Effects of coexisting neurochemicals on the release of serotonin from the intermediate area of rat thoracic spinal cord

Serotonin (5-HT), substance P (SP), neurokinin A (NKA), and thyrotropin-releasing hormone (TRH) coexist in the nerve terminals of the intermediolateral cell column (IML) of the thoracic spinal cord. The Ca2⁺-dependent release of 5-HT from the microdissected intermediate area (including the IML) of the rat thoracic spinal cord, and the 5-HT_1B autoreceptor regulator of 5-HT release, were previously demonstrated. In this paper, the effects of SP, NKA, TRH, and/or their analogs on the release of [³H]5-HT from the intermediate area were investigated using an in vitro superfusion system. Both SP (the endogenous ligand for neurokinin-1 (NK₁) receptor) and an NK₁, agonist (GR 73632) significantly increased the basal release of [³3H]5-HT. SP and GR 73632 did not change the K⁺-stimulated release of [³H]5-HT. The effect of the NK₁ agonist on the basal release of [³H]5-HT was dose-dependent, was reduced by an NK₁ antagonist (GR 82334), and was not dependent on extracellular Ca²⁺. Neither NKA, an NK₂, agonist (GR 64349), nor a TRH analog (MK-771) altered the basal or stimulated release of [³H]5-HT. These data suggest that basal release of 5-HT from the intermediate area of the rat thoracic spinal cord is regulated by SP (acting through an NK₁ receptor), but not by NKA or TRH. These results provide evidence for the role of SP as a modulator of serotoninergic neurons in the intermediate area of the thoracic spinal cord, and may help to clarify the role of coexisting neurochemicals in the spinal regulation of the sympathetic nervous system. © 1995 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Ionic and haemodynamic changes influence the release of the excitatory amino acid glutamate in the posterior hypothalamus

The push-pull technique was used to investigate the release of the excitatory amino acid glutamate in the posterior hypothalamic area of the conscious rat. The hypothalamus was superfused through the pushpull cannula with artificial cerebrospinal fluid (CSF), and the superfusate was collected in time periods of 10 min when ionic conditions in the CSF were changed, or in short periods of 3 min when blood pressure changes were evoked. The mean glutamate release rate was 2.8 + 0.7 pmol/min. Depolarization by hypothalamic superfusion with CSF containing 50 mM K⁺ enhanced the release of glutamate in the presence of Ca²⁺. The K⁺-induced release was attenuated by 40% when the hypothalamus was superfused with Ca²⁺-free CSF. Replacement of Ca²⁺ by Mg²⁺ abolished the K⁺-induced release of glutamate. Hypovolaemia elicited by haemorrhage enhanced the release rate of glutamate. Similarly, a hypotension elicited by i.v. injection of chlorisondamine (3 mg/kg) led to a pronounced and permanent enhancement in glutamate release. The effects of hypovolaemia and chlorisondamine on glutamate release were abolished in aortic denervated rats, indicating that this response is due to a decrease of impulse generation in baroreceptors. A hypovolaemia elicited by blood infusion did not affect the release of glutamate. Similarly, a pronounced pressor response to phenylephrine (15 /kg per minute) infused intravenously for 9 min was ineffective.The results show that the K⁺-induced release of glutamate in the hypothalamus is dependent on the presence of Ca²⁺. The increase in glutamate release rate by hypovolaemia or chlorisondamine suggests that the glutamatergic neurons in the posterior hypothalamic area respond to unloading of aortic baroreceptors and possess a counteracting, hypertensive function.     

Ionic mechanism of 4-aminopyridine action on leech neuropile glial cells

Extracellular 4-aminopyridine (4-AP), tetraethylammonium chloride (TEA) and quinine depolarized the neuropile glial cell membrane and decreased its input resistance. As 4-AP induced the most pronounced effects, we focused on the action of 4-AP and clarified the ionic mechanisms involved. 4-AP did not only block glial K+ channels, but also induced Na+ and Ca2+ influx via other than voltage-gated channels. The reversal potential of the 4-AP-induced current was -5 mV. Application of 5 mM Ni2+ or 0.1 mM d-tubocurarine reduced the 4-AP-induced depolarization and the associated decrease in input resistance. We therefore suggest that 4-AP mediates neuronal acetylcholine release, apparently by a presynaptic mechanism. Activation of glial nicotinic acetylcholine receptors contributes to the depolarization, the decrease in input resistance, and the 4-AP-induced inward current. Furthermore, the 4-AP-induced depolarization activates additional voltage-sensitive K+ and Cl- channels and 4-AP-induced Ca2+ influx could activate Ca2+-sensitive K+ and Cl- channels. Together these effects compensate and even exceed the 4-AP-mediated reduction in K+ conductance. Therefore, the 4-AP-induced depolarization was paralleled by a decreasing input resistance.     

Effects of local MAO inhibition in the locus coeruleus on extracellular serotonin and 5-HIAA during exposure to sensory and cardiovascular stimuli

Previously, we have shown that in the presence of pargyline the release of serotonin (5-HT) in the locus coeruleus is modulated by various sensory stimuli and blood pressure fluctuations. The aim of the present study was to investigate whether local inhibition of monoamine oxidase (MAO) influences basal and stimulus-induced release of 5-HT in the locus coeruleus. For this purpose, the locus coeruleus was superfused in the absence and in the presence of the MAO inhibitor pargyline. Additionally, we examined whether the release of the 5-HT metabolite 5-hydroxy-indole acetic acid (5-HIAA) in the locus coeruleus is altered in response to stimuli. The locus coeruleus of the conscious rat was superfused through a push-pull cannula with artificial cerebrospinal fluid (CSF). 5-HT and 5-HIAA were determined in the superfusate. The basal release rate of 5-HT and the basal outflow of 5-HIAA averaged 2.0 fmol/min and 69 fmol/min, respectively. The basal release rate of 5-HT and the 5-HIAA outflow were tetrodotoxin (TTX)-sensitive. In the absence of pargyline, the sensory stimuli noise stress or tail pinch, applied for 10 min, increased 5-HT and 5-HIAA outflow by 50–70%. In contrast, an experimentally induced rise in blood pressure for 10 min enhanced 5-HT release by 50%, but had no effect on 5-HIAA outflow. The release of 5-HT and/or 5-HIAA elicited by sensory stimuli or a blood pressure rise was abolished by TTX. Addition of pargyline to the CSF enhanced 5-HT release fourfold and slightly decreased 5-HIAA outflow. These levels remained stable throughout the entire observation period of 8 h. In the presence of pargyline, 5-HT release elicited by noise, tail pinch and increase in blood pressure was enhanced. It is concluded that superfusion with pargyline enhances 5-HT release and reduces 5-HIAA outflow in the locus coeruleus. Furthermore, the ability of sensory stimuli and baroreceptor activation to enhance 5-HT release is preserved during a prolonged pargyline-induced increase in extracellular 5-HT. Since sensory stimuli enhanced, while baroreceptor activation did not influence 5-HIAA outflow, 5-HIAA is not a reliable index for short-term changes in the activity of serotonergic neurons in the locus coeruleus. Received: 13 July 1998 / Accepted: 10 December 1998     

Cocaine's effects on speech sound identification and reaction times in baboons

The effects of cocaine on speech sound discriminations was examined to determine whether cocaine's previously demonstrated effect in reducing speech sound discriminability was dependent upon either the type of stimuli employed (simple tones versus complex speech) or the procedure (stimulus detection versus stimulus discrimination). Because of demonstrated similarities in the way that baboons and humans discriminate speech, and in the way the CNS is thought to encode and process speech sounds in these two species, baboons were trained to perform a choice procedure to identify the occurrence of different synthetic vowel sounds (/a/, /æ/, //, /U/, and /љ/). Animals held down a lever and released the lever only when one of four target vowels sounded, and not when a fifth, standard vowel sounded. Acute IM administration of cocaine (0.0032–1.0 mg/kg) produced dose-dependent decreases in vowel discriminability that were mostly due to elevations in false alarms (i.e., releases to the standard vowel) following cocaine. Cocaine also shortened reaction times to the stimuli in two of three baboons, but to a much lesser extent than observed previously. These results suggest that cocaine may interfere with the ability of the CNS to process the acoustic cues in speech sounds, and that the effects of cocaine on reaction times may depend upon the complexity of the reaction time procedure employed.     

Are the disparate pharmacological profiles of competitive and un-competitive NMDA antagonists due to different baseline activities of distinct glutamatergic pathways? (Hypothesis)

Summary In superfused mouse striatal slices preincubated with [³H] dopamine 25 nmol/l, the electrically (3 Hz) evoked tritium overflow was inhibited by histamine 10 mol/l by 18%. The degree of inhibition was increased to 38% by haloperidol but not affected by (1) atropine, (2) reducing the stimulation frequency to 0.3 Hz or (3) increasing the concentration of [³H]dopamine (used for preincubation) to 100 nmol/l. The effect of histamine was mimicked by the H₃ agonist R-(–)--methylhistamine; it was not affected by the H₁ antagonist dimetindene and the H₂ antagonist ranitidine but abolished by the H₃ antagonist thioperamide. Tritium overflow evoked by Ca²⁺ ions (introduced into Ca²⁺free, K⁺-rich medium containing tetrodotoxin) was not affected by histamine 10 mol/l in the absence, but inhibited (by 30%) in the presence of haloperidol; the effect of histamine was abolished by thioperamide. In conclusion, the dopaminergic nerve terminals in the mouse striatum are endowed with presynaptic H₃ receptors. Simultaneous blockade of dopamine autoreceptors increases the extent of the H₃ receptor-mediated inhibition of dopamine release.     

Mechanism of the enhancement in transmitter release from central and peripheral noradrenergic nerve terminals induced by the purified scorpion venom,tityustoxin

Summary In the isolated nerve-muscle preparation of the cat nictitating membrane exposure to 0.04 M of the scorpion venom tityustoxin (TsTX) increased significantly the overflow of ³H-noradrenaline and the responses elicited by postganglionic nerve stimulation (1200 pulses, 0.5 ms duration, supramaximal voltage). Concentration effect curves to exogenous (-)-noradrenaline were not affected in the presence of this concentration of TsTX.The enhanced release of ³H-noradrenaline obtained during nerve stimulation as well as the increase of the postsynaptic responses observed during exposure to TsTX were more pronounced at 4 Hz than at 20 Hz. The increase in the overflow of noradrenaline observed with the toxin was selective for nerve stimulation since the release evoked by tyramine was not affected by TsTX.TsTX did not increase further the enhancement of ³H-noradrenaline release obtained in the presence of 18 mM tetraethylammonium (TEA). On the other hand, both TsTX and TEA were able to increase further the overflow of ³H-noradrenaline after block of the presynaptic alpha-adrenoceptors with phenoxybenzamine 0.29 or 2.9 M.In slices of rat cerebral cortex, TsTX 0.04 M increased ³H-noradrenaline release induced by 10 mM and by 20 mM KCl. The increased release evoked by the toxin was more pronounced for the lower concentration of K⁺.An increased release of ³H-noradrenaline in the presence of the toxin was also observed in rat hypothalamic slices stimulated with 20 mM K⁺. The K⁺ stimulated induced release of ³H-noradrenaline was also increased by 1.8 mM TEA. As shown for the peripheral nervous, system the simultaneous addition of TEA and TsTX did not result in additive effects when compared with the effects of the two agents added separately. Tityustoxin did not modify the metabolic pattern of the neurotransmitter released by K⁺ from rat hypothalamic slices.It is concluded that TsTX increases the stimulation-induced release of ³H-noradrenaline from both peripheral and central noradrenergic nerve terminals. Tityustoxin appears to act on the nerve terminal by a mechanism similar to that of TEA, an agent known to enhance the amount of noradrenaline released by nerve stimulation by increasing the duration of the action potentials.     

Influence of selective phosphodiesterase inhibitors on human neutrophil functions and levels of cAMP and Cai

Summary Chromatographic analysis of 3,5-cyclic nucleotide phosphodiesterase (PDE) isoenzymes in the cytosol of human neutrophils shows the predominant presence of PDE IV (cAMP specific) and PDE V (cGMP specific). PDE IV is characterized by (1) cAMP selectivity, (2) a K_M for cAMP of 1.2 M and (3) a typical rank order of IC ₅₀-values for PDE inhibitors: 0.13, 0.17, 47 and 9.5 M for PDE IV selective rolipram, PDE III/IV selective zardaverine, PDE III selective motapizone and unselective 3-isobutyl-l-methylxanthine (IBMX), respectively. Functions of polymorphonuclear leukocytes (PMN) such as N-formylmethionyl-leucyl-phenylalanine (fMLP)-stimulated superoxide release and fMLP/thimerosal elicited leukotriene (LT) biosynthesis are inhibited by these PDE inhibitors with the same rank order and even lower IC₅₀-values. Measurements of changes in cytosolic Ca_i in Fura-2 loaded PMN demonstrate a transient Ca_i increase after stimulation with 0.1 M fMLP and an additional sustained elevation of Ca_i levels in the presence of thimerosal. PDE inhibitors suppress this sustained phase of Ca_i release with the same rank order of IC₅₀-values as LT biosynthesis. The correlation between fMLP/thimerosal-induced LT biosynthesis and Ca_i levels reveal a Ca_i threshold of 150 nM for arachidonic acid metabolism. cAMP levels in PMN were elevated by PDE inhibitors alone by less than 2-fold. In the presence of fMLP however, cAMP was increased up to 10-fold and the efficacy of PDE inhibitors to increase cAMP paralleled their potency to inhibit PDE IV. It is concluded that (1) suppression of PMN functions is achieved by PDE IV inhibition, (2) necessary cAMP elevations are within 50% increase, (3) superoxide release was affected by cAMP/protein kinase A (PKA) directly whereas (4) for inhibition of LT biosynthesis a cAMP related reduction of Ca-influx is involved. Send offprint requests to Ch. Schudt at the above address     

Species differences in presynaptic serotonin autoreceptors: mainly 5-HT1B but possibly in addition 5-HT1D in the rat, 5-HT1D in the rabbit and guinea-pig brain cortex

Summary The pharmacological properties of presynaptic serotonin autoreceptors were compared in slices of rat, rabbit, and guinea-pig brain cortex. The slices were preincubated with ³H-serotonin and then superfused with medium containing fluvoxamine 3 mol/l and stimulated four times by trains of four pulses delivered at 100 Hz. Cumulative concentration-response curves were determined and used for the calculation of agonist EC₅₀ values and maximal effects and antagonist K _B values.Unlabelled serotonin itself and the serotonin receptor agonists 5-carboxamidotryptamine (5-CT), 5-methoxy-3-(1,2,3,6-tetrahydro-4-pyridinyl)-1H-indole (RU 24969) and (±)-8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) reduced the stimulation-evoked overflow of tritium with a rank order of potency 5-CT = RU 24969 > serotonin > 8-OH-DPAT in the rat and 5-CT > serotonin > RU 24969 > 8-OH-DPAT in the rabbit and guinea-pig. Ipsapirone caused no change. Metitepine and metergoline antagonized the effect of 5-CT; the K _B values were lower in the rabbit and guinea-pig than in the rat. Yohimbine at up to 1 mol/1 did not reduce the evoked overflow of tritium and did not antagonize the inhibitory effect of 5-CT in the rat but reduced the evoked overflow in the rabbit and counteracted the effect of 5-CT in the guinea-pig. (–)-Propranolol, conversely, reduced the evoked overflow of tritium in the rat but neither reduced the evoked overflow nor antagonized the effect of 5-CT in the rabbit and guinea-pig. Isamoltane did not significantly change the effect of 5-CT in any species. In the rat, it also failed to antagonize the inhibitory effect of 8-OH-DPAT but did antagonize the effect of RU 24969. The inhibition caused by 8-OH-DPAT persisted in the presence of idazoxan but was attenuated by metitepine in all species.The experimental conditions used permit the determination of the constants of agonist and antagonist action undistorted by autoinhibition. The results confirm the view that the serotonin axons of rat brain possess 5-HT_1B autoreceptors. They show by direct comparison under identical conditions that the autoreceptors in rabbit and guinea-pig are very similar to each other but differ markedly from those in the rat. The results give additional credence to previous suggestions that, in the rabbit and guinea-pig, the autoreceptors are 5-HT_1D. The serotonin axons of rat brain cortex may possess 5-_1D in addition to 5-HT_1B autoreceptors. In many previous studies agonist potencies at, and antagonist affinities for, presynaptic serotonin autoreceptors have been underestimated due to the use of too intense stimuli to elicit serotonin release. Send offprint requests to N. Limberger at the above address     

Mu and kappa opioid receptor modulation of 5-HT3 and NK-3 receptor-evoked release of acetylcholine from the guinea-pig ileum myenteric plexus

Summary The effects of three different opioid agonists on contractions and [³H]-acetylcholine (ACh) release evoked by 5-hydroxytryptamine₃ (5-HT₃) and neurokinin-3 (NK-3) receptor activation were examined in the guinea-pig ileum longitudinal muscle-myenteric plexus strip (LMMP) preparation. The selective mu ()-opioid receptor agonist (d-Ala²,NMe-Phe⁴,Gly-ol]-enkephalin) (DAMGO; 1 nM–100 nM) and the selective kappa ()-opioid receptor agonist U50488 (10 nM -1 M) inhibited contractile responses to 5-HT and to the selective NK-3 receptor agonist senktide, producing a concentration-related progressive flattening of their concentration-response curves. IC₅₀ estimates for DAMGO and U50488 were somewhat higher for inhibition of 5-HT-evoked as compared to senktide-evoked contractions, and overall lay in the range 6 nM – 51 nM. The selective delta ()-opioid receptor agonist [d-Pen^2,5]-enkephalin (DPDPE) inhibited contractile responses only at the highest concentration used (1 M). ³H-overflow from LMMP preparations preincubated with [³H]-choline was measured as an indicator of [³H]-ACh release. DAMGO (1 nM –100 nM) and U50488 (10 nM -1 M) inhibited the increases in release of [³H]-ACh evoked by 5-HT (10 M) and by senktide (10 nM) in a concentration-dependant manner. IC₅₀ estimates for DAMGO and U50488 were not significantly different for inhibition of 5-HT as compared to senktide-evoked increases in [³H]-ACh release and lay in the range 6 nM –23 nM. DPDPE again only inhibited these responses at the maximum concentration used (1 M). The inhibitory effects of DAMGO, U50488 and DPDPE on contractions and [³H]-ACh release evoked by 5-HT and senktide were completely reversed by naloxone (10 M).These results show that ACh release in the guinea-pig ileum evoked by 5-HT and senktide can be modulated to a similar extent by the opioid agonists DAMGO and U50488, but not by DPDPE. This suggests that the pathways of excitation for 5-HT₃ and NK-3 receptors converge at some level susceptible to opioid inhibition, which may be mediated by - and -, but not -, opioid receptors.