GABAB receptor mediated inhibition of field stimulation-induced contractions of rabbit bladder muscle in-vitro

The effects of GABA and selective GABAA and GABAB receptor agonists and antagonists have been investigated on field stimulation-induced contractions (0.1 Hz) of rabbit urinary bladder strips in-vitro. Atropine inhibits twitches of bladder strips obtained from the bladder dome more effectively than those obtained from the bladder base. Both GABA and the selective GABAB receptor agonist, (+/-)-baclofen inhibited field stimulation-induced contractions to about the same extent, while the selective GABAA receptor agonist, homotaurine had no effect. In the presence of atropine, GABA failed to inhibit further the amplitude of twitches. The effects of either GABA or (+/-)-baclofen were antagonized by the GABAB receptor antagonists homotaurine and 5-aminovaleric acid, while the GABAA receptor antagonist picrotoxin had no effect. Neither GABA nor (+/-)-baclofen had any significant effect on acetylcholine-induced contractions of unstimulated bladder strips, but they were abolished by atropine. These results suggest that GABAB receptors inhibit field stimulation-induced contractions of rabbit bladder muscle by reducing the amount of acetylcholine released per nerve impulse.     

AN ATROPINE-LIKE INHIBITORY EFFECT OF DMPP ON RAT ISOLATED URINARY BLADDER

• 1 DMPP inhibits the nerve-mediated contractions of the rat isolated bladder, its effect being greater in preparations from newborn (2 day old) than adult animals. This effect of DMPP was unaffected by hexamethonium. In preparations from adult animals the effect of DMPP increased with frequency of stimulation and was fully prevented by the presence of atropine.
• 2 In bladders from newborn rats low concentrations of furthrethonium (FHR) (10 nM) activated a series of rhythmic contractions which were unaffected by tetrodotoxin and abolished by DMPP through an hexamethonium-insensitive action. On the other hand DMPP did not affect rhythmic contractions produced by a low concentration of eledoisin (60 nM).
• 3 In bladders from adult rats FHR (10 μM) and KCl (30 mM) produced contractures of comparable magnitude. DMPP inhibited, in concentration-related manner the FHR-induced tonic contraction but had little effect on that produced by KCl.
• 4 These findings indicate that in the rat bladder, DMPP antagonizes selectivity cholinergically-mediated contractions through a mechanism which is unaffected by hexamethonium or tetrodotoxin. An ‘atropine-like’ activity of DMPP should be considered.

     

EVIDENCE AGAINST PURINERGIC NERVE FIBRES IN THE HYPOGASTRIC NERVES OF THE CAT

• 1 Hypogastric nerve stimulation (HGS) produced a transient contraction of the cat urinary bladder and inhibition of pelvic nerve evoked bladder contractions.
• 2 Pretreatment by guanethidine (GUA) antagonized HGS inhibition of pelvic nerve evoked bladder contractions, but had no effect on the HGS-induced transient contractions.
• 3 GUA pretreatment had no effect on ATP or APPCP-induced contractions of the urinary bladder.
• 4 Pretreatment by 6-hydroxydopamine (6-OHDA) antagonized both the transient contraction and the inhibition of pelvic nerve evoked bladder contractions induced by HGS.
• 5 6-OHDA pretreatment had no effect on ATP or APPCP-induced contractions of the urinary bladder.

     

γ-AMINOBUTYRIC ACID AND POSTGANGLIONIC SYMPATHETIC TRANSMISSION IN THE PULMONARY ARTERY OF THE RABBIT

• 1 The effect of γ-aminobutyric acid (GABA) on postganglionic sympathetic neurotransmission was studied in strips of the rabbit pulmonary artery. The strips were preincubated with ³H-noradrenaline and then superfused with ³H-amine-free medium. They were stimulated either electrically at 2 Hz, or by 60 mM potassium, or by 1 μM tyramine.
• 2 GABA (1 – 1000 μM) did not change the basal outflow of tritium, but decreased the electrically evoked overflow as well as the contractile response. GABA 1 μM decreased the evoked overflow by 12%, and GABA 1000 μM, by 42%. The effect of GABA was not changed by yohimbine, propranolol, cocaine, corticosterone, or indomethacin. It was not antagonized by picrotoxin or bicuculline methiodide. GABA 100 μM also slightly reduced the potassium-evoked overflow of tritium but did not change the tyramineevoked overflow.
• 3 The results show that, in the pulmonary artery of the rabbit, GABA inhibits the release of noradrenaline. Its effect is independent of α- and β-adrenoreceptors and is not mediated by prostaglandins. The effect may be due to activation of presynaptic receptors which appear to differ from conventional GABA receptors inasmuch as they are insensitive to blockade by either picrotoxin or bicuculline.

     

THE EFFECTS OF CLONIDINE ON ELECTRICALLY-INDUCED CONTRACTIONS OF RAT DETRUSOR STRIPS IN VITRO

• 1 Clonidine (10^–9–3 times 10^–6M) produced a concentration dependent inhibition of field stimulation-induced contractions of rat detrusor muscle strip at 0.1 and 1 Hz which were completely abolished by tet-rodotoxin (5 times 10^–7M) but were unaffected by hexamethonium (10^–5M).
• 2 Pretreatment with yohimbine (10^–8–10^–7M) did not modify the amplitude of contractions but produced a rightward parallel shift of clonidine's cumulative response curve without a depression of the maximal response. The corresponding pA₂ value for yohimbine was 8.44 ± 0.1.
• 3 Atropine (3 times 10^–6M) produced a partial inhibition of contractions at both frequencies. In the presence of atropine the cumulative response curve of clonidine was significantly reduced at 1 but not at 0.1 Hz.
• 4 Indomethacin (5 times 10^–5M) and theophylline (2 times 10^–4M) produced a partial inhibition of amplitude of contractions at both frequencies without any interference with the effect of a supramaximal concentration of clonidine.
• 5 Prazosin (10^–6M), propranolol (10^–6M), chlorpheniramine (10^–6M), ranitidine (10^–6M), haloperidol (10^–7M), pizotifen (10^–6M), naloxone (10^–6M), quinidine (10^–6M), strychnine (10^–5M) or picrotoxin (10^–5M) neither affected the amplitude of contractions at either frequency nor antagonized clonidine effects.
• 6 The contractile response of non stimulated strips to acetylcholine (10^–5M), carbachol (3 times 10^–6M) and ATP (10^–3M) were not significantly influenced by pretreatment with clonidine (3 times 10^–6M). 7 These results suggest that stimulation of prejunctional α₂-adrenoreceptors located on postganglionic nerve endings might reduce the output of excitatory neurotransmitter(s) in rat urinary bladder.

     

MODULATION BY β-ADRENORECEPTORS OF SPONTANEOUS CONTRACTIONS OF RAT URINARY BLADDER

• 1 Propranolol (0.1 mg/kg i.v.) but not metoprolol (0.2 mg/kg i.v.) pretreatment increased the spontaneous motility triggered by progressive filling of rat urinary bladder without a concomitant effect on bladder capacity, except at high filling volumes. Compared to controls, the spontaneous motility of urinary bladder in propranolol pretreated rats displayed a higher frequency, indicating the existence of a tonic sympathetic inhibition.
• 2 β-adrenoreceptor stimulation by isoprenaline (0.1–10 μg/kg i.v.) or terbutaline (0.1–1 mg/kg i.v.) in vivo produced a dose dependent inhibition of bladder spontaneous motility which was antagonized by propranolol (0.1 mg/kg i.v.) but not by metoprolol (0.2 mg/kg i.v.). Propranolol (0.2 mg/kg i.v.) pretreatment did not antagonize the inhibition of bladder motility produced by intravenous papaverine (0.5 mg/kg).
• 3 Propranolol (0.1 mg/kg i.v.) significantly antagonized the isoprenaline-induced tachycardia (β₁ mediated) and fall in diastolic blood pressure (β₂ mediated) while metoprolol (0.2 mg/kg i.v.) antagonism was confined to β₁ mediated responses.
• 4 Isoprenaline (0.25–1.5 μM) inhibited in a concentration dependent manner field stimulation induced contractions of rat detrusor muscle strips as did tetrodotoxin (0.5 μM). Hexamethonium (50 μM) had no inhibitory effect.
• 5 Our in vivo findings support the view that β₂-adrenoreceptors are responsible for modulating bladder motility mainly by suppressing the onset of spontaneous contractions.

     

POSSIBLE INTERACTION OF CHOLINERGIC NERVES WITH TWO DIFFERENT (PRE AND POST) SITES OF THE NEUROMUSCULAR JUNCTION IN GUINEA-PIG VAS DEFERENS

• 1 Effects of various drugs on the mechanical responses of the longitudinal smooth muscle of guinea-pig vas deferens evoked by ACh and field nerve stimulation were examined.
• 2 ACh (28–280, μM) produced a contraction consisting of two phases. The first phase of the contraction was suppressed by guanethidine and by nicotinic antagonists. The second phase was suppressed only by atropine. Both phases were unaffected by TTX or prazosin.
• 3 Field stimulation (0.1 msec, 40 Hz) evoked contractions which also consisted of two (early and late) phases. Guanethidine (0.1-1 μM) suppressed both phases whilst prazosin (1 μM) suppressed only the late phase.
• 4 Atropine (0.1 μM) suppressed both phases whilst physostigmine (5 μM) potentiated both phases of field stimulation-evoked contractions.
• 5 Pentolinium suppressed both phases of field stimulation-evoked contractions at low concentrations (2–10 μM), but potentiated them at a higher concentration (100 μM).
• 6 dTC at a low concentration (0.5 mUM) suppressed the early phase, but slightly enhanced the late phase of field stimulation responses. At a higher concentration (20 μM), dTC potentiated both phases of the response.
• 7 Pentolinium and dTC did not affect the contractions induced by 90 mM-K ions, ATP or NA.
• 8 These results suggest that cholinergic nerves possess an excitatory action not only directly at the smooth muscle but also at the noradrenergic nerve terminals. The role of each receptor is discussed further.

     

Pharmacology of neurotransmission to the smooth muscle of the rat and the guinea-pig prostate glands

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Pharmacologically distinct GABAB receptors that mediate inhibition of GABA and glutamate release in human neocortex

1. The release of endogenous γ-aminobutyric acid (GABA) and glutamic acid in the human brain has been investigated in synaptosomal preparations from fresh neocortical samples obtained from patients undergoing neurosurgery to reach deeply located tumours.
2. The basal outflows of GABA and glutamate from superfused synaptosomes were largely increased during depolarization with 15 mM KCl. The K⁺-evoked overflows of both amino acids were almost totally dependent on the presence of Ca²⁺ in the superfusion medium.
3. The GABA_B receptor agonist (−)-baclofen (1, 3 or 10 μM) inhibited the overflows of GABA and glutamate in a concentration-dependent manner. The inhibition caused by 10 μM of the agonist ranged from 45–50%.
4. The effect of three selective GABA_B receptor antagonists on the inhibition of the K⁺-evoked GABA and glutamate overflows elicited by 10 μM (−)-baclofen was investigated. Phaclofen antagonized (by about 50% at 100 μM; almost totally at 300 μM) the effect of (−)-baclofen on GABA overflow but did not modify the inhibition of glutamate release. The effect of (−)-baclofen on the K⁺-evoked GABA overflow was unaffected by 3-amino-propyl (diethoxymethyl)phosphinic acid (CGP 35348; 10 or 100 μM); however, CGP 35348 (10 or 100 μM) antagonized (−)-baclofen (complete blockade at 100 μM) at the heteroreceptors on glutamatergic terminals. Finally, [3-[[(3,4-dichlorophenyl) methyl]amino]propyl] (diethoxymethyl) phosphinic aid (CGP 52432), 1 μM, blocked the GABA_B autoreceptor, but was ineffective at the heteroreceptors. The selectivity of CGP 52432 was lost at 30 μM, as the compound, at this concentration, inhibited completely the (−)-baclofen effect both on GABA and glutamate release.
5. It is concluded that GABA and glutamate release evoked by depolarization of human neocortex nerve terminals can be affected differentially through pharmacologically distinct GABA_B receptors.

     

Mechanisms involved in the spasmolytic effect of extracts from sabal serrulata fruit on smooth muscle

• 1.1. The effects of two extracts from Sabal serrulata fruits [total lipidic (L) and saponifiable (S)] on smooth muscle contractions have been assayed.
• 2.2. Both extracts (0.1–1 mg/ml) relaxed the tonic contraction induced by norepinefrine (30 nM) on rat aorta [EC₅₀, 0.53 ± 0.05 mg/ml (L) and 0.5 ± 0.04 mg/ml (S)] and by KCl (60 mM) on rat uterus. The Sabal extracts (0.3–1 mg/ml) also antagonized the dose-response curve of contractions induced by acetylcholine (0.1–100 μM) on urinary bladder.
• 3.3. dL-Propranolol (1 μM) but not the inactive (R)-(+)-propranolol (1 μM) potentiated the Sabal extracts relaxant effect by lowering the EC₅₀ (0.35 ± 0.2 vs 0.20 ± 0.01 mg/ml for L and 0.43 ± 0.02 vs 0.19 ± 0.02 mg/ml, P < 0.01, for S extract).
• 4.4. Cycloheximide (10 μg/ml) antagonized the effect of extracts from Sabal. However, actinomycin D (5 μg/ml) significantly (P ≤ 0.01) antagonized the effect of the total lipidic extract without modifying that of the saponifiable extract.
• 5.5. The relaxant effect of both extracts was not modified by the tyrosine kinase inhibitor genistein (10 μM) or the ornithine decarboxylase inhibitor α-difluoromethyl-ornithine (10 mM).

     

GABAA and GABAB receptor-mediated effects in guinea-pig ileum

1 The effects of γ-aminobutyric acid (GABA) and related substances were examined in guinea-pig ileum longitudinal muscle.

2 GABA at doses ranging from 10^-7 M to 3 × 10^-6 M elicited a relaxation while at higher doses (3 × 10^-6 M — 10^-4 M), as previously described, it caused a contraction followed by relaxation.

3 GABA-induced relaxation was bicuculline-insensitive, was mimicked by (-)-baclofen but not by homotaurine and muscimol. The effect of baclofen was stereospecific. GABA- and (-)-baclofen-induced relaxations were dose-dependent and their ED₅₀ values were similar. A specific cross-desensitization occurred between GABA and (-)-baclofen.

4 The bicuculline-insensitive relaxation induced by GABA and (-)-baclofen was prevented by tetrodotoxin and hyoscine but not by phentolamine plus propranolol, naloxone or theophylline.

5 In preparations in which the muscle tone was raised by histamine or prostaglandin F_2α, GABA and (-)-baclofen induced relaxation to the same extent as before increasing the tone. If the tone was raised by DMPP, a greater bicuculline-insensitive relaxation occurred.

6 Contraction caused by GABA was bicuculline-sensitive and was mimicked by homotaurine and muscimol. Contraction was dose-dependent and muscimol was about three times more potent than GABA or homotaurine. A specific cross-desensitization occurred between the contractile effects of GABA and those of homotaurine or muscimol.

7 Bicuculline competitively antagonized the contractile effects of GABA, homotaurine and muscimol and gave closely similar pA₂ values. The slope of the Schild plot for the above drugs was near 1, confirming the competitive nature of the antagonism.

8 The bicuculline-sensitive contraction induced by GABA, homotaurine and muscimol was abolished by tetrodotoxin and was non-competitively antagonized by hyoscine, while it was unaffected by hexamethonium, mepyramine and methysergide.

9 It is concluded that two receptors mediate the GABA effects in guinea-pig ileum: a bicuculline-sensitive GABA_A receptor, which elicits contraction through an excitatory action on cholinergic post-ganglionic neurones; and a bicuculline-insensitive GABA_B receptor which causes relaxation through an inhibitory presynaptic action on cholinergic post-ganglionic neurones. We confirm that GABA, homotaurine and muscimol are GABA_A agonists, while GABA and (-)-baclofen are GABA_B agonists.

     

The effect of peripherally administered GABA on spontaneous contractions of rat urinary bladder in vivo

• 1.1. Intravenous GABA suppresses the spontaneous contractions of rat urinary bladder produced by saline loading (micturition reflex) without possessing any significant inhibitory effect on spontaneous and stimulated (neuro-hormones and field stimulation) contraction of rat detrusor strips in vitro.
• 2.2. The in vivo effects of GABA, in view of the results obtained with atropine, hexamethonium and tetrodotoxin in the same experimental conditions, are likely to be ascribable to an action at pelvic ganglia level.

     

GABA-RELATED ACTIVITIES OF AMINO PHOSPHONIC ACIDS ON GUINEA-PIG ILEUM LONGITUDINAL MUSCLE

• 1 The effects of phosphonic analogues of GABA, β-alanine and glycine on guinea-pig ileum longitudinal muscle were measured.
• 2 Aminomethylphosphonic acid (AMPh) and 2-aminoethylphosphonic acid (2-AEPh) were devoid of any effect both in non-stimulated preparations and in electrically-stimulated preparations.
• 3 The phosphonic analogue of GABA, 3-aminopropylphosphonic acid (3-APPh) possessed a GABA_B agonistic effect (relaxation and inhibition of twitch response) at doses of 10^?3 M. No agonistic effect on GABA_a receptors was observed.
• 4 3-APPh at doses tested (2 × 10^?4 M and 10^?3 M) also displayed antagonistic action on the effects of GABA_B agonists producing a parallel shift of the log dose-effect curves of GABA- and (-)-baclofeninhibition of twitch responses. In contrast 3-APPh did not antagonize the inhibitory effect of morphine and noradrenaline.
• 5 The contractile effect of GABA, mediated via GABA_A receptors, was unaffected by 3-APPh (10^?3 M).
• 6 It is concluded that 3-APPh is a partial agonist at the GABA_B site in guinea-pig ileum.

     

GABAA- and GABAB-receptor-mediated modification of intestinal motility

The actions of γ-aminobutyric acid (GABA) and its analogues, 3-amino-1-propanesulphonic acid (3APS) and baclofen, have been investigated using isolated segments of the guinea-pig ileum and distal colon. GABA and 3APS, but not baclofen, induced GABA_A-receptor mediated effects; prompt, dose-dependent contractions of the ileum which were antagonised by bicuculline, picrotoxinin, piretanide, tetramethylenedisulphotetramine, atropine and tetrodotoxin. Baclofen and GABA, but not 3APS, induced a dose-dependent GABA_B-receptor mediated depression of electrically elicited twitch contractions of the ileum, unaffected by the GABA_A-receptor antagonists or by antagonism of adenosine, adrenergic, opiate or nicotinic receptors. In the distal colon, baclofen and GABA caused a bicuculline- and picrotoxinin-intensitive depression of spontaneous cholinergic contractions. Desensitization to GABA and baclofen, and cross-desensitization to both agonists was observed. Combined antagonism of GABA_A-receptors and desensitization to baclofen slowed pellet expulsion to the same extent as GABA desensitization alone, indicating that both GABA_A- and GABA_B-receptor sites are involved in this modification of peristalsis by GABA.     

Unique effect of tetrapentylammonium ions on sympathetic transmission in rat vas deferens

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Differential effects of two major neurosteroids on cerebellar and cortical GABA(A) receptor binding and function

Cerebellar and cerebrocortical A-type γ-aminobutyric acid (GABA_A) receptors were examined in mice and rats. In wild-type mouse cerebellum, the agonists GABA and gaboxadol exerted heterogeneous displacement of [³H]ethynylbicycloorthobenzoate (EBOB) binding with nanomolar and submicromolar affinities. In mouse cerebella lacking α6 subunits (α6KO), nanomolar displacement by GABA agonists was absent, while micromolar displacement was potentiated to 12-fold by 0.3 μM 5α-tetrahydrodeoxycorticosterone (5α-THDOC). In α6KO cerebellum, 60% of [³H]EBOB binding was neurosteroid-insensitive, while 5α-THDOC elicited enhancement with EC₅₀ = 150 nM instead of nanomolar displacement. In conclusion, nanomolar displacement of cerebellar [³H]EBOB binding by GABA agonists and neurosteroids can be attributed to GABA_A receptors containing α6 and δ subunits. In contrast, [³H]EBOB binding to rat cerebral cortex was affected by allopregnanolone and 5α-THDOC in bidirectional manner with nanomolar enhancement (EC₅₀ ~ 80 nM) and micromolar displacement. Nonequilibrium binding conditions with decreased incubation time tripled the maximal enhancement of [³H]EBOB binding by 5α-THDOC. 5ß-THDOC enhanced the cortical [³H]EBOB binding with EC₅₀ ~ 0.5 μM and it attenuated bidirectional modulation by 5α-THDOC. Allopregnanolone and 5α-THDOC produced biphasic enhancements of chloride currents elicited by 1 μM GABA in cerebellar granule cells, for 5α-THDOC with EC_50,1 ~ 16 nM and EC_50,2 ~ 1.3 μM. Differences in peak current enhancements in the absence minus presence of 0.1 mM furosemide corresponding to α6ßδ GABA_A receptors were augmented only by micromolar 5α-THDOC while the difference curve for allopregnanolone was polyphasic as without furosemide. Consequently, these neurosteroids differentially affected the binding and function of various GABA_A receptor populations.     

Multiple sensory and functional effects of non-phenolic aminodimethylene nonivamide: An approach to capsaicin antagonist

• 1.1. Hexylaminodimethylene nonivamide (CAPCNC6, 0.1–10 μM) inhibited the contractility of isolated guinea pig right atria, toxically revealed positive inotropic, chronotropic and then a cardiac arrest effect at 100 μM and inhibited capsaicin (1.0 μM)-induced cardiotonic effects.
• 2.2. CAPCNC6 (0.1–10 μM)-induced aorta contractions were inhibited in the presence of flunarizine, atropine, phentolamine, Ca²⁺-free solution and pre-treatment of the animal with capsaicin.
• 3.3. CAPCNC6 (1.0–300 μM)-induced trachea contractions were inhibited in the presence of capsaze. pine, ruthenium red, hCGRP_8–37 and pre-treatment of the animal with capsaicin.

     

Effect of exogenous adenosine and its monophosphate on the contractile response to acetylcholine in the human isolated detrusor muscle strips

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Electromechanical coupling in human vas deferens: effects of agents that modulate intracellular release of calcium

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Effects of propofol on GABAA channel conductance in rat-cultured hippocampal neurons

Channels were activated, in ripped-off patches from rat-cultured hippocampal neurons, by propofol alone, propofol plus 0.5 μM GABA (γ-aminobutyric acid) or GABA alone. The propofol-activated currents were chloride-selective, showed outward-rectification and were enhanced by 1 μM diazepam. The maximum propofol-activated channel conductance increased with propofol concentration from less than 15 pS (10 μM) to about 60 pS (500 μM) but decreased to 40 pS in 1 mM propofol. Fitting the data from 10 to 500 μM propofol with a Hill-type equation gave a maximum conductance of 64 pS, an EC₅₀ value of 32 μM and a Hill coefficient of 1.1. Addition of 0.5 μM GABA shifted the propofol EC₅₀ value to 10 μM and increased the maximum channel conductance to about 100 pS. The Hill coefficient was 0.8. The maximum channel conductance did not increase further when 1 μM diazepam was added together with a saturating propofol concentration and GABA. The results are compared to effects other drugs have on GABA_A channels conductance.