RESUMO
Flavonoid galetin 3,6-dimethyl ether (FGAL) has been isolated from the aerial parts of Piptadenia stipulaceae and has shown a spasmolytic effect in guinea pig ileum. Thus, we aimed to characterize its relaxant mechanism of action. FGAL exhibited a higher relaxant effect on ileum pre-contracted by histamine (EC50=1.9±0.4×10(-7) M) than by KCl (EC50=2.6±0.5×10(-6) M) or carbachol (EC50=1.8±0.4×10(-6) M). The flavonoid inhibited the cumulative contractions to histamine, as well as to CaCl2 in depolarizing medium nominally Ca(2+)-free. The flavonoid relaxed the ileum pre-contracted by S-(-)-Bay K8644 (EC50=9.5±1.9×10(-6) M) but less potently pre-contracted by KCl or histamine. CsCl attenuated the relaxant effect of FGAL (EC50=1.1±0.3×10(-6) M), but apamin or tetraethylammonium (1mM) had no effect (EC50=2.6±0.2×10(-7) and 1.6±0.3×10(-7) M, respectively), ruling out the involvement of small and big conductance Ca(2+)-activated K(+) channels (SKCa and BKCa, respectively). Either 4-aminopyridine or glibenclamide attenuated the relaxant effect of FGAL (EC50=1.8±0.2×10(-6) and 1.5±0.5×10(-6) M, respectively), indicating the involvement of voltage- and ATP-sensitive K(+) channels (KV and KATP, respectively). FGAL did not alter the viability of intestinal myocytes in the MTT assay and decreased (88%) Fluo-4 fluorescence, indicating a decrease in cytosolic Ca(2+) concentration. Therefore, the relaxant mechanism of FGAL involves pseudo-irreversible noncompetitive antagonism of histaminergic receptors, KV and KATP activation and blockade of CaV1, thus leading to a reduction in cytosolic Ca(2+) levels.
Assuntos
Cálcio/metabolismo , Flavonoides/farmacologia , Íleo/efeitos dos fármacos , Contração Muscular/efeitos dos fármacos , Canais de Potássio/agonistas , Éster Metílico do Ácido 3-Piridinacarboxílico, 1,4-Di-Hidro-2,6-Dimetil-5-Nitro-4-(2-(Trifluormetil)fenil)/farmacologia , 4-Aminopiridina/farmacologia , Animais , Apamina/farmacologia , Cloreto de Cálcio/antagonistas & inibidores , Cloreto de Cálcio/farmacologia , Carbacol/antagonistas & inibidores , Carbacol/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Césio/farmacologia , Cloretos/farmacologia , Flavonoides/antagonistas & inibidores , Glibureto/farmacologia , Cobaias , Histamina/farmacologia , Antagonistas dos Receptores Histamínicos/farmacologia , Íleo/fisiologia , Células Musculares/efeitos dos fármacos , Bloqueadores dos Canais de Potássio/farmacologia , Cloreto de Potássio/antagonistas & inibidores , Cloreto de Potássio/farmacologia , TetraetilamônioRESUMO
The monoterpenes are the main constituents of most essential oils and p-cymene is a monoterpene commonly found in various species of aromatic herbs, which has been reported for anti-inflammatory, antinociceptive, and antimicrobial activities. However, there is no report concerning its pharmacological activity on the vascular smooth muscle. The aim of current work was to investigate the effects of p-cymene in isolated rat aorta and also study its mechanism of action. In this work, we show that p-cymene has a relaxant effect, in a dose-dependent way, on the vascular smooth muscle, regardless of the presence of the endothelium. Using a nonselective potassium channel blocker, the CsCl, the relaxant effect of p-cymene was attenuated. In the presence of more selective potassium channels blockers, such as TEA or 4-AP, no change in the relaxant effect of p-cymene was evidenced, indicating that BKCa and KV channels are not involved in that relaxant effect. However, in the presence of glibenclamide or BaCl2, KATP and Kir blockers, respectively, the relaxant effect of p-cymene was attenuated. The data presented indicate that p-cymene has a relaxing effect on rat aorta, regardless of the endothelium, but with the participation of the KATP and Kir channels.
Assuntos
Monoterpenos/farmacologia , Canais de Potássio/efeitos dos fármacos , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologia , 4-Aminopiridina/farmacologia , Animais , Aorta/efeitos dos fármacos , Césio/farmacologia , Cloretos/farmacologia , Cimenos , Dimetil Sulfóxido/farmacologia , Glibureto/farmacologia , Masculino , Músculo Liso Vascular/efeitos dos fármacos , Fenilefrina/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio/fisiologia , Ratos , Ratos Wistar , Tetraetilamônio/farmacologiaRESUMO
The intrinsic properties of spherical neurons play a fundamental role in the sensory processing of self-generated signals along a fast electrosensory pathway in electric fish. Previous results indicate that the spherical neuron's intrinsic properties depend mainly on the presence of two resonant currents that tend to clamp the voltage near the resting potential. Here we show that these are: a low-threshold potassium current blocked by 4-aminopyridine and a mixed cationic current blocked by cesium chloride. We also show that the low-threshold potassium current also causes the long refractory period, explaining the necessary properties that implement the dynamic filtering of the self-generated signals previously described. Comparative data from other fish and from the auditory system indicate that other single spiking onset neurons might differ in the channel repertoire observed in the spherical neurons of Gymnotus omarorum.
Assuntos
Gimnotiformes/fisiologia , Potenciais da Membrana/fisiologia , Neurônios/fisiologia , 4-Aminopiridina/farmacologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Césio/farmacologia , Cloretos/farmacologia , Venenos Elapídicos/farmacologia , Técnicas In Vitro , Potenciais da Membrana/efeitos dos fármacos , Moduladores de Transporte de Membrana/farmacologia , Mesencéfalo/efeitos dos fármacos , Mesencéfalo/fisiologia , Neurônios/efeitos dos fármacos , Técnicas de Patch-Clamp , Potássio/metabolismo , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio/metabolismo , Tetraetilamônio/farmacologiaRESUMO
Recent evidence suggests a major role for ionic fluxes in apoptotic cell death and apoptotic volume decrease. Cerebellar granule neurons (CGN) undergo apoptosis when they are treated with staurosporine or camptothecin (CPT) or when cells are transferred from high extracellular potassium (25 mM KCl [K(+)](e), K25) to low potassium concentration (5 mM KCl [K(+)](e), K5). In this study we described that all three apoptotic conditions induced apoptotic volume decrease in CGN and that two different potassium channel blockers, cesium (Cs(+)) and tetraethylammonium (TEA(+)), prevented the apoptotic volume decrease, caspase-3 activation, nuclear condensation and cell death induced by K5 and CPT, but not by staurosporine. Cs(+) and TEA(+) also blocked membrane currents generated in K5 conditions in CGN. On the other hand, non specific Cl(-) channel blockers such as 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS) prevented loss of cell volume induced by K5 or staurosporine. Only the Cl(-) channels blocker but not the K(+) channels blockers protected from staurosporine-induced death of CGN. These data suggest that ionic fluxes play a key role in the activation of the apoptotic volume decrease and apoptotic death of CGN, but the fine mechanism seems to depend on the apoptotic condition.
Assuntos
Apoptose/efeitos dos fármacos , Cerebelo/citologia , Césio/farmacologia , Cloretos/farmacologia , Neurônios/citologia , Potássio/farmacologia , Tetraetilamônio/farmacologia , Animais , Camptotecina/farmacologia , Caspase 3/metabolismo , Cátions Monovalentes , Tamanho Celular , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Canais de Cloreto/antagonistas & inibidores , Ativação Enzimática , Potenciais da Membrana/efeitos dos fármacos , Técnicas de Patch-Clamp , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio/fisiologia , Ratos , Estaurosporina/farmacologiaRESUMO
Multifunctional central pattern generators (CPGs) are circuits of neurons that can generate manifold actions from a single effector system. This study examined a bilateral pair of pharyngeal motor neurons, designated B67, that participate in the multifunctional feeding network of Aplysia californica. Fictive buccal motor programs (BMPs) were elicited with four distinct stimulus paradigms to assess the activity of B67 during ingestive versus egestive patterns. In both classes of programs, B67 fired during the phase of radula protraction and received a potent inhibitory postsynaptic potential (IPSP) during fictive radula retraction. When programs were ingestive, the retraction phase IPSP exhibited a depolarizing sag and was followed by a postinhibitory rebound (PIR) that could generate a postretraction phase of impulse activity. When programs were egestive, the depolarizing sag potential and PIR were both diminished or were not present. Examination of the membrane properties of B67 disclosed a cesium-sensitive depolarizing sag, a corresponding I(h)-like current, and PIR in its responses to hyperpolarizing pulses. Direct IPSPs originating from the influential CPG retraction phase interneuron B64 were also found to activate the sag potential and PIR of B67. Dopamine, a modulator that can promote ingestive behavior in this system, enhanced the sag potential, I(h)-like current, and PIR of B67. Finally, a pharyngeal muscle contraction followed the radula retraction phase of ingestive, but not egestive motor patterns. It is proposed that regulation of the intrinsic properties of this motor neuron can contribute to generating a program-specific phase of motor activity.
Assuntos
Comportamento Alimentar/fisiologia , Neurônios Motores/fisiologia , Movimento/fisiologia , Rede Nervosa/fisiologia , Vias Neurais/fisiologia , Animais , Aplysia , Comportamento Animal , Carbacol/farmacologia , Césio/farmacologia , Bochecha/inervação , Agonistas Colinérgicos/farmacologia , Dopamina/farmacologia , Estimulação Elétrica/métodos , Gânglios dos Invertebrados/citologia , Potenciais Pós-Sinápticos Inibidores/efeitos dos fármacos , Potenciais Pós-Sinápticos Inibidores/fisiologia , Potenciais Pós-Sinápticos Inibidores/efeitos da radiação , Interneurônios/efeitos dos fármacos , Interneurônios/fisiologia , Interneurônios/efeitos da radiação , Movimento/efeitos dos fármacos , Rede Nervosa/efeitos dos fármacos , Rede Nervosa/efeitos da radiação , Vias Neurais/efeitos dos fármacos , Vias Neurais/efeitos da radiação , Técnicas de Patch-Clamp , Tempo de Reação/efeitos dos fármacos , Tempo de Reação/fisiologia , Tempo de Reação/efeitos da radiaçãoRESUMO
Electrocytes are muscle-derived cells that generate the electric organ discharge (EOD) in most gymnotiform fish. We used an in vitro preparation to determine if the complex EOD of Gymnotus carapo was related to the membrane properties of electrocytes. We discovered that in addition to the three Na(+)-mediated conductances described in a recent paper [Sierra F, Comas V, Buño W, Macadar O (2005) Sodium-dependent plateau potentials in electrocytes of the electric fish Gymnotus carapo. J Comp Physiol A 191:1-11] there were four K(+)-dependent conductances. Membrane depolarization activated a delayed rectifier (I(K)) and an A-type (I(A)) current. I(A) displayed fast voltage-dependent activation-inactivation kinetics, was blocked by 4-aminopyridine (1 mM) and played a major role in action potential (AP) repolarization. Its voltage dependence and kinetics shape the brief AP that typifies Gymnotus electrocytes. The I(K) activated by depolarization contributed less to AP repolarization. Membrane hyperpolarization uncovered two inward rectifiers (IR1 and IR2) with voltage dependence and kinetics that correspond to the complex "hyperpolarizing responses" (HRs) described under current-clamp. IR1 shows "instantaneous" activation, is blocked by Ba(2+) and Cs(+) and displays a voltage and time dependent inactivation that matches the hyperpolarizing phase of the HR. The activation of IR2 is slower and at more negative potentials than IR1 and is resistant to Ba(2+) and Cs(+). This current fits the depolarizing phase of the HR. The EOD waveform of Gymnotus carapo is more complex than that of other gymnotiform fish species, the complexity originates in the voltage responses generated through the interactions of three Na(+) and four K(+) voltage- and time-dependent conductances although the innervation pattern also contributes [Trujillo-Cenóz O, Echagüe JA (1989) Waveform generation of the electric organ discharge in Gymnotus carapo. I. Morphology and innervation of the electric organ. J Comp Physiol A 165:343-351].
Assuntos
Membrana Celular/metabolismo , Órgão Elétrico/metabolismo , Gimnotiformes/metabolismo , Músculo Esquelético/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Animais , Bário/farmacologia , Membrana Celular/efeitos dos fármacos , Césio/farmacologia , Órgão Elétrico/citologia , Eletricidade , Gimnotiformes/anatomia & histologia , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/fisiologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Músculo Esquelético/anatomia & histologia , Músculo Esquelético/efeitos dos fármacos , Técnicas de Cultura de Órgãos , Técnicas de Patch-Clamp , Potássio/metabolismo , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/efeitos dos fármacos , Canais de Sódio/efeitos dos fármacos , Canais de Sódio/metabolismo , Especificidade da EspécieRESUMO
BACKGROUND AND PURPOSE: Central anti-nociceptive actions of baclofen involve activation of K+ channels. Here we assessed what types of K+ channel might participate in the peripheral anti-nociception induced by baclofen. EXPERIMENTAL APPROACH: Nociceptive thresholds to mechanical stimulation in rat paws treated with intraplantar prostaglandin E2.(PGE2) to induce hyperalgesia were measured 3 h after PGE2 injection. Other agents were also given by intraplantar injection. KEY RESULTS: Baclofen elicited a dose-dependent (15 - 240 microg per paw) anti-nociceptive effect. An intermediate dose of baclofen (60 microg) did not produce antinociception in the contralateral paw, showing its peripheral site of action. The GABAB receptor antagonist saclofen (12.5 - 100 microg per paw) antagonized, in a dose-dependent manner, peripheral antinociception induced by baclofen (60 microg), suggesting a specific effect. This antinociceptive action of baclofen was unaffected by bicuculline, GABAA receptor antagonist (80 microg per paw), or by (1,2,5,6 tetrahydropyridin-4-yl) methylphosphinic acid, GABAC receptor antagonist (20 microg per paw). The peripheral antinociception induced by baclofen (60 microg) was reversed, in a dose-dependent manner, by the voltage-dependent K+ channel blockers tetraethylammonium (7.5 - 30 microg per paw) and 4-aminopyridine (2.5 - 10 microg per paw). The blockers of other K+ channels, glibenclamide (160 microg), tolbutamide (320 microg), charybdotoxin (2 microg), dequalinium (50 microg) and caesium (500 microg) had no effect. CONCLUSIONS AND IMPLICATIONS: This study provides evidence that the peripheral antinociceptive effect of the GABAB receptor agonist baclofen results from the activation of tetraethylammonium-sensitive K+ channels. Other K+ channels appear not to be involved.
Assuntos
Analgésicos/farmacologia , Baclofeno/farmacologia , Agonistas GABAérgicos/farmacologia , Agonistas dos Receptores de GABA-B , Canais de Potássio/efeitos dos fármacos , Tetraetilamônio/farmacologia , Animais , Baclofeno/análogos & derivados , Bicuculina/farmacologia , Césio/farmacologia , Charibdotoxina/farmacologia , Dequalínio/farmacologia , Glibureto/farmacologia , Masculino , Ratos , Ratos Wistar , Tolbutamida/farmacologiaRESUMO
In this paper we describe a simple and rapid protocol for DNA base composition determination by CsCl gradient in the presence of acrylamide. This method permits the determination of GC content in microgram amounts of DNA, and results are easily documented in photographs or graphs. The protocol was applied to the characterization of nematode DNA, but can be used for other organisms. Analyzing several experiments the mean standard deviation observed in the calculated GC content is near 1.3%.
Assuntos
Composição de Bases , Biofísica/métodos , Centrifugação com Gradiente de Concentração/métodos , Césio/farmacologia , Cloretos/farmacologia , Acrilamida/farmacologia , Animais , Caenorhabditis elegans , DNA/química , DNA/metabolismo , Escherichia coli/metabolismo , Temperatura Alta , Temperatura , UltracentrifugaçãoRESUMO
1. The use of molecular biology in combination with electrophysiology in the HEK-293 cell line has given fascinating insights into neuronal ion channel function. Nevertheless, to fully understand the properties of channels exogenously expressed in these cells, a detailed evaluation of endogenous channels is indispensable. 2. Previous studies have shown the expression of endogenous voltage-gated K+, Ca2+, and Cl- channels and this predicts that changes in membrane potential will cause intramembrane charge movement, though this gating charge translocation remain undefined. Here, we confirm this prediction by performing patch-clamp experiments to record ionic and gating currents. Our data show that HEK-293 cells express at least two types of K+-selective endogenous channels which sustain the majority of the ionic current, and exclude a significant contribution from Ca2+ and Cl- channels to the whole-cell current. 3. Gating currents were unambiguously resolved after ionic current blockade enabling this first report of intramembrane charge movement in HEK-293 cells arising entirely from endogenous K+ channel activity, and providing valuable information concerning the activation mechanism of voltage-gated K+ channels in these cells.
Assuntos
Membrana Celular/metabolismo , Ativação do Canal Iônico/fisiologia , Íons/metabolismo , Rim/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Canais de Cálcio/metabolismo , Cloreto de Cálcio/farmacologia , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Césio/farmacologia , Canais de Cloreto/metabolismo , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Rim/citologia , Rim/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Ácido Niflúmico/farmacologia , Técnicas de Patch-Clamp , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/antagonistas & inibidores , Cloreto de Potássio/farmacologia , Cloreto de Sódio/farmacologia , Tetraetilamônio/farmacologiaRESUMO
Population spikes associated with the paired pulse ratio protocol were used to measure the presynaptic inhibition of corticostriatal transmission caused by mu-opioid receptor activation. A 1 microM of [D-Ala(2), N-MePhe(4), Gly-ol(5)]-enkephalin (DAMGO), a selective mu-opioid receptor agonist, enhanced paired pulse facilitation by 44+/-8%. This effect was completely blocked by 2 nM of the selective mu-receptor antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-NH (CTOP). Antagonists of N- and P/Q-type Ca(2+) channels inhibited, whereas antagonists of potassium channels enhanced, synaptic transmission. A 1 microM of omega-conotoxin GVIA, a blocker of N-type Ca(2+) channels, had no effect on the action of DAMGO, but 400 nM omega-agatoxin TK, a blocker of P/Q-type Ca(2+)-channels, partially blocked the action of this opioid. However, 5 mM Cs(2+) and 400 microM Ba(2+), unselective antagonists of potassium conductances, completely prevented the action of DAMGO on corticostriatal transmission. These data suggest that presynaptic inhibition of corticostriatal afferents by mu-opioids is mediated by the modulation of K(+) conductances in corticostriatal afferents.