Binding properties of dipropyltryptamine at the human 5-HT1a receptor

Article abstract of DOI:10.1159/000085649

Dipropyltryptamine (DPT) is a synthetic indolealkylamine first characterized in the 1960s. Largely forgotten since the discovery of multiple serotonin receptor subtypes, some of the properties of DPT at the cloned human 5-HT1a receptor are described here. When [³H]8-OH-DPAT is bound to the receptor, DPT inhibits the interaction with an IC₅₀ of 0.1 μmol/l. This interaction is shown to be competitive when double-reciprocal plots of the DPT/agonist interaction are analyzed. DPT's effects in the signal transduction system are complex. While DPT alone (0.1-1,000 μmol/l) activates G_i when both cAMP and γ-S-GTP incorporation are measured, in the presence of 5-HT (0.1-10 μmol/l), DPT blocks the agonist effect. In combination, the findings suggest that DPT is a moderate affinity partial agonist at the human 5-HT1a receptor. These results provide evidence that DPT has potential as a versatile experimental tool at 5-HT1a receptors. Copyright

Full text of DOI:10.1159/000085649

Original Paper
Received: December 3, 2004
Accepted after revision: March 14, 2005
Published online: May 9, 2005
Pharmacology 2005;74:193–199
DOI: 10.1159/000085649
Binding Properties of Dipropyltryptamine
at the Human 5-HT1a Receptor
Harish V. Thiagaraj Ethan B. Russo Andrea Burnett Eric Goldstein
Charles M. Thompson Keith K. Parker
Department of Biomedical and Pharmaceutical Sciences, and the COBRE Center for Structural and Functional
Neuroscience, School of Pharmacy and Allied Health Sciences, The University of Montana, Missoula, Mont., USA
Key Words
Introduction
Serotonin ꢀ Hallucinogens ꢀ cAMP ꢀ ꢀ-S-GTP
Relationship of N,N-Dipropyltryptamine (DPT) to
N,N-Dimethyltryptamine (DMT)
Abstract
DPT was first synthesized in the 1950s. Unlike its
Dipropyltryptamine (DPT) is a synthetic indolealkyl- chemical relative DMT, which occurs naturally in plants
amine first characterized in the 1960s. Largely forgotten (e.g., Psychotria spp., Phalaris spp.), current understand-
since the discovery of multiple serotonin receptor sub- ing indicates that DPT only occurs as a synthetic com-
types, some of the properties of DPT at the cloned human pound [1, 2]. Although DPT was studied briefly in the
5-HT1a receptor are described here. When [3H]8-OH- 1960–1970s, it has been largely forgotten since the iden-
DPAT is bound to the receptor, DPT inhibits the interac- tification of multiple serotonin (5-hydroxytryptamine; 5-
tion with an IC50 of 0.1 ꢁmol/l. This interaction is shown HT) receptor (R) subtypes [3, 4] following the molecular
to be competitive when double-reciprocal plots of the biology revolution. DPT has been virtually unknown in
DPT/agonist interaction are analyzed. DPT’s effects in the the scientific literature since the mid-1970s when its use
signal transduction system are complex. While DPT as an adjunct to psychotherapy faded. Although this ear-
alone (0.1–1,000
and -S-GTP incorporation are measured, in the pres- was nevertheless obvious that the drug had substantial
ence of 5-HT (0.1–10 mol/l), DPT blocks the agonist ef- pharmacological activity [5]. DMT, often considered to
ꢁmol/l) activates Gi when both cAMP ly clinical experiment with DPT yielded mixed results, it
ꢀ
ꢁ
fect. In combination, the findings suggest that DPT is a be a prototypical psychedelic compound, is a Schedule I
moderate affinity partial agonist at the human 5-HT1a Drug under the Controlled Substances Act. While DPT
receptor. These results provide evidence that DPT has is not explicitly so recognized, it might, nevertheless, be
potential as a versatile experimental tool at 5-HT1a re- construed as such under the loose categorizations of the
ceptors.
Controlled Substance Analogue Enforcement Act [6].
The much better known DMT is a potent hallucinogen
[7–11], and its biochemical pharmacology has been at
least partially characterized. DMT is structurally related
to serotonin and the serotonin-like hallucinogens LSD,
psilocin, psilocybin, and bufotenin [12]. Many of the
Copyright © 2005 S. Karger AG, Basel
© 2005 S. Karger AG, Basel
Keith K. Parker
0031–7012/05/0744–0193$22.00/0
Department of Pharmaceutical Sciences (MPH 102)
School of Pharmacy, University of Montana
Fax +41 61 306 12 34
E-Mail karger@karger.ch
www.karger.com
Accessible online at:
www.karger.com/pha
32 Campus Drive 1552, Missoula, MT 59812-1552 (USA)
Tel. +1 406 243 4235, Fax +1 406 243 5228, E-Mail keith.parker@umontana.edu

pharmacological properties of these serotonin analogues, ligands that bind to the receptor can be monitored by
including hallucination, can be explained by interactions quantification of intracellular cyclic AMP (cAMP).
with 5-HTR. The structural relationship of DMT and
Additionally, since H5-HT1aR is a GPCR, receptor
DPT, coupled with the known activity of DMT, suggests activitycanbefollowedbyGTPincorporationtechniques.
considerable opportunity for exploration of the biological Recently, we have reported studies with H5-HT1aR using
potential of DPT in these serotonergic receptor systems peptide probes derived from the intracellular loop 2 and
[13].
3 regions of the receptor. These peptide studies are de-
Attention was directed at DPT in the current investi- signed to further understand the interfacial biology be-
gation due to an expectation that it, or structural ana- tween H5-HT1aR and Gi [24, 25]. Coupled with findings
logues, might represent possible candidates for migraine from other laboratories, we have developed a working
treatment. Field investigations among the Machiguenga model of the receptor/G protein interface [33]. The find-
tribe in the Peruvian Amazon indicated that they use eye ings about DPT interactions at H5-HT1aR’s ligand-bind-
drops from the leaves of Psychotria spp. (known to con- ing site and implications from these findings, in combina-
tain DMT) in the successful treatment of migraine head- tion with a large body of structure-activity observations
aches [14, 15]. DMT is known to display agonistic activ- at 5-HT1aR [34, 35], should increase the likelihood of re-
ity at 5-HT1aRs, and antagonistic activity at 5-HT2aRs finement of a comprehensive model of the receptor [36].
[11], precisely the same qualities suggested as pertinent Results reported here suggest that DPT is a high-affinity
to acute symptomatic and prophylactic treatment of mi- partial agonist at H5-HT1aR. These conclusions have im-
graine, respectively [14, 16–18].
plications for use of DPT or structural analogues at the
Subjective reports of DPT experiences are available in ligand-binding site of H5-HT1aR [21]. A preliminary re-
published form [1, 19]. These seem to indicate a psyche- port of these findings has appeared [37], including brief
delic (entheogenic) threshold of 1 mg/kg. Thus, DPT is observations about DPT’s activity at another 7TMD/
hallucinogenic, but is a much less potent hallucinogen GPC serotonin receptor [27, 42], the rat 5-HT2aR.
than DMT and especially LSD. This difference suggests
that serotonergic properties of DPT, potentially useful
Materials and Methods
in treatment of migraine, could possibly be obtained at
doses in which hallucinogenic potential is low. Explora-
tion of the biochemical pharmacology of DPT and com-
parison of its properties with DMT may shed light on
possible structural alterations [20] that could be exploited
in drug development [21]. Even if DPT’s pharmacology
proves to be less than useful in an applied sense, greater
understanding of its receptor activity could provide a
valuable experimental tool. With the recent placement of
5-methoxy-N,N-diisopropyltryptamine (foxy-methoxy)
into Schedule I [22] and the substantial lack of fundamen-
tal pharmacological studies for many of these active sub-
stances, it would seem an appropriate time to promote
studies of the psychoactive tryptamines.
Cell Culture
CHO cells expressing the H5-HT1aR [38–39] were cultured
in Ham’s F-12 medium fortified with 10% fetal calf serum and
200 ꢁg/ml geneticin. Cultures were maintained at 37°C in a hu-
midified atmosphere of 5% CO2. Cells were subcultured or assayed
upon confluency (5–8 days). Cloned H5-HT1aR was kindly pro-
vided by Dr. John Raymond (Medical University of South Caro-
lina).
Receptor Preparation
Cells were harvested by trypsinization and centrifuged at low
speed in ice-cold medium. The pellet was re-suspended in ice-cold
Earle’s balanced salt solution followed by centrifugation. Cells were
re-suspended in 10 ml of ice-cold binding buffer (50 mmol/l Tris,
4 mmol/l CaCl2, 10 ꢁmol/l pargyline, pH 7.4), homogenized with
Teflon-glass, and centrifuged at 450,000 g at 4°C. To produce a
crude membrane preparation, the pellet was re-suspended in 30 ml
of ice-cold binding buffer, and homogenized, first with Teflon-glass
and then with a Polytron (setting 4) for 5 s. The receptor prepara-
tion was stored on ice and assayed within the next 1.5 h.
Biochemical Pharmacology of DPT
The studies reported here represent the initial charac-
terization of DPT at cloned human (H) 5-HT1aR [23–
26]. H5-HT1aR [28] is a seven transmembrane domain
(7TMD), G-protein-coupled (GPC) receptor [29–31],
and has been linked to biomedical conditions such as de-
pression, anxiety, and migraine headache. Since H5-
HT1aR is negatively coupled to the adenylyl cyclase sig-
nal transduction system via Gi in the Chinese hamster
Assay of Receptor Activity
Binding of the agonist [3H]8-OH-DPAT ([3H]8-hydroxy-2-(di-
n-propylamino)tetralin) to H5-HT1aR followed well-characterized
in vitro protocols [25, 40]. Radioligands were purchased from New
England Nuclear (Boston, Mass., USA). 1-ml reaction mixtures, in
triplicate, were incubated for 30 min in a 30°C shaker bath. Com-
ovary (CHO) cells used here [32], functional attributes of position of the 1-ml reaction mixture was: 700
ꢁl of receptor prep-
Pharmacology 2005;74:193–199
194
Thiagaraj/Russo/Burnett/Goldstein/
Thompson/Parker

aration; 100
10 mol/l 5-HT (final concentration for non-specific binding),
100
lofthetritiatedagent(finalconcentrationof0.5nmol/l[3H]8-
OH-DPAT), and 100 l of diluted DPT or binding buffer in the
case of controls. In the case of experiments that varied in both DPT
concentration and [3H]8-OH-DPAT concentration (fig. 3), the fol-
lowing radioligand concentrations were used: 0.2, 0.4, 0.6, 0.8, and
1.0 nmol/l. To achieve double-reciprocal plots of the results, the
concentrations were represented as the following numbers respec-
tively: 5.0, 2.5, 1.67, 1.25, and 1.0 (see x-axis, fig. 3).
ꢁl of either binding buffer (for total binding) or
ꢁ
ꢁ
ꢁ
N
N
H
Reactions were stopped by addition of 4 ml of ice-cold
50 mmol/l Tris buffer, pH 7.4, and subsequent vacuum filtration
on glass fiber filters (Whatman GF/B). Filters were rinsed twice in
5 ml of ice-cold Tris buffer, dried, and counted in 5 ml of Ecoscint
(National Diagnostics) liquid scintillation fluid in a Beckman LS
6500 instrument. Homogenates were assayed for protein to main-
Fig. 1.Structureandsynthesisofdipropyltryptamine(DPT). Trypt-
amine (1.6 g; 10 mmol) and diisopropylethylamine (1.04 ml; 60
mmol) were dissolved in diethyl ether (60 ml) at 0°C. 1-Iodopro-
pane (1.02 g; 0.6 ml; 60 mmol) was added dropwise with stirring
over 1 h. The reaction stirred 16 h at room temperature, was filtered
to remove salts, and evaporated to an oil. The crude product, which
was contaminated with N-propyltryptamine and some unreacted
tryptamine, was chromatographed on silica to afford the product,
N,N-di-n-propyltryptamine in 46% yield. The purified product had
elemental and spectral characteristics consistent with literature val-
ues.
tain a nominal value of 50 ꢁg protein per filter over weekly assays
[41]. Total and non-specific binding tubes were run in triplicate.
cAMP Assay
CHO cells were cultured to confluency in 12- or 24-well plates.
Medium was aspirated and the cells were rinsed twice in warm se-
rum-free F-12 medium. Cells were then incubated for 20 min at
37°C in 0.5 ml of serum-free F-12 medium containing 100
isobutylmethylxanthine and the following substances (final concen-
trations) alone or in combination (see fig. 4): 30 mol/l forskolin
(for all treatments); 1–10 mol/l 5-HT; 0.1–1 mol/l DPT, and
0.1 mol/l 8-OH-DPAT. Reactions were stopped by aspiration of
medium and addition of 0.5 ml of 100 mmol/l HCl. After 10 min,
well contents were removed and centrifuged at 4,000 rpm. Super-
natants were diluted in 100 mmol/l HCl, and cAMP was quantified
[24] directly in a microplate format by colorimetric enzyme immu-
noassay with a kit from Assay Designs (Ann Arbor, Mich., USA).
Triplicate independent samples at a minimum were assayed in qua-
druplicate to increase precision.
ꢁmol/l
ꢁ
ꢁ
dures (including drug-receptor binding analysis) in the study were
conducted with PSI-Plot (Version 7) software (Poly Software Inter-
national), or on a Hewlett-Packard Graphing Calculator, HP48.
ꢁ
ꢁ
Results
Displacement of [3H]8-OH-DPAT from H5-HT1aR
DPT was synthesized at the University of Montana
(fig. 1) for the purpose of characterizing some of its bind-
ing and signal transduction characteristics at the H5-
HT1aR. The first series of experiments were designed to
determine the affinity of DPT at H5-HT1aR present in
membranes isolated from cultured CHO cells. Figure 2
shows that DPT produced concentration-dependent dis-
placement of the specific agonist [3H]8-OH-DPAT
(0.5 nmol/l) from the H5-HT1aR ligand-binding site.
The displacement of [3H]8-OH-DPAT at H5-HT1aR in-
dicates that DPT has moderately high affinity with an
ꢀ
-S-GTP Assay
H5-HT1aR membranes from transfected CHO cells were incu-
bated with 5-HT (0.1 mol/l) and/or DPT (0.1–1,000 mol/l; see
ꢁ
ꢁ
fig. 5), and the following incubation mixture: 20 mmol/l Hepes
buffer, pH 7.4, 5 mmol/l MgCl2, 1 mmol/l EDTA, 1 mmol/l DTT,
100 mmol/l NaCl, 100
ꢁ
mol/l GDP, 10
ꢁmol/l pargyline,
0.2 mmol/l ascorbate, and 0.1 nmol/l [35S]
ꢀ-S-GTP [43]. Mixtures
were incubated for 30 min at 30°C, and were terminated by dilu-
tion in cold buffer. The mixture was filtered on GF/C filters, rinsed
twice in buffer, followed by drying and liquid scintillation count-
ing. Negative control (basal incorporation) was the above mixture
minus DPT or 5-HT. Non-specific binding was determined in the
presence of cold ꢀ-S-GTP (10 ꢁmol/l). Positive control was H5-
apparent IC50 of about 0.1 ꢁmol/l.
HT1aR membranes in the same incubation mixture plus 5-HT. All
values reported in figure 5 are for specific binding (total – non-spe-
cific) of triplicates.
In figure 3, two different concentrations of DPT (40
and 200 nmol/l) were compared with a control lacking
DPT against various concentrations of [3H]8-OH-DPAT
(0.2, 0.4, 0.6, 0.8, and 1.0 nmol/l) bound to H5-HT1aR.
In this adaptation of the Lineweaver-Burke plot of en-
zyme kinetics, both agonist [3H]8-OH-DPAT concentra-
tions (x-axis) and agonist-receptor complex concentra-
tions (y-axis) are inverted. The resulting straight lines al-
Synthesis, Storage and Dilution of DPT; Data Analysis
DPT was synthesized as outlined in figure 1, and stored in the
dark at 4°C. Various spectral and physical analyses of DPT are
available on request. DPT is very soluble in water and dilutions
were made with distilled water. Final dilution was in the binding
bufferoftheassay. Allstatistics(means, standarderrorsofthemean
(SEM), and Pearson correlation coefficients) and graphical proce- low estimation of the nature of DPT/agonist interactions.
Pharmacology 2005;74:193–199
Pharmacology of DPT at the 5-HT1aR
195

120
100
80
60
40
20
0
1.2
1.0
0.8
0.6
0.4
0.2
0
Control
4 ꢂ 10– mol/l DPT
2 ꢂ 10–7 mol/l DPT
8
–2
–1
0
1
2
3
4
5
6
7
10
8.4
7.4
6.4
6
5.4
4.4
3
1/[L]
–0.2
-log DPT
Fig. 2. Inhibition of [3H]8-OH-DPAT binding by dipropyltrypt-
Fig. 3. Concentration dependency of [3H]8-OH-DPAT binding in
amine (DPT) in membrane preparations expressing the human 5- the presence of dipropyltryptamine (DPT) in membrane prepara-
HT1a receptor (H5-HT1aR). DPT concentrations are –log mol/l. tions of the human 5-HT1aR (H5-HT1aR). L is the concentration
Results are mean 8 SEM with n’s of 2–5. More detailed experi- of [3H]8-OH-DPAT. On the x-axis, 1 = 1 nmol/l; 5 = 0.2 nmol/l,
mental conditions of cell culture, membrane preparation, and drug- etc. On the y-axis, LR is the concentration of the [3H]8-OH-DPAT-
receptor binding are outlined in Methods.
H5-HT1aR complex. All points are triplicates of specific binding
with minimum n’s of 2. Detailed experimental conditions are out-
lined in Methods. Correlation coefficients exceed 0.99 for control
(lowest line) and 4 ! 10–8 mol/l DPT (middle line); correlation
coefficient for the upper line (2 ! 10–7 M DPT) is 0.89.
120
100
80
60
40
20
0
Fig. 4. Effects of serotonin (5-HT), 8-OH-
DPAT (DPAT) and dipropyltryptamine
(DPT) on forskolin-stimulated cyclic AMP
(cAMP) formation in whole cell prepara-
tions of the human 5-HT1a receptor (H5-
HT1aR). All conditions contain forskolin
(FSK) at 30 ꢁmol/l final concentration. Re-
sults are expressed as percentage of FSK
control as mean 8 SEM with n’s of 3
(DPAT), 7 (5-HT)–22 (FSK), with interme-
diate values for all other conditions. Final
molar concentrations for all conditions are
listed below the bars. Additional experi-
mental conditions are described in Meth-
ods.
10–6
10–5
10–7
10–6
DPT
+10–6
5–HT
+10–5
5–HT
FS
5–HT
DPT (10–7
)
DPAT (10–7
)
Interpretation of the experiments analyzed here is consis-
tent with DPT inhibiting agonist binding competitively
cAMP Determination
DPT’s ability to bind at the H5-HT1aR raised the
at H5-HT1aR’s ligand-binding site, as the lines intersect question of its potential to trigger the signal transduction
on the y-axis. Thus, the two concentrations of DPT yield system. We had preliminary data that suggested antago-
increases of [3H]8-OH-DPAT’s apparent Kd on the nega- nistic effects of DPT in this negatively coupled system.
tive x-axis.
Thus, determination of intracellular cAMP concentra-
Pharmacology 2005;74:193–199
196
Thiagaraj/Russo/Burnett/Goldstein/
Thompson/Parker

200
180
160
140
120
100
80
Fig. 5. Effects of serotonin (5-HT) and di-
propyltryptamine (DPT) on incorporation
of [35S]
ꢀ-S-GTP into membrane prepara-
tions of the human 5-HT1a receptor (H5-
HT1aR)-G protein complex. Control repre-
sents incorporation in the basal setting (buf-
60
40
fer). 5-HT concentration is 0.1 ꢁmol/l in all
20
cases. Molar DPT concentrations are listed
below the bars. Results are expressed rela-
tive to basal incorporation as mean 8 SEM
with n’s of 4 (1 mmol/l DPT)–16 (control).
Additional experimental conditions are de-
scribed in Methods.
0
7
6
5
4
3
7
6
10
10
10
10
10
+ 10 + 10
DPT DPT
7
7
Control
5-HT (10
)
DPT
5-HT (10
)
tions following DPT binding tested the hypothesis that concentration-dependent incorporation of [35S]
DPT is an antagonist. Figure 4 demonstrates that DPT From baseline at 0.1 mol/l, DPT increased incorpora-
reduced forskolin-stimulated cAMP concentrations. tion to a maximum of 76% above control at 1 mmol/l.
While at 0.1 mol/l DPT the effect is not significant, at When 5-HT (0.1 mol/l) and DPT (0.1 or 1.0 mol/l)
mol/l it clearly is. This action is consistent with ago- were administered together, the effect of 5-HT alone was
ꢀ-S-GTP.
ꢁ
ꢁ
ꢁ
ꢁ
1
ꢁ
nistic rather than antagonistic effects. The full agonist reduced by DPT. Together, DPT and 5-HT were not able
serotonin dramatically stimulated the receptor, reducing to exceed the effect of 5-HT alone or to even equal the 5-
cAMP levels in a concentration-dependent manner. Ad- HT effect alone. This would indicate that DPT is acting
ditionally, for comparison to the binding experiments, as an antagonist in the presence of the full agonist sero-
0.1
forskolin-stimulated cAMP by more than 95%. In com- By raising the DPT concentration to 1 mmol/l when ad-
bination with 5-HT, 0.1 mol/l DPT diminished the ef- ministered alone, the incorporation effect (76%) is almost
fects observed with 5-HT alone. This result could be con- aslargeasthatproducedby5-HTalone(92%)ata10,000-
sistent with the conclusion that DPT is an antagonist. fold lower concentration (0.1 mol/l). While the near full
However, any agent, agonist or antagonist that is compet- efficacy of DPT at 1 mmol/l compared to 5-HT at
ing with a higher affinity full agonist, could potentially 0.1 mol/l could suggest that DPT is a full agonist with
ꢁmol/l8-OH-DPAT(DPAT),afullagonist,decreased tonin, an effect consistent with that of a partial agonist.
ꢁ
ꢁ
ꢁ
reduce the full agonist’s effects. Such combinations of muchloweraffinitythan5-HT, theoverallimpressionleft
agents would produce a complicated set of effects, depen- by these observations is that DPT is a partial agonist.
dent on the relative concentrations and affinities of the
two agents. Thus, it is not possible to completely test the
hypothesis with these results. With another different but
related measure of the signal transduction system, we di-
rectly tested the intrinsic activity of DPT.
Discussion
DPT represents an interesting example of a drug that
has seen therapeutic and recreational uses while undergo-
ing few pre-clinical experimental analyses. While many
ꢀ -S-GTP Incorporation
Figure 5 summarizes another approach to testing closely related analogues of DPT have been carefully in-
DPT’s capacity to trigger signal transduction at H5- vestigated with respect to medicinal chemistry and in vi-
HT1aR. As anticipated, 5-HT (0.1
porated 92% more than the basal (buffer) incorporation sidelines.
of [35S]
-S-GTP. Additionally, DPT showed significant
ꢁmol/l) by itself incor- tro and in vivo pharmacology, DPT itself has been on the
ꢀ
Pharmacology 2005;74:193–199
Pharmacology of DPT at the 5-HT1aR
197

The results described in figures 2 and 3 provide strong
evidencethatDPTbindscompetitivelytotheH5-HT1aR range (IC 50 of 0.1
ligand-binding site. With an IC50 of 0.1 mol/l as mea- that a full agonistic effect should be achievable at 1,000-
With DPT’s binding affinity in the submicromolar
ꢁmol/l), binding theory would imply
ꢁ
sured by displacement of the high-affinity (subnanomolar fold higher concentration (0.1 mmol/l). However, even at
Kd) agonist [3H]8-OH-DPAT, DPT’s affinity is moder- 1 mmol/l, DPT still is not able to produce the agonistic
ate. This conclusion is in line with previously published effect of 5-HT, and as in the cAMP experiments, the high-
results for DMT at the 5-HT1aR [9, 11], where the IC50 er magnitude effect of 5-HT alone was never achieved
was in the range of 0.1–0.2
ꢁ
mol/l. Since [3H]8-OH- when 5-HT was in the presence of DPT. Thus, even
DPAT is a highly characterized, widely accepted specific though DPT comes close to fulfilling the requirements for
ligand at the 5-HT1aR ligand-binding pocket, the results a full agonist, it falls short. Interpretation of the binding
appearing in figure 3 provide compelling data that DPT data and two different measures of efficacy suggest that
and [3H]8-OH-DPAT compete with each other for this DPT is best classified as a moderate affinity partial ago-
site. We have conducted repeated Hill analyses at 5- nist at the H5-HT1aR.
HT1aR with [3H]8-OH-DPAT [42] and now with DPT
Published studies with DMT in rat brain [11] estab-
(data not shown); these results consistently demonstrate lished the full agonist properties of the drug using both
non-cooperative, unitary site binding, and addition of cAMP determinations and GTP inhibition studies. If re-
DPT to the mix changes nothing about this conclusion.
sults with DMT in rat 5-HT1aR and our findings with
With the additional bulkiness of DPT’s alkyl substitu- DPT in H5-HT1aR can be compared, the two drugs have
ents, we originally hypothesized that DPT would be an- similar affinities for both receptors; both drugs also show
tagonistic. Upon testing, it appears that this assumption agonistic properties at the receptors. However, while
is not correct, and we now believe that the data shown in DMT is a full agonist in comparison to the full agonist
figures 4 and 5 suggest that DPT is a partial agonist. Since [3H]8-OH-DPAT, DPT fails to achieve the full efficacy
H5-HT1aR is negatively coupled to AC via Gi in CHO, of the agonist, 5-HT, even at a very high concentration
agonists will activate Gi. In the experimental paradigm (1 mmol/l). Thus, it would appear that the concentration
used here, forskolin-stimulated cAMP levels should be re- and magnitude effect characteristics of DPT, while simi-
duced in the presence of agonist, as shown by 5-HT at two lar to those of DMT, better fit the definition of partial
different concentrations. 1 ꢁmol/l DPT, a concentration agonist at H5-HT1aR. It would be interesting to conduct
that should produce significant effect as judged by the thermodynamic studies with DMT and DPT to analyze
binding data shown in figures 2 and 3, also produced low- potential enthalpic and/or entropic differences in binding
er cAMP levels, an effect consistent with agonistic behav- parameters that may lead to the transition from full to
ior. The combination of 5-HT and DPT produced an ef- partial agonist properties at 5-HT1aR.
fect lower than that of 5-HT alone. Together these obser-
vations are consistent with the hypothesis that DPT is a previously shown that DPT is a low-affinity agonist or
partial agonist; however, the results are not conclusive. partial agonist (IC50 of about 4 mol/l). The results pro-
In preliminary results at the rat 5-HT2aR [37] we have
ꢁ
To more completely test the agonistic effects of DPT, vide a provocative contrast with the findings of Deliga-
we conducted concentration-effect experiments at the G nis et al. [11] with DMT at rat 5-HT2aR. That is, while
protein level. An agonist such as 5-HT should be able to DMT is an antagonist, DPT appears to be an agonist.
increase incorporation of [35S]
ꢀ
-S-GTP into Gi. The re- These differences cannot be explained on a species basis
sults presented in figure 5 demonstrate this effect at since both used rat receptors; however, the DMT work
0.1 mol/l serotonin. At a 10,000-fold concentration used rat cerebral cortex while our DPT studies utilized
range of DPT (from 0.1 mol/l to 1 mmol/l), graded in- cloned receptor. 5-HT1aR and 5-HT2aR are known to
creases in [35S]
-S-GTP incorporation were observed. A have very different binding determinants, and the struc-
ꢁ
ꢁ
ꢀ
lower-affinity full agonist should be able to achieve the tural changes from DMT to DPT, reducing agonism at
effectiveness of a higher-affinity full agonist, albeit at 5-HT1aR, as we have observed in this study, may well
higher concentration. Comparison of DPT at 1 mmol/l lead to the opposite transition at 5-HT2aR [44]. Addi-
with 5-HT at 0.1
ꢁmol/l almost allows this conclusion, tional binding, signal transduction, and thermodynamic
but not quite, as DPT produced a 76% incorporation in- studies with DMT and DPT at 5-HT2aR would be in-
crease compared to 92% for 5-HT. Judging from the bind- structive, especially with regard to potentially explaining
ing data of DPT (fig. 2, 3), 1 mmol/l is a very high, satu- the in vivo differences between DMT and DPT in hu-
rating concentration.
mans.
Pharmacology 2005;74:193–199
198
Thiagaraj/Russo/Burnett/Goldstein/
Thompson/Parker

Sciences and the Department of Biomedical and Pharmaceutical
Sciences, were instrumental in completion of this work. Todd Tal-
ley’s conceptual contributions to the work are greatly appreciated.
The following individuals made significant technical contributions
to the successful completion of the project: Tyra Spotted Elk, Ro-
Jon Grogans, Macy Hyvonen, Brian Hall, Caroline Macarah, Son-
ja Sakaske, Ben Seaver, Adam Spang, and Carol Todd.
Acknowledgements
The following NIH grants provided partial support for comple-
tion of this work: GM/OD 54302-02 and NCRR Grant # P20 RR
15583 to the NIH-COBRE Center for Structural and Functional
Neuroscience. AdditionalsupportfromtheUniversityofMontana-
Missoula, particularly the School of Pharmacy and Allied Health
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