Nitric Oxide and cGMP Mediate α1D-Adrenergic Receptor–Stimulated Protein Secretion and p42/p44 MAPK Activation in Rat Lacrimal Gland

Nitric oxide (NO) is a small, diffusible gaseous molecule that has been identified as a mediator in a variety of cellular functions including secretion, inflammation, and blood flow 1,2,3. NO is synthesized from arginine and molecular oxygen resulting in NO and L-citrulline by nitric oxide synthase (NOS). Nitric oxide synthase is a family of enzymes consisting of three known isoforms, endothelial nitric oxide synthase (eNOS or NOS-3), neuronal nitric oxide synthase (nNOS or NOS-1), and inducible nitric oxide synthase (iNOS or NOS-2). eNOS was first identified in endothelial cells as the enzyme which produced endothelial-derived relaxing factor, later identified as NO 4. It has since been shown to be present in a variety of tissues. nNOS was originally identified in neuronal cells, but has also been shown to be present in many different tissues. eNOS and nNOS are constitutively expressed hence they are referred to as constitutive enzymes. These two isoforms are activated by intracellular calcium and calmodulin. iNOS is not present in resting cells and is induced in a variety of cells by cytokines, infection, or lipopolysaccaride. This isoform is calcium independent and constitutively active 5. Induction of iNOS results in a large, rapid increase in NO that can have detrimental effects to surrounding tissue. In contrast, activation of eNOS and nNOS results in a smaller, slower increase in NO that interacts with a variety of signaling pathways. The effects of NO can be classified as either cGMP-dependent or cGMP-independent. NO interacts with many different types of proteins however interactions with heme-containing proteins such as hemoglobin, cytochrome P450, ryanodine receptors, or guanylate cyclase (GC) are well-documented 4,6. Ryanodine receptors are sarcoplasmic reticulum calcium-release channels whereas GCs convert GTP to cGMP. The family of GCs is divided into soluble GCs and particulate GCs 7. NO is a potent activator of soluble GCs but not of particulate GC 7. Signal transduction by cGMP is dependent upon its synthesis by GCs, its targeting, and its degradation by cGMP-dependent phosphodiesterases (PDEs). Once produced, cGMP interacts with protein kinase G (PKG) to phosphorylate downstream proteins, many which have yet to be identified that activate a variety of cellular functions. The lacrimal gland is an exocrine gland responsible for producing the majority of the aqueous portion of the tear film 8. Acinar cells are the major cell type present in the lacrimal gland. In addition, myoepithelial and ductal epithelial cells are also present. The function of the lacrimal gland is to secrete water, electrolytes and protein, which maintain, nourish, and protect the cells of the cornea and conjunctiva. If lacrimal gland secretion is altered in either amount or composition, a spectrum of diseases called dry eye syndromes results. Therefore secretion from the lacrimal gland is tightly regulated. As a result, parasympathetic and sympathetic nerves extensively innervate the lacrimal gland. Stimulation of the afferent sensory nerves in the cornea triggers tear secretion through the efferent parasympathetic and sympathetic nerves that innervate the lacrimal gland 8. Receptors for these neurotransmitters are located on the basolateral side of the acinar cells8. Activation of these receptors initiates signal transduction pathways that culminate in secretion of proteins, electrolytes, and water across the apical membranes into the lumen and onto the cornea and conjunctiva. We have previously shown that cholinergic agonists released from parasympathetic nerves and α1-adrenergic agonists released from sympathetic nerves are potent stimuli of protein secretion from the lacrimal gland. The signal transduction pathways used by cholinergic agonists have been well characterized 8. These agonists bind to the M3 muscarinic receptor to activate Gαq/11, which in turn activates phospholipase C (PLC). PLC causes the production of inositol trisphosphate, which causes the release of intracellular Ca2+. Diacylglycerol is also produced by PLC, which activates protein kinase C (PKC)α,-δ, and -ɛ9. In contrast, the pathways utilized by α1-adrenergic agonists have remained elusive. We have also shown that α1-adrenergic receptors mediate both protein secretion and activation of p42/p44 MAPK 10. In spite of this information, the G protein(s) involved in transduction of this signal are unknown, though Meneray et al have shown that inhibition Gαq is responsible for approximately 32% of the α1-adrenergic agonist-induced protein secretion 11. The phospholipase involved has also not been identified though it is known that neither phospholipase C nor D are involved 12,13. It is known that phenylephrine (an α1-adrenergic agonist in the lacrimal gland) activates PKC-ɛ, which stimulates protein secretion and PKCα and –δ to inhibit secretion. We have also shown that α1-adrenergic agonists transactivate the EGF receptor, recruiting Shc and Grb2 leading to activation of p42/p44 MAPK. Activation of p42/p44 MAPK inhibits protein secretion and may act to attenuate overall protein secretion from the lacrimal gland or to terminate stimulated secretion. It is known that the lacrimal gland contains many of the proteins necessary for the production of NO and cGMP. nNOS has been shown to be present in the lacrimal gland surrounding ducts, blood vessels, and acinar cells 14,15. Exogenous addition of NO, through the use of NO donors, has been shown to increase total protein secretion, which is inhibited by guanylate cyclase inhibitors in cultured lacrimal gland acinar cells 3,16. In addition, NO donors which increase cGMP induced a rise in intracellular [Ca2+] ([Ca2+]i) through the release of Ca2+ from intracellular stores 17. Jorgensen et al demonstrated that α1-adrenergic agonist-induced increase in [Ca ]i was inhibited by inhibitors of GC and cGMP-dependent protein kinase Ia 18. Although it is not known if activation of muscarinic receptors increases NO, it is known that when total IgGs and purified autoantibodies against M3 muscarinic receptors from patients suffering from the autoimmune disease Sjogren’s syndrome were added to freshly prepared rat lacrimal gland slices, NOS activity was increased. This activity was mediated via the M3 muscarinic receptor as the M3 muscarinic inhibitor, 4-DAMP, inhibited NOS activity 19. Thus evidence supporting a role for α1-adrenergic, β-adrenergic, or muscarinic agonists in stimulating lacrimal gland protein secretion via the NO/cGMP pathway is lacking. In the present study, we confirm the presence of eNOS and nNOS in the lacrimal gland and demonstrate that the α1-adrenergic agonist phenylephrine, using α1D-adrenergic receptors, activates eNOS, but not nNOS, to increase NO production. NO, in turn, activates GC to increase cGMP levels. α1-Adrenergic agonist activation of the NO/cGMP pathway leads to an increase in protein secretion and p42/p44 MAPK induction from freshly isolated rat lacrimal gland acinar cells.