Adrenocorticotropic Hormone and α‐Melanocyte‐Stimulating Hormone Induce Secretion and Protein Phosphorylation in the Rat Lacrimal Gland by Activation of a cAMP‐Dependent Pathway

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Abstract
In the present study a cAMP‐dependent pathway leading to exocytosis in the rat lacrimal gland was investigated in detail. Using a lobule system in vitro, adrenocorticotropic hormone (corticotropin) and α‐melano‐cyte‐stimulating hormone (α‐melanotropin) stimulated protein discharge almost equally effective as dibutyryl‐adenosine 3′,5′‐monophosphate (Bt2cAMP) and 3‐isobutyl‐1‐methylxanthine but less potent than carbamoyl‐choline. Maximal stimulation was obtained at a hormone concentration of 20 nM for both peptides indicating that the active sequence is part of the first 13 amino acids of the corticotropin molecule. 3‐Isobutyl‐1‐methyl‐xanthine (40 μM) potentiated the effect of corticotropin on secretion. In contrast to the action of carbamoyl‐choline, corticotropin‐induced protein discharge was not inhibited by omission of Ca2+ ions from the incubation medium.The tissue content of cAMP was not significantly affected, by corticotropin or 40 μM 3‐isobutyl‐1‐methyl‐xanthine. However, a combination of both agonists led to a rapid 2.5–5‐fold increase of the tissue cAMP level in vitro. Adenylate cyclase activity of a lacrimal membrane fraction was effectively stimulated by corticotropin as well as by α‐melanotropin. The following hormones and neurotransmitters were ineffective with respect to induction of secretion or activation of adenylate cyclase: isoproterenol (20 μM), dopamine (10 μM), secretin (0.1 μM) glucagon (10 nM), vasoactive intestinal peptide (0.1 μM), substance P (0.1 μM), pancreozymin (0.1 μM) and methioninenkephalin (0.1 μM).Corticotropin, α‐melanotropin, Bt2cAMP and 3‐isobutyl‐1‐methylxan thine induced the phosphorylation of three membrane‐bound proteins (Mr 35000, 26000 and 20000) which corresponded almost exactly to the EC‐protein and the proteins II and III respectively described previously for the parotid gland [Jahn, R., Unger, C. and Söling, H.D. (1980) Eur. J. Biochem. 112, 345–352]. In accordance with an earlier report [Jahn, R. and Soling, H.D. (1981) FEBS Lett. 131, 28–30] proteins II and III were not phosphorylated during cholinergic stimulation. The combination of submaximal concentrations of agonists acting via the same or via different mechanisms resulted in an additive enhancement of secretion as well as of phosphorylation of the EC‐protein.

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