Effect of penicillin on the increase in membrane conductance induced by γ-aminobutyric acid at the crab neuromuscular junction

The effects of penicillin and picrotoxin on the increase in membrane conductance produced by gamma-aminobutyric acid (GABA) at the hermit crab neuromuscular junction were investigated. Penicillin failed to block the effects of GABA, while picrotoxin proved to be a potent antagonist.     

Central cardiovascular actions of γ-aminobutyric acid

The vasomotor and cardiac effects of γ-aminobutyric acid (GABA) administered by different routes were studied in cats and dogs. The cat was resistant to the action of GABA administered intravenously or into a vertebral artery. Intrathecal injection of GABA into the cat depressed the vasomotor reponse to spinal compression. In dogs, intravenous, intrathecal or intraventricular injection, or topical application of GABA to the floor of 4th ventricle consistently produced hypotension and depressed the reflex and direct excitability of the vasomotor neurones, located at the supraspinal and spinal levels. Bradycardia observed after intravenous and intraventricular injections of GABA into dogs was abolished by stellate ganglionectomy but not by vagotomy. It has been attributed to depression of the central sympathetic neurones.     

Nonspecific depressant action of γ-aminobutyric acid on somatic reflexes

The effects of γ-aminobutyric acid (GABA) on central integration of somatic reflexes in the cat have been studied by topical application, and by intrathecal or intracerebroventricular injection. Intrathecal injection of GABA inhibited the monosynaptic patellar reflex. The facilitation of the patellar reflex induced by strychnine, leptazol, tubocurarine and tetanus toxin was also inhibited. Polysynaptic facilitation of the patellar reflex induced in the spinal cat by electrical stimulation of the contralateral sciatic nerve was depressed by intrathecal GABA. Similarly, the supraspinal facilitation of the patellar reflex by electrical stimulation of the brain stem reticular formation was inhibited by application of GABA to the floor of the 4th ventricle. The polysynaptic inhibition of the patellar reflex at both levels was intensified by GABA. The flexor (tibialis anterior) reflex was depressed in the same manner as the extensor patellar reflex. The polysynaptic linguomandibular reflex was depressed by intracerebroventricular GABA. The depressant action of GABA at spinal and supraspinal levels of the neuraxis is discussed in relation to the role of GABA as an inhibitory transmitter in the central nervous system.     

Two types of γ-aminobutyric acid receptor on embryonic sensory neurones

1 Embryonic sensory neurones of the chick grown in dissociated cell culture respond to application of low concentrations of gamma-aminobutyric acid (GABA) with a change in resting membrane resistance (R(in)) and/or a change in action potential duration (APD) (Dunlap & Fischbach, 1978; Choi & Fischbach, 1981). Intracellular microelectrode recording techniques were employed to determine if these two effects are mediated by the same, or different, GABA receptors.2 Cells responded, for the most part, with a change in either R(in) or APD, but 10% of the cells exhibited both effects. In the latter cells the two responses were clearly distinguishable as discussed below.3 The proportion of neurones exhibiting a GABA-induced decrease in R(in) declined during the first week in vitro while the proportion exhibiting a decrease in APD increased during that time.4 The two effects were pharmacologically distinct. Muscimol, a GABA analogue, produced only the change in R(in) (ED(50) = 5.5 muM) while baclofen, another analogue of GABA, produced only the change in APD (ED(50) = 1 muM). The analogues were approximately equipotent with GABA. Bicuculline, a GABA antagonist, blocked the muscimol-induced change in R(in) (but not the baclofen-induced change in APD) in a dose-dependent fashion with an ID(50) = 0.7 muM.5 The time courses of the two effects were different. The change in APD resulting from a brief application of GABA (or baclofen) was prolonged relative to the rapid return to control associated with the GABA- (or muscimol-) induced change in R(in).6 Desensitization of the two responses exhibited separate time courses. In the continual presence of the agonists, GABA- and muscimol-induced decreases in R(in) completely desensitized in ca. 10 s while GABA- and baclofen-induced decreases in APD persisted undiminished throughout a prolonged (1 min) application of the drugs and returned to control only after cessation of application.7 It is concluded that embryonic chick sensory neurones in culture exhibit two types of GABA receptor that differ in their functional and pharmacological properties. Implications of these results are discussed.     

Vasopressin release produced in anaesthetized cats by antagonists of γ-aminobutyric acid and glycine

1 In cats anaesthetized with chloralose, the central excitatory substances, tubocurarine, picrotoxin, bicuculline, leptazol and strychnine, were applied to the exposed ventral surface of the brain stem through paired Perspex rings placed across the medulla and their effects on vasopressin release and arterial blood pressure were examined.

2 The excitatory substances released large amounts of vasopressin when applied to an area 6-9 mm caudal to the trapezoid bodies. From this area vasopressin release was previously obtained with nicotine.

3 With nicotine, the vasopressin release occurred almost instantaneously and tachyphylaxis developed rapidly. With the excitatory substances the release increased gradually and there was no tachyphylaxis. When these substances were applied for several minutes, the release reached its maximum a considerable time after their removal, except with leptazol when release diminished at once after removal.

4 The excitatory substances had little or no effect on arterial blood pressure when applied to the vasopressin releasing area, but produced strong pressor responses when applied to a more rostrally situated area.

5 It is concluded that the excitatory substances release vasopressin and raise arterial blood pressure because they are antagonists of γ-aminobutyric acid and/or glycine and that numerous inhibitory neurones which release these amino-acids synapse at the ventral surface of the medulla. The physiological function of those which synapse at the vasopressin releasing area may be to act as a brake on vasopressin release, and of those which synapse at the more rostrally situated area to act as a brake on arterial blood pressure.

     

Influence of γ-aminobutyric acid on baclofen intestinal absorption

Since previous studies suggested that baclofen absorption in the rat middle intestine was inhibited by β-alanine and therefore mediated, at least in part, by the β-aminoacid carrier, we focused our new studies on the analysis of the possible inhibition of the drug by a γ-aminoacid model compound, γ-aminobutyric acid (GABA). A rat jejunum in situ study was undertaken in order to evaluate the effect of GABA on baclofen absorption and to establish the inhibition model. Assays using isotonic perfusion solutions of 0.5 mM baclofen with starting GABA concentrations ranging from 0 to 100 mM are reported. The results show that the absorption rate pseudoconstants of the drug decrease as the GABA concentration increases, with a limiting value of 0.65 h^?1 (±0.01). A partial competitive inhibition or complete competitive inhibition in the presence of a passive component could define the interaction phenomena between the two substances. Kinetic absorption parameters for GABA in the presence and absence of baclofen (K_i = 5.67 ± 1.54, K_m = 3.87 ± 0.63) suggest the existence of more than one intestinal carrier system for baclofen or GABA.     

Depolarization of neurones in the isolated olfactory cortex of the guinea-pig by γ-aminobutyric acid

1 Effects of γ-aminobutyric acid (GABA) on single neurones in slices of guinea-pig olfactory cortex maintained in vitro were recorded with single intracellular microelectrodes. The average resting potential of 52 cells was -75 mV and apparent input resistance ranged from 20 to 200 MΩ.

2 Superfusions of GABA over the slice invariably depolarized the neurones and reduced their input resistance. The minimum effective concentration was 50 to 200 μM.

3 The reversal potential for the depolarization produced by 0.1 mM GABA (E_g) was -66 ± 2 mV. At concentrations >0.1 mM the reversal potential became progressively more positive (-55 to -50 mV).

4 Reduction of external chloride, with isethionate as the substitute anion, increased the amplitude of the depolarization.

5 GABA reduced the amplitude of the excitatory postsynaptic potential produced by lateral olfactory tract stimulation, and occluded or reversed the subsequent depolarizing recurrent inhibitory postsynaptic potential.

6 Action potentials elicited by injection of depolarizing current or by focal antidromic stimulation were slowed and reduced in amplitude by GABA.

7 The effects of GABA on membrane conductance (potency = 1) were duplicated by 3-aminopropanesulphonic acid (potency = 20), β-alanine (0.5), β-amino-n-butyric acid (0.5), glycine (0.3) and L-2,4-diaminobutyric acid (0.2). For a given conductance change, 3-aminopropanesulphonic acid, glycine and β-alanine produced less depolarization than did GABA.

8 It is concluded that the action of GABA on the neurones is compatible with a role in mediating recurrent postsynaptic inhibition.

     

On the pharmacology of the γ-aminobutyric acid receptors on the cuneo-thalamic relay cells of the cat

1. gamma-Aminobutyric acid (GABA) and glycine applied by iontophoresis were equipotent depressants of cuneo-thalamic relay neurones isolated from the middle third of the cuneate nucleus of cats either decerebrated or anaesthetized with sodium pentobarbitone.2. Glycine 13+/-2 nA and GABA 20+/-2 nA were equipotent depressors of hair cells (n=22) and, bicuculline applied by iontophoresis caused a parallel shift to the right of the GABA but not the glycine log-current response curves. The GABA equipotent dose-ratio was 2.0+/-0.2 for bicuculline currents of approximately 144 nA lasting about 11 min in cells excited either transynaptically by peripheral stimulation or postsynaptically by glutamate.3. Although a maximal bicuculline current seldom caused a significant shift of the glycine-log current response curve, many of our records show the onset of the glycine response to be slowed by doses in excess of 84 nA.4. Bicuculline also antagonized depressions by beta-guanidinopropionic acid, and delta-aminovaleric acid which mimicked the action of GABA.5. When tested on the same neurone, bicuculline and picrotoxin applied by iontophoresis were equipotent and their effects appear to be additive.6. The GABA sensitivity was not modified by repetitive (5 or 6) doses of i.v. bicuculline (0.2 mg/kg).7. The antagonism of GABA by bicuculline and picrotoxin appears to be of sufficient specificity to enable the separate roles of GABA and glycine as putative inhibitory transmitters of cuneo-thalamic relay cells to be determined.     

Depolarizing actions of γ-aminobutyric acid and related compounds on rat superior cervical ganglia in vitro

1 Potential changes in rat superior cervical ganglia were recorded in vitro with surface electrodes.2 gamma-aminobutyric acid (GABA) produced a transient, low-amplitude ganglion depolarization at rest, and a transient hyperpolarization in ganglia depolarized by carbachol. Depolarization was not prevented by preganglionic denervation. The log dose-response curve for depolarization was sigmoid with a mean ED(50) of 12.5 muM.3 The ganglion was depolarized in similar manner by the following compounds (mean molar potencies relative to GABA (=1) in brackets): 3-aminopropane sulphonic acid (3.4), gamma-amino-beta-hydroxybutyric acid (0.27), beta-guanidino-propionic acid (0.12), guanidinoacetic acid (0.057), delta-aminovaleric acid (0.048), beta-alanine (0.01), 2,4-diaminobutyric acid, gamma-guanidinobutyric acid, taurine and N-methyl-GABA (all <0.01). The following compounds did not depolarize the ganglion at 10 mM concentrations: alpha- and beta-amino-n-butyric acids, alpha-amino-iso-butyric acid, glycine and glutamic acid.4 Depolarization declined in the continued presence of GABA. Ganglia thus ;desensitized' to GABA showed a diminished response to other amino acids but not to carbachol.5 The effect of GABA was not antagonized by hyoscine and hexamethonium in combination, in concentrations sufficient to block responses to carbachol.6 Responses to GABA were blocked more readily than those to carbachol by bicuculline (IC(50), 14 muM) and picrotoxin (IC(50), 37 muM). Strychnine (IC(50), 73 muM) was a relatively weak and less selective GABA-antagonist.7 It is concluded that sympathetic ganglion cells possess receptors for GABA and related amino acids which are (a) different from the acetylcholine receptors and (b) similar to GABA receptors in the central nervous system.     

Protective effect of γ-aminobutyric acid against glycerol-induced acute renal failure in rats

To investigate the effect of γ-aminobutyric acid (GABA) on acute renal failure, we used a rat model of acute tubular necrosis induced by glycerol. After deprivation of water for 6 h, the rats received an injection of 50% glycerol into the muscle of the rear limb at 10 ml/kg body weight. GABA was then administered orally to the rats (100 or 500 mg/kg body weight/day) once every 12 h for 3 days. The rats with acute renal failure showed arrested body weight gain and an increase of kidney weight, whereas oral administration of GABA attenuated the physiological changes induced by acute renal failure. However, GABA administration had no significant effect on increased urine volume. Oral administration of GABA at a dose of 100 or 500 mg/kg body weight/day for 3 days significantly improved the markedly elevated levels of blood urea nitrogen and creatinine and the reduced creatinine clearance related to progression of renal failure. Moreover, the rats with acute renal failure exhibited high levels of fractional excretion of sodium (FE_Na) due to alteration of tubule function following injection of glycerol. However, administration of GABA lowered the FE_Na levels dose-dependently. Furthermore, urine osmolarity was markedly reduced in control rats with acute renal failure as compared with normal rats, whereas it was significantly increased by administration of GABA at a dose of 500 mg/kg body weight/day. These results indicate that GABA has potential as a therapeutic agent against the renal damage involved in acute renal failure.     

Ca2+ and aminoguanidine on γ-aminobutyric acid accumulation in germinating soybean under hypoxia–NaCl stress

Gamma-aminobutyric acid (GABA), a nonproteinous amino acid with some benefits on human health, is synthesized by GABA-shunt and the polyamine degradation pathway in plants. The regulation of Ca²⁺ and aminoguanidine on GABA accumulation in germinating soybean (Glycine max L.) under hypoxia-NaCl stress was investigated in this study. Exogenous Ca²⁺ increased GABA content significantly by enhancing glutamate decarboxylase gene expression and its activity. Addition of ethylene glycol tetraacetic acid into the culture solution reduced GABA content greatly due to the inhibition of glutamate decarboxylase activity. Aminoguanidine reduced over 85% of diamine oxidase activity, and 33.28% and 36.35% of GABA content in cotyledon and embryo, respectively. Under hypoxia −NaCl stress, the polyamine degradation pathway contributed 31.61–39.43% of the GABA formation in germinating soybean.     

On the mechanism by which veratridine causes a calcium-independent release of γ-aminobutyric acid from brain slices

1 The mechanisms by which veratridine increases the release of γ-aminobutyric acid (GABA) from brain slices have been studied.

2 Exposure of superfused cerebro-cortical, nigral or cerebellar slices to veratridine (5 μM) or KCl (50 mM) caused large increases in the efflux of [³H]-GABA.

3 Reduction of the external Ca concentration [Ca]_o to zero had strikingly different effects on the veratridine and K-evoked release of [³H]-GABA. The K-evoked release from all three areas was greatly reduced in Ca-free medium, but the veratridine-evoked release from cerebeller slices was not affected, and the release of [³H]-GABA from cortical and nigral slices was increased three fold. The potentiation of the veratridine evoked release of GABA which occurred in Ca-free medium was not due to the reduction in divalent ions, because it still occurred in medium in which the Ca was replaced by an equivalent amount of Mg.

4 The veratridine-evoked release of [¹⁴C]-glycine from slices of spinal cord was also significantly increased in Ca-free medium. In contrast, the release of cortical [³H]-noradrenaline and [¹⁴C]-acetylcholine caused by the alkaloid was greatly diminished in Ca-free medium.

5 The veratridine but not the K-evoked release of [³H]-GABA was abolished when the external Na concentration [Na]_o was reduced to zero and by tetrodotoxin (TTX) (0.2 μM). Cl-free medium did not affect the veratridine-evoked release of [³H]-GABA or its potentiation by Ca-free medium.

6 Exposure of the tissue to depolarizing concentrations of external K ([K]_o = 120 mM) did not abolish the veratridine evoked release of [³H]-GABA or its potentiation by Ca-free medium.

7 Pre-incubation of cortical slices with L-2,4, diaminobutyric acid (DABA), or substitution of Na in the superfusion medium with Li, did not affect the veratridine-evoked release of [³H]-GABA, indicating that the alkaloid does not stimulate GABA efflux by a carrier-mediated transport process.

8 Exposure of the tissue to ruthenium red (10 μM) increased the veratridine evoked release of [³H]-GABA in both normal and in Ca-free medium but almost abolished the K-evoked release.

9 It is suggested that veratridine causes GABA release by increasing the permeability of the nerve terminals to Na. In normal medium, the resulting influx of Ca²⁺ ions through voltage-dependent Ca²⁺ channels may be involved in triggering the release of GABA. However, a major part of the GABA efflux appears to be triggered by the release of Ca²⁺ ions from intraterminal mitochondria, which results from the increase in[Na]_i. Since Ca²⁺ ions antagonize the action of veratridine, the potentiation of the drug-evoked release of GABA that occurs in Ca-free medium, might be due to the absence of the antagonistic Ca²⁺ ions. The resulting greater increase in Na entry and [Ca]_i caused by Ca release from intracellular stores, must presumably more than balance the contribution normally made by any influx of extracellular Ca²⁺.

     

A comparative study of some convulsant substances as γ-aminobutyric acid antagonists in the feline cerebral cortex

1. By the use of microiontophoretic techniques, quantitative estimates were obtained of the depressant effects of gamma-aminobutyric acid (GABA) on single feline cortical neurones.2. Picrotoxin, bicuculline, strychnine, (+)-tubocurarine, penicillin and leptazol were also applied microiontophoretically to single neurones. Sequential GABA applications were made before, during and after the microiontophoresis of these substances and any effects on the time course of the GABA depression were measured as an estimate of antagonism or potentiation of GABA.3. (+)-Tubocurarine was found to be a potent GABA antagonist. Picrotoxin and bicuculline were rather less potent and strychnine and penicillin only weakly active as GABA antagonists. Leptazol appeared to be inactive against GABA depressions.4. In addition, bicuculline and strychnine were found to be capable of potentiating the depressant action of GABA. This property was not shared by the other substances studied.5. All the substances studied produced changes in neuronal firing rate that did not correlate with GABA antagonism.6. In conclusion, several potent convulsants have been shown to be capable of GABA antagonism. It is not yet clear that this effect, rather than a direct effect on neuronal excitability, is the prime mechanism behind their convulsant properties.     

Baclofen (β-p-chlorophenyl-γ-aminobutyric acid) enhances [3H]γ-aminobutyric acid (3H-GABA) release from rat globus pallidus in vitro

The rat globus pallidus has been investigated as a possible model in which to study pre-synaptic G AB A mechanisms in vitro. (±)-Baclofen (300 μM-1 mM) significantly enhanced the release of radioactivity from superfused slices of rat globus pallidus prelabelled with ³H-GABA in vitro. This releasing action was specific to the (+)-isomer of baclofen: neither the (—)-isomer nor another neuronal depressant DL-α-∞-diaminopimelic acid had any significant effect. The releasing effect of baclofen appeared unrelated to the phenethylamine moiety of its structure as neither β–phenethylamine nor dopamine evoked release of ³H-GABA from pallidal slices. Baclofen increased the efflux of radioactivity from pallidal slices prelabelled with either [³H]β-alanine or [³H]diaminobutyric acid in vitro. The use of specific glial and neuronal GABA uptake blocking compounds (β-alanine and (±)-cis-1,3-amino-cyclohexanecarboxylic acid) did not permit resolution of the elements from which baclofen was evoking [³H]GABA release. Baclofen also inhibited uptake of [³H]GABA into pallidal slices with an IC50 value of 6 × 10^?4 M. The GABA-like properties of baclofen may be related to the (+)-isomer while non-specific neuronal depressant actions are an effect of the (—)-isomer. The potential of the (+)-isomer as an antipsychotic agent while (—)-baclofen remains the effective antispastic drug free from unwanted side-effects, is discussed.     

α- and β-Adrenoceptors in the detrusor muscle and bladder base of the pig and β-adrenoceptors in the detrusor muscle of man

1 The presence and type of adrenoceptors in the smooth muscle of the pig and human urinary bladder was assessed on the basis of the relative potency of α- and β-adrenoceptor agonists and antagonists.

2 In isolated, carbachol-contracted bladder strips from the pig detrusor muscle the relaxing potency of isoprenaline was four times that of salbutamol and ritodrine and thirty times that of noradrenaline.

3 Propranolol caused a parallel shift to the right of the noradrenaline dose-response curve which was not changed by phentolamine.

4 Propranolol and butoxamine showed, in contrast to practolol, a dose-dependent antagonism of the response to isoprenaline. A pA₂ value of 9.2 ± 0.2 and 6.8 ± 0.2 (mean ± s.e. mean) for the first two antagonists was calculated.

5 In the bladder base of the pig, propranolol caused a parallel shift to the right and phentolamine a shift to the left of the dose-response curve to noradrenaline.

6 In the human detrusor muscle the potency and maximum effect of isoprenaline and salbutamol were less than those in the pig detrusor muscle. The potency of isoprenaline was sixty times that of salbutamol.

7 Whereas a parallel shift to the right of the dose-response curve to isoprenaline was obtained with propranolol, no antagonism was obtained with butoxamine or practolol.

8 The results are interpreted as indicating the presence of β₂-adrenoceptors in the detrusor muscle of the pig and β-adrenoceptors with neither β₁- nor β₂-characteristics in the detrusor muscle of man. An indication of the presence of α-adrenoceptors in the bladder base but not in the detrusor muscle of the pig was obtained.