Effects ofPsychotria colorata alkaloids in brain opioid system

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Neurochemical Research, Vol. 21, No. 1, 1996, pp. 97-102

Effects of Psychotria colorata Alkaloids in Brain Opioid System T~nia Alves A m a d o r , 1,2 Elaine Elisabetsky, 1-3 a n d Diogo O n o f r e de S o u z a 2

(Accepted October 26, 1995)

An ethnopharmacological survey showed that home remedies prepared with flowers and fruits of Psychotria colorata are used by Amazonian peasants as pain killers. Psychopharmacological in vivo evaluation of alkaloids obtained from leaves and flowers of this species showed a marked dose-dependent naloxone-reversible analgesic activity, therefore suggesting an opioid-like pharmacological profile. This paper reports an inhibitory effect of P. colorata flower alkaloids on [3H]naloxone binding in rat striata as well as a decrease in adenylate cyclase basal activity. The alkaloids did not affect [3H] GMP-PNP binding. These findings provide a neurochemical basis for the opioid-like activity previously detected in vivo and point to Psychotria alkaloids as a potential source of new bioactive opiate derivatives. KEY WORDS: Naloxone; opioid; analgesia; alkaloids; binding.

INTRODUCTION

(7). The identification of useful natural products can follow a number of strategies, amongst which leads from traditional use of plants as homemade medicines (8,9). Surveys of medicinal plants used among Amazonian caboclos (non-indian rural populations) of the State of Par~-Brazil pointed to Psychotria colorata (Will. ex R. & S.) Muell. Arg., traditionally used for "treatment of earache" (10) and "calming abdominal pain" (11). We further reported that alkaloids present in leaves and flowers of P. colorata have marked analgesic activity, as evaluated through the acetic acid-induced writhing, formalin, and tail flick methods (12). Opioid receptors, which bind to the physiological peptide agonists enkephalins, endorphins and dynophims, exhibit a widespread distribution in central and peripheral nervous systems and participate in the modulation of pain as well as various physiological systems (13,14). The existence of at least three types of opioid receptors, namely ~t, K, and 8 has been well established, and many subtypes have been proposed (13,15). Pharmacological agents, as the agonist morphine and the antagonist naloxone, also bind to these receptors.

New analgesics have been intensively sought by the pharmaceutical industry (1). This lasting interest is related to the still significant range of undesired effects of presently available compounds and the billion dollar size of the market (2,3). The increasing recognition of the vast chemical diversity in tropical forests (4), associated with the development of revolutionary techniques for isolation and elucidation of chemical compounds and its pharmacological properties (5,6), justify the rekindled interest in natural products by pharmaceutical industries

Laborat6rio de Etnofarmacologia, Depto. de Farmacologia, Instituto de Bioci~ncias, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil. 2 Curso de P6s-gradua~o em Ci6ncias Biol6gicas-Bioquimica, Depto. de Bioquimica, Instituto de Bioci~ncias, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil. 3 Address reprint requests to: Elaine Elisabetsky, Laborat6rio de Etnofarmacologia, Departamento de Farmacologia, Instituto de Bioci6ncias, Universidade Federal do Rio Grande do Sul, Caixa Postal 5072, 90041970, Porto Alegre, RS, Brazil. Phone/FAX: (55)(051)226-7191

97 0364-3190/96/0100-0097509.50/0 9 1996PlenumPublishingCorporation

98 T h e m o d u l a t o r y effects o f m o s t n e u r o t r a n s m i t t e r s o n c e l l u l a r e v e n t s is o f t e n a c h i e v e d b y c o u p l i n g specific r e c e p t o r s to G p r o t e i n s (16,17). I n turn, p r o t e i n G activity is m o d u l a t e d b y g u a n i n e n u c l e o t i d e s ( 1 6 - 1 8 ) . It h a s b e e n d e m o n s t r a t e d t h a t o p i o i d r e c e p t o r s m o d u l a t e cell u l a r e f f e c t o r s s u c h as a d e n y l a t e c y c l a s e , i o n c h a n n e l s , a n d p h o s p h o l i p a s e s t h r o u g h c o u p l i n g to G p r o t e i n s (16,19). Specific t y p e s o f r e c e p t o r s m e d i a t e t h e p h a r macological actions of opioids, either important therap e u t i c t o o l s a n d / o r w i d e l y a b u s e d s o c i a l l y as a r e s u l t o f their euphorigenic and addictive properties. The detect i o n o f o p i o i d a g o n i s t s t h a t m a i n t a i n the a n a l g e s i c profile o f m o r p h i n e a n d l a c k its a d d i c t i v e p r o p e r t i e s is t h e r e f o r e highly desirable. T o f u r t h e r e x p l o r e the p o t e n t i a l o f Psychotria alk a l o i d s as n e w a n a l g e s i c a g e n t s , o u r s t u d y f o c u s e d in confirming the opioid nature of analgesia by investigating n e u r o c h e m i c a l c o r r e l a t e s c o m m o n to opioids. T h i s p a p e r r e p o r t s t h e effects o f a l k a l o i d s o b t a i n e d f r o m flowers o f Psychotria colorata o n specific b i n d i n g o f [ 3 H ] n a l o x o n e in r a t striata m e m b r a n e s , o n b i n d i n g o f [ 3 H ] G M P - P N P ( a n a n a l o g o f G T P ) to G p r o t e i n s a n d o n a d e n y l a t e c y c l a s e activity.

EXPERIMENTAL PROCEDURE Drugs' and Reagents. Morphine hydrochloride was kindly provided by Escola Panlista de Medicina. Naloxone, ATP, and cAMP protein binding were purchased from Sigma. Tween was acquired from Rhodia. [3H]Naloxone, [3H]cAMP, and [3H]GMP-PNP were purchased from Amersham Life Science. All other reagents were of analytical grade. Alkaloid Extracts. Extracts were prepared at the Laboratory of Natural Products Chemistry, Federal University of Pardi (Brazil) under the supervision of Dr. Domingos Nunes. Dried milled leaves (515 g) or flowers (64 g) were wetted with 6 N NH4OH and extracted with ethanol in a Soxhlet apparatus. After distillation of solvent, the crude ethanol extract was poured into a 2% acetic acid solution, left 12 h in refrigerator and filtered. The clear acidic solution was extracted with chloroform (residue discarded). The aqueous phase was adjusted to pH 7 with NaHCO 3 pH 7.0 and extracted with chloroform; the organic layer washed, dried (Na2SO4) and evaporated. TLC analysis shows a very similar composition for both alkaloids extracts (20). The HPLC/MS analyses identified a mixture of pyrrolidinoindoline alkaloids (e.g., quadrigemine C, calycanthine, isocalycanthine, among others not yet fully characterized). Animals'. Male adult Wistar rats (250-300 g) breed in our own animal house were used for neurochemical studies. In vivo experiments were done with male adult albino mice (25-35 g), food and water ad libitum, from our own stock. Tail-Flick Test. Analgesia was assessed with a Tail Flick apparatus (Albasch Electronics Equipments). Forty five minutes before testing, animals were placed individually in acrylic cages (20 • 20 x 20cm) which also served as observation chambers. The method of Ramabadran et al. (21) was adapted as follows. Baseline latency (reaction time) was obtained with three measures (after each measure

Amador, Elisabetsky, de Souza animals were retumed to the observation chambers for 2 min); the mean of these three measures was the pre-drug latency time. At this point, animals presenting two measures of 6 s or more were discarded. Alkaloids or drugs were administered (i.p) immediately after the third pre-drug measure. Thirty minutes later another set of three measures was taken and the mean considered as post-drug reaction time. A cutoff time of 10 s was used to prevent tissue damage. Reversibility by naloxone (i.p, 10.0 mg/kg) was tested by administering it 8 minutes before alkaloids or morphine. Differences between pre and post-drug latencies were analyzed by Wilcoxon test. Membrane Preparationfor Binding of [3H]Naloxone. Membranes were prepared as described by Lee et al (22). After decapitation, the striata was rapidly removed and homogenized in 50 vol. (ml/g) of 50 mM Tris-HC1 buffer (pH 7.4). The homogenate was centrifuged at 100,000 g for 30 minutes. The pellet was resuspended in the same volume of buffer and recentrifuged. The final pellet was resuspended as to provide a solution of 0.2 mg/ml of protein, used in all experiments. All steps were carried out at 4~

Membrane Preparation for Adenylate Cyclase Activity and for Binding ofpH]GMP-PNP. Membranes were prepared as described by Souza and Ramirez (23). Briefly, striata was homogenized in 20 vol. (ml/g) of 0.32 M sucrose containing 10 mM Tris/HC1 buffer, pH 7.4, and 1 mM MgC12. The homogenate was centrifuged twice at 1,000 g for 15 min and the final pellet discarded. Both supernatants were pooled and centrifuged at 27,000 g for 15 min. The resulting pellet was lysed for 30 min in 10 mM Tris/HCl buffer, pH 7.4 and washed three times in lysing buffer by centrifuging at 27,000 g for 15 min. The final pellet was used for the experiments. All steps were carried out at 4~ Binding of [3H]Naloxone. Membranes (200 gg of protein) were incubated for 5 min at 35~ in 0.5 ml of incubation medium containing 50 mM Tris/HC1 buffer, pH 7.4. Subsequently, 100 nM [3H]naloxone (specific activity, 60 Ci/mmol) was added to the reaction mixture and incubated for an additional 15 minutes period at 35~ Each experiment was performed in triplicates. Incubation was interrupted by filtration under vacuum with Sartorius GF/B filters and immediately washed with 18 ml of ice-cold 50 mM Tris-HCl buffer (pH 7.4). Filters were dried and immersed in scintillation liquid for radioactivity measurement. Specific naloxone binding was defined as the part of total binding displaced by a concentration of nonlabeled ligand 1,000 times greater than the corresponding radioligand concentration. The effect of alkaloids or morphine on [3H]naloxone binding was evaluated by adding these compounds to the incubation medium. Binding of ~H]GMP-PNP. Membranes (200-300 gg of protein) were incubated at 30~ in 0.5 ml of incubation medium containing 25 mM Tris/HC1 buffer, pH 7.4, 5 mM MgC12, 1 mM DTT and 40 nM of [3H]GMP-PNP. After 15 min., incubation was interrupted by cooling the tubes and further centrifuging for 2 rain. at 12,000 g. The supernatant was discarded. The walls of the tubes and the surface of the pellets were quickly and carefully rinsed with cold distilled water. Pellets were processed for radioactivity measurement. Specific binding was defined as the part of total binding displaced by a concentration of nonlabeled ligand 1,000 times greater than the corresponding radioligand concentration. The effect of alkaloids or morphine on [3H]GMP-PNP was evaluated by incubating the nucleotide with 5.0 and 50.0 gg of alkaloids or with 6 nM and 6,6 gM morphine. Experiments were always performed in triplicates. Adenylate CyclaseActivity. The procedure was carried out as described by Paz et al (24). Membranes were pre-incubated for 15 min at 30~ in incubation medium (final volume of 100 ml) containing 20 mM Tris/HC1 buffer, pH 7.4, 1 mM DTT, 10 mM MgC12 and 1 g% albumin, in the presence or absence of 10 mM GMP-PNP. Incubation

Naloxone Binding Inhibition by Alkaloids P. colorata was started by the addition of ATP (1 mM final concentration), and stopped after 1 min by boiling the tubes for 5 min. The tubes were cooled and centrifuged at 12,000 g for 5 min and the supernatants were used for measuring cAMP produced by the protein binding method (25). Protein Measurement. Protein content was measured according to the method of Lowry (26). Statistic Analysis. Data were analyzed for statistic significance by Student's-t test or compared through ANOVA followed by MSD, as specified in each experiment (see figure legends).

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RESULTS Fig. 1 compares in viva analgesic effects of alkaloids obtained from leaves (Fig. 1A) and flowers (Fig. 1B) of P. colorata and morphine. As it can be seen, all treatments produced analgesia reversible by previous administration of naloxone. Fig. 2 shows that the binding of [3H]naloxone increased with increasing [3H]naloxone concentrations. 100 nM [3H]naloxone was chosen for all other binding experiments. Fig. 3A shows that morphine inhibits the [3H]naloxone binding in a dose-dependent way (5.0; 10.0; 25.0; 50.0, and 100.0 nM). The effects of alkaloids is presented in Fig. 3t3: alkaloids from flowers inhibited [3H]naloxone binding in a dose-dependent pattern (0.3; 1.0; 3.0; 10.0 and 30.0 ktg), an inhibitory profile comparable with morphine. Fig. 4 shows the effects of morphine and flower alkaloids on adenylate cyclase (AC) activity. Although neither alkaloids nor morphine caused a statistically significant effect on the stimulation of AC activity by GMP-PNP, alkaloids inhibited AC basal activity. The effects of alkaloids and morphine o n [3H] GMP-PNP binding is presented in Fig. 5. No effects on the binding were seen with the conditions used.

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DISCUSSION Important analgesic prototypes (e.g., salicylic acid and morphine) were originally derived from the plant kingdom, yet the majority of plants traditionally used as pain killers have not been fully studied. P. colorata belongs to a plant genus commonly used medicinally by various peoples. For instance, P. poeppigiana Muel. Arg. and P. ulviformes Steyerm are used in South America to alleviate pain (27,28). It is known that thermal nociceptive tests are more sensitive to opioid g-agonists and non-thermal tests to opioid K-agonists (29,30); morphine effects on formalin

are supposedly mediated by activation of opioid K-agonists (30). Our previous data suggested the involvement of both p. and K opioid receptors in the analgesic activity of alkaloids from P. colorata. The specific binding of neurotransmitters and the coupling of their receptors to specific cellular effectors is useful to characterize neurochemical parameters relevant to in viva effects of new drugs (31). In the present study we report the inhibitory effect of alkaloids of P. colorata on [3H]naloxone binding in rat striata membranes (Fig. 3A, 3B). These results reinforce our previously reported suggestion that the analgesic activity of

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alkaloids present in leaves and flowers of P. colorata (12) is mediated by opioid receptors. It is relevant mentioning that a pharmacological study with extracts of Psychotria brachypoda (Mull. Arg.) Britton, provides evidence that its antinociceptive activity is also mediated by activation of opioid receptors (28). Although biochemical and pharmacological evidence indicate that It, K and 8 receptors are distinct molecular entities, all three classes of opioid receptors appear to be coupled to G proteins (32,19). Although some laboratories have confirmed that opioids inhibit AC activity in brain homogenates, others have failed to replicate this effect, while some even demonstrate that they actually stimulate enzyme activity (33). Some reports showed that guanine nucleotides modulate opioid binding (32,34). We evaluated the effects ofP. colorata alkaloids and morphine on binding of [3H]GMP-PNP to membrane preparations and its effect on AC activity. Our results indicated that the alkaloids do not affect GMP-PNP binding (Fig. 5). Basal AC activity was inhibited by alkaloids (Fig. 4). The meaning of these findings remains to be clarified. The effects of P. colorata alkaloids on the opioid system may be the neurochemical basis of the analgesic activity of these alkaloids. Ongoing investigations will further characterize compounds from Psychotria colorata and their analgesic profile, including interaction with

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specific opioid receptor subtypes. These findings provide a rational basis for the traditional medical use of P. colorata.

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sistance, Dr. Jos6 Roberto Leite for donation of morphine and Dingo Lara for revision of manuscript. This work was supported by grants from CNPq and CAPES.

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REFERENCES

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ACKNOWLEDGMENTS The authors wish to acknowledge Dr. Domingos S~vio Nunes and Ana do Carmo Carvalho for preparation of extracts and chemical as-

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