A distinct distribution of functional presynaptic 5-HT receptor subtypes on GABAergic nerve terminals projecting to single hippocampal CA1 pyramidal neurons.

5-HT is known to modify the excitability of GABAergic interneurons projecting to hippocampal CA1 neurons. In this study we investigate the presence and functionally characterize the 5-HT receptor subtypes found on the presynaptic nerve terminals of these GABAergic neurons. Using conventional whole-cell patch recording, we confirmed that the 5-HT(1A) agonist, 8-hydroxy-2-dipropylaminotetralin, presynaptically decreased electrically evoked GABA release while the 5-HT(3) agonist, m-chlorophenylbiguanide (mCPBG), presynaptically facilitated release. Using the 'synaptic bouton preparation', where CA1 neurons are acutely isolated with functional nerve terminals/boutons remaining adherent, we next showed that these receptor subtypes are found presynaptically. We next used the technique of focal stimulation of a single bouton in this preparation to further investigate the distribution of these 5-HT receptor subtypes. We found that all boutons contained inhibitory 5-HT(1A) receptors while a subset of boutons showed both 5-HT(1A) and excitatory 5-HT(3) receptors. No boutons were detected which contained only 5-HT(3) receptors. Our studies show that presynaptic 5-HT receptor subtypes are found presynaptically and are not uniformly distributed. This provides another potential mechanism whereby 5-HT can modulate GABA release and hence the excitability of hippocampal neurons.

The properties of ryanodine-sensitive Ca(2+) release in mouse gastric smooth muscle cells.

1. Under voltage-clamped conditions, gastric smooth muscle cells of BALB/c mice developed spontaneous (STOCs) and caffeine- (I(CAF)) and carbachol-induced (I(CCh)) transient outward currents. 2. In fura-2 microscopic measurements of intracellular Ca(2+) concentration ([Ca(2+)](i)), caffeine and carbachol (CCh) provoked similar transient [Ca(2+)](i) elevations. 3. Both I(CCh) and CCh-induced [Ca(2+)](i) elevation of single smooth muscle cells occurred in an 'all-or-nothing' fashion in contrast to the reproducible caffeine responses. 4. On the basis of the suppression of STOCs and I(CAF) by nicardipine, tetraethylammonium and iberiotoxin, but not by charybdotoxin nor apamin, it was suggested that both currents were generated by large conductance type Ca(2+)-activated K(+) channels. 5. In measurements of isometric tension, caffeine produced relaxation of gastric smooth muscle strips in a concentration-dependent manner (0.1 -- 3 mM). The concentration-dependent relaxation with caffeine was mimicked by dibutyryl cyclic AMP which produced potentiation of contraction triggered by 50 mM KCL. 6. At caffeine concentrations >3 mM, a transient contraction followed by relaxation was provoked as the quasi maximal response to caffeine. In the quasi maximal response, caffeine acted as a potent relaxant in smooth muscle strips precontracted with 50 mM KCl or 3 microM CCh. 7. The relaxation with caffeine was significantly accelerated in those strips precontracted with KCl or CCh. All these results suggest that ryanodine-sensitive Ca(2+) release, which is triggered by caffeine, is an important modifier of Ca(2+) homeostasis in the cytoplasm and the contractility of gastric smooth muscle cells of mice.

Noradrenaline receptor-mediated potentiation of caffeine-induced Ca( 2+)-activated K(+) currents in bovine ciliary muscle cells.

PURPOSE: Adrenoceptor-mediated modulation of a caffeine (CAF)-induced [Ca(2+)](i) elevation and resulting Ca(2+)-activated K(+) current (I(CAF)) in bovine ciliary muscle (CM) cells were investigated. METHODS: The nystatin-perforated patch clamp technique for the measurement of membrane currents and a microscope based fura-2 fluorescence imaging of [Ca(2+)](i) were applied to CM cells freshly dissociated with collagenase and identified with smooth muscle-specific alpha-isoactin. RESULTS: Under voltage-clamped conditions, noradrenaline (NA) potentiated I(CAF) in a NA concentration-dependent manner without producing current responses to NA when NA was applied alone. NA-induced potentiation of I(CAF) occurred within 20 sec after the application of NA, while the NA-potentiated I(CAF) gradually recovered to the control level within 30 min after removal of NA. Despite the little current response to NA applied alone, NA elicited a [Ca(2+)](i) elevation in a manner similar to that induced by CAF although the NA-induced [Ca(2+ )](i) elevation was smaller than the CAF-induced [Ca(2+ )](i) elevation. In contrast to the significant potentiation of I(CAF) with NA, NA produced little potentiation of the CAF-induced [Ca(2+)](i) elevation. The NA-induced potentiation of I( CAF) was antagonized by an alpha(1) adrenoceptor antagonist, prazosin. Neither clonidine nor isoproterenol had an effect on I(CAF), suggesting that alpha(2) and beta adrenoceptor are not involved in the response to NA. CONCLUSIONS: These results suggest that NA potentiates I( CAF) via alpha(1) adrenoceptor activation and that the NA-induced potentiation occurs at Ca(2+)-activated K(+) channels but not CAF-induced Ca(2+) releasing sites.

Alkalinization-induced K+ current of the mouse megakaryocyte.

We have recently found that mouse megakaryocytes responded to extracellular alkalinization to pH > 8.0, generating a K+ current under voltage-clamped conditions with the whole cell recording mode of the patch-clamp technique. The purpose of this study was to physiologically and pharmacologically characterize the alkaline-dependent K+ conductance of the megakaryocyte membrane. The alkalinization-induced K+ current (I(ALK)) did not seem to be Ca2+-dependent since I(ALK) was allowed to be generated under intracellularly Ca2+-buffered conditions with 10 mM EGTA, which completely prevented the generation of caffeine-induced Ca2+-activated currents of mouse megakaryocytes; and no [Ca2+]i elevation was evoked by the alkalinization protocol in contrast to a significant increase in [Ca2+]i in response to caffeine when [Ca2+]i was measured with a fura 2 ratiometry. I(ALK) was strongly suppressed with tetraethylammonium (TEA), 4-aminopyridine (4-AP) and streptomycin (SM), but was completely resistant to quinidine (QND). The values of IC50 for the suppression of I(ALK) with TEA, 4-AP and SM were 5.6, 0.47 and 1.5 mM, respectively. Voltage-gated K+ currents (I(K)) of the same megakaryocyte preparation were weakly suppressed with TEA and 4-AP, while they were significantly suppressed with either SM or QND. These results suggest that mouse megakaryocytes possess K+ conductance that was activated by extracellular alkalinization and that probably differs from conventional K+ conductance in its pharmacological properties.

The properties of caffeine- and carbachol-induced intracellular Ca2+ release in mouse bladder smooth muscle cells.

Freshly dissociated bladder smooth muscle cells of mice developed spontaneous, caffeine- (ICAF) and carbachol-induced (ICCh) currents under voltage-clamped conditions. Spontaneous currents, ICAF and ICCh were blocked with tetraethylammonium at 3 x 10(-4)-10(-2) M but were resistant to both charybdotoxin (10(-7)-10(-6) M) and iberiotoxin (10(-7)-10(-6) M). The reversal potential for each current indicated that K+ channels play a major role in the generation of each current. Both spontaneous currents and ICAF but not ICCh were abolished in nominally Ca2+-free and nicardipine (10(-6) M)-containing media. These results suggest that the activity of L-type voltage-sensitive Ca2+ channels is important in the generation and maintenance of spontaneous currents and ICAF but not ICCh. Ryanodine (10(-6) M) prevented spontaneous currents, ICAF and caffeine-induced [Ca2+]i elevation but not ICCh and carbachol-induced [Ca2+]i elevation, suggesting that the response of bladder smooth muscle cells to carbachol may involve a Ca2+ store distinct from that for caffeine. Pretreatment with carbachol suppressed ICAF to 22 +/- 7% (n = 7) and the caffeine-induced [Ca2+]i elevation to 25 + 3% (n = 6). Similarly, caffeine suppressed ICCh to 23 +/- 4% (n = 9) and the carbachol-induced [Ca2+]i elevation to 24 +/- 6% (n = 6).

Inhibition of sodium current by chloride channel blocker 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) in guinea pig cardiac ventricular cells.

The effects of 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), a potent anion transport blocker, on transmembrane action potentials (APs) and the sodium current (I[Na]) of guinea pig ventricular myocytes were examined by using conventional microelectrode and whole-cell patch-clamp recording techniques. In papillary muscle preparations, DIDS (> or =0.1 mM) suppressed the maximal upstroke velocity (.v[max]) of the AP without significant changes in other AP parameters. Extracellular application of DIDS on single cardiomyocytes isolated from the guinea pig ventricle markedly reduced the peak amplitude of the tetrodotoxin (TTX)-sensitive and voltage-activated sodium current. The concentration-dependent block of DIDS could be expressed by the Hill equation with a Hill coefficient of 0.97 and a dissociation constant of 0.15 mM at a holding potential of (VH) -120 mV. DIDS (0.1 mM) shifted the steady-state inactivation curve for I(Na) toward more negative potentials by 6.0 +/- 0.5 mV and the activation curve to more positive potentials by 5.0 +/- 1.0 mV, although the slope factors were unaffected. With repetitive depolarizing pulses from -120 mV, DIDS produced a use-dependent block on the I(Na). Recovery of I(Na) from inactivation was slowed (time constant = 245 ms, compared with 10 ms of control) in the presence of 0.1 mM DIDS. In the two-pulse experiments, DIDS produced two distinct phases of development of I(Na) block, the rapid phase (tau = 5 ms) caused by an open channel block, and the slower phase (tau = 382 ms) induced by an inactivated channel block. These results suggest that the Cl- transport blocker DIDS has a direct inhibitory effect on the cardiac sodium channel. DIDS-induced use dependence of I(Na) block may result from the interaction of the drug with sodium channels in both the open and inactivated channel states.

Cytoplasmic Ca2+ mobilization and Ca(2+)-dependent membrane currents in dispersed bovine ciliary muscle cells.

PURPOSE: The dependence of plasmalemma Ca2+ influx and Ca2+ release from intracellular stores on Ca2+ activated K+ channels of bovine ciliary muscle (CM) cells were examined. METHODS: The nystatin-perforated patch clamp technique for the measurement of membrane currents and a microscope based fura-2 fluorescence imaging of [Ca2+]i were applied to CM cells freshly dissociated with collagenase and identified with smooth muscle-specific alpha-isoactin. RESULTS: At holding voltages (VH) of > -60 mV, CM cells showed spontaneous transient outward currents (STOCs) and caffeine (> 10(-4) M) induced large transient outward currents (ICAF). Both STOCs and ICAF were abolished by tetraethylammonium chloride (10(-3) M) and charybdotoxin (10(-7) M), but not by apamin (10(-6) M), suggesting that both currents are mediated by Ca(2+)-activated K+ channels similar to those with medium (MK) or large (BK-type) conductance. Both STOCs and ICAF were gradually abolished in the nominally Ca(2+)-free and Co(2+)-containing solutions but were resistant to L-type Ca2+ channel blockers, including nicardipine, verapamil and diltiazem and a N-type channel blocker, omega-contoxin. The [Ca2+]i-elevation during high K+ (100 mM)-depolarization was prevented by Ca(2+)-free and Co(2+)-containing solutions but not by nicardipine. CONCLUSIONS: These results suggest that CM cells possess MK or BK type-like Ca(2+)-activated K+ channels and that L-type Ca2+ channels play minor roles for the maintenance of Ca(2+)-dependent responses in contrast to other types of smooth muscle cells.

Impairment of Kit-dependent development of interstitial cells alters contractile responses of murine intestinal tract.

We examined developmental changes in responses of the isolated segment of the ileum of BALB/c mice treated with a monoclonal antibody (ACK2) to the receptor tyrosine kinase (Kit) for 4 days postnatally to pharmacological agents in vitro. Rhythmic contraction and relaxation of the isolated ileum started to appear on day 4 postpartum, and the sensitivity to acetylcholine (ACh) decreased gradually after birth. Treatment with ACK2 induced augmentation of contractile responses and receptor sensitivity of the longitudinal muscle of the ileum to ACh, bradykinin, and prostaglandin F2 alpha. ACh induced larger depolarization in smooth muscle cells of the ileum in the ACK2-treated mice than in the control. Circular muscle responses to these substances, as measured by changes in intraluminal pressure, were not altered by ACK2 treatment. Results suggest that interstitial cells play an important role not only in the development of the pacemaking system of the small intestine but also in the functional development of the contractile properties of the intestinal smooth muscle.

Different effects of baclofen and GTP gamma S on voltage-activated Ca2+ currents in rat hippocampal neurons in vitro.

Reduction of voltage-activated Ca2+ currents by intracellular application of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) through ultraviolet (UV) photolysis of the caged compound, is followed by a re-augmentation to control levels within 10 min, independently of the divalent cation used. The Ca2+ current inhibition by the gamma-aminobutyric acid type B (GABAB) receptor agonist baclofen, which is also thought to be mediated by a GTP-binding protein (G-protein), is potentiated when GTP gamma S is uncaged during agonist superfusion. The authors suggest that GTP gamma S activates G-protein-dependent pathways that are not activated by the baclofen receptor.

Rhythmic Cl- current and physiological roles of the intestinal c-kit-positive cells.

Chronic injection of an anti-c-KIT receptor tyrosine kinase monoclonal antibody (ACK2) results in the disruption of the normal motility patterns of young BALB/c mice intestine. This effect is accompanied by a drastic decrease in the number of intestinal c-kit-expressing (c-kit+) cells when studied immunohistochemically with the fluorescence-labelled antibody. In order to clarify the mechanism underlying the ACK2 action and the physiological roles of intestinal c-kit+ cells, we studied the excitability of intestinal c-kit+ cells in primary culture by use of the nystatin perforated-patch-clamp technique. Under voltage-clamp at -40 mV, the majority of c-kit+ cells tested (59/70) elicited rhythmic current waves with an amplitude and frequency of 263 +/- 24 pA and 2.30 +/- 0.25 cycles/min (mean +/- SEM), respectively. Intracellular perfusion of the c-kit+ cells with ethylenebis (okonitrilo) tetraacetate (EGTA) as well as a nominally Ca(2+)-free external solution or low holding voltage (< -60 mV) prevented the rhythmic current. The reversal potential of the rhythmic current was close to the equilibrium potential for Cl-(ECl). Moreover the rhythmic current was depressed by a Cl- channel blocker, 4-acetoamido-4-isothiocyanat-ostilbene-2,2'-disulphoni c acid (SITS). The smooth muscle cells freshly dissociated from the same intestinal specimen revealed a Ca(2+)-activated K+ current, as has been described in a variety of smooth muscle cells. Cultured smooth muscle cells from the ileum preparation lacked neither the Ca(2+)-activated K+ nor rhythmic Cl- currents. Smooth muscle cells freshly dissociated from the same ileum preparation and those in culture showed no immunoreactivity with the labelled ACK2, which was consistent with our previous in situ study. Results provided direct evidence that the intestinal c-kit+ cells, but not the smooth muscle cells, possess a rhythmic Cl- current oscillation, suggesting their participation in pacemaker activity for the peristaltic gut movement.

Effects of plant diterpenes on the neuronal GABAA receptor-operated chloride current.

The effects of plant diterpenes, horminone (HMN) and taxodione (TXN), on the GABAA receptor-operated Cl-current (IGABA) were investigated in voltage-clamped and internally perfused neurones dissociated from frog dorsal root ganglia. Both diterpenes depressed IGABA in a concentration-dependent manner similar to that of picrotoxin. Concentrations required to elicit 50% depression of the IGABA were 10(-6) M for picrotoxin, 10(-5) M for HMN and 10(-4) M for TXN. Blocking and restoration kinetics of the IGABA with HMN were also similar to those of picrotoxin. Time constants for both the blockade and restoration of the IGABA with TXN were more than five times greater than those with HMN.

Bradykinin-evoked non-specific cationic current in neuroblastoma-glioma hybrid (NG108-15) cells and its down-regulation through differentiation.

Effects of bradykinin (BK) on the membrane conductance and level of cytoplasmic free Ca2+ in undifferentiated and differentiated neuroblastoma-glioma hybrid (NG108-15) cells were studied using the nystatin-perforated patch-clamp technique and fura-2 fluorometry. Under voltage clamp at -20 mV, undifferentiated cells responded to BK at > 10(-9) M, producing a biphasic current composed of an apamin-sensitive Ca(2+)-activated K+ outward current and non-specific cationic inward current. Both current components corresponding to a biphasic elevation of [Ca2+]i were completely prevented by an intracellular perfusion with EGTA (1 mM) under conventional whole cell recording condition. Undifferentiated cells revealed almost no voltage sensitive Ca2+ current. In NG108-15 cells differentiated with 8-Br-cyclic AMP (1 mM) or rolipram (1 mM), an inhibitor of type IV phosphodiesterase, BK concentration required for the non-specific cationic current with amplitude of > 100 pA was much greater than that of undifferentiated cells. This suggests that the differentiated cells decreased BK-sensitivity in induction of the non-specific cationic current. The non-specific cationic channel is suggested to play roles as a source of Ca2+ entry in undifferentiated NG108-15 cells.

Effects of lindane (gamma-BHC) and related convulsants on GABAA receptor-operated chloride channels in frog dorsal root ganglion neurons.

Effects of lindane (gamma-benzenehexachloride; gamma-BHC) on GABA-evoked Cl- current (IGABA) in freshly dissociated frog sensory (dorsal root ganglion) neurons were studied and compared with those of tert-butylbicycloortho benzoate (TBOB) and picrotoxin by the use of the suction-pipette method [13]. Drugs were applied with a rapid drug-application method, "Concentration-clamp" technique. At concentration of GABA of > 3 x 10(-6) M, at least two components of the IGABA were recognized distinct degree of desensitization. Those were defined as the peak and plateau components in the text. At low concentration (3 x 10(-7) M) of gamma-BHC, only the plateau component of IGABA at 10(-5) M were depressed without changing the peak amplitude. While gamma-BHC at high concentration (3 x 10(-5) M) depressed both the peak and plateau current components. The gamma-BHC-induced depression of IGABA seemed to be IGABA-component-dependent. A detailed analysis of the gamma-BHC action in the concentration-response relationship for GABA revealed that the IGABA with strong desensitization was preferentially blocked by gamma-BHC (3 x 10(-5) M). The rate of recovery of the IGABA from gamma-BHC-induced block depended on the concentration of GABA. The lower the concentration of GABA, the slower the recovery. The GABAA receptor Cl- channels were proposed to be classified into two types of the gamma-BHC-sensitive and -resistant ones.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein kinase A-mediated phosphorylation reduces only the fast desensitizing glycine current in acutely dissociated ventromedial hypothalamic neurons.

Modulation of glycine receptor-ionophore complex by internally perfused cyclic AMP was investigated and compared to that of GABA in the acutely dissociated ventromedial hypothalamic neurons using whole-cell and outside-out patch-clamp techniques. Cyclic AMP significantly reduced both GABA- and glycine-gated chloride currents. The reduction in glycine-induced chloride current was specific in that only the fast-desensitizing one gated by high concentrations of glycine (30-100 microM) was affected. Cyclic AMP did not modulate the non-desensitizing current induced by lower concentrations (6-10 microM). Addition of N-[-2-(methylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride, a protein kinase A inhibitor, did not have a significant effect on its own but prevented the attenuation of fast desensitizing glycine current induced by cyclic AMP. Both the reversal potential and inactivation kinetics of glycine current were not affected by the activation of protein kinase A, suggesting that cyclic AMP-mediated attenuation is not due to an enhancement of desensitization. In outside-out patch studies intracellular perfusion of cyclic AMP reduced the open probability of the 100 microM glycine-activated channels without affecting that of the 6 microM glycine-activated channels. In conclusion, cyclic AMP selectively modulates the channel open frequency of the glycine receptor when activated at higher concentrations through a protein kinase A-mediated phosphorylation.

Penicillin-induced triphasic modulation of GABAA receptor-operated chloride current in frog sensory neuron.

Effects of penicillin-G (PCN) on GABA-evoked Cl- current (IGABA) were investigated in freshly dissociated frog sensory neurons by the use of the concentration-clamp technique combined with the suction-pipette method. Under conditions where the internal and external solutions allowed only Cl- permeability, PCN elicited triphasic modulation on IGABA, consisting of two modes of blockade on IGABA and a following rebound (rebound-like transient IGABA). Simultaneously applied PCN and GABA depressed IGABA immediately (phasic blockade), with the depressed IGABA slightly recovering in amplitude to achieve a stable level of blockade (tonic blockade). When a solution containing a mixture or PCN and GABA was quickly replaced by one containing GABA alone, a rebound-like transient Cl- current (IR) was evoked. Each component of the PCN actions on IGABA was PCN- and GABA-concentration-dependent. The reversal potential for each component of the PCN actions on IGABA was close to the chloride equilibrium potential (ECl) calculated using the Nernst equation. The current-voltage (I-V) relations for both the phasic and tonic blockade revealed inward rectification, while I-V curves for the control IGABA and the IR were outwardly rectified. The degree of IGABA-desensitization and the amplitude of the IR correlated well. The data suggest that partial removal of the GABAA receptor-desensitization may result in generation of the IR.

Penicillin-induced potentiation of glycine receptor-operated chloride current in rat ventro-medial hypothalamic neurones.

1. Effects of penicillin G (PCN) on glycine (Gly)-evoked Cl- current (IGly) were investigated in acutely dissociated rat ventro-medial hypothalamic (VMH) neurones by the whole cell mode of patch clamp technique. 2. When PCN was applied simultaneously with Gly, PCN depressed IGly like a Cl- channel blocker. 3. The PCN-induced blocking action was clearly observed at a low PCN concentration (30 u), while the maximal blockade was achieved by 600 u (units per 10 ml) PCN. 4. When tested solution containing both PCN and Gly was quickly substituted with one containing Gly only, a new rebound-like transient current (I(T)) which also passed through Cl- channel, was elicited. 5. The peak amplitude of I(T) induced by PCN at concentrations higher than 100 u was greater than that induced by glycine alone. We termed this phenomenon PCN-induced potentiation of IGly. In all cells tested, PCN potentiated IGly. 6. At a lower PCN concentration below 30 u, I(T) generation was not clear in the presence of 10(-5) M gamma-aminobutyric acid. With PCN a higher concentration than 300 u, I(T) amplitude was greater than that of the original peak IGly. This was observed in 18 neurones out of 21. The maximal amplitude of the I(T) was achieved with 600 u PCN.

Augmentation of GABA-induced current in frog sensory neurons by pentobarbital.

The effect of pentobarbital sodium (PB) on the gamma-aminobutyric acid (GABA)-induced macroscopic and microscopic Cl- currents (ICl and iCl, respectively) was studied in the soma membrane of isolated frog sensory neurons using a rapid concentration-jump and patch-clamp technique. The GABA-induced ICl was composed of a transient peak and a steady plateau. PB shifted the concentration-response curve of the GABA-induced peak ICl to the left without affecting the maximum value. The apparent dissociation constant (KD) decreased from 13 microM at control to 8.0, 4.8, and 2.9 microM by adding 10, 30, and 100 microM PB, respectively. PB also shifted the concentration-response curve for the plateau ICl to the left and augmented the maximum value of the plateau, indicating an increase in the available receptor-channel complex. The Hill coefficient (n = 2) in concentration-response curves of both peak and plateau responses was not changed by adding PB. Both the activation and desensitization phases of GABA-induced ICl consisted of two exponential components. PB significantly increased the time constant of slow desensitization component at all concentrations of GABA used. In the "inside-out" configuration, PB markedly increased the open probability (Po) of a GABA-gated single Cl- channel having a conductance of 14.57 +/- 2.3 pS (n = 123) without affecting the single-channel conductance. The increase of Po was due to the prolongation of mean open time (tau of the tau os) and shortening of mean closed time (tau cf and tau cs), resulting in the increase of channel-opening events.(ABSTRACT TRUNCATED AT 250 WORDS)

What confers specificity on glycine for its receptor site?

1. The structural requirements for activation of the glycine receptor were studied in isolated ventromedial hypothalamic neurones of rats by use of a 'concentration-clamp' technique under single-electrode voltage-clamp conditions. 2. alpha-Amino acids (L-alpha-alanine, and D-alpha-alanine, and L-serine), and glycine-methylester, glycine-ethylester and beta-amino acids (beta-alanine and taurine) produced a transient inward Cl- current, which was similar to that induced by glycine. 3. The responses to individual alpha- and beta-amino acids were selectively antagonized by strychnine, but were not affected by bicuculline, picrotoxin or the taurine antagonist, TAG (6-aminomethyl-3-methyl-4H,1,2,4-benzothiadiazine-1,1-dioxide hydrochloride), suggesting that alpha- and beta-amino acids activate the same glycine receptor. 4. beta-Amino acids were slightly more potent than the alpha-amino acids in causing cross-desensitization of the glycine response. 5. From the results of the structure-activity analysis of the optical isomers of alpha-alanine, serine and cysteine, a tentative structure of the glycine receptor is proposed.

Augmentation of GABA-induced chloride current in frog sensory neurons by diazepam.

The effect of diazepam (DZP) on the GABA-induced macroscopic and microscopic Cl- current was investigated in isolated frog sensory neurons using both 'concentration-clamp' and patch-clamp techniques. At concentration range between 10(-9) and 10(-4) M, DZP itself evoked no response but potentiated time- and dose-dependently the subthreshold GABA responses, though at high DZP concentrations beyond 10(-5) M the potentiation ratio decreased. The potentiation effect was long-lasting and desensitized slowly over the course of several 10 minutes after washing-out of DZP. DZP potentiated GABA response without shifting the GABA reversal potential. The entire GABA dose-response curve was shifted in a parallel manner to the left by adding DZP without changing cooperatively: the Hill slope was 2.0. The potentiation of GABA response by DZP did not depend on either inward or outward direction of the Cl- current but slightly on the membrane potential. The time constants of activation of desensitization of GABA-gated Cl- current consisted of fast and slow components, respectively. The slow components were concentration-dependent, and significantly changed in the presence of DZP, while DZP had little effects on fast components. In the 'inside-out' configuration, the addition of DZP activated GABA-receptor ionophore complexes under subthreshold without changing the single Cl- channel conductance. It is concluded that DZP may act at a site to modulate GABA binding, in which DZP increases GABA binding affinity and also affects the kinetics of GABA-gated Cl- channels, indicating that DZP has dual action on the GABA-induced responses.

Kinetic analysis of acetylcholine-induced current in isolated frog sympathetic ganglion cells.

1. Kinetic properties of activation and inactivation phases of the ACh-gated nicotinic current were investigated in isolated frog sympathetic ganglion cells using "concentration-clamp" technique under voltage-clamp conditions. This technique combines internal perfusion with a rapid change of the external solution within a few milliseconds. 2. The dose-response curve for the peak current induced by ACh showed a sigmoidal increase, in which the apparent dissociation constant Kd and Hill coefficient were 2.6 x 10(-5) M and 2.0, respectively. 3. The current-voltage relationship of ACh-induced currents were linear at potentials more negative than the reversal potential (EACh) of -5.5 +/- 1.3 mV (mean +/- SE) but showed a slight inward rectification at positive potentials over +20 mV. Since this current could be generated predominantly by an increase of Na+ and K+ conductances, the value of EACh was close to the theoretical potential, -1.3 mV, for the total amount of both Na+ and K+ or Cs+ in the extracellular and intracellular solutions. 4. There was no difference between the dose-response curves of ACh- and nicotine-induced currents. The ACh-induced current was suppressed in a competitive manner by the nicotinic antagonists, d-tubocurarine and hexamethonium, but muscarine did not induce any response, indicating that the ACh-gated current might be mediated by the nicotinic ACh receptor-ionophore complex. 5. There was a latent period of the order of milliseconds in the ACh receptor activation phase before attaining exponential increase of activation process.(ABSTRACT TRUNCATED AT 250 WORDS)