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G protein-coupled receptors in trigeminal ganglion (TG) neurons are often associated with sensory mechanisms, including nociception. We have previously reported the expression of P2Y12 receptors, which are Gi protein-coupled receptors, in TG cells. Activating P2Y12 receptors decreased the intracellular free Ca2+ concentration ([Ca2+]i). This indicated that intracellular adenosine 3′,5′-cyclic monophosphate (cAMP) levels can mediate Ca2+ signaling in TG cells. Here, we report more extensive-expression patterns of Gs protein-coupled receptors in primary cultured TG neurons isolated from 7-day-old newborn Wistar rats and further examine the roles of these receptors in cAMP signaling using the BacMam sensor in these neurons. To identify TG neurons, we also measured [Ca2+]i using fura-2 in TG cells and measured intracellular cAMP levels. TG neurons were positive for Gαs protein-coupled receptors, beta-2 adrenergic (β2), calcitonin gene-related peptide (CGRP), adenosine A2A (A2A), dopamine 1 (D1), prostaglandin I2 (IP), and 5-hydroxytriptamine 4 (5-HT4) receptor. Application of forskolin (FSK), an activator of adenylyl cyclase, transiently increased intracellular cAMP levels in TG neurons. The application of a phosphodiesterase inhibitor augmented the FSK-elicited intracellular cAMP level increase. These increases were significantly suppressed by the application of SQ22536, an adenylyl cyclase inhibitor, in TG neurons. Application of agonists for β2, CGRP, A2A, D1-like, IP, and 5-HT4 receptors increased intracellular cAMP levels. These increases were SQ22536-sensitive. These results suggested that TG neurons express β2, CGRP, A2A, D1, IP, and 5-HT4 receptors, and the activations of these Gαs protein-coupled receptors increase intracellular cAMP levels by activating adenylyl cyclase. [ABSTRACT FROM AUTHOR]

Odontoblasts are involved in sensory generation as sensory receptor cells and in dentin formation. We previously reported that an increase in intracellular cAMP levels by cannabinoid 1 receptor activation induces Ca2+ influx via transient receptor potential vanilloid subfamily member 1 channels in odontoblasts, indicating that intracellular cAMP/Ca2+ signal coupling is involved in dentinal pain generation and reactionary dentin formation. Here, intracellular cAMP dynamics in cultured human odontoblasts were investigated to understand the detailed expression patterns of the intracellular cAMP signaling pathway activated by the Gs protein-coupled receptor and to clarify its role in cellular functions. The presence of plasma membrane Gαs as well as prostaglandin I2 (IP), 5-hydroxytryptamine 5-HT4 (5-HT4), dopamine D1 (D1), adenosine A2A (A2A), and vasoactive intestinal polypeptide (VIP) receptor immunoreactivity was observed in human odontoblasts. In the presence of extracellular Ca2+, the application of agonists for the IP (beraprost), 5-HT4 (BIMU8), D1 (SKF83959), A2A (PSB0777), and VIP (VIP) receptors increased intracellular cAMP levels. This increase in cAMP levels was inhibited by the application of the adenylyl cyclase (AC) inhibitor SQ22536 and each receptor antagonist, dose-dependently. These results suggested that odontoblasts express Gs protein-coupled IP, 5-HT4, D1, A2A, and VIP receptors. In addition, activation of these receptors increased intracellular cAMP levels by activating AC in odontoblasts. [ABSTRACT FROM AUTHOR]

An inflammatory response following dental pulp injury and/or infection often leads to neurogenic inflammation via the axon reflex. However, the detailed mechanism underlying the occurrence of the axon reflex in the dental pulp remains unclear. We sought to examine the intracellular cyclic adenosine monophosphate (cAMP) signaling pathway in odontoblasts via the activation of Gs protein-coupled receptors and intercellular trigeminal ganglion (TG) neuron–odontoblast communication following direct mechanical stimulation of TG neurons. Odontoblasts express heterotrimeric G-protein α-subunit Gαs and calcitonin receptor-like receptors. The application of an adenylyl cyclase (AC) activator and a calcitonin gene-related peptide (CGRP) receptor agonist increased the intracellular cAMP levels ([cAMP]i) in odontoblasts, which were significantly inhibited by the selective CGRP receptor antagonist and AC inhibitor. Mechanical stimulation of the small-sized CGRP-positive but neurofilament heavy chain-negative TG neurons increased [cAMP]i in odontoblasts localized near the stimulated neuron. This increase was inhibited by the CGRP receptor antagonist. In the mineralization assay, CGRP impaired the mineralization ability of the odontoblasts, which was reversed by treatment with a CGRP receptor antagonist and AC inhibitor. CGRP establishes an axon reflex in the dental pulp via intercellular communication between TG neurons and odontoblasts. Overall, CGRP and cAMP signaling negatively regulate dentinogenesis as defensive mechanisms. [ABSTRACT FROM AUTHOR]

Bradykinin (BK) and its receptors, B.sub.1 and B.sub.2, in trigeminal ganglion (TG) neurons are involved in the regulation of pain. Recent studies have revealed that B.sub.1 receptors are expressed in neonatal rat TG neurons; however, the intracellular signaling pathway following B.sub.1 receptor activation remains to be elucidated. To investigate the mechanism by which B.sub.1 receptor activation leads to intracellular Ca.sup.2+ mobilization, we measured the intracellular free Ca.sup.2+ concentration ([Ca.sup.2+].sub.i) in primary-cultured TG neurons. The application of Lys-[Des-Arg.sup.9]BK (B.sub.1 receptor agonist) increased the [Ca.sup.2+].sub.i in these TG neurons even in the absence of extracellular Ca.sup.2+. Pretreatment with inhibitors of ryanodine receptors or sarco/endoplasmic reticulum Ca.sup.2+-ATPase suppressed the increase in Lys-[Des-Arg.sup.9]BK-induced [Ca.sup.2+].sub.i. The Lys-[Des-Arg.sup.9]BK-induced [Ca.sup.2+].sub.i increase was unaffected by phospholipase-C inhibitor. B.sub.1 receptor activation-induced [Ca.sup.2+].sub.i increase was suppressed by phosphodiesterase inhibitor and enhanced by adenylyl cyclase inhibitor. These results suggest that B.sub.1 receptor activation suppresses intracellular cAMP production via adenylyl cyclase inhibition and mobilizes intracellular Ca.sup.2+ via ryanodine receptors that access intracellular Ca.sup.2+ stores. Keywords: Adenylyl cyclase, Ca.sup.2+ mobilization, Cyclic AMP, G protein, Ryanodine receptors, Trigeminal ganglion neuron, Author(s): Reiko Terashima[sup.1,2], Maki Kimura[sup.2], Asuka Higashikawa[sup.2], Yuki Kojima[sup.2], Tatsuya Ichinohe[sup.1], Masakazu Tazaki[sup.2] and Yoshiyuki Shibukawa[sup.2] Introduction Bradykinin (BK) and related peptides play important roles in the modulation of physiological [...]