TITLE: Enhancement of Brain Membrane Chloride Uptake

by the Major Psychoactive Factor in Marijuana. 1991


AUTHORS: Bruce E. Morton and Derek T. Shirae


INSTITUTION: Department of Biochemistry and Biophysics, University of Hawaii School

of Medicine, Honolulu, HI 96822, U.S.A. (808) bemorton@hawaii.edu



In humans, (-)-Delta-9-tetrahydrocannabinol (THC), the major psychoactive component of marijuana, causes many effects similar to those produced by benzodiazepines, barbiturates, and alcohol, each of which act on the chloride channel regulated by the gamma-aminobutyric acid (GABA) type A receptor. Here, using rat brain synaptoneurosomes, we have found that THC significantly increased basal and GABA-mediated chloride-36 ion uptake in a manner which could be blocked by picrotoxin or bicuculline. This provides evidence of significant GABA-A receptor-complex involvement in cannabis intoxication.


Key Words: Cannabis savita; Cannabinoids; GABA A receptor;

Chloride channel complex; Chloride uptake; (Rat)



(-)-Delta-9-tetrahydrocannabinol (THC) is the major psychoactive compound present in marijuana (Cannabis savita) (Pradham, 1984; Razdan, 1986). In human subjects, THC alters anxiety, muscle tension, thermoregulation, memory, the sense of time, nausea, pain, and a number of other functions (Bachman et al., 1979; Hollister, 1986). The prevalent view was that THC, a very hydrophobic molecule, produced its CNS functional alterations by changing general fluidity and permeability characteristics of neuronal membranes (Hillard et al., 1985; Martin, 1986). Although reasonable, this view could not explain why compounds with similar lipid solubilities as THC had little effect on the CNS (Kriwacki and Makriyannis, 1989; Razdan, 1986), while such drugs benzodiazepines (BZ), barbiturates, and muscimol, affecting specific receptors produced many of the behavioral changes caused by THC.

In the 1980s researchers began to find evidence for some type of receptor-mediated

mechanism for THC (Pertwee and Greentree, 1988; Sethi et al., 1986; Watanabe et al., 1987; Revuelta et al., 1979). Now, the recent discovery of a "cannabinoid" receptor for THC (Devane et al., 1988; Herkenham et al., 1990) has made a receptor-mediated mechanism for THC seem very likely. This cannabinoid receptor appears to act via a Gi protein transducer to inhibit adenylate cyclase (Howlett and Fleming, 1984; Howlett et al., 1986; Howlett et al., 1988).

Many of the behavioral effects of THC are similar to those produced by benzodiazepines, barbiturates, muscimol, and alcohol. Namely, each of these has antianxiety, euphorogenic, muscle relaxant, sedative-hypnotic, analgetic, cardiovascular, thermoregulatory, and memory altering properties. It is therefore relevant that all of the above compound classes act on the chloride channel regulated by the gamma aminobutyric acid (GABA) type A receptor complex to increase inhibitory chloride influx, and also interact synergistically with THC in the production of certain behaviors (Olsen, 1982; Harris and Allan, 1989; Sanna et al., 1990; Pertwee et al., 1988).

However, we and others have observed that THC does not compete directly for the GABA, the BZ, or the barbiturate-picrotoxin-hypnotic steroid receptors of this complex (Koe et al., 1985; Majewska et al., 1986; Lawrence et al., 1985). For example, we found that 1 mM (-)-delta-9-THC displaced neither [3H]muscimol, [3H]flunitrazepam, nor [3H]t-butylbicycloorthobenzoate (TBOB) (B.E. Morton et al., unpublished). This suggests either that additional unrecognized receptors exist as part of the GABA-A receptor complex, or that some form of cascade coupling exists between the cannabinoid receptor and the chloride channel. Thus, it was of interest that the regional distribution of the newly discovered cannabinoid receptor was remarkably similar to that of the GABA-A receptor complex (Herkenham et al., 1991a; de Blas et al., 1988; Herkenham et al., 1991b).

Here, we report that THC causes an increase in GABA-muscimol mediated 36-chloride uptake into brain synaptoneurosomes. The effects of THC to increase chloride uptake were blocked by the GABA-A chloride channel antagonists, bicuculline and picrotoxin. These results suggest that THC in some way increases chloride influx-mediated

inhibitory hyperpolarization.



In brief, synaptoneurosomes were prepared from whole rat brain, and the effects of several cannabinoid compounds and other drugs upon synaptosomal 36-chloride (36Cl) uptake was assayed using slightly modified versions of standard test tube and filtration procedures (Harris and Allan, 1985; McQuilkin and Harris, 1990).

Preparation of Synaptoneurosomes (SNs): The method of Harris (Harris and Allan, 1985; McQuilkin and Harris, 1990) was adapted for use with rats. Fresh (<1 hr) rat brain was washed with buffer and homogenized in 5 ml assay buffer with 10 up and down strokes of a glass-teflon homogenizer. The resulting crude homogenate was placed in a 15 ml polystyrene tube and centrifuged for 15 minutes at 1000 x g (SS34 rotor). The supernatant fraction was discarded and the pellet was gently resuspended in 10 ml assay buffer with a glass-teflon homogenizer. Assay buffer was 145 mM NaCl, 5 mM KCl, 1 mM MgCl2, 10 mM D-glucose, 1 mM CaCl2, and 10 mM HEPES, adjusted to pH 7.4 with Tris base. The suspension was placed in a 15 ml polystyrene tube and vortexed, followed by centrifugation (15 minutes, 1000 x g). The pellet was resuspended with a glass-teflon homogenizer in enough buffer to create 10 ml total volume. 100 ul of this was assayed for protein content. After assaying the amount of protein present, enough buffer was added to make a final SNs preparation of 2.5 mg/ml protein. Properties of similar osmotically sensitive vesicular preparations from guinea pig brain have described (Hollingsworth et al. 1985).

Synaptoneurosome Preincubation: To each of one set of tubes, 300 ul SNs were added. Another set of tubes contained 36-Cl and various drugs. All tubes were preincubated in a 34 degree C water bath for 5 minutes, and then incubated in the same bath as below. An alternative approach was that THC was sometimes added to SNs just before placement in the water bath. This was done in order to see the effect of THC preincubation on chloride channel function.

The hydrophobicity of THC prevents its easy solubilization in aqueous media (Garrett and Hunt, 1974). Use of PVP-40 promoted dispersal of THC, creating an opaque emulsion which could be used in the examination of THC's effect on chloride influx in synaptoneurosomes. To avoid the effects of rapid cannabinoid decomposition, fresh THC solutions were utilized. With these precautions, we were able to observe the effect of THC on chloride influx. THC, prepared in a colloidal suspension with 3%, w/v, polyvinylpyrrolidone-40 (PVP40), was active in our hands over the concentration range of 100-1000 uM.

Synaptoneurosome Incubation-Separation Methods: The following two methods were used interchangeably: Test tube method: A tube with the 36-Cl/drug and a tube with synaptoneurosomes (SNs) were removed from the 34 degrees C water bath. The tube with SNs was vortexed and 200 ul of the SNs was removed. The tube with the 36-Cl/drug was then vortexed, and while vortexing, the 200 ul of the SNs was rapidly delivered into the drug tube. After 3 seconds, over which time chloride uptake has been shown to be linear (Harris and Allan, 1985), 4 ml of ice cold 100 uM picrotoxin/buffer was rapidly added into the drug tube, to quench the influx. Tube contents were poured rapidly into a presoaked GB100R filter (Fisher Scientific) on a Hoefer FH224V filtration unit (manifold on) at a

vacuum of 15 inches of water. After initial filtration, 2 consecutive washes, lasting about 1

second each, of 4 ml 100 uM picrotoxin/buffer were added to the filter (manifold off).

Filter Method: 1 ml of preincubated SNs (0.5 mg protein/ml) were poured on a filter with the manifold valve of the filtration unit opened and manifold tower removed. The valve was closed and 1 ml of 36-Cl/drug added to the filter. After 3 seconds, the manifold valve was reopened and 3 ml assay buffer containing ice cold 100 uM picrotoxin was poured upon the filter, which was then washed with 8 ml ice-cold assay buffer containing 100 uM picrotoxin.

Scintillation Counting: After washing, filters were placed in glass scintillation vials. 5 ml of ScintiVerse scintillation fluid (Fisher Scientific) was added to each vial. Vial radioactivity was then measured with a scintillation counter. All methods used here sought to be identical or equivalent to those developed in R. A. Harris' laboratory (Harris and Allan, 1985; McQuilkin and Harris, 1990).

Statistical Treatment of Data: Significant 36-Cl uptake differences between control synaptosomal samples and drug treated samples were confirmed by computer assisted analysis of variance.



Using the test tube 36-Cl assay, 200 uM THC enhanced 36-Cl uptake over that of the

reagent blank by an average of 20% (Figure 1). When the filter assay was used, 200 uM THC increased the uptake an average of 42% above control (Figure 1). Analysis of variance (ANOVA) confirmed both of these increases to be highly significant (p=<0.05).

(600 uM), or 100 uM of the GABA-A antagonist, bicuculline methiodide, reduced both GABA-muscimol and THC induced chloride uptake increases to basal levels,

confirming that THC was acting to increase chloride flux through the GABA-A mediated chloride channel complex (Figure 2). These results were also highly significant (p=<0.05).

When the effects of GABA on basal 36-Cl uptake were examined, half maximal stimulation was found at 15 uM GABA with a maximum being reached by 100 uM at which point uptake was at least doubled over that occurring in the absence of exogenous GABA (Figure 3). In the presence of 1000 uM THC, there was a leftward shift of GABA stimulation so that uptake was enhanced by at least 20% for both 3 (p=<0.05) and 10 uM GABA (p=<0.05), falling off at 30 uM GABA (p=<0.05), so that at the 100 uM maxima, no significant increase was observed (Figure 3). Similar results were observed repeatedly with muscimol (data not shown).



The elevation of basal chloride uptake in synaptoneurosomes by THC was highly

reproducible. Cannabinol or cannabidiol (1 mM) were without effect, while 1 mM (+)-THC was somewhat active, as had been reported in the case of the cloned cannabinoid receptor (Matsuda et al., 1990). Since the THC-induced elevation in 36-chloride uptake was blocked by picrotoxin, it demonstrates that the flux of chloride through a chloride channel was affected by THC (Lawrence et al., 1985; Lawrence and Cassida, 1983). Since bicuculline also blocked the THC-induced increase in chloride uptake, this indicates that the GABA-A receptor complex is involved (Mohler and Okada, 1978; Olsen and Snowman, 1983). Although the Ki of THC at the cannabinoid receptor is about 0.5 uM (Devane et al., 1988), THC was not active in less than micromolar concentrations here. Difficulties in solubilizing it, preventing its degradation, and avoiding its adsorption to containers make it probable that the absolute THC concentration present in our assays was significantly lower than calculated.

The increase in 36-chloride uptake by THC, suggests three mechanistic interpretations: 1. THC mobilizes some of the large endogenous GABA pool to increase chloride influx indirectly. 2. THC binds to a presently unidentified allosteric activator site on the GABA-regulated chloride channel to cause an increase in GABA affinity, as occurs in the case of benzodiazepine and barbiturate-steriod binding to this complex. 3. THC binds to the cannabinoid receptor, thereby inhibiting cyclic AMP synthesis by adenylate synthetase via a Gi transducer (Howlett, Qualy, and Katachatrian, 1986). Lowered levels of cyclic AMP then reduce protein kinase A activity to phosphorylate, the chloride channel of the GABA-A complex. In the absence of phosphorylation which narrows this channel, chloride influx is enhance (Browning et al., 1990; Porter et al., 1990). Although, each of these mechanisms would result in chloride uptake, hyperpolarization, and neuronal inhibition to produce the GABA-A-like intoxication symptoms of THC, the mechanism involving the cannabinoid receptor is at present most attractive.

As this work was nearing completion, an abstract appeared which described an increase of whole-cell ion current in cultured rat hippocampal neurons that was produced by THC which was mimicked by GABA addition (Hampson et al. 1990). A preliminary report of our own work has also appeared (Shirae and Morton, 1991).



We thank R. A. Harris for assistance in setting up the synaptoneurosome chloride uptake assay, The Department of Biochemistry and Biophysics for some supplies and a graduate stipend (to D.T.S.), and W. K. Ogata for technical aid on statistics and graphics.



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FIGURE 1 The effect of (-)-delta-9-THC on basal 36-chloride uptake into rat whole brain synaptoneurosomes as measured by test tube and filter assay methods.

Legend: The first two columns utilized the test tube assay: column 1 (n=14) was the control; column 2 (n=15) contained 200 uM THC. The last two columns used the filter assay: column 3 (n=15) was the control; column 4 (n=15) contained 200 uM THC. The THC dispersant, 3% PVP-40, but no GABA, was present in all tubes. (*) indicates data where p=<0.05.


FIGURE 2. Separate effects of picrotoxin and bicuculline on THC-stimulated 36-chloride influx into rat whole brain synaptoneurosomes.

Legend: The first and last three columns (black) utilized test tube assays: column 1 (n=9) contained 200 uM THC; column 2 (n=8), 240 uM picrotoxin; column 3 (n=8), 200 uM THC and 240 uM picrotoxin. The middle three columns (hatched) were from filter assays: column 4 (n=5) contained 100 uM THC; column 5 (n=4), 600 uM picrotoxin; column 6 (n=5), 100 uM THC and 600 uM picrotoxin. Continuing with test tube assays, column 7 (n=7) contained 1000 uM THC; column 8 (n=6), 100 uM bicuculline methiodide; column 9 (n=4), 1000 uM THC and 100 uM bicuculline methiodide. *p=<0.05. Assays were conducted as described in Figure 1. PVP-40 (3%), but no GABA, was present in all tubes.


FIGURE 3. The effect of (-)-delta 9-THC on GABA-stimulated 36-chloride influx into rat whole brain synaptoneurosomes.

Legend: Circular data points depict the effects of GABA alone. Triangular data points show the effects of GABA in the presence of 1000 uM THC. *p=<0.05. 3%PVP-40 was present in all tubes. Other conditions were as described in the test tube assay in the Figure 1 legend.