The novel phenylpropiophenone derivates induced relaxation of isolated rat aorta.

Our aim was to define how different chemical properties of newly developed phenylpropiophenone derivates (PhPds) influenced their potency and efficacy to relax rat aorta. A contribution of ion channels in the PhPds and propafenone mechanism of vasodilatation was tested. PhPds were syntethysed by substitution in the benzyl moiety with -F, -CH3 or -CF3 groups on the ortho or para position. The vasodilatation by PhPds was examined on the rings of rat aorta precontracted with phenylephrine. In order to test involvement of voltage-gated Na+ and K+ channels and L-type Ca2+ channels in a mechanism of action of PhPds, we used their blockers: lidocaine, nifedipine and 4-aminopiridine, respectively. Aorta was more sensitive to 5-ortho-trifluoromethyl derivate than to propafenone and other PhPds. The 5-para-methyl derivate had lower potency and efficacy than propafenone and other PhPds. Lidocaine did not influenced relaxation induced by PhPds, but slightly inhibited the effect of propafenone. The 4-aminopiridine only inhibited relaxation induced by 5-para-methyl derivate. Nifedipine inhibited relaxation of the rat aorta induced by 5-ortho-trifluoromethyl derivate and by propafenone. Introduction of 5-ortho-trifluoromethyl and 5-para-methyl group in the benzyl moiety of propafenone molecule changed its potency, efficacy and mechanism of action in the rat aorta. The 4-aminopiridine- and nifedipine sensitive ion channels are involved in mechanism of action of 5-para-methyl and 5-ortho-trifluoromethyl derivate. The introduction of other tested groups in the benzyl moiety does not affect pharmacological properties of the PhPds in relation to propafenone.

Xylazine, an alpha 2-adrenergic agonist, induces apoptosis of rat thymocytes and a thymocyte hybridoma line in vitro.

In our previous experiments, we demonstrated that xylazine, an alpha 2-adrenergic agonist, stimulated proliferation of thymocytes triggered by concanavalin A. In contrast, higher concentrations of xylazine were inhibitory. In this work, we studied the mechanisms involved in immunosuppression of xylazine and found that the compound at concentrations between 100 microM and 500 microM induced apoptosis of rat thymocytes in vitro. In addition, xylazine at concentrations higher than 50 microM also induced apoptosis of a thymocyte hybridoma (BWRT8) and increased apoptosis of the line triggered by T cell receptor (TCR) cross-linking. Apoptosis was confirmed by morphological analysis staining with merocyanine 540 and propidium iodide and in cases of BWRT8 by fragmentation of DNA. The mechanisms of xylazine-induced apoptosis of the BWRT8 hybridoma were further examined. We demonstrated that the process in both nonactivated and activated (TCR cross-linking) BWRT8 cells was not prevented by yohimbine (a selective alpha-adrenergic antagonist) and by antibodies to Fas and Fas-L. In contrast, cell death was completely blocked by a caspase inhibitor, z-Val-Ala-Asp (OMe)-CH2F. Cyclosporine, a calcineurin blocker, partly inhibited the xylazine-induced apoptosis of activated BWRT8 cells.

Immunomodulatory effect of xylazine, an alpha(2) adrenergic agonist, on rat spleen cells in culture.

Xylazine is an adrenergic alpha(2) agonist, which is used in veterinary medicine as a sedative and anesthetic agent. In this work we found that xylazine administered in vivo at a dose of 2.5 mg/kg enhanced spleen cell proliferation and interleukin 2 (IL-2) production in cultures stimulated with concanavalin A (Con A), whereas doses of 10 and 25 mg/kg were inhibitory. A similar stimulatory (10 microM) and inhibitory (50-500 microM) effect on splenocyte proliferation and IL-2 production was observed in vitro. Clonidine, another alpha(2) adrenergic agonist, only had a stimulatory proliferative effect on splenocytes. Yohimbine, an alpha(2) adrenergic antagonist, abrogated the stimulatory action of both clonidine and xylazine, but not the suppressive proliferative activity of xylazine in vitro. The inhibited proliferation of splenocytes to Con A correlated with increased apoptosis of T cells. The apoptosis was not blocked by yohimbine or antibodies to Fas and Fas-L. N-Nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide (NO) synthase, enhanced proliferation of splenocytes to Con A, partly abrogated the inhibitory effect of xylazine in the proliferation assay, and, only at high concentration (1000 microM), partly suppressed apoptosis of lymphocytes. The enhancing effect of L-NAME on the Con A-induced proliferation of splenocytes correlated with decreased NO production. However, decreased NO production observed in cultures with xylazine was followed by both decreased lymphocyte proliferation and apoptosis. Cumulatively, these results suggest that the immunosuppressive properties of xylazine on splenocytes in vitro are due to increased apoptosis of lymphocytes, predominantly involve NO-independent pathways, and are probably independent of its action through alpha(2) adrenoreceptors.

Xylazine, an alpha 2-adrenergic agonist, modulates proliferation of rat thymocytes in vivo and in vitro.

The effect of xylazine, an alpha 2-adrenergic agonist, on proliferation of rat thymocytes in vivo and in vitro was examined. It was found that the agonist administered to rats in vivo at doses of 2.5 mg/kg and 5 mg/kg stimulated thymocyte proliferation to suboptimal (0.625 microgram/ml) concentrations of concanavalin A (Con A). A similar effect was confirmed in vitro when lower concentrations of xylazine (5 microM) were added to cultures of thymic cells from intact animals in the presence of both suboptimal and optimal (2.5 micrograms/ml) Con A concentrations. Higher doses in vivo (25 mg/kg) and in vitro (50 microM, 100 microM and 250 microM) significantly inhibited proliferation of thymocytes to Con A. The phenomenon was followed by a decrease in interleukin-2 (IL-2) production (in vivo and in vitro) and down-regulation of IL-2 receptor alpha (IL-2R alpha) expression (in vitro). The exogenous IL-2 completely restored the inhibitory effect of xylazine in vivo on thymocyte proliferation. However, a minimal influence of the cytokine on the xylazine-inhibited thymocyte proliferation in vitro was observed. Stimulatory effect of xylazine on proliferation of thymocytes was probably mediated through alpha 2-adrenoreceptors since it was blocked by yohimbine, an alpha 2-adrenoreceptor antagonist. It seems that the pathways involved in inhibition of thymocyte proliferation by xylazine are more complex because the xylazine-suppressed thymocyte proliferation was potentiated by yohimbine.

The effects of excitatory amino acids on isolated gut segments of the rat.

Glutamate and aspartate are excitatory neurotransmitters in both central and peripheral nervous systems, acting on ionotropic and metabotropic receptors. In our study we have examined the effects of glutamate, aspartate, N-methyl-d-aspartate (NMDA), kainic acid and (+/-)-1-aminocyclopentane-cis-1,3-dicarboxylic acid (ACPD) on tone and spontaneous activity of isolated rat gastric fundus, jejunum, ileum, ascending colon and rectum. Both glutamate and aspartate produced concentration-dependent tonic contractions of rat fundus and rectum; the other gut segments used in the study were not responsive. While only NMDA and kainic acid produced concentration-dependent tonic contractions of isolated rat gastric fundus, all three type-selective agonists of glutamate receptors (NMDA, kainic acid and ACPD) produced tonic contractions of isolated rat rectum. The results of our study suggest that glutamate and aspartate in rat gastric fundus activate excitatory intrinsic neurons through only ionotropic receptors (NMDA and non-NMDA receptors), while the same action in rat rectum is mediated through both ionotropic and metabotropic receptors.