Abstract
α1-Adrenoceptor subtypes mediate many of the actions of the renal nerve, but their locations along the nephron are unknown. We investigated the distribution of α1-adrenoceptor subtype mRNA and protein in rat proximal tubules and medullary thick ascending limbs (MTAL) using reverse transcription combined with polymerase chain reaction (PCR) and radioligand binding methods. Complementary primers were designed to span cDNA sequences in each of the third intracellular loops of the rat α1B-and α1D-adrenoceptors. Expression of the mRNA of α1B- and α1D-adrenoceptors was first detected in total RNA from whole rat kidney, and the PCR product identity was confirmed by sequencing. Endogenous expression of α1B- and/or α1D-adrenoceptor mRNA was then investigated in microdissected segments of the rat proximal convoluted tubule (S2 segments) and the MTAL. mRNA was reverse-transcribed directly from permeablized microdissected segments and the resulting cDNA was subjected to PCR with the α1-adrenoceptor primers. In proximal convoluted tubules, amplification of both α1B- and α1D-adrenoceptor mRNA was observed. In MTAL segments, only α1D-adrenoceptor mRNA was detected. We also measured receptor protein using [3H]prazosin in saturation and competition binding experiments. Proximal tubular membranes contained 3.3-fold more α1-adrenoceptor than did MTAL membranes (163 ± 21 versus 49 ± 3 fmol/mg of protein). When the alkylating agent chloroethylclonidine (CEC) (10 μM, 10 min) was used to define α1-adrenoceptor subtypes, proximal tubules were found to contain primarily CEC-insensitive (α1A) sites (68 ± 4%) and MTAL primarily CEC-sensitive sites (75 ± 3%). Most [3H]prazosin binding sites (72 ± 2%) in MTAL segments were also sensitive to the alkylating agent SZL-49, consistent with their identification as α1D-adrenoceptors. In competition studies with the antagonists WB4101, 5-methylurapidil, and (+)-niguldipine, both high and low affinity sites were observed in proximal tubules. WB4101 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 α1-adrenoceptor subtypes in small amounts of tissue. Results from our experiments suggest that α1A-, α1B-, and α1D-adrenoceptors are all expressed in proximal tubules and that α1D-adrenoceptors are the primary α1-adrenoceptor subtype expressed in MTAL. The distinct anatomical distribution of each of these adrenoceptor subtypes suggests that they serve different functions in the kidney.