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 a-melanocyte-stimulating hormone (cr-melanotropin) stimulated protein discharge almost equally effective as dibutyryladenosine 3',5'-monophosphate (Bt2cAMP) and 3-isobutyl-I-methylxanthine but less potent than carbamoylcholine. 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-I-methylxanthine (40 pM) potentiated the effect of corticotropin on secretion. In contrast to the action of carbamoylcholine, corticotropin-induced protein discharge was not inhibited by omission of Ca2+ ions from the incubation medium.

Stimulation of secretion in exocrine cells is associated with the incorporation of up to 3 to 4 phosphates into the ribosomal protein S6. This occurs with secretagogues involving either cAMP or free calcium as second messenger. An analysis of the phosphorylation pattern of S6 from stimulated guineapig parotid glands reveals 3 phosphopeptides (termed A,B,C). The phosphopeptide pattern was identical for cAMP‐ or calcium‐mediated stimulation, whereas phosphorylation of the S6 protein in vitro with catalytic subunit of cAMP‐dependent protein kinase resulted only in the formation of phosphopeptides A and C. Therefore, secretagogue‐mediated phosphorylation is not or not exclusively catalyzed by cAMP‐dependent protein kinase even when cAMP is the second messenger.

The present study was undertaken to investigate a possible involvement of phospholipid methyltransferases in the coupling of receptor-mediated stimulation to secretion. Phospholipid methyltransferases were assayed in isolated parotid acini in the presence of carbamoylcholine or isoprenaline. Carbamoylcholine reduced the incorporation of methyl groups into phospholipids, whereas isoprenaline showed no effect. Amylase secretion stimulated either by carbamoylcholine or by isoprenaline could not be affected by inhibitors of methyltransferases (3-deaza-adenosine alone or plus homocysteine thiolactone) under conditions where phospholipid methylation was strongly inhibited. The activity of adenylate cyclase in isolated parotid microsomal membranes was not inhibited or stimulated by S-adenosyl-homocysteine or -methionine respectively. These results indicate that phospholipid methylation does not play an essential role in stimulus-secretion coupling in the parotid gland.

The ribosomal protein S6 in exocrine cells is phosphorylated during stimulated during stimulation of exocytosis by cAMP‐dependent or calcium‐dependent agonists. Under both conditions the same tryptic S6 phosphopeptides (termed A, B, and C) were found [Padel, Kruppa, Jahn & Söling (1983) FEBS Lett. 159, 112–118]. Studies have now been made of the phosphorylation pattern of protein S6 from purified guinea pig parotid ribosomes following in vitro phosphorylation with calmodulin‐dependent, phospholipid‐dependent, and cAMP‐dependent protein kinases. Only the phospholipid‐dependent enzyme led to the phosphorylation of peptides A, B, and C, while the cAMP‐dependent enzyme phosphorylated only peptides A and C, and the calmodulin‐dependent enzyme did not phosphorylate any of the phosphopeptides found in S6 from unstimulated or stimulated intact cells. Guinea pig parotid microsomes contain substantial phospholipid‐dependent protein kinase activity. Stimulation of intact parotid glands with tetradecanolyphorbol acetate led to a significant phosphorylation of S6 and a similar tryptic S6 phosphopeptide pattern as seen with carbamoylcholine. It is concluded that activation of phospholipid‐dependent protein kinase is responsible for the phosphorylation of protein S6 during stimulation with calcium‐dependent and cAMP‐dependent secretagogues.

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