 34) and 154 +/- 7% (n = 41) of control rates in differentiated and undifferentiated cells, respectively. Empty stores caused Ba2+ entry to increase to 282 +/- 20% (n = 8) of its basal rate in differentiated cells and to 187 +/- 20% (n = 8) in undifferentiated cells. Rates of Ca2+ extrusion, measured after rapid removal of extracellular Ca2+ from cells in which capacitative Ca2+ entry had been activated, were similar in differentiated (t1/2 = 23 +/- 2 s, n = 7) and undifferentiated (23 +/- 1 s, n = 6) cells. The different relationships between capacitative Ca2+ and Mn2+ signals are not, therefore, a consequence of more active Ca2+ extrusion mechanisms in differentiated cells, nor are they a consequence of different fura 2 loadings in the two cell types. We conclude that during differentiation of BC3Hl cells, the cation selectivity of the capacitative pathway changes, becoming relatively more permeable to Mn2+ and Ba2+. The change may result either from expression of a different capacitative pathway or from modification of the permeation properties of a single pathway.
Assuntos
Cálcio/metabolismo , Cátions Bivalentes/metabolismo , Diferenciação Celular , Inibidores Enzimáticos/farmacologia , Terpenos/farmacologia , Trifosfato de Adenosina/farmacologia , Animais , Bário/metabolismo , Transporte Biológico/efeitos dos fármacos , Cafeína/farmacologia , ATPases Transportadoras de Cálcio/antagonistas & inibidores , Linhagem Celular , Citosol/metabolismo , Histamina/farmacologia , Cinética , Manganês/metabolismo , Camundongos , Músculo Liso , Transdução de Sinais/efeitos dos fármacos , Especificidade por Substrato , TapsigarginaRESUMO
The present study investigated the characteristics of D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) binding sites in crude membrane preparations of rabbit aortic smooth muscle. A particular aim was to demonstrate if increases in cytoplasmic cyclic guanosine 3':5' monophosphate (cGMP), which mediates the effect of nitrovasodilators, may cause smooth muscle relaxation in part by the displacement of Ins(1,4,5)P3 binding. Negligible Ins(1,4,5)P3 binding was observed at pH < 7, while maximum binding occurred over the pH range 8-9. Saturation analysis of isotopic dilution binding data revealed an apparently homogenous population of Ins(1,4,5)P3 binding sites with a KD of 4.02 +/- 0.53 nM and a Bmax of 27.7 +/- 4.6 fmol/mg protein. Heparin, an Ins(1,4,5)P3 receptor antagonist, inhibited binding with an IC50 of 11.43 +/- 2.81 micrograms/ml. The ability of other polyphosphate compounds to inhibit Ins(1,4,5)P3 binding in this preparation was also examined. D-myo-Inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4), adenosine 5'-triphosphate (ATP) and guanosine 5'-triphosphate (GTP) inhibited Ins(1,4,5)P3 binding, although each was significantly less potent that Ins(1,4,5)P3. In contrast, cyclic guanosine 3':5' monophosphate (cGMP) did not significantly alter Ins(1,4,5)P3 binding in rabbit aortic smooth muscle. This observation suggests that competitive inhibition of Ins(1,4,5)P3 receptor binding is not an important consideration in cGMP-mediated vascular smooth muscle cell relaxation.