Electrophysiological evidence for oxytocin receptors on sympathetic preganglionic neurones--an in vitro study on the neonatal rat.

The action of oxytocin (0.01-1 microM) on sympathetic preganglionic neurones was studied by intracellular recording in slices of neonatal rat thoracic spinal cord. In 85% of the cells superfusion induced a slow tetrodotoxin-insensitive depolarization accompanied by the appearance or increase in frequency of repetitive discharges. Oxytocin also caused some cells to switch from silent neurones to spontaneously active ones. These effects were reversibly blocked by a specific oxytocin antagonist.

Localization of oxytocin binding sites in the thoracic and upper lumbar spinal cord of the adult and postnatal rat: a histoautoradiographic study.

Oxytocin binding sites were detected by autoradiography on films and emulsion-coated sections in the spinal cord of adult and postnatal rats from C8 to L2, using a highly selective 125I-labelled oxytocin antagonist. Oxytocin binding sites were detected on all transverse sections in the dorsal horn, where labelling was scattered over laminae I and II. The autonomic areas, i.e. the intermediolateral cell column, the central grey (lamina X) and the nucleus intercalatus were labelled. Binding in the intermediolateral cell column was most frequently observed on sections from T9 to T11 in adult and T7 to T8 in postnatal rats. In this location, oxytocin binding sites were highly concentrated on cell bodies of putative sympathetic preganglionic neurons; however, not all of these cells were labelled. Diffuse labelling occurred on the dorsal part of the central grey, mainly between T8 and L2. Isolated labelled cells belonging to the nucleus intercalatus were scattered between the central canal and the intermediolateral cell column. In addition, oxytocin binding sites were found on some motoneurons of the lateral group of T12-T13, but only in postnatal rats. The distribution of oxytocin binding sites in the rat spinal cord coincides with that of the oxytocin innervation and strongly suggests a modulatory role of this peptide in sensory and autonomic functions.

Caffeine-induced Ca2+ release inhibits GABAA responsiveness in rat identified native primary afferents.

It has been shown previously that an increase in cytoplasmic Ca2+ concentration depresses the GABA-A response. However, little attention has been paid to the Ca2+ source involved. In the present study, we show that the Ca2+ increase triggered by caffeine-induced Ca2+ release from the intracellular pool inhibits the GABA-A response, whereas Ca2+ influx through voltage-activated Ca2+ channels has no effect on this response.

Synthesis and biochemical evaluation of baclofen analogues locked in the baclofen solid-state conformation.

The synthesis of six close analogues of baclofen [3-(4-chlorophenyl)-4-aminobutyric acid] (BAC), a potent GABAB agonist, are reported. The compounds were designed starting from the structural informations contained in the solid state of BAC, regarded as a possible bioactive conformation, in which the p-chlorophenyl ring is perpendicular to the GABA backbone. A similar conformational situation was created by rigidifying the BAC structure by means of methylene (1), ethylene (2 and 6), or propylene (3) units, or by introducing chlorine atoms (4 and 5) into the ortho positions ("ortho effect"). Only compound 5 showed affinity for the GABAB receptor. Compound 6 [1-(aminomethyl)-5-chloro-2,3-dihydro-1H-indene-1-acetic acid], which was initially considered as representing the optimal mimic of the solid-state conformation of BAC, was surprisingly found inactive. An extensive conformational analysis was performed on compounds 1-6 in order to evaluate their flexibility and the overlap of their conformational population with respect to BAC. For this purpose a distance map was generated from three possible pharmacophoric groups: the amino and the carboxylic functions, and the phenyl ring. Finally, several explanations are proposed to account for the poor affinities of the prepared compounds such as steric hindrance or flexibility demand of the receptor.

Quantitative evaluation of the properties of a pyridazinyl GABA derivative (SR 95531) as a GABAA competitive antagonist. An electrophysiological approach.

We have investigated the effects of an aryl-aminopyridazine derivative of GABA (SR 95531) on dose-response curves of GABA-induced depolarizations from dorsal root ganglion neurones recorded intracellularly. The reversible shift to the right of the dose-response curves in a parallel fashion and the dissociation constant (KB) value of 0.13 +/- 0.02 microM (n = 15) indicate that this compound is a potent competitive GABAA antagonist. The competitive nature of SR 95531-induced antagonism was confirmed by single channel analysis. In excised membrane patches from bovine chromaffin cells (outside out configuration), 0.2-0.5 microM SR 95531 did not alter the mean open time of GABA-activated channels and did not introduce further short closing gaps within bursts. Whole cell recordings from cultured nodose ganglion neurones indicated that SR 95531 (10 microM) did not modify significantly any of the 3 types of calcium currents already reported in sensory neurones. This result might be of importance for further studies of presynaptic GABA actions on transmitter release.

Low concentrations of GABA reduce accommodation in primary afferent neurons by an action at GABAB receptors.

The pattern of accommodation of spike activity during sustained membrane depolarization was investigated in primary afferent neurons recorded intracellularly in vitro in the rat. We show that gamma-aminobutyric acid (GABA) and baclofen reduce accommodation in some fast conducting dorsal root ganglion neurons. This effect was restricted to those A delta cells with axons displaying a rather fast conduction velocity (15-25 m/s). GABA-induced blockade of accommodation was not observed in large A beta neurons. Pharmacological studies with baclofen, as opposed to isoguvacine, indicate that this effect is due to GABAB receptors activation. The effect is also shown to be resistant to bicuculline antagonism. In slow conducting afferents, GABAB receptor activation is known to shorten the CA2+ component of action potentials. By contrast, no such component was observed in the A delta cells studied. Furthermore, Ca2+-activated K+ conductances are not implicated in the reduction of accommodation caused by GABAB receptor activation. In conjunction with the actual knowledge about the distribution of GABA receptors on primary afferents, our result indicates that GABAA and GABAB receptors coexist on all categories of A delta and C primary afferents in the rat.

Electrophysiological study of SR 42641, a novel aminopyridazine derivative of GABA: antagonist properties and receptor selectivity of GABAA versus GABAB responses.

A new arylamino-pyridazine gamma-aminobutyric acid (GABA) derivative, SR 42641, has been tested for its ability to antagonize the actions of GABA on mammalian sensory neurones. SR 42641 and bicuculline reversibly decreased GABAA-induced depolarizations and currents recorded intracellularly from dorsal root ganglion neurons (DRG). Dose-response curves were shifted to the right in a parallel fashion. KB values (determined under voltage clamp conditions) were respectively 0.12 +/- 0.05 and 0.38 +/- 0.08 microM. Similar values were obtained with current clamp recording conditions. The study of the GABA-induced Cl- current under voltage-clamp conditions did not show any voltage-dependency of the antagonist effect of SR 42641. In nodose ganglion neurones, SR 42641 (0.4-4.5 microM) did not alter the (-)-baclofen-induced shortening of the calcium component of action potentials. At concentrations higher than 10 microM, SR 42641 itself prolonged calcium-dependent action potentials. Patch-clamp recordings from DRG cultured neurones indicated that SR 42641 did not affect the calcium current responsible for sustained calcium entry into cells. We conclude that SR 42641 is a potent competitive GABA antagonist, specific for the GABAA receptor. It does not act at the level of the chloride ionophore.

Properties of the GABA receptors located on spinal primary afferent neurones and hypophyseal neuroendocrine cells of the rat.

Electrophysiological techniques have been used to study the pharmacological characteristics of GABA receptors in two in vitro preparations likely to provide the ionic basis for GABAergic inhibition of excitation-secretion coupling. The shortening of Ca2+ spikes duration by GABAB receptors was shown to occur in slow conducting dorsal root ganglion cells, independently of marked depression of inward calcium currents. Ion-selective electrodes (K+ or Ca2+) were used to show the presence of both GABAA and GABAB receptors on the neurosecretory terminals and gland cells from hypophyseal neuro-intermediate lobe (NIL). In this latter preparation, potentiation of hormone release was observed under GABAA receptor activation, whilst inhibition was seen with GABAB agonists.

Dual population of GABAA and GABAB receptors in rat pars intermedia demonstrated by release of alpha MSH caused by barium ions.

We have studied the effects of selective GABAA and GABAB agonists on alpha-melanophore stimulating hormone (alpha MSH) release from intact rat neurointermediate lobes (NIL) in vitro. Agonist effects were tested against either basal alpha MSH output or BaCl2 (5 mM)-evoked release. GABA (50 microM) produced a biphasic effect on basal release, with an enhancement followed by inhibition of release. The enhancement but not the inhibition was blocked by bicuculline methiodide (100 microM). Baclofen (10 microM), a specific GABAB agonist, reduced the basal and Ba2+-evoked hormonal release in a stereospecific manner. (-)-Baclofen (5 microM) was active whereas the (+)-isomer was inactive at the same concentration. Isoguvacine (50 microM) a specific GABAA agonist, potentiated the Ba2+-evoked release of alpha MSH. GABA (50 microM) mimicked this effect, and its action was antagonized by bicuculline methiodide (200 microM). The results suggest that both GABAA and GABAB receptors are present on the endocrine cells of the intermediate lobe.

Different effects of phenylephrine and clonidine on 86Rb efflux and on contraction in the rat caudal artery.

A large transitory efflux of 86Rb was induced by concentrations of phenylephrine (10(-6) and 10(-4)M) or clonidine (10(-4)M) which were able to produce phasic contraction. By contrast, a lower concentration of clonidine (10(-6) M), which could only induce a slower (tonic) contraction, only produced a weak but sustained 86Rb efflux. These results show qualitative and quantitative differences in the effects of the two alpha-agonists. They further support the view that phasic and tonic alpha-adrenergic responses may be related to different mechanisms.

Electrophysiological study of GABAA versus GABAB receptors on excitation-secretion coupling.

Inwardly-directed Ca++-currents are caused by numerous types of action potentials which would not otherwise cause secretion. This process is regulated electrically and by neurotransmitters. We have studied in vitro the ionic mechanisms of GABA-mediated presynaptic inhibition and thereby the distinctive characteristics of GABAA and GABAB receptors: i.e., the GABAA system, which produces such short-lasting changes that there is an instantaneous reduction of spike amplitudes, in particular by opening Cl- -conductance, and the GABAB system, which results directly in inhibition of secretion due to a tonic depression of Ca++-currents. The principal aim was to determine if GABAB/Ca++ receptors could coexist on a membrane already possessing a large number of GABAA/Cl- sites available for presynaptic inhibition. Intracellular recordings of A delta and C dorsal root ganglion cell bodies were used as a model for the study of preterminal axonal membranes. Results were tentatively correlated with those obtained extracellularly by recording Ca++ and K+ movements (Cl- being assessed indirectly) from a set of other cells which also secrete neuropeptides by exocytosis: e.g., endings of unmyelinated fibres in the neurohypophysis and clusters of innervated gland cells in the pars intermedia. In recent years there has been a growing interest in receptors for neurotransmitters (e.g., monoamines, peptides, gamma-aminobutyric acid) modulating Ca++-dependent secretion in various biological systems (14-16, 24, 25, 29, 34). Modulation is possible either by changing the basic characteristics of the ionic currents during a standard action potential (Na+/K+ voltage transients being markedly altered by repolarizing K+ currents) or by any kind of direct action on voltage-dependent Ca++-channels: the latter will allow Ca++ to enter the cell in graded amounts not only as parts of well-defined Ca++-spikes (16, 14, 39; see also 24, 26, 28) but of well-defined Ca++-spikes (16, 14, 39; see also 24, 26, 28) but possibly also along complex sequences of tail-currents (for the biophysics, see 28, 29). Accordingly, it is essential to study presynaptic actions of transmitters in systems where spikes can be recorded, though presynaptic receptor activation can sometimes be identified by other means than spiking patterns (e.g., membrane effects in relation to other well-defined ionophores). The study of cells which are in contact with synapses synthesizing and releasing gamma-aminobutyric acid (GABA) has drawn much of the attention given to these problems.(ABSTRACT TRUNCATED AT 400 WORDS)

Electrophysiological study with K+- and Ca2+-sensitive micropipettes of GABA receptors in the rat neurointermediate lobe in vitro.

An efflux of K+ and a decrease in extracellular Ca2+ activity are to be expected when GABA markedly depolarizes the membrane of unmyelinated axons or secretory cells. Accordingly, we used extracellular recordings of ionic movements to specify GABA receptor presence in parts of the pituitary having GABAergic innervation: the neurohypophysis and intermediate lobe (NIL). We identified a site of action having the same desensitization characteristics and pharmacological criteria as the GABA A receptor which modulates Cl- -conductance. Baclofen, a GABA B agonist, was without effect. The possible distribution of receptors and role of GABAergic synapses in modulating neurotransmitter and hormone release by the NIL are discussed.

A comparative study of the action of frusemide and methyclothiazide on renin release by rat kidney slices and the interaction with indomethacin.

1 The effects of frusemide (a diuretic acting on the loop of Henle) and methyclothiazide (a thiazide diuretic) on renin release were studied on rat kidney slices. 2 Frusemide at concentrations of 1.5 and 7.5 mmol/l produced significant increases in renin release but had no effect at 0.15 mmol/l. 3 Methyclothiazide in a similar concentration range did not increase renin release; instead, at the highest concentration used, methyclothiazide (3.5 mmol/l) inhibited renin release. 4 Indomethacin (25 mumol/l) did not inhibit the increase of renin induced by frusemide. 5 Our limited study in vitro is consistent with the findings of other workers who have shown in vivo, in the absence of systemic electrolyte depletion, that only "loop diuretics" increase renin secretion. Under our experimental conditions, it is suggested that frusemide exerts a direct action either upon the epithelioid cells or upon the macula densa since the renal prostaglandin system does not intervene.

Evidence for the participation of beta1-adrenoceptors in isoprenaline-induced renin release from rat kidny slices in vitro.

1. The inhibitory effects were studied of 4 beta-adrenoceptor antagonists against renin release induced by isoprenaline (0.5 mumol/1) in rat kidney slices. Additionally the pA2 values of these 4 drugs were measured against isoprenaline in guinea-pig isolated atria and trachea (against beta1- and beta2-adrenoceptors respectively). 2 When employed at a concentration of 2 mumol/1 propranolol and atenolol significantly inhibited renin release (P less than 0.001 and P less than 0.01) whereas practolol and IPS 339 [t-butyl-amino-3 ol-2 propyl) oximino-9 fluorene] had little effect. 2 A positive correlation was shown between the degree of inhibition of renin release and the pA2 of the antagonists at the beta1-adrenoceptors. 4 When practolol and IPS 339 were used in equipotent molar concentrations to propranolol for the beta1-adrenoceptors they inhibited renin release. 5 The results suggest that the adrenoceptor involved in the renin release induced by isoprenaline in the rat kidney is of the beta1-type.

Effect of catecholamines on renin release in vitro.

Beta receptor activation by isoproterenol stimulates renin release by kidney slices "in vitro". This effect is dose-dependent and blocked by propranolol. High norepinephrine concentration has an inhibitory effect on renin secretion which is reversed by phentolamine: activation of alpha receptors probably decreases renin release.

beta-blocking drugs and renin secretion in the anaesthetized dog.

1. The effect of propranolol and sotalol on renin secretion was studied in the anaesthetized dog. 2. beta-adrenergic blockade did not modify basal renin secretion and did not affect the rise of renin secretion induced by haemorrhage or aortic constriction. 3. beta-adrenergic blockade diminished the rise in renin secretion induced by stimulation of the renal nerves.