Luteinizing hormone-releasing hormone-binding sites in the rat thymus: characteristics and biological function.
The present study was designed to explore the effects of LHRH and its agonists on immune system function. As a first step, to identify a putative site of action, the very potent and stable LHRH agonist (LHRH-A), [D-Ser(TBU6)] des-Gly10-LHRH ethylamide (buserelin), was used as an iodinated ligand to characterize LHRH receptors in a membrane preparation of rat thymus, a key organ of the immune system. The effects of LHRH and LHRH-A were then investigated on the proliferative capacity of rat thymocytes exposed in vitro to a mitogen and on ornithine decarboxylase specific activity. In addition, to determine whether LHRH-A treatment in vivo might directly influence thymic function, we treated hypophysectomized (hypox) rats with a moderately high dose of LHRH-A for a period of 2 weeks, and thymocyte mitogenic capacity, thymus weight, and the histological and functional appearance of the thymus were then assessed. Specific binding of LHRH-A to rat thymic membrane preparations is a saturable process, depending on both time and temperature of incubation, but differs markedly from binding to the rat pituitary or ovarian LHRH receptor in its low binding affinity. Binding is optimal in the absence of chelating agents (EDTA) or divalent metal ions, and increases linearly with increasing protein concentration. Binding is specific for LHRH, LHRH-A, and antagonists. Both the C-terminal amide and N-terminal regions of the LHRH molecule were required for binding, and amino acid substitutions at position 6 markedly enhanced and at position 8 markedly reduced binding potencies in rat thymic tissue. A number of peptides, proteins, and other agents had no effect on the specific binding of LHRH-A to thymic membrane preparations. The binding affinity (Ka) of the membrane receptor of the rat thymus for the LHRH superagonist buserelin was 8.4 x 10(8) M-1, while a higher binding affinity (Ka = 2.8 x 10(9) M-1) was calculated for the ovarian LHRH-binding site. Preincubation of rat thymocytes with LHRH-A for 20 h induced a significant dose-dependent increase in the proliferative response to the mitogen Concanavalin-A, monitored by [3H]thymidine incorporation. Using native LHRH, it was also possible to elicit stimulatory effects on the same parameter, although much higher concentrations were required than with LHRH-A. Furthermore, simultaneous addition of a LHRH antagonist, abolished the LHRH effect on thymocytes. Ornithine decarboxylase specific activity under lectin stimulation was also significantly increased by LHRH-A in cultures of rat thymocytes.(ABSTRACT TRUNCATED AT 400 WORDS)
Marchetti
B
B
Department of Pharmacology, Medical School, University of Catania, Italy.
Guarcello
V
V
Morale
M C
MC
Bartoloni
G
G
Farinella
Z
Z
Cordaro
S
S
Scapagnini
U
U
D000818:Animals; D001665:Binding Sites; D002064:Buserelin / Q000378:metabolism / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D002455:Cell Division / Q000187:drug effects; D005260:Female; D005835:Genitalia / Q000187:drug effects; D007987:Gonadotropin-Releasing Hormone / Q000378:metabolism* / Q000494:pharmacology / Q000502:physiology; D007016:Hypophysectomy; D037102:Lectins / Q000494:pharmacology; D008297:Male; D009929:Organ Size; D009955:Ornithine Decarboxylase / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D011919:Rats, Inbred Strains; D011966:Receptors, LHRH / Q000032:analysis / Q000502:physiology*; D012964:Sodium / Q000494:pharmacology; D013950:Thymus Gland / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure
D037102:Lectins; D011966:Receptors, LHRH; D007987:Gonadotropin-Releasing Hormone; D012964:Sodium; D009955:Ornithine Decarboxylase; D002064:Buserelin; D002118:Calcium