RESUMO
Exocytosis of secretory vesicles results in the release of insulin from pancreatic beta-cells, although little is known about this process in humans. We examined the exocytosis of single secretory vesicles and their associated fusion pores in human beta-cells by cell-attached capacitance and conductance measurement. Unitary capacitance steps were observed, consistent with the exocytosis of single secretory vesicles. These were often coincident with increases in patch conductance representing the presence of a stable fusion pore. In some events, the fusion pore closed, mediating kiss-and-run, which contributed 20% of the exocytotic events. The cAMP-raising agent forskolin (5 microM) doubled the relative contribution of kiss-and-run. This effect was confirmed visually in MIN6 cells expressing a fluorescent granule probe. Thus, we demonstrate the unitary capacitance steps and fusion pores during single vesicle exocytosis in human beta-cells. Furthermore, these secretory vesicles can undergo rapid recycling by kiss-and-run, and this process is up-regulated by cAMP.
Assuntos
Exocitose/fisiologia , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Vesículas Secretórias/metabolismo , Adulto , Animais , Colforsina/farmacologia , Capacitância Elétrica , Exocitose/efeitos dos fármacos , Humanos , Secreção de Insulina , Masculino , Pessoa de Meia-IdadeRESUMO
A large array of voltage-gated K(+) channel (Kv) genes has been identified in vascular smooth muscle tissues. This molecular diversity underlies the vast repertoire of native Kv channels that regulate the excitability of vascular smooth muscle tissues. The contributions of different Kv subunit gene products to the native Kv currents are poorly understood in vascular smooth muscle cells (SMCs). In the present study, Kv subunit-specific antibodies were applied intracellularly to selectively block various Kv channel subunits and the whole-cell outward Kv currents were recorded using the patch-clamp technique in rat mesenteric artery SMCs. Anti-Kv1.2 antibody (8 microg/ml) inhibited the Kv currents by 29.2 +/- 5.9% (n = 6, P < 0.05), and anti-Kv1.5 antibody (6 microg/ml) by 24.5 +/- 2.6% (n = 7, P < 0.05). Anti-Kv2.1 antibody inhibited the Kv currents in a concentration-dependent fashion (4-20 microg/ml). Co-application of antibodies against Kv1.2 and Kv2.1 (8 microg/ml each) induced an additive inhibition of Kv currents by 42.3 +/- 3.1% (n = 7, P < 0.05). In contrast, anti-Kv1.3 antibody (6 microg/ml) did not have any effect on the native Kv current (n = 6, P > 0.05). A control antibody (anti-GIRK1) also had no effect on the native Kv currents. This study demonstrates that Kv1.2, Kv1.5, and Kv2.1 subunit genes all contribute to the formation of the native Kv channels in rat mesenteric artery SMCs.
Assuntos
Artérias Mesentéricas/metabolismo , Músculo Liso Vascular/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Canais de Potássio/metabolismo , Animais , Especificidade de Anticorpos , Western Blotting , Canais de Potássio de Retificação Tardia , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G , Técnicas In Vitro , Canal de Potássio Kv1.2 , Canal de Potássio Kv1.5 , Masculino , Artérias Mesentéricas/citologia , Músculo Liso Vascular/citologia , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-Dawley , Canais de Potássio ShabRESUMO
This study was designed to evaluate the contribution of ATP-dependent potassium (KATP) channels to the changes in vascular reactivity and spontaneous tone observed in vessels isolated from deoxycorticosterone acetate (DOCA)-salt hypertensive rats. In phenylephrine preconstricted aortic rings, cromakalim induced concentration-dependent, glibenclamide-sensitive relaxation. The concentration response curve to cromakalim was shifted to the right in DOCA-salt hypertensive rats (EC50: 0.850 +/- 0.100 microM) compared with SHAM-normotensive rats (0.108 +/- 0.005 microM), and the maximum relaxation (Emax) evoked by cromakalim was significantly lower in aortic rings from the DOCA group (68 +/- 2%) compared with the SHAM group (108 +/- 5%). The results were similar in endothelium-denuded rings. Spontaneous tone was observed in aortic rings (5 g preload) from DOCA-salt but not SHAM rats. Cromakalim abolished spontaneous tone and the effect was blocked by glibencamide. In whole cell patch clamp studies, increasing extracellular K concentrations from 5.4 to 140 mM and the administration of cromakalim evoked dramatic increases in KATP channel currents in aortic cells isolated from SHAM rats. In contrast, in aortic cells from DOCA-salt hypertensive rats, KATP channel currents were either absent or weak in response to challenges by elevated extracellular K and by cromakalim. These findings suggest that the function of KATP channels is impaired in smooth muscle cells from aorta of DOCA-salt hypertensive rats, which may contribute to the impaired vasodilatation and spontaneous tone observed in these rats.