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GABA dilates cerebral arteries in vitro and increases regional cerebral blood flow in vivo
Brnin Resuwch
Vol. 5, Suppl.2, pp. 335-339. Printed in the U.S.A.
Bdlrtin,
GABA Dilates Cerebral Arteries In Vitro and Increases Regional Cerebral Blood Flow In Vivo LARS EDVINSSON, Department
DIANA
N. KRAUSE,’
of Clinical
Pharmacology, S-22185 Lund,
BENGT LARSSON University Sweden
AND TOR SK;iRBY
Hospital
of Lund
B. LARSSON AND T. SKARBY. GABA dilates cerebral arteries in vitro and in vivo. BRAIN RES. BULL. 5: Suppl. 2, 335-339, 1980.-We have studied two aspects of GABA interaction with the cerebral circulation which are consistent with the existence of cerebrovascular GABA receptors. In vitro vasomotor responsiveness to GABA receptor agonists and antagonists were examined in isolated cat and dog cerebral artery segments. GABA and known GABA agonists produced a dose-dependent dilation which could be blocked by the GABA antagonists bicuculline and picrotoxin. No effect of GABA on extracranial blood vessels was observed. In vivo regional blood flow also was measured in anesthetized rats using the r4C-ethanol technique. Injection (IV) of muscimol, a potent GABA agonist, resulted in a significant increase in blood flow in cortical areas, while no effect was seen in cerebellum, thalamus, or caudate nucleus. The effect of muscimol on cortical blood flow is consistent with an involvement of GABA mechanisms in cerebral flow regulation. EDVINSSON,
increases
L., D. N. KRAUSE,
regional cerebra/
bloodflow
Cerebral arteries Cerebral blood flow GABA Cerebrovascular GABA receptor Cerebral circulation
of overwhelming interest in the mechanisms involved in the control of the cerebral circulation, few studies have been concerned with involvement of the inhibitory neurotransmitter y-aminobutyric acid (GABA). An association of GABA with cerebral blood vessels has been indicated by the presence of a non-neuronal form of the enzyme which synthesizes GABA, glutamate decarboxylase (GAD), in relatively high concentrations in pial arteries as compared to peripheral vessels [13,14]. GABA transaminase (GABA-T), the degradative enzyme for GABA also has been demonstrated histochemically in large concentrations around brain vessels. A possible involvement of GABA mechanisms in the physiology of the cerebral circulation is supported by the finding that GABA may dilate canine cerebral blood vessels [8] and reduce cerebral arterial resistance and increase cerebral blood flow [ 131. In this study, we have further characterized the receptor mediating GABA-induced dilation by examining the effects of various known GABA agonists and antagonists on the mechanical response of cat pial arteries in vitro. In order to assess a possible involvement of GABA receptors in the cerebral circulation we have utilized the rat in viva preparation to measure regional cerebral blood flow with the 14Cethanol technique before and after treatment with the potent GABA receptor agonist muscimol.
GABA-mediated
IN SPITE
*Division of Neurosciences,
Copyright
vasodilation
Muscimol
METHOD
Vascular Preparation for In Vitro Studies Thirteen cats of both sexes (2.tS4.5 kg) were anesthetized with sodium pentobarbital(30 mg/kg IP) and killed by exsanguination. The brain was removed, and the middle cerebral, posterior communicating and basilar arteries (3W6Ofl pm in dia.) were dissected out and placed immediately in an oxygenated Krebs-bicarbonate solution. Branches of similar caliber were taken from the external maxillary or lingual arteries. The morphology of the vessels and the in vitro conditions for studying their mechanical responses have been determined previously [4,5]. Pieces of intra- and extracranial arteries (about 5 mm long each) were used immediately for testing; the remainder were kept in a refrigerator at 4°C for later use (up to 24 hr). The two types of arteries were mounted in a 50 ml temperaturecontrolled organ bath with two separate systems of L-formed metal holders for recording isometric circular contractions as described previously [4]. The bath contained a solution of the following composition (mM): NaCl, 118; KCl, 4.5; CaCl,.H,O, 2.5; MgS04.7H20, 1.0; NaHCO,,, 25; KH, PO1, 1.0; and glucose, 6.0. The bath and the stock solution were maintained at 37.5 t 0.5”C (range) and aerated con-
City of Hope National Medical Center, Duarte, CA.
0 1980 ANKHO
International
in vivo
Inc.-0361-9230/80/080335-05$01.00/O
336
FIG. 1. Dose-response curve for the dilator effect of GABA tested on segments of cat middle cerebral arteries actively-contracted by either 2.5x 10e6 M PGF,a or 3 x 1Om8M 5-HT. Points represent the means f SEM for thirteen experiments.
tinuously with a mixture of 95% 0, and 5% CO,. Shortly after the arterial preparations had been mounted in the organ bath, each was subjected to a load of 400 dynes and allowed to stabilize; the tension decreased approximately 50-200 dynes. The test drugs were administered after 1.5 hr of equilibration. preliminary tests showed that the GABA agonists dilate cerebral vessels. Under our experimental conditions [5], the vessels were almost completely relaxed, and dilator responses were thus difficult to obtain. Therefore vessels were given a resting tone by addition of either 2.5 x 10e6 M prostaglandin F,cw (PGF,ol) or 3x 1Om8M 5-hydroxytryptamine (5-I-IT). The contraction by either of these two agents, an average of 200 dynes in this study, remained at a steady level for at least 30 min. During this time increasing doses of various GABA agonists were added cumulatively. In blockade experiments, the antagonists were added 15-20 min before the agonist doses and remained in the bath during agonist application. The effect of the agonists was plotted in terms of responses against log dose. The maximum response obtained, EAm(expressed in dynes), and the ED, (molar concentration of agonist at which half-maximum response occurs) were used as measures of agonist potency. The results are generally expressed as mean values f standard error of the mean.
EDVINSSON
c‘7’ Al..
FIG. 2. Effect of picrotoxin on the dose-response curve for the dilator effect of GABA. Isolated cat middle cerebral arteries were pretreated with either 3 x IO-’ M, or 3 x lo-@ M picrotoxin for 15 min and then given active tone with 2.5x WR M PGF,(Y and tested for responses to increasing concentrations of GABA. Each point represents the mean for two experiments.
technique of Ekliif et al. [7]. In brief, saline containing tracer amounts of “C-ethanol was continuously infused IV for 30 set, and arterial blood was sampled every 3 set for 14Cactivity. Following infusion, the animal was sacrificed by decapitation, and the brain frozen immediately by immersion into liquid nitrogen. Later tissue samples from various brain regions were removed and measured for 14C-activity. The blood flow was calculated according to formulas described by Ekliif et al. [7] using a Wang desk computer. The results were statistically compared using Student’s t-test. Drugs Bicucilline (Chemicals Procurement Labs), imidazoleacetic acid (Calbiochem), muscimol (Merck Frosst Laboratories), prostaglandin F,u (Astra) and taurine (Eastman) were used in this study as well as the following compounds obtained from Sigma: p-alanine, y-aminobutyric acid (GABA), (+-)y-amino+-hydroxybutyric acid, &aminovaleric acid, glutamic acid, glycine, 5-hydroxytryptamine creatine sulfate (5-HT), and picrotoxin.
Cerebral Blood Flow Measurements Male Sprague-Dawley rats (25g-300 g) were anesthetized with 2-3% halothane, tracheotomized, immobilized (tubocurarine 0.5 m&g IV) and artificially ventilated with 70% NzO in O2 to give arterial pCOZ values of between 35-40 mm Hg and pOZ values exceeding 100 mm Hg. Body temperature was adjusted to 37°C. The femoral arteries were cannulated for measurement of blood pressure, anaerobic sampling for blood gas measurements, and blood sampling to determine IT-ethanol activity. Injections were made in a femoral vein. When these procedures were completed, the animals were left undisturbed for 10-15 min to stabilize their condition. The animals were then injected with muscimol (0.43 n&kg) or served as controls. Ten minutes later regional cerebral blood flow was measured, using the *4C-ethanol
RESULTS
Response of Cat Cerebral Arteries GABA was found to dilate in a dose-dependent manner intracranial vessels actively contracted by either PGF,~Y or 5-HT (Fig. 1). This response, however, could not be detected in every vessel; GABA f&ed to dilate any of the intracranial arteries tested from 4 of the 12 cats studied. In those vessels which did respond (15 out of 33 vessels examined), the GABA dilatory effect, while not strong, was consistent. Characteristically, GABA produced a threshold of response at a bath concentration of about 0.33 PM, a half maximum response (ED,) at 0.8 phi, and a maximum response of approximately 60 dynes dilation at 10-100 PM. Relaxation in
GABA-MEDIATED
337
VASODILATION TABLE DILATORY
1
EFFECTS OF VARIOUS GABAERGIC MIDDLECEREBRALARTERY
Agonist
N
Muscimol GABA Imidazoleacetic acid y-Aminovaleric acid y-Amino-/3-hydroxybutyric P-Alanine
6 13 4 2 3 3
acid
AGONISTS
ON THE CAT
E,, (dynes)
ED,,, (M) 3.8 7.7 1.1 2.0 2.3 3.5
? t + + + ?
2.5 2.4 0.5 1.4 1.5 3.3
x x x x x x
54 + 59+ 49? 51+ 472 47 +
lo--’ lo-’ lO-fi 10~” lo-” 10-e
10 11 11 4 9 12
Mechanical responses of isolated segments of cat middle cerebral arteries were recorded as described in METHOD. Increasing doses of a GABA agonist were added cumulatively to the bath of vessel segments which were tonically contracted by approximately 200 dynes with either 2.5 x 10mfiM PGF2a or 3 X IO-” M 5-HT. Results are given as means t SEM except for n=2 which is the mean + standard deviation. n=number of experiments, ED,,,=dose producing a half-maximumdilation, and EAm=maximum dilation.
response to GABA was reversible and rapid, being complete within l-2 min. Similar GABA effects were observed with basilar and middle cerebral artery preparations, although most experiments were performed with the latter vessel type. GABA was never found to affect extracranial vessels, nor did it relax intracranial vessels which had been actively contracted with saturated KC1 (3 vessel segments tested). The effects of the convulsants picrotoxin and bicuculline, two relatively specific antagonists of GABA responses, were examined on the GABA-induced dilation of cat middle cerebral arteries. Pretreatment of an intracranial vessel with either picrotoxin or bicuculline at concentrations of 0.3 PM and 3 PM caused a dose-dependent inhibition of the vessel’s response to GABA. The antagonists by themselves had no obvious effect on the tonic, active contraction induced by PGF,(Y. Picrotoxin was examined in more detail and appeared to produce parallel shifts in the GABA dose-response curve towards higher agonist doses (Fig. 2), an action characteristic of competitive antagonists. Table 1 summarizes the effects on cat intracranial vessels of several compounds structurally related to GABA that have been well characterized as GABA agonists in neuronal preparations. Muscimol, imidazoleacetic acid, &aminovaleric acid, (a)-y-amino-P-hydroxybutyric acid, and p-alanine each caused a dose-dependent relaxation when tested on actively contracted, GABA-responsive vessels at bath concentrations of 10-H-10-4 M. The maximum dilatory response was similar for all agonists tested, including GABA. When compared on the same vessel preparation, the potency of muscimol was greater than or equal to that of GABA. The other compounds were found to be somewhat less potent than GABA, with @alanine generally being the least potent agonist. Other putative amino acid neurotransmitters, glycine, taurine, and glutamic acid, were also tested at 1O-4 M on actively contracted vessels, but no response of the vessels could be detected. Cerebral
Blood Flow
Earlier results from this laboratory have determined the steady state regional cerebral blood flow of control animals [21. The present control flow results, as well as the other
Reglonal Cerebral Blood Flow (ml x 100.‘a x mm’) 250 -
*
cl
Control Musclmol
I
I
*
1
T
200
150-
100-
SO-
0 Fro::1
Panetotemp cortex
Occlpltol Caudate cortex nucleus
Thalam
II
Mesenc
Cerebel
FIG. 3. Regional cerebral blood flow (rCBF) in control rats (open bars) and after IV injection of 0.43 mg/kg of muscimol (tilled bars). For information about the number of animals and the associated physiological parameters, see text. Mean values ? SEM. Student’s t-test: *p
physiological parameters measured, agree well with those previously obtained. The injection of muscimol did not change blood gases, pH or blood pressure during the 10-15 min period before the cerebral blood flow was measured.
33x
EDVINSSON
The effect of muscimol on regional cerebral blood flow is illustrated in Fig. 3. In general, the cortical flow was enhanced; the regional flow increase was 88% in occipital cortex (p
While the existence of a cerebrovascular GABA receptor suggests a role for this substance in the control of cerebral circulation, the in Iitro relaxation produced by GABA and its analogs was not as pronounced as that of other known mediators of vasodilation such as adenosine compounds 191, pH reduction [6], or P-receptor agonists [5]. Under certain in viva conditions, however, GABAmediated
DISCUSSION
A recent in vitro study on dog pial arteries [8] suggests that GABA may effect the cerebral vasculature in addition to its known inhibitory neurotransmitter actions on nerve cells and invertebrate striated muscle [ 151. Our studies [ 31 support a direct vasodilatory action of GABA, observed here in isolated cat pial arteries. Similarly, human cerebral arteries have also been shown to respond to GABA [3]. Fujiwara et al. [8] found that most of their isolated dog pial arteries only responded to high GABA concentrations; however, we obtained from all vessels which responded to GABA fairly consistent dose-response relationships with an average ED,, of 7.7 * 2.4~10~~ M. In our studies the maximum response to GABA was lower in both cat (59 * 11) and dog (63 c 11) vessels than the previously reported value for dog vessels of approximately 200 dynes dilation. No GABA responses were ever detected in extracranial vessels. Our results are consistent with the presence of a specific GABA receptor on the cerebral vessels which appears analogous to the GABA receptor on neurons and invertebrate striated muscle. The GABA response was blocked in a dose-dependent manner by picrotoxin and bicuculline, known GABA antagonists [15]. In our studies, picrotoxin appeared to act as a competitive antagonist, producing parallel shifts to the right of the GABA dose-response curve. The relative potency for the agonists tested on the cerebral arteries was consistent with that found for other known GABA receptors. Muscimol was generally the most potent agonist; this has been demonstrated in many preparations. Imidazoleacetic acid (?)y-amino-&hydroxybutyric acid, and &aminovaleric acid have been reported to be somewhat less potent than GABA, while @alanine has been shown to be an even less effective GABA agonist. A similar pharmacology is found for the cerebrovascular GABA receptor site when it is identified using 3H-muscimol binding assays [ 101.
E:l i\l..
vasodilation
may be enhanced.
GABA
has been
reported to increase cerebral blood flow in l~ivo [13]; and muscimol, when administered intravenously to rats, also produces an increase in local cerebral blood flow as determined by the Y-ethanol technique. The cortical flow enhancement observed here with muscimol is in accordance with the findings of Mirzoyan and Akopyan [ 131. In this earlier study which used the technique of thermistors and 0, polarography, an increment of the cerebral blood flow was measured simultaneously with a rise in brain tissue Ot, tension after GABA administration to cats, rabbits or dogs. The effect on cerebral blood flow seen with muscimol administration is consistent with a direct dilation of brain vessels via the cerebrovascular GABA receptors described previously [3. 8, IO]. However, muscimol is rapidly deaminated and the product formed may be effective in the cerebral circulation. Cerebral vessels are exposed normally to several sources of GABA in vivo. Brain vessels contain GABA-related enzymes 112, 14, 171 and thus are capable of producing GABA themselves. Direct GABAergic innervation of the vessels has never been seen with the specific GAD immunocytochemical technique, although GABA is released into the extracellular fluid of the brain by neurons and possibly glia [ 161. According to recent reports [ 11,191, the cerebrospinal fluid (CSF), which surrounds pial arteries in vivcr, contains in humans from about 0.1 to 0.8 PM GABA, concentrations which correspond to the lower range of the dose-response relationship reported here. Interestingly, elevated CSF levels of GABA have been measured in human patients during certain pathological conditions involving altered blood flow such as migraine headache 1181and cerebral anoxia due to cerebrovascular disease [ 11. ACKNOWLEDGEMENTS
This research was supported by the Swedish Medical Research Council (Grant No. 04X-7323 and USPHS Grants NS-05695 and NS-12116.
REFERENCES 1. Anchar, V. S., K. M. A. Welch, E. Chabi, K. Bartosh and J. S. Meyer. Cerebrospinal fluid gamma-aminobutyric acid in neurologic disease. Neurology 26: 777-780, 1976. 2. Edvinsson, L., P. Lacombe, C. Owman, A.-M. Reynier-
Rebuffel and J. Seylaz. Quantitative changes in regional cerebra1 blood flow of rats induced by a- and P-adrenergic stimulants. Acra physiol. wand. 107: 28%2%, 1979. 3. Edvinsson, L. and D. N. Krause. Pharmacological characterization of GABA receptors mediating vasodilation of cerebral arteries in vitro. Brain Res. 173: 8%97, 1979. 4. Edvinsson. L.. K. C. Nielsen and C. Owman. Influence of initial tension a&l changes in sensitivity during amine-induced contractions of pial arteries in vitro. Arch. int. Pharmacodyn. 2Ogz235-242. 1974.
5. Edvinsson, L. and C. Owman. Pharmacological characterization of adrenergic alpha and beta receptors mediating vasomotor response of cerebral arteries in vitro. Circa/at. Res. 35:
835-849, 1974. 6. Edvinsson, L. and R. Sercombe. Influence of pH and pC0, on mediated contraction in brain vessels. Actu 97: 325-331, 1976. 7. Ekliif, B., N. A. Lassen, B. Nilsson, K.Norberg, B. K. Siesja and P. Torliif. Regional cerebral blood flow in the rat measured by the tissue sampling technique: critical evaluation using four indicators (Yantipyrine, CY-ethanol, H3-water and Xenon’33. Acta physiol. scan>. 91: l-10, 1974. 8. Fujiwara; M., I. Muramatsu and S. Shibata. y-Aminobutyric acid receptor on vascular smooth muscle of dog cerebral arteries. Br. J. Phhrmac. 55: 561-562, 1975. alpha-receptor
physiol. stand.
GABA-MEDIATED
VASODILATION
9. Hardebo, .I. E. and L. Edvinsson. Adenine compounds: cerebrovascular effects in vitro with reference to their possible involvement in migraine. Stroke 10: 58-62, 1979. 10. Krause, D. N., E. Wong, P. Degener and E. Roberts. GABA receptors in bovine cerebral blood vessels: binding studies with RH-muscimol. Brain Res. 185: 51-57. 1980. 11. Huizinga, J. D., A. W. Teelken, F. A. J. Muskiet, J. V. D. Meulen and B. G. Wolthers. Identification of GABA in human CSF by gas liquid chromatography and mass spectrometry. Neus Engl. .I. Med. 296: 692, 1977. 12. Kuriyama, K., B. Haber and E. Roberts. Occurrence of a new L-glutamic acid decarboxylase in several blood vessels of the rabbit. Brain Res. 23: 111-123, 1970. 13. Mirzoyan, S. A. and V. P. Akopyan. The effect produced by gamma-aminobutyric acid on the cerebral circulation and oxygen tension in the brain. Farmak. Toks. 5: 572-574, 1967.
339
14. Mirzoyan, S. A., V. A. Kazaran and V. P. Akopyan. The glutamic decarboxylase activity in blood vessels of the brain. Dokl. Acad. Nauk SSSR 190: 1241-1243, 1970. 15. Roberts, E., T. N. Chase and D. B. Tower (Eds). GABA in Nervous System Function. New York: Raven Press, 1976. 16. Swagel, M. W., K. Ikeda and E. Roberts. Effects of GABA, imidazoleacetic acid, and related substances on conductance of crayfish abdominal stretch receptor. Nature New Biol. 246: 91-92, 1973. 17. Van Gelder,
y-aminobutyric
N. M. A possible enzyme barrier for acid in the central nervous system. Progr. Brain
Res. 28: 259-268,
1%8.
18. Welch, K. M. A., E. Chabi, K. Bartosh, V. S. Achar and J. S. Meyer. Cerebrospinal fluid y-aminobutyric acid levels in migraine. Br. med. J. 3: 516-517, 1975. 19. Wood, J. H., B. S. Glaeser, S. J. Enna and T. A. Hare. Veritication and quantification of GABA in human cerebrospinal fluid. J. Neurochem. 20: 291-293, 1978.
Vol. 5, Suppl.2, pp. 335-339. Printed in the U.S.A.
Bdlrtin,
GABA Dilates Cerebral Arteries In Vitro and Increases Regional Cerebral Blood Flow In Vivo LARS EDVINSSON, Department
DIANA
N. KRAUSE,’
of Clinical
Pharmacology, S-22185 Lund,
BENGT LARSSON University Sweden
AND TOR SK;iRBY
Hospital
of Lund
B. LARSSON AND T. SKARBY. GABA dilates cerebral arteries in vitro and in vivo. BRAIN RES. BULL. 5: Suppl. 2, 335-339, 1980.-We have studied two aspects of GABA interaction with the cerebral circulation which are consistent with the existence of cerebrovascular GABA receptors. In vitro vasomotor responsiveness to GABA receptor agonists and antagonists were examined in isolated cat and dog cerebral artery segments. GABA and known GABA agonists produced a dose-dependent dilation which could be blocked by the GABA antagonists bicuculline and picrotoxin. No effect of GABA on extracranial blood vessels was observed. In vivo regional blood flow also was measured in anesthetized rats using the r4C-ethanol technique. Injection (IV) of muscimol, a potent GABA agonist, resulted in a significant increase in blood flow in cortical areas, while no effect was seen in cerebellum, thalamus, or caudate nucleus. The effect of muscimol on cortical blood flow is consistent with an involvement of GABA mechanisms in cerebral flow regulation. EDVINSSON,
increases
L., D. N. KRAUSE,
regional cerebra/
bloodflow
Cerebral arteries Cerebral blood flow GABA Cerebrovascular GABA receptor Cerebral circulation
of overwhelming interest in the mechanisms involved in the control of the cerebral circulation, few studies have been concerned with involvement of the inhibitory neurotransmitter y-aminobutyric acid (GABA). An association of GABA with cerebral blood vessels has been indicated by the presence of a non-neuronal form of the enzyme which synthesizes GABA, glutamate decarboxylase (GAD), in relatively high concentrations in pial arteries as compared to peripheral vessels [13,14]. GABA transaminase (GABA-T), the degradative enzyme for GABA also has been demonstrated histochemically in large concentrations around brain vessels. A possible involvement of GABA mechanisms in the physiology of the cerebral circulation is supported by the finding that GABA may dilate canine cerebral blood vessels [8] and reduce cerebral arterial resistance and increase cerebral blood flow [ 131. In this study, we have further characterized the receptor mediating GABA-induced dilation by examining the effects of various known GABA agonists and antagonists on the mechanical response of cat pial arteries in vitro. In order to assess a possible involvement of GABA receptors in the cerebral circulation we have utilized the rat in viva preparation to measure regional cerebral blood flow with the 14Cethanol technique before and after treatment with the potent GABA receptor agonist muscimol.
GABA-mediated
IN SPITE
*Division of Neurosciences,
Copyright
vasodilation
Muscimol
METHOD
Vascular Preparation for In Vitro Studies Thirteen cats of both sexes (2.tS4.5 kg) were anesthetized with sodium pentobarbital(30 mg/kg IP) and killed by exsanguination. The brain was removed, and the middle cerebral, posterior communicating and basilar arteries (3W6Ofl pm in dia.) were dissected out and placed immediately in an oxygenated Krebs-bicarbonate solution. Branches of similar caliber were taken from the external maxillary or lingual arteries. The morphology of the vessels and the in vitro conditions for studying their mechanical responses have been determined previously [4,5]. Pieces of intra- and extracranial arteries (about 5 mm long each) were used immediately for testing; the remainder were kept in a refrigerator at 4°C for later use (up to 24 hr). The two types of arteries were mounted in a 50 ml temperaturecontrolled organ bath with two separate systems of L-formed metal holders for recording isometric circular contractions as described previously [4]. The bath contained a solution of the following composition (mM): NaCl, 118; KCl, 4.5; CaCl,.H,O, 2.5; MgS04.7H20, 1.0; NaHCO,,, 25; KH, PO1, 1.0; and glucose, 6.0. The bath and the stock solution were maintained at 37.5 t 0.5”C (range) and aerated con-
City of Hope National Medical Center, Duarte, CA.
0 1980 ANKHO
International
in vivo
Inc.-0361-9230/80/080335-05$01.00/O
336
FIG. 1. Dose-response curve for the dilator effect of GABA tested on segments of cat middle cerebral arteries actively-contracted by either 2.5x 10e6 M PGF,a or 3 x 1Om8M 5-HT. Points represent the means f SEM for thirteen experiments.
tinuously with a mixture of 95% 0, and 5% CO,. Shortly after the arterial preparations had been mounted in the organ bath, each was subjected to a load of 400 dynes and allowed to stabilize; the tension decreased approximately 50-200 dynes. The test drugs were administered after 1.5 hr of equilibration. preliminary tests showed that the GABA agonists dilate cerebral vessels. Under our experimental conditions [5], the vessels were almost completely relaxed, and dilator responses were thus difficult to obtain. Therefore vessels were given a resting tone by addition of either 2.5 x 10e6 M prostaglandin F,cw (PGF,ol) or 3x 1Om8M 5-hydroxytryptamine (5-I-IT). The contraction by either of these two agents, an average of 200 dynes in this study, remained at a steady level for at least 30 min. During this time increasing doses of various GABA agonists were added cumulatively. In blockade experiments, the antagonists were added 15-20 min before the agonist doses and remained in the bath during agonist application. The effect of the agonists was plotted in terms of responses against log dose. The maximum response obtained, EAm(expressed in dynes), and the ED, (molar concentration of agonist at which half-maximum response occurs) were used as measures of agonist potency. The results are generally expressed as mean values f standard error of the mean.
EDVINSSON
c‘7’ Al..
FIG. 2. Effect of picrotoxin on the dose-response curve for the dilator effect of GABA. Isolated cat middle cerebral arteries were pretreated with either 3 x IO-’ M, or 3 x lo-@ M picrotoxin for 15 min and then given active tone with 2.5x WR M PGF,(Y and tested for responses to increasing concentrations of GABA. Each point represents the mean for two experiments.
technique of Ekliif et al. [7]. In brief, saline containing tracer amounts of “C-ethanol was continuously infused IV for 30 set, and arterial blood was sampled every 3 set for 14Cactivity. Following infusion, the animal was sacrificed by decapitation, and the brain frozen immediately by immersion into liquid nitrogen. Later tissue samples from various brain regions were removed and measured for 14C-activity. The blood flow was calculated according to formulas described by Ekliif et al. [7] using a Wang desk computer. The results were statistically compared using Student’s t-test. Drugs Bicucilline (Chemicals Procurement Labs), imidazoleacetic acid (Calbiochem), muscimol (Merck Frosst Laboratories), prostaglandin F,u (Astra) and taurine (Eastman) were used in this study as well as the following compounds obtained from Sigma: p-alanine, y-aminobutyric acid (GABA), (+-)y-amino+-hydroxybutyric acid, &aminovaleric acid, glutamic acid, glycine, 5-hydroxytryptamine creatine sulfate (5-HT), and picrotoxin.
Cerebral Blood Flow Measurements Male Sprague-Dawley rats (25g-300 g) were anesthetized with 2-3% halothane, tracheotomized, immobilized (tubocurarine 0.5 m&g IV) and artificially ventilated with 70% NzO in O2 to give arterial pCOZ values of between 35-40 mm Hg and pOZ values exceeding 100 mm Hg. Body temperature was adjusted to 37°C. The femoral arteries were cannulated for measurement of blood pressure, anaerobic sampling for blood gas measurements, and blood sampling to determine IT-ethanol activity. Injections were made in a femoral vein. When these procedures were completed, the animals were left undisturbed for 10-15 min to stabilize their condition. The animals were then injected with muscimol (0.43 n&kg) or served as controls. Ten minutes later regional cerebral blood flow was measured, using the *4C-ethanol
RESULTS
Response of Cat Cerebral Arteries GABA was found to dilate in a dose-dependent manner intracranial vessels actively contracted by either PGF,~Y or 5-HT (Fig. 1). This response, however, could not be detected in every vessel; GABA f&ed to dilate any of the intracranial arteries tested from 4 of the 12 cats studied. In those vessels which did respond (15 out of 33 vessels examined), the GABA dilatory effect, while not strong, was consistent. Characteristically, GABA produced a threshold of response at a bath concentration of about 0.33 PM, a half maximum response (ED,) at 0.8 phi, and a maximum response of approximately 60 dynes dilation at 10-100 PM. Relaxation in
GABA-MEDIATED
337
VASODILATION TABLE DILATORY
1
EFFECTS OF VARIOUS GABAERGIC MIDDLECEREBRALARTERY
Agonist
N
Muscimol GABA Imidazoleacetic acid y-Aminovaleric acid y-Amino-/3-hydroxybutyric P-Alanine
6 13 4 2 3 3
acid
AGONISTS
ON THE CAT
E,, (dynes)
ED,,, (M) 3.8 7.7 1.1 2.0 2.3 3.5
? t + + + ?
2.5 2.4 0.5 1.4 1.5 3.3
x x x x x x
54 + 59+ 49? 51+ 472 47 +
lo--’ lo-’ lO-fi 10~” lo-” 10-e
10 11 11 4 9 12
Mechanical responses of isolated segments of cat middle cerebral arteries were recorded as described in METHOD. Increasing doses of a GABA agonist were added cumulatively to the bath of vessel segments which were tonically contracted by approximately 200 dynes with either 2.5 x 10mfiM PGF2a or 3 X IO-” M 5-HT. Results are given as means t SEM except for n=2 which is the mean + standard deviation. n=number of experiments, ED,,,=dose producing a half-maximumdilation, and EAm=maximum dilation.
response to GABA was reversible and rapid, being complete within l-2 min. Similar GABA effects were observed with basilar and middle cerebral artery preparations, although most experiments were performed with the latter vessel type. GABA was never found to affect extracranial vessels, nor did it relax intracranial vessels which had been actively contracted with saturated KC1 (3 vessel segments tested). The effects of the convulsants picrotoxin and bicuculline, two relatively specific antagonists of GABA responses, were examined on the GABA-induced dilation of cat middle cerebral arteries. Pretreatment of an intracranial vessel with either picrotoxin or bicuculline at concentrations of 0.3 PM and 3 PM caused a dose-dependent inhibition of the vessel’s response to GABA. The antagonists by themselves had no obvious effect on the tonic, active contraction induced by PGF,(Y. Picrotoxin was examined in more detail and appeared to produce parallel shifts in the GABA dose-response curve towards higher agonist doses (Fig. 2), an action characteristic of competitive antagonists. Table 1 summarizes the effects on cat intracranial vessels of several compounds structurally related to GABA that have been well characterized as GABA agonists in neuronal preparations. Muscimol, imidazoleacetic acid, &aminovaleric acid, (a)-y-amino-P-hydroxybutyric acid, and p-alanine each caused a dose-dependent relaxation when tested on actively contracted, GABA-responsive vessels at bath concentrations of 10-H-10-4 M. The maximum dilatory response was similar for all agonists tested, including GABA. When compared on the same vessel preparation, the potency of muscimol was greater than or equal to that of GABA. The other compounds were found to be somewhat less potent than GABA, with @alanine generally being the least potent agonist. Other putative amino acid neurotransmitters, glycine, taurine, and glutamic acid, were also tested at 1O-4 M on actively contracted vessels, but no response of the vessels could be detected. Cerebral
Blood Flow
Earlier results from this laboratory have determined the steady state regional cerebral blood flow of control animals [21. The present control flow results, as well as the other
Reglonal Cerebral Blood Flow (ml x 100.‘a x mm’) 250 -
*
cl
Control Musclmol
I
I
*
1
T
200
150-
100-
SO-
0 Fro::1
Panetotemp cortex
Occlpltol Caudate cortex nucleus
Thalam
II
Mesenc
Cerebel
FIG. 3. Regional cerebral blood flow (rCBF) in control rats (open bars) and after IV injection of 0.43 mg/kg of muscimol (tilled bars). For information about the number of animals and the associated physiological parameters, see text. Mean values ? SEM. Student’s t-test: *p
physiological parameters measured, agree well with those previously obtained. The injection of muscimol did not change blood gases, pH or blood pressure during the 10-15 min period before the cerebral blood flow was measured.
33x
EDVINSSON
The effect of muscimol on regional cerebral blood flow is illustrated in Fig. 3. In general, the cortical flow was enhanced; the regional flow increase was 88% in occipital cortex (p
While the existence of a cerebrovascular GABA receptor suggests a role for this substance in the control of cerebral circulation, the in Iitro relaxation produced by GABA and its analogs was not as pronounced as that of other known mediators of vasodilation such as adenosine compounds 191, pH reduction [6], or P-receptor agonists [5]. Under certain in viva conditions, however, GABAmediated
DISCUSSION
A recent in vitro study on dog pial arteries [8] suggests that GABA may effect the cerebral vasculature in addition to its known inhibitory neurotransmitter actions on nerve cells and invertebrate striated muscle [ 151. Our studies [ 31 support a direct vasodilatory action of GABA, observed here in isolated cat pial arteries. Similarly, human cerebral arteries have also been shown to respond to GABA [3]. Fujiwara et al. [8] found that most of their isolated dog pial arteries only responded to high GABA concentrations; however, we obtained from all vessels which responded to GABA fairly consistent dose-response relationships with an average ED,, of 7.7 * 2.4~10~~ M. In our studies the maximum response to GABA was lower in both cat (59 * 11) and dog (63 c 11) vessels than the previously reported value for dog vessels of approximately 200 dynes dilation. No GABA responses were ever detected in extracranial vessels. Our results are consistent with the presence of a specific GABA receptor on the cerebral vessels which appears analogous to the GABA receptor on neurons and invertebrate striated muscle. The GABA response was blocked in a dose-dependent manner by picrotoxin and bicuculline, known GABA antagonists [15]. In our studies, picrotoxin appeared to act as a competitive antagonist, producing parallel shifts to the right of the GABA dose-response curve. The relative potency for the agonists tested on the cerebral arteries was consistent with that found for other known GABA receptors. Muscimol was generally the most potent agonist; this has been demonstrated in many preparations. Imidazoleacetic acid (?)y-amino-&hydroxybutyric acid, and &aminovaleric acid have been reported to be somewhat less potent than GABA, while @alanine has been shown to be an even less effective GABA agonist. A similar pharmacology is found for the cerebrovascular GABA receptor site when it is identified using 3H-muscimol binding assays [ 101.
E:l i\l..
vasodilation
may be enhanced.
GABA
has been
reported to increase cerebral blood flow in l~ivo [13]; and muscimol, when administered intravenously to rats, also produces an increase in local cerebral blood flow as determined by the Y-ethanol technique. The cortical flow enhancement observed here with muscimol is in accordance with the findings of Mirzoyan and Akopyan [ 131. In this earlier study which used the technique of thermistors and 0, polarography, an increment of the cerebral blood flow was measured simultaneously with a rise in brain tissue Ot, tension after GABA administration to cats, rabbits or dogs. The effect on cerebral blood flow seen with muscimol administration is consistent with a direct dilation of brain vessels via the cerebrovascular GABA receptors described previously [3. 8, IO]. However, muscimol is rapidly deaminated and the product formed may be effective in the cerebral circulation. Cerebral vessels are exposed normally to several sources of GABA in vivo. Brain vessels contain GABA-related enzymes 112, 14, 171 and thus are capable of producing GABA themselves. Direct GABAergic innervation of the vessels has never been seen with the specific GAD immunocytochemical technique, although GABA is released into the extracellular fluid of the brain by neurons and possibly glia [ 161. According to recent reports [ 11,191, the cerebrospinal fluid (CSF), which surrounds pial arteries in vivcr, contains in humans from about 0.1 to 0.8 PM GABA, concentrations which correspond to the lower range of the dose-response relationship reported here. Interestingly, elevated CSF levels of GABA have been measured in human patients during certain pathological conditions involving altered blood flow such as migraine headache 1181and cerebral anoxia due to cerebrovascular disease [ 11. ACKNOWLEDGEMENTS
This research was supported by the Swedish Medical Research Council (Grant No. 04X-7323 and USPHS Grants NS-05695 and NS-12116.
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