D4 and D1A dopamine receptor (DR) mRNAs are expressed in the rat cortical collecting duct (CCD)

Academic Article

Abstract

  • Dopamine inhibits vasopressin (AVP)-dependent water permeability and Na+ transport in the rat CCD, and clozapine reverses the effect suggesting that a D4 DR is involved (Am J Physiol 271:F391, 1996). We used RT-PCR of total RNA extracted from microdissected CCD segments to determine the DR mRNAs expressed. After RT 1/20 of the resulting cDNA was used per PCR reaction (48 cycles). With a few exceptions, there was no detectable product for extracts from <50 mm CCD, indicating low abundance messages; therefore, extracts of 100-200 mm were used so that cDNA from the equivalent of 5-10 mm of CCD were amplified. Products were verified by automated DNA sequencing of TA cloned products. Three primer pairs gave amplification of D4 regions covering most of the total sequence: transmembrane 2 (tm-2) to tm-3, the second cytoplasmic loop (c-2) to tm-6, and c-3 to tm-7 with, respectively, 100%, 100% and 98% nucleotide identity to the known rat D4 sequence; however, there was an extra six-base insert in the first product at the 3′ end of tm-2 that was identical to the human and mouse but not the rat sequences. Primers designed for the D1 DR amplified regions: tm-7 into the 3′-UTR and tm-6 to tm-6 (the third cytoplasmic loop) with, respectively, 100% and 92% identity to the known rat D1 sequence. We conclude that both DR isoforms are expressed in the rat CCD but the physiological effect appears to be attributable to D4, as also supported by our studies of the effect of dopamine on cAMP generation (Li et al., FASEB J., in press, 1997).
  • Authors

    Author List

  • Sun D; Wilborn TW; Schafer JA
  • Volume

  • 11
  • Issue

  • 3