Splice isoforms of α(1a)-adrenoceptor in rabbit

Abstract

1. Two splice isoforms of rabbit α(1a)-adrenergic receptor (AR), (named α(1a)-OCU.2-AR and α(1a)- OCU.3-AR) have been isolated from the liver cDNA library in addition to the previously reported isoform (α(1a)-OCU.1-AR). Although they have the identical splice position with human α(1a)-AR isoforms, the C-terminal sequences are distinct from those of human isoforms. 2. Among these rabbit α(1a)-AR isoforms, there are no significant differences in pharmacological properties: high affinity for prazosin, WB4101, KMD-3213 and YM617 and low affinity for BMY7378, using COS-7 cells expressing each isoform by radioligand binding assay. 3. Competitive reverse transcription-polymerase chain reaction (RT - PCR) analysis revealed that mRNA of α(1a)-ARs was expressed in liver, thoracic aorta, brain stem and thalamus of rabbit. The splice isoforms exhibited a distinct distribution pattern in rabbit: α(1a)-OCU.1-AR was expressed most abundantly in those tissues. 4. CHO clones, stably expressing each isoforms with receptor density 740 fmol mg-1 protein in α(1a)- OCU.1-AR, 1200 fmol mg-1 in α(1a)-OCU.2-AR and 570 fmol mg-1 in α(1a)-OCU.3-AR, respectively, showed a noradrenaline-induced increase in inositol trisphosphate which was suppressed by prazosin. 5. Noradrenaline elicited a concentration-dependent increase in extracellular acidification rate (EAR) in the CHO clones with pEC50 values of 6.19 for α(1a)-OCU.1-AR, 6.49 for α(1a)-OCU.2-AR and 6.58 for α(1a)-OCU.3-AR, respectively. 6. Noradrenaline caused a concentration-dependent increase in intracellular Ca2+ concentration ([Ca2+](i)) in the CHO clones with pEC50 values of 6.14 for α(1a)-OCU.1-AR, 7.25 for α(1a)-OCU.2-AR and 7.70 for α(1a)-OCU.3-AR, respectively. 7. In conclusion, the present study shows the occurrence of three splice isoforms of rabbit α(1a)-AR, which are unique in C-terminal sequence and in tissue distribution. They show similar pharmacological profiles in binding studies but α(1a)-OCU.3-AR had the highest potency of noradrenaline in functional studies in spite of the lowest receptor density. These findings suggest that the structure of C-terminus of α(1a)-ARs may give the characteristic functional profile.