INTRACELLULAR MECHANISMS FOR ALPHA-1-ADRENERGIC REGULATION OF THE TRANSIENT OUTWARD CURRENT IN RABBIT ATRIAL MYOCYTES

被引:65
作者
BRAUN, AP
FEDIDA, D
CLARK, RB
GILES, WR
机构
[1] UNIV CALGARY, DEPT MED BIOCHEM, CALGARY T2N HN1, ALBERTA, CANADA
[2] UNIV CALGARY, DEPT MED PHYSIOL & MED, CALGARY T2N 4N1, ALBERTA, CANADA
来源
JOURNAL OF PHYSIOLOGY-LONDON | 1990年 / 431卷
关键词
D O I
10.1113/jphysiol.1990.sp018355
中图分类号
Q189 [神经科学];
学科分类号
071006 ;
摘要
1. The intracellular mechanism(s) underlying the decrease of a transient outward K+ current (It) induced by alpha 1‐adrenergic agonists was studied in isolated adult rabbit atrial myocytes using whole‐cell voltage clamp and cell‐attached patch clamp techniques. Experiments were carried out at 22‐23 degrees C. 2. Application of the specific alpha 1‐adrenergic agonist, methoxamine, produced a decrease in It which was irreversible after the non‐hydrolysable GTP analogues, GTP gamma S and Gpp(NH)p, had been introduced into cells via the recording micropipette. 3. Pre‐treatment of cells with 0.1‐0.15 microgram/ml pertussis toxin (PT) for 8‐9 h at 30‐34 degrees C did not prevent the alpha 1‐induced decrease in It. Yet, this protocol, as measured by the PT‐catalysed incorporation of [32P]ADP‐ribose in membrane‐associated 40 and 41 kDa proteins, effectively caused the ADP‐ribosylation of approximately 70% of the PT‐sensitive GTP‐binding proteins (i.e. Gi) in these treated cells. After taking into account the proportion of non‐viable cells (20‐30%), the effectiveness of this treatment probably approaches 100% in the viable myocytes from which electrophysiological recordings were made. 4. Cell‐attached patch recordings showed that bath application of methoxamine altered the single‐channel events underlying It by decreasing their opening probability. Averaged currents from ensemble single‐channel openings recorded in the presence of 0.2 mM‐methoxamine outside the patch reproduced the features of alpha 1‐adrenergic modulation of the macroscopic It observed during whole‐cell voltage clamp measurements. This observation provides evidence for the involvement of a diffusible intracellular second messenger in the alpha 1‐adrenergic modulation of It. 5. The protein kinase C (PKC) activators, 4 beta‐phorbol 12‐myristate 13‐acetate (PMA) and 1‐oleoyl‐2‐acetylglycerol (OAG) increased It, when included in the bath perfusate, whereas the inactive analogues, 4 alpha‐phorbol and 4 alpha‐phorbol 12,13‐didecanoate, had no effect on It. 6. Exposure of cells to the PKC inhibitors, staurosporine and H‐7, either by bath superfusion or intracellularly, via the recording micropipette, did not block the decrease in It produced by methoxamine. 7. Prolonged stimulation of atrial myocytes for 7‐9 h at 22 degrees C with 500 nM‐PMA produced a ‘down‐regulation’ of endogenous PKC activity, as well as a physical loss of the immunoreactive enzyme, as measured by an in vitro assay, and an anti‐PKC monoclonal antibody, respectively.(ABSTRACT TRUNCATED AT 400 WORDS) © 1990 The Physiological Society
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页码:689 / 712
页数:24
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