The effects of continuous and single-dose radiation on choline uptake in organotypic tissue slice cultures of rabbit hippocampus.

The objective of the present study was to determine the time-dependent course of choline uptake in mature organotypic slice cultures of rabbit hippocampal formation and to assess the effects of continuous and single high-dose irradiation on choline uptake in cultivated slices in vitro. Transverse slices of hippocampus were dynamically incubated in a cerebrospinal fluid-like culture medium for 72 h. To study the changes in choline uptake longitudinally, the slice cultures were processed with 0.1 microM [3H]-choline, and tritium accumulation was counted. Two different gamma irradiation sources (125I seeds and a clinical 60Co source) were used as representative models of interstitial radiosurgery and other radiosurgical techniques. A total dose of approximately 6000 cGy was delivered to the brain slices in one session or in a continuous, relatively low-dose rate fashion, and their effects on high-affinity choline uptake were examined. In another set of experiments with 125I, 5 microM hemicholinium-3 was used in choline uptake procedures as a competitive high-affinity choline uptake inhibitor. The results can be summarized as follows: (1) in the control group of the hippocampal tissue culture, there was a significant increase in tritium accumulation values from 0 to 48 h and a decrease thereafter; (2) continuous 125I irradiation caused a highly significant depression of the accumulation of tritium compared to that observed in the control group throughout its application for 72 h; (3) there was no significant change in the accumulation of tritium in the slices after single high-dose rate irradiation with a 60Co source; and (4) 5 microM hemicholinium significantly depressed the accumulation of tritium in both the control and the 125I-irradiated groups, and there was no longer a difference between 125I-irradiated and control groups when both groups were treated with hemicholinium. These results demonstrate that the delivery of continuous but relatively low-dose rate gamma irradiation is more efficacious than single high-dose external irradiation on high-affinity choline uptake in hippocampal nervous tissue. The results also indicate that continuous irradiation specifically affected the high-affinity energy-dependent choline uptake mechanism, whereas nonspecific choline uptake did not seem to be disturbed.

GABAergic inhibition of nitric oxide-mediated relaxation of guinea-pig ileum.

The effects of GABA receptor agonists were investigated on guinea-pig isolated ileum longitudinal muscle with intact myenteric plexus. Electrical field stimulation (1 Hz, 10 s) of the histamine (1 microM)-precontracted preparation caused a contraction followed by a relaxation. Relaxations were inhibited by L-N(G)-nitroarginine (L-NA; EC50 3 microM) in a concentration-dependent manner. The inhibitory action of 10 microM L-NA was blocked by 10 microM L-arginine but not by D-arginine, which indicates that the relaxation was largely mediated by endogenous nitric oxide (NO). Tetrodotoxin (1 microM) reduced the relaxation only by about 50%. GABA and the GABA(B) agonist, baclofen, inhibited the field stimulation-induced longitudinal muscle relaxation in a concentration-dependent manner. The GABA(B) receptor antagonist, saclofen (10 microM), antagonised the effect of baclofen (apparent pA2 of saclofen: 5.6). Muscimol (10 microM) similarly inhibited the relaxation, and this inhibition was prevented by bicuculline (1 microM). It is concluded that nitrergic nerves of the guinea-pig myenteric plexus are endowed with GABA(A) and GABA(B) receptors which mediate inhibition of NO release.

Inhibition by nitric oxide and cyclic GMP of 5-hydroxytryptamine release from the vascularly perfused guinea-pig small intestine.

The effects of nitric oxide (NO) on the spontaneous release of 5-hydroxytryptamine (5-HT) were studied in the in vitro vascularly perfused guinea-pig small intestine. The NO donor SIN-1 concentration-dependently decreased 5-HT release with an EC50 of 1.34 microM, whereas the NO synthase inhibitor N(G)-nitro-L-arginine (100 microM) was without effect. The inhibition by SIN-1 of 5-HT release was enhanced by superoxide dismutase (150 U/ml) and antagonized by the selective inhibitor of soluble guanylyl cyclase, ODQ (1 microM). Tetrodotoxin (1 microM) prevented the inhibition by SIN-1 of 5-HT release, which suggests that the effect of SIN-1 is indirectly mediated via release of an inhibitory neurotransmitter. Substance P could be excluded as inhibitory transmitter because the effect of SIN-1 remained unchanged in the presence of the NK1 receptor antagonist CP 99994 (100 nM). The cyclic GMP analogue, 8-bromo cyclic GMP (300 microM), also decreased basal release of 5-HT, but this decrease was not tetrodotoxin-sensitive. It is concluded that NO inhibits the release of 5-HT from enterochromaffin cells via release of an enteric neurotransmitter. Acetylcholine (via nicotinic receptors) and substance P (via NK1 receptors) are not involved in the NO-mediated inhibition. The inhibition of 5-HT outflow by NO is due to the activation of soluble guanylyl cyclase. 8-Bromo cyclic GMP inhibited 5-HT release by a direct effect on the enterochromaffin cells.

NK1- and NK3-receptor mediated inhibition of 5-hydroxytryptamine release from the vascularly perfused small intestine of the guinea-pig.

The effects of tachykinins on the spontaneous release of 5-hydroxytryptamine (5-HT) from the enterochromaffin cells into the portal circulation was investigated in vitro using the vascularly perfused isolated guinea-pig small intestine. 5-HT was determined by HPLC with electrochemical detection. Test substances were applied intraarterially. Substance P (SP) caused a concentration-dependent decrease in 5-HT outflow with an EC50 of 50 pmol/l. Similarly, the selective NK1 receptor agonist SP methyl ester (1 nmol/l) significantly inhibited 5-HT outflow (to 51 +/- 3%). When tetrodotoxin (1 mumol/l) was added to the arterial perfusion medium, the inhibition by SP of 5-HT outflow was not affected. The selective NK1 receptor antagonist CP 99994 [(+)-(2S,3S)-3-(2-methoxybenzylamino)-2-phenylpiperidine] (0.1 mumol/l) prevented the inhibitory effect of SP (0.1 mumol/l). Neither GR 94800 (PhCO-Ala-Ala-DTrp-Phe-DPro-Pro-NleNH2) (0.1 mumol/l) nor SR 142801 [(S)-(N)-(1-(3-(1-benzoyl-3-(3,4-dichlorophenyl) piperidin-3-yl)propyl)-4-phenylpiperidin-4-yl)-N- methylacetamide] (10 nmol/l), which are selective NK2 and NK3 receptor antagonists, changed the SP-mediated inhibition. The selektive NK3 receptor agonist senktide (10 nmol/l) also decreased the 5-HT outflow (to 57 +/- 5%). This inhibition was prevented by SR 142801 (10 nmol/l) and by tetrodotoxin. CP 99994 (0.1 mumol/l) significantly antagonized the senktide-mediated inhibition of 5-HT outflow. The outflow of 5-HT was unaffected when CP 99994, GR 94800 or SR 142801 alone were added to the perfusion medium. It is concluded that the release of 5-HT from enterochromaffin cells is directly inhibited by NK1 receptors, and indirectly by neuronal NK3 receptors whose stimulation leads to the release of SP.

Inhibition by ascorbic acid of NMDA-evoked acetylcholine release in rabbit caudate nucleus.

The interaction of ascorbic acid and of dehydroascorbic acid with acetylcholine (ACh) release in rabbit caudate nucleus was investigated. The presence of ascorbic acid in the superfusion medium decreased the release of ACh evoked by N-methyl-D-aspartate (NMDA), but not by electrical stimulation. The pH of the buffer was always maintained at 7.4. Inhibition occurred even at 570 mumol/l ascorbic acid, a concentration which is widely employed in transmitter release experiments. In vivo this concentration may be reached extracellularly in brain tissue. Both ascorbic acid and dehydroascorbic acid inhibited the NMDA-evoked ACh release to the same degree in a non-competitive manner. The nearly identical action of ascorbic acid and dehydroascorbic acid makes a mode of action by lipid peroxidation or by redox phenomena unlikely. The mechanism of action underlying the described effects is unknown.

The non-dihydropyridine L-type voltage-sensitive calcium channel activator FPL 64176 enhances K(+)-evoked efflux of [3H]norepinephrine from rat neocortical slices.

The non-dihydropyridine FPL 64176 (methyl-2,5-dimethyl-4-(2-phenylmethyl)benzoyl-[1-H]pyrrole-3-carboxy la te) was tested for an interaction with neuronal L-type voltage-sensitive calcium channels (L-VSCCs) by using a [3H]isradipine ([3H]ISR) binding assay, and for its ability to enhance K(+)-evoked [3H]norepinephrine ([3H]NE) release from rat neocortical slices. The classical L-VSCC activator, the dihydropyridine (DHP) BAY K 8644, was also used for comparative purposes. FPL 64176 and BAY K 8644 both produced a similar concentration-dependent enhancement of 15 mM K(+)-evoked [3H]NE release which could be completely blocked by the L-VSCC blocker ISR (0.1 microM). FPL 64176, in contrast to BAY K 8644, was a very weak inhibitor of [3H]ISR binding to L-VSCCs. These findings indicate that FPL 64176 is a novel non-dihydropyridine L-VSCC activator, most probably by acting on a site different from the DHP binding site.