Investigation of the refractive index repeatability for tantalum pentoxide coatings, prepared by physical vapor film deposition techniques.

Random effects in the repeatability of refractive index and absorption edge position of tantalum pentoxide layers prepared by plasma-ion-assisted electron-beam evaporation, ion beam sputtering, and magnetron sputtering are investigated and quantified. Standard deviations in refractive index between 4*10-4 and 4*10-3 have been obtained. Here, lowest standard deviations in refractive index close to our detection threshold could be achieved by both ion beam sputtering and plasma-ion-assisted deposition. In relation to the corresponding mean values, the standard deviations in band-edge position and refractive index are of similar order.

Serum KL-6 as a novel disease marker in adolescent and adult cystic fibrosis.

BACKGROUND: Cystic fibrosis (CF) is a chronic progressive disease leading to obstructive pulmonary impairment, fibrosis and shortened life expectancy. Serum levels of KL-6, high molecular weight human MUC1 mucin, are increased in the majority of patients with various interstitial lung disorders. Whether they are also elevated in CF has not been investigated before. OBJECTIVE: To evaluate whether serum KL-6 levels are elevated and correlate with pulmonary function variables in CF. DESIGN: Serum KL-6, lactate dehydrogenase (LDH) and C-reactive protein (CRP) levels were measured in 72 consecutive CF and 80 age- and sex-matched healthy control subjects. The relationship between serum KL-6 levels and pulmonary function variables was analyzed. RESULTS: Serum KL-6 levels in CF patients were significantly increased compared to healthy subjects. Receiver operating characteristic curve analysis revealed that the diagnostic accuracy of KL-6 was better than that of LDH and CRP. Serum KL-6 levels showed an inverse relationship with vital capacity (VC) % predicted and forced expiratory volume in one second (FEV1) % predicted. CONCLUSIONS: Serum KL-6 levels are elevated and appear to be correlated with pulmonary function variables in CF. These results suggest that KL-6 may be a useful noninvasive marker to monitor disease severity.

P2-receptor-mediated inhibition of serotonin release in the rat brain cortex.

The possibility of a P2-receptor-mediated modulation of the release of serotonin in the rat brain cortex was investigated in occipito-parietal slices preincubated with [3H]serotonin and then superfused and stimulated electrically (10 pulses, 1 Hz). Adenosine receptor agonists decreased the stimulation-evoked overflow of tritium at best slightly; the selective A1 agonist N6-cyclopentyl-adenosine caused no change. Several nucleotides had more marked effects: ATP (3-1000 microM), adenosine-5'-O-(3-thiotriphosphate) (3-300 microM) and P1,P5-di(adenosine-5')-pentaphosphate (3-300 microM) decreased the evoked overflow by up to ca 35%. AMP, alpha,beta-methylene-ATP and UTP produced smaller decreases and 2-methylthio-ATP and UMP caused no change. The inhibition by ATP was attenuated both by the P1-receptor antagonist 8-(p-sulphophenyl)-theophylline (100 microM) and by the P2-receptor antagonist suramin (300 microM) but was not changed by indomethacin (10 microM) and NG-nitro-L-arginine (10 microM). We conclude that the release of serotonin in the rat brain cortex is inhibited through presynaptic P1-receptors (which are not A1) as well as P2-receptors. Inhibition of release via P2-receptors has been previously shown for noradrenaline (brain cortex and hippocampus) and dopamine (neostriatum) and, hence, may be widespread. Differences between transmitter systems exist, however, in the degree of their sensitivity to presynaptic P2-receptor-mediated modulation.

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.

Tetrodotoxin-resistant release of 3H-noradrenaline from the mouse vas deferens by high intensity electrical stimulation.

Vasa deferentia of mice were preincubated with 3H-noradrenaline and then superfused with a medium containing cocaine 10 microM and phentolamine 30 microM. The tetrodotoxin-resistant outflow of tritium evoked by high intensity electrical field stimulation (0.5 Hz, 200 mA current strength, 2 ms pulse width) was studied and, in some experiments, compared with the tetrodotoxin-sensitive outflow evoked by low intensity electrical field stimulation (0.5 Hz, 50 mA, 1 ms). In contrast to the outflow evoked by low intensity stimulation, the outflow evoked by high intensity stimulation was increased in Na+-free medium, and was only partly dependent on the external Ca2+ concentration. The Ca2+-dependent fraction consisted mainly of 3H-noradrenaline. Again, in contrast to the outflow caused by stimulation at low intensity, that caused by stimulation at high intensity was not reduced by Mg2+ 20 mM, Co2+ 5 mM or normorphine 40 or 100 microM, and was not enhanced by tetraethylammonium 5 mM or 4-aminopyridine 1 mM. It is concluded that high intensity electrical stimulation elicits a tetrodotoxin-resistant, calcium-dependent release of noradrenaline which differs in mechanism from the release elicited by action potentials.

Release and modulation of release of serotonin in rabbit superior colliculus.

The release of previously incorporated [3H]serotonin and its presynaptic modulation were studied in slices of rabbit superior colliculus. Electrical stimulation at frequencies of 0.017-3 Hz greatly increased the outflow of tritiated compounds; this response was almost abolished by tetrodotoxin and in a low calcium medium. Unlabelled serotonin, when added in the presence of nitroquipazine, an inhibitor of high-affinity neuronal serotonin uptake, reduced the electrically evoked overflow of tritium, an effect antagonized by metitepin. Given alone, metitepin caused an increase. The evoked overflow was also decreased by clonidine, and the effect of clonidine was counteracted by phentolamine. Phentolamine itself increased the overflow response. However, this was probably not due to antagonism against an inhibitory effect of endogenous noradrenaline because, first, the selective alpha 2-adrenoceptor antagonist idazoxan did not share with phentolamine the overflow-enhancing effect, second, phentolamine continued to increase the overflow after noradrenergic axons had been destroyed by 6-hydroxydopamine, and third, the facilitatory effects of metitepin and phentolamine were not additive. Phentolamine, like metitepin, antagonized the presynaptic inhibitory effect of serotonin, indicating that it may increase the evoked overflow of tritium by blocking serotonin receptors rather than alpha-adrenoceptors. Ethylketocyclazocine decrease the electrically evoked overflow, and its effect was prevented by naloxone: peptides selective for opioid mu- or delta-receptors caused no change. Nicotine increased the basal outflow of tritium (in the absence of electrical stimulation); the increase was attenuated by hexamethonium and low calcium medium. No or minimal changes in tritium outflow were obtained with beta-adrenoceptor, dopamine receptor, muscarine receptor and GABA receptor ligands or with substance P and glutamate. In conjunction with our previous studies, these results indicate that serotonin is a neurotransmitter in the superior colliculus. Its release is modulated through presynaptic autoreceptors (probably 5-HT1), alpha 2-adrenoceptors, opioid kappa-receptors and nicotine receptors, of which only the autoreceptors receive an endogenous input, at least under the experimental conditions chosen. Each of the three groups of collicular monoamine axons that we have studied recently (cholinergic, noradrenergic, serotoninergic) possesses a specific pattern of presynaptic, release-modulating receptors. A physiological role seems likely only for the alpha 2-autoreceptors at the noradrenergic and the 5-HT1-autoreceptors at the serotoninergic axons.

Are presynaptic dopamine autoreceptors and postsynaptic dopamine receptors in the rabbit caudate nucleus pharmacologically different?

Slices of the rabbit caudate nucleus were preincubated with [3H]dopamine or [3H]choline and then superfused and stimulated electrically. Apomorphine reduced the stimulation-evoked overflow of tritium over the same concentration range, independently of whether slices had been pre-incubated with [3H]dopamine or with [3H]choline. Each of three antagonists--molindone, sulpiride and metoclopramide--increased the evoked overflow of tritium over the same concentration range in experiments with [3H]dopamine and those with [3H]choline. For each antagonist, the pA2 values against apomorphine obtained in [3H]dopamine experiments and in [3H]choline experiments were very similar. This study is a functional in vitro approach to receptor characterization, as opposed to radioligand binding studies or in vivo investigations. The results show that the dopamine receptor agonist apomorphine and three antagonists are unable to distinguish between the presynaptic, release-inhibiting dopamine autoreceptors and those postsynaptic dopamine receptors which, when activated, depress the release of acetylcholine. Although there are certainly more dopamine receptors in the caudate nucleus, these two physiologically important groups seem to be closely related.

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.

Axon terminal P2-purinoceptors in feedback control of sympathetic transmitter release.

Extracellular ATP acts on P2-purinoceptors of peripheral effector cells, and this is the basis for its function as a (co-)transmitter in peripheral efferent neurons. ATP also acts on P2-receptors of neuronal cell bodies or dendrites, and this is the basis for its function as a fast excitatory transmitter at neuroneural synapses. A third site of action is axon terminals. In the vas deferens of the mouse, noradrenaline and ATP are postganglionic sympathetic co-transmitters, and exogenous ATP acts on P2-purinoceptors of the sympathetic terminals to inhibit release of noradrenaline. Here we show that two P2 antagonists, suramin and Reactive Blue 2, increase the release of noradrenaline in mouse vas deferens. The increase is only obtained when there has been preceding nerve activity and is largely independent of the postjunctional response. These findings indicate a physiological function for axon terminal P2-purinoceptors: they mediate a novel prejunctional negative feedback in which released ATP inhibits subsequent transmitter release.

Co-release of noradrenaline and ATP from cultured sympathetic neurons.

The vesicles of postganglionic sympathetic axons store both noradrenaline and ATP. The theory of noradrenaline-ATP co-transmission implies that both compounds are released by nerve action potentials and elicit postjunctional effects. Many properties of postjunctional responses support the theory. However, neural release of ATP has been difficult to detect biochemically: by far the major part of the overflow of ATP from intact tissues upon sympathetic nerve stimulation comes from non-neural elements, especially smooth muscle and endothelial cells. Here we describe a parallel electrically evoked overflow of [3H]noradrenaline and endogenous ATP from cultured chick sympathetic neurons. The overflow was abolished by tetrodotoxin, omega-conotoxin and withdrawal of Ca2+, was increased by tetraethylammonium and 4-aminopyridine, and was not changed by prazosin or suramin. The results demonstrate directly the action potential-evoked, Ca(2+)-dependent and presumably vesicular and exocytotic release of ATP from postganglionic sympathetic neurons. They support the co-transmitter theory and suggest that cultured sympathetic neurons are a preparation in which noradrenaline-ATP co-release can be examined free from postjunctional components.

Inhibition of GABAergic inhibitory postsynaptic currents by cannabinoids in rat corpus striatum.

Electrophysiological consequences of activation of cannabinoid receptors have been mostly investigated on neuronal cell lines and on cells transfected with cannabinoid receptors. The aim of the present experiments was to study cannabinoid effects on identified neurons in situ. Electrically-evoked postsynaptic currents and voltage-dependent calcium currents were investigated in the principal neurons of the corpus striatum, the medium spiny neurons, with the patch-clamp method for brain slices. These neurons were chosen because they produce messenger RNA for cannabinoid receptors and because the density of cannabinoid binding sites in the striatum is high. Activation of muscarinic receptors by carbachol (10(-5) M) reduced inhibitory postsynaptic current amplitude by 67%. The synthetic cannabinoid receptor agonist R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4- benzoxazin-yl]-(1-naphtalenyl)methanone (WIN55212-2; 10(-8) to 10(-5) M) dose-dependently reduced striatal inhibitory postsynaptic currents; the maximum effect, inhibition by 52%, was observed at 10(-6) M. Another cannabinoid agonist, (-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydr oxypropyl)cyclohexanol (CP55940; 10(-6) M), also reduced inhibitory postsynaptic currents, by 50%. The CB1 cannnabinoid receptor antagonist N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)4-methyl-3-pyra zolecarboxamide (SR141716A; 10(-6) M) had no effect when given alone but abolished the effect of WIN55212-2 (10(-6) M). WIN55212-2 (10(-6) M) did not change the current evoked by the GABA(A)-receptor agonist muscimol (10(-6) M). Activation of muscarinic receptors by carbachol (10(-5) M) inhibited voltage-dependent calcium currents by 21%, but the cannabinoid receptor agonist WIN55212-2 (10(-6) M) was without effect. The results show that activation of CB1 cannabinoid receptors reduces GABAergic inhibitory postsynaptic currents in medium spiny neurons of the corpus striatum: the likely mechanism is presynaptic inhibition of GABA release from terminals of recurrent axons of the medium spiny neurons themselves.

Differences in the mode of stimulation of cultured rat sympathetic neurons between ATP and UDP.

Postganglionic sympathetic neurons possess at least two excitatory receptors for nucleotides: P2X-purinoceptors and separate receptors for uracil nucleotides. In cultured neurons from rat superior cervical ganglia (SCG), both receptors, when activated, induce release of noradrenaline. Here we describe marked differences between the responses of cultured neurons from rat thoracolumbal paravertebral ganglia to ATP and UDP. ATP elicited release of previously taken up [3H]noradrenaline, induced an inward current, and increased the intra-axonal free calcium level, over the same range of micromolar concentrations. UDP was more potent than ATP in releasing [3H]noradrenaline but induced an inward current only at a concentration of 1 mM and caused much smaller increases in intraaxonal free calcium. The mechanism of action of ATP presumably consists of P2X-purinoceptor activation followed by depolarization, calcium entry through voltage-sensitive channels and exocytosis. The mode of action of UDP is different. It probably activates a G-protein-coupled pyrimidinoceptor. The pyrimidinoceptor then possibly mediates mobilization of intracellular calcium close to the sites of transmitter release and in addition an increase in the calcium sensitivity of the exocytotic apparatus.

Stimulation of mouse cultured sympathetic neurons by uracil but not adenine nucleotides.

Cultured neurons from the paravertebral sympathetic chain of rats possess excitatory P2X as well as excitatory uracil nucleotide-sensitive P2Y receptors. Preliminary observations had indicated that the analogous neurons of mice lacked P2X receptors. This difference was now investigated. Thoracolumbar sympathetic neurons from one- to three-day-old mice were cultured for seven days. When the neurons were preincubated with [3H]noradrenaline and then superfused, ATP failed to cause any change in tritium outflow. UTP (3-300 microM) and UDP (30-100 microM), in contrast, caused marked increases, and so did nicotine (3-100 microM). The effect of UTP was not changed by suramin but abolished by tetrodotoxin and in the absence of calcium. The effect of nicotine was antagonized by hexamethonium and also abolished by tetrodotoxin and in the absence of calcium. Pre-exposure to UDP prevented the effect of UTP. In neurons studied by means of whole-cell patch-clamp techniques under current clamp, ATP lacked any effect. UTP (100 microM), UDP (100 microM) and nicotine (10 microM) caused depolarization accompanied by action potentials. Pre-exposure to UDP prevented the effect of UTP. In neurons studied under voltage clamp, ATP, UTP and UDP failed to cause any detectable current. Nicotine (10 microM), in contrast, elicited inward currents. Neither UTP nor UDP reduced the M-type potassium outward current. These results demonstrate a pronounced difference between cultured sympathetic neurons from the mouse and the rat paravertebral chain. Neurons from both species possess the nicotinic acetylcholine receptor. Neurons from both species also possess uracil nucleotide-sensitive P2Y receptors which, when activated, mediate depolarization, action potential firing and noradrenaline release; these effects are not due to inhibition of M-type potassium channels. Only the rat but not the mouse neurons, however, possess P2X receptors which, when activated, mediate cation entry, depolarization, action potential generation and transmitter release. The absence of functional P2X receptors makes the mouse neurons suitable for further study of the uracil nucleotide-sensitive P2Y receptors.

A study of the mechanism of the release of ATP from rat cortical astroglial cells evoked by activation of glutamate receptors.

Glutamate and the selective agonists at ionotropic glutamate receptors N-methyl-D-aspartate, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) and kainate release ATP from superfused primary cultures of rat cortical astrocytes. The mechanism of this release was investigated. The release of ATP elicited by N-methyl-D-aspartate and kainate was abolished or greatly reduced in the absence of external calcium as well as in the presence of cadmium (1 mM) and nicardipine (10 microM). The release of ATP elicited by AMPA, in contrast, was not changed by these interventions. The calcium ionophore ionomycin (5 microM) released ATP in the presence but not in the absence of external calcium. No release was obtained with alpha-latrotoxin. Of several compounds tested as potential blockers of ATP transporters or channels only glibenclamide (100 microM) and diphenylamine-2-carboxylate (500 microM), which block the cystic fibrosis transmembrane conductance regulator, caused any change: both reduced the effect of AMPA without changing the effects of N-methyl-D-aspartate and (only glibenclamide tested) kainate. Lithium (1 mM) abolished the release of ATP evoked by glutamate and AMPA and significantly reduced the release evoked by N-methyl-D-aspartate and kainate. The three glutamate receptor agonists did not increase the release of lactate dehydrogenase. The results confirm the previous observation that activation of N-methyl-D-aspartate, AMPA and kainate receptors induces release of ATP from astrocytes in culture. Two different mechanisms seem to be involved. The N-methyl-D-aspartate- and kainate-induced release of ATP requires an influx of calcium, is not due to neuron-like exocytosis, is not mediated by cystic fibrosis transmembrane conductance regulator or a mechanism regulated by cystic fibrosis transmembrane conductance regulator, and is reduced (by an unknown mechanism) but not abolished by lithium. The AMPA-induced release does not require extracellular calcium, may be mediated by cystic fibrosis transmembrane conductance regulator or a mechanism regulated by cystic fibrosis transmembrane conductance regulator, and is abolished (by an unknown mechanism) by lithium. The ability of astrocytes to both release ATP and respond to ATP suggests that ATP may act as an autocrine or paracrine messenger between these glial cells.

Release of ATP from cultured rat astrocytes elicited by glutamate receptor activation.

The release of ATP was studied in cultures of astrocytes derived from the brain hemispheres of newborn rats. There was a basal efflux of ATP, which was increased up to 19-fold by glutamate (300-1000 microM). N-methyl-D-aspartate (20-500 microM), alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA; 30-100 microM) and kainate (20 microM). The N-methyl-D-aspartate receptor-selective antagonist 2-amino-5-phosphonopentanoate (100 microM) blocked the effect of N-methyl-D-aspartate but not the effects of AMPA, kainate and glutamate. The AMPA receptor-selective antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline (30 microM) blocked the effect of AMPA and also of glutamate and N-methyl-D-aspartate, but not the effect of kainate. The kainate receptor-selective antagonist D-glutamyl-amino-methanesulfonate (30 microM) blocked the effect of kainate but not of glutamate. Glutamate (1000 microM) did not increase the release of lactate dehydrogenase from astrocytes. Excitatory amino acids are known to release adenyl compounds in the brain. The present results identify one adenyl compound thus released, namely ATP, and identify astrocytes as one source. The release is brought about by activation of any of the three ionotropic glutamate receptor types-N-methyl-D-aspartate, AMPA and kainate receptors. AMPA receptors seem to mediate at least a part of the effect of glutamate itself, but the involvement of other receptors cannot be ruled out. ATP and its degradation products, such as adenosine, once released, may exert acute as well as trophic effects on neurons and glial cells.

An electrophysiological study of presynaptic alpha-adrenoceptors in the vas deferens of the mouse.

1--Effects of clonidine and alpha-adrenoceptor antagonists were studied on sympathetic neuroeffector transmission in the mouse vas deferens. The amplitude of excitatory junction potentials (e.j.ps) was taken as a measure of transmitter release per impulse. 2--At a concentration of 0.5 microM, prazosin abolished depolarizations evoked by iontophoretically applied noradrenaline, but changed neither spontaneous nor nerve stimulation-evoked e.j.ps. 3--Yohimbine 0.1 and 1 microM, rauwolscine 1 microM and corynanthine 1 microM di not change the e.j.p. amplitudes elicited by the first 2-3 pulses in trains of 15 pulses at 3 Hz, but increased the e.j.ps elicited by the subsequent pulses. Corynanthine 1 microM was much less effective than yohimbine 1 microM or rauwolscine 1 microM, and corynanthine 0.1 microM had no effect. 4--Clonidine 0.01 microM reduced the e.j.p. amplitudes evoked by single pulses and its effect was counteracted by yohimbine 1 microM. 5--In vasa deferentia from reserpine-treated mice the e.j.p. trains were changed in much the same way as by yohimbine and rauwolscine. Yohimbine 1 microM did not further increase the e.j.p. amplitudes in these organs, whereas clonidine 0.01 microM caused a marked inhibition. 6--It is concluded that the release of the motor transmitter in the mouse vas deferens is inhibited by activation of presynaptic alpha-adrenoceptors, and that these receptors are normally activated by neurally released noradrenaline.

Alpha-adrenoceptor antagonists and the release of noradrenaline in rabbit cerebral cortex slices: support for the alpha-autoreceptor hypothesis.

Slices of rabbit cerebral cortex were preincubated with [3H]-noradrenaline and then superfused and stimulated electrically twice for 2 min each (S1, S2) at various frequencies (0.2-3 Hz). The stimulation-evoked overflow of tritium (S1) increased with increasing frequency and was higher when cocaine (10 microM) was present. In the absence of cocaine, tetraethylammonium (TEA; 100 and 300 microM), added before S2, increased the stimulation-evoked overflow of tritium to about the same extent, irrespective of the frequency. In contrast, rauwolscine (0.1 and 1 microM) and idazoxan (0.1-10 microM) increased the evoked overflow much more, the higher the frequency of stimulation. Phentolamine (0.1 and 1 microM) reduced the overflow elicited at 0.3 and 1 Hz, and (1 microM) caused an increase only at 3 Hz. In slices superfused throughout with cocaine 10 microM, rauwolscine (1 microM) and idazoxan (1 and 10 microM) again increased the evoked overflow of tritium more, the higher the frequency of stimulation. For a given frequency, rauwolscine and idazoxan enhanced the evoked overflow to a greater extent in the presence than in the absence of cocaine. Idazoxan (1 and 10 microM) and rauwolscine (1 microM) counteracted the inhibition that phentolamine (0.1 microM) produced at low frequency. The increases caused by rauwolscine (1 microM) and TEA (300 microM) were approximately additive, but those caused by rauwolscine (1 microM) and idazoxan (10 microM) were not. The effects of rauwolscine, idazoxan and phentolamine depend on the experimental conditions (frequency, cocaine) in a manner compatible with the operation of a presynaptic alpha 2-adrenoceptor-mediated autoinhibition of noradrenaline release. When given at sufficient concentrations, these antagonists enhance the release of noradrenaline more, the higher the biophase concentration of the transmitter and the stronger, hence, the autoinhibition. In the case of the partial alpha 2-adrenoceptor agonist phentolamine, a low perineuronal noradrenaline concentration even reverses facilitation to inhibition. This pattern differs markedly from the pattern of effects of TEA which increases the release of noradrenaline by a mechanism other than alpha-adrenoceptor blockade.

Blockade by 4,4'-diisothiocyanatostilbene-2,2'-disulphonate (DIDS) of P2X-purinoceptors in rat vas deferens.

1. The possibility of an antagonist effect of 4,4'-diisothiocyanatostilbene-2,2'-disulphonate (DIDS) at P2X-purinoceptors was studied in rat vas deferens. 2. DIDS reduced contractions elicited by alpha,beta-methylene ATP 3 microM, IC50 1.6 microM, but did not change contractions elicited by K+ 35 mM. DIDS 3.2 microM slightly shifted the concentration-response curve of alpha,beta-methylene ATP to the right and reduced the maximum. DIDS 10 microM markedly decreased and DIDS 32 microM abolished contractions over the entire range of the alpha, beta-methylene ATP concentration-response curve. DIDS 32 microM also abolished contractions elicited by ATP but did not change contractions elicited by noradrenaline. The antagonist effect of DIDS was only slowly reversible. 3. The presence of either suramin 320 microM or alpha,beta-methylene ATP 10 microM during the exposure to DIDS protected the tissue from the long-lasting blocking effect of DIDS. 4. 4,4'-Diisothiocyanatodihydrostilbene-2,2'-disulphonate (H2DIDS) was equipotent with DIDS whereas several analogues in which one or both of the isothiocyanate residues were replaced were less effective or without effect against alpha,beta-methylene ATP. 5. DIDS attenuated the purinergic component of neurogenic contractions elicited by electrical field stimulation, IC50 3.9 microM, but did not change the adrenergic component. 6. It is concluded that DIDS causes a selective, long-lasting, non-equilibrium blockade of P2X-purinoceptors in rat vas deferens. Due to this effect it also selectively blocks the purinergic component of neurogenic contractions.

Chloroethylclonidine: an irreversible agonist at prejunctional alpha 2-adrenoceptors in rat vas deferens.

1. The possibility that chloroethylclonidine (CEC) activates prejunctional alpha 2-adrenoceptors was studied in the isolated vas deferens of the rat. Tissues were stimulated electrically and both the stimulation-evoked overflow of tritium (after preincubation with [3H]-noradrenaline) and the purinergic contraction component (isolated by prazosin 0.3 microM) were measured. 2. CEC (0.1-3 microM) concentration-dependently reduced the overflow of tritium evoked by trains of 6 pulses/100 Hz. The inhibition by CEC was not altered by prazosin (0.3 microM) but was prevented by pre-exposure to rauwolscine (0.3 microM). The inhibition, once established, did not fade upon washout of CEC, even when the washout fluid contained rauwolscine (0.3 microM). 3. CEC (0.1-3 microM) concentration-dependently reduced the purinergic component of contractions elicited by single pulses. The inhibition, again, was prevented by pre-exposure to rauwolscine (0.3 microM) and once established, did not fade upon washout of CEC, even when the washout fluid contained rauwolscine (0.3 microM). 4. CEC (3 microM) reduced the overflow of tritium evoked by 20 pulses/10 Hz, did not alter the overflow evoked by 100 pulses/10 Hz and increased the overflow evoked by 500 pulses/10 Hz. 5. CEC (3 microM) reduced the early peak, but increased the late plateau phase, of purinergic contractions elicited by 100 pulses/10 Hz. 6. It is concluded that CEC reduces the release of noradrenaline and a purinergic co-transmitter by irreversible activation of prejunctional alpha 2-adrenoceptors. CEC seems to be a partial alpha 2-agonist with an efficacy lower than that of noradrenaline. The prejunctional inhibitory effect limits the suitability of CEC for the characterization of postjunctional alpha 1-adrenoceptors mediating responses to sympathetic nerve stimulation.

Effects of narcotic analgesics and their antagonists on the rabbit isolated heart and its adrenergic nerves.

1. In isolated perfused hearts of rabbits, the effects of morphine, methadone, pethidine, fentanyl, levallorphan and naloxone on heart rate, the spontaneous outflow of noradrenaline, the uptake of infused noradrenaline and the overflow of noradrenaline in response to stimulation of the accelerans nerves were investigated.2. At concentrations of 10-100 muM, methadone, fentanyl, levallorphan and naloxone, but not morphine and pethidine, decreased the heart rate. Only pethidine and levallorphan (100 muM) augmented the spontaneous outflow of noradrenaline.3. With the exception of naloxone, all drugs diminished the neuronal uptake of noradrenaline from the perfusion fluid. Methadone and pethidine (1 muM) were the most effective inhibitors. The inhibitory effect of morphine was not antagonized by naloxone.4. All drugs increased the overflow of noradrenaline evoked by stimulation of the accelerans nerves at 5 Hz. Simultaneously, the positive chronotropic effect of stimulation was usually enhanced. Morphine also augmented the response to stimulation at 2.5 and 10 Hz. The effect of morphine was prevented by pre-infusion of cocaine. The response to stimulation was never depressed.5. It is concluded that the adrenergic nerves of the rabbit heart lack specific morphine receptors which in some other sympathetic nerves mediate an inhibition of the stimulation-induced secretion of noradrenaline. The mechanism of the enhancement of adrenergic neurotransmission by relatively high concentrations of the drugs is discussed.