通过注射环磷酸腺苷和对藤壶肌纤维中的钠外流进行外部酸化来刺激的方式。

Mode of stimulation by injection of cyclic AMP and external acidification of the sodium efflux in barnacle muscle fibres.

作者信息

Bittar E E, Demaille J, Fischer E H, Schultz R

出版信息

J Physiol. 1979 Nov;296:277-89. doi: 10.1113/jphysiol.1979.sp013005.

DOI:10.1113/jphysiol.1979.sp013005

PMID:43391

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC1279078/

Abstract

A study has been made in single barnacle muscle fibres of the effect of micro-injected pure protein kinase inhibitor (PKI) on the response of the Na efflux to injection of cyclic AMP and external acidification. 2. (i) Injection into fibres of 1.6 x 10(-4) M-pure PKI is without effect on the resting Na efflux. (ii) Injection of 1.6 x 10(4) M-pure PKI before 0.03 M-cyclic AMP causes a marked reduction in the magnitude of the response of the Na efflux to the nucleotide. The same is true when 10(-4) M-cyclic AMP is injected after PKI. (iii) Injection of partially pure catalytic subunits causes a sustained stimulation of the ouabain-insensitive Na efflux, which is almost completely reversed by injecting PKI. (iv) Injection of 100 mM-EGTA before PKI fails to alter the lowered response of the ouabain-insensitive Na efflux to injection of 10(-4) M-cyclic AMP. (v) Ouabain (10(-4) M) when applied following the injection of 10(-4) M-cyclic AMP causes a drastic fall in the stimulated Na efflux. 3. (i) Injection of 1.6 x 10(-4) M-pure PKI before or after external acidification fails to abolish or reduce the stimulatory response to acidification. (ii) Injection of 1.6 x 10(-4) M-pure PKI before acidification practically abolishes the response of the ouabain-insensitive Na efflux to 0.03 M-cyclic AMP in the presence of acidification. (iii) Radioimmunoassay of total cyclic AMP and cyclic GMP content in single fibres before and after acidification shows no appreciable alteration in nucleotide content following acidificiation. (iv) Injection of 100 mM-EGTA before acidification enhances the stimulatory response to acidification. (v) External application of Dantrolene (10(-5) M) fails to alter the size of the stimulatory response to acidification. 4. (i) Prior external application of 5 x 10(-4) M-benzolamide results in a marked reduction in the magnitude of the response of the ouabain-insensitive Na efflux to the injection of 3 x 10(-4) M-cyclic AMP. (ii) Benzolamide totally abolishes the response of the ouabain-insensitive Na efflux to the injection of catalytic subunits. 5. The evidence brought forward is compatible with the view that (a) The mechanism by which cyclic AMP stimulates the Na efflux involves activation by cyclic AMP of the cyclic AMP-dependent protein kinase system, and hence release of the catalytic subunit, and (b) the mechanism by which external acidification leads to stimulation of the Na efflux involves activation of a benzolamide-sensitive system, possibly carbonic anhydrase, rather than the adenyl cyclase system. The actions of cyclic AMP and catalytic subunits on the Na efflux are closely linked to activation of the benzolamide sensitive system.

摘要

已对藤壶单肌纤维进行了一项研究，观察微量注射纯蛋白激酶抑制剂（PKI）对Na外流对环磷酸腺苷（cAMP）注射及细胞外酸化反应的影响。2. （i）向纤维中注射1.6×10⁻⁴M的纯PKI对静息Na外流无影响。（ii）在注射0.03M cAMP之前注射1.6×10⁻⁴M的纯PKI会导致Na外流对该核苷酸反应的幅度显著降低。在PKI之后注射10⁻⁴M cAMP时情况相同。（iii）注射部分纯化的催化亚基会导致哇巴因不敏感的Na外流持续受到刺激，而注射PKI几乎可使其完全逆转。（iv）在PKI之前注射100mM乙二醇双乙醚四乙酸（EGTA）不会改变哇巴因不敏感的Na外流对注射10⁻⁴M cAMP的降低反应。（v）在注射10⁻⁴M cAMP后应用10⁻⁴M哇巴因会导致受刺激的Na外流急剧下降。3. （i）在细胞外酸化之前或之后注射1.6×10⁻⁴M的纯PKI不会消除或降低对酸化的刺激反应。（ii）在酸化之前注射1.6×10⁻⁴M的纯PKI实际上会消除在酸化存在时哇巴因不敏感的Na外流对0.03M cAMP的反应。（iii）对酸化前后单纤维中总cAMP和环磷酸鸟苷（cGMP）含量的放射免疫测定表明，酸化后核苷酸含量无明显变化。（iv）在酸化之前注射100mM EGTA会增强对酸化的刺激反应。（v）细胞外应用丹曲林（10⁻⁵M）不会改变对酸化的刺激反应大小。4. （i）事先细胞外应用5×10⁻⁴M苯磺酰胺会导致哇巴因不敏感的Na外流对注射3×10⁻⁴M cAMP反应的幅度显著降低。（ii）苯磺酰胺完全消除了哇巴因不敏感的Na外流对注射催化亚基的反应。5. 所提出的证据与以下观点相符：（a）cAMP刺激Na外流的机制涉及cAMP对环磷酸腺苷依赖性蛋白激酶系统的激活，从而释放催化亚基；（b）细胞外酸化导致Na外流受到刺激的机制涉及激活一个苯磺酰胺敏感系统，可能是碳酸酐酶，而非腺苷酸环化酶系统。cAMP和催化亚基对Na外流的作用与苯磺酰胺敏感系统的激活密切相关。