Interaction of histamine and calcitonin gene-related peptide in the formalin induced pain perception in rats.

Histamine and calcitonin gene-related peptide (CGRP) contribute to the pain perception. The aim of the present study is to clarify the interaction of histamine and CGRP in the perception of inflammatory pain. The effects of a histamine H1 receptor antagonist (pyrilamine, i.p.), an H2 receptor antagonist (ranitidine, i.p.) and a CGRP antagonist (CGRP 8-37, i.t.) on the formalininduced pain was studied in rats. Pyrilamine and ranitidine produced a dose-dependent antinociceptive response in the first and the second phases of the formalin test. A single administration of pyrilamine (1 mg/kg, i.p.), ranitidine (10 mg/kg, i.p.) or CGRP 8-37 (10 µg/µL, i.t.) had no significant effects on the pain perception in the second phase. A combination of CGRP 8-37 and pyrilamine or ranitidine at these sub-effective doses, however, showed nociceptive response in the second phase. Moreover, a histamine (i.t.)-induced hyperalgesia was completely prevented by treatment with GGRP 8-37 at this dose. Our findings have raised the possibility that the CGRP system has interaction with histamine in the perception of inflammatory pain.

Potentiation of potassium currents by beta-adrenoceptor agonists in human urinary bladder smooth muscle cells: a possible electrical mechanism of relaxation.

We examined the effects of beta-adrenoceptor agonists on the membrane currents of smooth muscle cells from the human urinary bladder using a whole-cell patch clamp to investigate the involvement of Ca(2+)-activated K(+) (K(Ca)) channels in relaxation by beta-adrenergic agonists. With 0.05 mmol/l EGTA in the patch pipette, depolarizing pulses evoked outward rectifying currents. Isoproterenol (1 micromol/l) significantly increased the membrane currents by 75% at +80 mV with 0.05 mmol/l EGTA pipette solution. BRL 37344 (1 micromol/l) significantly increased the membrane currents by 44% at +80 mV. Iberiotoxin (100 nmol/l) significantly decreased the membrane currents by 60% at +80 mV. In the presence of iberiotoxin, the potentiation of the outward currents by isoproterenol was greatly suppressed and, in the presence of iberiotoxin and apamin (1 micromol/l), the potentiation by isoproterenol was totally abolished. On the other hand, with 5 mmol/l EGTA pipette solution, depolarizing pulses evoked smaller outward currents. Isoproterenol (1 micromol/l) did not change the membrane currents with 5 mmol/l EGTA pipette solution. The real-time PCR analysis revealed the expression of beta(2)-adrenoceptors in the cells. These results suggest that Ca(2+)-activated and iberiotoxin- and apamin-sensitive currents via both large-conductance and small-conductance K(Ca) channels could be increased by stimulation of beta(2)-adrenoceptors.

Modified behavioral characteristics following ablation of the voltage-dependent calcium channel beta3 subunit.

Voltage-dependent calcium channels are important for calcium influx and the ensuing intracellular calcium signal in various excitable membranes. The beta subunits of these channels modify calcium currents through pore-forming alpha1 subunits of the high-voltage- activated calcium channels. In the present study, beta3 subunit-null mice were used to investigate the importance of the beta3 subunit of the voltage-dependent calcium channel, which couples with the CaV2.2 (alpha1B) subunit to form the major component of neuronal N-type calcium channels in the brain. Western blot analysis revealed a significant decrease in N-type calcium channels in beta3 subunit-null mice, while protein levels of other high-voltage-activated calcium channel alpha1 subunits were unchanged. Immunoprecipitation analysis with an anti-CaV2.2 antibody showed that reshuffling of the assembly of N-type channels had occurred in the beta3 subunit-null mice. Ablation of this subunit resulted in modified nociception, decreased anxiety, and increased aggression. The beta3 subunit-null mice also showed impaired learning ability. These results suggest the importance of voltage-dependent calcium channels and the key role of the beta3 subunit in memory formation, nociceptive sensory transduction, and various neurological signal transduction pathways.

Effects of activation of central nervous histamine receptors in cardiovascular regulation; studies in H(1) and H(2) receptor gene knockout mice.

To elucidate the central roles of histamine receptors in cardiovascular regulatory system, systolic, mean, and diastolic blood pressures (BPs) and heart rate (HR) were examined in conscious H(1) receptor gene knockout (H(1)KO) mice, H(2) receptor gene knockout (H(2)KO) mice, H(1) and H(2) receptor gene double knockout (DKO) mice, and their respective control mice by the tail-cuff system. Histamine, histamine-trifluoromethyl-toluidine derivative (HTMT, an H(1) agonist), dimaprit (an H(2) agonist), and immepip (an H(3) agonist) were intrathecally administered to these KO mice and control mice. Basal BPs and HR were not different among these three KO mice and their control or wild-type mice. Intrathecal administration of histamine significantly increased BPs and decreased HR in control mice. The increases in BPs were produced by histamine in H(1)KO and H(2)KO mice and by HTMT and dimaprit in C57BL mice. The pressor responses by HTMT and dimaprit in C57BL mice were greater than those by histamine in H(1)KO and H(2)KO mice, although the same decreases in HR were induced by histamine in C57BL and H(1)KO mice and by dimaprit in C57BL mice. The selective stimulation of H(3) receptors by immepip produced a consistent decrease in BPs in control mice. These results obtained with the exogenous selective agonists of three histamine receptors suggest that the pressor responses to histamine are mediated through the stimulation of both H(1) and H(2) receptors, whereas the atropine-sensitive decrease in heart rate is mainly due to H(2) receptors which activate the vagal output to the heart.

Differential inhibition of transient outward currents of Kv1.4 and Kv4.3 by endothelin.

The effects of endothelin on the transient outward K(+) currents were compared between Kv1.4 and Kv4.3 channels in Xenopus oocytes expression system. Both transient outward K(+) currents were decreased by stimulation of endothelin receptor ET(A) coexpressed with the K(+) channels. Transient outward current of Kv1.4 was decreased by about 85% after 10(-8) M ET-1, while that of Kv4.3 was decreased by about 60%. By mutagenesis experiments we identified two phosphorylation sites of PKC and CaMKII in Kv1.4 responsible for the decrease in I(to) by ET-1. In Kv4.3 a PKC phosphorylation site was identified which is in part responsible for the decrease in I(to). Differences in the suppression of I(to) could be ascribed to the difference in intracellular signaling including the number of phosphorylation sites. These findings might give clues for the understanding of molecular mechanism of ventricular arrhythmias in heart failure, in which endothelin is involved in the pathogenesis.

[Basic arrhythmogenic mechanisms in both inherited and acquired long QT syndrome].

The heterologous expression system will provide clues for understanding the basic mechanism of arrhythmogenicity in both inherited and acquired long QT syndrome, which are reviewed here, with emphasis on the K+ channels. Endothelin is implicated in the morphological and electrical remodeling of cardiac muscles in heart failure. The effects of endothelin on the transient outward K+ currents (Ito) were compared between Kv1.4 (rich in endocardial muscle) and Kv4.3 (rich in epicardial muscle) channels in the Xenopus oocytes expression system. Both Itos were decreased by stimulation of endothelin receptor ETA coexpressed with the K+ channels. Ito of Kv1.4 was decreased by about 85% after 10(-8) M ET-1, whereas that of Kv4.3 was decreased by about 60%. By mutagenesis experiments, we identified two phosphorylation sites of PKC and CaMKII in Kv1.4 responsible for the decrease in Ito by ET-1. In Kv4.3 we identified a PKC phosphorylation site that is partly responsible for the decrease. Differences in the suppression of Ito could be due to the differences in intracellular signaling including the number of phosphorylation sites. These findings show some of the molecular mechanisms of ventricular arrhythmias in heart failure, resulting in dispersion and prolongation of action potential which elicit reentry and after depolarization.

Identification and characterization of novel rat and human gonad-specific organic anion transporters.

We have isolated three novel organic anion transporter cDNAs designated rat GST-1 (gonad-specific transporter), rat GST-2, and human GST, expressed at high levels in the testis. Rat GST-1, GST-2, and human GST consist of 748, 702, and 719 amino acids, respectively, and all molecules possess the 12 predicted transmembrane domains, which is a common structure of organic anion transporters. Northern blot analyses and in situ hybridization revealed that both of the rat molecules are highly expressed in the testis, especially in Sertoli cells, spermatogonia, and Leydig cells. Weak signals are also detected in the epididymis and ovary in adult rat. The exclusive expression of human GST mRNA in the testis was confirmed by RT-PCR. The pharmacological experiments of Xenopus laevis oocytes injected with the respective rat GST-1- and GST-2-cRNAs revealed that both rat GST-1 and GST-2 transport taurocholic acid, dehydroepiandrosterone sulfate, and T4 with Michaelis-Menten kinetics (taurocholic acid, Km = 8.9 and 2.5 microm, dehydroepiandrosterone sulfate, Km = 25.5 and 21.microm, and T4, Km = 6.4 and 5.8 for rat GST-1 and GST-2, respectively). T3 was also transported by rat GST-1 and GST-2. These data suggest that rat GST-1 and GST-2 might be one of the molecular entities responsible for transporting dehydroepiandrosterone sulfate and thyroid hormones involved in the regulation of sex steroid transportation and spermatogenesis in the gonad.