RESUMO
Metabolic changes are critical in the regulation of Ca2+ influx in central and peripheral neuroendocrine cells. To study the regulation of L-type Ca2+ channels by AMPK we used biochemical reagents and ATP/glucose-concentration manipulations in rat chromaffin cells. AICAR and Compound-C, at low concentration, significantly induce changes in L-type Ca2+ channel-current amplitude and voltage dependence. Remarkably, an overlasting decrease in the channel-current density can be induced by lowering the intracellular level of ATP. Accordingly, Ca2+ channel-current density gradually diminishes by decreasing the extracellular glucose concentration. By using immunofluorescence, a decrease in the expression of CaV1.2 is observed while decreasing extracellular glucose, suggesting that AMPK reduces the number of functional Ca2+ channels into the plasma membrane. Together, these results support for the first time the dependence of metabolic changes in the maintenance of Ca2+ channel-current by AMPK. They reveal a key step in Ca2+ influx in secretory cells.
Assuntos
Proteínas Quinases Ativadas por AMP , Aminoimidazol Carboxamida , Canais de Cálcio Tipo L , Células Cromafins , Glucose , Animais , Células Cromafins/metabolismo , Células Cromafins/efeitos dos fármacos , Canais de Cálcio Tipo L/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Ratos , Glucose/metabolismo , Glucose/farmacologia , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Trifosfato de Adenosina/metabolismo , Ribonucleotídeos/farmacologia , Pirimidinas/farmacologia , Cálcio/metabolismo , Pirazóis/farmacologia , Células Cultivadas , Ratos Wistar , Ativação do Canal Iônico/efeitos dos fármacosRESUMO
BACKGROUND AND PURPOSE: ATP is highly accumulated in secretory vesicles and secreted upon exocytosis from neurons and endocrine cells. In adrenal chromaffin granules, intraluminal ATP reaches concentrations over 100 mM. However, how these large amounts of ATP contribute to exocytosis has not been investigated. EXPERIMENTAL APPROACH: Exocytotic events in bovine and mouse adrenal chromaffin cells were measured with single cell amperometry. Cytosolic Ca2+ measurements were carried out in Fluo-4 loaded cells. Submembrane Ca2+ was examined in PC12 cells transfected with a membrane-tethered Ca2+ indicator Lck-GCaMP3. ATP release was measured using the luciferin/luciferase assay. Knockdown of P2X7 receptors was induced with short interfering RNA (siRNA). Direct Ca2+ influx through this receptor was measured using a P2X7 receptor-GCamp6 construct. KEY RESULTS: ATP induced exocytosis in chromaffin cells, whereas the ectonucleotidase apyrase reduced the release events induced by the nicotinic agonist dimethylphenylpiperazinium (DMPP), high KCl, or ionomycin. The purinergic agonist BzATP also promoted a secretory response that was dependent on extracellular Ca2+. A740003, a P2X7 receptor antagonist, abolished secretory responses of these secretagogues. Exocytosis was also diminished in chromaffin cells when P2X7 receptors were silenced using siRNAs and in cells of P2X7 receptor knockout mice. In PC12 cells, DMPP induced ATP release, triggering Ca2+ influx through P2X7 receptors. Furthermore, BzATP, DMPP, and KCl allowed the formation of submembrane Ca2+ microdomains inhibited by A740003. CONCLUSION AND IMPLICATIONS: Autocrine activation of P2X7 receptors constitutes a crucial feedback system that amplifies the secretion of catecholamines in chromaffin cells by favouring submembrane Ca2+ microdomains.
Assuntos
Trifosfato de Adenosina , Catecolaminas , Células Cromafins , Exocitose , Receptores Purinérgicos P2X7 , Animais , Receptores Purinérgicos P2X7/metabolismo , Células Cromafins/metabolismo , Células Cromafins/efeitos dos fármacos , Bovinos , Trifosfato de Adenosina/metabolismo , Camundongos , Catecolaminas/metabolismo , Exocitose/efeitos dos fármacos , Células PC12 , Ratos , Cálcio/metabolismo , Comunicação Autócrina , Camundongos Endogâmicos C57BL , Células Cultivadas , MasculinoRESUMO
Pannexin-1 (Panx1) forms plasma membrane channels that allow the exchange of small molecules between the intracellular and extracellular compartments, and are involved in diverse physiological and pathological responses in the nervous system. However, the signaling mechanisms that induce their opening still remain elusive. Here, we propose a new mechanism for Panx1 channel activation through a functional crosstalk with the highly Ca2+ permeable α7 nicotinic acetylcholine receptor (nAChR). Consistent with this hypothesis, we found that activation of α7 nAChRs induces Panx1-mediated dye uptake and ATP release in the neuroblastoma cell line SH-SY5Y-α7. Using membrane permeant Ca2+ chelators, total internal reflection fluorescence microscopy in SH-SY5Y-α7 cells expressing a membrane-tethered GCAMP3, and Src kinase inhibitors, we further demonstrated that Panx1 channel opening depends on Ca2+ signals localized in submembrane areas, as well as on Src kinases. In turn, Panx1 channels amplify cytosolic Ca2+ signals induced by the activation of α7 nAChRs, by a mechanism that seems to involve ATP release and P2X7 receptor activation, as hydrolysis of extracellular ATP with apyrase or blockage of P2X7 receptors with oxidized ATP significantly reduces the α7 nAChR-Ca2+ signal. The physiological relevance of this crosstalk was also demonstrated in neuroendocrine chromaffin cells, wherein Panx1 channels and P2X7 receptors contribute to the exocytotic release of catecholamines triggered by α7 nAChRs, as measured by amperometry. Together these findings point to a functional coupling between α7 nAChRs, Panx1 channels and P2X7 receptors with physiological relevance in neurosecretion.
Assuntos
Células Cromafins/metabolismo , Conexinas/metabolismo , Exocitose/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Receptor Cross-Talk/fisiologia , Receptores Purinérgicos P2X7/metabolismo , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Animais , Quelantes de Cálcio/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Bovinos , Linhagem Celular Tumoral , Células Cromafins/efeitos dos fármacos , Exocitose/efeitos dos fármacos , Humanos , Camundongos , Receptor Cross-Talk/efeitos dos fármacosRESUMO
Venoms of medically important scorpions from Buthidae family have been intensively studied, in contrast to non-buthid venoms, for which knowledge is scarce. In this work, we characterized the venom of a Diplocentridae species, Didymocentrus krausi, a small fossorial scorpion that inhabits the Tropical Dry Forest of Central America. D. krausi venom soluble fraction contains proteases with enzymatic activity on gelatin and casein. Mass spectrometry and venomic analysis confirmed the presence of elastase-like, cathepsin-O-like proteases and a neprilysin-like metalloproteinase. We did not detect phospholipase A2, C or D, nor hyaluronidase activity in the venom. By homology-based venom gland transcriptomic analysis, NDBPs, a ß-KTx-like peptide, and other putative toxin transcripts were found, which, together with a p-benzoquinone compound present in the venom, could potentially explain its direct hemolytic and cytotoxic effects in several mammalian cell lines. Cytotoxicity of D. krausi venom was higher than the effect of venoms from two buthid scorpion species distributed in Costa Rica, Centruroides edwardsii and Tityus pachyurus. Even though D. krausi venom was not lethal to mice or crickets, when injected in mouse gastrocnemius muscle at high doses it induced pathological effects at 24â¯h, which include myonecrosis, weak hemorrhage, and inflammatory infiltration. We observed an apparent thrombotic effect in the skin blood vessels, but no in vitro fibrinogenolytic activity was detected. In crickets, D. krausi venom induced toxicity and paralysis in short periods of time.
Assuntos
Sobrevivência Celular/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Venenos de Escorpião/química , Venenos de Escorpião/toxicidade , Escorpiões/fisiologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/metabolismo , Proteínas de Artrópodes/toxicidade , Linhagem Celular , Células Cromafins/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Gryllidae/efeitos dos fármacos , Humanos , Camundongos , Mioblastos/efeitos dos fármacos , Coelhos , RatosRESUMO
It is known that chronic ethanol (EtOH) consumption leads to hypertension development and has been associated with deleterious effects on the cardiovascular system. Whether this condition alters calcium (Ca2+) signaling and exocytosis in adrenal chromaffin cells (CCs) as the case is for genetic hypertension, is unknown. We explored this question in four randomized experimental groups, male Wistar Kyoto (WKY/EtOH) and Spontaneously Hypertensive (SHR/EtOH) rats were subjected to the intake of increasing EtOH concentrations (5-20%, for 30 days) and their respective controls (WKY/Control and SHR/Control) received water. WKY/EtOH developed hypertension and cardiac hypertrophy; blood aldehyde dehydrogenase (ALDH) and H2O2 were also augmented. In comparison with WKY/Control, CCs from WKY/EtOH had the following features: (i) depolarization and higher frequency of spontaneous action potentials; (ii) decreased Ca2+ currents with slower inactivation; (iii) decreased K+ currents; (iv) augmented K+-elicited cytosolic Ca2+ transients ([Ca2+]c); (v) enhanced K+-elicited catecholamine release. These cardiovascular, blood and CCs changes were qualitatively similar to those undergone by SHR/Control and SHR/EtOH. The results suggest that the hypertension elicited by chronic EtOH has pathogenic features common to genetic hypertension namely, augmented [Ca2+]c transients and catecholamine release from their CCs.
Assuntos
Consumo de Bebidas Alcoólicas/efeitos adversos , Catecolaminas/metabolismo , Células Cromafins/efeitos dos fármacos , Células Cromafins/metabolismo , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Hipertensão/induzido quimicamente , Hipertensão/patologia , Potenciais de Ação/efeitos dos fármacos , Animais , Cálcio/metabolismo , Células Cromafins/patologia , Citosol/efeitos dos fármacos , Citosol/metabolismo , Etanol/farmacologia , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Masculino , Potássio/metabolismo , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Fatores de TempoRESUMO
Adrenal chromaffin cells (CCs) from spontaneously hypertensive rats (SHRs) secrete more catecholamine (CA) upon stimulation than CCs from normotensive Wistar Kyoto rats (WKY). Unitary CA exocytosis events, both spontaneous and stimulated, were amperometrically recorded from cultured WKY and SHR CCs. Both strains display spontaneous amperometric spikes but SHR CCs produce more spikes and of higher mean amplitude. After a brief stimulation with high K(+) or caffeine which produces voltage-gated Ca(2+) influx or intracellular Ca(2+) release, respectively, more spikes and of greater mean amplitude and unitary charge were recorded in SHR CCs. Consequently, peak cumulative charge was ~2-fold higher in SHR CCs. Ryanodine (10 µM), a specific blocker of the ryanodine receptors reduced depolarization-induced peak cumulative charge by ~10 % in WKY and ~77 % in SHR CCs, suggesting, a larger contribution of Ca(2+)-induced Ca(2+) release to CA exocytosis in SHR CCs. Accordingly, Ca(2+) imaging showed larger [Ca(2+)]i signals induced both by depolarization and caffeine in SHR CCs. Distribution amplitude histograms showed that small amperometric spikes (0-50 pA) are more frequent in WKY than in SHR CCs. Conversely, medium (50-190 pA) and large (190-290 pA) spikes are more numerous in SHR than in WKY CCs. This study reveals that the enhanced CA secretion in SHR CCs results from a combination of (1) larger depolarization-induced Ca(2+) transients, due to a greater Ca(2+)-induced intracellular Ca(2+) release, (2) more exocytosis events per time unit, and (3) a greater proportion of medium and large amperometric spikes probably due to a higher mean CA content per granule. Enhanced CA release by excessive amplification by Ca(2+) induced Ca(2+) release and larger granule catecholamine content contributes to the increased CA plasma levels and vasomotor tone in SHRs.
Assuntos
Glândulas Suprarrenais/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Cálcio/metabolismo , Cálcio/farmacologia , Catecolaminas/metabolismo , Células Cromafins/metabolismo , Glândulas Suprarrenais/efeitos dos fármacos , Animais , Pressão Sanguínea/efeitos dos fármacos , Cafeína/farmacologia , Células Cultivadas , Células Cromafins/efeitos dos fármacos , Exocitose , Frequência Cardíaca/efeitos dos fármacos , Masculino , Inibidores de Fosfodiesterase/farmacologia , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Rianodina/farmacologia , Canal de Liberação de Cálcio do Receptor de Rianodina/efeitos dos fármacosRESUMO
The cortical actin network is dynamically rearranged during secretory processes. Nevertheless, it is unclear how de novo actin polymerization and the disruption of the preexisting actin network control transmitter release. Here we show that in bovine adrenal chromaffin cells, both formation of new actin filaments and disruption of the preexisting cortical actin network are induced by Ca2+ concentrations that trigger exocytosis. These two processes appear to regulate different stages of exocytosis; whereas the inhibition of actin polymerization with the N-WASP inhibitor wiskostatin restricts fusion pore expansion, thus limiting the release of transmitters, the disruption of the cortical actin network with cytochalasin D increases the amount of transmitter released per event. Further, the Src kinase inhibitor PP2, and cSrc SH2 and SH3 domains also suppress Ca2+-dependent actin polymerization, and slow down fusion pore expansion without disturbing the cortical F-actin organization. Finally, the isolated SH3 domain of c-Src prevents both the disruption of the actin network and the increase in the quantal release induced by cytochalasin D. These findings support a model where a rise in the cytosolic Ca2+ triggers actin polymerization through a mechanism that involves Src kinases. The newly formed actin filaments would speed up the expansion of the initial fusion pore, whereas the preexisting actin network might control a different step of the exocytosis process.
Assuntos
Actinas/metabolismo , Células Cromafins/metabolismo , Quinases da Família src/metabolismo , Citoesqueleto de Actina/efeitos dos fármacos , Animais , Cálcio/farmacologia , Bovinos , Células Cultivadas , Células Cromafins/citologia , Células Cromafins/efeitos dos fármacos , Citocalasina D/farmacologia , Exocitose/efeitos dos fármacos , Cinética , Pirazóis/farmacologia , Pirimidinas/farmacologia , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismo , Quinases da Família src/química , Quinases da Família src/genéticaRESUMO
AIM: After exocytosis, neuroendocrine cells and neurones keep constant the plasma membrane and the releasable vesicle pools by performing endocytosis and vesicular cycling. Patch-clamp capacitance measurements on chromaffin cells showed that strong Ca(+2) entry activates excess retrieval: a rapid endocytosis process that retrieves more membrane than the one fused by preceding exocytosis. The main purpose of the present experiments was to study the recycling pathway that follows excess retrieval, which is unknown. METHODS: Membrane recycling after exocytosis-endocytosis can be studied by fluorescence imaging assays with FM1-43 (Perez Bay et al. Am J Physiol Cell Physiol 2007; 293, C1509). In this work, we used this assay in combination with fluorescent dextrans and specific organelle-targeted antibodies to study the membrane recycling after excess retrieval in mouse chromaffin cells. RESULTS: Excess retrieval was observed after the application of high-K(+) or cholinergic agonists during 15 or 30 s in the presence of FM1-43. We found that the excess retrieval membrane pool (defined as endocytosis-exocytosis) was associated with the generation of a non-releasable fraction of membrane (up to 30% of plasma membrane surface) colocalizing with the lysosomal compartment. The excess retrieval membrane pool followed a saturable cytosolic Ca(2+) dependency, and it was suppressed by inhibitors of L-type Ca(2+) channels, endoplasmic reticulum Ca(2+) release and PKC. CONCLUSION: Excess retrieval is not associated with the cycling of releasable vesicles, but it is related to the formation of non-releasable endosomes. This process is activated by a concerted contribution of Ca(2+) entry through L-channels and Ca(2+) release from endoplasmic reticulum.
Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Membrana Celular/metabolismo , Células Cromafins/metabolismo , Endocitose , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/efeitos dos fármacos , Canais de Cálcio Tipo L/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Agonistas Colinérgicos/farmacologia , Células Cromafins/efeitos dos fármacos , Capacitância Elétrica , Endocitose/efeitos dos fármacos , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Endossomos/metabolismo , Exocitose , Corantes Fluorescentes/metabolismo , Fusão de Membrana , Potenciais da Membrana , Camundongos , Microscopia de Fluorescência , Técnicas de Patch-Clamp , Potássio/metabolismo , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Compostos de Piridínio/metabolismo , Compostos de Amônio Quaternário/metabolismo , Fatores de TempoRESUMO
Ca(2+) currents (I(Ca)) recorded from adrenal chromaffin cells (CCs) of spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats are similar to one another, but different from those recorded in other rodent species. I(Ca) in WKY/SHR CCs comprises an early, transient (I(Ca(e))) and a late, sustained component (I(Ca(s))). In Wistar CCs, I(Ca(e)) is absent, and I(Ca(s)) is of greater amplitude. Activation and steady-state inactivation of I(Ca(e)) and I(Ca(s)) in WKY/SHR CCs suggest the recruitment of at least two populations of Ca(2+) channels with different voltage dependence and kinetics. In WKY/SHR CCs, I(Ca(e)) is inhibited by nifedipine, enhanced by BAY K 8644, is not blocked by the mibefradil analog NNC 55-0396, and displays Ca(2+)-dependent inactivation and fast deactivation kinetics, suggesting that it results from the opening of L-type rather than T-type Ca(2+) channels. I(Ca(e)) properties suggest that it originates from the opening of Ca(2+) channels formed with the short splice variant (Ca(V)1.3(42A)). RT-PCR showed that expression of Ca(V)1.3(42A) mRNA is similar in both Wistar and WKY/SHR, but that the long variant (Ca(V)1.3(42)) is virtually absent in WKY/SHR. Thus I(Ca(e)) corresponds to the recruitment of Ca(V)1.3(42A) channels, unmasked by the absence of Ca(V)1.3(42) channels. Studies in WKY CCs do not report major functional alterations, despite the unusual expression pattern of Ca(V)1.3 splice variants. It remains to be established if more subtle functional alterations exist, and if the atypical splicing pattern of Ca(V)1.3 could be related to the functional and behavioral alterations reported in WKY/SHR rats, including their susceptibility to develop hypertension.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Cálcio/metabolismo , Células Cromafins/metabolismo , Éster Metílico do Ácido 3-Piridinacarboxílico, 1,4-Di-Hidro-2,6-Dimetil-5-Nitro-4-(2-(Trifluormetil)fenil)/farmacologia , Animais , Benzimidazóis/farmacologia , Agonistas dos Canais de Cálcio/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/genética , Células Cromafins/efeitos dos fármacos , Ciclopropanos/farmacologia , Células HEK293 , Humanos , Masculino , Naftalenos/farmacologia , Nifedipino/farmacologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKYRESUMO
Spontaneously hypertensive rats (SHR) are widely used as model to investigate the pathophysiological mechanisms of essential hypertension. Catecholamine plasma levels are elevated in SHR, suggesting alterations of the sympathoadrenal axis. The residual hypertension in sympathectomized SHR is reduced after demedullation, suggesting a dysfunction of the adrenal medulla. Intact adrenal glands exposed to acetylcholine or high K+ release more catecholamine in SHR than in normotensive Wistar Kyoto (WKY) rats, and adrenal chromaffin cells (CCs) from SHR secrete more catecholamines than CCs from WKY rats. Since Ca2+ entry through voltage-gated Ca2+ channels (VGCC) triggers exocytosis, alterations in the functional properties of these channels might underlie the enhanced catecholamine release in SHR. This study compares the electrophysiological properties of VGCC from CCs in acute adrenal slices from WKY rats and SHR at an early stage of hypertension. No significant differences were found in the macroscopic Ca2+ currents (current density, IV curve, voltage dependence of activation and inactivation, kinetics) between CCs of SHR and WKY rats, suggesting that Ca2+ entry through VGCC is not significantly different between these strains, at least at early stages of hypertension. Ca2+ buffering, sequestration and extrusion mechanisms, as well as Ca2+ release from intracellular stores, must now be evaluated to determine if alterations in their function can explain the enhanced catecholamine secretion reported in CCs from SHR.