Sympathetic histamine exerts different pre- and post-synaptic functions according to the frequencies of nerve stimulation in guinea pig vas deferens

Histamine (HA) was found to be present in the sympathetic nerve terminals of guinea pig hearts and vasa deferentia in our previous study; however, little is known about the functions of this neurogenic HA. In this study, we used guinea pig vasa deferentia to investigate the pre- and post-synaptic functions of HA evoked by different frequencies of sympathetic nerve stimulation. We found that sympathetic nerve stimulation could evoke HA release, which was independent to mast cell degranulator compound 48/80 and mast cell stabilizer cromolyn, but was highly sensitive to Na⁺ channel blocker tetrodotoxin and chemical sympathectomy with 6-hydroxydopamine. The neurogenically released HA evoked by 12.5 Hz of nerve stimulation activated only pre-synaptic H₃ receptors and mediated pre-synaptic inhibitory effects, while under 25 or 50 Hz stimulation condition, HA simultaneously activated both pre-synaptic H₃ receptors and post-synaptic H₁ receptors. However, the direct contractile responses evoked by sympathetic HA via H₁ receptors were observed at 50 Hz. HA release and HA-mediated contractile responses upon sympathetic nerve stimulation were significantly inhibited by pre-treatment of histidine decarboxylase inhibitor α-fluoromethylhistidine. Furthermore, application of exogenous HA could mimic these pre- and post-synaptic effects. Our findings indicate that HA in sympathetic neurons acts as a neurotransmitter and its functions vary from pre-synaptic inhibition, to post-synaptic facilitation, to direct post-synaptic contractile responses according to sympathetic nerve stimulation frequencies.     

Cytochemical localization of membrane-bound Mg2+-dependent ATPase activity in guinea pig sperm head before and during the acrosome reaction

The distribution of ATPase activity in the heads of uncapacitated, capacitated, and acrosome-reacting guinea-pig spermatozoa was examined cytochemically using the Wachstein-Meisel's technique. In uncapacitated spermatozoa, the reaction products of the enzyme activity were localized on both the inner surface of the plasma membrane and the outer surface of the outer acrosomal membrane. The activity was Mg²⁺-dependent and inhibited by both Ca²⁺ and SH-blocking agents. This Mg²⁺-dependent ATPase activity was also demonstrated at the same sites in capacitated spermatozoa, whereas it was completely absent in acrosome-reacting spermatozoa. Although we did not determine the exact time of inactivation of the enzyme, it appeared to occur before the plasma membrane fused with the underlying outer acrosomal membrane. The abrupt loss of the Mg²⁺-dependent ATPase activity in the plasma and outer acrosomal membranes immediately before the onset of the acrosome reaction seems to suggest that inactivation of this enzyme by Ca²⁺ is one of the important biochemical events involved in the acrosome reaction.     

Functional interactions between the SK2 channel and the nicotinic acetylcholine receptor in enteric neurons of the guinea pig ileum

The neurotransmitter acetylcholine (ACh) plays a critical role in gastrointestinal function. The role of the small conductance Ca²⁺-activated K⁺ (SK) channel in ACh release was examined using myenteric plexus preparations of guinea pig ileum. Apamin, an inhibitor of the SK channel, significantly enhanced nicotine-induced ACh release, but neither electrical field stimulation- nor 5-hydroxytryptamine-induced ACh release, suggesting that SK channels might be selectively involved in the regulation of nicotine-induced ACh release. Therefore, we investigated the distribution of SK2 and SK3 subunits and the interaction between SK2 channels and nicotinic ACh receptors (nAChRs) in the guinea pig ileum. The immunoreactivity of SK2 subunits was located in enteric neuronal cells. Furthermore, SK2-immunoreactive cells stained with an antibody for choline acetyltransferase, a marker for cholinergic neurons, and with an antibody for the α3/5 subunits of nAChR. In contrast, immunoreactivity of SK3 subunits was not found in enteric neurons. A co-immunoprecipitation assay with Triton X-100-soluble membrane fractions prepared from the ileum revealed an association of the SK2 subunit with the α3/5 subunits of nAChR. These results suggest that SK2 channels negatively regulate the excitation of enteric neurons via functional interactions with nAChRs.     

Stereoselectivity of the aliphatic hydroxylation of 6-n-propylchromone-2-carboxylic acid in rat and guinea pig

Following administration of 6-n-propylchromone-2-carboxylic acid (6-n-PCCA) (500 μmol/kg) to male rats, three metabolic products were detected and isolated from the 0–24 h urine. All were identified as resulting from oxidation exclusively along the 6-n-propyl moiety. Some 66% of the dose was excreted in the 0–24 h urine, 55% of which was 6-PCCA, with 15% as (6-1′-hydroxypropyl)chromone-2-carboxylic acid (6-1′-HPCCA), 22% as 6-(2′-hydroxypropyl)chromone-2-carboxylic acid (6-2′-HPCCA), and 4% as (6-3′-carboxypropyl)chromone-2-carboxylic acid (6-3′-CPCCA). Derivatization of the methyl esters of the hydroxylated metabolities with S-α-methoxy-α-(trifuloromethyl)-phenylacetyl chloride (Mosher's reagent) allowed the evaluation of urinary enantiomeric composition by HPLC and assignment of their absolute configurations by NMR. This was found to be 90:10 (R/S) for 6-2′-HPCCA, and 7:93 (R/S) for 6-1′-HPCCA. When rats were dosed with the racemic 1′- and 2-hydroxy metabolites; no stereoselective metabolism or excretion was observed. Administration of 6-n-PCCA to male guinea pigs revealed that this species was unable to metabolise this compound. © 1993 Wiley-Liss, Inc.     

Locoregional therapy with polyethylene-glycol-modified interleukin-2 of an intradermally growing hepatocellular carcinoma in the guinea pig induces T-cell-mediated antitumor activity

Therapy with repeated intratumoral and perilymphatic administration of relatively low doses of polyethylene-glycol(PEG)-modified interleukin-2 (IL-2) in the syngeneic guinea pig line 10 (L10) hepatocarcinoma results in significant local tumor growth inhibition and a delay in development of regional lymph node metastases of more than 3 weeks when compared to controls. Occasionally animals are cured of tumor. The mechanism of this antitumor activity was studied. The antitumor activity of locoregionally administered PEG-IL-2 was abrogated by pretreatment with polyclonal anti-thymocyte serum, indicating that the observed tumor growth inhibition was a T-cell-mediated phenomenon. Besides the locoregional tumor growth inhibition, a systemic effect was recorded as the growth of a second tumor cell inoculum at the contralateral side was inhibited as well. Furthermore, those animals cured after PEG-IL-2 therapy developed specific immunity against the L10 tumor and this immunity could be transferred to naive animals by spleen cells. Immunohistological observations of the tumor site revealed a slight increase of helper and cytotoxic T cell subpopulations after PEG-IL-2 therapy. More pronounced, however, was the rise in number of eosinophilic granulocytes present in the stroma surrounding the tumor cells. Involvement of cytotoxic cells in the antitumor effects of PEG-IL-2 could not be demonstrated: regional lymph node cells and spleen cells obtained immediately after therapy (day 15) or on day 21 showed no cytotoxic activity in vitro against L10, K562, Daudi and line 1 (L1) target cells.In conclusion, locoregional therapy with PEG-IL-2 induced a systemic T-cell-mediated antitumor response. As no cytotoxic T cell activity was measured, however, the underlying mechanism is most likely a T-helper response. Eosinophils at the tumor site may be tumoricidal but further experiments must reveal the role of these cells in the PEG-IL-2-induced tumor regression.