Reversal of gamma-aminobutyric acid inhibition by carbon dioxide

[email protected]. Phpiol., 1973,Voi. 444, pp. 829 to 850. Pergamun Press. Printed in Great Britain

REVERSAL OF GAMMA-AMINOBUTYRIC ACID INHIBITION BY CARBON DIOXIDE PHILIP KASHIN Department of Biology, Queens College, C.U.N.Y.,

Flushing, New York 11367

(Receioeii 8 June 1972) Abstract-l. Gamma-aminobutyric acid (GABA) potentiates the stimulatory action of CO* on the spontaneous bioelectric activity of the isolated central nervous system of the cockroach, Periplaneta americana (LJ. 2. When GABA reacts with CO*, an unstable carbamino-GARA compound is formed that might be ph~iolo~~y active. 3. Go-hy~o~bu~c acid, which cannot form a carbamino compound with COe, reduces the sensitivity of the isolated cockroach CNS to CO*. 4. Bem-hydroxy-gamma-aminobutyrate, which has less ability than GABA to form a carbamino compound, is less effective than GABA in potentiating the stimulatory action of CO,. 5. IV-Acetyl-GABA and L-glutamate are compounds that are structurally similar to carbamino-GABA, and stimulate the preparation at high concentrations. 6. These findings suggest that carbamino-GABA is a physiologically active excitatory substance in the insect CNS.

INTRODUCTlON

inhibitor action of g~~~-~~obu~ric acid (GABA) has been studied in vertebrate and invertebrate nervous systems, where it may play a role as a functional chemical mediator of inhibition (Otsuka et al., 1966; Kravitz, 1967). In animalsGABA, as well as the specific decarboxylasethat catalyzesits production from glutamic acid, occurs exclusively in nervous tissue (Awapara et al., 1950; Roberts et aZ., 1950; Wing0 & Awapara, 1950). Both GABA and the natural inhibitor exert similar inhibitory actions in crustaceansby virtue of stabilizingthe postsynaptic neuronal membrane potential (Boistel & Fatt, 1958; Brinley et al., 1960; Takeuchi & Takeuchi, 1967). GABA and the neuronal inhibitor exert a dual role, since in addition to the postsynaptic effect they both diminish the quanta1 output of the excitatory transmitterfrom presynaptic terminals (Dude1 & Kuffler, 1961; Takeuchi & Takeuchi, 1966). The actions of both GABA and the neuronal inhibitor are abolished by picrotoxin (Robbins & Van Der Kloot, 1958; Kufller, 1959). GABA may also act as an inhibitory substance in insects: it is found in higher concentrations in the head of the housefly than in any other part of the animal (Price, 1961), and has also been found in the central nervous system of Periplaneta ff~~icun~ (Ray, 1964) and in the heads and bodies of yellow fever mosquitoes

THE

SYNAPTIC

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&SHIN

(Kashin, 1969). Relatively high ~oncen~atio~ of GABA depress the electrical activity of ganglia of the pine moth caterpillar (Vereshtchagin et aZ., 19611, inhibit the auditory synapses in the prothoracic ganglion of the grasshopper (Suga & Katsuki, 1961), and inhibit excitatory synapses affecting ascending giant fiber activity in P. americana (Gahery & Boistel, 1965). The action of the peripheral inhibitory transmitter is mimicked by GABA in grasshopper and locust muscle (Usherwood & Grundfest, 1964, 1965), and in cockroach central neurons (Pitman & Kerkut, 1970), so it is a strong candidate for the role of a neuronal inhibitory transmitter. The amino and carboxyl end groups of GABA were found to be essential for the retention of maximal inhibitory activity on the crayfish stretch receptor preparation; compounds in which either group was modified had reduced inhibitory potency (Edwards & Kuffler, 1959). Any agent that interacts chemically with GABA may therefore alter GABA-mediated synaptic transmission. GABA, like other amino acids as well as primary and secondary amines and proteins, spontaneously and rapidly forms a carbamino compound through association of molecular CO, with its amino group (Ed&l& Wyman, 1958; Kashin, 1969). It has been suggested that the inhibitory properties of GABA might be modulated by carbamino formation (Kashin, 1969). The physiologic~ identity of cells sensitive to CO, in vertebrate and invertebrate nervous systems is completely unknown. CO, sensitivity might be a general characteristic of cells exhibiting pH sensitivity, or it might involve special mechanisms that are peculiar to CO2 sensitive cells. COs stimulates bioelectric activity in isolated cockroach ganglia (Boistel & Coraboeuf, 1954; Farley et al., 1967), has a specific action in inhibiting contraction of the locust spiracular muscle (Hoyle, 1960), and affects the discharge frequency in respiratory neurons of the mammalian brain (Cohen, 1968). Since GABA appears to be wideiy distributed in nervous systems of the animal kingdom and may play a role in synaptic transmission, it seemed wo~hwhile to explore the possibili~ that GABA action can indeed be modulated by COB, and that carba~no-GABA might be of physiological importance in mediating the effects of CO, in specialized nervous tissue. MATERIALS

AND METHODS

A segment of the central nervous system dissected from male Periplanetu americana (L.) cockroaches colonized according to estabhshed procedures (Smittle, 1966) was used throughout this work. The insect was first cooled to inactivate it sufficiently for ease in handling, then pinned ventral side up to a cork dissecting board. Under a dissecting microscope, the basisterna and furcasternum covering the metathoracic ganglion were removed, together with the frrst four segments of the abdominal sterna, thus exposing the metathoracic ganglion and four abdominal ganglia. The central nerve cord was severed anterior to the metathoracic ganglion and posterior to the fourth abdominal ganglion. Peripheral connectives in the region comprising the metathoracic ganglion and the first four abdominal ganglia were severed, and this segment of the central nervous system was removed. The preparation was placed in a watch-glass containing saline solution and cleansed of adhering tissue debris. A small 7” fashioned from 30-gauge platinum wire (weighing about 6 mg) was inserted between

GARA

AND co,

831

the two interganglionic connectives just anterior to the fourth abdominal ganglion to serve as a weight. The apparatus consisted of a differential preamplifier (Grass Model P4 with a high impedence probe), a dual beam cathode ray oscilloscope (CRO) (Hewlett-Packard Model 132A), a 6 circuit multi-cam timer (Type MC-2, with rack and gear assembly Type E-15 to give an overall time cycle of 12.5 set; Industrial Timer Corp., Parsippany, N.J.), and an events-per-unit time counter (Hewlett-Packard Model 5221A, Electronic Frequency Counter). Terminals Gl and G2 of the high impedance probe were connected across a l-Ma potentiometer, with the center tap of the potentiometer connected to the ground terminal of the probe. The electrode leads consisted of a short piece of two-strand shielded cable. One strand was connected to Gl, the other strand to G2, and the shield to the ground terminal of the probe. The recording electrode was a piece of 30-gauge platinum wire 2 cm long. One end of this platinum wire was soldered to the shielded strand connected to Gl of the probe, and the other end terminated in a small hook. The reference electrode was a straight piece of 30-gauge platinum wire about 3.5 cm long, soldered at one end to the shielded strand connected to G2 of the probe. In order to record spontaneous action potentials extracellularly from the nerve cord preparation, the hook of the recording electrode was placed between the interganglionic connectives just posterior to the metathoracic ganglion. The hook was then gently pressed around the ganglion with a fine forceps so that the preparation was firmly held. The preparation was thus suspended from the recording electrode, and kept in an extended position by the small platinum weight at the bottom. The reference electrode was about 05 cm from the recording electrode. Action potentials were observed upon immersion of the preparation and the reference electrode in saline. The parameter measured was the change in the spontaneous firing rate of the preparation in response to various treatments. Each data point represents a 5-set counting period (controlled by the multi-cam timer), with 1 set between counting periods to permit recording of the data and recycling of the counter. Electrical balance between reference and recording electrodes was adjusted in order to eliminate as far as possible in-phase signals. After the electrode balance was adjusted by means of the l-MQ potentiometer, the zero level of the electronic counter was adjusted by short circuiting the electrodes in the saline solution, and setting the counter sensitivity control to just barely show no counts when the amplifier amplification control was set one step higher (more amplification) than the setting that was used during the experiment. (Each amplification step on the preamplifier is a multiple of 1.5.) The preamplifier amplification control was then set to the level at which the experimental data was taken (i.e. one amplification step lower than the setting at which the zero level was adjusted). When thus adjusted, a vertical excursion on the CR0 of about 0.5 cm, equivalent to 20 ~LV,was necessary before an event was tallied on the counter. A block diagram of the experimental setup is shown in Fig. 1. The basic physiological saline used throughout was essentially that described by Smit et al. (1967), but with the omission of bicarbonate and phosphate salts, and the addition of 0.01 M of the buffer N-2-hydroxyethyl-piparazine-N’-2-ethanesulfonic acid (HEPES), described by Good et al. (1966). The composition of the buffered saline per liter was: 9.1 g NaCl, 0.9 g KCI, O-51 g CaCI,, O-8 g MgCl,.6H,O, 4.0 g glucose and 2.383 g HEPES, titrated to pH 7.60 with NaOH. This buffered saline solution kept the preparation in good condition for many hours. All test solutions with which the nerve cord was treated were made up with this buffered saline and adjusted to pH 7.60 before use. The test solution was placed in a ~-CC beaker, and the beaker was raised by means of a jack so that the reference electrode, metathoracic ganglion and recording electrode were immersed in the test solution for 5 sec. After the 5-set immersion period, the beaker was slowly lowered by means of the jack until the recording electrode just broke through the

832

PHILIP

KASHIN

PREAMPLIFIER

1

I

IORACIC LION

DIGITAL

COUNTER

COUNTER

CONTROL

Pt.WEIOHT HAONETIC STIRRING

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BAR

MAGNETIC STIRRER

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FIG. 1. Method for recording spontaneous bioelectric activity from a segment of the isolated central nervous system of the cockroach. surface tension of the solution, thus unshorting the electrodes and permitting the gathering of data. This method proved reproducible and accurate, and permitted the same amount of the nerve cord to be removed from the solution for each recording. When the data gathering period ended, the test solution was replaced with a beaker containing about 10 cc of saline that was stirred to wash the preparation before a new test. In the Figures of the Results section, an arrow pointing to a double circle designates the last control saline point before the test solution was applied. Immediately after this point there is a slight discontinuity during which time the saline solution was replaced with the test solution into which the preparation was immersed. CO, treatment of solutions consisted of rapidly bubbling a mixture of 10% COP in air (pre-mixed compressed gas, The Matheson Co., Inc., Joliet, Ill.) for 2 min into 5 cc of the solution in a test tube (20 x 150 mm). The CO,-treated solution was then decanted into a S-cc beaker, and immediately applied to the preparation as described above. The effects of various substances were also tested on single cells of the second thoracic spiracular closer muscle of the locust, Schistocercu gregaria. For this purpose, the centrally generated EPSPs were monitored with 3 M KCl- or 2 M potassium acetate-filled glass microelectrodes (10-50 Ma) in single cells of this muscle during topical perfusion with the test substances. The opening and closing of the spiracle itself was also simultaneously observed with a binocular dissecting microscope. RESULTS

The eflects of pH and CO, When 10% CO2 was bubbled through the buffered saline solution for 2 min, the pH of the solution decreased from 7.60 to about 6.2-6.3. In order to distinguish

GA&d AND coa

833

between possible stimulator

effects due to pH shift alone from those of CO,, a prep~ation was treated with buffered saline solutions at various pH values, and the change in spontaneous discharge rate was compared with that caused by CO, treatment. The results are shown in Fig. 2. There was no change in the discharge rate when the ganglion chain was treated with the saline solution at pH 6.0, but the discharge rate of the preparation increased slowly when treated with a saline solution at pH 5.75. The stimulatory effect on the same preparation of the saline solution treated with CO, can be seen to far exceed the pH effect. Since the pH of the buffered saline solution never decreases below pH 6.2 when treated with the 10% CO, mixture, it can be concluded that the stimulation observed with the CO,treated solution is not complicated by pH effects.

cl

I

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3

2 MIN

4

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FIG. 2. Comparison of the effects of pH and CO@on the spontaneousdischarge rate pH 6.0; O0, pH 5.75; x-x, of the preparation. 0 -0, co, treatment.

The eflects of variozcs GABA concentrations with and without CO, Experiments were performed to test the effects of various concentrations of GABA with and without CO, on the spontaneous discharge rate of a preparation. A CO,-treated saline solution alone has a stimulatory effect (Fig. 3a). lo-8 M GABA alone did not exert an i~ibito~ action, but when CO, was bubbled into it there was a stimulatory action very similar to that of the CO,-saline solution alone (Fig. 3b). GABA (lob6 M) also did not alter the electrical discharge rate of the preparation, and when the 10T5 M GABA solution was equilibrated with 10% CO,, the discharge rate remained similar to that in the CO,-treated saline solution alone (Fig. 3~). With 1O-4 M GABA alone, slight inhibition occurred (Fig. 3d), but when CO, was added to this solution the preparation was more stimulated than previously (cf. Fig. 3a-d).

834

PHILIP

KA.SiIN

(0)

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2 MIN

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fb)

(cl

2cd

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MIN

FIG. 3.

(a-c)

Treatment of the preparationwith 5 x 10”’ M GABA again appeared to slightly reduce the discharge rate, but when 10% CO, was bubbled through thii GABA solution the preparation was highly stimulated (Fig. 3e). Application of 10” M GABA caused an immediate and precipitous decline in the electrical activity of the preparation, but this GABA solution became a potent stimulator when treated with 10% CO, (Fig. 3f).

GABA

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BIG. 3. Effects of CO9 alone and various concentrations of GABA alone and GABAfCO8 on the spontaneous discharge rate of the preparation. (a) CO8 alone; (b) GABA, 10-6 M with and without CO,; (c) GABA, lo-’ M with and without CO,; (d) GABA, lo-$ M with and without CO,; (e) GABA, 5 x IO-$ M with and without CO,; (f) GABA, 10-s M with and without CO*; (g) GABA 5 x lo-* M with and without CO,. In all cases, closed circles represent GABA alone, open circles represent GABA + CO,.

Continuing these observations, treatment of the preparation with 5 x 10~~ M GABA again caused an immediite decline in the firing rate; when the solution was then treated with CO%the onset of inhibition was slower, but the inhibitor action of GABA predominated over the stimulation due to CO, (Fig. 3g). The inhibitory

836

?%iILIP

&SHIN

GABA effect also predominated at higher GABA concentrations, largely masking the CO, effect. After the initial stimulation, a decline in the discharge rate was observed in all of the CO,-treated GABA solutions. The amount of decline seen in the CO,treated solutions having GABA concentrations of 5 x 10” M or less was approximately equal to the control levels, while the decline after initial stimulation in the CO,-treated GABA solutions having higher GABA concentrations was to below control level. This decline probably resulted from the rapid escape of CO, after the removal of the third thoracic ganglion from the saline solution to permit the recording of data. At the higher GABA concentrations the inhibitory action of GABA can be expressed after the escape of CO,.

The effects of gamma-hydroxybutyric acid (GHB) with and without COs were tested in a series of experiments comparable to those carried out with GABA. This compound is of interest because it has a chemical structure similar to that of GABA, with the exception that a hydroxyl group is on carbon-4 instead of an amino group. GHB therefore cannot form a carbamino compound. GHB has been reported to have inhibitory action on the crayfish stretch receptor, although it is less effective than GABA (Edwards & Rufller, 1959). If the mech~ism of stimulatory action of CO, is not through carbamino-GABA formation, but instead through an independent receptor site for COs, then the results of the GHB experiments should be qualitatively similar to those of the GABA series. The results of these experiments are described below. No inhibitory response was evident at GHB concentrations below 5 x lOmaM GHB (Fig. 4a-e), but an inhibitory effect occurred at this concentration (Fig. 4f). The stimulator action of COs at the lowest concentration used (lo* M) is shown in Fig. 4a. When lo-+ M GHB with lOo/0 COa was tested on the preparation, a decline in the stimulatory action of CO, occurred (Fig. 4b). The stimulatory effectiveness of CO, was even further reduced in 1O-4 M GHB (Fig. 4c), but no longer evident in lO+j M GHB (Fig. 4d), or in any of the higher concentrations of GHB (Fig. 4e and f). Thus, the stimulatory action of CO, in this series of experiments was maximal at the lowest GHB concentration, and declined steadily until a GHB concentration of 10-O M was reached. It was totally abolished at GHB con~ntrations greater than lOa M. Furthermore, the addition of CO, to the GHB solution where inhibition was first observed (5 x 10e2 M) has little modifying effect on the inhibition (Fig. 4f). The effects of a mixture of GHB, GABA and CO, In view of the results of the above experiments, it was of interest to observe the responses of a preparation to a solution containing a mixture of GHB, GABA and CO,. The presence of GHB in a solution containing GABA and CO2 could modify the responses of the preparation to the stimulatory effects of GABA with

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CO,, and also perhaps provide some degree of insight into the identity of the sites of action of GABA, GHB and carbamino-GABA. The results are described below. Both 1W’ M and 5 x 10q M GABA were highly effective in potentiating the stimulator effects of CO, (Fig. 3d and e>. GHB (lOA M) did not completely abolish the CO, effect, but considerably modified it (Fig. 4~). Mixtures of these solutions were used in these experiments.

838

PHILIP KASHIN

FIG. 4. Effects of various concentrations of GHB alone and GHB + COs on the spontaneous discharge rate of the preparation, (a) GHB, lo-* M with and without CO,; (b) GHB, lo-’ M with and without CO,; (c) GHB, lo+ M with and without CO*; (d) GHB, 10” M with and without CO%; (e) GHB, 5 x lOa M with and without CO,,; (f) GHB, 5 x lOWaM with and without CO*. Closed circles represent GHB alone, open circles represent GHB + COs.

The stimulatory effect of CO,-treated saline alone is shown in Fig. 5(a). A GABA concentration of 104 M barely altered the discharge rate of the preparation (Fig. 5b), but treatment with 5 x 1V M GABA had an inhibitory effect Fig. 5(c). When this same preparation was subsequently treated with lOA M GABA+ 10% CO, (upper curve, Fig. 5d), the stimulatory effect was very nearly the same as that previously seen with CO, alone (Fig. Sa). When the preparation was treated with a mixture containing lOA M GABA and lo+ M GHB+ 10% COs (lower curve, Fig. Sd), there was a definite depression in the response of the preparation. The preparation was then washed and treated with 5 x lOA M GABA+ 10% CO,. The stimulatory effect of CO, was in no way reduced by the presence of this inhibitory concentration of GABA, but perhaps even enhanced (Fig. 5e, upper curve). When 104 M GHB was also present in the CO,-treated solution containing 5 x 10-P M GABA, the response of the preparation was again considerably depressed (Fig. Se, lower curve).

DL-beta-hydro~-~a~~a-~inobu~~c acid (BHGA) was shown by Hayashi (1958) to be a more potent inhibitor of cerebral activity than GABA; Florey (1961) demonstrated that BHGA, like GABA, depressed acetyicholine-induced contractions in the hind gut of a crayfish. This compound was of interest in this work because it is capable of forming a carbamino compound through the amino group on carbon-4, but it also contains a hydroxyl group on carbon-3. Thus, although BHGA can form a carbamino compound, it probably would not do so as avidly as GABA due to mutual repulsion between the negatively charged carboxyl group

GABA

AND

CO2

839

(a)

lb)

FIG. 5 (a-e)

of the carbamino moiety and the proximal electronegative hydroxyl group. This compound thus provided the opportunity to study the stimulatory action of CO, in the presence of a compound that is intermediate between GABA and GHB in terms of carbamino formation. The results are described below. A comparison is made between various concentrations of BHGA and corresponding concentrations of GABA with and without CO, on the spontaneous firing rate of the preparation. A CO,-treated saline solution alone has a stimulatory action (Fig. 6a). BHGA at 10-s M is inhibitory without CO, (Fig. 6b, lower curve), but unlike GHB at this con~ent~tion permits the stimulatory action of CO, to be expressed (Fig. 6b, upper curve), The inhibitory effect of 1O-5 M

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PHILIP

KASHIN (e)

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dt-4

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FIG. 5. Effects of mixtures of GABA + GHB + CO, on the spontaneous discharge rate of the preparation. (a) CO2 alone; (b) 10v4 M GABA; (c) 5 x lo-* M GABA; (d) lO+M GABA+C02 (a), and a mixture of lo-“M GABA+lO-“M GHB+COp (0); (e) 5 x lo-’ M GABA+COa (a) and a mixture of 5 x 10e4 MGABA+SxlO-*M GHB+CO,(o).

GABA is shown in Fig. 6c (lower curve). In the presence of CO,, 10ms M GABA is more stimulatory than 1O-s M BHGA with CO,. BHGA and GABA were then tested at 5 x 1O-s M, with and without CO,. There is not very much difference between the inhibitory action of BHGA (Fig. 6d, lower curve) and GABA (Fig. 6e, lower curve), but the stimulatory action of CO, is altered by the presence of these compounds at this concentration. The CO,treated 5 x lo4 M BHGA solution causes at most only a slightly greater spontaneous discharge rate than in the presence of 5 x lOTa M BHGA alone (Fig. 6d, upper curve), but the inhibitory action of 5 x 10e8 M GABA alone is largely overcome in the presence of CO2 (Fig. 6e, upper curve). The inhibitory effect of lo-* M BHGA on the preparation (Fig. 6f, lower curve) is again similar to that of the 1O-8 M GABA solution (Fig. 6g, lower curve). The difference between the stimulatory effect of the CO,-treated lo-* M BHGA solution (Fig. 6f, upper curve) and the CO,-treated 10m2M GABA solution (Fig. 6g, upper curve) is not as great as seen at the respective 5 x 10Va M concentrations since inhibition apparently predominates, but the CO,-treated 10ea GABA solution still shows greater stimulatory potency than the CO,-treated 10m2M BHGA solution.

GABA AND CO,

841

(a)

FIG. 6 (a-c) The eflects of N-acetyl-GABA

and L-glutamate

If carbamino-GABA is indeed an excitatory substance, then it was of interest to determine whether N-acetyl-GABA (NAG), a substance that has a structure similar to that of carbamino-GABA (Fig. 7) is also excitatory. A high NAG concentration of O-1 M caused a considerable increase in the discharge rate (Fig. Sa), but lower concentrations (10-2-10-6 M) did not alter the discharge rate.

PHKLIPK&SHIN

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FIG. 6. Effects of various concentrations of GABA and BHGA, with and without CO,, on the spontaneous discharge rate of the preparation. (a) CO, alone;

(b) 10” M BHGA (0) and lo-* M BHGA+COP (0); (c) lo-* M GABA (0) and 10”M GABAfCOp (0); (d) SX~O-~M BHGA (a) and 5x 10-*M BHGA+CO, (0); (e) 5 x lo-* M GABA (e) and 5 x 10” M GABA+CO8 (0); (f) lo-* M BHGA (0) and IO-’ M BHGA-k CO, (0); (g) IO-’ M GABA (a) and lo-” M GABA-t CO, (0). In invertebrates, L-glutamate may be either a naturally occurring excitatory synaptic transmitter, or act presynaptically to cause increased quantal transmitter release (Takeuchi & Takeuchi, 1964; Kerkut et al., 1965; Kerkut & Walker, 1966, 1967; Beranek & Miller, 1968; Usherwood & ~achili, 1966, 1968; Florey & Woodcock, 1968). Since L-glutamate also has a structure similar to that of carbamino-GABA (Fig. 7), it was also of interest to test the effect of L-glutamate on the

843

GABA ANDCO2

,:-,,l-f’ C-&IiC-i-l 3

+H2

H

H

2l

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FIG. 7. Comparison of the structures of carbamino-GABA, N-ace@ GABA and glutamic acid at physiological pH.

spontaneous firing rate. As in the NAG experiment, 0.1 M glutamate, but not lower concentrations, caused a large increase in the firing rate (Fig. gb). No attempt was made to remove the perineural sheath in any of these preparations, so its barrier action (Twarog & Roeder, 1956) might account for the low effectiveness of these compounds. Locust spiraculur muscle Gaseous CO2 has a direct and specific action in causing relaxation of the closer muscle that controls the second thoracic spiracle of the locust, S. gregaria, resulting in spiracular opening (Hoyle, 1960). In order to determine whether carbaminoGABA plays a role in this mechanism, a preliminary investigation of some of the pharmacological properties of this muscle was made. The spontaneous centrally generated excitatory postsynaptic potentials (EPSPs) were monitored with intracellular glass microelectrodes, and the opening and closing of the spiracle itself simultaneously observed. When the muscle was topically perfused with 10m3M glutamate, the EPSPs disappeared, the muscle cell was depolarized by about 5-15 mV, and the spiracle closed ; i.e. the muscle appeared to go into contracture. These effects were easily reversed by perfusing with saline. Perfusion with GABA, NAG or GHB had no discernible effect whatever on the normal electrical or mechanical activity of the muscle, even at concentrations as high as O-1 M. Treatment of the GABA solutions or saline solution with CO, made no difference. Only when a pure CO, gas stream was directed on the muscle did the EPSPs diminish in size concomitant with muscular relaxation and spiracular opening. GHB did not interfere with the action of gaseous CO,. DISCUSSION Carbamino compounds of hemoglobin have long been recognized to have physiological importance in contributing to the transport of CO, in the blood

(Roughton, 1944). The evidence presented here suggests that the carbamino compound of GABA might have physiological importance in the nervous system.

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PHILIP KASHIN

The effects of application of CO, to abdominal ganglia of ApZ@u were attributed solely to the decrease of extracellular pH produced by the addition of CO,, and it was suggested that this mechanism could account for the effects of CO, on other systems such as the hyperpolarizing effects on cortical, phrenic and lumbar

(a)

FIG. 8. Effect of (a) 0.1 M NAG, and (b) 0.1 M L-glutamate on the spontaneous discharge rate of two different preparations.

neurons and the depolarizing effects on arterial chemoreceptors and respiratory center neurons (Walker & Brown, 1970). The effects of CO2 on the ‘respiratory center of cockroach ganglia could be mimicked by weakly dissociated acids in reversibly initiating respiratory rhythms (Case, 1961).

GABA

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The above data show that the CO, effect on the isolated cockroach central nervous system cannot be mimicked by lowering the pH of the extracellular medium. While the possibility has not been ruled out that CO, might produce its effect by lowering the pH at some site not directly accessible to hydrogen ions from the medium, these findings are more readily explained by a direct and specific action of CO,. The results of these experiments support the idea that GABA is directly involved in the stimulatory action of CO, on cockroach ganglia. Although GABA alone is inhibitory, the presence of all but the highest and lowest concentrations of GABA used potentiates the stimulatory effect of CO,, with maximum effect at about 10-d M GABA. This potentiation is apparent even at a GABA concentration of 10-s M, which is inhibitory in the absence of CO,. Only at the highest GABA concentrations did the inhibitory effect predominate over this apparent potentiation. These data suggest that the actual stimulatory agent might be carbaminoGABA. The stimulatory effect of CO, in the absence of exogenous GABA might result from the interaction of CO, with endogenous GABA. If carbamino-GABA is indeed the active species in CO, stimulation, it is probably active at very low concentrations. At physiological pH, pCO,, and temperature, the fraction of CO, (in all forms) carried by blood as carbaminohemoglobin is about S-10 per cent; a small though physiologically important fraction of total blood CO, (Rossi-Bernardi et al., 1969). CO, will react only with uncharged amino groups, and an amino acid must be in the anionic form for reaction to occur (Edsall & Wyman, 1958). At pH values near neutrality, the ratio of anionic GABA to its zwitterion is very small (about 2.8 x 10m4). Therefore under physiological conditions the concentration of carbamino-GABA will necessarily be very small. Although no studies of the kinetics or equilibria of the reaction of GABA with CO, are yet available, the rate constant of carbamino formation and decomposition for other amino acids is exceedingly rapid (Jensen & Faurholt, 1952), and the equilibrium is highly pH dependent (Edsall & Wyman, 1958). Since neither the pH nor the GABA concentration at the effector site on the synaptic membrane are known, little can be said about the physiologically active concentration. The stimulatory action of CO, is continuously depressed in the presence of increasing concentrations of GHB. There is no potentiation or maximum of the CO, effect as was observed in the GABA experiments. These results are expected if carbamino-GABA is a stimulatory form of CO,, since carbamino formation with GHB is impossible. The findings of the GHB experiments are consistent with the hypothesis that carbamino-GABA is a neurostimulatory compound, but seem to be more compatible with this hypothesis if it is assumed that GHB competes with carbamino-GABA for a second excitatory site that is distinct from the GABA inhibitory site. The idea of a second site seems attractive since it would be difficult to understand how very low carbamino-GABA concentrations could be effective in the presence of excess free GABA if the two compounds had opposite actions at a single site. The observation that the concentration of GHB at which 28

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PHILIP

KASHIN

the CO, effect is annulled (10~~ M) is about 15 orders of magnitude less than that which produces inhibition (5 x 10e2 M) can also be explained if two sites are assumed; one inhibitory and one excitatory. GHB can bind at both sites, but more GHB is required at the inhibitory site to block spontaneous bioelectric activity than at the excitatory site to block the action of CO, or carbamino-GABA. If only one membrane receptor site were involved, it would have to be nearly totally saturated with GHB to completely block the action of CO,. Yet, if this site were indeed saturated, GHB at a concentration of 1O-3 M should have inhibited spontaneous bioelectric activity. No inhibition was observed at 1O-3 M GHB (Fig. 4d). The stimulatory effects of solutions containing both GABA and CO, were suppressed in the presence of GHB even though the GHB concentration used (10U4 M) was not in itself inhibitory (Fig. 5d and e). These results again suggest that GHB and carbamino-GABA compete for an excitatory site on the synaptic membrane that is different from the inhibitory site. Nearby electronegative groups reduce the tendency of amino groups to form carbamino compounds due to mutual repulsion between the electronegative group and the carboxylate moiety of the carbamino compound (Edsall& Wyman, 1958). For instance, alpha-analine reacts about half as rapidly with CO, as beta-alanine (Jensen & Faurholt, 1952). On this basis it was correctly predicted that BHGA would be less effective than GABA in potentiating the effects of CO, because of the decreased capability of BHGA to form a carbamino compound resulting from the proximity of the electronegative hydroxyl group to the amino group. The results of the BHGA experiments again suggest that carbamino formation might be the mechanism by which CO, exerts its neurostimulatory action in specialized receptors. As shown previously, treatment of the isolated cockroach central nervous system with L-glutamate or NAG at high concentrations increases its spontaneous bioelectric activity. A comparison of the structures of glutamate, carbamino-GABA and NAG is shown in Fig. 7. All three compounds bear resemblances in that each has an amino group juxtaposed to a carboxyl or carbonyl moiety at one end of the molecule separated by three carbon atoms from another carboxyl moiety at the other end of the molecule. Thus, a structural rationale can be adduced to explain the apparent pharmacological similarities of these compounds. The results of the preliminary experiments on the locust spiracle muscle however clearly suggest the existence of other mechanisms underlying the physiological action of CO,. CO, was ineffective in solution, but elicited immediate relaxation and diminution of EPSPs when the gas was directed on the muscle surface. These results are in agreement with those of Hoyle (1960), who suggested that the gas may either cause presynaptic inhibition of the release of excitatory transmitter, or compete postsynaptically with the transmitter substance in a curare-like fashion. Although the possibility that GABA or carbamino-GABA receptors in the spiracular muscle are not accessible to the perfusate has not been ruled out, it was not possible to demonstrate in these experiments that GABA has any effect on the

GABA

AND

CO2

847

electrical or mechanical properties of the muscle, or that carbamino-GABA is involved in the mechanism of action by which CO, causes this muscle to relax. It may be that in order for the carbamino-GABA effect to be observed, as well as the pharmacological actions of the other compounds utilized in this investigation, a tissue must be sensitive to both GABA and glutamate. The cockroach central nervous system apparently has both of these sensitivities. It is possible that carbamino-GABA may be effective either through a “co-operative” interaction between GABA and glutamate receptors that are very close to each other and thus form a new active site, or through specific receptors for carbamino-GABA. It does not appear likely that CO, acts pre- or postsynaptically in the release of or competition with excitatory or inhibitory transmitters in the cockroach central nervous system, since neither of these assumptions could explain the distinct potentiation observed with a constant concentration of CO, in the face of increasing GABA concentrations (up to about 5 x lo4 M GABA), nor the continuous loss of sensitivity to CO, as a direct function of GHB concentration. These results could also not be explained if GHB is simply considered to be more effective than GABA in blocking the stimulatory action of CO,, while BHGA has an intermediate blocking action. The evidence is more conducive to the interpretation that carbamino-GABA is in itself a substance of physiological importance. The extremely rapid and spontaneous formation and decomposition of carbamino-GABA as a direct function of CO, concentration with an attendant reversal of the neurophysiological action of the GABA molecule could have fundamental implications for neural control mechanisms in animals that may govern, for instance, respiration. Molecular CO,, which appears to be the immediate product of enzymatic decarboxylations (Rothberg & Steinberg, 1957), difIuses much more rapidly through tissues and cells than polar bicarbonate ions, and is the molecular species active in carbamino formation. When threshold concentrations of carbamino-GABA are formed through the periodic accumulation of metabolic CO,, spontaneous respiratory rhythms could be initiated with periodicities that are directly related to the rate of production and diffusion of CO, into GABAcontaining synapses, and to the specific sensitivities of hypothetical carbaminoGABA receptors. GABA-bound CO, will be released as tissue pC0, decreases with ventilation, just as carbamino-hemoglobin readily yields its bound CO, in lung alveolar capillaries. SUMMARY

Gamma-aminobutyric acid (GABA) potentiates the stimulatory action of CO, on the spontaneous bioelectric activity of the isolated central nervous system of the cockroach, P, americana. The usual inhibitory effect of GABA predominates in non-CO,-treated GABA solutions, and in CO,-treated GABA solutions having GABA concentrations above that which maximally potentiates the CO, action. Since GABA forms an unstable carbamate through spontaneous and rapid association of its amino group with molecular CO,, the possibility was explored that an unstable carbamino-GABA compound produced in the presence of CO,

848

PHILIP KA~HIN

might account for the observed potentiation of the CO, effect. Various closely related substances were therefore tested for physiological activity. Gammahydroxy butyric acid (GHB) cannot form a carbamate; its presence in CO,-treated solutions caused a steady decline in the st~ulato~ action of CO, as a direct function of GHB concentration. zeta-hydroxy-gag-~~obu~ic acid has less ability than GABA to form a carbamate, probably due to its proximal electronegative hydroxyl group; it is less effective than GABA in potentiating the stimuIatory action of CO,. L-Glutamate and N-acetyl-GABA are compounds that are structurally similar to carbamino-GABA; both stimulate the preparation at high concentrations. These results suggest that carbamino-GABA is a physiologically active substance in specialized nervous tissue, and implicates GABA in a mechanism by which CO, exerts its stimulatory effect. A~k~~ledg~~~-~~~ work on the isolated cockroach CNS was supported by the U.S. Army Medical Research and Development Command, Office of the Surgeon General, under Research Contract No. DA-49-193-MD-2281, and conducted at I.I.T. Research Institute. The investigation is from a thesis submitted by the author in partial fuhilhnent of the research requirements for the Ph.D. degree at Illinois Institute of Technology. I am grateful to Dr. William F. Danforth for the insights and discussions he provided throughout the course of this work. The investigations dealing with the locust spiracle muscle were conducted during the author’s tenure of an N.I.H. Special Postdoctoral Fellowship (1 FlO HS 02235-01, NINDS), in the laboratory of Dr. Graham Hoyle, University of Oregon. I wish to thank Dr. Hoyle for his discussions and critical reading of the manuscript. REFERENCES J., LANDIJAA. J., FUERSTR. & SEALEB. (1950) Freegamma-aminobutyric acid in brain. J. biol. Chem. 187, 35-39. BI&NEK R. & MILLER P. L. (1968) Sensitivity of insect muscle fibers to L-glutamate. J. Physiol., Lond. 3, 7lP-72P. BOISTELJ. & CORABOEUF E. (1954) Action excitante de l’anhydride carbonique sur l’activitti dlectrique du nerf is016 d’insecte. J. PhysioZ., Paris 46, 258-261. BOISTELJ. & FATT P. (1958) Membrane permeability change during inhibitory transmitter action in crustacean muscle. J. Physiol., Lond. 144, 176-191. BRINL~Y F. J., HANDELE. R. & MARSHALLW. H. (1960) Effect of gull-~obu~ic acid (GABA) on K4a outflux from rabbit cortex. 3. N~r~hy~o~. 23, 237-245. CASE J. F. (1961) Effects of acids on an isolated insect respiration center. BioE. Bull. 121, 385. COHEN M. I. (1968) Discharge patterns of brain-stem respiratory neurons in relation to carbon dioxide tension. r. Neurophysiol. 31, 142-165. DUDEL J. & KIJFFLER S. W. (1961) Presynaptic inhibition at the crayfish neuromuscular junction. J. Physiol., Lond. 155, 543-562. EDSALL J. T. & WYMAN J. (1958) BiophysicaE Chemistry, Vol. I, pp. 550-590. Academic Press, New York. EDWARDSC. & KUFFLER S. W. (1959) The blocking effect of gamma-aminobutyric acid (GABA) and the action of related compounds on single nerve cells. J. Neurochem. 4, 19-30. FARLEY R. D., CASEJ. F. & ROEDERK. D. (1967) Pacemaker for tracheal ventilation in the cockroach, Peripbneta americana (L.). J. Insect Physiol. 13, 1713-1728. AWAPARA

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FLOES E. (1961) A new test preparation for bio-assay of Factor I and gamma-aminobutyric acid. J. Physiol., Lond. 156, 1-7. FLOREY E. & WOODCOCK B. (1968) Presynaptic excitatory action of glutamate applied to crab nerve-muscle preparations. Camp. Biochem. Physiol. 26, 651-661. GAHERYY. & BOISTELJ. (1965) Study of some pharmacological substances which modify the electrical activity of the sixth abdominal ganglion of the cockroach, Periplaneta americana. In The Physiology of the Insect Central Nervous System (Edited by TRE~ERNIZ J. E. & BEAMENTJ. W, L.), pp. 73-78. Academic Press, London. Goon N. E., WINGET G. D., WINTER W., CONNOLLYT. N., IZAWAS. & SINGH R. M. M. (1966) Hydrogen ion buffers for biological research. Biochem. 5, 467-477. HAYASHI T. (1958) Inhibition and excitation due to gamma-aminobutyric acid in the central nervous system. Nature, Land. 182,1076-1077. HOYLE G. (1960) The action of carbon dioxide gas on an insect spiracular muscle. J. § Physiol. 4, 63-79. JENSEN A. & FAURHOLTC. (1952) Studies on carbamates-V. The carbamates of aZphaalanine and beta-alanine. Actu them. &and. 6, 385-394. KA~HIN P. (1969) The role of gamma-aminobutyric acid in mosquito-host interactions: a hypothesis. Ann. ent. Sot. Am. 62, 695-702. KERIWT G. A., LEAKIZL. D., SHAPIRAA., COWANS. & WALKERR. J. (1965) The presence of glutamate in nerve-muscle per&sates of Helix, Cmcinus and Periplaneta. Comp. Biochem. Physiol. 15,485-502. K~RKUTG. A. & WALKERR. J. (1966) The effect of L-glutamate, acetylcholine and gammaaminobutyric acid on the miniature end-plate potentials and contractures of the coxal muscles of the cockroach, Periplaneta americana. Comp. Biochem. Physiol. 17,435-454. KERKUTG. A. & WALKERR. J. (1967) The effect of iontophoretic injection of glutamic acid and gamma-~~o-N-bu~ric acid on the miniature end-plate potentials and contractures of the coxal muscles of the cockroach Periptaneta americana L. Camp. Biochem. Physiol. 20,999-1003. KRAVITZE. A. (1967) Acetylcholine, gamma-aminobutyric acid, and glutamic acid: physiological and chemical studies related to their roles as neurotransmitter agents. In The Nearosciences; A Study Program (Edited by QUARTONG. C., MELNECHUKT. & SCHMITT F. O.), pp. 433-444. Rockefeller University Press, New York. KUFFLER S. W. (1959) Excitation and inhibition in single nerve cells. Ifarvey Lect. 54, 176-218. Academic Press, New York. OTSUKAM., IVERSONL. L., HALL 2. W. & KRAVITZE. A. (1966) Release of gamma-aminobutyric acid from inhibitory nerves of lobster. PYOC.natn. Acad. Sci. U.S. 56, lllO1115. PITMAN R. M. & KERKUT G. A. (1970) Comparison of the actions of iontophoretically applied acetylcholine and gamma aminobutyric acid with the EPSP and IPSP in cockroach central neurons. Camp. getr. Pharmac. 1,221-230. PRICE G. M. (1961) Some aspects of amino acid metabolism in the adult housefly, Musca domestica. Biochem. J 80, 420-428. RAY J. W. (1964) The free amino acid pool of the cockroach (Periplaneta americana) central nervous system and the effect of insecticides. J. Insect Physiol. 10,587-597. ROBRINSJ. & VANDERKLOOTW. G. (1958) The effect of picrotoxin on peripheral inhibition in the crayfish. 3. PhysioZ., Land. 143, 541-552. ROBERTSE., FRANKELS. & HAR~ P. J. (1950) Amino acids of nervous tissue. PYOC. Sot. exp. Biol. Med. 74, 383-387. ROSSI-BERNARDI L., PACE M., ROUCHTONF. J. W. AZVANKEMPENL. (1969) The estimation of hemoglobin-CO* compounds by gel filtration and ion-exchange chromatography. In CO,: Chemical, Biochemical, and Physiological Aspects (Edited by FORSTER R. E., EDSALLJ. T., OTIS A. B. & ROUGHTONF. J. W.), pp. 65-71. National Aeronautics and Space A~inistration publication NASA SP-188, Washington, D.C.

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ROTHBERCS. & STEINBERGD. (1957) Studies on the mechanism of enzymatic decarboxylation. J. Am. Chem. Sot. 79, 3274-3278. ROUGHTONF. J. W. (1944) Some recent work on the chemistry of carbon dioxide transport by the blood. Harvey Lect. 39, 96142. Academic Press, New York. SMIT W. A., BBCHT G. & BEENAKKER~ A. M. Th. (1967) Structure, fatigue, and enzyme activities in “fast” insect muscles. J. Insect Physiol. 13, 1857-1868. SMITTLE B. J. (1966) Cockroaches. In Insect Colonization and Mass Production (Edited by SMITH C. N.), pp. 240-277. Academic Press, New York and London. SUCAN. & KATSUKIY. (1961) Pharmacological studies on the auditory synapses in a grasshopper. J. exp. Biol. 38, 759-770. TAKEUCHIA. & TAKEUCHI N. (1964) The effect on crayfish muscle of iontophoretically applied glutamate. J. Physiol., Lond. 170, 296-317. TAKEUCHIA. & TAKEUCHIN. (1966) On the permeability of the presynaptic terminal of the crayfish neuromuscular junction during synaptic inhibition and the action of gammaaminobutyric acid. J. Physiol., Lond. 183, 433-449. TAKEUCHIA. & TAKEUCHIN. (1967) Electrophysiological studies of the action of GABA on the synaptic membrane. Fedn Proc. Fedn Am. Sots exp. Biol. 26, 1633-1638. TWAROGB. M. & ROEDW K. D. (1956) Properties of the connective tissue sheath of the cockroach abdominal nerve cord. Biol. Bull. 111, 278-286. USHERWOOD P. N. R. & GRUNDFESTH. (1964) Inhibitory post synaptic potentials in grasshopper muscle. Science, N. Y. 143, 817-818. USHERWOODP. N. R. & GRUNDFESTH. (1965) Peripheral inhibition in skeletal muscles of insects. J. Neurophysiol. 28, 497-518. USHERWOODP. N. R. 81 MACHILI P. (1966) Chemical transmission at the insect excitatory neuromuscular synapse. Nature, Lend. 210, 634-636. USHERWOOD P. N. R. & MACHILI P. (1968) Pharmacological properties of excitatory neuromuscular synapses in the locust. g. exp. Biol. 49, 341-361. VSRBSHTCHAGIN S. M., SYTINSKYI. A. & TYSHCHENKOV. P. (1961) The effect of gammaaminobutyric acid and beta-alanine on bioelectrical activity of nerve ganglia of the pine moth caterpillar (Dindrolimus pini). J. Insect Physiol. 6, 21-25. WALKERJ. L., JR. & BROWNA. M. (1970) Unified account of the variable effects of carbon dioxide on nerve cells. Science 167, 1502-1504. WINGO W. J. 81 AWAPARAJ. (1950) Decarboxylation of L-glutamic acid by brain. r. biol. Chem. 187, 267-271. Key Word Index-Gamma-aminobutyric acid; carbamate; carbamino-GABA; synaptic inhibition; cockroach nerve cord; carbon dioxide; nervous stimulation; ventilatory rhythm; glutamate; inhibitory transmitter; insect; central nervous system.