Secretion of Epidermal Growth Factor, Nerve Growth Factor, and Renin-Like Enzymes by Dispersed Male Mouse Submandibular Gland Cells in Vitro*

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Abstract
The submandibular gland of the mouse contains a variety of hormone-like proteins and enzymes, such as epidermal growth factor (EGF), nerve growth factor (NGF), and submandibular gland renin-like enzymes (SGR). Dispersed cells from the submandibular gland of male mice were prepared by treatment with collagenase to study the mechanisms by which these specific proteins are secreted in vitro. All three proteins were secreted concomitantly in vitro by the dispersed submandibular gland cells. Norepinephrine, epinephrine, and phenylephrine, all α-adrenergic agonists, stimulated release of these three proteins in a dose-dependent manner, whereas isoproterenol, a β-adrenergic agonist, and carbamylcholine, a cholinergic agonist, did not. The effect of α-adrenergic agonists was inhibited either by removal of calcium ions from the incubation medium or by the addition of verapamil, a selective blocker of extracellular calcium influx. The divalent cation ionophore, A-23187, stimulated release of all three proteins in a dose-dependent manner only in the presence of calcium ion, but not in the presence of magnesium ion. Thus, the ionophore mimicked the action of α-adrenergic agonists. Dibutyryl cAMP, dibutyryl cGMP, methylisobutylxanthine, and a variety of hormones or other biologically active peptides found in the alimentary tract, brain, and/or skin had no effect on the release of EGF, NGF, or SGR. The apparent molecular sizes of all three proteins secreted into the medium were similar to those found in mouse saliva; that is, they were similar to those of the respective highly purified standard. However, submandibular gland extract contained high molecular weight EGF and NGF components which dissociated under conditions of extreme pH to yield standard small molecular weight EGF and NGF, respectively. The secretion of EGF, NGF, and SGR appears to be similarly initiated by activation of α-adrenergic receptors and apparently involves a mechanism independent of cyclic nucleotides but dependent upon influx of extracellular calcium ion.