Assessment of rat brain alpha1-adrenoceptor binding and activation of inositol phospholipid turnover following chronic imipramine treatment

Chronic (21 days) treatment of rats with imipramine (10 mg/kg) did not change the density or affinity of alpha₁-adrenoceptors as measured by the specific binding of [³H]prazosin in rat cortical membranes, but produced the expected significant decrease in the density of beta-adrenoceptors labeled by [¹²⁵I]iodocyanopindolol. The functional status of brain alpha₁-adrenoceptors was also assessed by measuring the noradrenaline (NA)-induced accumulation of [³H]inositol 1-phosphate (IP₁) in brain slices from these animals. No apparent change was observed in the concentration-response relationship between NA and [³H]IP₁ accumulation in rat cerebral cortex after chronic treatment with imipramine. At concentrations higher than 1 M in vitro, imipramine and its metabolite, desipramine, produced a concentration-dependent decrease in the [³H]IP₁ accumulation elicited by NA. This inhibitory effect is likely mediated by direct blockade of alpha₁-adrenoceptors by these drugs. As the endogenous drug concentration would not reach 1 M in our preparation, the lack of changes in alpha₁-adrenoceptor response following chronic imipramine treatment are not likely attributable to residual imipramine or desipramine retained in the tissues. In conclusion, the above findings do not support previous suggestions that brain alpha₁-adrenoceptors are upregulated following chronic imipramine administration.     

NMDA and AMPA Receptors Evoke Transmitter Release from Noradrenergic Axon Terminals in the Rat Spinal Cord

N-methyl-D-aspartate (NMDA) stimulated release of [³H]noradrenaline (NA) from prelabelled rat spinal cord slices. The release was partially insensitive to tetrodotoxin (TTX) and was inhibited by the NMDA antagonist MK-801. Alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) also evoked release of [³H]NA, which was enhanced by blocking AMPA receptor desensitization with cyclothiazide. AMPA-evoked release was inhibited by the non-NMDA antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)-quinoxaline (NBQX) but was not affected by TTX. NMDA and AMPA showed synergistic effects, indicating co-existence of NMDA and AMPA receptors on noradrenergic terminals. Kainate evoked [³H]NA release only at high concentrations and the release was not potentiated by blocking kainate receptor desensitization with concanavalin A. Thus, the results indicate that there are stimulatory presynaptic NMDA and AMPA receptors on noradrenergic axon terminals in the spinal cord and that they interact synergistically to evoke release of [³H]NA.     

Evoked secretion of [3H]noradrenaline and ATP from nerve varicosities isolated from the myenteric plexus of the guinea pig ileum

Neuronal varicosities, isolated from the myenteric plexus of guinea pig ileum longitudinal muscle, were incubated with [3H]noradrenaline to label the contents of the noradrenergic secretory vesicles. Exposure of these varicosities to KCl, nicotine, or acetylcholine resulted in the Ca2+ -dependent release of [3H]noradrenaline. Veratridine also evoked a large efflux of [3H] from this preparation, but this release was only partially Ca2+ dependent. The alpha 2-adrenoceptor agonist, clonidine, inhibited the K+-, nicotine-, and acetylcholine-induced release of [3H]noradrenaline. Similarly, exogenously administered (-)noradrenaline was an effective inhibitor of the K+ -evoked release of [3H]noradrenaline. The alpha 2-adrenoceptor antagonist, yohimbine, antagonized the inhibitory actions of both clonidine and (-)noradrenaline on the K+ -evoked release of [3H]noradrenaline from myenteric varicosities. Nicotine, acetylcholine, KCl, and veratridine also released ATP from these guinea pig ileal myenteric varicosities. However, the evoked release of ATP was unaffected by clonidine. These results indicate that myenteric varicosities can take up and release [3H]noradrenaline and that they possess presynaptic alpha 2-adrenoceptors which, when activated, inhibit the release of [3H]noradrenaline. These receptors may play a role in modulating the release of noradrenaline in the myenteric plexus in vivo. In addition, the present results suggest that ATP and [3H]noradrenaline may not be released from the same population of secretory vesicles in neuronal varicosities isolated from guinea pig ileum longitudinal muscle.     

Presynaptic effects of neuropeptide Y on [3H]noradrenaline and [3H]acetylcholine release

The electrically evoked release of radioactivity from mouse vas deferens and rat hypothalamic slices preloaded with [3H]noradrenaline was measured. In addition the release of [3H]acetylcholine from longitudinal muscle strip of guinea-pig ileum was also measured. Neurochemical evidence has been obtained that neuropeptide Y (NPY), although it co-exists and is released with (-)-noradrenaline (NA), it behaves differently as far as its effect on presynaptic modulation of chemical neurotransmission is concerned. It exerts a frequency-dependent presynaptic inhibitory effect on noradrenaline release from mouse vas deferens but has no effect on the electrically evoked release of NA from rat hypothalamus. Unlike NA, NPY does not influence the release of [3H]acetylcholine from the longitudinal muscle strip of guinea-pig ileum and does not potentiate the presynaptic effect of NA. It seems very likely, that the inhibitory effect of NPY is mediated via receptors. Its action is concentration dependent. While exogenous noradrenaline inhibited the release of noradrenaline by 91%, the maximum inhibition reached with NPY was not higher than 60%, indicating that either the intrinsic activity of NPY is lower or much less axon terminals are equipped with NPY receptors. Peptide YY (PYY) also reduced the release of NA from mouse vas deferens.     

Effects of muscarinic toxins MT1 and MT2 from green mamba on different muscarinic cholinoceptors

MT1 and MT2, polypeptides from green mamba venom, known to bind to muscarinic cholinoceptors, behave like muscarinic agonists in an inhibitory avoidance task in rats. We have further characterised their functional effects using different preparations. MT1 and MT2 behaved like relatively selective muscarinic M₁ receptor agonists in rabbit vas deferens, but their effects were not reversed by washing or prevented by muscarinic antagonists, although allosteric modulators altered responses to MT1. Radioligand binding experiments indicated that both toxins irreversibly inhibited [³H]N-methylscopolamine binding to cloned muscarinic M₁ and M₄ receptors, and reduced binding to M₅ subtype with lower affinity, while they reversibly inhibited the binding of [³H]prazosin to rat cerebral cortex and vas deferens, with 20 fold lower affinity. High concentrations of MT1 reversibly blocked responses of vas deferens to noradrenaline. MT1 and MT2 appear to irreversibly activate muscarinic M₁ receptors at a site distinct from the classical one, and to have affinity for some -adrenoceptors.     

Functional and neurochemical evidence that neurotensin-induced release of acetylcholine from Auerbach's plexus of guinea-pig ileum is presynaptically controlled via α2-adrenoceptors

The effect of neurotensin (NT) on [³H]acetylcholine release and contraction from isolated longitudinal muscle strip of guinea-pig ileum was examined. Neurotensin dose-dependently enhanced the release of [³H]-acetylcholine. This effect of neurotensin was inhibited by stimulation of ₂-adrenoceptors: noradrenaline, clonidine, xylazine or dexmedetomidine (₂-adrenoceptor agonists) inhibited neurotensin-induced release of acetycholine (ACh) as well as the contractions, while CH-38083 or yohimbine (₂-adrenoceptor antagonist) prevented this inhibitory effect. Our findings suggest that neurotensin may play a neuromodulatory role in the regulation of cholinergic neuronal activity in the gut and this modulatory effect is continuously controlled by the tonic activity of the sympathetic nervous system: endogenous noradrenaline release is capable of reducing the release of ACh and the consequent contraction of the gut enhanced by neurotensin.     

Effect of Noradrenergic Lesions on Subtypes of α2-Adrenoceptors in Rat Brain

Abstract: The binding of [³H]rauwolscine to α_2A- (also referred to as α_2D-) and α_2C-adrenoceptors in homogenates of rat cerebral cortex was measured by exploiting the selectivity of oxymetazoline for α_2A-adrenoceptors. Inhibition of [³H]rauwolscine binding by oxymetazoline was modeled best assuming binding to two sites (p < 0.001). Competition curves for oxymetazoline were shifted rightward by the addition of GTP (250 µM) but were still fit best by a two-site model (p < 0.001). A concentration of oxymetazoline was calculated that would optimally antagonize [³H]rauwolscine binding (with GTP present) to oxymetazoline-sensitive α_2A-adrenoceptors, minimally inhibiting binding to α_2C-adrenoceptors. Subsequently, [³H]rauwolscine binding to α_2A- and α_2C-adrenoceptors in cortex was examined 3 weeks after destruction of noradrenergic terminals. Binding to α_2C-adrenoceptors was increased significantly after treatment with 6-hydroxydopamine (6-OHDA) compared with vehicle-treated controls, whereas binding to α_2A-adrenoceptors was unchanged. Pretreatment of rats with desipramine before 6-hydroxydopamine, to protect noradrenergic neurons, resulted in no changes in binding to either α_2A- or α_2C-adrenoceptors. Thus, α_2C-adrenoceptors are regulated by changes in synaptic availability of norepinephrine. α_2A-Adrenoceptors are either not regulated by synaptic norepinephrine or are located both post- and presynaptically so that up-regulation of postsynaptic α_2A-adrenoceptors is offset by a loss of presynaptic α_2A-adrenoceptors.     

The effect of cocaine on kinetics of α<Subscript>1</Subscript>-adrenergic contractile response in different portions of the rat vas deferens

The effect of cocaine (10 μM) on the kinetics of contractile response to noradrenaline (NA) was studied in the rat epididymal and prostatic vas deferens. Cocaine caused an acute increase in vas deferens adrenergic sensitivity primarily due to blockage of NA neuronal capture. The presynaptic action prevailed in the epididymal half: the EC 50 value decreased 32-fold with a slight increase of the maximum adrenergic response more evident in the prostatic half. In the presence of cocaine, the prostatic contraction to NA was mediated not by a single pool of α₁-adrenoceptors like in epididymal vas deferens but by two. Its high affinity pool had the same affinity as α₁-adrenoceptors of the epididymal half, the affinity value of the low one was 36-fold less, and the total maximal response of both pools increased 4.5-fold. The differences in cocaine effect on the rat epididymal and prostatic vas deferens contractions to NA appear to be caused by the different sources of Ca²⁺ involved in these responses.     

Role of Adenylate Cyclase in Presynaptic α2-Adrenoceptor- and μ-Opioid Receptor-Mediated Inhibition of [3H]Noradrenaline Release from Rat Brain Cortex Slices

Rat brain cortex slices, prelabelled with [3H]noradrenaline, were superfused and exposed to electrical biphasic block pulses (1 Hz; 12 mA, 4 ms) or to the Ca2+ ionophore A 23187 (10 microM) in the presence of 1.2 mM Ca2+. Forskolin (10 microM), 8-bromo-cyclic AMP (300 microM), and dibutyryl-cyclic AMP (300 microM) facilitated both the electrically evoked and A 23187-induced [3H]noradrenaline release, whereas the phosphodiesterase inhibitors 3-isobutyl-1-methylxanthine (IBMX, 300 microM) and 4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidone (ZK 62771, 30 microM) enhanced the electrically evoked release only. The inhibitory effects of clonidine (1 nM-1 microM) and the facilitatory effect of phentolamine (0.01-10 microM) on the electrically evoked [3H]noradrenaline release were strongly reduced in the presence of 8-bromo-cyclic AMP. Clonidine (1 microM) reduced and phentolamine (3 microM) enhanced A 23187-induced [3H]noradrenaline release, provided that the slices were simultaneously exposed to forskolin. The inhibitory effects of morphine (1 microM) and [D-Ala2-D-Leu5]enkephalin (DADLE, 0.3 microM), like that of the Ca2+ antagonist Cd2+ (15 microM), on the electrically evoked release of [3H]noradrenaline were not affected by 8-bromo-cyclic AMP. Moreover, morphine and DADLE did not inhibit A 23187-induced release in the absence or presence of forskolin. These data strongly suggest that in contrast to presynaptic mu-opioid receptors, alpha 2-adrenoceptors on noradrenergic nerve terminals are negatively coupled to adenylate cyclase and may thus reduce neurotransmitter release by inhibiting the feed-forward action of cyclic AMP on the secretion process.     

The role of various calcium and potassium channels in the regulation of somatodendritic serotonin release

We prepared slices from midbrain containing the raphe nuclei and from hippocampus of rats. The brain slices were loaded with [³H]serotonin and superfused in order to measure the release of radioactivity at rest and in response to electrical stimulation. No difference was observed in the resting and stimulated fractional release of tritium in the somatodendritic and axon terminal parts of serotonergic neurons. The selective 5-HT_1A receptor agonist 8-OH-DPAT decreased the electrically induced tritium effux from raphe nuclei slices preloaded with [³H]serotonin, and this inhibition was reversed by 5-HT_1A receptor antagonist (+)WAY-100135. The 5-HT_1B receptor agonist CGS-12066B but not 8-OH-DPAT, inhibited the stimulation-evoked tritium efflux from hippocampal slices after labeling with [³H]serotonin. The electrical stimulation-evoked tritium efflux in raphe nuclei slices incubate with [³H]serotonin was completely external Ca²⁺-dependent, and omega-conotoxin GVIA and Cd²⁺, but not diltiazem, inhibited the tritium overflow. In raphe nuclei slices 4-aminopyridine enhanced the electrical stimulation-induced trititum release in a concentration-dependent manner. The inhibition of tritium efflux by 8-OH-DPAT was abolished with 4-aminopyridine. Glibenclamide or tolbutamide proved to be ineffective. These data indicate that (1) different 5-HT receptor subtypes (5-HT_1A and 5-HT_1B) regulate dendritic and axon terminal 5-HT release; (2) serotonin release from the dendrites may be regulated by the voltage-sensitive N-type Ca²⁺ channels; (3) the 5-HT_1A receptor-mediated inhibition of serotonin release may be due to opening of voltage-sensitive K⁺ channels.     

Effect of Presynaptic P2 Receptor Stimulation on Transmitter Release

Because ATP is degraded to adenosine, its effect could be mediated by both P1 and P2 receptors. Hence, the actions of an ATP analogue, resistant to enzymatic breakdown (alpha, beta-methylene ATP), were studied on the resting and electrically evoked release of radioactivity from longitudinal muscle strips of guinea pig ileum, preloaded either with [3H]choline or with [3H]noradrenaline. Their effects were compared with the actions of adenosine and ATP. Although adenosine and ATP markedly decreased the [3H]acetylcholine release evoked by field stimulation, alpha,beta-methylene-ATP, a potent and selective agonist of P2x receptors, enhanced this release. However, 2-methyl-2-thio-ATP, an agonist of the P2y receptors, neither enhanced nor inhibited the [3H]-acetylcholine release. 8-Phenyltheophylline, an antagonist of P1 receptors, increased the stimulation-evoked release of acetylcholine, indicating that the release of acetylcholine is tonically controlled by endogenous adenosine via P1 receptors. When alpha,beta-methylene-ATP and 8-phenyltheophylline were added together, their potentiating effect on the acetylcholine release proved to be additive. Because alpha,beta-methylene-ATP failed to antagonize the presynaptic effect of adenosine on P1 purinoceptors, it seems very likely that its effect to enhance transmitter release is mediated via separate receptors, i.e., via P2x receptors, located on the axon terminals. Similarly, the stimulation-evoked release of [3H]noradrenaline was enhanced slightly by alpha,beta-methylene-ATP. Our results suggest that both cholinergic and noradrenergic axon terminals are equipped with P2 receptors through which the stimulation-evoked release of transmitter can be modulated by ATP in a positive manner.(ABSTRACT TRUNCATED AT 250 WORDS)     

Imipramine-Induced Changes in 5-HT2 Receptor Sites and Inositoltrisphosphate Levels in Rat Brain

The effect of chronic administration of Imipramine on [³H]Spiperone binding to 5-HT₂ sites and inositoltrisphosphate (IP₃) levels in rat cerebral cortex was studied. Our data shows that treatment with imipramine (5 mg/kg body weight, intraperitoneally) for 30 days significantly down regulates 5-HT₂ receptors sites (262 ± 29 fmol/mg protein) in cerebral cortex (38%), compared to control rats (425 ± 60 fmol/mg protein., P < 0.001). However there was no significant change in the affinity of [³H]-Spiperone binding (kd) to 5-HT₂ sites in cerebral cortex after exposure to imipramine (Kd = 0.84 ± 0.11 nM). It is also observed that imipramine treatment significantly reduces 5-HT stimulated [³H]IP₃ formation in cerebral cortex (6,411 ± 708 dpm/mg protein), compared to the saline treated rats (12,238 ± 1,544 dpm/mg protein; P < 0.001), with concomitant decrease in Pdtlns-4–5-P₂. This study suggests that the therapeutic action of imipramine in brain might be by reducing hypersensitivity of 5-HT₂ receptors by down regulation, which leads to reduced levels of inositolphospholipids. This inturn reduces the levels of IP₃. In conclusion, imipramine acts at presynaptic site by blocking the reuptake of serotonin and at post synaptic site it downregulates 5-HT₂ sites with decreased IP₃ levels after chronic exposure.     

A quantitative autoradiographic study of muscarinic cholinergic receptor subtypes in the brains of pyrithiamine-treated rats

Previous studies describe decreased acetylcholine synthesis in brain as well as neurobehavioural evidence for a central muscarinic cholinergic deficit in pyrithiamine-induced thiamine-deficient rats. In order to further evaluate this possibility, quantitative autoradiographic procedures using [³H]quinuclidinyl benzilate (for total muscarinic binding sites), [³H]pirenzepine (for muscarinic M₁ sites) and [³H]AF-DX 384 (for muscarinic M₂ sites) were performed at early (presymptomatic) and late (symptomatic) stages of thiamine deficiency induced in rats by administration of the central thiamine antagonist, pyrithiamine. No significant alterations in densities of M₁, M₂ or total muscarinic binding sites were observed in any brain structure evaluated at either early or late stages of thiamine deficiency. These findings do not support a major role for modifications of muscarinic cholinergic function in the pathogenesis of the neurological symptoms of thiamine deficiency.     

Spontaneous and evoked release of [3H]taurine from a P2 subcellular fraction of the rat retina

The effects of spontaneous and evoked [³H]taurine release from a P₂ fraction prepared from rat retinas were studied. The P₂ fraction was preloaded with [³H]taurine under conditions of high-affinity uptake and then examined for [³H]taurine efflux utilizing superfusion techniques. Exposure of the P₂ fraction to high K⁺ (56 mM) evoked a Ca²⁺-independent release of [³H]taurine. Li⁺ (56 mM) and veratridine (100 M) had significantly less effect (8–15% and 15–30%, respectively) on releasing [³H]taurine compared to the K⁺-evoked release. 4-Aminopyridine (1 mM) had no effect on the release of [³H]taurine. The spontaneous release of [³H]taurine was also Ca²⁺-independent. When Na⁺ was omitted from the incubation medium K⁺-evoked [³H]taurine release was inhibited by approximately 40% at the first 5 minute depolarization period but was not affected at a second subsequent 5 minute depolarization period. The spontaneous release of [³H]taurine was inhibited by 60% in the absence of Na⁺. Substitution of Br^– for Cl^– had no effect on the release of either spontaneous or K⁺-evoked [³H]taurine release. However, substitution of the Cl^– with acetate, isethionate, or gluconate decreased K⁺-evoked [³H]taurine release. Addition of taurine to the superfusion medium (homoexchange) resulted in no significant increase in [³H]taurine efflux. The taurine-transport inhibitor guanidinoethanesulfonic acid increased the spontaneous release of [³H]taurine by approximately 40%. These results suggest that the taurine release of [³H]taurine is not simply a reversal of the carrier-mediated uptake system. It also appears that taurine is not released from vesicles within the synaptosomes but does not rule out the possibility that taurine is a neurotransmitter. The data involving chloride substitution with permeant and impermeant anions support the concept that the major portion of [³H]taurine release is due to an osmoregulatory action of taurine while depolarization accounts for only a small portion of [³H]taurine release.     

Autoradiography of muscarinic cholinergic receptors in cortical and subcortical brain regions of C57BL/6 and DBA/2 mice

Mice of the inbred strains C57BL/6 and DBA/2 show strain-dependent behavioural differences which have been correlated with variations in brain cholinergic systems. In the present study, the density of muscarinic cholinergic receptors in both strains of mice was determined by autoradiographic methods using [³H]quinuclidinyl benzilate (QNB) and [³H]pirenzepine as ligands. C57BL/6 mice showed a significantly lower [³H]QNB binding level in the frontal cortex by one third as compared to DBA/2 mice. In the striatum and the cholinergic pontomesencephalic nucleus laterodorsalis tegmenti the [³H]QNB binding was lower in C57BL/6 by 28% and 31%, respectively. The [³H]pirenzepine binding level was found to be significantly higher in C57BL/6 temporal cortex (by 22%). These results are discussed in relation to interstrain differences in cholinergic cell density and in the activity of cholinergic enzymes.     

Immunohistochemical characteristics of nerve fibres supplying the porcine vas deferens. A colocalisation study

 Double-labelling immunofluorescence was used to investigate the coexistence of the catecholamine-synthesising enzymes, tyrosine hydroxylase and dopamine-β-hydroxylase and several neuropeptides including neuropeptide Y, vasoactive intestinal polypeptide, Leu⁵-enkephalin, somatostatin, calcitonin gene-related peptide and substance P in nerve fibres supplying the vas deferens in juvenile and adult pigs. The study has revealed three major populations of nerve terminals innervating the organ: (1) noradrenergic fibres; (2) non-noradrenergic (putative cholinergic) fibres containing vasoactive intestinal polypeptide, neuropeptide Y and somatostatin, supplying almost exclusively the lamina propria; and (3) non-noradrenergic, presumably sensory fibres, containing calcitonin gene-related peptide and substance P. The population of noradrenergic nerves can be divided into three subpopulations: a somatostatin-containing, a Leu⁵-enkephalin-containing and a subpopulation immunonegative to the peptides investigated, in descending order of magnitude. Coexistence patterns of the substances existing within nerve fibres supplying the vas deferens blood vessels are clearly different from those found in nerve fibres innervating the organ wall. The majority of the noradrenergic fibres associated with blood vessels contain neuropeptide Y only, while non-noradrenergic perivascular nerves contain predominantly vasoactive intestinal polypeptide. The possibility of different sources of origin of the particular nerve fibre subpopulations supplying the porcine vas deferens and its blood vessels is discussed. Accepted: 23 October 1996     

Involvement of phospholipase A2 and arachidonic acid in the depolarization-evoked accumulation of Ca2+ in hippocampal mossy fiber nerve endings

Depolarization-evoked increases in intraterminal free Ca²⁺ are required for the induction of neurotransmitter release from nerve terminals. Although the mechanisms that regulate the voltage-induced accumulation of presynaptic Ca²⁺ remain obscure, there is evidence that the phospholipase-dependent accumulation of arachidonic acid, or its metabolites, may be involved. Therefore, fura-2 loaded hippocampal mossy fiber nerve endings were used to investigate the relationships between membrane depolarization, lipid metabolism and presynaptic Ca²⁺ availability. It was observed that depolarization of the nerve terminals with KCl induced an increase in intraterminal free calcium that was inhibited more than 90% by a combination of voltage-sensitive Ca²⁺ channel blockers. In addition, the K⁺-dependent effects on Ca²⁺ concentrations were attenuated in the presence of phospholipase A₂ inhibitors, but were mimicked by the phospholipase A₂ activator melittin and exogenous arachidonic acid. Both the melittin- and arachidonic acid-induced increases in presynaptic Ca²⁺ were reduced by voltage-sensitive Ca²⁺ channel blockers. The stimulatory effects of arachidonic acid appeared to be independent of its further metabolism to prostaglandins. In fact, inhibition of either cyclooxygenase or lipoxygenase pathways resulted in a potentiation of the depolarization-evoked increase in intraterminal free Ca²⁺. From these results, we propose that some portion of the depolarization-evoked increase in intraterminal free calcium depends on the activation of phospholipase A₂ and the subsequent accumulation of unesterified arachidonic acid.     

Alpha2 adrenergic receptors are located prejunctionally in the Auerbach's plexus of the guinea pig small intestine: Direct demonstration by radioligand binding

We provide direct evidence that alpha₂-receptors in the guinea pig small intestine are localized prejunctionally in neurons of the Auerbach's plexus. The alpha₂-agonist ligand [³H]clonidine bound to a single saturable class of sites with a K_d of 1–2 nM and a capacity of approximately 70 fmol/mg protein in membranes from the innervated longitudinal and circular muscle layers of the intestine. By a special dissection technique the Auerbach's plexus could be completely removed from the longitudinal muscle. In these denervated preparations the clonidine binding sites were virtually completely removed whereas the expected binding was observed in innervated controls. The innervated preparations also contained a small number of alpha₁-receptors as revealed by binding with [³H]prazosin (capacity approximately 18 fmol/mg protein with a K_d of 0.4-0₃7 nM). Thus, the present study suggests that alpha₂-receptors ([³H]clonidine binding sites) are localized in neurons (i.e., prejunctionally) in the Auerbach's plexus of the guinea pig small intestine.     

Heterogeneity,position, and functional capability of the macrophages in Peyer's patches

Summary Radioenzymatic assays and light microscope radioautographic studies performed on photophores of Porichthys notatus demonstrated (1) significant amounts of catecholamines (dopamine, noradrenaline, adrenaline) and 5-hydroxytryptamine (serotonin) in these organs; (2) selective uptake and storage of [³H]noradrenaline ([³H]NA) by axon terminals innervating the photocytes, and (3) strong accumulation of [³H]5-hydroxytryptamine ([³H]5-HT) within the photocytes. Uptake and storage of [³H]NA in the nerve fibers were seemingly unaffected by the addition of ten-fold molar concentrations of unlabelled serotonin. Accumulation of [³H]5-HT by the photocytes was dose-dependent and diminished markedly in the presence of ten-fold molar concentrations of non-radioactive noradrenaline. Neither neuronal uptake of [³H]5-HT or [³H]A, nor photocytic accumulation of [³H]A were detectable under the conditions of the present experiments. This information should provide a framework for further investigations of the regulation of photophore luminescence by the biogenic amines.Supported by grants from the National Research Council and Medical Research Council of CanadaJacques de Champlain and Lise Farley provided facilities and expertise with the radioenzymatic techniques. The technical assistance of Sylvia Garcia and Marie-Hélène Parizeau was also appreciated     

The Na+-Ca2+ Exchange Inhibitor KB-R7943 Inhibits High K+-Induced Increases in Intracellular Ca2+ Concentration and [3H]Noradrenaline Release in the Human Neuroblastoma SH-SY5Y

The effects of the Na⁺-Ca²⁺ exchange inhibitor 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulfonate (KB-R7943) on depolarization-induced Ca²⁺ signal and [³H]noradrenaline release were examined in SH-SY5Y cells. KB-R7943 at 10 M significantly inhibited high K⁺-induced increase in intracellular Ca²⁺ concentration. KB-R7943 also inhibited high K⁺-evoked release of [³H]noradrenaline from the cells. These findings suggest that the Na⁺-Ca²⁺ exchanger in the reverse mode is involved at least partly in depolarization-induced transmitter release.