HETEROGENEOUS EXPRESSION OF ALPHA(1)-ADRENOCEPTOR SUBTYPES AMONG RAT NEPHRON SEGMENTS

Alpha1-Adrenoceptor subtypes mediate many of the actions of the renal nerve, but their locations along the nephron are unknown. We investiga ted the distribution of alpha1-adrenoceptor subtype mRNA and protein i n rat proximal tubules and medullary thick ascending limbs (MTAL) usin g reverse transcription combined with polymerase chain reaction (PCR) and radioligand binding methods. Complementary primers were designed t o span cDNA sequences in each of the third intracellular loops of the rat alpha1B- and alpha1D-adrenoceptors. Expression of the mRNA of alph a1B- and alpha1D-adrenoceptors was first detected in total RNA from wh ole rat kidney, and the PCR product identity was confirmed by sequenci ng. Endogenous expression of alpha1B- and/or alpha1D-adrenoceptor mRNA was then investigated in microdissected segments of the rat proximal convoluted tubule (S2 segments) and the MTAL. mRNA was reverse-transcr ibed directly from permeablized microdissected segments and the result ing cDNA was subjected to PCR with the al-adrenoceptor primers. In pro ximal convoluted tubules, amplification of both alpha1B- and alpha1D-a drenoceptor mRNA was observed. In MTAL segments, only alpha1D-adrenoce ptor mRNA was detected. We also measured receptor protein using [H-3]p razosin in saturation and competition binding experiments. Proximal tu bular membranes contained 3.3-fold more alpha1-adrenoceptor than did M TAL membranes (163 +/- 21 versus 49 +/- 3 fmol/mg of protein). When th e alkylating agent chloroethylclonidine (CEC) (10 muM, 10 min) was use d to define alpha1-adrenoceptor subtypes, proximal tubules were found to contain primarily CEC-insensitive (alpha1A) sites (68 +/- 4%) and M TAL primarily CEC-sensitive sites (75 +/- 3%). Most [H-3]prazosin bind ing sites (72 +/- 2%) in MTAL segments were also sensitive to the alky lating agent SZL-49, consistent with their identification as alpha1D-a drenoceptors. In competition studies with the antagonists WB4101, 5-me thylurapidil, and (+)-niguldipine, both high and low affinity sites we re observed in proximal tubules. WB41 01 interacted with only one site in MTAL membranes, intermediate in affinity between those sites found in proximal tubules. We conclude that reverse transcription-PCR is a useful method for demonstrating the expression of alpha1-adrenoceptor subtypes in small amounts of tissue. Results from our experiments sugg est that alpha1A-, alpha1B-, and alpha1D-adrenoceptors are all express ed in proximal tubules and that alpha1D-adrenoceptors are the primary alpha1-adrenoceptor subtype expressed in MTAL. The distinct anatomical distribution of each of these adrenoceptor subtypes suggests that the y serve different functions in the kidney.