A MOLECULAR MAP OF G-PROTEIN ALPHA-CHAINS IN MICRODISSECTED RAT NEPHRON SEGMENTS

Membrane-associated guanine nucleotide binding proteins regulate many receptor-mediated signals. Heterogeneity of biochemical and functional properties in nephron segments could be due to differences in G prote in expression. To ascertain whether such heterogeneity of G proteins i s present in various nephron segments, this study examines the distrib ution and relative abundance of G protein alpha chains in microdissect ed medullary thick ascending limb, cortical collecting tubules, outer medullary collecting tubules, proximal inner medullary tubules, and di stal inner medullary tubules. Reverse transcription and polymerase cha in reactions were employed using oligonucleotides encoding highly cons erved regions of all known alpha chains. The cDNA was sequenced for al pha chain identification. The alpha(i2) versus alpha(s), distribution was different in the outer medullary collecting tubules, when compared with the medullary thick ascending limb (P < 0.001) or the cortical c ollecting tubule, the proximal inner medullary tubules, and the distal inner medullary tubules (P < 0.05). These latter four segments did no t significantly differ from each other. A similar analysis was applied to the frequently used line of kidney cells, LLC-PK1, whose exact cel lular origin remains unclear. Interestingly, we detected both alpha(i2 ), and alpha(i3), While only alpha(i2) was detected in the rat distal nephron. No alpha(o) or alpha(z) reverse transcription PCR products we re detected. In contrast alpha11 and alpha14 members of the more recen tly described alpha(q) family were detected in the outer medullary col lecting tubules and the proximal inner medullary tubules, respectively . We conclude that the majority of nephron segments have a relatively constant distribution of G protein alpha chains.