Experimental concept for examination of biological effects of magnetic field concealed by gravity.

Space is not only a place to study biological effects of gravity, but also provides unique opportunities to examine other environmental factors, where the biological actions are masked by gravity on the ground. Even the earth's magnetic field is steadily acting on living systems, and is known to influence many biological processes. A systematic survey and assessment of its action are difficult to conduct in the presence of dominant factors, such as gravity. Investigation of responses of biological systems against the combined environment of zero-gravity and zero-magnetic field might establish the baseline for the analysis of biological effects of magnetic factors. We propose, in this paper, an experimental concept in this context, together with a practical approach of the experiments, both in orbit and on the ground, with a thin magnetic shielding film. Plant epicotyl growth was taken as an exemplar index to evaluate technical and scientific feasibility of the proposed system concept.

Nicotinic receptors involved in gastric noradrenaline release evoked by electrical stimulation of the splanchnic nerve in rats.

In the present experiment, we tried to compare the functional nicotinic receptors activated by electrical stimulation of the greater splanchnic nerve (containing preganglionic sympathetic nerves) to those activated by (-)-nicotine, using the isolated rat stomach. The stomach was perfused with Krebs-Ringer solution and endogenous noradrenaline released into the perfusate was electrochemically measured using high-performance liquid chromatography. The release of noradrenaline evoked by repeated application of 30 mM (-)-nicotine rapidly declined. However, the release of noradrenaline evoked by electrical stimulation of the splanchnic nerve at 2.5 Hz was not disturbed by the appearance of tachyphylaxis for (-)-nicotine. The (-)-nicotine-induced release of noradrenaline was abolished by diltiazem, but this reagent had no effect on the electrically evoked release of noradrenaline. The electrically evoked release of noradrenaline was not influenced by atropine, but was reduced to approximately 50% by hexamethonium. This electrically evoked release of noradrenaline was not influenced by alpha-bungarotoxin, alpha-conotoxin ImI (blockers of alpha 7 nicotinic receptors) or dihydro-beta-erythroidine (a blocker of alpha 4 beta 2 nicotinic receptors), but was reduced to about 50% by mecamylamine (a blocker of alpha 3 beta 4 nicotinic receptors). The (-)-nicotine-induced release of noradrenaline has already been shown to be partially blocked by dihydro-beta-erythroidine and to be abolished by mecamylamine as shown by Yokotani et al. [Eur. J. Pharmacol. 402 (2000) 223.]. These results suggest that the gastric release of noradrenaline in response to electrical stimulation of the greater splanchnic nerve is mediated by cholinergic (probably ganglionic alpha 3 beta 4 nicotinic receptor-mediated) and non-cholinergic mechanisms in rats. However, the functional nicotinic receptor activated by electrical stimulation of the splanchnic nerve seems to be different in character from that activated by (-)-nicotine.

Role of brain arachidonic acid cascade on central CRF1 receptor-mediated activation of sympatho-adrenomedullary outflow in rats.

The present experiments were designed to characterize the mechanisms involved in the corticotropin releasing factor (CRF)-induced activation of central sympatho-adrenomedullary outflow in rats. Intracerebroventricularly (i.c.v.) administered CRF and urocortin (0.5, 1.5 and 3.0 nmol/animal) effectively and dose-dependently elevated plasma levels of adrenaline and noradrenaline, and the effect of urocortin was almost the same as that of CRF. The elevation of catecholamines induced by CRF and urocortin (1.5 nmol/animal) was reduced by CP-154,526(butyl-ethyl-(2,5-dimethyl-7-(2,4,6trimethylphenyl)-7H-pyrrolo [2,3-d] pyrimidin-4-yl]amine), a selective CRF1 receptor antagonist, in a dose dependent manner (1.2 and/or 2.4 micromol/animal, i.c.v.), and abolished by indomethacin (1.2 micromol/animal, i.c.v.), an inhibitor of cyclooxygenase. Furegrelate (1.8 micromol/animal, i.c.v.), an inhibitor of thromboxane A2 synthase, abolished the CRF-induced elevation of adrenaline, but had no effect on the evoked release of noradrenaline. These results suggest that activation of brain CRF1 receptor facilitates the central sympathetic and adrenomedullary outflow in distinct central pathways in rats; brain thromboxane A2 is involved in the central adrenomedullary outflow; an active metabolite of arachidonic acid other than thromboxane A2 (probably prostaglandin E2) may be involved in the central sympathetic outflow.

A species-selective allelopathic substance from germinating sunflower (Helianthus annuus L.) seeds.

From the exudate of germinating sunflower (Helianthus annuus L.) seeds was isolated a stereoisomer of diversifolide, 4, 15-dinor-3-hydroxy-1(5)-xanthene-12,8-olide (designated sundiversifolide) as determined by analysis of its IR, APCI-, ESI- and HR-MS and 13C and 1H NMR spectra. This substance inhibited shoot and root growth of cat's-eyes by about 50% at a concentration of 30 ppm. It also showed species-selective activity on the shoot and root growth of tested plants. When cat's-eyes seeds were incubated together with sunflower seeds, the cat's-eyes growth was inhibited. Furthermore, it was detected from an extract of river sand when sunflower seeds were incubated on the sand. These results indicate that sundiversifolide has an allelopathic function in sunflower plants.

Characterization of nicotinic acetylcholine receptor-mediated noradrenaline release from the isolated rat stomach.

We characterized nicotinic acetylcholine receptor-mediated noradrenaline release from the isolated, vascularly perfused rat stomach. The stomach was perfused via the coeliac artery with Krebs-Ringer solution at a constant flow rate of 4 ml per minute. Endogenous noradrenaline released into the perfusate was electrochemically measured using high-performance liquid chromatography. Nicotinic receptor agonists were applied once into the perfusion medium for 2 min and nicotinic receptor antagonists were administered throughout the experiments. The (-)-nicotine (3x10(-5) M)-induced noradrenaline release was abolished by tetrodotoxin and hexamethonium and partially blocked by dihydro-beta-erythroidine (up to 10(-5) M) (a relatively selective antagonist of alpha4beta2 nicotinic receptors) and abolished by mecamylamine (10(-5) M) (a relatively selective antagonist of alpha3beta4 nicotinic receptors), but not influenced by alpha-bungarotoxin (3x10(-7) M) or alpha-conotoxin ImI (10(-6) M) (antagonists of alpha7 nicotinic receptors). (+/-)-Epibatidine (3x10(-7) M) (a very potent, but non-selective agonist) and (-)-cytisine (3x10(-4) M) (an agonist of beta4 nicotinic receptors) effectively evoked the release of noradrenaline, while (E)-N-methyl-4-(3-pyridinyl)-3-butene-1-amine (RJR-2403) (up to 10(-4) M) (an agonist of alpha4beta2 nicotinic receptors) had no effect. The potency of these agonists was as followed; (+/-)-epibatidine>>(-)-nicotine>(-)-cytisine>>>RJR -2403. These results are compatible with the published view that alpha3beta4 nicotinic receptors are predominant in other parts of the autonomic nervous system. These receptors (probably located on the gastric sympathetic ganglia) are involved in the release of noradrenaline from the rat stomach.

Nicotine-induced noradrenaline release from the isolated rat stomach by activation of L- and N-type calcium channels.

We examined the effect of nicotine on the release of endogenous noradrenaline (NA) from the isolated, vascularly perfused rat stomach. The stomach was perfused via the coeliac artery with Krebs-Ringer solution containing 10 microM pargyline at a constant flow rate of 4 ml per minute. Nicotine was once applied in the perfusion medium for 2 min. Nicotine (10(-6) - 10(-4) M) evoked NA release in a concentration-dependent manner. The nicotine (3 x 10(-5) M)-evoked NA release was abolished by hexamethonium and tetrodotoxin. Diltiazem and isradipine [blockers of L-type voltage-activated calcium channel (VACC)] and omega-conotoxin GVIA (a blocker of N-type VACC) also abolished this nicotine-evoked NA release. Previously we reported that N-type, but not L-type, VACCs are located on the gastric postganglionic sympathetic nerve terminals, since the NA release evoked by electrical stimulation of periarterial nerves around the left gastric artery (postganglionic sympathetic nerves) was abolished by omega-conotoxin GVIA, but not by diltiazem (Yokotani et al., Jpn. J. Pharmacol. 78, 75- 77, 1998). From these results, it was suggested that nicotine activates nicotinic acetylcholine receptors located on the sympathetic ganglia, thereby evoking NA release by activation of L-type VACC located on the gastric sympathetic ganglia and N-type VACC probably located on the sympathetic nerve terminals in the rat stomach.

U-46619, a selective thromboxane A2 mimetic, inhibits the release of endogenous noradrenaline from the rat hippocampus in vitro.

Possible roles of thromboxane A2 (TXA2) in the release mechanism of hippocampal noradrenaline (NA) were examined in vitro. Slices or crude synaptosomes prepared from the rat hippocampus were superfused with modified Krebs-Ringer solution. Application of 20 mM KCl for 5 min increased the release of NA from the slices, and this release was consistently reproduced. Application of U-46619 (9,11-dideoxy-9alpha,11alpha-methanoepoxy prostaglandin F2alpha), a specific TXA2 mimetic, just before the second KCl (20 mM) stimulation decreased the KCl-evoked NA release in a concentration-dependent manner (10-100 microM). This U-46619 (50 microM)-induced inhibition of NA release was abolished by 10 microM SQ29548, a specific TXA2 receptor antagonist. In experiments with hippocampal crude synaptosomes, however, KCl (20 and 40 mM)-evoked release of NA was not attenuated by U-46619 (100 microM). Furthermore, the inhibitory effect of U-46619 (50 microM) in the sliced preparations was not modified by 100 microM (-)-bicuculline, a GABA(A)-receptor antagonist. The present results indicate that U-46619 inhibits the release of NA from the rat hippocampus by activation of TXA2 receptors. Activation of TXA2 receptors probably excites an unidentified but not GABAergic neuron system, thereby inhibiting the NA release from the rat hippocampus.

Brain phospholipase A(2)-arachidonic acid cascade is involved in the activation of central sympatho-adrenomedullary outflow in rats.

The present experiments were designed to explore the role of the brain phospholipase A(2)-arachidonic acid cascade in the activation of central sympatho-adrenomedullary outflow in rats, using melittin (an activator of phospholipase A(2)) and arachidonic acid. Intracerebro-ventricularly administered melittin (2.5, 10, and 25 microg/animal) or arachidonic acid (75, 150, 300 microg/animal) effectively and dose dependently elevated plasma levels of adrenaline and noradrenaline. The elevation of both catecholamines induced by melittin (10 microg/animal) was abolished by centrally administered mepacrine (an inhibitor of phospholipase A(2)), but not by neomycin (an inhibitor of phospholipase C). However, mepacrine had no effect on the increase induced by arachidonic acid (150 microg/animal). Indomethacin (an inhibitor of cyclooxygenase) abolished all responses induced by melittin and arachidonic acid. Furegrelate (an inhibitor of thromboxane A(2) synthase) abolished the elevation of adrenaline induced by melittin and arachidonic acid, but had no effect on the elevation of noradrenaline induced by these compounds. These results suggest that activation of the brain phospholipase A(2)-arachidonic acid cascade facilitates the central sympatho-adrenomedullary outflow in rats. Brain thromboxane A(2) is involved in the activation of central adrenomedullary outflow and an active metabolite of arachidonic acid other than thromboxane A(2) may be involved in activation of the central sympathetic outflow.

Histamine H(3) receptor-mediated inhibition of endogenous acetylcholine release from the isolated, vascularly perfused rat stomach.

We studied the effects of histamine H(3) receptor ligands on the release of endogenous acetylcholine from the isolated, vascularly perfused rat stomach. The stomach was perfused via the celiac artery with modified Krebs-Ringer solution containing physostigmine. Released acetylcholine from the portal vein was electrochemically measured using high-performance liquid chromatography and an enzyme system. Vagus nerves were electrically stimulated twice for 2 min (0.5 or 2.5 Hz). Acetylcholine release evoked at 2.5 Hz was slightly inhibited by histamine and effectively potentiated by thioperamide, a histamine H(3) receptor antagonist. Acetylcholine release evoked at 0.5 Hz in the presence of atropine was not influenced by thioperamide, but effectively inhibited by histamine, R-alpha-methylhistamine or imetit, histamine H(3) receptor agonists. These inhibitory effects were abolished by thioperamide or pertussis toxin. These results suggest that histamine attenuates acetylcholine release from vagus nerves through histamine H(3) receptor-mediated and pertussis toxin-sensitive mechanisms in the rat stomach.

Perfusion of the hypothalamic paraventricular nucleus with N-methyl-D-aspartate produces thromboxane A2 and centrally activates adrenomedullary outflow in rats.

We applied a microdialysis technique for the measurement of hypothalamic thromboxane B2, a stable metabolite of thromboxane A2, in urethane-anesthetized rats. Perfusion with N-methyl-D-aspartate (1.5 and 2.5mM) of the paraventricular nucleus by microdialysis probe concentration-dependently elevated the levels of thromboxane B2 in this region and plasma levels of catecholamines. The elevation of adrenaline was much more marked than that of noradrenaline. Pretreatment with dizocilpine maleate (0.1 mM), a non-competitive antagonist of N-methyl-D-aspartate receptors, of the paraventricular nucleus by microdialysis probe attenuated the N-methyl-D-aspartate (1.5 mM)-induced elevations of both thromboxane B2 and plasma catecholamines. Intracerebroventricular administration of furegrelate (250 microg/animal), a thromboxane A2 synthase inhibitor, also abolished the responses evoked by N-methyl-D-aspartate. These results indicate that N-methyl-D-aspartate applied into the paraventricular nucleus produces thromboxane A2 in this region and elevates plasma levels of catecholamines, especially adrenaline. Thromboxane A2 produced in this hypothalamic nucleus is probably involved in the N-methyl-D-aspartate-induced central adrenomedullary outflow.

Neurotransmitter contents in the retina of RCS rat.

BACKGROUND: Retinitis pigmentosa is a hereditary disease characterized by gradually developing degeneration of photoreceptors. The Royal College of Surgeons (RCS) rat is an experimental model of retinitis pigmentosa. However, there is a paucity of information concerning neurotransmitter contents in the retina of RCS rats. Thus, we determined the retinal contents of neurotransmitters in RCS rats at 4 and 23 weeks postnatally and in age-matched congenic control rats. METHODS: Dopamine (DA) and acetylcholine (ACh) were electrochemically measured by high-performance liquid chromatography (HPLC). Neuroactive amino acids, including gamma-aminobutyric acid (GABA) and taurine, were determined by means of an HPLC-precolumn derivatization method. RESULTS: Contents of DA, ACh, glutamate, aspartate and GABA in the retina of RCS rats 4 weeks postnatally were within normal ranges. At 23 weeks, the retinal contents of DA, glutamate and aspartate in the RCS rats were significantly lower than in the age-matched control rats, while the contents of ACh and GABA were unaffected even at this later stage. On the other hand, the retinal content of glycine in the RCS rats at 23 weeks was significantly higher than that in the age-matched control rats. It is interesting to note that the content of taurine in the RCS rats had already decreased at 4 weeks postnatally and the decrease was more marked at 23 weeks. CONCLUSION: The decrease in taurine content is probably the first sign of degeneration revealed by the retinal neurotransmitters of RCS rats.

Melatonin inhibits the central sympatho-adrenomedullary outflow in rats.

Central effects of melatonin on the sympatho-adrenomedullary outflow were investigated in urethane-anesthetized rats. In the intact animals, intracerebroventricularly (i.c.v.) administered interleukin-1beta (IL-1beta) (100 ng/animal) slightly, but significantly, elevated the plasma level of noradrenaline (NA), but not the level of adrenaline (Ad). Melatonin (100 microg/animal, i.c.v.) did not modulate the effects of IL-1beta on plasma levels of catecholamines. In the pinealectomized animals, however, the same dose of IL-1beta markedly elevated plasma levels of both Ad and NA, and the elevation of Ad was more potent than that of NA. In these pinealectomized animals, the serum level of melatonin was significantly lower than that in the sham-operated control animals. Furthermore, the IL-1beta-induced elevations of plasma catecholamines in these pinealectomized animals were attenuated by i.c.v. administered melatonin. These results suggest that melatonin plays an inhibitory role in the central regulation of sympatho-adrenomedullary outflow in rats.

Involvement of N-type voltage-activated Ca2+ channels in the release of endogenous noradrenaline from the isolated vascularly perfused rat stomach.

We characterized the voltage-activated Ca2+ channels involved in noradrenaline (NA) release from gastric sympathetic neurons using isolated, vascularly perfused rat stomach. The evoked NA release by electrical stimulation of periarterial nerves was abolished by calcium removal from the perfusion medium and by cadmium. Omega-conotoxin GVIA (N-type Ca2+-channel blocker) effectively and omega-conotoxin MVIIC (N/P/Q-type blocker) slightly inhibited the evoked NA, while omega-agatoxin IVA (P-type blocker) had no effect. These results suggest that omega-conotoxin GVIA and omega-conotoxin MVIIC-sensitive N-type Ca2+ channels are involved in NA release from the rat gastric sympathetic nerve terminals.

Involvement of mu-receptor in endogenous opioid peptide-mediated inhibition of acetylcholine release from the rat stomach.

We examined the effect of endogenous opioid peptides on vagally evoked release of acetylcholine (ACh) from the isolated, vascularly perfused rat stomach. The vagus nerves were electrically stimulated twice at 2.5 Hz for 2 min, and test substances were administered during the second stimulation. beta-Endorphin (10(-7) and 3 x 10(-7) M), an endogenous nonselective agonist of mu-receptors, inhibited the release of ACh. However, [Leu5]-enkephalin, an endogenous nonselective agonist of delta-receptors, and U-50488, a kappa-receptor agonist, had no effect at a higher dose of 10(-6) M. Beta-endorphin-induced inhibition was abolished by naloxone. Endomorphins 1 and 2 (3 x 10(-7) and 10(-6) M), endogenous selective agonists of mu-receptors, also inhibited the release of ACh. These results suggest that the mu-receptor is involved in the endogenous opioid peptide-induced inhibition of the release of ACh from the rat stomach.

Thromboxane A2 is involved in the nitric oxide-induced central activation of adrenomedullary outflow in rats.

The central effect of 3-morpholinosydnonimine, a nitric oxide donor, on the sympatho-adrenomedullary system was investigated in urethane-anesthetized rats. Intracerebroventricular administration of 3-morpholinosydnonimine (100, 250 and 500 microg/animal) induced a marked elevation of adrenaline levels and a slight elevation of noradrenaline levels in the plasma. These 3-morpholinosydnonimine (250 microg/animal)-induced elevations of catecholamines were abolished by intracerebroventricular treatments with 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-l-oxyl 3-oxide (750 microg/animal), a nitric oxide scavenger, and indomethacin (500 microg/animal), a cyclo-oxygenase inhibitor, but not with superoxide dismutase (250 units/animal), a superoxide anion scavenger. Furthermore, the 3-morpholinosydnonimine (250 microg/animal)-induced elevation of plasma adrenaline levels was abolished by intracerebroventricular treatments with thromboxane A2 synthase inhibitors [furegrelate (100, 250 and 1000 microg/animal) and carboxyheptyl imidazole (500 microg/animal)], and also with thromboxane A2 receptor blockers [(+)-S-145 (100, 250 and 1000microg/animal) and SQ29548 (8microg/animal)]. The elevation of noradrenaline levels was, however, not attenuated by these thromboxane A2-related test agents. The present results indicate that nitric oxide but not peroxynitrite markedly activates central adrenomedullary outflow. Thromboxane A2 in the brain is probably involved in this central activation of adrenomedullary outflow.

Properties of calcium channels coupled to endogenous glutamate release from the vascularly perfused rat stomach in vitro.

We have demonstrated that both high-K+ and electrical stimulation of the vagus nerves release endogenous glutamate from the vascularly-perfused rat stomach in a calcium-dependent manner. In the present study, we examined properties of calcium channel subtypes mediating endogenous glutamate release from the stomach. Application of 50 mM KCl elicited a release of glutamate, and this release was abolished in calcium-free medium. The release of glutamate was significantly inhibited by both omega-agatoxin IVA, a P/Q-type calcium channel antagonist, and isradipine, an L type calcium channel antagonist. Omega-conotoxin GVIA, an N type calcium channel antagonist and flunarizine, a nonselective T-type calcium channel antagonist were without effect. In contrast to this case of glutamate, omega-conotoxin GVIA induced a marked inhibition in the release of gastric noradrenaline. The combined treatment with omega-agatoxin IVA plus isradipine produced a marked synergistic inhibition of the glutamate release. This inhibition was, however, much less than that by cadmium. The present results suggest that P/Q and L type calcium channels coexist to regulate the release of gastric glutamate. Furthermore, it is possible that unidentified calcium channels other than P/Q and L type channels are also involved in the release of glutamate in the stomach.

Centrally applied nitric oxide donors inhibit vagally evoked rat gastric acid secretion: involvement of sympathetic outflow.

ntracerebroventricularly (i.c.v.) administered nitric oxide (NO) donors, 3-morpholinosydnonimine (SIN-1) (100-500 microg/animal) and sodium nitroprusside (SNP) (100-250 microg/animal) dose-dependently inhibited the rat gastric acid secretion evoked by vagal stimulation at 3 Hz. Furthermore, the inhibitory effect of SIN-1 (250 microg/animal) was more marked and its onset was more rapid than that of SNP (250 microg/animal). The SIN-1 (250 microg/animal)-induced antisecretory effect was abolished by both splanchnicotomy and phentolamine (5 mg/kg, i.m.), and also by indomethacin (500 microg/animal, i.c.v.). These results suggest that i.c.v. administered NO donors inhibit vagally evoked gastric acid secretion by activation of central sympathetic outflow. Central prostaglandin is probably implicated in this NO-mediated antisecretory effect.

Brain histamine mediates the bombesin-induced central activation of sympatho-adrenomedullary outflow.

Intracerebroventricular (i.c.v.) administration of bombesin (0.3 nmol) increased plasma levels of both adrenaline and noradrenaline in urethane anesthetized rats. These bombesin-induced increases were inhibited by i.c.v. pretreatment with pyrilamine, an H1-receptor antagonist. Ranitidine, an H2-receptor antagonist also inhibited the increase of adrenaline, however, its effective dose was much larger than that of pyrilamine. Furthermore, the bombesin-induced increase of noradrenaline was not effectively inhibited by ranitidine. In the next series, turnover of histamine was assessed by measuring accumulation of tele-methylhistamine (t-MH), a major metabolite of brain histamine. I.c.v. administration of bombesin (0.3-3 nmol) increased turnover of hypothalamic histamine, while its intravenous administration was without effect. The present results suggest that the bombesin-induced central activation of sympatho-adrenomedullary outflow is probably, at least in part, mediated through brain histaminergic neurons.

Nitric oxide mediates central activation of sympathetic outflow induced by interleukin-1 beta in rats.

The excitatory mechanism of central sympathetic outflow induced by interleukin-1 beta was investigated in urethane-anesthetized rats. Intracerebroventricular administration of interleukin-1 beta induced a gradually developing elevation of plasma noradrenaline levels in a dose-dependent manner (50, 100 and 200 ng/animal), while the levels of adrenaline were not affected. The elevation of noradrenaline levels induced by interleukin-1 beta (100 ng/animal i.c.v.) was abolished by the following treatments with: (1) a chemical sympathectomizer, 6-hydroxydopamine (15 mg/kg i.v., 3 days before); (2) a prostaglandin synthesis inhibitor, indomethacin (500 micrograms/animal i.c.v.); (3) a nitric oxide synthase inhibitor, L-NG-nitroarginine methyl ester (100 micrograms plus 10 micrograms/min i.c.v.); and (4) a nitric oxide scavenger, oxyhemoglobin (32.3 micrograms plus 3.23 micrograms/min i.c.v.). In contrast to these results, D-NG-nitroarginine methyl ester, an inactive isomer of L-NG-nitroarginine methyl ester, and methemoglobin, a metabolite of oxyhemoglobin, were without effect. Furthermore, prostaglandin E2 (100 ng/animal i.c.v.) rapidly and markedly elevated the plasma level of noradrenaline but not adrenaline. This prostaglandin E2-induced elevation of plasma noradrenaline levels was not attenuated by L-NG-nitroarginine methyl ester (100 micrograms plus 10 micrograms/min i.c.v.). The present results suggest that nitric oxide is involved in the interleukin-1 beta-induced central activation of sympathetic outflow. Furthermore, there probably exists nitric oxide-linked prostaglandin-generating system in the brain.