RESUMO
Thymocyte maturation is governed by antigen-T cell receptor (TCR) affinity and the extent of TCR aggregation. Signals provided by coactivating molecules such as CD4 and CD28 also influence the fate of immature thymocytes. The mechanism by which differences in antigen-TCR avidity encode unique maturational responses of lymphocytes and the influence of coactivating molecules on these signaling processes is not fully understood. To better understand the role of a key second messenger, calcium, in governing thymocyte maturation, we measured the intracellular free calcium concentration ([Ca2+]i) response to changes in TCR avidity and costimulation. We found that TCR stimulation initiates either amplitude- or frequency-encoded [Ca2+]i changes depending on (a) the maturation state of stimulated thymocytes, (b) the avidity of TCR interactions, and (c) the participation of specific coactivating molecules. Calcium signaling within immature but not mature thymocytes could be modulated by the avidity of CD3/CD4 engagement. Low avidity interactions induced biphasic calcium responses, whereas high avidity engagement initiated oscillatory calcium changes. Notably, CD28 participation converted the calcium response to low avidity receptor engagement from a biphasic to oscillatory pattern. These data suggest that calcium plays a central role in encoding the nature of the TCR signal received by thymocytes and, consequently, a role in thymic selection.
Assuntos
Antígenos CD4/fisiologia , Antígenos CD8/fisiologia , Cálcio/metabolismo , Receptores de Antígenos de Linfócitos T/fisiologia , Transdução de Sinais/fisiologia , Linfócitos T/imunologia , Animais , Anticorpos Monoclonais/farmacologia , Antígenos CD28/fisiologia , Complexo CD3/fisiologia , Células Cultivadas , Feto , Imunofenotipagem , Linfonodos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Modelos Imunológicos , Complexo Receptor-CD3 de Antígeno de Linfócitos T/fisiologia , Timo/imunologiaRESUMO
Esophageal striated myogenesis progresses differently from appendicular myogenesis, but the mechanism underlying this process is incompletely understood. Early theories of transdifferentiation of smooth muscle into striated muscle are not supported by transgenic fate-mapping experiments; however, the origin of esophageal striated muscle remains unknown. To better define the process of striated myogenesis, we examined myogenesis in murine fetal cultured esophageal whole-organ explants. Embryonic day 14.5 (E14.5) esophagi maintained a functional contractile phenotype for up to 7 days in culture. Striated myogenesis, as evidenced by myogenin expression, proceeded in a craniocaudal direction along the length of the esophagus. Esophageal length did not change during this process. Complete, but not partial, mechanical disruption of the rostral esophagus inhibited myogenesis distally. Addition of fibroblast growth factor-2 (FGF-2) to the culture media failed to inhibit striated myogenesis, but attenuated smooth muscle actin expression and reduced peristaltic activity. Inhibition of c-kit failed to inhibit peristalsis. These results suggest that striated myogenic precursors are resident along the entire length of the esophagus by day 14.5 and do not migrate along the esophagus after E14.5. Induction of myogenesis craniocaudally appears to require physical continuity of the esophagus and is not inhibited by FGF-2. Finally, peristalsis in E14.5 esophagi appears not to be regulated by interstitial cells of Cajal.
Assuntos
Colo/citologia , Desenvolvimento Muscular/fisiologia , Peristaltismo/fisiologia , Animais , Movimento Celular , Esôfago/citologia , Esôfago/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
Stimulation of calcium-activated potassium (KCa) channels in airway smooth muscle cells by phosphorylation-dependent and membrane-delimited, G protein actions has been reported (Kume, H. A. Takai, H. Tokuno, and T. Tomita. 1989. Nature [Lond.]. 341:152-154; Kume, H., M. P. Graziano, and M. I. Kotlikoff. 1992. Proc. Natl. Acad. Sci. USA. 89:11051-11055). We show that beta-adrenergic receptor/channel coupling is not affected by inhibition of endogenous ATP, and that activation of KCa channels is stimulated by both alpha S and cAMP-dependent protein kinase (PKA). PKA stimulated channel activity in a dose-dependent fashion with an EC50 of 0.12 U/ml and maximum stimulation of 7.38 +/- 2.04-fold. Application of alpha S to patches near maximally stimulated by PKA significantly increased channel activity to 15.1 +/- 3.65-fold above baseline, providing further evidence for dual regulatory mechanisms and suggesting that the stimulatory actions are independent. Analysis of channel open-time kinetics indicated that isoproterenol and alpha S stimulation of channel activity primarily increased the proportion of longer duration events, whereas PKA stimulation had little effect on the proportion of short and long duration events, but resulted in a significant increase in the duration of the long open-state. cAMP formation during equivalent relaxation of precontracted muscle strips by isoproterenol and forskolin resulted in significantly less cAMP formation by isoproterenol than by forskolin, suggesting that the degree of activation of PKA is not the only determinant of tissue relaxation. We conclude that beta-adrenergic stimulation of KCa channel activity and relaxation of tone in airway smooth muscle occurs, in part, by means independent of cyclic AMP formation.
Assuntos
Agonistas Adrenérgicos beta/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Isoproterenol/farmacologia , Músculo Liso/fisiologia , Canais de Potássio/fisiologia , Traqueia/fisiologia , Adenilil Imidodifosfato/farmacologia , Animais , Cálcio/metabolismo , Cálcio/farmacologia , Colforsina/farmacologia , Eletrofisiologia/métodos , Proteínas de Ligação ao GTP/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Guanosina Trifosfato/farmacologia , Cavalos , Ativação do Canal Iônico/efeitos dos fármacos , Cinética , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Músculo Liso/efeitos dos fármacos , Canais de Potássio/efeitos dos fármacos , Traqueia/efeitos dos fármacosRESUMO
Large conductance, calcium-activated potassium (maxiK) channels are expressed in nerve, muscle, and other cell types and are important determinants of smooth muscle tone. To determine the mechanisms involved in the transcriptional regulation of maxiK channels, we characterized the promoter regions of the pore forming (alpha) and regulatory (beta) subunits of the human channel complex. Maximum promoter activity (up to 12.3-fold over control) occurred between nucleotides -567 and -220 for the alpha subunit (hSlo) gene. The minimal promoter is GC-rich with 5 Sp-1 binding sites and several TCC repeats. Other transcription factor-binding motifs, including c/EBP, NF-kB, PU.1, PEA-3, Myo-D, and E2A, were observed in the 5'-flanking sequence. Additionally, a CCTCCC sequence, which increases the transcriptional activity of the SM1/2 gene in smooth muscle, is located 27 bp upstream of the TATA-like sequence, a location identical to that found in the SM1/2 5'-flanking region. However, the promoter directed equivalent expression when transfected into smooth muscle and other cell types. Analysis of the hSlo beta subunit 5'-flanking region revealed a TATA box at position -77 and maximum promoter activity (up to 11.0-fold) in a 200 bp region upstream from the cap site. Binding sites for GATA-1, Myo-D, c-myb, Ets-1/Elk-1, Ap-1, and Ik-2 were identified within this sequence. Two CCTCCC elements are present in the hSlo beta subunit promoter, but tissue-specific transcriptional activity was not observed. The lack of tissue-specific promoter activity, particularly the finding of promoter activity in cells from tissues in which the maxiK gene is not expressed, suggests a complex channel regulatory mechanism for hSlo genes. Moreover, the lack of similarity of the promoters of the two genes suggests that regulation of coordinate expression of the subunits does not occur through equivalent cis-acting sequences.
Assuntos
Canais de Potássio Cálcio-Ativados , Canais de Potássio/genética , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Biblioteca Genômica , Humanos , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta , Canais de Potássio Ativados por Cálcio de Condutância Alta , Dados de Sequência Molecular , Músculo Esquelético/metabolismo , Músculo Liso/metabolismo , Canais de Potássio/química , Regiões Promotoras GenéticasRESUMO
The effects of sulfhydryl reduction/oxidation on the gating of large-conductance, Ca(2+)-activated K+ (maxi-K) channels were examined in excised patches from tracheal myocytes. Channel activity was modified by sulfhydryl redox agents applied to the cytosolic surface, but not the extracellular surface, of membrane patches. Sulfhydryl reducing agents dithiothreitol, beta-mercaptoethanol, and GSH augmented, whereas sulfhydryl oxidizing agents diamide, thimerosal, and 2,2'-dithiodipyridine inhibited, channel activity in a concentration-dependent manner. Channel stimulation by reduction and inhibition by oxidation persisted following washout of the compounds, but the effects of reduction were reversed by subsequent oxidation, and vice versa. The thiol-specific reagents N-ethylmaleimide and (2-aminoethyl)methanethiosulfonate inhibited channel activity and prevented the effect of subsequent sulfhydryl oxidation. Measurements of macroscopic currents in inside-out patches indicate that reduction only shifted the voltage/nP0 relationship without an effect on the maximum conductance of the patch, suggesting that the increase in nP0 following reduction did not result from recruitment of more functional channels but rather from changes of channel gating. We conclude that redox modulation of cysteine thiol groups, which probably involves thiol/disulfide exchange, alters maxi-K channel gating, and that this modulation likely affects channel activity under physiological conditions.
Assuntos
Cálcio/fisiologia , Músculo Liso/fisiologia , Canais de Potássio/fisiologia , Animais , Citosol/metabolismo , Condutividade Elétrica , Metanossulfonato de Etila/análogos & derivados , Metanossulfonato de Etila/farmacologia , Etilmaleimida/farmacologia , Cavalos , Indicadores e Reagentes/farmacologia , Músculo Liso/citologia , Oxirredução/efeitos dos fármacos , Técnicas de Patch-Clamp , Canais de Potássio/efeitos dos fármacos , Compostos de Sulfidrila/farmacologiaRESUMO
Calcium-induced calcium release (CICR) has been observed in cardiac myocytes as elementary calcium release events (calcium sparks) associated with the opening of L-type Ca(2+) channels. In heart cells, a tight coupling between the gating of single L-type Ca(2+) channels and ryanodine receptors (RYRs) underlies calcium release. Here we demonstrate that L-type Ca(2+) channels activate RYRs to produce CICR in smooth muscle cells in the form of Ca(2+) sparks and propagated Ca(2+) waves. However, unlike CICR in cardiac muscle, RYR channel opening is not tightly linked to the gating of L-type Ca(2+) channels. L-type Ca(2+) channels can open without triggering Ca(2+) sparks and triggered Ca(2+) sparks are often observed after channel closure. CICR is a function of the net flux of Ca(2+) ions into the cytosol, rather than the single channel amplitude of L-type Ca(2+) channels. Moreover, unlike CICR in striated muscle, calcium release is completely eliminated by cytosolic calcium buffering. Thus, L-type Ca(2+) channels are loosely coupled to RYR through an increase in global [Ca(2+)] due to an increase in the effective distance between L-type Ca(2+) channels and RYR, resulting in an uncoupling of the obligate relationship that exists in striated muscle between the action potential and calcium release.
Assuntos
Potenciais de Ação/fisiologia , Cálcio/metabolismo , Músculo Liso/metabolismo , Potenciais de Ação/efeitos dos fármacos , Animais , Canais de Cálcio Tipo L/fisiologia , Sinalização do Cálcio/fisiologia , Quelantes/farmacologia , Ativação do Canal Iônico/fisiologia , Masculino , Contração Muscular/fisiologia , Músculo Liso/química , Técnicas de Patch-Clamp , Coelhos , Canal de Liberação de Cálcio do Receptor de Rianodina/fisiologia , Sarcômeros/química , Sarcômeros/metabolismo , Bexiga Urinária/química , Bexiga Urinária/metabolismoRESUMO
Potassium channels are an important determinant of smooth muscle excitability and force generation. Two potassium channels have been fully described in airway smooth muscle: large conductance, calcium-activated potassium channels and voltage-dependent delayed rectifier channels. This article will review the biophysics and pharmacology of these channels and discuss what is currently known with respect to their regulation and physiological significance.
Assuntos
Cálcio/farmacologia , Músculo Liso/fisiologia , Canais de Potássio/fisiologia , Fenômenos Fisiológicos Respiratórios , Agonistas Adrenérgicos beta/farmacologia , Animais , Eletrofisiologia , Potenciais da Membrana/fisiologia , Proteínas de Membrana/fisiologia , Músculo Liso/metabolismo , Potássio/metabolismo , Canais de Potássio/efeitos dos fármacos , Canais de Potássio/metabolismo , Sistema Respiratório/efeitos dos fármacos , Sistema Respiratório/metabolismoRESUMO
We report the generation of transgenic mice designed to facilitate the study of vascular and nonvascular smooth muscle biology in vivo. The smooth muscle myosin heavy chain (smMHC) promoter was used to direct expression of a bicistronic transgene consisting of Cre recombinase and enhanced green fluorescent protein (eGFP) coding sequences. Animals expressing the transgene display strong fluorescence confined to vascular and nonvascular smooth muscle. Enzymatic dissociation of smooth muscle yields viable, fluorescent cells that can be studied as single cells or sorted by FACS for gene expression studies. smMHC/Cre/eGFP mice were crossed with ROSA26/lacZ reporter mice to determine Cre recombinase activity; Cre recombinase was expressed in all smooth muscles in adult mice, and there was an excellent overlap between expression of the recombinase and eGFP. Initial smooth muscle-specific expression of fluorescence and Cre recombinase was detected on embryonic day 12.5. These mice will be useful to define smooth muscle gene function in vivo in mice, for the study of gene function in single, live cells, and for the determination of gene expression in vascular and nonvascular smooth muscle.
Assuntos
Integrases/genética , Músculo Liso/metabolismo , Proteínas Virais/genética , Animais , Animais Recém-Nascidos , Embrião de Mamíferos/metabolismo , Feminino , Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Genótipo , Proteínas de Fluorescência Verde , Integrases/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia de Fluorescência , Cadeias Pesadas de Miosina/genética , Regiões Promotoras Genéticas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Tempo , Proteínas Virais/metabolismoRESUMO
We isolated and characterized genomic clones of the human P2X1 receptor (hP2X1) gene in an effort to understand its tissue specific expression. The hP2X1 gene contains 12 exons spanning 20 kb, with exon sizes ranging from 59 to 143 bp. A 385 bp upstream fragment promoted hP2X1 gene expression in smooth muscle (A7R5 and primary trachealis) and fibroblast (NIH3T3) cell lines, and mutation of a consensus E box sequence (CACCTG) within this fragment (-340 to -345) did not alter basal promoter activity. However, co-transfected bHLH factors regulated activity of the 385 bp minimal P2X1 promoter in a tissue-specific manner. E12 expression inhibited and ITF2b augmented activity in A7R5 cells, but had no effect in NIH3T3 cells. ITF2a, Myo-D, and Id1 proteins had no effect on either cell line, but co-expression of ITF2a blocked E12 inhibition in A7R5 cells, while ITF2b failed to reverse the inhibition. Northern analysis of A7R5 RNA identified high levels of E12 and ITF2b transcripts, and gel shift assays using A7R5 and NIH3T3 nuclear extracts indicated the formation of a protein-DNA complex with an oligonucleotide corresponding to -330 and -348, which was abolished by base substitutions within the E box motif. Our results identify a critical E box response element in the hP2X1 promoter that binds bHLH factors and demonstrate smooth muscle specific transcriptional regulation by E proteins.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Sequências Hélice-Alça-Hélice , Músculo Liso Vascular/metabolismo , Proteína MyoD/metabolismo , Proteínas do Tecido Nervoso , Regiões Promotoras Genéticas , Receptores Purinérgicos P2/genética , Proteínas Repressoras , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Células 3T3 , Sequência de Aminoácidos , Animais , Sequência de Bases , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Sítios de Ligação , Clonagem Molecular , DNA Complementar , Proteínas de Ligação a DNA/genética , Perfilação da Expressão Gênica , Humanos , Proteína 1 Inibidora de Diferenciação , Camundongos , Dados de Sequência Molecular , Músculo Liso Vascular/citologia , Proteína MyoD/genética , Receptores Purinérgicos P2X , Fatores de Transcrição TCF , Transativadores/genética , Fator de Transcrição 4 , Proteína 1 Semelhante ao Fator 7 de Transcrição , Proteína 2 Semelhante ao Fator 7 de Transcrição , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
ATP acts as a fast excitatory neurotransmitter by binding to a large family of membrane proteins, P2X receptors, that have been shown to be ligand-gated, non-selective cation channels. We report the cloning of a full-length and alternatively spliced form of the human P2X4 gene. Clones were identified from a human stomach cDNA library using a rat P2X4 probe. Nucleotide sequence analysis of positive clones identified the full-length human P2X4 cDNA, which codes for a 388-residue protein that is highly homologous (82%) to the rat gene, and an alternatively spliced cDNA. In the alternatively spliced cDNA, the 5'-untranslated region and the first 90 amino acids in the coding region of full-length human P2X4 are replaced by a 35 amino acid coding sequence that is highly homologous with a region of chaperonin proteins in the hsp-90 family. The open reading frames of the full-length and splice variant clones were confirmed by in vitro translation. Northern analysis indicated expression of the full-length P2X4 message in numerous human tissues including smooth muscle, heart, and skeletal muscles. Alternatively spliced RNAs were identified in smooth muscle and brain by RT-PCR and confirmed by RNAse protection assays using a 710 bp anti-sense RNA probe that spanned the alternatively spliced and native P2X4 regions. Injection of full-length, but not alternatively spliced, cRNA into Xenopus oocytes resulted in the expression of ATP gated non-selective cation currents.
Assuntos
Processamento Alternativo , Receptores Purinérgicos P2/genética , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Células Cultivadas , Clonagem Molecular , DNA Complementar , Mucosa Gástrica/metabolismo , Expressão Gênica , Humanos , Dados de Sequência Molecular , RNA Mensageiro/metabolismo , Ratos , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2X4 , Homologia de Sequência de Aminoácidos , Xenopus laevisRESUMO
We have demonstrated that stimulation of airway smooth muscle by muscarinic agonists results in a coordinated modulation of two membrane ion channel proteins. Both channels are modulated in a similar way, although their effects on open-channel probability are opposite. The voltage-dependence of channel activity is shifted to more positive potentials in the case of KCa, and to more negative potentials in the case of the voltage-dependent calcium channels. Similarly, KCa channel dwell-time kinetics are shifted to short open lifetimes, whereas the long open state is favored for the large-amplitude voltage-dependent calcium channel. Although little is known about the molecular coupling of calcium channels, muscarinic inhibition of KCa channels is mediated through a pertussis toxin-sensitive guanine nucleotide binding protein.
Assuntos
Canais Iônicos/efeitos dos fármacos , Parassimpatomiméticos/farmacologia , Sistema Respiratório/efeitos dos fármacos , Animais , Di-Hidropiridinas/farmacologia , Guanosina Trifosfato/farmacologia , Humanos , Canais Iônicos/metabolismo , Potenciais da Membrana , Cloreto de Metacolina/farmacologia , Músculo Liso/efeitos dos fármacos , Sistema Respiratório/metabolismoRESUMO
M2 receptor stimulation results in the gating of nonselective cation channels in several smooth muscle cell types. However the requirement for current activation includes a rise in cytosolic calcium mediated by M3 receptor induced calcium release. This complex signaling system confers substantial complexity on the interpretation of pharmacological experiments. M2 and M3 receptor stimulation has also been linked to the inhibition of potassium channels in smooth muscle. These signaling events are likely to play important roles in excitation/contraction coupling.
Assuntos
Músculo Liso/fisiologia , Receptores Muscarínicos/fisiologia , Transdução de Sinais , Animais , Cálcio/metabolismo , Ativação do Canal Iônico , Músculo Liso/inervação , Canais de Potássio/fisiologia , Receptor Muscarínico M2RESUMO
The effects of IV histamine infusion and beta-adrenergic blockade on pulmonary function were studied in neonatal Holstein calves, beta-Adrenergic blockade with IV propranolol increased total pulmonary resistance, but was without effect on lung compliance, lung volumes, and gas exchange. beta-Adrenergic blockade enhanced the histamine-induced increase in total pulmonary resistance and the decrease in dynamic and quasistatic compliance, lung volumes, and arterial oxygen tension. These data indicate that beta-adrenergic receptors may be present in the large and small airways of cattle and they modulate the effects of bronchoconstrictor agents as in other species.
Assuntos
Antagonistas Adrenérgicos beta/farmacologia , Animais Recém-Nascidos/fisiologia , Bovinos/fisiologia , Histamina/farmacologia , Pulmão/efeitos dos fármacos , Animais , Complacência Pulmonar/efeitos dos fármacos , Medidas de Volume Pulmonar/veterinária , Masculino , Propranolol/farmacologia , Receptores Adrenérgicos beta/efeitos dos fármacos , Receptores Adrenérgicos beta/fisiologiaRESUMO
Membrane-associated receptors for rapid, steroidal neuromodulation remain elusive. Estradiol has been reported to facilitate activation of voltage- and Ca(2+)-dependent BK potassium channels encoded by Slo, if associated with beta1 subunits. We show here that 1) multiple members of the beta family confer sensitivity to multiple steroids on BK channels, 2) that beta subunits differentiate between steroids, and 3) that different betas have distinct relative preferences for particular steroids. Expressed in HEK 293 cells, inside-out patches with channels composed of Slo-alpha alone showed no steroid sensitivity. Cells expressing alphabeta4 exhibited potent, rapid, reversible, and dose-dependent potentiation by corticosterone (CORT; a glucocorticoid), and were potentiated to a lesser degree by other sex and stress steroids. In contrast, alphabeta2 channels were potentiated more strongly by dehydroepiandrosterone (DHEA; an enigmatic, stress-related adrenal androgen), and to a lesser extent by CORT, estradiol, testosterone, and DHEA-S. Cholesterol had no effect on any BK channel compositions tested. Conductance-voltage plots of channels composed of alpha plus beta2 or beta4 subunits were shifted in the negative direction by steroids, indicating greater activation at negative voltages. Thus our results argue that the variety of Slo-beta subunit coexpression patterns occurring in vivo expands the repertoire of Slo channel gating in yet another dimension not fully appreciated, rendering BK gating responsive to dynamic fluctuations in a multiple of steroid hormones.
Assuntos
Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/fisiologia , Potenciais da Membrana/efeitos dos fármacos , Esteroides/farmacologia , Animais , Northern Blotting/métodos , Células Cultivadas , Células Cromafins/efeitos dos fármacos , Células Cromafins/fisiologia , Células Cromafins/efeitos da radiação , Corticosterona/farmacologia , Desidroepiandrosterona/farmacologia , Relação Dose-Resposta a Droga , Relação Dose-Resposta à Radiação , Interações Medicamentosas , Estimulação Elétrica/métodos , Humanos , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/classificação , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Potenciais da Membrana/fisiologia , Técnicas de Patch-Clamp/métodos , Subunidades Proteicas/fisiologia , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Esteroides/química , Esteroides/classificação , Transfecção/métodosRESUMO
Canine tracheal smooth muscle cells were enzymatically dissociated, and individual myocytes were voltage clamped through use of the whole cell, patch-clamp method. Cells dialyzed with solutions high in potassium and bathed in physiological saline demonstrated brief inward currents, followed by large outward currents that inactivated very slowly. When outward currents were blocked, a voltage-activated inward current was observed that activated with depolarizations to voltages positive to -45 mV, with an apparent reversal potential greater than 110 mV, and a peak current at 15 mV. This current was identified as a calcium current on the basis of 1) its presence under conditions in which calcium was the only permeant cation, 2) the lack of a blocking effect of 2 microM tetrodotoxin, and 3) block of the current by Mn2+, Cd2+, and CO2+. Increases in external calcium concentration from 2 to 20 mM resulted in an increase in current amplitude and a shift of voltage activation toward more positive potentials. The current displayed a rapid inactivation phase with a time constant of 16-52 ms, which was well fit by a single exponential. Steady-state inactivation of the calcium current was sigmoidal, with a voltage of half inactivation of -21 mV in 20 mM Ca2+. The principle component of the calcium current was further identified as a transient current on the basis of its rapid inactivation, current-voltage characteristics, and relative insensitivity to dihydropyridine calcium channel blocking agents.
Assuntos
Cálcio/metabolismo , Canais Iônicos/metabolismo , Músculo Liso/metabolismo , Animais , Cádmio/metabolismo , Células Cultivadas , Di-Hidropiridinas/farmacologia , Cães , Condutividade Elétrica , Feminino , Masculino , Potenciais da Membrana , TraqueiaRESUMO
The electrical properties of dissociated canine tracheal smooth muscle cells were examined using the whole cell patch-clamp technique. In current clamp mode, current clamp steps did not initiate action potentials but showed clear outward rectification, which was abolished when cells were loaded with Cs+ ions and when tetraethylammonium (TEA+) ions replaced Na+ in the bath solution. In voltage-clamp experiments, depolarizations positive to -45 mV evoked brief voltage-dependent inward Ca2+ currents [Am. J. Physiol. 254 (Cell Physiol. 23): C793-C801, 1988], followed by sustained outward currents, which did not completely inactivate. Outward currents were identified as K+ currents on the basis of the reversal potential of the current and by ion-substitution experiments. The currents were further defined as Ca2(+)-insensitive delayed rectifier currents, since they were unaltered under conditions in which 1) the Ca2+ current was completely blocked by Mn2+ or nifedipine (10 microM); 2) Ba2+ ions were substituted for Ca2+ as the inward current charge carrier; or 3) charybdotoxin (40 nM) or TEA+ (up to 10 mM) were added to the bath. A Ca2(+)-activated potassium [K(Ca)] current was activated by application of methacholine (100 microM), or A23187 (1 microM), under conditions of low Ca2+ buffering capacity in the internal solution [0.3 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA)]. The K(Ca) current was blocked by 10 mM TEA+ and was not observed under conditions of high intracellular Ca2+ buffering (11 mM EGTA). These data indicate that canine airway smooth muscle cells contain voltage-dependent delayed rectifier channels that underlie membrane rectification and K(Ca) channels that are activated by agents which release intracellular Ca2+ stores.
Assuntos
Músculo Liso/fisiologia , Canais de Potássio/fisiologia , Traqueia/fisiologia , Animais , Calcimicina/farmacologia , Cálcio/farmacologia , Cães , Condutividade Elétrica , Eletrofisiologia/métodos , Feminino , Técnicas In Vitro , Cinética , Masculino , Potenciais da Membrana/efeitos dos fármacos , Cloreto de Metacolina/farmacologia , Canais de Potássio/efeitos dos fármacosRESUMO
1. Fluorescence measurements of intracellular calcium concentrations ([Ca2+]i) were made on cultured human airway smooth muscle cells using the dye Fura-2. The response to either histamine (100 microM) or bradykinin (1 microM) was biphasic, with a transient increase in [Ca2+]i followed by a sustained [Ca2+]i increase lasting many minutes. The average steady-state (plateau) [Ca2+]i following agonist activation was 267 +/- 5 nM, whereas the average basal [Ca2+]i was 148 +/- 4 nM. 2. The sustained rise in [Ca2+]i required the continued presence of either histamine or bradykinin and was dependent on extracellular Ca2+. The magnitude of the transient rise in [Ca2+]i was not dependent on extracellular Ca2+. Sustained, receptor-activated rises in [Ca2+]i were rapidly abolished by chelation of extracellular Ca2+, or addition of non-permeant polyvalent cations, whereas these agents had minor effects in the absence of agonist. These data indicate that the sustained increase in [Ca2+]i was dependent on receptor-activated Ca2+ influx. 3. Receptor-activated Ca2+ influx was not affected by treatment with organic Ca2+ channel antagonists (nifedipine (10 microM), nisoldipine (10 microM) or diltiazem (10 microM] or agonists (Bay K 8644 (500 nM to 10 microM) or Bay R 5417 (500 nM]. The magnitude of the sustained rise was also not affected by pre-treatment with ouabain (100 microM) indicating little involvement of Na(+)-Ca2+ exchange in the influx mechanism. 4. Receptor-activated Ca2+ influx could be completely inhibited by several polyvalent cations (Co2+, Mn2+, Ni2+, -Cd2+ or La3+). Quantitative estimates of the potency of block were obtained for Ni2+ and La3+. These measurements indicate that the pKi for Ni2+ was 3.6 and for La3+ was 3.5. 5. Both Mn2+ and Co2+ ions caused a time-dependent quench of intracellular Fura-2; however, permeation of neither ion was increased following receptor activation, indicating that the influx pathway is not permeable to these cations. 6. Fura-2 was used to monitor the rate of Ba2+ entry into airway smooth muscle cells by monitoring the Ca(2+)-Fura-2 and Ba(2+)-Fura-2 isosbestic points as well as the 340 and 380 nm signals. Cell activation did not increase the rate of Ba2+ entry indicating that the Ca2+ influx pathway was poorly permeant to Ba2+ ions. Ba2+ (2 mM) was able to inhibit Ca2+ entry as shown by its effects on the Ba(2+)-independent, Ca(2+)-dependent wavelength (371 nm). 7. The voltage dependence of Ca2+ influx was examined before and after agonist-induced activation. The effect of KCl-induced depolarization prior to cell activation was to cause a slight increase in [Ca2+]i.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Cálcio/metabolismo , Músculo Liso/metabolismo , Bário/metabolismo , Bradicinina/fisiologia , Brônquios/metabolismo , Cálcio/fisiologia , Canais de Cálcio/fisiologia , Cátions/metabolismo , Células Cultivadas , Histamina/fisiologia , Humanos , Sódio/metabolismo , Traqueia/metabolismoRESUMO
Application of a muscarinic agonist to the extracellular surface of membrane patches from airway smooth muscle cells resulted in an inhibition of calcium-activated potassium (KCa) channels in outside-out patches. Methacholine (50 microM) inhibited channel activity at physiological cytosolic calcium concentrations and resulted in a marked shift in channel open-time kinetics. In inside-out patches, KCa channels were inhibited upon addition of GTP (100 microM) when methacholine was present in the patch pipette. Muscarinic inhibition was blocked when guanosine 5'-O-(2-thiodiphosphate) was used to compete with endogenous GTP in outside-out or inside-out experiments. Pretreatment of dissociated cells with pertussis toxin (0.1 micrograms/ml) blocked muscarinic inhibition of the channel in a time-dependent fashion. These results demonstrate, at the single-channel level, a coupling between muscarinic receptor stimulation and inhibition of KCa in smooth muscle and demonstrate the guanine nucleotide dependence of this coupling.
Assuntos
Proteínas de Ligação ao GTP/fisiologia , Músculo Liso/metabolismo , Toxina Pertussis , Canais de Potássio/metabolismo , Receptores Muscarínicos/metabolismo , Fatores de Virulência de Bordetella/farmacologia , Animais , Cálcio/metabolismo , Bovinos , Células Cultivadas , Cães , Proteínas de Ligação ao GTP/efeitos dos fármacos , Cinética , Potenciais da Membrana , Cloreto de Metacolina/farmacologia , Músculo Liso/citologia , Potássio/metabolismo , Canais de Potássio/efeitos dos fármacosRESUMO
We have identified and characterized single voltage-dependent calcium channels in both acutely dissociated rat bronchial and cultured human tracheobronchial smooth muscle cells using the patch-clamp technique. In both cell types, on-cell membrane patches displayed unitary currents selective for barium ions and exhibited one conductance level (21-26 pS), and the open state probability was increased by membrane depolarization. Unitary barium currents were enhanced by the calcium channel selective agonist, BAY R 5417, and inhibited by the dihydropyridine calcium channel antagonist, nisoldipine (apparent inhibition constant less than 100 nM). Moreover, the degree of nisoldipine inhibition of the rat bronchial smooth muscle channels was increased with membrane depolarization in a manner consistent with the drug interacting with highest affinity to the inactivated channel state. In addition, the sensitivity to BAY R 5417 augmentation and nisoldipine inhibition of depolarization-induced tonic force of intact rat bronchial ring segments was in close agreement to the single channel results. Thus these data suggest that activation of voltage-dependent calcium channels can influence airway contraction and that dihydropyridines may be effective modulators of depolarization-induced increases in bronchial tone. We conclude that both rat and human airway smooth muscle cells have high-conductance voltage-dependent calcium channels that interact in a predictable manner with dihydropyridines and are similar to voltage-dependent calcium channels observed in other smooth muscle cells.