Cell Tissue Res (1984) 235:107-115 Cell and Tissue Research 9 Springer-Verlag 1984 Immunohistochemical localization of enkephalin- and ACTH-related substances in the pituitary of the lamprey Robert M. Dores, Thomas E. Finger, and Michael R. Gold Departments of Physiology and Anatomy, University of Colorado School of Medicine, Denver, Colorado, USA Summary. The distributions of ACTH-, aMSH-, flLPH-, and enkephalin-related substances were determined im- munohistochemically in the pituitary of the brook lamprey, Lampetra lamotenii. An antiserum directed against the mid- dle region of ACTH reacted chiefly with cells in the pro- adenohypophysis. An antiserum specific for aMSH reacted with all of the cells of the meta-adenohypophysis, but did not react with any of the middle ACTH-positive cells in the pro-adenohypophysis. Several antisera which crossreact with both flLPH and fl-endorphin did not react with any region of the lamprey pituitary. However, an antiserum di- rected against 7LPH did react with a small population of cells in the meso-adenohypophysis. This reactivity could be blocked following pre-absorption with mouse flLPH but was not blocked by synthetic fl-endorphin(1-31). Antisera directed against either met-enkephalin or leu- enkephalin reacted with fibers in the anterior neurohypoph- ysis, cells in the pro-adenohypophysis, and all the cells of the meta-adenohypophysis. This crossreactivity could be blocked following pre-absorption with the appropriate en- kephalin, but not by pre-absorption with synthetic fl-endor- phin(1-31) or dynorphin(1-13). In addition, the enkepha- lin-like reactivity in the adenohypophysis of the lamprey was coincident with middle ACTH-like immunoreactivity in the pro-adenohypophysis and with aMSH-like immuno- reactivity in the meta-adenohypophysis. The absence of flLPH/fl-endorphin immunoreacfivity coincident with ACTH immunoreactivity, and the presence of enkephalin-like material in the adenohypophysis are un- ique to the lamprey. Key words: ACTH - aMSH - Enkephalin - Adenohypo- physis - Lamprey The ACTH/flLPH family of adenohypophyseal hormones is a collection of polypeptides related by sequence homolo- gy (Baker 1980) and a common biosynthetic pathway (Eip- per and Mains 1980). In mammals, ACTH (adrenocortico- tropin) and flLPH (beta lipotropin) are synthesized as ele- ments of a larger (30 K) common precursor protein (pro- ACTH/endorphin or proopiomelanocortin) both in cortico- tropic cells of the pars distalis and in melanotropic cells Send offprint requests to: Dr. Robert M. Dores, University of Mi- chigan School of Medicine, Mental Health Research Institute. 205 Washtenaw Place, Ann Arbor, Michigan 48109, USA of the pars intermedia (Eipper and Mains 1978; Nakanishi et al. 1979; Herbert et al. 1980; Krieger et al. 1980). Post- translational processing of the precursor in the pars distalis yields 16K fragment (the NH2-terminal third of pro- ACTH/endorphin), ACTH, flLPH, ?LPH (the NH2-termi- nal two thirds offlLPH), and fl-endorphin (the COOH ter- minal third of flLPH) as end products (Mains et al. 1978; Roberts and Herbert 1977; Eipper and Mains 1978; Mains and Eipper 1978). The initial proteolytic processing of pro- ACTH/endorphin in the pars intermedia is identical to that in the pars distalis. However, ACTH is processed further to yield ~MSH-sized molecules (a-melanotropin) and CLIP (corticotropin-like intermediate lobe peptide), while nearly all of the flLPH is cleaved to yield 7LPH and fl-endorphin- size molecules (Eipper and Mains 1978; Gianoulakis et al. 1979; Mains and Eipper 1979, 1981; Eipper and Mains 1981). In some species 7LPH and 16 K fragment may be cleaved to smaller peptides (Pederson and Brownie 1979; Brownie et al. 1981 ; Estivariz et al. 1982). Among non-mammalian vertebrates a similar biosyn- thetic pathway has been reported in amphibians (Loh and Gainer 1977; Loh 1979; Pezella et al. 1978) and repitles (Dores 1982a, b). Furthermore, in birds, bony fishes, and cartilaginous fishes, end products of the ACTH/flLPH sys- tem have been isolated and, in some instances, fully charac- terized (Kawauchi and Muramoto 1979; Baker 1980; Kaw- auchi et al. 1980a, b, c; McLean and Lowry 1981 ; Kawau- chi et al. 1982). However, little is known about the distribu- tion of peptides related to ACTH and flLPH in agnathans (lampreys and hagfish), the most ancient lineage of extant vertebrates. The present study will focus on the distribution of these peptides in lampreys. The pituitary of the lamprey is similar in organization to the pituitary of other vertebrates (Hardisty 1981). The adenohypophysis is divisible into the pro- and meso-adeno- hypophyses (homologs of pars distalis) and the meta-adeno- hypophysis (homolog of the pars intermedia; Fig. 1). The neurohypophysis is divided into two regions: the anterior neurohypophysis, the homolog of the infundibulum, and the posterior neurohypophysis, the homolog of the pars nervosa. The latter region is the storage site for a vasotocin- like peptide (Rurak and Perks 1976, 1977). With respect to the ACTH/flLPH system, ACTH-like bioactivity and ACTH-related immunoreactivity have been detected in whole pituitary homogenates (Scott et al. 1971; Eastman and Portanova 1982). Furthermore, MSH-like bioactivity has been localized in the meta-adenohypophysis (Lanzing