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Transactivation of EGF receptors by G protein-coupled receptors is a well-known phenomenon. This process involves the ectodomain shedding of growth factors in the EGF family by matrix metalloproteinases. However, many of these studies employ transformed and/or cultured cells that overexpress labeled growth factors. In addition, few studies have shown that EGF itself is the growth factor that is shed and is responsible for transactivation of the EGF receptor. In this study, we show that freshly isolated, nontransformed lacrimal gland acini express two of the three known [[alpha].sub.1]-adrenergic receptors (ARs), namely, [[alpha].sub.1B]- and [[alpha].sub.1D]-ARs. [[alpha].sub.1D]-ARs mediate phenylephrine (an [[alpha].sub.1]-adrenergic agonist)-induced protein secretion and activation of p42/p44 MAPK, because the [[alpha].sub.1D]-AR inhibitor BMY-7378, but not the [[alpha].sub.1A]-AR inhibitor 5-methylurapidil, inhibits these processes. Activation of p42/p44 MAPK occurs through transactivation of the EGF receptor, which is inhibited by the matrix metalloproteinase ADAM 17 inhibitor TAPI-1. In addition, phenylephrine caused the shedding of EGF from freshly isolated acini into the buffer. Incubation of freshly isolated cells with conditioned buffer from cells treated with phenylephrine resulted in activation of the EGF receptor and p42/p44 MAPK. The EGF receptor inhibitor AG1478 and an EGF-neutralizing antibody blocked this activation of p42/p44 MAPK. We conclude that in freshly isolated lacrimal gland acini, [[alpha].sub.1]-adrenergic agonists activate the [[alpha].sub.1D]-AR to stimulate protein secretion and the ectodomain shedding of EGF to transactivate the EGF receptor, potentially via ADAM17, which activates p42/p44 MAPK to negatively modulate protein secretion. epidermal growth factor ectodomain shedding; protein secretion; signal transduction

The intestinal brush border (BB) [Na.sup.+]/[H.sup.+] exchanger isoform 3 (NHE3) is acutely inhibited by elevation in the concentration of free intracellular [Ca.sup.2+] ([[[Ca.sup.2+]].sub.i]) by the cholinergic agonist carbachol and [Ca.sup.2+] ionophores in a protein kinase C (PKC)-dependent manner. We previously showed that elevating [[[Ca.sup.2+]].sub.i] with ionomycin rapidly inhibited NHE3 activity and decreased the amount of NHE3 on the plasma membrane in a manner that depended on the presence of the PDZ domain-containing protein E3KARP (NHE3 kinase A regulatory protein, also called NHERF2). The current studies were performed in PS120 fibroblasts (NHE-null cell line) stably transfected with NHE3 and E3KARP to probe the mechanism of PKC involvement in [Ca.sup.2+] regulation of NHE3. Pretreatment with the general PKC inhibitor, GF109203X prevented ionomycin inhibition of NHE3 without altering basal NHE3 activity. Similarly, the [Ca.sup.2+]-mediated inhibition of NHE3 activity was blocked after pretreatment with the conventional PKC inhibitor Go-6976 and a specific PKC[alpha] pseudosubstrate-derived inhibitor peptide. [[[Ca.sup.2+]].sub.i] elevation caused translocation of PKC[alpha] from cytosol to membrane. PKC[alpha] bound to the PDZ1 domain of GST-E3KARP in vitro in a [Ca.sup.2+]-dependent manner. PKC[alpha] and E3KARP coimmunoprecipitated from cell lysates; this occurred to a lesser extent at basal [[[Ca.sup.2+]].sub.i] and was increased with ionomycin exposure. Biotinylation studies demonstrated that [[[Ca.sup.2+]].sub.i] elevation induced oligomerization of NHE3 in total lysates and decreased the amount of plasma membrane NHE3. Treatment with PKC inhibitors did not affect the oligomerization of NHE3 but did prevent the decrease in surface amount of NHE3. These results suggest that PKC[alpha] is not necessary for the [Ca.sup.2+]-dependent formation of the NHE3 plasma membrane complex, although it is necessary for decreasing the membrane amounts of NHE3, probably by stimulating NHE3 endocytosis. Na absorption; PDZ domains; signal complex

Conjunctival goblet cells are the primary source of mucins in the mucous layer, the innermost layer of the tear film. Conjunctival goblet cell mucin secretion is under neural control because exogenous addition of parasympathetic agonists stimulates goblet cell secretion. To elucidate the intracellular signal pathways used by cholinergic agonists to stimulate goblet cell mucin secretion, we determined whether p42/p44 mitogen-activated protein kinase (MAPK) is activated during cholinergic agonist-stimulated mucin secretion. Rat conjunctiva was removed, preincubated with or without antagonists, and stimulated with the cholinergic agonist carbachol ([10.sup.-4] M). Carbachol statistically significantly stimulated the phosphorylation of MAPK in a time- and concentration-dependent manner. U-0126, an inhibitor of MAPK activation, completely inhibited both the activation of MAPK and goblet cell secretion stimulated by carbachol. The [M.sub.1] muscarinic antagonist pirenzepine, the [M.sub.2] muscarinic antagonist gallamine, and the [M.sub.1]/[M.sub.3] muscarinic receptor antagonist N-(3-chloropropyl)-4-piperidinyl diphenylacetate (4-DAMP) also inhibited carbachol-stimulated MAPK activation. Increasing the intracellular [Ca.sup.2+] concentration with a [Ca.sup.2+] ionophore increased MAPK activation, and chelation of extracellular [Ca.sup.2+] inhibited carbachol-stimulated activation. Carbachol also increased tyrosine phosphorylation of Pyk2, p60Src, and the epidermal growth factor receptor (EGFR). The Src inhibitor PP1 and the EGFR inhibitor AG-1478 completely inhibited carbachol-stimulated MAPK activation. AG-1478 also inhibited goblet cell secretion. We conclude that carbachol transactivates the EGFR to activate MAPK, leading to conjunctival goblet cell secretion. In addition, carbachol also activates Pyk2 and p60Src that could play a role in the transactivation of the EGFR. signal transduction; muscarinic receptors; conjunctiva; Pyk2; p60Src

The purpose of this study was to determine the role of p42/p44 mitogen-activated protein kinase (MAPK) in [[alpha].sub.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].sub.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.sup.Src], and possibly Pyk2, to activate MAPK. We conclude that, in the lacrimal gland, activation of MAPK plays an inhibitory role in [[alpha].sub.1]-adrenergically and cholinergically stimulated protein secretion and that these agonists use different signaling mechanisms to activate MAPK. signal transduction; Pyk2; Src

The role protein kinase C on the protein secretion in rat lacrimal gland was analyzed after the synthesis of N-myristoylated Myr-PKC-alpha-(15-28), Myr-PKC-delta-(15-28) and Myr-PKC-epsilon (142-153). Phorbol ester-mediated protein secretion in rat lacrimal gland was mediated by Myr-PKC-alpha (15-28) and Myr-PKC-epsilon (142-153). Furthermore, the three PKC isozymes are also utilized by cholinergic agonist during protein secretion.

The unique ability of phospholipase D (PLD) activity is utilized to determine the presence of phospholipase D (PLD) activity in rat lacrimal gland acini. The investigation reveals that the activity is present in the acini and is independent of intracellular calcium ions and PKC.

The effects of phorbol 12-myristate 13-acetate (PMA) with 4beta-phorbol 12,13-dibutyrate (PdBu) in stimulating protein discharge in the lacrimal glands of rats were studied. Results show that PMA is less efficient compared to PdBu. Tests proved that neither sphingosine, chelerythrine, staurosporrine failed to inhibit both PMA and PdBu. On the other hand, trifluoperazine produced complete inhibition of PMA-induced protein discharge. The study questions the identification of protein kinase C as sole effector of PMA-stimulated protein secretion in rat lacrimal glands.