Characteristics of GABA binding sites in bovine pineal gland

Bruin Rcwcrrrh

Bullrrin.

Vol. 5, Suppl.2, pp. 179-187.

Printed

in the U.S.A

Characteristics of GABA Binding Sites in Bovine Pineal Gland MANUCHAIR Department

EBADI AND ALBERT

CHAN

of Pharmacology, The University of Nebraska, College of Medicine 42nd Street and Dewey Avenue, Omaha, Nebraska 6810.5

EBADI, M. AND A. CHAN. Characteristics c>fGABA binding sites in bovine pinral gland. BRAIN RES. BULL. 5: Suppl. 2, l7%187, 1980.-In the absence of any known function of GABA in pineal gland, the possibility of its neuroinhibitory effects on pineal biochemical parameters including serotonin N-acetyltransferase (NAT) was tested. GABA (5-20 PM), but not other biogenic amines (histamine, dopamine, acetylcholine or melatonin). inhibited the norepinephrine (10 PM)-induced stimulation of NAT activity in bovine pineal explants in culture without altering the basal NAT activity. The inhibitory effect of GABA, which was dose-dependent, could be blocked by N-methylbicuculline and was not seen in Cl-free medium. Binding studies on synaptic membranes prepared from bovine pineal gland revealed the presence of sodium dependent and sodium independent binding sites. The sodium independent GABA binding site had a K,, value of 16.5 nM and a receptor density of 121 + 3 fmoles per mg protein. The sodium dependent GABA binding site had a K,, value of 0.677 FM and a receptor density of 4.47 * 0.42 pmolesimg protein. Sodium independent or sodium dependent binding sites exhibited neither positive nor negative cooperactivity. The exact functional interaction between GABAergic and adrenergic neurons in pineal gland, which may vary among mammalian species, is not clear and awaits further studies. Norepinephrine

Serotonin N-acetyltransferase

GABA

N-acetyltransferase (NAT) that acetylates serotonin to N-acetylserotonin regulates the circadian rhythm of melatonin synthesis in pineal gland through mechanisms which are only partially understood (for review, see [10,21]). In general, the light signals which originate from the retinal photoreceptors are transmitted via suprachiasmatic nuclei of hypothalamus and superior cervical ganglion to pineal gland, mediating the release of norepinephrine, which in turn stimulates P-adrenergic receptors on pinealocytes to initiate a group of events that result in stimulation of NAT activity. In addition to having large numbers of terminals containing norepinephrine and serotonin, the pineal gland also contains GABA, whose function is subject to much speculation. Mata ef al. [33] reported that glutamic acid decarboxylase (GAD) and GABA are associated with cell structure intrinsic to the pineal gland and not with nerve processes whose cell bodies are extra-pineal. Nevertheless, they mentioned “A small decrease in GAD activity with ganglionectomy and some loss of the activity during organ culture make it impossible rigorously to rule out a contribution from sympathetic nerve endings or their associated Schwann cells.” On the other hand, Waniewski and Suria [3 1] reported that the concentration of GABA demonstrated a diurnal variation and the uptake of :sH-GABA was significantly reduced following superior cervical ganglionectomy. These results were interpreted to indicate that “GABA may have some functional role in pineal gland.” In order to gain additional information on the possible function of GABA in pineal gland, we have explored the possibility of culturing bovine pineal explants and of studying the nature of adrenergic stimulation of NAT which has been shown to vary drastically in various species. For SEROTONIN

Copyright

8 1980 ANKHO

International

GABA binding sites in pineal glands

example, norepinephrine stimulates NAT activity in rat; whereas, it has the opposite effect in chicken [4]. Using bovine pineal explants, we found that norepinephrine stimulated NAT activity from OS-5 fold through an interaction with a P-adrenergic receptor and this stimulation was blocked by GABA. Similarly, bovine pineal glands weighing 150 mg each encouraged us to investigate and detect stereospecific, saturable and methylbicuculline sensitive binding sites. The results of these investigations which have appeared as preliminary communications [7,12] are detailed in this paper. METHOD

Chemiculs rend Other Materials Culture media 199 and BGJh (Fitton-Jackson modification) were purchased from Grand Island Biological Company (Grand Island, NY). Nylon mesh (CMN-1000) was obtained from Small Parts, Inc. (Miami, FL). Plastic tissue culture trays (Model FB-16-24-TC) were purchased from Linbro Chemical Company Inc., (New Haven, CT). (Acetyl-RH)acetyl Coenzyme A was purchased from ICN Pharmaceuticals Inc. (Irvine, CA). y-(2,3-3H)-Aminobutyric acid (34.5 Ci/mmole) was obtained from New England Nuclear Corp. (Boston, MA). N-methylbicuculline was synthesized with methyl iodide according to the method of Pong and Graham [26]. Carbachol chloride was obtained from Parke, Davis and Co. (Detroit, MI). Bicuculline, chlorpromazine, 2,4-DABA, ,f3-alanine, imidazole-6acetic acid, tryptamine, acetyl-CoA, I-norepinephrine, dopamine, histamine, GABA, serotonin and melatonin were obtained from Sigma Chemical Co. (St. Louis, MO). All other chemicals used were of the purest grade available.

Inc.-0361-9230/80/080179-09$01.40/O

Fresh bovine pineal glands were obtained from John Roth and Son Meat Packing Co. in Omaha, NE usually about II:00 a.m. and no later than 15 min after slaughter. The pineai glands were sliced and transported to the laboratory in culture media maintained at 4°C which was pre-equilibrated with 95% 0, and 5% CO,. For comparative studies with other mammalian pineal glands, Sprague-Dawley rats (260-280 g) were used and obtained from Sasco, Inc., Omaha, NE, Bovine Pined Explant in Culture Medium 199 was supplemented with glutamine (final concentration 2 mM), 0.1% BSA, streptomycin (100 ~gfml), penicillin (100 unit/ml). and 0.1 mg/ml of ascorbic acid. The pineal glands were cut into 1 mm3 explants in Dulbecco’s phosphate buffered saline. Two or three explants were placed on a piece of lens paper which was in turn supported by a piece of nylon mesh (CMN-1000). The explants were then incubated in 1 ml of culture medium in the well of a plastic tissue culture tray (Model FB-16-24-TC). All explants were adapted to the culture medium at room temperature for 30 min and at 37°C for an additional 30 min under an atmosphere of 95% 0, and 5% CO,. All drugs were prepared fresh in sterile water and added to culture media in concentrated solutions so that they would not significantly alter the final volume of 1 ml. After completion of the studies, the control and experimental pineal explants were immediately frozen in microculture tubes (50x6 mm) and stored at -80°C for enzyme determination. The chloride-free culture medium was prepared according to the procedure of Merchant rf al. [24], with the exception that CaCl, and KC1 were replaced with equal concentrations of Ca(H,PO,), and KH2P0,, respectively. Similarly, NaCl was replaced with 62 mM NhHPO, and 62 mM glucose. The pH of the culture medium was adjusted to pH 7.3 with HNO,,. The culture was incubated under 92% 0, and 8% CO,. Rut Pineai Gland Culture The rat pineal glands were cultured in medium BGJ, (Fitton-Jackson) according to the method of Trowel1 [29] as modified by Par&t and Klein 1251.The rat pineal glands were preincubated at room temperature for half an hour and at 37°C for 2 hr prior to experiments. Assay of’Serotonin

N-acetyltransferase

NAT activity was determined according to the method of Deguchi and Axelrod [9] as modified by Howd et al. [20]. The bovine pineal explants were homogenized in 40 ~1 of 10 mM sodium phosphate buffer (pH 6.5) cont~ning 4 mM penicillamine. An aliquot of IO ~1 of homogenate was used for protein determination and 25 ~1 was added to an assay mixture containing 0.35 M sodium phosphate buffer (pH 6.5), 35 mM tryptamine, 122.5 FM acetyl CoA (0.25-0.50 &i/assay) in a final volume of 35 ~1. When serotonin was used as a substrate, the product was extracted with isoamyl alcohol. Assay I$ GABA ~e~a~ol~zin~ Enzymes Glutamic acid decarboxylase was assayed according to the method of Wu and Roberts [32] and GABA-transaminase was measured according to the method of Gonnard et al. r151.

Forty-two bovine pineal glands, weight 8.3 g were dissected free of connective tissues, blood vessels and pineal stalks. The synaptic membranes were prepared according to the method of De Robertis [ll] as modiFted by Enna and Snyder [ 131. For sodium-independent binding study, the pellets were stored at -20°C for 18 hr before use. For sodium-dependent binding studies, the freshly prepared membranes were used.

The frozen pellets were thawed and washed with 50 mM ice cold Tris-citrate buffer (pH 7.1) prior to GABA binding assay which was carried out according to the method of Enna and Snyder [ 131. fn general, aliquots of synaptic membrane suspensions (0.2-0.5 mg of protein per ml) were incubated in triplicate for 5 min at 4°C in 1 ml of 50 mM Tris-citrate buffer (pH 7.1) in test tubes containing 35-60 nM of (3H)GABA alone or in the presence of 1 mM GABA. However, the low receptor density for GABA in pineal gland necessitated substituting the filtration for centrifugation technique in terminating the reactions [S]. These reactions were terminated by filtration under a uniform vacuum system (230 mm Hg pressure) through Whatman glass-fiber filter circles (GFiC-2.4 cm), the filter was washed with 3 ml of ice cold 50 mM Ttis-citrate acid buffer (pH 7.1) and the washing was completed within 3 sec. The filters were then shaken with 10 ml of scintiverse cocktail (Fisher Chemical Co.) for 1 hr, allowed to remain in the cocktail for 24 hr and then the radioactivity was determined by liquid scintillation spectrometry. No binding of (“H)GABA to the filter was noted. Sodium-Dependent

Binding

The sodium-dependent binding was carried out according to the method of Enna and Snyder [ 131. In general, aliquots of fresh synaptic membranes (0.5-2.5 mg protein/ml) were incubated in triplicate at 4°C for 5 min in 1 ml of 50 mM Tris-citrate buffer (pH 7.1) in polypropylene minivials (Fisher Scientific Co., St. Louis, MO) containing 0.15 M NaCl, and 25-860 nM (3H)GABA alone or in the presence of 1 mM GABA. After incubation, the reactions were terminated by centrifugation at 48,000 g for 10 min. The supernatant fluid was decanted and the pellet rinsed rapidly and superficially with 5 to 10 ml of ice cold distilled water. The pellets were solubilized by shaking them in 0.4 ml of NCS solubilizer for 2 hr. Then, 5.0 ml of scintillation fluid (0.5% w/v PPO and 0.01% w/v POPOP in toluene) was added to each vial and the samples were counted.

In order to determine the radiochemical identity of the bound ligand, the radioactive materials bound to synaptic membrane were extracted with 0.1 M HCl. The extract was then applied to an Eastman chromatogram sheet (6061 silica gel) using a solvent system (%% aqueous ethanol: 34% aqueous NH,OH, 7:3 v/v) which enabled us to separate GABA from its deaminated products [S]. Protein was determined as described by Lowry et al. 1221, Bovine serum albumin was used as a standard. Statistics Statistical

analyses

were carried out by the use of the

PINEAL

181

GLAND AND GABA TABLE

1

THE INHIBITION BY GABA OF NOREPINEPHRINE-STIMULATED ACTIVITY OF SEROTONIN N-ACETYLTRANSFERASE (NAT)IN BOVINE PINEAL EXPLANTS IN CULTURE NAT in pmolesimg protein/mm

Treatment

% inhibition

1.57 t 0.31

Control

3.60 t 0.53

p
4.06 t 0.34

p <0.05

0

GABA, 7.5 FM +Norepinephrine. 10 WM

2.59 ?Z0.54

JKo.05

50

GABA, 20 FM + Norepinephrine,

1.61 2 0.24

Norepinephrine, GABA, .5 /.iM f Norepinephrine.

10 pM IO FM

10 g*M

100

The explants serving as control were incubated in medium 199 containing 2 mM glutamine, 0.1% BSA. streptomycin (100 r&ml) and penicilline (100 units/ml) under an atmosphere of 95% 0, and 5% CO,. The norepineph~netreated group received this medium plus IO FM of norepinephrine. The GABA-treated group received this medium plus S-20 PM of GABA preincubated for 1 hr before the addition of norepinephrine (IO PM). Two hr after addition of norepinephrine, the control and experimental pineal explants were frozen immediately in microculture tubes and stored for determination of NAT according to a method discussed in METHOD. Each control or experimental culture contained 2-3 explants. The data, which are the average of at least 4 separate cultures, are presented as mean + SE.

Student’s t-test, and the significance of the differences between means was indicated by the value of p. The null hypothesis was rejected at the 5% level of confidence [31.IMa are expressed as mean k SE.

TABLE 2 THE NULLI~CA~ON OF THE INHIBITORY EFFECTS OF GABA ON NOREPINEPHRINE-STIMULATED SEROTONIN-N-ACETYLTRANSFERASE(NAT) ACTIVITY IN Cl-FREE MEDIUM

RESULTS

The activity of GABA metabolizing enzymes have not been reported in bovine pineal glands. Our studies have shown that the specific activity of glutamic acid decarboxylase was 54 * 10 pmoles of GABA formed/mg proteinimin; whereas the activity of GABA transaminase was 140 f 10 pmoles GABA metaboiized/mg protein/min. By using 8 culture media including Trowel1 Tx, DMEM, CMRL, BGJh and NCTC 135, we found that media alpha MEM and 199 maintained 100% of the basal NAT activity. Therefore, medium 199 was used throughout these experiments. The dist~bution of NAT in bovine pineai gland was assessed by dividing one gland in 21 explants and by determining the NAT activity in each explant. The results indicated that the mean activity of NAT was 0.86 pmoles of N-acetyltryptamine produced/mg protein/min, with a range of activity varying from 0.69 to 1.04 units with less than 1% variation. However, considerable interglandular variation in NAT activity did occur which ranged from 0.50-2.0 pmoles product/mg protein/min. Consequently, in many studies, explants obtained from a single pineal gland was used to serve as controls for experimental groups. GABA inhibited the norepineph~ne-induced stimuiation of NAT activity in a dose-related fashion (Table 1) without having any direct effect on enzymatic assay or altering the basal NAT activity. Similarly, the effect of GABA was specific since it was not seen with other amines such as dopamine, serotonitt, histamine and meiatonin. (Data not

Treatment Control Norepinephrine, 10 FM GABA. 20 PM + Norepineph~ne. IO PM

NAT in pmoles/mg protein/min 1.83 rt 0.08 4.17 t 0.39

p
3.30 Lt 0.21

p
The explants serving as control were incubated in chloride-free culture medium which was prepared according to the procedure of Merchant rt (II. (241 as described in METHOD. With the exception of chloride, the other constituents of medium 199 remained the same containing 2 mM glutamine, 0.1% BSA, streptomycin (100 &ml) and penicilline (100 units/ml) under an atmosphere of 95% 0, and 5% CO<. The norepinephrine-treated groups received chloride-free medium plus 10 FM of norepinephrine. The GABA-treated groups received chloride-free medium plus 20 FM of GABA preincubated for 1 hr before the addition of norepinephrine (IO FM). Two hr after addition of norepinephrine, the control and experimental pineal explants were frozen immediately in microculture tubes and stored for determination of NAT activity according to a method discussed in METHOD. Each control or experimental culture contained 2-3 explants. The data, which are the average of at least 7 separate cultures, are presented as mean + SE.

shown.) In addition, the inhibitory effect of GABA on norepinephrine-induced stimulation of NAT activity was not seen in chloride free medium (Table 2). Furthermore, the

EBADI AND CHAN TABLE 3 THE BLOCKADE BY N-METHYLBICUCULLINE OF THE INHIBITIONBY GABA OF NOREPINEPHRINE-STIMULATEDACTIVITYOF SEROTONIN-N-ACETYLTRANSFERASE (NAT) IN BOVINE PINFAI.EXPLANTS IN CULTURE NAT in pmoles/mg proteinimin

Section

Treatment

One

None (Control) Norepineph~ne, 10 PM GABA. 20 mM + Norepineph~ne, 10 PM N-methylbicuculiine, 250 PM, GABA, 20 mM + Norepineph~ne, 10 PM

0.48 -r 0.05 1.98 i 0.12 O.Si i 0.03

/>“O.oOS

2.03 i_ 0.10

p
Control N-methylbicuculline,

0.82 t 0.04 0.80 ,c 0.03

Two

250 PM

The explants serving as control were incubated in medium 199 containing 2 mM glutamine, 0.1% BSA, streptomycin (100 &ml) and penicilline (100 units/ml) under an atmosphere of 92% Oz and 8% CO,. In Section One, the norepinephrine-treated group received this medium plus 10 PM norepinephrine. The GABA-treated group received this medium plus 20 FM of GABA, preincubated for 112 hr before the addition of norepinephrine. The bicuculline-treated group received this complete medium plus 250 FM of bicuculline, preincubated for 112hr, and 20 mM of GABA preincubated for an additional 112hr before receiving norepineph~ne (IO &Ml. Two hr after addition of norepineph~ne, the control and experimental pineal explants were frozen immediately and stored for determination of NAT according to a method discussed in METHOD. Each control or ex~~rnent~ culture contained 2-3 explants. The data, which are the average of at least 6 separate cultures, are presented as mean r SE. The lack of effects of N-methylbicuculline on NAT which was carried out in a separate group of experiments, is shown in Section Two.

inhibitory effect of GABA was blocked by N-methylbicuculline; which did not have any effect on basal NAT activity (Table 3). Encouraged by these results that the inhibitory effect of GABA may be mediated via GABA receptor sites, we exarnined the presence of sodium dependent and independent GABA binding sites in synaptic membrane. The binding of (3H)GABA to synaptic membrane was linear between 0.5 and 2 mg of membrane proteins per ml (Fig. 1). Therefore, all binding studies were performed within this linear range. Specific sodium-independent (3H)GABA binding site was saturable; whereas, the non-specific (3H)GABA binding was not (Fig. 2). The studies on the subcellular distribution revealed that the highest concentrations of Na-independent (3H)GABA binding sites were located in crude synaptic membrane (Table 4). Scatchard plot analysis indicated that the sodium independent binding had a KD value of 16.5 nM and receptor density of 121 2 3 fmoles/mg protein (Fig. 3). Analysis of the membrane-Lund radioactivity by thin layer chromato~phy confirmed that ail of the bound material was GABA and not its metabolites (data not shown). When the Hill plot was performed for the sodium independent binding site, the slope was linear with n= 1.13 suggesting that neither positive nor negative cooperactivity was prominent feature of the sodium-independent binding sites (Fig. 4). The cdculated K,, value from the Hill plot equaled 17.18 nM (Fig. 4) which was not significantly different from the K,, of 16.5 nM obtained from the Scatchard plot (Fig. 3). Studies with freshly prepared synaptic membrane showed NaCIdependent (Fig. 5) and saturable binding sites (Fig. 6). From Scatchard plot, the sodium dependent binding had a K,, value of 0.677 FM and receptor density of 4.47 t 0.42

pmoles/mg protein (Fig. 7). From Hill plot the slope was linear with n= 1.02, indicating neither positive nor negative cooperactivity was prominent feature of the sodiumdependent (3H)GABA binding site. The KI, value obtained from the Hill plot equaled to 0.616 PM which was not significantly different from 0.677 FM obtained from Scatchard plot (Fig. 8). N-methylbicucu~ine was shown to be a potent inhibitor of Na-independent binding sites (Table 5); whereas, @alanine, a known inhibitor of glial uptake [28] inhibited Na-dependent binding sites in pineal gland (Table 6). DISCUSSION

Evidence gathered mostly if not entirely in rats [21] indicates the serotonin N-acetyltransferase (NAT) is the rate limiting step for the synthesis of melatonin in pineal glands. The activity of this enzyme has been closely linked to a classical beta adrenergic receptor site, since it is stimulated by norepineph~ne and the norepinephrine-induced stimulation of NAT is reduced, bfocked or reversed by classical beta adrenergic blocking agents such as propranoIo1 1211. By using bovine pineal gland, we have demonstrated that similar to rat, the activity of NAT is stimulated by norepinephrine and the norepinephrine-induced stimulation of NAT is blocked by propranolol [ 121. However, the factors controlling the biology of rat and bovine pineal glands are not identical. For example, in rats, norepinephrine stimulated NAT activity 30-50 fold and the maximum stimulation is seen 4 hr after the addition of norepinephrine. In bovine pineal plands, norepinephrine stimulates NAT activity modestly and between 2-5 fold, and the maximum stimulation is seen 2 hr after the addition of norepinephrine. In bovine pineal glands,

PINEAL

183

GLAND AND GABA

between specific Na-independent FIG. 2. The relationship (RH)GABA binding and non-specific GABA binding. Binding assay contained synaptic membrane (0.7 mg protein/ml) in 50 mM Triscitrate buffer (pH 7.1), 25-60 nM (3H)GABA alone or in the presence of 1 mM GABA in the total volume of 1 ml as described in the section on Method. Specific (3H)GABA binding was obtained by subtracting non-specific binding from total binding. The data represent mean 2 SE of at least three determinations.

mg of

membrane FIGURE

Protein/ml 1

FIG. 1. The relationship between various concentrations of synaptic membrane proteins and the degree of specific (3H)GABA binding. Binding assay contained synaptic membranes (0.5-2.0 mg per ml) in 50 mM T&-citrate buffer (pH 7.1), 60 nM (“H)GABA alone or in the presence of 1 mM GABA in the total assay volume of 1 ml, as described in the section on Method. Data represent mean 2 SE for at least three determinations.

the norepinephrine-stimulated NAT activity is blocked by GABA in a dose related fashion [7]; whereas, this does not occur in rat. In bovine pineal gland, the activity of GABA transaminase is considerably higher than GAD; whereas, in rat the reverse is the case [27]. It has also been shown that the NAT activity can be inhibited by its end-product melatonin [20]; whereas, melatonin has no effect on NAT activity in rat pineal gland [23]. These observations suggest that in bovine pineal gland the synthesis of melatonin may be more complex and under the influence of both adrenergic and GABAergic neurons. Anatomically, in addition to adrenergic fibers derived from the superior cervical ganglion [2], the bovine pineal gland is innervated by a bundle of nerve fibers derived from the central structures through the pineal stalk [l]. These nerve fibers pass deep into pineal gland, in which their myelin sheaths terminate in a heminode ranvier, and the naked axon continues among the pinealocytes and glial cells [I]. These nerve terminals contain clear vesicles [I], whose compositions are not known and may contain GABA. In addition to the above cited factors, the biological variation between the rat, a nocturnal mammal and a cow, a diurnal one, should not be overlooked. The association among pineal gland, NAT and biological clock has been documented. Binkley in her review article [4] entitled A Timekeeping Enzyme in the Pineal Gland makes

references to NAT and states: “At present I view the vertebrate mechanism for generating circadian rhythms as a hierarchy of structures capable of oscillation. 1 believe these structures are organized so that one of them acts as a pacemaker, keeping all of them synchronized. Future should disclose exactly how they interact. I suspect that these interactions differ from species to species, depending on the various ways the species have adapted to their environment.” Several isolated observations gathered in other laboratories are indicative of a definite function for GABA in mammalian pineal glands. Firstly, GABA and its metabolizing enzymes have been shown to exist in mammalian pineal glands including human [27,301. In rats, the concentration of GABA in pineal gland rises to a peak level at 3:00 p.m. and declines thereafter [31]. Also, there exists an inverse relationship between the concentrations of GABA and the activity of NAT. Furthermore, superior cervical ganglionectomized rats take up and accumulate less (“H)GABA. In addition, the GAD activity in these animals is considerably lower [23]. These observations, however impressive, precluded the assignment of a neurotransmitter role to GABA in rat pineal gland. One criterion for assigning a transmitter function to any putative molecule is the presence of a specific postsynaptic receptor molecule to which the transmitter could bind with high affinity. Naturally, the rat’s pineal gland weighing only 1 mg did not allow searching for such receptor sites for GABA. Bovine pineal glands, weighing 150-220 mg each, encouraged us to examine this parameter. Our observations lead us to believe that the inhibition by GABA of norepinephrine-stimulated NAT activity in bovine pineal gland may be mediated through a receptor-GABA interaction. We base our conclusions upon the following observations. (1) GABA did not interfere with NAT assay nor did it influence the basal NAT activity. (2) The inhibitory effect of GABA on norepinephrineinduced NAT activity was blocked by N-methylbicuculline. (3) The inhibitory effects of GABA on norepinephrinestimulated NAT activity was not seen in chloride free medium. (4) The inhibitory dose of GABA (20 PM) on norepinephrine-stimulated NAT activity corresponds with and is in keeping with the inhibitory doses of GABA observed under

EBADI TABLE SUBCELLULAR

4

DISTRIBUTION OF SODIUM-INDEPENDENT IN BOVINE PINEAL GLAND

CWGABA

Specific PHIGABA

Fraction Whole Crude Crude Crude

homogenate nuclear pellet (P,) mitochondrial pellet (P,) microsomal pellet (P:J

Osmotically shocked P, subfractions Mitochond~al-myelin pellet Crude synaptic membrane pellet

AND CHAN

Binding (fmoleimg protein) 32.81 19.39 64.81 9.76

-c 3.01 I 4.20 ” 4.35 rt 0.08

33.32 -r 2.10 74.18 Z!Z 4.24

BINDING

SITES

binding ‘Total binding (fmoleifractionl

6898 244s 3913 32.83

t 630 1 528 + 240 z+ 0.28

425 3204

i- 27 + 183

The subcellular fractions were prepared according to the method of Gray and Whittaker [16] and were frozen at -20°C for 18 hr. The frozen pellets were thawed and washed with 50 mM ice-cold Tris-citrate buffer (pH 7.1) prior to GABA binding assay which was carried out according to the method of Enna and Snyder [13]. In general, aliquots of synaptic membrane suspensions (0.7-l .2 mg of protein per ml) were incubated in triplicate for 5 min at 4°C in I ml of 50 mM Tris-citrate buffer, pH 7.1 in test tubes containing 60 nM (3H)GABA alone or in the presence of 1 mM GABA. The reactions were stopped by vacuum filtration. Specitic pH)GABA binding was determined by subtracting the amounts of rH)GABA in the pellet in the presence of 1 mM unlabelled GABA from the total amount bound in the absence of unlabelled GABA. Total binding represents the amounts of specific binding multiplied by the total amounts of protein. Data represent mean it SE of five determinations.

FIG. 3. Scatchard plot depicting the K,, value and the receptor density for sodium dependent (“H)GABA binding sites in bovine pineal glands.

FIG. 4. Hill plot depicting the K,) and n values for sodium independent (aH)GABA binding sites in bovine pineal glands. The total number af binding sites obtained from Scatchard plot has been used in graphing the Hill plot.

PINEAL GLAND AND GABA

I

N&l FIGURE

(Ml 5

FIG. 6. The relationship between specific Na dependent (3H)GABA binding and non-specific GABA binding. Binding assay contained synaptic membrane (0.5 mg protein/ml) in 50 mM Tris-citrate buffer (pH 7.1) NaCl (0.15 M), and 25-860 nM PH)GABA alone or in the presence of 1 mM GABA in the total volume of 1 ml as described in the section on Teflon. Specific (3H)GABA binding was obtained by subtracting non-specific binding from total binding. The data represent mean i SE of at least three determinations.

FIG. 5. The relationship between the concentration of NaCl and the degree of specific sodium dependent (3H)GABA binding in freshly prepared synaptic membrane from bovine pineal glands. Binding assay contained aliquots of synaptic membrane (0.4 mg protei~ml), NaCl ranging from 0.023-0.3 M and 25 nM (“H)GABA alone or in the presence of 1 mM GABA. Data represent mean lr SE of at least three determinations.

5.0

4.0

RD

q

0.677pM

KG= 0616~1tM

Brnax = 4.47 pmoles/mg protein

n-102

3.0

2.0

1.0

BOUND/FREE (fmole/nM) FIGURE 7

FIG. 7. Scatchard plot depicting the Kn value and the receptor density for sodium independent (3H)GABA binding sites in bovine pineal gland.

~oc,~ (th-GABA] FIGURE

x 109) 8

FIG. 8. Hi11plot depicting the K,, and n values for sodium dependent (“HI GABA binding sites in bovine pineal glands. The total number of binding sites obtained from Scatchard plot has been used in graphing the Hill plot.

IX6

EBADI

TABLE 5 THE INHIBITION

OF SODIUM-INDEPENDENT BY N-METHYLBICUCULLINE

BINDING

Potency index

Agents tested GABA Imidazole-4-acetic acid N-methylbicuculline p-alanine Chloropromazine 2,4-DABA

TABLE (‘H)GABA

0.0165 0.0140 0.130 14.0 29.0 )100.0

I 0.85 8.33 897.40 1757.60 16000.00

The frozen pellets containing synaptic membranes were thawed and washed with 50 mM ice-cold Tris-citrate buffer (pH 7.1) prior to

GABA binding assay which was carried out according to the method of Enna and Snyder [ 131.In general, aliquots of synaptic membrane suspensions (0.7 mg of protein per ml) were incubated in triplicate for 5 min at 4°C in 1 ml of 50 mM Tris-citrate buffer (pH 7. I) in test tubes containing five concentrations of the agents listed, 35 nM of (?H)GABA alone or in the presence of 1 mM GABA. The reactions were terminated by filtration. I&,,, which depicts the concentration of agents which inhibited GABA binding by 50%, was calculated by plotting the degree of specific binding versus the logarithm of various concentrations of difFerent agents. Potency index has been obtained by dividing the IC,,, of the agents listed by the IQ,, of GABA for at least three determinations.

in vivo

situation. For example, the concentration of GABA necessary for half-maximal depolarization of rat superior cervical ganglion was reported to be 12.5 /IM [6]. Similarly, GABA at 1 PM concentration inhibits the spontaneous firing of Purkinje cells in explants of rat cerebellum [ 141. Although biochemical evidence for two classes of GABA receptors have been reported [ 171, the limitation of the existing techniques and the low receptor density in pineal gland precluded detection of any low affinity GABA binding sites. Our studies indicate that the bovine pineal gland is endowed with sodium independent and sodium dependent binding sites. The sodium independent binding site had a KD value of 16.5 nM and receptor density of 121 ? 3 fmoles per mg protein. From displacement studies, one may conclude that imidazole-4-acetic acid and N-methylbicuculline had high affinity for these binding sites which we believe may represent the postsynaptic GABA receptors. The sodium-

AND CHAN

6

THE INHIBITION OF SODIUM-DEPENDENT (%ALANINE

V’H)GABA BINDING BY -Potency

Compound

IC-,,,(FM)

index

GABA Imidazole-4-acetic acid Chloropromazine L,-2,4-DABA p-alanine N-methylbicuculline

0.67 65 II 650 23.5 .a 1000.0

1.o 97.0 16.0 970.1 35. I 3 1500.0

The sodium-dependent binding was carried out according to the method of Enna and Snyder (131. In general, aliquots of fresh synaptic membranes (1.7 mg protein/ml) were incubated in triplicate at 4°C for 5 min in 1 ml of 50 mM T&citrate buffer (pH 7.1) in polypropylene minivials (Fisher Scientific Co., St. Louis, MO) containing 0.15 M NaCI, five concentrations of the agents listed, 35 nM of (JH)GABA alone or in the presence of 1 mM GABA. The reactions were terminated by ffitration. IC,,,, which depicts the concentration of agents which inhibited GABA binding by 5%. was calculated by plotting the degree of specific binding versus the logarithm of various concentrations of different agents. The potency index has been obtained by dividing the IC,,, of the agents listed by the IC,,, of GABA for at least three determinations.

dependent binding site had a K, value of 0.677 /.LMand receptor density of 4.47 r 0.42 pmoleslmg protein. From displacement studies, N-methylbicuculline had a very low affinity for such binding sites and /3-alanine depicted 28 times higher affinity than DABA. These sites may indeed correspond to glial uptake for GABA as previous autoradiographic studies had indicated [27]. Neither sodium independent nor dependent binding sites exhibited positive nor negative cooperactivity. The precise interrelationship between GABAergic and adrenergic neurons in mediating and perhaps in controlling the biological events in pineal gland is not clear and awaits study and reexamination. ACKNOWLEDGEMENTS

The authors express their gratitude to the expert assistance Mrs. Margaret McCall in preparing this manuscript.

of

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