Alpha 2-adrenoceptor agonist-mediated inhibition of [3H]noradrenaline release from rat hippocampus is reduced by 4-aminopyridine, but that caused by an adenosine analogue or omega-conotoxin is not

The inhibitory effect of an adenosine analogue, R-PIA, and an alpha 2-adrenoceptor agonist, UK 14,304, on [3H]NA efflux from field-stimulated rat hippocampal slices was examined. The effect of 0.1 microM UK 14,304 was mimicked by 30 nM omega-conotoxin and by 10 microM cadmium chloride, inhibitors of N- and L-type Ca2+ channels. R-PIA (1 microM) had no effect per se, but caused a clear-cut inhibition after blockade of the pre-synaptic alpha 2-receptor by yohimbine. 4-Aminopyridine (4-AP) caused a dose-dependent increase in evoked transmitter release. At 30 microM 4-AP did not affect the actions of omega-conotoxin or cadmium chloride. The pre-synaptic effect of R-PIA was similarly unaffected by 30 microM 4-AP. The pre-synaptic effect of UK 14,304 was virtually abolished by 4-AP (30 microM). The effect of UK 14,304 (0.1 microM) could be partly restored by reducing the Ca2+ concentration during treatment with 4-AP (22% inhibition compared to 42% with normal Ca2+). The magnitude of increase in evoked [3H]NA efflux by yohimbine (1 microM) was decreased by 4-AP in a concentration-dependent manner from 142% increase in controls to 21% at 100 microM 4-AP. The present results indicate that NA release is reduced by somewhat different mechanisms by pre-synaptic alpha 2- and adenosine A1-receptors. Furthermore, the results indicate that pre-synaptic A1-receptors on hippocampal NA neurons do not primarily regulate 4-AP-dependent potassium channels, but they might act directly on a Ca2+ conductance.     

Adenosine as a modulator of sympathetic nerve-stimulation-induced release of noradrenaline from the isolated rabbit heart

The ability of adenosine to inhibit sympathetic nerve-stimulation-induced overflow of noradrenaline was studied in isolated rabbit hearts with intact sympathetic innervation. Noradrenaline in the heart effluents was measured by HPLC with electrochemical detection. The drugs used, adenosine, theophylline, and 8-parasulphophenyltheophylline, were administered via the perfusion fluid. Adenosine (1-100 microM) dose-dependently inhibited stimulation-evoked outflow of noradrenaline from the heart, by up to 47%: the inhibition was completely antagonized by theophylline (200 microM), and by 8-parasulphophenyltheophylline (100 microM). Neither theophylline nor 8-parasulphophenyltheophylline did per se affect basal or stimulation-evoked outflow of noradrenaline. Simultaneous infusion of adenosine (100 microM) and theophylline (200 microM) significantly increased the outflow of noradrenaline during nerve stimulation, by almost 40%. No such effect was observed by adenosine (100 microM) and 8-parasulphophenyltheophylline (100 microM), indicating that theophylline may facilitate transmitter release by an action dissociated from purinoceptor antagonism. It is concluded that (a) adenosine inhibits depolarization-induced liberation of sympathetic transmitter in the rabbit heart, (b) this inhibition is mediated by activation or purinoceptors, probably located on the presynaptic nerve terminals, and (c) brief periods of sympathetic stimulation in the normoxic heart does not release sufficient amounts of adenosine to cause significant inhibition of transmitter release.     

The metabolism of [3H]noradrenaline released by electrical stimulation from the isolated nictitating membrane of the cat and from the vas deferens of the rat

1. The noradrenaline (NA) stores of the isolated medial muscle of the cat's nictitating membrane were labelled with [(3)H]NA and the tissue was set up in an isolated organ bath for field stimulation. The tritiated NA and its metabolites released spontaneously and by field stimulation were determined by scintillation counting following chromatographic separation.2. NA represented 11.8 +/- 1.0% of the total radioactivity of the spontaneously released tritiated compounds. The rest was accounted for by NA metabolites: (a) normetanephrine (NMN), 35%; (b) 4-hydroxy-3-methoxymandelic acid (VMA), 20%; (c) 3,4-dihydroxyphenylglycol (DOPEG), 10%; (d) 3,4-dihydroxymandelic acid (DOMA), 10%; (e) 4-hydroxy-3-methoxyphenylglycol (MOPEG), 14%.3. Field stimulation at 25 shocks/sec with supramaximal stimuli of 1 msec duration increased the outflow of NA six to eightfold and that of NMN, DOPEG and VMA two to threefold. The increase in outflow of DOMA and MOPEG was small. NA represented 35.8 +/- 4.1% of the total increase in radioactivity.4. After pargyline pretreatment field stimulation increased the outflow of NA and NMN. Stimulation in the presence of pyrogallol raised the release of NA, DOMA and DOPEG. Neither pargyline nor pyrogallol affected the total release induced by stimulation.5. Cocaine 0.3 mug/ml. increased the release due to stimulation at 4 shocks/sec but not at 25 shocks/sec. Cocaine did not affect the metabolism of the released transmitter.6. Phenoxybenzamine (10 mug/ml.) increased release by stimulation at 4 and 25 shocks/sec. Metabolism of the released transmitter was prevented in the presence of phenoxybenzamine.7. Phentolamine (3 mug/ml.), like phenoxybenzamine, blocked responses to field stimulation, but failed to modify release and subsequent metabolism of NA liberated by field stimulation.8. The main NA metabolites in the rat vas deferens were DOPEG, DOMA and MOPEG. Stimulation at 4 shocks/sec resulted in an increased outflow of NA and of DOPEG. Fifty per cent of the total increase of radioactive compounds was accounted for by NA metabolites.9. These experiments show that for the calculation of the actual output of transmitter it is important to include the metabolites and not to rely on the determination of [(3)H]NA alone.     

Catechol and noradrenaline facilitate the neuromuscular transmission via different mechanisms

Using curarized frog sartorius muscles, selective presynaptic facilitatory effects of two structurally related chemicals, noradrenaline (NA) and catechol (pyrocatechol, PC), were compared by calculating the fractional release (P) from the rundown of endplate potential during a train-of-three stimulation. Below 10 microM, both NA and PC increased P in a similar manner. At higher concentrations, however, the effects of NA were found to be quite different from those of PC. NA never increased further the value of P (0.11-0.13) whereas PC augmented P in a dose dependent fashion up to about 0.76. Thus, the present data suggested that NA and PC facilitate the transmitter release via different mechanisms. The effects of the latter were identical to the case of "chemical potentiation or accelerated evoked release" by 4-aminopyridine.     

Adenosine modulation of neuroeffector transmission in guinea-pig uterine smooth muscle.

Effects of adenosine and adenosine analogues on neuroeffector transmission in separate layers of the guinea-pig uterine smooth muscle during different phases of the oestrus cycle were studied. Adenosine (ADO), N6-[(R)-1-methyl-2-phenylethyl]- adenosine (R-PIA) and 5'-N-ethylcarboxamideadenosine (NECA) enhanced spontaneous contractile activity as well as contractile responses to nerve stimulation or direct muscle stimulation in the longitudinal and circular muscle layers both at the time of ovulation and implantation. The agonist potency order was R-PIA greater than or equal to NECA greater than ADO. Furthermore, in the circular muscle layer inhibitory effects were seen at time of implantation and the agonist potency for this inhibitory effect was NECA greater than R-PIA greater than or equal to ADO. 8-p-sulphophenyltheophylline (8-PSOT) antagonized both the stimulatory and inhibitory effects of the purines and 8-p-sulphophenyltheophylline applied to untreated preparations inhibited nerve-induced contractile activity. NECA inhibited stimulation-induced release of 3H in preparations incubated with [3H]noradrenaline. We suggest that adenosine and its analogues can modulate adrenergic neuroeffector transmission in guinea-pig uterine smooth muscle via action at postjunctional receptors stimulating contractile activity and prejunctional receptors inhibiting transmitter release. In the circular muscle layer postjunctional receptors inhibiting contractile activity were evident at time of implantation. Furthermore, endogenous purines exerting a stimulatory action on the neuroeffector transmission were likely formed during the present experimental conditions.     

Relationship between the overflow of endogenous and radiolabelled noradrenaline from canine blood perfused gracilis muscle

The effects of sympathetic nerve stimulation (SNS) on the overflow of endogenous noradrenaline (NA) and on vasoconstrictor responses were studied in blood perfused canine gracilis muscle in situ. A conventional tracer technique with 3H-labelled NA (3H-NA) was used in parallel. At rest there was a net extraction of endogenous NA and adrenaline across the tissue. The SNS evoked overflow of endogenous NA was frequency-dependent and logarithmically correlated to the vasoconstrictor responses. The neuronal uptake inhibitor desipramine doubled the SNS induced overflow of endogenous NA without enhancing the vasoconstrictor responses. A further fourfold increase in NA overflow was caused by a dose of the alpha-blocker phenoxybenzamine which reduced the vasoconstrictor responses by 50-75%. Less than 10% of the spontaneous 3H efflux was recovered as unmetabolized 3H-NA, whereas virtually all 3H overflow evoked by SNS was 3H-NA. The fractional release of NA or 3H-NA per nerve impulse increased with increasing frequencies of SNS under all conditions studied. Although there was a preferential release of the newly stored radiolabelled transmitter, results concerning endogenous NA and 3H-NA overflow were qualitatively similar, also under conditions with marked changes in transmitter overflow. Endogenous NA gave a more reproducible index of transmitter overflow than did 3H-NA and, in particular, total 3H. The overflow of endogenous NA closely reflects SNS evoked neuronal release of NA in blood perfused skeletal muscle and seems more suitable than conventional radiotracer techniques for studies of NA release under in vivo conditions.     

Role of the coronary endothelium in the regulation of sympathetic transmitter release in isolated rabbit hearts

The roles of endothelium-derived relaxing factor (EDRF) and endothelin in the regulation of vascular tone are intensely studied at present. Since factors which directly affect vascular tone also frequently modulate sympathetic transmitter release, we found it of interest to study whether EDRF or endothelin displays such modulatory activity as well. Isolated rabbit hearts were perfused according to Langendorff, and the release of transmitter induced by sympathetic nerve stimulation was estimated by analysis of the effluent content of noradrenaline (NA) with liquid chromatography. The activity of EDRF spontaneously formed in the heart was counteracted by addition of haemoglobin (Hb, 2.4-15 g l-1) or facilitated by addition of superoxide dismutase (SOD, 14-140 U ml-1), to the perfusion solution. In other experiments authentic endothelin (0.1-10 nm) was given to the heart. Nerve stimulation (5 Hz for 30 s) elicited a release of NA into the cardiac effluent amounting to 319 +/- 28 pmol (n = 53). Hb lowered the coronary flow to 68 +/- 9% (P less than 0.01) and impaired the outflow of NA to 62 +/- 9% of control (P less than 0.01). SOD facilitated coronary flow by 11 +/- 4% (P less than 0.005), and augmented the outflow of NA by 15 +/- 6% (P less than 0.05). Endothelin dose-dependently inhibited the coronary flow, with an IC50 of about 1 nM, and in parallel decreased the efflux of NA. Mechanical obstruction of the coronary flow induced an attenuation of the efflux of NA that was quantitatively similar to the flow reduction.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Role of presynaptic L-type Ca2+ channels in GABAergic synaptic transmission in cultured hippocampal neurons

Using dual whole cell patch-clamp recordings of monosynaptic GABAergic inhibitory postsynaptic currents (IPSCs) in cultured rat hippocampal neurons, we have previously demonstrated posttetanic potentiation (PTP) of IPSCs. Tetanic stimulation of the GABAergic neuron leads to accumulation of Ca2+ in the presynaptic terminals. This enhances the probability of GABA-vesicle release for up to 1 min, which underlies PTP. In the present study, we have examined the effect of altering the probability of release on PTP of IPSCs. Baclofen (10 microM), which depresses presynaptic Ca2+ entry through N- and P/Q-type voltage-dependent Ca2+ channels (VDCCs), caused a threefold greater enhancement of PTP than did reducing [Ca2+]o to 1.2 mM, which causes a nonspecific reduction in Ca2+ entry. This finding prompted us to investigate whether presynaptic L-type VDCCs contribute to the Ca2+ accumulation in the boutons during spike activity. The L-type VDCC antagonist, nifedipine (10 microM), had no effect on single IPSCs evoked at 0.2 Hz but reduced the PTP evoked by a train of 40 Hz for 2 s by 60%. Another L-type VDCC antagonist, isradipine (5 microM), similarly inhibited PTP by 65%. Both L-type VDCC blockers also depressed IPSCs during the stimulation (i.e., they increased tetanic depression). The L-type VDCC "agonist" (-)BayK 8644 (4 microM) had no effect on PTP evoked by a train of 40 Hz for 2 s, which probably saturated the PTP process, but enhanced PTP evoked by a train of 1 s by 91%. In conclusion, the results indicate that L-type VDCCs do not participate in low-frequency synchronous transmitter release, but contribute to presynaptic Ca2+ accumulation during high-frequency activity. This helps maintain vesicle release during tetanic stimulation and also enhances the probability of transmitter release during the posttetanic period, which is manifest as PTP. Involvement of L-type channels in these processes represents a novel presynaptic regulatory mechanism at fast CNS synapses.     

Neurochemical evidence for the activation of the 'efferent' function of capsaicin-sensitive nerves by lowering of the pH in the guinea-pig urinary bladder

Lowering of the pH of the superfusion solution (physiological phosphate buffers derived from normal Krebs solution) of mucosa-free muscle strips of the guinea-pig urinary bladder from 7.4 to 6 or 5, produced a marked increase of calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) outflow (total evoked release being 1677 +/- 287 fmol/g/35 min and 5283 +/- 484 fmol/g/35 min, respectively). The increase in CGRP-LI outflow evoked by exposure to pH 5 was abolished in strips pre-exposed to capsaicin (10 microM for 20 min), and was reduced by approximately 80% when superfusion was performed with Ca2(+)-free media. After application of pH 5 solution, capsaicin (10 microM) evoked a CGRP-LI release that amounted to 21% of controls. It is concluded that lowering of the pH induces a Ca2(+)-dependent release of CGRP-LI from capsaicin-sensitive nerves in the guinea-pig urinary bladder.     

Differential release of [3H]acetylcholine from the rat phrenic nerve-hemidiaphragm preparation by electrical nerve stimulation and by high potassium

Neuronal transmitter stores of the phrenic nerve were labelled under different conditions. Subsequently, transmitter release evoked by electrical nerve stimulation and by a high potassium-low sodium solution was studied. Incubation of the end-plate preparation with [³H]choline at rest led to the synthesis of [³H]acetylcholine which could not be released by electrical nerve stimulation but it was released by high potassium-low sodium solution, independent of the presence of extracellular calcium. When the end-plate preparation was labelled during stimulation at 1 Hz, prolonged periods of electrical nerve stimulation released 83% of the total releasable [³H]transmitter pool in a completely calcium-dependent manner. After exhaustion of the electrically releasable pool, high potassium-low sodium solution still caused a significant outflow. Without a preceding exhaustion of the [³H]acetylcholine pool, high potassium-low sodium solution released a similar amount in the absence of extracellular calcium or after pretreatment with the intracellular calcium chelating substance, Quin-2. When evoked transmitter release was studied at different temperatures (36, 26 and 16°C) Q 10 values of 1.6 and 1.0 were found for the release caused by electrical nerve stimulation and high potassium-low sodium solution (calcium-independent effect), respectively. After labelling during a short interval (2 min) but at a high stimulation rate (50 Hz), only 72% of the releasable [³H]transmitter could be released by electrical nerve stimulation, whereas the outflow due to the calcium-independent effect of high potassium-low sodium solution increased from 17 (labelling during stimulation at 1 Hz) to 28%.It is suggested that the calcium-independent effect of high potassium-low sodium solution reflects the release of acetylcholine from the cytoplasmic compartment, as this outflow occurred after labelling at rest and increased when cytoplasmic synthesis was enhanced by a high loading stimulation. In contrast to high potassium-low sodium solution, propagated nerve activity cannot release acetylcholine synthesized at rest (presumed to be cytoplasmic), but only [³H]acetylcholine synthesized during quantal release (presumed to be vesicular). The absolute requirement of extracellular calcium for electrically stimulated release suggests an exocytotic release mechanism. The low Q 10 value of 1.6 does not fit into the concept of a carrier- or channel-operated release mechanism. High potassium-low sodium solution triggers both calcium-dependent release (exocytosis) and calcium-independent liberation of acetylcholine, which escapes from the cytoplasmic compartment by a diffusion-like mechanism. Accordingly, it is important to consider that propagated nerve activity and high potassium-low sodium solution can cause the release of transmitter by different mechanisms from different compartments.     

Spontaneous release of endogenous 5-hydroxytryptamine and 5-hydroxyindoleacetic acid from the isolated vascularly perfused ileum of the guinea-pig

The spontaneous release of 5-hydroxytryptamine and its metabolite 5-hydroxyindoleacetic acid from the enterochromaffin cells of the small intestine into the portal circulation was investigated in vitro using the vascularly perfused ileum of the guinea-pig. The release of 5-hydroxytryptamine decreased by 70% in a calcium-free medium and by 35% in the presence of tetrodotoxin. Inhibition of monoamine oxidase activity by pargyline (100 microM) had no effect on the spontaneous release of 5-hydroxytryptamine although it caused a 75% reduction in the outflow of 5-hydroxyindoleacetic acid. Imipramine (1 microM), an inhibitor of neuronal uptake of 5-hydroxytryptamine, reduced the 5-hydroxyindoleacetic acid outflow by 57% and increased the release of 5-hydroxytryptamine by 66%. The combination of both drugs showed no additional effect. The tissue content of 5-hydroxytryptamine and its metabolite was not changed after perfusion with the precursor L-tryptophan or monofluoromethyldopa, an inhibitor of the L-aromatic amino acid decarboxylase. The results show that the spontaneous release of 5-hydroxytryptamine and its metabolite reflects largely calcium-dependent exocytotic release of the amine. "Neuronal uptake" (into aminergic and/or enterochromaffin cells) followed by deamination appears to be the main pathway of 5-hydroxytryptamine catabolism in the guinea-pig ileum.     

Potency of depolarization-induced transmitter release is determined by divalent cation influx in PC 12 cells.

Evoked release of [3H]dopamine ([3H]DA) from pheochromocytoma cells (PC 12) is dependent on extracellular calcium ([Ca2+]ex), but it can take place if calcium ions (Ca2+) are substituted by other divalent ions such as strontium (Sr2+) and barium (Ba2+). The potency of the divalent cations at supporting release varies with the cell type; in PC 12 cells the order of potency is Ba2+ > Sr2+ > Ca2+. The close correlation between depolarization-evoked Ca2+ entry and depolarization-evoked transmitter release prompted us to examine whether the higher evoked transmitter release in the presence of Sr2+ correlates with an increased evoked Sr2+ influx. Influx studies were conducted on PC12 cells using a radioactive tracer (45Ca2+ or 85Sr2+, < 1 microM) in the presence of either Sr2+ (0.5 mM) or Ca2+ (0.5 mM). Depolarization with K Cl (60 mM) increased evoked 45Ca2+ influx 2-fold when Ca2+ was substituted with Sr2+. Similarly, evoked 85Sr2+ influx increased 1.87-fold by substituting Ca2+ for Sr2+. Thus the amount of evoked cation influx is determined by the type of divalent ion which is accessible in the extracellular medium, independently of the radioactive tracer used. Increased evoked transmitter release in the presence of Sr2+ was associated with increased evoked Sr2+ influx. This suggests that the potency of evoked transmitter release is determined predominantly by the influx of divalent cations. Furthermore, the steps subsequent to cation influx in the release process are equally efficient for both cations.     

Noradrenaline modulates GABA-mediated synaptic transmission in neurones of the mediolateral part of the guinea pig lateral septum via local circuits

The response to bath-applied noradrenaline (NA, 50 microM) was tested with conventional intracellular recordings in neurones of the mediolateral part of the lateral septum (LSml) by using guinea-pig brain slices. NA elicited direct hyperpolarizations and depolarizations and affected the frequency of tetrodotoxin (TTX)- and bicuculline-sensitive inhibitory post-synaptic potentials (IPSPs). Decrease or increase in IPSPs frequency was observed in 37.6 and 18.2% neurones respectively. Frequency of bicuculline-sensitive miniature IPSPs recorded under TTX was unchanged. Increase in IPSPs frequency was blocked by TTX and was observed during iontophoretic application of NA in the LSml. Taken together, these data suggest that NA hyperpolarizes or depolarizes local presynaptic GABAergic neurones to cause alterations in action potential-dependent transmitter release. NA-induced decrease or increase in IPSPs frequency were respectively mimicked by clonidine and L-phenylephrine and blocked by yohimbine and prazosin, indicating that NA modifies release of GABA by activation of alpha(2) and alpha(1) adrenoceptors at the somato-dendritic region of presynaptic GABAergic neurones. These findings show that, in addition to their direct input from NA afferents, LSml neurones receive a indirect input by way of GABAergic interneurones. These interneurones may serve as inhibitory relays for noradrenergic afferents originating from the locus coeruleus and may be involved in the NA control of LSml functions.     

Low pH-induced release of calcitonin gene-related peptide from capsaicin-sensitive sensory nerves: mechanism of action and biological response.

Protons can release in a Ca(2+)-dependent manner, calcitonin gene-related peptide (CGRP)-like immunoreactivity from peripheral endings of capsaicin-sensitive afferents. Here we have studied the mechanism by which proton promotes CGRP-like immunoreactivity release and whether the neuropeptide released might exert a biological action. In muscle slices of guinea-pig urinary bladder high pH (pH 8 or 9) media neither enhanced CGRP-like immunoreactivity outflow nor affected the capsaicin-evoked CGRP-like immunoreactivity release. The CGRP-like immunoreactivity release evoked by superfusion with pH 5 medium was not affected by tetrodotoxin (0.3 microM) indomethacin (10 microM) or the protein kinase C inhibitor H-7 (30 microM). However, it was reduced by 35% in the presence of the voltage-sensitive Ca(2+)-channel antagonists nifedipine (1 microM) and omega-conotoxin (0.1 microM) and by 80% in presence of the capsaicin "antagonist" Ruthenium Red (10 microM). The CGRP-like immunoreactivity release by capsaicin (10 microM) was reduced by 80% in the presence of Ruthenium Red, and not affected by voltage-sensitive Ca(2+)-channel blockers, while that evoked by 80 mM K+ was decreased by 82% in the presence of nifedipine and omega-conotoxin. The Ca(2+)-channel agonist Bay K 8644 enhanced the high K(+)-evoked CGRP-like immunoreactivity release but not that induced by capsaicin or pH 5 medium. Exposure to pH 6 solution of one half of the neck of guinea-pig urinary bladder induced a slowly developing inhibition of electrically evoked contractions, that was absent in the half pre-exposed in vitro to a desensitizing dose of capsaicin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adenosine inhibits action potential-dependent release of calcitonin gene-related peptide- and substance P-like immunoreactivities from primary afferents in rat spinal cord.

Electrical field stimulation (5 Hz) evoked a prompt outflow of calcitonin gene-related peptide- and substance P-like immunoreactivities (CGRP-LI and SP-LI, respectively) from superfused slices of the dorsal but not ventral half of the rat spinal cord. The evoked outflow was abolished by tetrodotoxin, calcium-free medium or previous exposure to capsaicin, indicating that it is produced through action potentials invading the central terminals of capsaicin-sensitive primary afferents. Adenosine as well as gamma-aminobutyric acid (GABA) or the GABAB receptor agonist (-)-baclofen produced a concentration-dependent inhibition of the evoked CGRP-LI outflow. Adenosine also inhibited the evoked SP-LI outflow. These findings demonstrate that inhibition of transmitter release from primary afferent neurons should be considered as a possible mechanism of the antinociceptive action of adenosine and adenosine analogs.     

低氧时钙离子对冠状动脉内皮细胞释放ET-1、NO、PGI2的影响

目的:探讨低氧引起冠状动脉内皮细胞(CAEC)释放ET-1、NO和PGI₂改变的机制。方法:运用放射性[⁴⁵Ca²⁺]掺入、放免和比色等方法, 测定常氧组、低氧组冠状动脉内皮细胞钙摄取率, 以及常氧组、低氧组、低氧+维拉帕米组冠状动脉内皮细胞培养液中ET-1、NO和PGI₂含量。结果:低氧组CAEC[⁴⁵Ca²⁺]摄取率为(29.6±3.7)ng/gprotein/30min, 常氧对照组为(15.2±1.1)ng/gprotein(P<0.01).低氧+维拉帕米组ET-1分泌量显著多于低氧组(P<0.01).低氧+维拉帕米组分泌的NO显著低于低氧组(P<0.05), 与常氧组相差不显著。低氧+维拉帕米组分泌的PGI₂显著多于低氧组, 与常氧组相差不显著。结论:低氧时引起的NO、ET-1、PGI₂分泌改变与低氧引起的冠状动脉内皮细胞Ca²⁺内流增加有关。     

The influence of salsolinol on dopaminergic system activity within the mediobasal hypothalamus of anestrous sheep: a model for studies on the salsolinol-dopamine relationship

Salsolinol with its derivatives has been considered as a potential neurotoxin for the dopaminergic system in the human and rat brain. Investigating a sheep model for studies on the action of salsolinol within the central nervous system we examined whether this compound is able to affect the hypothalamic neuroendocrine dopaminergic (NEDA) system during its high seasonal activity, when sheep entered to anestrus under the long day conditions. Therefore, salsolinol was infused into the third ventricle of the brain in combination with the in vivo push-pull perfusion of the mediobasal hypothalamus/median eminence (MBH/ME). The effects of this drug on either perfusate noradrenaline (NA) or plasma prolactin concentration were also studied. The infusion of salsolinol resulted in rapid and permanent diminution in dopamine (DA) release into the extracellular spaces of the MBH/ME up to an undetectable level and in the 57% decrease in DA metabolite 3,4-dihydroxyphenylacetic acid concentration, compared to the control. This effect of salsolinol was accompanied by the significant enhancement of the pituitary prolactin release into circulation. The concentration of other DA metabolite, homovanillic acid, as well as NA in the MBH/ME was not affected. Thus, our results in the anestrous sheep underline the role played by salsolinol as a neuromodulator for the hypothalamic NEDA system and as a signal transmitter for the pituitary prolactin release. We suggest that the hypothalamic NEDA system of anestrous sheep during its high secretory activity may be set as a model for studies on the salsolinol-dopamine relationship.     

Neuropeptide Y is released together with noradrenaline from the human heart during exercise and hypoxia

The myocardial release of neuropeptide Y-like immunoreactivity (NPY-LI) and noradrenaline (NA) during exercise with and without arterial hypoxia was measured in 18 healthy men by arterial (a) and coronary sinus (cs) catheterization. Exercise was performed in the supine position on a cycle ergometer at a load, selected to produce a heart rate during air breathing of 120 beats min-1. Coronary sinus blood flow (CSBF) was measured and a and cs samples for NPY-LI, NA, oxygen and lactate analyses were taken at rest and after 6 min exercise. The inspiratory gas was then switched to 15% (n = 8) or 12% (n = 10) oxygen in nitrogen, exercise continued at the same load and measurements repeated after 6 min. At rest no significant release and during normoxic exercise a very small myocardial release of NPY-LI and NA was detected. During hypoxia compared to normoxia the cardiac NPY-LI release increased four-fold and the NA net release doubled at the same time as the arterial NPY-LI remained unaltered or only slightly increased. Both the NPY-LI and the NA net release from the heart correlated with the heart rate and the arterial but not the cs oxygen tension. The NPY-LI release was correlated with the NA net release. The findings suggest that arterial hypoxia stimulates cardiac NPY together with NA release which derives from local sympathetic nerves. The release from the heart seems to be greater than from other tissues.     

Stimulus-specific signaling pathways in rabbit carotid body chemoreceptors

The carotid body is an arterial chemosensory organ which responds to multiple natural and pharmacological stimuli, including hypoxia and nicotine. Numerous studies have investigated the initial molecular events which activate chemosensory type I cells in the carotid body, but less attention has been focused on later steps in the transduction cascade, which mediate neurotransmitter release from type I cells and excitation of chemoreceptor afferent fibers in the carotid sinus nerve. In the present study, we examined the effects of a highly specific inhibitor of calcium/calmodulin-dependent kinase II, KN-62, and a calmodulin inhibitor, trifluoperazine, on carotid sinus nerve activity and catecholamine release evoked from rabbit carotid bodies superfused in vitro. KN-62 did not alter sinus nerve activity and catecholamine release evoked by hypoxia, but this agent significantly reduced nerve activity and neurotransmitter release evoked by 100 microM nicotine. Trifluoperazine (10 microM), likewise inhibited activity evoked by nicotine, as well as hypoxia. Basal levels of nerve activity and catecholamine release (established in superfusate equilibrated with 100% O2) were unaffected by all drug treatments. Separate biochemical experiments showed that Ca2+/calmodulin-dependent incorporation of 32P into carotid body particulate proteins is significantly reduced following incubation of intact carotid bodies in nicotine, but not following exposure to hypoxia. Our observations suggest that excitation of the carotid body by diverse stimuli may involve the activation of distinct, stimulus-specific transduction pathways. Furthermore, these data correlate with our previous findings which showed that hypoxia, on the one hand, and nicotine on the other, evoke the preferential release of either dopamine or norepinephrine, respectively, from carotid bodies incubated in vitro.