Abstract
Purpose: :
Carbachol (a cholinergic agonist), phenylephrine (an alpha–1 adrenergic agonist) and epidermal growth factor (EGF) stimulate MAPK activity in the lacrimal gland. In this study, we determined the effect of increasing the cellular level of cyclic AMP on MAPK activity in lacrimal gland.
Methods: :
Both exorbital lacrimal glands were removed from male Sprague–Dawley rats. Acini were isolated by collagenase digestion in Krebs–Ringer bicarbonate (KRB) buffer at 37 0C. Cellular cAMP levels were increased by using the cell permeant cAMP analogs, dibutyryl cAMP (dbcAMP) and 8–pCPT–2Me–cAMP. The former activates protein kinase A (PKA) and Epac a guanine–nucleotide exchange factor; the latter specifically activates Epac. Acini were incubated with increasing concentrations of dbcAMP or 8pCPT–2Me–cAMP for 0–60 min. In addition, acini were preincubated with or without increasing concentrations of H89, a specific inhibitor of PKA, for 10 min and then with dbcAMP (10–3M) for 30 min prior to stimulation for 5 min with carbachol (10–4M), phenylephrine (10–4M) or EGF (10–7M). Acini were sonicated in RIPA buffer and western blot analysis were performed by using primary antibodies specific to phosphorylated (activated) p42/44 MAPK, total p42–MAPK. Phosphorylated cyclic AMP response binding protein (CREB) and total CREB were also detected as an indicator of cAMP activity. The films were digitally scanned and analyzed with NIH Image software. Epac proteins (Epac1 and Epac2) were detected by western blotting using Epac and Epac2 specific antibodies.
Results: :
DbcAMP (10–3M) inhibited basal MAPK activity a maximum of 40 % during a 30 min incubation, as well as carbachol– and EGF–stimulated MAPK activity by about 50 % H89 (10–4M) significantly reversed the dbcAMP–induced inhibition of basal and agonist–stimulated MAPK activity. CREB activity was increased 1.8 fold by dbcAMP at 10–30 min of incubation and this was prevented by H89 (10–4M). Epac proteins were expressed in rat lacrimal gland acinar cells. In contrast to dbcAMP, 8–pCPT–2Me–cAMP (10–4M) incubated for 30 min induced a 1.4 fold increase in MAPK activity over basal.
Conclusions: :
In the lacrimal gland, cAMP activates PKA to inhibit the MAPK pathway and, in contrast, activates Epac to stimulate MAPK activity.
Keywords: lacrimal gland • signal transduction • neurotransmitters/neurotransmitter systems