Prcg. Neu?-c#sychopharmacol. &Bid psychint. 1999, Vol. 23, pp. 1063-1077
Copyright 0 1999 Elsevier Science Inc.
Printed in the USA. All rights reserved
0278-5846/99/$+x front matter
mPREBsIOR OF CABBABIBOID BEcxPToRs A?m
THEIR GENE TRABSCBIPTS IB HUMAB BLOOD CELLS
EMMANUEL S. 0NAIV11~2, GAUTAM CHAUDHURi4, ASLI S.
ABAC15, MONICA PARKER3, DONALD H. MANIER’, PETER
R. MARTIN112 AND JOHN R. HUBBARD115
Department of ‘Psychiatry, 2Pharmacology and 3Cell Biology,
Vanderbilt University School of Medicine, Nashville, TN, USA
4Department of Microbiology, Meharry Medical College,
Nashville, TN, USA
‘Department of Veterans Affairs Medical Center, Nashville,
(Final form, July 1999)
Onaivi, Emmanuel S., Gautam Chaudhuri, Ask S. Abaci, Monica Parker, Donald H. Manier,
Peter R. Martin and John R. Hubbard: Expression of cannabinoid receptors and their gene
transcripts in human blood cells. Prog. Neuro-Psychopharmacol. & Biol. Psychiat.
19%,2& pp. 1063-1077.81999 Elswier Science Inc.
1. This study shows that the human cannabinoid receptors and their gene transcripts can be
analyzed in blood samples when combined with polymerase chain reaction. The results
also demonstrate that the expression of the cannabinoid receptors is dependent on
gender and ethnic background. 1063
1064 E.S. Onaivi ei al.
2. Normal human volunteers who do not use marijuana have genes that encode for the
marijuana (cannabinoid) receptor proteins.
3. Primer pairs from CBI and CB2 cDNA coding region sequences showed identical
amplified DNA band sizes in both DNA-PCR and reverse PCR, with human templates.
This suggests that the CBI and CB2 genes are intronless at least in their coding regions.
4. An advantage of the coding region being intronless may be that the expression of these
genes will have one major RNA processing event to skip, thus making the conditions of
their expression relatively quick and simple. This advantage may have implications
related to the biological functions of these proteins.
5. We therefore concluded that the existence of human cannabinoid receptors and genes
along with the discovery of endogenous cannabinoids (endocannabinoids) may be useful
markers in elucidating the role(s) and mechanism(s) of action of cannabinoids.
m: cannabinoid receptors, cannabinoid receptor genes, gender differences, genetic
variation, human blood, marijuana, polymerase chain reaction,
Abbrewatlons: acquired immune deficiency syndrome (AIDS), cannabinoid receptor subtype
1 (CBI), cannabinoid receptor subtype 2 (CB2), deoxyribonucleic acid (DNA),
electroconvulsive therapy (ECT), institutional review board (IRB), natural killer cells (NK),
polymerase chain reaction (per), polymorphonuclear neutrophils (PMN), Ribonucleic acid,
(RNA), urine drug screen (UDS).
Cannabis (including marijuana) is one of the most widely used drugs in the world.
Cannabinoids are constituents in the Cannabis sativa of which A’-tetrahydrocannabinol (A’-
THC) is the major psychoactive component. Endogenous cannabinoid ligands
(endocannabinoids) have been discovered in the human nervous system. The discovery of
endocannabinoids (Devane, 1992), and the genes encoding the cannabinoid receptors
(Matsuda et al., 1990, Munro et al., 1993 and Chakrabarti et al., 1995) suggest that the
cannabinoid system represents a previously unrecognized elaborate network in the human
peripheral and central nervous systems whose biological role is not well understood.
Although the physiologi& role or cannabinoids is largely unknown, a high conservation
between the genes encoding the human, rat, mouse, fish and bovine cannabinoid receptors
suggests an important biological function. As many of the actions of cannabinoids are
known to be receptor or non-receptor nrediated, understanding the neurochemistry,
regulation and molecular basis of cannabinoid receptor activities is of significant clinical and
scientific interest. A recent report demonstrated robust acute effects of marijuana on
subjective and physiological measures, including smooth pursuit eye tracking performance
which returned to baseline levels after 3.5 h. (Fant et al 1998). Such acute effects were not
Cannabinoid receptors and gene expression in human blood cells 1065
present the day following the smoking, indicating that the residual effects of smoking a
single marijuana cigarette are minimal (Fant et al 1998).
There is now a considerable body of evidence to suggest that cannabinoids may have a
number of medicinal uses: for the relief of pain in paraplegia, neuralgia and spasticity in
multiple sclerosis; as an appetite stimulant in treating AIDS patients with wasting disease,
cachexia; for the prevention of nausea and vomiting associated with cancer chemotherapy;
in glaucoma: as immunomodulators for autoimmune diseases, inflammation, allergies and
nephritis. In addition, the discovery of transporters and enzymes involved in the synthesis
and degradation of endocannabinoids and the cardiovascular effects of cannabinoids may
provide new therapeutic drug targets. The rapid progress in cannabinoid research and the
development of new research tools which has now transformed cannabis research into
mainstream science may contribute to understanding the mechanism(s) of action of
cannabinoids which has been slow to emerge.
Marijuana is frequently used for its effects of euphoria, relaxation and sensory changes.
The precise reason(s) for use varies with each individual (Hubbard et al., 1993). Previous
studies by us and others indicate that the expression of genes encoding cannabinoid
receptors can be evaluated in blood and brain samples in animals and humans (Bouaboula
et al., 1993 and Onaivi et al., 1996a). In human leukocytes the expression of cannabinoid
receptor mRNA has been reported to be lower than that found in brain tissue. The
message has, however, been detected in all subsets of leukocytes examined. The
message levels are greatest in S cells, followed sequentially by natural killer cells (NK),
polymorphonuclear neutrophils (PMN), T8 cells, monocytes and T4 cells. The objective of
this study was to evaluate the measurement of cannabinoid receptors in human, and to
determine the level of expression of cannabinoid receptor genes in blood samples of non-
marijuana using subjects for subsequent comparison to marijuana using subjects. We also
evaluated whether or not gender and ethnic background plays a role in the expression of
cannabinoid receptors and their transcripts in the human blood cells.
1066 E.S. Onaivi et al.
This report presents findings in white, black and Asian normal human controls from the
Vanderbilt University community. This study was approved by Vanderbilt IRB and informed
consent was obtained from participants. Twenty three healthy male and female subjects
who did not meet DSM-IV criteria (American Psychiatric Association, 1994) for alcohol or
marijuana dependence participated in the study. Subjects were excluded from participation
for non-substance-related AXIS 1 psychiatric disorder(s) other than adjustment disorders or
dysthymia and for severe AXIS 11 disorder, such as severe borderline personality disorder.
and RNA Isol&ton from Blood SarrtpLes
Blood samples were obtained for cannabinoid receptor (CBI) protein and mRNA (CBI
and CB2) determinations in leukocytes from each human subject. Blood (6 ml) was
collected in heparinzed tubes for immunoblotting and in TRIZOL LS Reagent solution (10
ml) (GIBCO BRL) for RNA isolation for northern blotting. Human RNA was isolated from
leucocytes according to the methods used by Lin et al., 1993. For the protein isolation from
blood samples for immunoanalysis using CBI polyclonal antibody, 2 ml of the blood was
centrifuged at 5000 rpm at 4% for 5 min and the supematant discarded. The pellet was
resuspended in 400 ul of lysis buffer containing protease inhibitors and homogenized. After
rinsing with 200 PI lysis bufffer into a tube, the homogenate was centrifuged at 15000 rpm
at 4OC for 15 min. The supernatant was aliquoted and stored at -80°C for immunoblotting.
Samples were prepared for immunoblotting using 50 11 of the homogenate and 2X
Laemille’s buffer and boiled for 10 min. After cooling, the mixture was centrifuged at 15000
rpm, at 4°C for 15 min and the supernatant used for Western blotting.
. . .
Reaction (PCR) and Riot Hybnr&&n
Probes for human CBI and CB2 for the northern blot analysis were obtained and
prepared. Human CBI receptor cDNA (clone # HFBCF53, ATCC; Genebank/EMBL #
M77952) was used as a probe. The cDNA insert was excised from the pBluescript SK-
clone with EcoR I that yielded two fragments of 2.15 kb and 0.6 kb. These fragments were
Cannabinoid receptors and gene expression in human blood cells 1067
Oligodeoxyribonucleotide Primer Pairs Used For The Polymerase Chain Reaction
Primer Namea Positionb Nucleotide Sequences@‘->3’) Expected Size
Specific (bp) of the Amp
to cDNA lified Band”
CBI Fl 2344
CBI F4 1200-1222 GAGGAGCAAGGACCTGAGACATG
84 1438-1415 CCACMAAGCAGCAGCTCACAGAG 239
CB2 F5 5-28
CB2 F7 464482
CB2 F8 706-723
CB2 F5 5-28
“F = Forward primer; B = Reverse or backward primer. bThe ‘A’ of the protein initiation
codon, ATG, in the cDNA sequence is designated as 1. The length of the cDNA coding
regions of rat CBI and human CB2 are 1440 bp and 1134 bp, respectively. ‘The size of the
amplified band is given for a specific pair of forward and reverse primers.
1068 E.S. Onaivi et al.
purified from low melting point agarose gels with freeze-spin columns (Stratagene, La Jolla,
CA). The probe was radiolabeled with 32P using radom primer labeling method (Sambrook
et al., 1989) and was purified by Nick-spin columns (Pharmacia LKB Biotechnology,
Piscataway, NJ). Radiolabeled probe was denatured by boiling for IO min, followed by
rapid cooling in ice. The CB2 probe primer pair for PCR , Table 1, was designed from the
human CB2 gene sequence (Munro et al., 1993) using MacVector program (IBI-Kodak,
New Haven, CT). The forward primer sequence was 5’-
AGGAATGCTGGGTGACAGAGATAG-3’ and reverse or backward primer was 5’-
GGGAGTGAACTGATTTCTGACTTG-3’. The primer pair used for PCR with genomic DNA
(500 ng) as template for CBI was 5’-TTGCAGACACCACCTTCCGTAC-3’ as forward and
Y-CCACAAAAGCAGCAGCTCACAGAG-3’ as reverse primer. These oligonucleotide
primers were synthesized by the phosphoramidate method using the Applied Biosystem
Model 380A automated DNA synthesizer. The oligonucleotides were purified by
electrophoresis through a denaturing polyacrylamide gel, followed by Sep-Pak Cl8
(Waters, Millipore Corp., Milford, MA) reversed-phase column chromatography (Sambrook
et al., 1989). Human CB2 cDNA was amplified from total blood RNA (1 kg) using the above
forward and reverse primer pair into pCR-SCRIPT(+) plasmid (Stratagene, La Jolla, CA)
and the manufacturers kit reagents. The CB2 insert was cut out from purified recombinant
plasmid with Xba I/Barn H I and was used to probe the blots with amplified human CB2
RNA (20 pg) was separated by electrophoresis through a 1% (w/v) agarose/ 6.7% (v/v)
formaldehyde gel in MOPS buffer [20 mM MOPS, pH 7.0, 5 mM sodium acetate, 1 mM
EDTA] (Sambrook et al., 1989). Separated RNA molecules were transferred to
nitrocellulose membranes (Schleicher & Schuell, Keene, NH) with 20 x SSC for northern
hybridization. The blots were neutralized and air dried. Blots were prehybridized using
standard protocols (Sambrook et al., 1989). Hybridization of the prehybridized blots with
the denatured radiolabeled probes was done at 42°C for 20 h in fresh prehybridization
solution with IO6 cpm of probe per ml. Blots were washed four times, 30 min each, at 55°C
with SSC buffer and 0.1% sodium dodecyl sulphate. Washed blots were exposed to Kodak
X-OMAT AR-5 film at room temperature for 20 h (Sambrook et al., 1989). Amplified DNA
fragments from agarose gels were transferred to Nytran nylon membrane (Schleicher &
Schuell, Keene, NH) following the alkaline transfer method (Sambrook et al., 1989). The
conditions for the Southern hybridization were identical to that described above for Northern
Cannabinoid receptors and gene expression in human blood cells 1069
Equal amounts of protein were loaded and separated by 10% SDS-PAGE and then
transferred to nitrocellulose. The blots were blocked in freshly prepared PBS containing 3%
non fat milk for 20 minutes at 20-25°C with constant agitation. After incubation with a
rabbit-anti-human CBI antibody (Calbiochem, Cambridge, MA) for overnight at 4”C, a goat
anti-rabbit IgG linked to horseradish peroxidase (Amersham Life Science products,
Arlington HTs, IL) was added for additional 1.5 hr. Blots were developed using enhanced
chemiluminescence (Amersham Life Science products, Arlington HTs, IL). For quantitation,
films were scanned in Pharmacia LKB Ultroscan XL enhanced laser densitometer and the
results reported in relative units.
ston and Analysis of Human CBI and CB2 Gene
The ethidium bromide stained agarose gel (2%) and the corresponding autoradiogram of
the blot that was hybridized with human CBI cDNA as a probe is shown in Figs. IA and 1 B.
This shows the PCR amplification of DNA segments from total genomic DNA isolated from
human leucocytes using the forward and reverse primer pair designed from rat CBl cDNA
sequence. The single band corresponding to the expected size of the coding region of the
CBI cDNA is indicated. Figures 2A and 2B represent the ethidium bromide stained
agarose gel (2%) (A and B) and the corresponding autoradiogram (A and B’) of the blot
hybridized with human CB2 cDNA as probe. The PCR amplification of DNA segments from
human blood and its autoradiogram is shown by (A and A) while the RT-PCR
autoradiogram is shown by (B an 8’). The insets in A’ and B’ shows the 48 h over exposure
in lanes 3 and 4.
n CR1 Cannabinoid Receotor Protein
Figures 3 and 4 represent the representative immunoblots from the blood of individual
White, Black and Asian individual controls. The CBI antibody recognized a 58 kDa protein
from the individuals and some other currently unknown bands were visible from the blot.
The relative levels of the cannabinoid receptor proteins as determined from densitometric
analysis appear to vary with gender and race. The relative levels of the 58 kDa CBI protein
1070 E.S. Onaivi et al.
from the male volunteers were, 47.4%; 39.0% and 13.6% for the White, Black and Asian
blood samples respectively as shown in Fig. 3. The relative levels of the Cl31 protein in the
male and female volunteers were 49.6%, 32.2% and 18.2% for the white and black females
in comparison to the black male blood samples respectively as shown in Fig. 4. Therefore
in both males and females, the cannabinoid receptors appear to vary by gender and
ethnicity, for example Fig. 3 show white male > black male > Asian male and Fig. 4 show
white female > black female > black male. The mean arbitrary values were analyzed by the
students t test.
The emergence of novel research tools has accelerated cannabis research in the last
decade, more so than at any time in the thousands of years of marijuana use in human
history, Onaivi et al., 1996b and Matsuda, 1997 and Pertwee, 1997). Although, it is not yet
known why the cannabinoid system is so abundant in the nervous system, the analysis of
the receptor proteins and the genes encoding these cannabinoid receptors may shed some
light on the mode of action of cannabinoids, and the biological role of these genes in the
nervous system. In this study, we have analyzed both CBI and CB2 cannabinoid receptor
genes in normal humans who do not use marijuana. Furthermore, the authors compared
the expression of the cannabinoid receptor proteins in different human population according
to gender and ethnic background in Asians, blacks and whites.
s of Cannabrnord Receptor lCB1 and CB7) Gene Struw
While rapid progress is being made in cannabis research, there is presently little in
literature on cannabinoid receptor genetics and its regulation. The present study is
therefore unique as it extends the analysis of gene structure and function to genes
encoding cannabinoid receptors in humans. These preliminary results on human marijuana
receptor gene expression indicate that human blood can be used to determine the
cannabinoid receptor gene expression when combined with polymerase chain reaction
(PCR). In addition, data from the Southern hybridizations of the blots from gels with the
human CBI and CB2 cDNA probe indicate the expected transcript size for CBI and CB2
genes and two unexpected additional bands for the CB2 receptor genes. Whether these
additional bands represent CB2 subtypes in human cells or are mere PCR artifacts remains
to be determined. The authors tested the hypothesis that if the amplified DNA fragment
Cannabinoid receptors and gene expression in human blood cells 1071
Fig 1. Analysis and the structure of human CBI gene. Ethidium bromide-stained agarose
gel (2%) (A) and the corresponding autoradiogram (B) of the blot hybridized with human
CBl cDNA as probe, showing PCR amplification of DNA segments from total genomic
DNA isolated from human leukocytes using primer pair designed from rat CBI cDNA
sequence. M: DNA size standards containing a mixture of HIND Ill-digested h- DNA and
Hae Illdigested fX174 RF DNA. The arrow heads in the figure indicate the highest size
DNA fragment (1.4 kb) of the fX174 RF DNA. The bands that follow (downwards) are 1 .l
kb, 0.9 kb, 0.6 kb, 0.31 kb, 0.28 kb, 0.28 kb, 0.27 kb, 0.23 kb, 0.19 kb, 0.12 kb and 0.07 kb
in size, respectively.
1072 E.S. Onaivi et ai.
Fig 2. Analysis and the structure of human CB2 gene. Ethidium bromide-stained agarose
gel (2%) (A and B) and corresponding autoradiogram (A’ and B’) of the blot hybridized with
human CB2 cDNA as probe, showing PCR amplification of DNA segments from human
blood DNA (A, A) and RNA (6, B’). The insets in A’ and B’ show the over exposed (48 h)
lanes 3 and 4. M: DNA size standards containing a mixture of Hind Ill-digested h -DNA
and Hae- Ill-digested fX174 RF DNA. The arrow heads in the figure A and B indicate the
highest size DNA fragment (1.4 kb) of the fX174 RF DNA. The bands that follow
(downwards)are 1.1 kb, 0.9 kb, 0.6 kb, 0.31 kb, 0.28 kb, 0.27 kb, 0.23 kb, 0.19 kb, 0.12 kb
and 0.07 kb in size, respectively. Lane 1: Primer pairs F5/B5; Lane 2: Primer pairs F6/B6;
Lane 3: Primer pairs F7/B6; Lane 4: Primer pairs F8/B8; Lane 5: Primer pairs F5/B8.
Cannabinoid receptors and gene expression in human blood cells 1073
Fig 3. lmmunoblots from human blood. lmmunoblots following the transfer to a
nitrocellulose membrane, blocked with blotto and the CBI antibody and goat anti-rabbit
IgG-HRP conjugate. lmmunoblots were detected by chemiluminescence. The CBI anti-
body recognized the expected 58 kDa protein of cannabinoid receptor subtype 1 in the
human blood shown by the arrow. The relative levels of the CBl receptor appear to vary
with race as the densitometric analysis indicated in lane C = 47.4%; lane B = 39.0% and
lane A = 13.6%, in white, black and Asian blood samples respectively.
Fig 4. lmmunoblots from human blood. lmmunoblots following the transfer to a
nitrocellulose membrane, blocked with blotto and the CBI antibody and goat anti-rabbit IgG-
HRP conjugate. lmmunoblots were detected by chemiluminescence. The CBI anti-body
recognized the expected 58 kDa protein of cannabinoid receptor subtype 1 in the human
blood shown by the arrow. The relative levels of the CBI receptor appear to vary with
gender as the densitometric analysis indicated in lane C = 49.6%; lane B = 32.2% and lane
A = 18.2% , in white female, black female and black male blood samples respectively.
1074 E.S. Onaivi et al.
sizes were identical with DNA and cDNA templates used for the PCR, then the genes are
intronless, otherwise, the intron location, size and structure can be determined. To this end
we used computer generated multiple primer pairs spanning the cDNA sequences of CBI
and CB2 genes to test whether DNA fragments amplified by these primer pairs were
identical with both the genomic DNA and cDNA template. The results suggest that the CBI
and CB2 genes are intronless at least in their coding regions. The data obtained with the
CBI is in agreement with those of Bouaboula et al., (1993). These workers using either
human genomic DNA or cDNA derived from brain RNA as template with the primers specific
for cannabinoid receptor gene showed single bands corresponding to the predicted length
for the cDNA, indicating that there is no intron in the amplified DNA segments.
In order to verify the intronlessness of the human CBI and CB2 genes, the authors
screened human genomic library. Five independent clones for CBI and two independent
clones for CB2 gene were isolated after screening of 300,000 plaques of the lambda EMBL-
3 library. All of those clones contained full length copy of the respective gene as was
revealed by restriction mapping, PCR and probing with 5’- or 3’- segments of the
corresponding cDNAs (data not shown). As expected, those clones contained intronless
genes. Cannabinoids are known to induce numerous behavioral alterations after smoking
marijuana or following THC administration (Onaivi et al., 1990, 1996b and Chakrabarti et al,
1998). This has led us to hypothesize that other subtypes of cannabinoid receptors exist in
the nervous system. Alternative splicing of the precursor mRNA is a possible mechanism
for generation of different subtypes of a receptor coded by multiple-exon gene (Green,
1991, Maniatis, 1991 and Rio, 1992). It is interesting to find that both of the presently
known subtypes of the cannabinoid receptors are coded by single-exon genes. There is, of
course, the possibility of these genes having intron(s) at the upstream or downstream non-
coding regions. We did not test this possibility but in other systems like the monoamines,
the dopamine Dl receptor gene is reported to have an intron at the 5’ non-coding region
(O’Dowd, 1993). The advantages or disadvantages of being intronless are subject to
speculation (Lambowitz and Belfcrt, 1993). One obvious advantage is that the expression
of these genes have one major RNA processing event to skip, thus making the conditions
of their expression relatively quick and simple. This advantage may have implications
related to the biological functions of these receptor proteins. Thus, the existence of genes
encoding cannabinoid receptors in human blood cells may also provide a molecular basis to
elucidate how cannabinoid compounds may mediate their immunologic, endocrine and
Cannabinoid receptors and gene expression in human blood cells
rmtors and Gene Fxpressron
The present finding that the expression of cannabinoid receptors in humans varies
according to gender and ethnic differences among whites, blacks and Asian population is a
significant first finding if confirmed in a larger sample size. The implication and
physiological relevance of this finding are only speculative and premature if unconfirmed.
However, this is not surprising as numerous studies have linked genetic determinants and
differences to the neurobehavioral responses of abused drugs in man and animals (Le et
al., 1994 and Harada et al., 1996 ). For example genetic differences in alcohol and
compulsive drug taking behavior has been demonstrated in animals and man (Le et al.,
1994 and Harada et al., 1996 ). Genetic variation in some receptor and enzyme systems
e.g. cholecystokinin and serotonin IA receptors and liver enzymes, alcohol and aldehyde
dehydrogenase, may be associated with alcohol dependence due to the modified function
in physiological and behavioral responses (Thomasson et al., 1991 and Harada et al.,
1996). Thus the implication and relevance of the differential expression of cannabinoid
receptors in humans according to ethnic background remains to be determined. If it turns
out that these levels are relevant to the psychoactivity, toxicity and perhaps therapeutic
efficacy then determination of the expression of cannabinoids in human blood may be used
to predict the outcome of their actions. As endogenous cannabinoid ligands have also
been discovered, they may play important roles in the regulation and activation of the
cannabinoid genes and receptors in vivo.
This study indicates that both the genes encoding marijuana (cannabinoid) receptors and
the resultant cannabinoid receptor proteins can be analyzed from human blood when
combined with PCR. The present results also showed that the expression of the
cannabinoid receptors varies according to ethnic background. These findings support the
existence of an elaborate human cannabinoid system that could be exploited
1076 ES. Onaivi et al.
This work was supported by grants from NHLBI KOI HL 03319 to ESO, DHM was
supported by NIMH grant MH 29228 (PI, Dr. Fridolin Suiser) and MH 52339 (PI, Dr.
Shelton), The authors also acknowledge the assistance of Dr. Barbara Fingleton for the
scanning of the immunoblots, MS Margaret Gilchrist, the clinical nurse specialist for
phelbotomy services, Dr. Amitabha Chakrabarti for technical support and the Vanderbilt
clinic for pharmacologic treatment of addiction.
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Inquires and reprint requests should be addressed to:
Dr. Emmanuel S. Onaivi
Department of Psychiatry (MCN-2125)
Nashville, TN 37232