Lipopolysaccharides enhance the action of bradykinin in enteric neurons via secretion of interleukin-1beta from enteric glial cells.

Functional changes of the enteric nervous system have been observed under inflammatory states of inflammatory bowel disease increasing the endotoxin level. The aim of the present study was to determine the effect of lipopolysaccharides (LPS) on myenteric neuron-glia interaction in vitro. We examined the increase of the intracellular Ca(2+) concentration ([Ca(2+)](i)) and the release of interleukin-1beta (IL-1beta) or prostaglandin E(2) (PGE(2)) and COX-2 expression in myenteric plexus cells from the rat intestine induced by LPS. LPS potentiated BK-induced [Ca(2+)](i) increases in both myenteric neurons and enteric glial cells, which were suppressed by a B1R antagonist. Only in enteric glial cells, a B1R agonist increased [Ca(2+)](i). The effects of LPS were blocked by pretreatment with an interleukin-1 receptor antagonist or by reducing the density of enteric glial cells in culture. LPS prompted the release of IL-1beta from enteric glial cells. The augmenting effects of IL-1beta on the BK-induced neural [Ca(2+)](i) increase and PGE(2) release from enteric glial cells were abolished by a phospholipase A(2) (PLA(2)) inhibitor and a COX inhibitor, and partly suppressed by a COX-2 inhibitor. IL-1beta up-regulated the COX-2 expression in enteric glial cells. LPS promotes IL-1beta secretion from enteric glial cells, resulting in augmentation of the neural response to BK through PGE(2) release via glial PLA(2) and COX-2. The alteration of the regulatory effect of glial cells may be the cause of the changes in neural function in the enteric nervous system in inflammatory bowel disease.

Potentiation of transient receptor potential V1 functions by the activation of metabotropic 5-HT receptors in rat primary sensory neurons.

5-Hydroxytryptamine (5-HT) is one of the major chemical mediators released in injured and inflamed tissue and is capable of inducing hyperalgesia in vivo. However, the cellular mechanisms of 5-HT-induced hyperalgesia remain unclear. Transient receptor potential V1 (TRPV1) plays a pivotal role in nociceptive receptors. In the present study, we determined whether 5-HT changes TRPV1 functions in cultured dorsal root ganglion (DRG) neurons isolated from neonatal rats, using Ca(2+) imaging and whole-cell patch-clamp techniques. In more than 70% of DRG neurons, 5-HT potentiated the increases of [Ca(2+)](i) induced by capsaicin, protons and noxious heat. Capsaicin-induced current and depolarizing responses, and proton-induced currents were also augmented by 5-HT. RT-PCR analysis revealed the expression of 5-HT(2A) and 5-HT(7) receptors in rat DRG neurons. Agonists for 5-HT(2A) and 5-HT(7) receptors mimicked the potentiating effect of 5-HT, and their antagonists decreased it. In DRG ipsilateral to the complete Freund's adjuvant-injected inflammation side, expression levels of 5-HT(2A) and 5-HT(7) mRNAs increased, and the potentiating effect of 5-HT was more prominent than in the contralateral control side. These results suggest that the PKC- and PKA-mediated signalling pathways are involved in the potentiating effect of 5-HT on TRPV1 functions through the activation of 5-HT(2A) and 5-HT(7) receptors, respectively. Under inflammatory conditions, the increases of the biosynthesis of these 5-HT receptors may lead to further potentiation of TRPV1 functions, resulting in the generation of inflammatory hyperalgesia in vivo.

P2X2 receptors are essential for [Ca2+]i increases in response to ATP in cultured rat myenteric neurons.

We characterized ATP-induced changes in intracellular Ca2+ concentration ([Ca2+]i) and membrane current in cultured rat myenteric neurons using ratiometric Ca2+ imaging with fura-2 and the whole cell patch-clamp technique, respectively. Neuronal cells were functionally identified by [Ca2+]i responses to high K+ and nicotine, which occurred only in cells positive for neuron-specific protein gene product 9.5 immunoreactivity. ATP evoked a dose-dependent increase of [Ca2+]i that was greatly decreased by the removal of extracellular Ca2+ concentration ([Ca2+]o). The amplitude of the [Ca2+]i response to ATP was reduced by half in the presence of voltage-dependent Ca2+ channel blockers. In [Ca2+]o-free solution, ATP produced a small transient rise in [Ca2+]i similar to that induced by P2Y agonists. At -60 mV, ATP evoked a slowly inactivating inward current that was suppressed by the removal of extracellular Na+ concentration. The current-voltage relation for ATP showed an inward rectification with the reversal potential of about 0 mV. The apparent rank order of potency for the purinoceptor agonist-induced increases of [Ca2+]i was ATP > or = adenosine 5'-O-3-triphosphate > or = CTP > or = 2-methylthio-ATP > benzoylbenzoyl-ATP. A similar potency order was obtained with current responses to these agonists. P2 antagonists inhibited inward currents induced by ATP. Ca2+ and Mg2+ suppressed the ATP-induced current, and Zn2+, Cu2+, and protons potentiated it. RT-PCR and immunocytochemical studies showed the expression of P2X2 receptors in cultured rat myenteric neurons. These results suggest that ATP mainly activates ionotropic P2X2 receptors, resulting in a [Ca2+]i increase dependent on [Ca2+]o in rat myenteric neurons. A small part of the ATP-induced [Ca2+]i increase may be also mediated via a P2Y receptor-related mechanism.