Compartmentalisation of cAMP-dependent signalling by caveolae in the adult cardiac myocyte
Compartmentalisation of cAMP-dependent signalling by caveolae in the adult cardiac myocyte
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DOI：
10.1016/j.yjmcc.2008.04.004
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发表时间：
2008-07-01
影响因子：
5
通讯作者：
White, Ed
中科院分区：
文献类型：
--
作者：
Calaghan, Sarah;Kozera, Lukasz;White, Ed
环状AMP表现出局部(肌膜)和全局(胞浆)的信号模式，允许单个第二信使产生受体特异性信号。在这里，我们确定凹陷的脂筏是否负责将β(2)肾上腺素能受体(AR)cAMP信号限制到肌膜间隔。用去胆固醇药物甲基-β-环糊精(M-βC)处理心肌细胞以破坏小凹。通过免洗涤剂蔗糖梯度分级法分离出含有小窝的膜组分。测定肌浆网蛋白磷蛋白(PLB)和肌丝蛋白肌钙蛋白I(TnI)对β(2)AR刺激的细胞缩短和磷酸化程度(沙丁胺醇加1 mU阿替洛尔)。PLB的Ser(16)磷酸化(PPLB)、TnI的Ser(22，23)磷酸化(PTnI)和正性lusitroy被用作整体cAMP信号的指标。MβC破坏小窝的能力通过选择性地耗尽小窝的两个基本成分--胆固醇和小窝蛋白3的浮动膜部分来证实。在对照细胞中，β(2)AR刺激并未记录到pPLB、pTnI或半松弛时间的变化(P>；0.05)，但在腔隙阻断后，这两种蛋白的磷酸化水平增加了60%-70%，并伴有正性松弛(P
Cyclic AMP exhibits local (sarcolemmal) and global (cytosolic) patterns of signalling, allowing receptor-specific signals to be generated by a single second messenger. Here we determine whether caveolae, invaginated lipid rafts, are responsible for confining the beta(2) adrenoceptor (AR) cAMP signal to the sarcolemmal compartment. Myocytes were treated with the cholesterol-depleting agent methyl-beta-cyclodextrin (M beta C) to disrupt caveolae. Caveolae-containing membrane fractions were isolated by detergent-free sucrose gradient fractionation. Cell shortening and phosphorylation of the sarcoplasmic reticular protein phospholamban (PLB) and the myofilament protein troponin I (TnI) were measured in response to beta(2) AR stimulation (with salbutamol in the presence of 1 mu M atenolol). Ser(16) phosphorylation of PLB (pPLB), Ser(22,23) phosphorylation of TnI (pTnI), and positive lusitropy were used as indices of global cAMP signals. The ability of M beta C to disrupt caveolae was confirmed by selective depletion of the buoyant membrane fractions of cholesterol and caveolin 3, the 2 essential components of caveolae. In control cells, no change in pPLB, pTnI or time to half relaxation was recorded with beta(2) AR stimulation (P>0.05), but following caveolar disruption a 60-70% increase in phosphorylation of both proteins was seen, accompanied by positive lusitropy (P