Effects of estradiol on release and disposition of norepinephrine from nerve endings

Studies were performed to determine whether estradiol (E2) has a direct action on the release and disposition of norepinephrine (NE) from adrenergic nerve endings in isolated superfused canine saphenous veins. [3H]NE and is labeled metabolites were separated by column chromatography with measurement by liquid scintillation spectrometry. An increase in the spontaneous overflow of total 3H, [3H]NE, and [3H]dihydroxyphenylglycol ([3H]DOPEG) that occurred with 1 and 10 microgram/ml E2 in the superfusing medium suggested that E2 either induced NE release or interfered with intraneuronal NE storage. During electrical stimulation (ES), release of [3H]NE and [3H]DOPEG exceeded controls with 1 and 10 microgram/ml E2 in the superfusate, yet total 3H was little changed. The evoked release of [3H]DOPEG showed less of an elevation over its spontaneous efflux in E2-treated veins than in nontreated veins, suggesting that E2 may block neuronal reuptake of released NE. The efflux of O-methylated deaminated metabolites during and after ES was decreased by E2 treatment, suggesting also an inhibition of extraneuronal uptake of NE.     

Norepinephrine metabolism in canine saphenous vein: prevalence of glycol metabolites

To examine the disposition of [3H]norepinephrine ([3H]NE) in adrenergically innervated veins, helical strips of canine saphenous veins were incubated in Krebs-Ringer solution containing D,L[3H]NE (2 X 10(-7) M) for 2 h. [3H]NE and its metabolites were measured in extracts of veins and in superfusate (Krebs-Ringer) collected during basal conditions and during release of [3H]NE evoked by electrical stimulation (1-8 Hz), tyramine (5 X 10(-6) to 5 X 10(-4) M), or high concentrations of potassium (35-100 meq/liter). During basal conditions, the efflux from veins comprised mainly metabolits of [3H]NE, especially 3,4-dihydroxphenylglycol (DOPEG) and 3-methoxy-4-hydroxyphenylglycol (MOPEG); this pattern was unchanged by cocaine treatment, and monoamine oxidase inhibition reduced the formation of DOPEG. During evoked release of NE, the major metabolites in the perfusate were DOPEG, MOPEG, and normetanephrine, and their proportions differed with the stimulus used: O-methylated metabolites in the perfusate always increased more than did the deaminated catechol compounds; DOPEG and MOPEG were released in greater amounts than the corresponding acids; and cocaine treatment caused a higher content of all metabolites except DOPEG. 3-Methoxy-4-hydroxymandelic acid was also formed by the vein but was retained in the tissue.     

Simultaneous secretion of catecholamines from the adrenal medulla and of [3H]norepinephrine from sympathetic nerves from a single test preparation: different effects of agents on the secretion

The uptake and release of catecholamines was investigated in the isolated perfused adrenal gland of the rat after preloading the preparation with [3H]norepinephrine, and the effects of various agents were examined on the stimulation-evoked secretion of catecholamines and total tritium. Large quantities of tritium were found in the adrenal medulla after either intravenous injection of [3H]norepinephrine to the rat, or perfusion of the isolated adrenal gland with Krebs-bicarbonate solution containing [3H]norepinephrine. The retention of the tritium was inhibited 90% by desipramine. Acute treatment with guanethidine and chronic treatment with 6-hydroxydopamine abolished the secretion of tritium without affecting the secretion of catecholamines evoked at 1 Hz. Nicotine, muscarine and acetylcholine enhanced the secretion of catecholamines but not tritium, whereas tyramine and ephedrine enhanced the secretion of tritium but not catecholamines. It is concluded that chromaffin cells do not possess the norepinephrine uptake mechanism and that the uptake of [3H]norepinephrine occurs mainly in sympathetic nerve terminals present in the adrenal gland and the surrounding blood vessels (adrenal and renal veins). The differential localization of [3H]norepinephrine and catecholamines allowed us to test the effects of a variety of pharmacological agents that alter neurotransmitter release by acting on receptors on the neuronal membrane, acting on sodium and potassium channels, or acting to alter the intracellular concentrations of adenosine 3',5'-cyclic monophosphate and protein kinase C. Transmural stimulation (1 Hz for a total of 300 pulses) markedly enhanced the release of catecholamines and tritium which was blocked by tetrodotoxin (sodium channel-blocker) and potentiated by tetraethylammonium and gallamine (potassium channel-blockers). Phentolamine, an alpha adrenergic blocking agent which acts on both alpha-1 and alpha-2 receptors, caused a 3- to 4-fold facilitation of the tritium secretion while inhibiting catecholamine secretion by 45%. [Met]enkephalin almost completely inhibited the evoked-secretion of tritium but had very little effect on the secretion of catecholamines. Forskolin inhibited the tritium secretion by 80% but produced more than a 2-fold facilitation of catecholamine secretion. Phorbol 12,13-dibutyrate caused facilitation of evoked secretion of both catecholamines and tritium. A combination of phorbol ester and forskolin had a synergistic effect on stimulation-evoked secretion of catecholamines, whereas phorbol ester partially reversed the inhibitory effects of forskolin on the tritium secretion.(ABSTRACT TRUNCATED AT 400 WORDS)     

Evidence for a neurotransmitter function of acetylcholine in rabbit superior colliculus

Acetylcholinesterase staining and studies on the uptake of [3H]choline into the subsequent efflux of tritium from collicular slices were carried out in order to provide evidence for a neurotransmitter function of acetylcholine in rabbit superior colliculus. Acetylcholinesterase staining was dense and homogeneous in superficial layers whereas the staining was arranged in patches with slightly higher density caudally than rostrally in the intermediate layers. The accumulation of tritium in slices incubated with [3H]choline depended on time, temperature and concentration, and was inhibited by hemicholinium-3. Accumulation was slightly higher in caudal than in rostral slices. Electrical stimulation enhanced tritium outflow from slices preincubated with [3H]choline. Tetrodotoxin and a low calcium medium inhibited the evoked overflow whereas hemicholinium-3 caused an enhancement. Oxotremorine decreased the evoked overflow; atropine prevented this effect. The opioids [D-Ala2, MePhe4, Glycol5]enkephalin, [D-Ala2, D-Leu5]enkephalin and ethylketocyclazocine caused an inhibition. The effects of the latter two agonists were antagonized by naloxone. The GABAB-receptor-agonist (-)-baclofen decreased the evoked overflow at lower concentrations than GABA, whereas the GABAA-receptor-agonist muscimol was ineffective. Serotonin produced an inhibition which was prevented by metitepin, alpha- and beta-adrenoceptor as well as dopamine-receptor ligands caused no change. It is concluded that in the rabbit superior colliculus the pattern of acetylcholinesterase staining is comparable, but not identical to the distribution in other species. The accumulation of [3H]choline, as well as the tetrodotoxin-sensitive and calcium-dependent overflow of tritium upon electrical stimulation (reflecting presumably release of [3H]acetylcholine) indicate that acetylcholine has a neurotransmitter function in this tissue. The release of [3H]acetylcholine was modulated by various transmitter substances and related compounds. The pattern of modulation of release differed from the pattern in other cholinergically innervated tissues.     

Effects of cocaine on monoamine uptake as measured ex vivo

The increase in extracellular dopamine (DA) following cocaine administration plays a major role in cocaine abuse. In vitro, cocaine binds to DA transporters (DAT) and blocks DA uptake. Moreover, cocaine can increase extracellular DA concentration as measured by in vivo neurochemical methods. The present study examined the effects of cocaine and other drugs on DA, NE and 5-HT uptake using an ex vivo assay. Rats were injected i.v. with saline or drug and sacrificed at various time points after injections. Brains were dissected for regional monoamine uptake studies ex vivo. In most brain regions, cocaine given in vivo blocked monoamine uptake as expected. [3H]DA uptake in nucleus accumbens was inhibited with an ED50=22.3 micromol/kg. Cocaine fully inhibited [3H]NE uptake (ED50=4.58 micromol/kg) in the occipital cortex and partially inhibited [3H]5-HT uptake (33% at 30 micromol/kg) in the midbrain. However, under the same conditions [3H]DA uptake in the striatum was not inhibited after injections of cocaine up to 56 micromol/kg. Although the mechanism for this discrepancy is unclear, DA binding and uptake sites may be distinct and/or there may be regional differences in DA transporters.     

Uptake, release, and modulation of release of noradrenaline in rabbit superior colliculus

The noradrenaline content, the uptake of [3H]noradrenaline, and the release of previously incorporated [3H]noradrenaline were studied in slices of rabbit superior colliculus. The concentration of endogenous noradrenaline was higher in superficial than in deep layers of the superior colliculus. Upon incubation with [3H]noradrenaline, tritium was accumulated by a mechanism that was strongly inhibited by oxaprotiline but little inhibited by 6-nitroquipazine. Electrical stimulation at 0.2 or 3 Hz increased the outflow of tritium from slices preincubated with [3H]noradrenaline; the increase was almost abolished by tetrodotoxin or a low calcium medium. Clonidine reduced the evoked overflow of tritium, whereas yohimbine increased it and antagonized clonidine. The evoked overflow was also reduced by the dopamine D2-receptor-selective agonists apomorphine and quinpirole, an effect antagonized by sulpiride. The preferential opioid kappa-receptor agonist ethylketocyclazocine produced an inhibition that was counteracted by naloxone. Nicotine accelerated the basal outflow of tritium; part of the acceleration was blocked by hexamethonium. The muscarinic agonist oxotremorine slightly diminished the electrically evoked overflow, and its effect was abolished by atropine. The oxaprotiline-sensitive uptake of [3H]noradrenaline as well as the tetrodotoxin-sensitive and calcium-dependent overflow of tritium upon electrical stimulation (presumably reflecting the release of [3H]noradrenaline) indicate that noradrenaline is a neurotransmitter in the superior colliculus. The release of [3H]noradrenaline is modulated through alpha 2-adrenoceptors as well as dopamine D2-receptors, opioid kappa-receptors and nicotine and muscarine receptors. No clear evidence was found for modulation through beta-adrenoceptors, D1-receptors, serotonin receptors, opioid mu- or delta-receptors or receptors for GABA or glutamate. Only the alpha 2-adrenoceptors receive an endogenous agonist input, at least under the conditions of these experiments. The pattern of presynaptic modulation resembles that found for noradrenaline release in other rabbit brain regions, suggesting that all noradrenergic axons arising in the locus coeruleus possess similar presynaptic receptor systems.     

Choline transport by lung epithelium

The uptake of [3H]choline was investigated using isolated perfused rat lungs and primary cultures of granular pneumocytes isolated by tryptic digestion of rat lungs. Metabolic products were separated from free choline by chloroform:methanol extraction and column chromatography. Tissue-associated [3H]choline increased progressively in the perfused lung, and estimated mean intracellular concentration at 2 h was 12 times the extracellular concentration (5 microM). Choline uptake was inhibited by ventilation with CO and by perfusion with the choline analog, hemicholinium-3 (HC-3). Isolated granular pneumocytes also accumulated choline against a concentration gradient by an energy-dependent process. The concentration for half-maximal uptake, after correction for the diffusion component, was estimated at 18 +/- 4 microM (mean +/- SE; n = 3), and the estimated maximal rate of uptake was 213 +/- 44 pmol/min/microliter cell water. HC-3 inhibited uptake by approximately 50% at a concentration of 10(-4) M. There was no effect on uptake when Na+ in the medium was replaced by Li+ or N-methylglucamine+. These results indicate that granular pneumocytes possess a transport system that results in accumulation of choline against a concentration gradient. The characteristics of uptake indicate that this system is similar to the low affinity choline transport system of other organs.     

Apparent [3H]1,25-dihydroxyvitamin D3 uptake by canine and rodent brain

The brain uptake of [3H]1,25-dihydroxyvitamin D3 ([3H]1,25(OH)2D3) was studied during steady-state conditions using the multiple-indicator dilution technique in dogs. The fractional [3H]1,25(OH)2D3 uptake was evaluated at 0.8 +/- 0.15% during a single passage through the dog brain. Evaluation of the [3H]1,25(OH)2D3 uptake by the vitamin D-replete and vitamin D-depleted rat brain indicated that 30 min after its injection, the fractional uptake was not influenced by the vitamin D status of the animals or by the amount of [3H]-1,25(OH)2D3 injected. In the rodent the fractional [3H]-1,25(OH)2D3 brain accumulation was between 0.16 and 0.20%, whereas the brain-to-serum ratio varied between 5 and 6%. Protein-binding studies of serum [3H]1,25(OH)2D3 indicated that, at 37 degrees C, 94.8 +/- 0.4% of the hormone was protein bound 30 min after its intravenous injection. These observations suggest that 1,25(OH)2D3 is able to cross the blood-brain barrier. However, its limited brain uptake in relation to its serum concentration suggests that the hormone does not penetrate freely into the central nervous system and that its brain uptake may be related to the free circulating 1,25(OH)2D3 concentration perfusing the blood-brain barrier.     

Uptake and metabolism of [3H]norepinephrine in uterine nerves of pregnant guinea pig.

Myometrial tissue slices from virgin, pregnant (uni- or bilateral pregnancy), and puerperal animals were incubated in media containing [3H]norepinephrine ([3H]NE), and the neuronal and extraneuronal uptake was estimated. The metabolic fate of [3H]NE was elucidated by the chromatographic separation of [3H]NE, [3H]normetanephrine and 3H-labeled acid metabolites. An early and extensive reduction in both total and neuronal uptakes occurred in the myometrial regions surrounding the fetuses, and at term pregnancy no neuronal [3H]NE uptake at all was found in tissue slices from the fetus-containing horns. Concomitantly, the fraction of unchanged [3H]NE decreased with a corresponding increment in its metabolites. Similar changes, though less extensive, also occurred in the empty horn in unilateral pregnancy. The total amount of neuronal uptake in the cervix seemed unchanged compared to virgin animals. Earlier and present data support the following suggestion that in uterine horns harboring fetuses, an extensive axonal degeneration occurs; in those devoid of fetuses (unilateral pregnancy), the nerve terminals are to a great extent structurally intact, but functionally damaged. Recovery of the pregnancy-induced impairment of the adrenergic nerve plexus is slow and incomplete.     

Histamine H3 receptor activation inhibits dopamine D1 receptor-induced cAMP accumulation in rat striatal slices

In striatal membranes bearing significant levels of histamine H3 receptors (72 +/- 14 fmol/mg protein), the H3 agonist immepip (1 microM) increased [35S]GTPgammaS binding to 119 +/- 2% of basal, an effect prevented by the H3 antagonist clobenpropit and by pre-treatment with pertussis toxin. In slices labelled with [3H]adenine and in the presence of 1 mM isobutylmethylxantine (IBMX), the selective dopamine D1-like (D1/D5) receptor agonist SKF-81297 stimulated cyclic [3H]AMP ([3H]cAMP) accumulation (maximal stimulation 205 +/- 24% of basal, EC50 113 +/- 12 nM), an effect fully blocked by the D1/D5 antagonist SCH-23390. The accumulation of [3H]cAMP induced by 1 microM SKF-81297 was inhibited in a concentration-dependent manner by the selective H3 receptor agonist immepip (maximal inhibition 60+/-5%, IC50 13 +/- 5 nM). The inhibitory action of 100 nM immepip was reversed in a concentration-dependent manner by the H3 antagonist thioperamide (EC50 13 +/- 3 nM, Ki 1.4 +/- 0.3 nM). Forskolin-induced [3H]cAMP accumulation (726 +/- 57% of basal) was also reduced by H3 receptor activation, although to a lesser extent (19.1 +/- 3.2% inhibition), an action not affected by the absence of either IBMX or Ca2+ ions in the incubation medium. Neither the density of [3H]SCH-23390 binding sites (D1 receptors) nor the inhibition by SKF-81297 were affected by 1 microM immepip, ruling out a direct interaction between D1 and H3 receptors. These results indicate that through H3 receptors coupled to Galphai/o proteins, histamine modulates cAMP formation in striatal neurones that possess D1 receptors, most probably GABAergic striato-nigral neurones.     

肺血管内皮细胞对交感神经末梢去甲肾上腺素释放的影响

ｃＧＭＰ升高抑制肾上腺能神经末梢释放去甲肾上腺素（ＮＥ），内皮细胞释放的舒张因子（ＥＤＲＦ）增加ｃＧＭＰ，故其对交感神经ＮＥ的释放可能有调节作用。本文观察去内皮细胞或抑制ＥＤＲＦ合成后肺血管对跨膜交感神经刺激（ＴＮＳ）的反应及对２－〔１４Ｃ〕－ＮＥ的摄取和释放。利用磨擦损伤内皮细胞后或使用ＬＮＭＭＡ抑制ＥＤＲＦ合成后，肺血管对ＴＮＳ刺激的反应明显增加，尤以低频率刺激为甚。肺血管对２－〔１４Ｃ〕－Ｎ     

Uptake and release of [3H]dopamine by the median eminence: evidence for presynaptic dopaminergic receptors and for dopaminergic feedback inhibition

The accumulation and release of [3H]dopamine by the median eminence in vitro was studied after treatments with different pharmacological agents, to determine whether such a procedure would be useful for measuring neuronal activity in the tuberoinfundibular dopaminergic system. The accumulation of [3H]dopamine was temperature, time, and sodium dependent, and reduced by unlabelled dopamine and by a potent dopamine uptake blocker, nomifensine. The outflow of tritium was studied after blocking the oxidative deamination of dopamine by nialamide. The outflow of tritium was elicited consistently by biphasic square wave electrical pulses and by high molarity potassium ions. The response to electrical stimulation was dependent largely on calcium and partially on sodium. The response to high molarity potassium ions was reduced in the absence of calcium ions. The response to electrical stimulation was increased by nomifensine and by a dopaminergic antagonist, haloperidol, and was reduced by dopamine and by a dopaminergic agonist, piribedil. The inhibitory action of dopamine was antagonized by haloperidol. These results indicate the existence of uptake and release mechanisms in the tuberoinfundibular dopamine neurons, and suggest that dopamine may inhibit its own release via dopaminergic receptors. This in vitro method may be useful for measuring dopamine uptake and release by tuberoinfundibular dopaminergic neurons.     

Uptake and metabolism of [3H]choline mustard by cholinergic nerve terminals from rat brain

The objective of this study was to measure the uptake and metabolism of [3H]choline mustard aziridinium ion in rat brain synaptosomes. In previous investigations, we showed that this compound binds irreversibly to the choline carrier thereby inhibiting choline transport into nerve terminals; it also acts as both a substrate and inhibitor of the acetylcholine biosynthetic enzyme choline acetyltransferase. We now report that [3H]choline mustard aziridinium ion was transported into purified rat brain synaptosomes by a hemicholinium-sensitive mechanism, but at only a fraction of the rate of uptake of [3H]choline. Following 5 min incubation with the nerve terminal preparation, uptake of [3H]choline mustard aziridinium ion was 20% of that of [3H]choline transport, but this fell to 10% of [3H]choline accumulation at 30 min incubation. Apparent Michaelis constants derived from double reciprocal plots of velocity of transport versus substrate concentration revealed that the apparent affinity constants (Km) of the high-affinity choline carrier for [3H]choline mustard aziridinium ion and [3H]choline were not different (1.44 +/- 0.15 and 2.14 +/- 0.80 microM for choline and choline mustard aziridinium ion, respectively). Increasing the incubation time from 5 to 30 min, during which time a proportion of the high-affinity choline carriers were irreversibly inactivated by choline mustard aziridinium ion, did not alter the binding affinity for this compound. The maximum velocity of transport (Vmax) for the two compounds were significantly different with the maximum uptake of [3H]choline mustard aziridinium ion being 19.5% of that for choline at 5 min incubation, and falling to only 10.6% of the maximum rate of choline transport by 30 min incubation. [3H]Choline mustard aziridinium ion transported into synaptosomes on the high-affinity choline carrier was metabolized, with 27% being recovered as [3H]acetylcholine mustard aziridinium ion, 27% as [3H]phosphorylcholine mustard aziridinium ion, 7% as unmetabolized [3H]choline mustard aziridinium ion and 16% recovered as an unidentified metabolite. In parallel samples, [3H]choline taken up into synaptosomes was recovered as [3H]acetylcholine (71%) and unmetabolized [3H]choline (18%) with no net production of [3H]phosphorylcholine. Acetylation of [3H]choline mustard aziridinium ion amounted to only 7.6% of [3H]acetylcholine synthesized under the same conditions. These results show clearly that choline mustard aziridinium ion was accumulated into the cholinergic nerve terminals by the high-affinity choline carrier, but the amount was small relative to the uptake of choline and probably restricted by progressive inactivation of the transporters through covalent bond formation.     

Release and metabolism of [3H]dopamine in the neurointermediate lobe of the rat pituitary gland

Neurointermediate lobes of the rat pituitary gland were incubated with [3H]dopamine in the presence of desipramine and then superfused with radioactivity-free medium. The outflow of tritium was studied and in most experiments [3H]dopamine and its metabolites were separated by column chromatography. After 60-70 min of superfusion, the spontaneous rate of tritium outflow was 1.2%/min. The spontaneously released radioactivity consisted of 52% O-methylated and deaminated metabolites, 28% 3,4-dihydroxyphenylacetic acid, 18% dopamine and 2% 3-methoxytyramine. In the presence of pargyline (10 microM) the spontaneous rate of total tritium outflow decreased by 46%, that of the O-methylated and deaminated metabolites by 72% and that of 3,4-dihydroxyphenylacetic acid by 79%. The spontaneous rate of outflow of dopamine was unchanged and that of 3-methoxytyramine increased 3-fold. Further addition of nomifensine (10 microM) doubled the rate of outflow of dopamine and 3-methoxytyramine, but had no effect on the other metabolites. Electrical stimulation of the pituitary stalk (0.2 ms, 80 V, 3 Hz, 2 min) caused a tritium release of 8.5% of the tissue tritium. The evoked tritium release was only partially dependent on the extracellular calcium and not affected by tetrodotoxin. In contrast, vasopressin release evoked by stimuli of the same strength was completely calcium-dependent and blocked by tetrodotoxin. After modification of the stimulation conditions (1 ms, 10 V, 10 Hz, 2 min) the evoked tritium release was 4.1% of the tissue tritium. This tritium release was reduced by 73% in the presence of tetrodotoxin. The total evoked tritium release was decreased by 30% in the presence of pargyline and increased by 150% after further addition of nomifensine. Under the latter conditions, tetrodotoxin reduced the evoked tritium release by 67%, but nearly all of the tetrodotoxin-resistant tritium release could be identified as dopamine metabolites. Thus, the electrical stimulation appears to liberate some [3H]dopamine metabolites from an extraneuronal compartment. In conclusion, oxidative deamination and O-methylation are important pathways of the catabolism of dopamine in the neurointermediate lobe of the pituitary gland. After labelling of the transmitter stores with [3H]dopamine, the total tritium release is a poor indicator of [3H]dopamine release from the nerve terminals. Only the isolated [3H]dopamine fraction appears to reflect the release of neuronal [3H]dopamine.     

Vitamin B12 uptake in dog kidney: its use as an extracellular reference marker.

The renal handling of [3H]cyanocobalamin has been investigated in dogs in vivo using the pulse injection multiple-indicator dilution technique. Simultaneous renal vein and urine outflow curves were obtained for [3H]cyanocobalamin relative to T-1824-albumin (plasma reference) and creatinine (extracellular reference). The renal vein recovery of [3H]cyanocobalamin relative to creatinine was significantly less than unity and increased with increasing intraarterial dose. This indicates a saturable postglomerular uptake mechanism. The urine recovery of [3H]cyanocobalamin relative to simultaneously filtered creatinine is also less than unity and increases with increasing intra-arterial doses. This indicates a saturable interaction at the luminal surface of the nephron. Saturation interaction at the luminal surface of the nephron. Saturation of postglomerular and luminal interactions can also be achieved by systemic preloading with unlabeled vitamin B12. After saturation the renal vein and urine recoveries for [3H]cyanocobalamin and creatinine become equal. However, significant differences can be observed between their renal vein mean transit times, depending on experimental conditions. During hydropenia [3H]cyanocobalamin behaves as a true extracellular marker and emerges in renal vein and urine outflows, superimposing on creatinine. During mannitol diuresis vitamin B12 still behaves as a glomerular marker, but its renal vein mean transit time is now significantly less than that of creatinine and its calculated postglomerular volume of distribution is reduced by approximately 15% of the available interstitial space.     

N-methyl-D-aspartate-evoked release of acetylcholine from the medial septum/diagonal band of rat brain

N-Methyl-D-aspartate (NMDA)-evoked release of [3H]acetylcholine (ACh) from slices of rat brain medial septum/vertical limb of the diagonal band (ms/vdB) was examined. NMDA increased the release of tritium in a concentration-dependent manner and the specific non-competitive NMDA antagonist, MK-801, and the competitive NMDA antagonist kynurenic acid inhibited this release. Tetrodotoxin inhibited the NMDA-evoked release suggesting the [3H]ACh released arises from collaterals of the cholinergic septohippocampal neurons. Basal release of tritium was significantly increased by glycine alone and strychnine inhibited this response while having no effect on NMDA-evoked release. However, glycine, although not affecting the NMDA-evoked release, did enhance release of tritium in the presence of NMDA and blocking concentrations of Kynurenic acid. Together, these findings suggest that under the conditions of these experiments sufficient concentrations of glycine permit the full expression of NMDA-evoked modulation of [3H]ACh release, and that the predominant actions of glycine were mediated by a specific, strychnine-sensitive receptor.     

In vitro uptake and autoradiographic localization of tritiated gossypol inTaenia taeniaeformis metacestodes

Gossypol, a natural polyphenolic compound, induces growth-inhibitory and antiparasitic effects inTaenia taeniaeformis metacestodes in vivo and in vitro. We investigated the uptake and localization of [³H]-gossypol in this parasite. Metacestodes were incubated in 10^–5 M [³H]-gossypol at 37°C. Parasites steadily took up tritium activity over the first 3 h of incubation, after which a plateau was maintained for the duration of the experiment. Tissue: medium radioactivity ratios revealed that intralarval tritium activity matched extralarval activity within 30 min of incubation and continued to increase with time. Reverse-phase high-performance liquid chromatographic (HPLC) analysis confirmed tissue incorporation of tritium activity that manifested as a single radioactive species. Autoradiography localized [³H]-gossypol to the tegument, calcareous corpuscles, and parenchyma over the first 2 h of incubation. By 6 h, parenchymal radioactivity had disappeared.T. taeniaeformis metacestodes rapidly take up and accumulate [³H]-gossypol in vitro. This accumulation is apparently selective for specific sites, which may have implications for gossypol's metacestocidal action.     

Progesterone decreases mating and estradiol uptake in preoptic areas of male monkeys.

Synthetic progestins such as medroxyprogesterone acetate (MPA) are used widely in the treatment of male sex offenders. In male cynomolgus monkeys (Macaca fascicularis) treated with testosterone (T), both MPA and progesterone (P) had comparable inhibitory effects on male sexual motivation and behavior. To determine if P, like MPA, decreases endogenous T levels, plasma T and P levels were analyzed in weekly blood samples (N=186) from eight intact males, each paired with a sexually receptive female before, during, and after treatment with subcutaneous Silastic P implants (336 behavior tests). P treatment decreased sexual activity but not plasma T levels. To ascertain if P, like MPA, acts by decreasing the nuclear uptake of T by brain, four P-treated and four control males were euthanized 60 min after intravenous injection of 3 mCi of [3H]T. The nuclear uptake of unchanged [3H]T and its metabolites [3H]E(2) and [3H]DHT was measured in samples of brain, pituitary gland, genital tract, and liver. P, unlike MPA, did not affect the nuclear uptake of [3H]androgens by brain, but reduced by 80% the nuclear accumulation of [3H]E(2) in tissue samples containing preoptic area and the anterior part of the bed nucleus of stria terminalis, although not in samples from hypothalamus or amygdala.     

Cationic proteins inhibit L-arginine uptake in rat alveolar macrophages and tracheal epithelial cells. Implications for nitric oxide synthesis.

Eosinophil-derived cationic proteins play an essential role in the pathogenesis of bronchial asthma. We tested whether cationic proteins interfere with the cationic amino-acid transport in alveolar macrophages (AMPhi) and tracheal epithelial cells, and whether L-arginine-dependent pathways were affected. The effect of cationic polypeptides on cellular uptake of [(3)H]-L-arginine, nitrite accumulation, and the turnover of [(3)H]-L-arginine by nitric oxide (NO) synthase and arginase (formation of [(3)H]-L-citrulline and [(3)H]-L-ornithine, respectively) were studied. Poly-L-arginine reduced [(3)H]-L-arginine uptake in rat AMPhi and tracheal epithelial cells in a concentration-dependent manner (at 300 microgram/ml by 70%). Poly-L-lysine, protamine, and major basic protein (each up to 300 microgram/ml) tested in rat AMPhi inhibited [(3)H]-L-arginine uptake by 35 to 50%. During 6 h incubation in amino acid-free Krebs solution, rat AMPhi, precultured in the absence or presence of LPS (1 microgram/ml), accumulated 1.4 and 3.5 nmol/10(6) cells nitrite, respectively. Addition of 100 microM L-arginine increased nitrite accumulation by 70 and 400% in control and lipopolysaccharide-treated AMPhi, respectively. Nitrite accumulation in the presence of L-arginine was reduced by poly-L-arginine and poly-L-lysine (100 and 300 microgram/ml) by 60 to 85% and 20 to 30%, respectively. Poly-L-arginine, but not poly-L-lysine, inhibited nitrite accumulation already in the absence of extracellular L-arginine. Poly-L-arginine (300 microgram/ml) inhibited [(3)H]-L-citrulline formation by AMPhi stronger than that of [(3)H]-L-ornithine. We conclude that cationic proteins can inhibit cellular transport of L-arginine and this can limit NO synthesis. Poly-L-arginine inhibits L-arginine uptake more effectively than other cationic proteins and exerts additional direct inhibitory effects on NO synthesis.     

Sindbis virus infection increases hexose transport in quiescent cells

Sindbis virus infection of baby hamster kidney cells or chick embryo cells resulted in a significant increase in the rate of uptake of [2-3H]deoxy-D-glucose ([3H]dGlu). Stimulation of hexose transport in Sindbis virus-infected cells occurred only if the cells were rendered quiescent by culturing at high density or by serum starvation. In contrast, Sindbis virus-induced inhibition of potassium transport, measured as a decrease in the uptake of 86Rb+, was independent of cell growth state. Stimulation of [3H]dGlu uptake in Sindbis virus-infected cells was the result of an increase in the Vmax of the hexose transporter, but not a change in the Km. The stimulation of [3H]dGlu uptake induced by Sindbis virus was insensitive to the drug actinomycin D, but was blocked by cordycepin. The stimulation was also insensitive to treatment with tunicamycin, which prevented the virally induced inhibition of the plasma membrane-associated Na+/K+ ATPase and termination of host protein synthesis.