EXPRESSION OF ALPHA(2)-ADRENOCEPTOR AND BETA-ADRENOCEPTOR SUBTYPES INHUMAN ISLETS OF LANGERHANS

Sequences from cDNA molecules encoding alpha(2)-adrenoceptor subtype g enes were subcloned into prokaryotic vectors and riboprobes generated to hybridise selectively with each of the human alpha(2)C2-, alpha(2)C 4- and alpha(2)C10-adrenoceptor subtype mRNA species. The riboprobes w ere labelled with either P-32 or digoxigenin and used to study the exp ression of alpha(2)-adrenoceptor subtypes in sections of human pancrea s, in isolated human islets of Langerhans and in clonal HIT-T15 pancre atic beta-cells. Using a ribonuclease protection assay protocol, expre ssion of mRNA species encoding both alpha(2)C2 and alpha(2)C10 was dem onstrated in preparations of isolated human islets of Langerhans. mRNA encoding alpha(2)C4 was also detected in human islet RNA, using rever se transcription coupled with the polymerase chain reaction. In situ h ybridisation was then employed to examine the distribution of each alp ha(2)-adrenoceptor subtype in sections of human pancreas. All three su btypes of alpha(2)-adrenoceptor mRNA were identified in sections of fo rmalin-fixed, paraffin-embedded human pancreas using riboprobes labell ed with digoxigenin. Although some labelling of the three alpha(2)-adr enoceptor mRNA subtypes was seen in the islets, the labelling was most intense in the exocrine tissue of the pancreas for each receptor subt ype. The specificity of the digoxigenin-labelled RNA probes was confir med in several control tissues and by in situ hybridisation studies us ing sense probes in the pancreas. The integrity of the pancreas sectio ns was confirmed by in situ hybridisation with an antisense riboprobe derived from human insulin cDNA. The results demonstrate that multiple alpha(2)-adrenoceptor subtypes are expressed in human pancreas. Both the exocrine and endocrine cells express more than one receptor subtyp e, although the islets stain less intensely than the bulk of the tissu e suggesting that the islet cells may have lower levels of expression than the acinar tissue. The presence of alpha(2)-adrenoceptor subtype mRNA species in pancreatic beta-cells was confirmed by Northern blotti ng of RNA extracted from the clonal beta-cell line, HIT-T15. Transcrip ts encoding each of the three cloned alpha(2)-adrenoceptor subtypes we re detected in HIT-T15 cells. Hybridisation of sections of human pancr eas with oligodeoxynucleotide probes designed to hybridise with beta(2 )-adrenoceptor mRNA revealed expression of this species in islet beta- cells but not in the exocrine tissue of the pancreas.