Mandon B, Siga E, Champigneulle A, Imbert-Teboul M, Elalouf J M
Départment de Biologie Cellulaire et Moléculaire, Commissariat à l'Energie Atomique Saclay, Gif sur Yvette, France.
Am J Physiol. 1995 Jun;268(6 Pt 2):F1070-80. doi: 10.1152/ajprenal.1995.268.6.F1070.
Expression and functional properties of beta-adrenergic receptors (beta-ARs) were studied in rat collecting tubules isolated by microdissection. Reverse transcription-polymerase chain reaction experiments demonstrated that the beta 1- and beta 2-AR mRNAs, but not the beta 3-subtype, are expressed in the cortical collecting duct (CCD). Quantitation of mRNAs, carried out using mutant RNAs as internal standards, further showed that beta 1- and beta 2-ARs transcripts are present at comparable amounts in CCD (3,000-4,000 copies/mm of tubular length), but reach 6-8 times lower levels in the outer medullary collecting duct (OMCD: beta 1, 480 +/- 180; beta 2, 590 +/- 110 copies/mm of tubular length). Functional studies, carried out in CCD, corroborated the expression of these two receptor subtypes. The rank order of potency of beta-agonists for stimulating adenosine 3',5'-cyclic monophosphate (cAMP) accumulation was isoproterenol > norepinephrine = epinephrine, and similar efficiencies were found for a beta 1- and a beta 2-antagonist to inhibit isoproterenol-dependent cAMP formation. Fura 2 fluorescence measurements revealed that isoproterenol (10 microM) induces a biphasic rise of intracellular free Ca2+ concentration ([Ca2+]i), consisting of an initial fast increase (delta [Ca2+]i = 122 nM) followed by a plateau phase (delta [Ca2+]i = 58 nM). In the absence of basolateral Ca2+, the initial peak was still observed, suggesting intracellular Ca2+ release. Norepinephrine and epinephrine, as well as selective beta 1- and beta 2-agonists, also increased [Ca2+]i in CCD. Only slight [Ca2+]i variations were produced by isoproterenol in the OMCD (delta [Ca2+]i = 21 nM) and the cortical thick ascending limb (delta [Ca2+]i = 25 nM). These results show that both beta 1- and beta 2-ARs are expressed in the collecting tubule and that they predominate in the CCD. The two receptor subtypes contribute to cAMP accumulation induced by beta-agonists. They also trigger [Ca2+]i variations, indicating their possible coupling to several transduction pathways in the rat CCD.
通过显微切割分离出大鼠集合管，研究了β-肾上腺素能受体（β-ARs）的表达及功能特性。逆转录-聚合酶链反应实验表明，β1-和β2-AR mRNA在皮质集合管（CCD）中有表达，但β3-亚型无表达。以突变RNA作为内标进行mRNA定量分析，结果进一步显示，β1-和β2-AR转录本在CCD中的含量相当（每毫米肾小管长度有3000 - 4000个拷贝），但在外髓集合管（OMCD）中的含量则低6 - 8倍（β1，480±180；β2，590±110个拷贝/毫米肾小管长度）。在CCD中进行的功能研究证实了这两种受体亚型的表达。β-激动剂刺激腺苷3',5'-环磷酸（cAMP）积累的效能顺序为异丙肾上腺素>去甲肾上腺素 = 肾上腺素，并且发现β1-和β2-拮抗剂抑制异丙肾上腺素依赖性cAMP形成的效率相似。Fura 2荧光测量显示，异丙肾上腺素（10μM）可诱导细胞内游离Ca2+浓度（[Ca2+]i）呈双相升高，包括最初的快速升高（Δ[Ca2+]i = 122 nM），随后是平台期（Δ[Ca2+]i = 58 nM）。在无基底外侧Ca2+的情况下，仍可观察到初始峰值，提示细胞内Ca2+释放。去甲肾上腺素、肾上腺素以及选择性β1-和β2-激动剂也可使CCD中的[Ca2+]i升高。异丙肾上腺素在OMCD（Δ[Ca2+]i = 21 nM）和皮质厚升支（Δ[Ca2+]i = 25 nM）中仅引起轻微的[Ca2+]i变化。这些结果表明，β1-和β2-ARs在集合管中均有表达，且在CCD中占主导地位。这两种受体亚型均参与β-激动剂诱导的cAMP积累。它们还引发[Ca2+]i变化，表明它们可能与大鼠CCD中的多种转导途径偶联。
