Abstract. Glutamate (Glu) is the major excitatory neurotransmitter in the central nervous system. The role of peripheral Glu and Glu receptors (GluRs) in nociceptive transmission is, however, still unclear. In the present study, we examined Glu levels released in the subcutaneous perfusate of the rat hind instep using a microdialysis catheter and the thermal withdrawal latency using the Plantar Test following injection of drugs associated with GluRs with / without capsaicin into the hindpaw. The injection of capsaicin into the rat hind instep caused an increase of Glu level in the s.c. perfusate. Capsaicin also significantly decreased withdrawal latency to irradiation. These effects of capsaicin were inhibited by pretreatment with capsazepine, a transient receptor potential vanilloid receptor 1 (TRPV1) competitive antagonist. Capsaicin-induced Glu release was also suppressed by combination with each antagonist of ionotropic GluRs (iGluRs: NMDA / AMPA receptors) and group I metabotropic GluR (mGluR), but not group II and group III mGluRs. Furthermore, these GluRs antagonists showed remarkable inhibition against capsaicin-induced thermal hyperalgesia. These results suggest that Glu is released from the peripheral endings of small-diameter afferent fibers by noxious stimulation and then activates peripheral iGluRs and group I mGluR in development and/ or maintenance of nociception. Furthermore, the activation of peripheral NMDA / AMPA receptors and group I mGluR may be important in mechanisms whereby capsaicin evokes nociceptive responses.

Prostaglandin I2 (PGI2, prostacyclin), an eicosanoid of the cyclooxygenase pathway, causes relaxation of vascular smooth muscle in most blood vessels and inhibits platelet aggregation. PGI2 and its stable analogues activate a specific cell-surface receptor (IP receptor, IPR), which is coupled to adenylyl cyclase through G(s)-protein. Elevation of 3': 5'-cyclic monophosphate (cyclic AMP, cAMP) levels has been considered to be a key cellular event to trigger blood vessel relaxation by IP agonists; however, its exclusive role has been recently challenged. Downstream effectors of the IP agonist metabolic cascade are plasma membrane K+ channels that upon activation would cause smooth muscle cell hyperpolarization and relaxation. The K+ channel candidates include ATP-sensitive K+ (KATP) channel and large conductance, Ca2+ -activated K+ (MaxiK, BK) channel. The contribution of each K+ channel subtype would be governed by their relative expression and/or particular co-localization with different proteins of the IPR signaling cascade in each vascular bed. Scrutiny of the cellular mechanisms underlying IPR-activated vascular relaxation of a large conduit artery revealed that relaxation by an IP agonist, beraprost, is elicited through cAMP-independent pathway as well as by a cAMP-dependent route. Both mechanisms include activation of MaxiK channels. The cAMP-independent vasorelaxant mechanism is partly attributed to a direct activation of MaxiK channel by G(s)-protein. In this review article, we discuss cAMP-dependent and -independent mechanisms by which IPR stimulation activates MaxiK channel. Our recent work demonstrates a functional tight coupling between IPR and MaxiK channel through a cAMP-independent, G(s)-protein mediated mechanism(s) in vascular smooth muscle.

The present study was aimed to elucidate the cellular pathway(s) controlling vascular relaxation triggered by stimulation of prostaglandin I2 (PGI2, IP) receptor with a stable PGI2 analog, beraprost. Beraprost caused a concentration-dependent relaxation in de-endothelialized guinea-pig aorta contracted with prostaglandin F2alpha (PGF2alpha). Beraprost-induced relaxation was almost abolished in high-KCl-contracted tissue, indicating a major role of K+ conductances. In contrast to other PGI2 analogs (e.g. cicaprost and iloprost), beraprost-induced relaxation was practically abolished by a selective voltage and Ca2+-activated K+ (MaxiK, BK) channel blocker Iberiotoxin (10(-7) M) or by tetraethylammonium (2 x 10(-3) M). The relaxation induced by beraprost was not significantly affected by other K+ channel blockers glibenclamide (10(-6) M) or Ba2+ (10(-5) M), but was slightly attenuated by 4-aminopyridine (10(-4) M). Beraprost increased intracellular cyclic AMP levels, suggesting a role for cyclic AMP-dependent pathways. A selective inhibitor of cyclic AMP-specific phosphodiesterase, RO-20-1724 (10(-4) M), significantly potentiated beraprost-induced relaxation. Iberiotoxin (10(-7) M) completely counteracted this potentiation. Moreover, tension decrement due to forskolin (3 x 10(-7) M) or 8-bromo-cyclic AMP (10(-2) M) was thoroughly restored by Iberiotoxin (10(-7) M), confirming a role for a cyclic AMP-dependent mechanism. However, SQ 22,536 (10(-4) M), an adenylyl cyclase inhibitor, did not affect beraprost-induced relaxation though it almost totally inhibited the elevation of cyclic AMP contents induced by beraprost, suggesting the existence of an additional mechanism that is cyclic AMP-independent. Moreover, cholera toxin (CTX, 1 microg/ml for 6 h), which activates the stimulatory G protein of adenylyl cyclase (Gs), significantly suppressed PGF2alpha-induced contraction both in the absence and presence of SQ 22,536 (10(-4) M). Iberiotoxin (10(-7) M) was also capable of restoring the relaxation induced by CTX. These findings suggest that MaxiK channel plays a primary role in mediating smooth muscle relaxation following stimulation of IP receptor with beraprost in guinea-pig aorta. Both cyclic AMP-dependent and -independent pathways contribute to the MaxiK channel-mediated relaxation following IP receptor stimulation in this vascular tissue. Direct regulation of MaxiK channels by Gs may partly account for the cyclic AMP-independent relaxant mechanism.

We examined the contribution of large-conductance, Ca 2+ -sensitive K + (MaxiK) channel to β2-adrenoceptor-activated relaxation to isoprenaline in guinea-pig tracheal smooth muscle focusing on the role for cAMP in the coupling between β2-adrenoceptor and MaxiK channel. Isoprenaline-elicited relaxation was confirmed to be mediated through β2-type of adrenoceptor since the response was antagonized in a competitive fashion by a β2-selective adrenoceptor antagonist butoxamine with a pA2 value of 6.56. Isoprenaline-induced relaxation was significantly potentiated by a selective inhibitor of cyclic AMP-specific phosphodiesterase, Ro-20-1724 (0.1-1 µM). cAMP-dependent mediation of MaxiK channel in the relaxant response to isoprenaline was evidenced since the potentiated response to isoprenaline by the presence of Ro-20-1724 (1 µM) was inhibited by the channel selective blocker, iberiotoxin (IbTx, 100 nM). This concept was supported by the finding that the relaxation to a membrane permeable cAMP analogue, 8-bromo-cAMP (1 mM), was susceptible to the inhibition by IbTx. On the other hand, isoprenaline-induced relaxation was not practically diminished by an adenylyl cyclase inhibitor SQ 22,536 (100 µM). However, isoprenaline-induced relaxation in the presence of SQ 22,536 was suppressed by IbTx. Characteristics of isoprenaline-induced relaxant response, i.e., impervious to SQ 22,536 but susceptible to IbTx, were practically mimicked by cholera toxin (CTX, 5 µg/ml), an activator of adenylyl cyclase coupled-heterotrimeric guanine nucleotide-binding regulatory protein Gs. These findings indicate that in guinea-pig tracheal smooth muscle: 1) MaxiK channel substantially mediates β2-adrenoceptor-activated relaxation; 2) both cAMP-dependent andindependent mechanisms underlie the functional coupling between β2-adrenoceptor and MaxiK channel to induce muscle relaxation; and 3) direct regulation of MaxiK channel by Gs operates in cAMP-independent coupling between β2-adrenoceptor and this ion channel.

Isoprenaline is known to produce vascular relaxation through activation of β-adrenoceptors. In recent years, β-adrenoceptor-activated vascular relaxation has been the focus of pharmacological study in terms of both the receptor subtypes and the intracellular signaling mechanisms which trigger smooth muscle mechanical functions. In addition, the possible contribution of the endothelium to β-adrenoceptor-activated relaxation of vascular beds has provoked considerable discussion, with consensus still to be established. In the present study, we examined the effects of isoprenaline on isolated mouse aortic smooth muscles to determine whether the presence of the endothelium plays a substantial role in the relaxation it produces. A possible role for nitric oxide (NO) as a primary endothelium-derived factor released in response to isoprenaline was also elucidated pharmaco-mechanically. In isolated thoracic and abdominal aortae precontracted with phenylephrine (3 × 10 -7 -10 -6 M), isoprenaline elicited relaxation in a concentration-dependent fashion (10 -9 -10 -5 M). In endothelium-denuded preparations, isoprenaline-elicited relaxation was reduced to 40~50% of the response obtained in endothelium-intact preparations. In the preparations treated with N G -nitro-L-arginine methyl ester (L-NAME, 3 × 10 -4 M; an NO synthase inhibitor) or 1H-[1,2,4]-oxadiazolo-[4,3-a]-quinoxalin-1-one (ODQ, 10 -5 M; a soluble guanylyl cyclase inhibitor), isoprenalineelicited relaxation was attenuated almost to the same degree as the response in endothelium-denuded preparations. The degree of endothelium-dependency in isoprenaline-elicited relaxation was largely diminished when treated with propranolol (3 × 10 -6 M). The present findings indicate that isoprenaline substantially relaxes the mouse aorta with both endothelium-dependent and -independent mechanisms. The endothelium-dependent component seems to correspond to about 50% of the isoprenaline-elicited relaxation, and is almost entirely due to endothelium-derived NO. Activation of propranolol (3 × 10 -6 M)-inhibitable β-adrenoceptors seems to be primarily responsible for the NO-mediated endothelium-dependent pathway in isoprenaline-elicited Correspondence to: