Tsuyako Ohkubo

Modified formalin test: characteristic biphasic pain response

&NA; A modified formalin test in mice was investigated. The pain response curve induced by 0.5% formalin was biphasic, having 2 peaks, from 0 to 5 min (first phase) and from 15 to 20 min (second phase). A low concentration of formalin was used, allowing the effects of weak analgesics to be detected. Centrally acting drugs such as narcotics inhibited both phases equally. Peripherally acting drugs such as aspirin, oxyphenbutazone, hydrocortisone and dexamethasone only inhibited the second phase. Aminopyrine and mefenamic acid which acted on both central and peripheral sites inhibited both phases, but the second phase was inhibited by lower doses. Thus, this method enables one to easily distinguish the site of action of analgesics. Furthermore, pain response in the first phase was inhibited by capsaicin‐treated desensitization and Des‐Arg9‐(Leu8)‐bradykinin (bradykinin inhibitor). The second phase was inhibited by compound 48/80 pretreatment, indomethacin and bradykinin inhibitor. Therefore, it is suggested that substance P and bradykinin participate in the manifestation of the first phase response, and histamine, serotonin, prostaglandin and bradykinin are involved in the second phase. These results indicate that the first and second phase responses induced by formalin have distinct characteristic properties, and it is a very useful method for examining pain, nociception and its modulation by pharmacological or other means.

Regulation of substance P release mediated via prejunctional histamine H3 receptors.

The involvement of the histamine H3 receptor in the regulation of substance P release in neurogenic inflammation was studied by using rat hindpaw skin. R-(-)-alpha-Methylhistamine, a specific histamine H3 receptor agonist, significantly inhibited the increased vascular permeability induced by antidromic electrical stimulation of the sciatic nerve in a dose-dependent manner at doses of 0.5-3 mg/kg (i.v.), and thioperamide (2 mg/kg i.p.), a specific histamine H3 receptor antagonist, prevented the inhibitory effect of R-(-)-alpha-methylhistamine. The antidromic stimulation also caused a significant increase in immunoreactive substance P release in the subcutaneous (s.c.) perfusate in the rat hindpaw. R-(-)-alpha-Methylhistamine (0.25-2 mg/kg) dose dependently inhibited the increase in release of immunoreactive substance P, and thioperamide (2 mg/mg i.p.) antagonized it. Perfusion of histamine (10(-3) M) elicited a significant increase of immunoreactive substance P release in the perfusate, which was reduced by R-(-)-alpha-methylhistamine and the antagonism of thioperamide was also observed. Histamine (in the presence of histamine H1 and H2 receptor antagonists) had an inhibitory effect on the electrically evoked release of immunoreactive substance P. These results strongly support the hypothesis that histamine regulates substance P release via prejunctional histamine H3 receptors that are located on peripheral endings of sensory nerves.

Inhibition of T-type calcium channels and hydrogen sulfide-forming enzyme reverses paclitaxel-evoked neuropathic hyperalgesia in rats.

Hydrogen sulfide (H₂S), a gasotransmitter, facilitates pain sensation by targeting Ca(v)3.2 T-type calcium channels. The H₂S/Ca(v)3.2 pathway appears to play a role in the maintenance of surgically evoked neuropathic pain. Given evidence that chemotherapy-induced neuropathic pain is blocked by ethosuximide, known to block T-type calcium channels, we examined if more selective T-type calcium channel blockers and also inhibitors of cystathionine-γ-lyase (CSE), a major H₂S-forming enzyme in the peripheral tissue, are capable of reversing the neuropathic pain evoked by paclitaxel, an anti-cancer drug. It was first demonstrated that T-type calcium channel blockers, NNC 55-0396, known to inhibit Ca(v)3.1, and mibefradil inhibited T-type currents in Ca(v)3.2-transfected HEK293 cells. Repeated systemic administration of paclitaxel caused delayed development of mechanical hyperalgesia, which was reversed by single intraplantar administration of NNC 55-0396 or mibefradil, and by silencing of Ca(v)3.2 by antisense oligodeoxynucleotides. Systemic administration of dl-propargylglycine and β-cyanoalanine, irreversible and reversible inhibitors of CSE, respectively, also abolished the established neuropathic hyperalgesia. In the paclitaxel-treated rats, upregulation of Ca(v)3.2 and CSE at protein levels was not detected in the dorsal root ganglia (DRG), spinal cord or peripheral tissues including the hindpaws, whereas H(2)S content in hindpaw tissues was significantly elevated. Together, our study demonstrates the effectiveness of NNC 55-0396 in inhibiting Ca(v)3.2, and then suggests that paclitaxel-evoked neuropathic pain might involve the enhanced activity of T-type calcium channels and/or CSE in rats, but not upregulation of Ca(v)3.2 and CSE at protein levels, differing from the previous evidence for the neuropathic pain model induced by spinal nerve cutting in which Ca(v)3.2 was dramatically upregulated in DRG.

Endogenous and exogenous hydrogen sulfide facilitates T-type calcium channel currents in Cav3.2-expressing HEK293 cells

Hydrogen sulfide (H2S), a gasotransmitter, is formed from l-cysteine by multiple enzymes including cystathionine-γ-lyase (CSE). We have shown that an H2S donor, NaHS, causes hyperalgesia in rodents, an effect inhibited by knockdown of Cav3.2 T-type Ca(2+) channels (T-channels), and that NaHS facilitates T-channel-dependent currents (T-currents) in NG108-15 cells that naturally express Cav3.2. In the present study, we asked if endogenous and exogenous H2S participates in regulation of the channel functions in Cav3.2-transfected HEK293 (Cav3.2-HEK293) cells. dl-Propargylglycine (PPG), a CSE inhibitor, significantly decreased T-currents in Cav3.2-HEK293 cells, but not in NG108-15 cells. NaHS at 1.5mM did not affect T-currents in Cav3.2-HEK293 cells, but enhanced T-currents in NG108-15 cells. In the presence of PPG, NaHS at 1.5mM, but not 0.1-0.3mM, increased T-currents in Cav3.2-HEK293 cells. Similarly, Na2S, another H2S donor, at 0.1-0.3mM significantly increased T-currents in the presence, but not absence, of PPG in Cav3.2-HEK293 cells. Expression of CSE was detected at protein and mRNA levels in HEK293 cells. Intraplantar administration of Na2S, like NaHS, caused mechanical hyperalgesia, an effect blocked by NNC 55-0396, a T-channel inhibitor. The in vivo potency of Na2S was higher than NaHS. These results suggest that the function of Cav3.2 T-channels is tonically enhanced by endogenous H2S synthesized by CSE in Cav3.2-HEK293 cells, and that exogenous H2S is capable of enhancing Cav3.2 function when endogenous H2S production by CSE is inhibited. In addition, Na2S is considered a more potent H2S donor than NaHS in vitro as well as in vivo.

Blockade of TRPM8 activity reduces the invasion potential of oral squamous carcinoma cell lines

Several members of the transient receptor potential (TRP)-channel family are expressed in cancer cells. One, cold/menthol-sensitive TRPM8, is reportedly an important player in carcinogenesis in human prostate cancer, although its involvement in oral squamous cell carcinoma (SCC) remains unclear. The present immunohistochemistry and RT-PCR results revealed intense TRPM8 expression in two SCC cell lines, HSC3 and HSC4, derived from the human tongue. Menthol, icilin, and a more specific TRPM8 agonist (WS-12) induced non-specific cation currents, with Ca2+ permeability being greater than that of Na+ or K+. The novel TRPM8 antagonist RQ-00203078 (RQ) profoundly reduced such agonist-induced cation currents. Intracellular Ca2+ imaging revealed that menthol induced both intracellular Ca2+ release and store-operated Ca2+ entry, with RQ inhibiting each effect. To assess the possible pathophysiological role of TRPM8 in oral SCC, we performed motility and invasion assays, and gelatin zymography. Menthol augmented the migration and invasion abilities of both HSC3 and HSC4 cells by potentiating MMP-9 activity. RQ suppressed all of these effects. These results may aid understanding of the pathophysiological implications of TRPM8 channels in the oral SCC cells, support TRP proteins as valuable targets for pharmaceutical intervention, and inform the targeting of oral SCC in which the prognosis is poor.

T-type voltage-activated calcium channel Cav3.1, but not Cav3.2, is involved in the inhibition of proliferation and apoptosis in MCF-7 human breast cancer cells

T-type voltage-gated Ca2+ channels have unique electrophysiological properties, suitable for generating Ca2+ oscillations and waves and thus controlling the proliferation of various tumor cells. In the present study, we investigated the role of Cav3.1, a candidate tumor suppressor gene, in neoplastic processes, and compared the differences between Cav3.1 with Cav3.2 channels. While the overexpression of a full-length Cav3.1 clone suppressed cell proliferation, the knockdown of the Cav3.1 gene by siRNA, or treatment with ProTx-I, a relatively selective inhibitor for Cav3.1, promoted the cell proliferation of MCF-7 cells (a human breast adenocarcinoma cell line). Although Cav3.1 and Cav3.2 channels possess comparable biophysical properties and are often co-expressed in various tissues, gene knockdown or the overexpression of Cav3.2 channels exhibited no effect on cell proliferation. Using immunocytochemical co-staining, the Cav3.1 channels were specifically visualized in the plasma membranes of apoptotic cells, identified by Annexin V and terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) assays and nuclear condensation. On the contrary, Cav3.2 channels were expressed at the membrane of large portions of cells, with no likely relation to Cav3.1 expression or apoptosis. An apoptosis assay revealed that the overexpression of the Cav3.1 clone caused an increase in the number of apoptotic cells. Furthermore, Cav3.1 knockdown blocked cyclophosphamide-induced apoptosis. These results suggest that Cav3.1 channels may contribute to the repression of tumor proliferation and the promotion of apoptosis mediated via Cav3.1-specific Ca2+ influx.

Identification and electrophysiological characteristics of isoforms of T-type calcium channel Ca(v)3.2 expressed in pregnant human uterus.

Electrophysiological characteristics were compared among four cloned human α1H isoforms transcripted by alternative splicings of exons 25B and 26 [Δ25B/+26 (native form; α1H-a), Δ25B/Δ?6 (α1H-b), +25B/Δ26, and +25B/+26] in the intracellular loop between domains III and IV (III-IV linker) of a human T-type calcium channel (Cav3.2). The native isoform Δ25B/+26 predominated in ovary and non-pregnant uterus, while isoform Δ25B/Δ26 (α1H-b) predominated in pregnant uterus and testis. Expressions of the newly identified +25B/Δ26 and +25B/+26 isoforms were greater in the uterus at gestation than in the non-pregnant uterus. When expressed in Xenopus laevis oocytes, all isoforms produced transient inward currents with low voltage-dependent activation and inactivation characterized in typical T-type Ca2+ currents. Each isoform possessing exon 25B (+25B/?Δ26 or +25B/+26) showed current activation and inactivation at a more negative membrane potential than the respective isoform (Δ25B/Δ26 or Δ25B/+26) lacking it. Moreover, the current activation and inactivation rates were faster for the two isoforms possessing exon 25B than for the respective isoforms lacking it. By itself, exon 26 seemed not to affect any electrophysiological characteristics. Increasing the net positive charge (relative to the native form), as occurred in isoforms Δ25B/Δ26, +25B/Δ26, and +25B/+26, caused recovery from short-term inactivation to become faster. Our results show that molecular-structure variations within the III-IV linker influence the voltage-dependence and kinetics of both activation and inactivation. Although the role of T-type Ca2+ channels in uterine tissue remains unknown, changes in the uterine expression of these α1H isoforms may influence physiological functions during pregnancy.

Role of substance P in neurogenic inflammation in the rat incisor pulp and the lower lip

Vascular permeability was significantly increased in the incisor pulp and skin of the lower lip in the rat after antidromic electrical stimulation of the inferior alveolar nerve, and this response was significantly inhibited by a substance-P antagonist. The content of substance P in the pulp and lip was also increased after stimulation. The permeability response was reduced by aspirin and bradykinin antagonists (both B1- and B2-receptor types) in the pulp and lip, indicating that prostaglandins and bradykinin may be involved. Mepyramine and methysergide inhibited the vascular response in the lip but not the pulp; the roles of histamine and serotonin differ in the two tissues. Injection of substance P into the incisor pulp and the lip skin caused dye leakage. This response was inhibited by pretreatment with compound 48/80 in the lip but not the pulp. Lip histamine content was decreased significantly after antidromic stimulation of the inferior alveolar nerve and pretreatment with compound 48/80, but was not changed in the pulp. The results suggest that substance P in the lip, after being released from the peripheral sensory-nerve endings, may act on the vascular system via histamine release from mast cells; but in the pulp may cause vascular response directly because of the scarcity of mast cells.

Spinally delivered N-, P/Q- and L-type Ca2+-channel blockers potentiate morphine analgesia in mice.

We studied the antinociceptive effects induced at the spinal level by N-, P/Q- and L-type voltage-dependent Ca2+-channel (VDCC) blockers given alone or in combination with morphine, the test responses being the algesic ones induced by acute thermal and mechanical stimuli. When given alone, intrathecal omega-agatoxin IVA (P/Q-type blocker) produced a potent dose-dependent inhibition in the tail-flick and tail-pressure over the dose range 0.33-33 pmol/mouse. Omega-conotoxin GVIA (N-type blocker) also produced dose-dependent inhibitions, but its antinociception against thermal stimuli was weaker than against mechanical stimuli. Calciseptine (L-type blocker) slightly reduced both nociceptive responses, but only at 33 pmol. At their subthreshold doses, intrathecal omega-agatoxin IVA, omega-conotoxin GVIA and calciseptine each significantly enhanced morphine analgesia in the tail-flick and tail-pressure tests, the rank order of potencies being N-> or =P/Q->L-type. These results indicate that combining a low-dose VDCC blocker, especially the N- or P/Q-type, with morphine may be a very useful way of minimizing the dose of morphine and may reduce side effects.

ATP-sensitive K+ channels mediate regulation of substance P release via the prejunctional histamine H3 receptor

Perfusion of histamine (10(-3) M) elicited a significant increase of immunoreactive substance P release in the subcutaneous perfusate in the rat hindpaw. The active L-enantiomer of cromakalim, lemakalim (50 micrograms/kg, i.v.), a selective K+ channel activator, significantly inhibited the immunoreactive substance P release. Glibenclamide (10 mg/kg, i.v.), an ATP-sensitive K+ channel blocker, abolished the response to lemakalim on the release of immunoreactive substance P. R(-)-alpha-methylhistamine (1 mg/kg, i.v.), a specific histamine H3 receptor agonist, significantly inhibited the release of immunoreactive substance P. Glibenclamide (10 mg/kg, i.v.) antagonized the inhibitory effect of R(-)-alpha-methylhistamine. Tetraethylammonium (10 mg/kg, i.p.), a K+ channel blocker, also reduced the inhibitory effect significantly. These results suggest that the inhibition of substance P release from sensory nerve endings via prejunctional histamine H3 receptors may be achieved by activating the ATP-sensitive K+ channel coupled to the histamine H3 receptor in the rat skin.