Effect of overexpression of constitutively active PKCalpha on rat lacrimal gland protein secretion.

PURPOSE: The lacrimal gland secretes water, electrolytes, and protein into the tear film. Decreased secretion from the lacrimal gland can lead to dry eye syndromes with deleterious effects on vision. Protein kinase C (PKC)-alpha plays a major role in cholinergic- and alpha1-adrenergic-induced protein secretion from the lacrimal gland. This study was undertaken to determine whether activation of PKCalpha alone would induce lacrimal gland protein secretion by examining the effects of overexpression of constitutively active PKCalpha. METHODS: Rat lacrimal gland acini were transduced with an adenovirus containing a gene for constitutively active PKCalpha. Protein secretion was measured in response to cholinergic and alpha1-adrenergic agonist stimulation. RESULTS: More than 84% of acinar cells were transduced, and PKCalpha expression was increased 176-fold. Western blot analysis using an antibody to phosphorylated (activated) PKCalpha indicated that the overexpressed PKCalpha was active, and basal secretion was increased. Cholinergic agonist-stimulated protein secretion was not stimulated above basal secretion, whereas alpha1-adrenergic-agonist-stimulated protein secretion was increased in transduced acini. CONCLUSIONS: Basal lacrimal gland protein secretion can be stimulated by bypassing the release of neurotransmitters and activating PKCalpha, possibly leading to the development of new treatments for dry eye syndromes.

Alpha 1-adrenergic and cholinergic agonists activate MAPK by separate mechanisms to inhibit secretion in lacrimal gland.

The purpose of this study was to determine the role of p42/p44 mitogen-activated protein kinase (MAPK) in alpha(1)-adrenergically and cholinergically stimulated protein secretion in rat lacrimal gland acinar cells and the pathways used by these agonists to activate MAPK. Acini were isolated by collagenase digestion and incubated with the alpha(1)-adrenergic agonist phenylephrine or the cholinergic agonist carbachol, and activation of MAPK and protein secretion were then measured. Phenylephrine and carbachol activated MAPK in a time- and concentration-dependent manner. Inhibition of MAPK significantly increased phenylephrine- and carbachol-induced protein secretion. Inhibition of EGF receptor (EGFR) with AG1478, an inhibitor of the EGFR tyrosine kinase activity, significantly increased phenylephrine- but not carbachol-induced protein secretion. Whereas phenylephrine-induced activation of MAPK was completely inhibited by AG1478, activation of MAPK by carbachol was not. Phenylephrine stimulated tyrosine phosphorylation of the EGFR, whereas carbachol stimulated p60(Src), and possibly Pyk2, to activate MAPK. We conclude that, in the lacrimal gland, activation of MAPK plays an inhibitory role in alpha(1)-adrenergically and cholinergically stimulated protein secretion and that these agonists use different signaling mechanisms to activate MAPK.