The melanocortin receptor in the rat lacrimal gland: a model system for the study of MSH (melanocyte stimulating hormone) as a potential neurotransmitter.
The melanocortin receptors in intraorbital and extraorbital rat lacrimal glands were studied with [125I][Nle4,D-Phe7]alpha MSH as radioligand and with several unlabeled melanocortin peptides. The pharmacological properties of the melanocortin receptor in both tissues appeared to be essentially identical. Receptor binding was studied in a membrane fraction sedimented at 12,000-100,000 X g, establishing for [125I][Nle4,D-Phe7]alpha MSH a KD of 0.76 and 2.2 nM for the intra- and extraorbital glands, respectively. Binding of the radioligand was competitively inhibited by alpha MSH (alpha-melanocyte stimulating hormone) and ACTH-(1-24) with IC50 values in the submicromolar range. MSH binding in both tissues was abolished by EGTA and was increased dose dependently with elevation of free Ca2+ ion concentration. The half-maximal effect on MSH binding was obtained around 200 microM Ca2+ and maximal binding was reached at nearly 2 mM free Ca2+ in membrane preparations from both tissues. The calmodulin-binding peptides, melittin, mastoparan and M5, the latter being the 18-amino acid synthetic analogue of the C-terminal calmodulin-binding domain of myosin light chain kinase, inhibited MSH binding in the concentration range of 1-20 microM. Macroscopic autoradiographic analysis of cryosections prepared from either lacrimal gland to which [125I][Nle4,D-Phe7]alpha MSH was subsequently bound, showed the melanocortin receptor to be uniformly distributed within the acinar lobes. At the microscopic level, MSH was found to be associated with the acinar cells, primarily at the basal perinuclear region. Peroxidase secretion from extraorbital lacrimal slices was stimulated by MSH, epinephrine and carbamylcholine to a similar extent. The response of the tissue to stimulation by MSH was however not blocked by alpha/beta-adrenoceptor blockers or by atropine, suggesting that MSH acts as a primary secretagogue in this tissue. Thus, this system seems to be uniquely suited to serve as a model for the study of both the molecular and pharmacological details of the action of MSH and other melanocortins in a non-melanogenic tissue.
Leiba
H
H
Department of Hormone Research, Weizmann Institute of Science, Rehovot, Israel.
Garty
N B
NB
Schmidt-Sole
J
J
Piterman
O
O
Azrad
A
A
Salomon
Y
Y
D000818:Animals; D001345:Autoradiography; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D002478:Cells, Cultured; D005088:Exocrine Glands / Q000033:anatomy & histology / Q000187:drug effects / Q000378:metabolism*; D066298:In Vitro Techniques; D007765:Lacrimal Apparatus / Q000033:anatomy & histology / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D018377:Neurotransmitter Agents; D010544:Peroxidases / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D011956:Receptors, Cell Surface / Q000187:drug effects / Q000378:metabolism*; D000521:alpha-MSH / Q000031:analogs & derivatives / Q000378:metabolism / Q000502:physiology*
D002147:Calmodulin; D018377:Neurotransmitter Agents; D011956:Receptors, Cell Surface; D000521:alpha-MSH; D010544:Peroxidases; D002118:Calcium