Kazuichi Sakamoto

Suppression of Prostaglandin E Receptor Signaling by the Variant Form of EP1 Subtype

A cDNA clone of prostaglandin (PG) E receptor EP1 subtype (rEP1) was isolated from a rat uterus cDNA library. It encodes 405 amino acid residues with seven transmembrane-spanning domains and couples to Ca2+ mobilization. In addition, three cDNA clones encoding a variant form of rEP1 were isolated. The open reading frame can code a 366-amino acid protein carrying a specific change of 49 amino acids from the middle of transmembrane segment VI to COOH terminus; it possesses a transmembrane segment VII-like structure lacking an intracellular COOH-terminal tail. Southern blot analysis of rat genomic DNA and genomic polymerase chain reaction demonstrated that these cDNAs were derived from a single copy gene. Northern blot analysis and ribonuclease protection assay revealed that both rEP1 and rEP1-variant receptor mRNAs were highly expressed in the kidney. Immunoblot with an antibody directed toward the specific region of rEP1-variant receptor showed that rEP1-variant receptor protein was expressed in the membrane of the kidney and Chinese hamster ovary (CHO) cells transfected with rEP1-variant cDNA. Thus, the rEP1-variant receptor is translated from mRNA which is not spliced at nucleotide position 952 in the segment VI transmembrane region. rEP1-variant receptor retained the ligand binding activity with affinity and specificity similar to rEP1 receptor, but lost the coupling of signal transduction systems by itself. However, when rEP1-variant receptor was stably co-expressed with rEP1 receptor in CHO cells, the Ca2+ mobilization mediated by EP1 receptor was significantly suppressed. Furthermore, when rEP1-variant receptor was expressed in CHO cells, cAMP formation by activation of endogenous EP4 receptor was strongly blocked. These results suggest that the rEP1-variant receptor may affect the efficiency of signal coupling of PGE receptors and attenuate the action of PGE2 on tissues.

Nitric oxide mediates allodynia induced by intrathecal administration of prostaglandin E2 or prostaglandin F2α in conscious mice

&NA; We recently reported that intrathecal (i.t.) administration of prostaglandin (PG) E2 or PGF2&agr; in conscious mice induced allodynia through a pathway that includes the glutamate receptor system. Allodynia induced by PGE2 and PGF2&agr; was blocked by antagonists for NMDA and metabotropic glutamate receptor subtypes, respectively. In the present study, we examined the possibility for the involvement of nitric oxide (NO) in the PG‐evoked allodynia. Allodynia was assessed once every 5 min by light stroking of the flank of mice with a paintbrush. Intrathecal administration of L‐arginine, a substrate of nitric oxide synthase (NOS), in conscious mice resulted in allodynia. Dose dependency of L‐arginine for allodynia showed a bell‐shaped pattern (1–10 &mgr;g/mouse). The maximal allodynic effect was observed with 5.0 &mgr;g at 10–15 min after i.t. injection, similar in time course and magnitude to that induced by L‐glutamate. L‐Arginine‐induced allodynia was dose‐dependently reduced by the NOS inhibitor N&ohgr;‐nitro‐L‐arginine methyl ester (L‐NAME) and the soluble guanylate cyclase inhibitor methylene blue with IC50 values of 7.68 and 8.70 pg/mouse, respectively. PGE2 induced allodynia was also dose‐dependently inhibited by L‐NAME and methylene blue with IC150. values of 94.7 and 74.9 pg/mouse. PGF2&agr;‐induced allodynia was inhibited by methylene blue with an IC50. value of 40.6 pg/mouse, but not by L‐NAME at doses up to 1.0 ng. These results demonstrate that PGEZ‐induced allodynia is mediated through the NO‐generating system and that PGF2&agr;‐induced allodynia may be mediated by interactions with the NO system at a site different from the NO‐generating site in the spinal cord.

Blockade by ONO-NT-012, a unique prostanoid analogue, of prostaglandin E2-induced allodynia in conscious mice

1 Intrathecal (i.t.) administration of prostaglandin E2 (PGE2) to conscious mice was reported to induce allodynia, a state of discomfort and pain evoked by innocuous tactile stimuli through prostaglandin E receptor subtype EP1 and hyperalgesia through prostaglandin E receptor subtypes EP2 and/or EP3. In the present study, we investigated the effects of an EP1 antagonist on these sensory disorders by use of ONO‐NT‐012 or AH6809. 2 ONO‐NT‐012 dose‐dependently antagonized the PGE2‐induced allodynia but had no effect on the PGE2‐induced hyperalgesia by the hot plate test. On the other hand, AH6809 blocked the PGE2‐induced hyperalgesia at the highest dose examined (50 μ kg−1) but had no effect on the PGE2‐induced allodynia. The i.t. injection of AH6809 or ONO‐NT‐012 alone did not have any effect on the response to noxious or innocuous stimuli. 3 Increasing doses (5 pg kg−1‐500 ng kg−1) of ONO‐NT‐012 produced parallel shifts to the right of the dose‐response curves to PGE2. The Schild plot regression line was linear and the slope was close to unity. The pA2 value against PGE2 was calculated to be 9.96. 4 The present study demonstrates that i.t. administration of PGE2 exerts allodynia through EP1 in the mouse spinal cord and that ONO‐NT‐012 is a highly potent, simple competitive antagonist for the PGE2‐induced allodynia.

Cyclic-AMP-dependent Ca2+ influx elicited by prostaglandin D2 in freshly isolated nonchromaffin cells from bovine adrenal medulla

We previously reported that prostaglandin D2 (PGD2) specifically elevates intracellular cyclic AMP in nonchromaffin cells isolated from bovine adrenal medulla (Biochim. Biophys. Acta (1989) 1011, 75-80). Here we again found that PGD2 increased intracellular Ca2+ concentration ([Ca2+]i) in freshly isolated nonchromaffin cells and investigated the cellular mechanisms of PGD2-induced [Ca2+]i increase using the Ca2+ indicator fura-2 and a fluorescence microscopic imaging system. Treatment of the cells with PGD2 receptor agonists BW245C and ZK110841 resulted in both marked stimulation of cyclic AMP formation and an increase in [Ca2+]i. The [Ca2+]i response was also induced by bypassing of the receptor with forskolin, a direct activator of adenylate cyclase, but not by PGE2 or PGF2 alpha both of which are devoid of the ability to generate cyclic AMP in the cells. These cyclic AMP and [Ca2+]i responses induced by PGD2 were completely blocked by the PGD2 receptor antagonist BWA868C. The time-course of cyclic AMP production stimulated by PGD2 coincided with that of the [Ca2+]i increase. While the Ca(2+)-mobilizing hormone bradykinin stimulated a rapid inositol phosphate accumulation in nonchromaffin cells, PGD2 did not stimulate it significantly. Removal of extracellular Ca2+ markedly reduced the Ca2+ response to PGD2 in magnitude and duration, but did not alter the peak [Ca2+]i response to bradykinin. These results demonstrate that PGD2 receptor activation induces the increase in [Ca2+]i via cyclic AMP mainly by increasing the Ca2+ influx from the outside, unlike inositol trisphosphate which causes release of Ca2+ from internal stores.