Lysophosphatidic acid stimulates calcium transients in enteric glia.

The enteric nervous system plays an integral role in the gastrointestinal tract. Within this intricate network, enteric glia are crucial in the maintenance of normal bowel function, yet their signaling mechanisms are poorly understood. Enteric glia, and not enteric neurons, selectively responded to lysophosphatidic acid (LPA), a product of phosphatidylcholine metabolism, with dose-dependent calcium (Ca(2+)) signaling over a range from 100 pM to 10 microM. The elicited calcium transients involved both the mobilization of intracellular Ca(2+) stores and the influx of extracellular Ca(2+) as LPA signals were obliterated following the depletion of intracellular Ca(2+) and attenuated by the removal of Ca(2+) from the perfusion buffer. Pretreatment with pertussis toxin (100 ng/ml) reduced the magnitude of LPA Ca(2+) transients (95+/-20 nM vs 168+/-17 nM for controls). Repetitive exposure yielded diminished responsiveness, with a 25% reduction in [Ca(2+)](i) between first and second exposures. Inhibition of the inositol 1,4,5-trisphosphate (IP(3)) receptor with 200 microM 2-aminoethoxydiphenylborate (2APB) abolished LPA signals. RT-PCR analysis demonstrated the presence of two LPA-coupled endothelial differentiation gene (EDG) receptor mRNAs (EDG-2 and EDG-7) in myenteric plexus primary cultures. EDG-2 expression in glial cells of the ENS was confirmed immunocytochemically.

Stimulation of rat pancreatic exocrine secretion by cocaine- and amphetamine-regulated transcript peptide.

Cocaine- and amphetamine-regulated transcript (CART) peptide is a recently described neuropeptide that has been localized to areas of the central and peripheral nervous systems. CART has been shown to be involved in feeding behavior when injected centrally, however, its effects upon peripheral tissues have not been studied. This report describes the effects of CART peptide on rat pancreatic exocrine secretion. Infusion of CART peptide caused four-fold increases in amylase secretion from anesthetized rats that had been fashioned with a bile-pancreatic duct cannula. CART peptide-induced increases in pancreatic secretion appear to involve pathways that are sensitive to both acetylcholine (ACh) and cholecystokinin (CCK) since pre-treatment with atropine (ACh receptor antagonist) or L-364,718 (CCK-A receptor antagonist) inhibited the effects of CART peptide on amylase secretion. Pre-treatment with a combination of atropine and L-364,718 abolished the effects of CART peptide. When isolated rat pancreatic acini were exposed to varying doses of CART peptide, no increase in amylase secretion was observed. The results of the present study suggest that CART peptide has stimulatory effects upon pancreatic exocrine secretion. CART peptide-induced increases in pancreatic secretion appear to be indirectly mediated as no direct effect upon pancreatic acini was shown. CART peptide likely acts upon either peripheral or central regulators of pancreatic secretory function that are distant from the acinar unit.

Cholinergic stimulation of rat acinar cells increases c-fos and c-jun expression via a mitogen-activated protein kinase-dependent pathway.

Acetylcholine release from cholinergic neurons regulates pancreatic exocrine function through pathways that are still under investigation. Pancreatic AR42J acinar cells were studied to determine intracellular calcium ([Ca(2+)](i)) release, enzyme activation, and gene expression in response to the acetylcholine analog carbachol (CCh). CCh stimulated dose-dependent increases in [Ca(2+)](i) that were inhibited by atropine and by specific inhibitors to the muscarinic receptor subtypes m1 and m3. Polymerase chain reaction analysis was performed, which sequenced products corresponding to the m1 and m3 receptor subtypes but not the m2 subtype. CCh also stimulated mitogen-activated protein kinase activity. CCh induced time-and dose-dependent increases in the c-fos and c-jun early-response genes, which were blocked by m1 and m3 inhibition but not by m2 inhibition.

Cholinergic intrapancreatic neurons induce Ca²+ signaling and early-response gene expression in pancreatic acinar cells.

Pancreatic exocrine function has been demonstrated to be under neuronal regulation. The pathways responsible for this effect, and the long-term consequences of such interactions, are incompletely described. The effects of neuronal depolarization on pancreatic acinar cells were studied to determine whether calcium signaling and c-fos expression were activated. In pancreatic lobules, which contain both neurons and acinar cells, agonists that selectively stimulated neurons increased intracellular calcium in acinar cells. Depolarization also led to the expression of c-fos protein in 24% +/- 4% of the acinar cells. In AR42J pancreatic acinar cells, cholinergic stimulation demonstrated an average increase of 398 +/- 19 nmol/L in intracellular calcium levels, and induced c-fos expression that was time and dose dependent. The data indicate that intrapancreatic neurons induce Ca²+ signaling and early-response gene expression in pancreatic acinar cells.

ATP induces c-fos expression in C6 glioma cells by activation of P(2Y) receptors.

BACKGROUND: Extracellular ATP functions in the enteric nervous system as a neurotransmitter, and recent evidence suggests ATP may regulate development through effects on cellular proliferation. METHODS: The action of ATP at purinoceptors and the role of second messenger pathways in c-fos mRNA expression in C6 glioma cells were investigated using the techniques of Northern and Western blotting. RESULTS: Treatment of C6 cells with ATP caused a time- and dose-dependent increase in c-fos expression. The rank order of agonist potency was ATP = ADP > gammasATP > alphabetaATP > betagammaATP > AMP = UTP. The ATP-induced c-fos increment was inhibited by three P(2Y) receptor antagonists-suramin, reactive blue, and DIDS-by 99+/-3, 89+/-7, and 61+/-14%, respectively. The ATP-stimulated c-fos expression was attenuated by phospholipase C inhibitor (U73122), protein kinase C (PKC) down-regulation (4alpha-phorbol 12-myristate 13-acetate and chelerythrine), mitogen-activated protein (MAP) kinase inhibition (apigenin), an inhibitor of MAP kinase kinase (PD98059), down-regulation of adenylate cyclase (SQ22536), and inhibition of type II protein kinase A (8-(4-chlorophenylthio)adenosine-3',5'-cyclic monophosphorothioate), but was not affected by inhibition of type I protein kinase A (8-bromoadenosine-3',5'-cyclic monophosphorothioate) and inhibitors of calmodulin kinase (KN93 and KN62). Phosphorylated MAP kinase was increased in cells exposed to ATP. This effect was suppressed by chelerythrine. CONCLUSIONS: These studies demonstrate that ATP-induced c-fos mRNA expression is under multifactorial regulation.

Functional overlap of IP(3)- and cADP-ribose-sensitive calcium stores in guinea pig myenteric neurons.

In myenteric neurons two different receptor subtypes govern the intracellular Ca(2+) stores: the inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R) and the ryanodine receptor (RyR). Their degree of functional overlap was determined by examining Ca(2+) release in these cells through both superfusion techniques and intracellular microinjection. Microinjection of IP(3) (50 microM) and cADP-ribose (cADPr, 50 microM), specific ligands for the IP(3)R and RyR, respectively, demonstrated mobilization of intracellular Ca(2+) stores. Perfusion with cinnarizine (50 microM) or dantrolene (10 microM), antagonists of the IP(3)R and RyR, respectively, eliminated the Ca(2+) response to microinjected IP(3) and cADPr. Superfusion of the neurons with 100 microM ATP, an IP(3)-mediated Ca(2+)-mobilizing agonist, caused intracellular Ca(2+) increments, which were antagonized by cinnarizine, and the RyR antagonists dantrolene, procaine (5 mM), and ryanodine (1 microM). Caffeine (10 mM) was applied repetitively in Ca(2+)-free conditions to deplete RyR-sensitive stores; subsequent perfusion with ATP demonstrated a Ca(2+) response. Conversely, caffeine caused a Ca(2+) response after repetitive ATP exposures. The internal Ca(2+) stores of myenteric neurons are governed by two receptor subtypes, IP(3)R and RyR, which share partial functional overlap.

Inhibition of pancreatic protein secretion by ghrelin in the rat.

1. The role of ghrelin in the regulation of pancreatic protein secretion was investigated in vivo using anaesthetized rats with pancreatic ductal cannulas, and in isolated pancreatic acinar cells and pancreatic lobules in vitro. 2. In vivo, pancreatic protein output stimulated by CCK-8 (400 pmol kg(-1) h(-1)) was dose-dependently inhibited by continuous ghrelin infusion (1.2 and 12 nmol kg(-1) h(-1)) by 45 +/- 8 and 84 +/- 7 %, respectively. 3. In rats with acute subdiaphragmatic vagotomy, ghrelin (12 nmol kg(-1) h(-1)) significantly inhibited CCK-stimulated pancreatic protein secretion by 75 +/- 18 %. 4. Infusion of ghrelin (12 nmol kg(-1) h(-1)) abolished pancreatic protein secretion caused by the central vagal stimulant 2-deoxy-D-glucose (75 mg kg(-1)), whereas bethanechol-stimulated pancreatic protein output was inhibited by only 59 +/- 7 %. 5. In vitro, ghrelin (10(-11)-10(-7) M) produced no change in basal amylase release from dispersed, purified acinar cells. Co-incubation of ghrelin (10(-11)-10(-7) M) with CCK-8 (10(-10) M) demonstrated no inhibition of CCK-stimulated amylase release from dispersed acini. In contrast, ghrelin (10(-9)-10(-7) M) dose-dependently inhibited amylase release from pancreatic lobules exposed to 75 mM potassium. 6. Our results show that (1) ghrelin is a potent inhibitor of pancreatic exocrine secretion in anaesthetized rats in vivo and in pancreatic lobules in vitro; and (2) the actions of ghrelin are indirect and may be exerted at the level of intrapancreatic neurons.

Sphingosine-1-phosphate mediates calcium signaling in guinea pig enteroglial cells.

The enteric nervous system, which regulates multiple aspects of digestive activity, is composed of two major cell types, neurons and glial cells. Enteric glia, but not enteric neurons, respond to bioactive lipids with calcium signaling. The sphingomyelin metabolite sphingosine-1-phosphate (S1P) caused dose-dependent calcium (Ca(2+)) signaling using extracellular and intracellular Ca(2+). The signal transduction cascade was pertussis toxin-insensitive and involved an extracellular receptor since repetitive exposure yielded diminished responsiveness. Inhibition of either phospholipase C or the inositol 1,4,5-trisphosphate receptor abolished S1P effects. RT-PCR analysis demonstrated the presence of S1P-coupled endothelial differentiation gene (EDG) receptor mRNAs (EDG-1, EDG-3, and EDG-5) within the enteric nervous system. Immunocytochemical analysis demonstrated strong expression of both EDG-1 and EDG-3 and weak expression of EDG-5 in enteric glial cells. Other sphingomyelin cycle components, including sphingomyelin, sphingomyelinase, and sphingosine caused Ca(2+) transients in enteric glia. Related lipids lysophosphatidic acid and sphingosylphosphorylcholine also induced Ca(2+) signaling in enteric glia, suggesting that multiple lipid-activated signaling mechanisms exist in these cells.