β1/β2肾上腺素能受体基因缺失对肾脏球旁器颗粒细胞肾素分泌的影响

Effect of β1/β2 adrenergic receptor gene deficient on the expression of renin in juxtaglomerular granular cells

目的观察β肾上腺素能受体对肾脏球旁器颗粒（JG）细胞肾素分泌的调节作用。方法密度梯度离心原代分离野生型和131／132肾上腺素受体基因敲除小鼠肾小球球旁器（JGA）颗粒细胞（JGC）。实时PCR明确JG细胞上存在β1/β2肾上腺素能受体基因表达。激光共聚焦显微镜下动态观察喹林和LysoTracker-Red染色条件下异丙肾上腺素促使JG活细胞分泌肾素的动态过程。尾静脉取血，用放射免疫方法测定清醒状态野生型和β1/β2肾上腺素能受体基因敲除小鼠基础肾素水平。不同浓度前列腺素E2（PGE2）、异丙肾上腺素和福斯高林刺激野生型和β1/β2肾上腺素能受体基因缺失小鼠JG细胞，放免法测定细胞内和上清中肾素活性。实时PCR测定肾皮质及JG细胞中肾素mRNA水平，以及不同浓度福斯高林对JG细胞内cAMP浓度的影响。结果成功分离的JG细胞上存在13肾上腺素能受体基因表达。激光共聚焦显微镜能观察到异丙肾上腺素明显刺激JG细胞分泌包含肾素的酸性颗粒。β1/β2肾上腺素能受体基因敲除小鼠血浆基础肾素降低，与肾脏皮质肾素mRNA降低平行。原代培养的β1/β2肾上腺素能受体基因敲除小鼠的JG细胞，肾素产生和分泌都明显降低，肾素mRNA也降低。PGE2、异丙肾上腺素和福斯高林呈剂量依赖性增加野生型小鼠JG细胞肾素转录、合成和分泌。β1/β2肾上腺素能受体基因敲除小鼠JG细胞对13肾上腺素能受体激动剂没有反应，而大剂量福斯高林能刺激其肾素分泌。福斯高林呈剂量依赖性上调JG细胞内cAMP水平。结论JG细胞上存在的肾上腺素能β受体，通过上调JG细胞内cAMP，促使JG细胞合成和分泌肾素。

Objective To study the regulation of renin in juxtaglomerular granular cells （JG cells） cultured from β1/β2 adrenergic receptor-deficient mice. Methods Real-time PCR was used to demonstrate the expression of β1-adrenergic receptors in primary cultures of JG cells. JG cells were stained with quinacrine and Lyso Tracker-Red and observed by confocal microscopy in order to observe the effect of isoproterenol on renin release in real time. Blood was taken from conscious wild type （WT） and β1/β2 adrenergic receptor knockout mice （β1/β2 ADR-/-） by tail vein puncture. Plasma renin concentration was measured by radioimmunoassay. Forskolin, isoproterenol and PGE2 were used to stimulate the renin activity in supernatant and cell lysate of primary cultures of JG cells from WT and β1/β2 ADR-/- mice. The renin mRNA expression ofkidney cortex and JG cells was measured by real-time PCR. Results Real-time PCRconfirmed the expression of 13-adrenergic receptor mRNA in JG cells of WT mice, but not in β1/β2 ADR-/- mice. Isoproterenol stimulated granule exocytosis and the emptying of granule content. Basal plasma renin concentration and renin mRNA expression was low in β1/β2 ADR-/- mice. PGE2, isoproterenol and forskolin increased the renin activity in the JG cell lysate and supernatant in cultures of WT mice, but isoproterenol had no effect on renin expression in JG cells from β1/β2 ADR-/- mice while higher doses of forskolin were needed to stimulate renin secretion. This effect was accompanied by increased renin mRNA expression of JG cells. Forskolin stimulated the cAMP content of JG cells. Conclusions 13-adrenergic receptors play an important role in regulating renin secretion in primary cultures of JG cells. This effect is associated with the activation of the cAMP pathway.