Abstract
Salivary glands are innervated by sympathetic and parasympathetic neurons, which release neurotransmitters that promote fluid secretion and exocytosis when they bind to muscarinic and beta-adrenergic receptors, respectively. Signaling pathways downstream of these receptors are mainly distinct, but there is cross-talk that affects receptor-dependent events. Here we report that the beta-adrenergic ligand isoproterenol blocks increases in extracellular signal-related kinase (ERK) phosphorylation, a protein kinase C-dependent event promoted by the muscarinic receptor ligand carbachol in freshly dispersed rat parotid acinar cells. The inhibitory action of isoproterenol was reproduced by cAMP stimuli (forskolin) and mimetics (dibutyryl-cAMP, 8-(4-chlorophenylthio)-cAMP), including one highly selective for protein kinase A (N(6)-benzoyl-cAMP). In contrast, Epac (exchange proteins directly activated by cAMP)-selective activators did not mimic the blockade of ERK by isoproterenol, suggesting that inhibition involved protein kinase A. Isoproterenol also blocked ERK downstream of phorbol 12-myristate 13-acetate and the P2X(7) and epidermal growth factor receptors. Isoproterenol and forskolin blocked MEK phosphorylation, reduced RAF phosphorylation on a stimulatory site (Ser-338), and increased RAF phosphorylation on an inhibitory site (Ser-259). Inhibitory effects on ERK were also observed in freshly dispersed rat submandibular acinar cells but not in three immortalized/cancer salivary cell lines (Par-C10, HSY, HSG), indicating significant differences between native cells and cell lines. Notably, in native parotid cells isoproterenol enhanced the carbachol-promoted increases in [Ca(2+)](i) and oxygen consumption, events that initiate and accompany, respectively, the stimulation of fluid secretion by muscarinic ligands. Thus, isoproterenol produces opposite effects on prominent events downstream of the muscarinic receptor second messengers diacylglycerol (decrease in ERK phosphorylation) and inositol trisphosphate (increase in [Ca(2+)](i) and fluid secretion).
Highlights
Grant DE-10877 (NIDCR). 1 To whom correspondence should be addressed: Beth Israel Deaconess and -adrenergic receptor are G-protein-coupled receptors that affect the physiological activities of many cells, and neurotransmitters binding to these receptors produce well defined and mainly separate functional effects in salivary glands, which are innervated by both sympathetic and parasympathetic neurons [1]
We examined the effects of isoproterenol on multiple physiological indices of fluid secretion as well as on Extracellular signal-related kinase (ERK) phosphorylation downstream of P2X7 and epidermal growth factor (EGF) receptors, neither of which is coupled to G-proteins
Isoproterenol Rapidly Blocks ERK Phosphorylation Promoted by Muscarinic Stimulation—Because parotid acini are innervated by both sympathetic and parasympathetic nerves and because there is a history of functional interactions between muscarinic and -adrenergic receptors in parotid glands, we evaluated the effects of isoproterenol itself on the activation of ERK as well as its effects on the activation of ERK by carbachol in rat parotid acinar cells
Summary
M3R, muscarinic M3 receptor; 6-Bnz-cAMP, N6-benzoyl-cAMP; 8-Br-cAMP, 8-bromo-cAMP; BzATP, 2Ј(3Ј)-O-(4-benzoylbenzoyl)adenosine 5Ј-triphosphate; [Ca2ϩ]i, intracellular calcium concentration; 8-CPT-cAMP, 8-(4-chlorophenylthio)-2Ј-O-cAMP; 8-CPT-2Ј-Me-cAMP, 8-(4-chlorophenylthio)-2Ј-O-methyl-cAMP; EGF, epidermal growth factor; EGFR, EGF receptor; Epac, exchange proteins directly activated by cAMP; ERK, extracellular signal-related kinase; H-89, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide; InsP3, inositol 1,4,5-trisphosphate; MEK, mitogen-activated protein kinase/ERK kinase; 8-pMeOPT-2Ј-O-MecAMP, 8-(4-methoxyphenylthio)-2Ј-O-methyl-cAMP; PKA, protein kinase A; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; QO2, oxygen consumption rate; Rp-cAMPS, Rp-adenosine-3Ј,5Ј-cyclic monophosphothioate. Isoproterenol and the adenylyl cyclase activator forskolin increased the release of Ca2ϩ from intracellular stores by the muscarinic and ␣-adrenergic receptor stimulation in rodent parotid acinar cells, and this was due to the phosphorylation of the InsP3 receptor in a PKA-dependent manner (14 –16). This offers an explanation for the stimulatory effect of cAMP production/PKA activation on fluid secretion in parotid and submandibular glands [17, 18] and other epithelial glands [19]. The results indicate that isoproterenol produces both positive and negative effects on muscarinic signaling within the same cell and can block ERK activation downstream of multiple types of receptors
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
