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Keywords:

  • cannabinoid;
  • gastrointestinal motility;
  • radiology;
  • rat;
  • tolerance

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Competing interests
  8. Conflict of Interest
  9. References

Abstract  The use of cannabinoids to treat gastrointestinal (GI) motor disorders has considerable potential. However, it is not clear if tolerance to their actions develops peripherally, as it does centrally. The aim of this study was to examine the chronic effects of the cannabinoid agonist WIN 55,212-2 (WIN) on GI motility, as well as those in the central nervous and cardiovascular systems. WIN was administered for 14 days, at either non-psychoactive or psychoactive doses. Cardiovascular parameters were measured in anaesthetized rats, whereas central effects and alterations in GI motor function were assessed in conscious animals using the cannabinoid tetrad and non-invasive radiographic methods, respectively. Tests were performed after first (acute effects) and last (chronic effects) administration of WIN, and 1 week after discontinuing treatment (residual effects). Food intake and body weight were also recorded throughout treatment. Blood pressure and heart rate remained unchanged after acute or chronic administration of WIN. Central activity and GI motility were acutely depressed at psychoactive doses, whereas non-psychoactive doses only slightly reduced intestinal transit. Most effects were reduced after the last administration. However, delayed gastric emptying was not and could, at least partially, account for a concomitant reduction in food intake and body weight gain. The remaining effects of WIN administration in GI motility were blocked by the CB1 antagonist AM 251, which slightly accelerated motility when administered alone. No residual effects were found 1 week after discontinuing cannabinoid treatment. The different systems show differential sensitivity to cannabinoids and tolerance developed at different rates, with delayed gastric emptying being particularly resistant to attenuation upon chronic treatment.

Cannabinoids are being proposed for the treatment of an ever-increasing number of pathologies, many of them requiring repeated administration of the drug.1 Their beneficial potential, though, could be reduced by the development of adverse effects.2 The presence of central side effects is the most frequently cited reason that limits their usefulness.3 In experimental animals (mice and rats), central depression induced by cannabinoids is characterized by four typical signs: hypothermia, analgesia, catalepsy and deficits in motor performance (cannabinoid tetrad4,5).

In addition to these central effects, cannabinoids can affect both cardiovascular (CV) function and gastrointestinal (GI) motility. Cannabinoids induce hypotension in both normotensive and hypertensive rats6,7 and a vasorelaxant effect in rat resistance and conduit vessels.8 Marked ortostatic hypotension is also occasionally described in humans.9,10 Regarding the possible mechanisms involved, cannabinoids not only interact with the nerve fibres innervating the organ, but they also exert direct actions on the vascular tissue.11

In the GI tract, gastric emptying and intestinal transit are delayed upon cannabinoid administration in both rodents12–18 and human.19,20 This effect primarily involves a peripheral site of action18,21 and is mainly due to the presynaptic inhibition of acetylcholine release from myenteric neurones,22–24 which express cannabinoid CB1 receptors.25,26

Whereas tolerance develops to the central effects of cannabinoids upon chronic (daily) administration,27 tolerance to peripheral (CV and GI) effects is less well known. In the CV system, most data refer to the use of Δ9-tetrahydrocannabinol (THC) and are controversial. Thus, while some authors describe tolerance only to THC-induced modifications in the heart rate,28 others have demonstrated that tolerance may also develop to alterations in blood pressure.29

Regarding the GI tract, only one early report has specifically addressed the development of tolerance to the cannabinoid antitransit effect in vivo,30 whereas the remaining studies on development of tolerance to the effect of cannabinoid drugs in the GI tract have been carried out using in vitro preparations.17,31,32

Therefore, the aim of the present study was to examine the effect of daily administration of the mixed cannabinoid agonist WIN 55,212-2 (WIN) on GI motility (gastric emptying and intestinal transit, using a non-invasive radiographic method recently developed in our laboratory33), and compare this with actions in the central nervous system (CNS), using the cannabinoid tetrad, and CV function by measuring blood pressure and heart rate.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Competing interests
  8. Conflict of Interest
  9. References

The experiments were designed and performed in strict accordance with the EC regulations for care and use of experimental animals (EEC Nº 86/609) and were approved by the Ethical Committee at the Universidad Rey Juan Carlos.

Animals

Male Wistar rats (240–300 g) were obtained from Harlan-Iberica (Barcelona, Spain) and housed (4–6 per cage) in standard transparent cages (60 cm × 40 cm × 20 cm) that were furnished with wood shaving bedding, which was changed every 1–2 days. Cages were placed adjacent to each other under environmentally controlled conditions (temperature = 20 °C; humidity = 60%) with a 12 h light/12 h dark cycle (lights on between 08:00 and 20:00 hours). Animals had free access to standard laboratory rat chow (Harlan-Iberica) and tap water.

Chronic treatment  Experiments were started 1 week after the animals arrived at the laboratory. Every day for 14 consecutive days, rats received an intraperitoneal injection of saline (0.9% NaCl weight/volume), vehicle (see below) or WIN. Two doses of the cannabinoid were tested: a low one (0.5 mg kg−1, low-WIN), which prevents the development of neuropathic signs induced by antitumoral drugs34 and which is likely devoid of central effects,5,35 and a high dose (5 mg kg−1, high-WIN), capable of inducing psychoactive effects.5 Drug volumes were adjusted to a maximum of 4–5 mL kg−1. Central effects and alterations in CV parameters and GI motility were measured after the first (acute effect) and last (chronic effect) drug administrations. In addition, central and GI effects were also evaluated 1 week after last administration (residual effects). Finally, the CB1 antagonist AM 251 (1 mg kg−1, i.p.) was administered 30 min prior to the last high-WIN dose in some animals in order to determine whether or not CB1 receptors are involved in the remaining GI chronic effects of WIN.

Evaluation of body weight and food ingestion  Body weight and food intake were measured each day between 9:00 and 11:00 hours, immediately before drug administration. Food consumption was measured by subtracting the amount remaining from the amount provided the day before for each cage (50 g per animal). Care was taken to collect all particles, which were weighed to correct the values of food consumption to the nearest 1 g. Food consumption represented the combined data from four rats, and at least two cages (eight rats) were included for each treatment. Individually recorded weights served as an indirect indicator of individual food consumption.

Psychoactive effects of WIN 55,212-2 (cannabinoid tetrad)  The classical cannabinoid tetrad test evaluates temperature, antinociception, catalepsy and motility in the same animal after cannabinoid administration.4 The test values were recorded by an observer unaware of the treatments, as previously reported,35 with slight modifications.

Heat-antinociception was tested 20 min after drug administration using a 37 370 plantar test apparatus (Ugo Basile, Comerio, VA, Italy). The withdrawal latency from a focused beam of radiant heat applied to the mid plantar surface of the hind paws was recorded. The intensity of the light was adjusted at the beginning of the experiment so that the control average baseline latencies were about 8 s, and a cut-off latency of 25 s was imposed. The withdrawal latency of each paw was measured during three trials separated by 2 min intervals, and the mean of the three readings was used for data analysis.

To measure catalepsy, the rats were hung by their front paws from a rubber-coated metal ring (12 cm diameter) fixed horizontally at a height that allowed their hind paws to just touch the bench. The time taken for the rat to move off the ring was measured with a cut-off limit of 30 s. Latencies were measured approximately 25 min after drug or vehicle administration.

Rectal temperature was recorded 30 min after drug administration using a P6 thermometer and a lubricated rectal probe (Cibertec S.A., Madrid, Spain) inserted into the rectum to a constant depth of 5 cm.

Spontaneous locomotor activity was evaluated using individual photocell activity chambers (Cibertec S.A.). Rats were placed in the recording chambers (55 × 40 cm, with a 3 cm spacing between beams) 40 min after drug administration, and the number of interruptions of photocell beams was recorded over a 10-min period.36 The mean number of crossings of the photocell beams was used for comparison.

Evaluation of cardiovascular parameters  After anaesthesia with equithesin (3 mL kg−1 i.p.: chloral hydrate 2.1 g, sodium pentobarbital 0.46 g, MgSO4 1.06 g, propylene glycol 21.4 mL, ethanol (90%) 5.7 mL, H2O 23 mL),37 a catheter coupled to a pressure transducer was inserted into the right carotid artery of the animals for direct measurements of mean arterial blood pressure (MBP) and heart rate (HR) using a PowerLab/4e system (PanLab S.L., Barcelona, Spain). Recording of CV parameters started 1 h after drug administration and lasted for 20 min.

Evaluation of GI motor function  Gastrointestinal motor function was studied by radiographic methods previously described.33 Immediately after drug injection, 2.5 mL of a suspension of barium sulphate (Barigraf® AD, Juste SAQF, Madrid, Spain, 2 g mL−1, tº=22 °C) were administered per os. Plain facial radiographs of the GI tract were performed using a Helident DS X-ray apparatus (Sirona Iberica, Barcelona, Spain; 60 kV, 7 mA) with a focus distance manually fixed to 50 ± 1 cm. Exposure time was adjusted to 0.06 s. Immobilization of the rats in prone position was achieved by placing them inside hand-made transparent plastic tubes, which were adjusted to the size of the rat so that they could not move, escape or turn around. Habituation to the recording chamber prior to commencement of the study did not significantly alter GI motility.33 In order to further reduce stress, rats were released immediately after each shot (immobilization lasted for 2–3 min). X-rays were recorded on Kodak Ektavision G film (15 × 30 cm) housed in a hand-made cassette provided with regular intensifying screen at different times (immediately and 1, 2, 4, 6 and 8 h: T0–T8) after administration of the contrast medium. The film cassette was located directly beneath the restraining tube. While taking the radiographs, the qualified investigator remained at least 2 m away from the X-ray source, where radioactivity while shooting was not different from environmental readings. Films were developed in a Kodak X-omat 2000 automatic processor. Analysis of the radiographs was performed by a trained investigator blind to the drug administered. Alterations in GI motility were semiquantitatively determined from the images by assigning a compounded value to each region of the GI tract, from 0 to 12 points, considering the following parameters: percentage of the GI region filled with contrast (0–4); intensity of contrast (0–4); homogeneity of contrast (0–2); and sharpness of the GI region profile (0–2). Qualitative differences in gastric and intestinal shape, size, tone and peristaltic activity were also recorded.

Compounds and drugs  Barium sulphate (Barigraf® AD) was suspended in tap water and continuously hand-stirred until administration. WIN was obtained from Tocris Cookson (Bristol, UK) and dissolved in ethanol 1 mg : 1 mL and, subsequently, in ethanol and Tween 80 (1 : 2), after which, the ethanol was evaporated and saline added to reach the final concentration.22

Statistical analysis

Data are presented as the mean values ± SE differences between groups were analyzed using unpaired Student’s t-test, with Welch’s correction where appropriate or one- or two-way anova followed by post hoc Bonferroni multiple comparison test. Values of P < 0.05 were regarded as being significantly different.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Competing interests
  8. Conflict of Interest
  9. References

Body weight gain and food ingestion

After 14 days, saline-treated rats had gained 3.44 ± 0.22% of body weight and eaten 21.24 ± 0.15 g per day (n = 14). These values were not significantly modified by chronic administration of vehicle (3.71 ± 0.26% and 20.86 ± 0.20 g, n = 14) or low-WIN (3.21 ± 0.22% and 21.30 ± 0.22 g, n = 14), but they were significantly reduced by daily administration of high-WIN (2.16 ± 0.27%, P < 0.001 vs control and 19.46 ± 0.42 g, P < 0.01 vs control, n = 14).

Psychoactive effects (cannabinoid tetrad)

As shown in Fig. 1, neither acute nor chronic administration of the vehicle used to deliver the cannabinoids induced any of the signs of the cannabinoid tetrad as compared with saline-treated animals.

image

Figure 1.  Psychoactive effects of the cannabinoid agonist WIN 55,212-2 in the rat. Cannabinoid psychoactive effects were evaluated using the cannabinoid tetrad: A-plantar test (for heat-analgesia); B-ring test (for catalepsy); C-rectal temperature (for hypothermia); D-spontaneous locomotor activity (for hypolocomotion). Rats were injected i.p. daily for 14 days with: saline (4–5 mL kg−1, black bars, n = 8), vehicle (white bars, n = 8), WIN 55,212-2 (WIN) at 0.5 (hatched bars, n = 8) or 5 mg kg−1 (grey bars, n = 8). Values were obtained after first administration (acute), last administration (chronic) or one week after last administration (residual). Bars show mean values ± SE *P < 0.05, ***P < 0.001 vs vehicle (Two-Way anova followed by Bonferroni post hoc test); + P < 0.05, ++ P < 0.01, +++ P < 0.001 vs acute WIN 5 mg kg−1 (unpaired t-test).

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After the first administration (acute effect), low-WIN did not modify the tetrad values in a significant manner, although it tended to induce some catalepsy (Fig. 1B). However, high-WIN induced significant analgesia (Fig. 1A), catalepsy (Fig. 1B), hypothermia (Fig. 1C) and a decrease in spontaneous locomotor activity (Fig. 1D).

After daily treatment (chronic effect), low-WIN was still without effect. In those animals treated with high-WIN, tolerance had fully developed to hypothermia (Fig. 1C) and hypolocomotion (Fig. 1D), whereas catalepsy (Fig. 1B) and analgesia (Fig. 1A) were still present to some extent. No residual central effect was detected 1 week after the end of chronic treatment (Fig. 1).

Cardiovascular effects (MBP and HR)

Neither acute nor chronic administration of the cannabinoid vehicle induced any modification in MBP or HR as compared with saline-treated animals.

Irrespective of the time point for recording (after first or last injection), administration of WIN (0.5 or 5 mg kg−1) did not induce any modification of either MBP (acute: low-WIN : 116.61 ± 14.77 mmHg, n = 8; high-WIN : 101.54 ± 12.75 mmHg, n = 8; chronic: low-WIN : 107.06 ± 8.41 mmHg, n = 8; high-WIN : 105.34 ± 5.78 mmHg, n = 5) or HR (acute: low-WIN : 397.08 ± 20.00 beats min−1, n = 8; high-WIN : 371.65 ± 24.81 beats min−1, n = 8; chronic: low-WIN : 383.88 ± 16.09 beats min−1, n = 8; high-WIN : 337.14 ± 24.96 beats min−1, n = 5), as compared with saline-treated animals (acute: MBP: 109.53 ± 5.55 mmHg, n = 7; HR: 342.66 ± 14.42 beats min−1, n = 7; chronic: MBP: 116.85 ± 8.91 mmHg, n = 8; HR: 358.68 ± 8.15 beats min−1, n = 8).

Gastrointestinal motor function

Neither acute nor chronic administration of vehicle induced any significant alteration in gastric emptying as compared with saline-treated animals (Fig. 2). Gastric emptying was delayed after acute administration of high- but not low-WIN (Fig. 2A,D,E), and this effect was still present after the last daily administration (chronic effect, Fig. 2B,F,G). One week after chronic treatment was ceased, the stomach motility curves were practically identical in all animals (Fig. 2C,H,I).

image

Figure 2.  Effect of the cannabinoid agonist WIN 55,212-2 on gastric emptying in the rat. Gastric emptying was measured by radiological methods (see text). Saline (4–5 mL kg−1, open circles, n = 8), vehicle (closed circles, n = 8), or WIN 55,212-2 (WIN) at 0.5 (open triangles, n = 8) or 5 mg kg−1 (closed triangles, n = 8) were administered i.p. daily for 14 days. Barium sulphate (2.5 mL, 2 g mL−1) was intragastrically administered to test the following effects: acute (after first administration, A), chronic (after last administration, B), and residual (1 week after treatment had ceased, C). X-rays were taken 0, 1, 2, 4, 6, and 8 h after barium administration. Data represent mean ± SE *P < 0.05, **P < 0.01, ***P < 0.001 vs vehicle (Two-Way anova followed by Bonferroni post hoc test). D–I: representative X-rays of rats treated with vehicle or WIN 5 mg kg−1 (WIN 5) taken 2 h after barium administration. Scale bar: 4 cm.

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The motility curves for the small intestine are shown in Fig. 3. As in the stomach, saline- and vehicle-treated animals showed similar curves after the first (Fig. 3A) and last daily administration (Fig. 3B) as well as 1 week after discontinuing treatment (Fig. 3C). Thus, maximum filling and half emptying were reached 1–2 and 4 h, respectively, after contrast administration in these control animals. Low-WIN did not significantly or consistently alter these curves (Fig. 3). However, both intestinal filling (it took at least 4 h to reach its maximum) and emptying (it needed at least 6 h to start) were significantly delayed after acute administration of high-WIN (Fig. 3A). Some tolerance was developed to this effect because maximum filling was already reached 2 h after the last high-WIN administration, and emptying started immediately after that time point, although it was still incomplete at the end of the experiment (Fig. 3B). No residual effect was detected in small intestinal motility 1 week after discontinuing treatment (Fig. 3C).

image

Figure 3.  Effect of the cannabinoid agonist WIN 55,212-2 on motor function of small intestine in the rat. Motor function was measured by radiological methods (see text). Saline (4–5 mL kg−1, open circles, n = 8), vehicle (closed circles, n = 8), or WIN 55,212-2 (WIN) at 0.5 (open triangles, n = 8) or 5 mg kg−1 (closed triangles, n = 8) were administered i.p. daily for 14 days. Barium sulphate (2.5 mL, 2 g mL−1) was intragastrically administered to test the following effects: acute (after first administration, A), chronic (after last administration, B), and residual (1 week after treatment had ceased, C). X-rays were taken 0, 1, 2, 4, 6, and 8 h after barium administration. Data represent mean ± SE *P < 0.05, **P < 0.01, ***P < 0.001 vs vehicle (Two-Way anova followed by Bonferroni post hoc test). D–I: representative X-rays of rats treated with vehicle or WIN 5 mg kg−1 (WIN 5) taken 1 h after barium administration. Scale bar: 4 cm.

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As described for the previous GI regions, motility curves for caecum in saline- and vehicle-treated rats were not significantly different (Fig. 4). However, acute administration of WIN dose-dependently delayed filling of the caecum (Fig. 4A,D,E). Although some effect was still present after the last administration of WIN at either dose, tolerance had clearly developed, at least to the effect of the highest dose (Fig. 4B,F,G). One week after treatment was finished, the animals that had received high-WIN, caecum filling was slightly faster than in control or low WIN-treated rats (Fig. 4C,H,I).

image

Figure 4.  Effect of the cannabinoid agonist WIN 55,212-2 on motor function of caecum in the rat. Motor function was measured by radiological methods (see text). Saline (4–5 mL kg−1, open circles, n = 8), vehicle (closed circles, n = 8), or WIN 55,212-2 (WIN) at 0.5 (open triangles, n = 8) or 5 mg kg−1 (closed triangles, n = 8) were administered i.p. daily for 14 days. Barium sulphate (2.5 mL, 2 g mL−1) was intragastrically administered to test the following effects: acute (after first administration, A), chronic (after last administration, B), and residual (1 week after treatment had ceased, C). X-rays were taken 0, 1, 2, 4, 6, and 8 h after barium administration. Data represent mean ± SE *P < 0.05, **P < 0.01, ***P < 0.001 vs vehicle (Two-Way anova followed by Bonferroni post hoc test). D–I: representative X-rays of rats treated with vehicle or WIN 5 mg kg−1 (WIN 5) taken 4 h after barium administration. Scale bar: 4 cm.

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Motility curves of the colorectal region were also similar for saline- and vehicle-treated animals. As in the caecum, filling of this region was dose-dependently delayed after the first dose of WIN (Fig. 5A,D,E), and tolerance developed upon daily treatment (Fig. 5B,F,G). No significant or consistent residual effect was found 1 week after last drug administration (Fig. 5C,H,I).

image

Figure 5.  Effect of the cannabinoid agonist WIN 55,212-2 on motor function of colorectum in the rat. Motor function was measured by radiological methods (see text). Saline (4–5 mL kg−1, open circles, n = 8), vehicle (closed circles, n = 8), or WIN 55,212-2 (WIN) at 0.5 (open triangles, n = 8) or 5 mg kg−1 (closed triangles, n = 8) were administered i.p. daily for 14 days. Barium sulphate (2.5 mL, 2 g mL−1) was intragastrically administered to test the following effects: acute (after first administration, A), chronic (after last administration, B), and residual (1 week after treatment had ceased, C). X-rays were taken 0, 1, 2, 4, 6, and 8 h after barium administration. Data represent mean ± SE *P < 0.05, **P < 0.01, ***P < 0.001 vs vehicle (Two-Way anova followed by Bonferroni post hoc test). D–I: representative X-rays of rats treated with vehicle or WIN 5 mg kg−1 (WIN 5) taken 8 h after barium administration. Scale bar: 4 cm.

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Involvement of CB1 receptors in the remaining GI effects after daily WIN

When the CB1 antagonist AM 251 (1 mg kg−1) was administered 30 min prior to the last high-WIN administration, the remaining delay in gastric emptying and intestinal motility was completely blocked, and a slight, although significant, accelerating effect was detected in the motility curve of the colorectum. When a single dose of the antagonist was administered alone, gastric emptying and filling of the caecum and the colorectum were slightly but significantly accelerated as compared with vehicle-only injected animals (Fig. 6).

image

Figure 6.  Effect of the cannabinoid CB1 antagonist AM 251 on gastrointestinal motor function in the rat. Motor function was measured by radiological methods (see text). Rats were injected i.p. daily for 14 days with vehicle (closed circles, n = 8) or WIN 55,12-2 (WIN) at 5 mg kg−1 (closed triangles, n = 8). In some animals, AM 251 (1 mg kg−1) was administered 30 min prior to the last dose of vehicle (open squares, n = 8) or WIN 5 mg kg−1 (closed squares, n = 8). Barium sulphate (2.5 mL, 2 g mL−1) was intragastrically administered immediately after last administration. X-rays were taken 0, 1, 2, 4, 6, and 8 h after barium administration. Data represent mean ± SE for motor function in stomach (A), small intestine (B), caecum (C) and colorectum (D). *P < 0.05, **P < 0.01, ***P < 0.001 vs vehicle (Two-Way anova followed by Bonferroni post hoc test). E-H: representative X-rays of rats treated with vehicle, WIN (WIN 5), WIN and AM 251 (WIN 5+ AM) or AM 251 only (AM) taken 6 h after barium administration. Scale bar: 4 cm.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Competing interests
  8. Conflict of Interest
  9. References

In the present study, the acute and chronic effects of cannabinoid agonist administration in three different systems (central nervous, CV and GI) were analyzed in parallel in the rat. Under similar experimental conditions, the different systems show differential sensitivity to the effects of the mixed cannabinoid agonist WIN and the development of tolerance. Thus, although no alteration in CV parameters was detected after acute administration of WIN, it induced the typical central signs (cannabinoid tetrad), as well as depression of GI motility. After chronic administration, no CV effects, hypolocomotion, or hypothermia were detected, whereas delayed gastric emptying, analgesia and some catalepsy were still present, suggesting that these effects are relatively resistant to the development of tolerance.

Effects of acute administration of WIN

In the early 1970’s, several authors reported that THC reduced defecation in rats38,39 and delayed passage of a charcoal meal in mice.40 However, this is the first time that the effect of a cannabinoid on GI motility has been analyzed in experimental animals using radiographic techniques, which allow the analysis of different regions of the GI tract at several time points in the same individual.33

In agreement with previous reports,13,15,16,18,21 when acutely administered, the cannabinoid agonist WIN exerted its depressive action in both stomach and intestine, and this effect was transient.30 Thus, the stomach of high WIN-treated animals was practically inactive for 2 h, but afterwards, gastric emptying returned to normal, and the slope of the curve was similar to that for control animals (Fig. 2A).

Although the reduction in small intestinal transit described here may be influenced by delayed gastric emptying, the fact that low cannabinoid doses were effective in reducing intestinal transit and not gastric emptying supports the notion that cannabinoids depress intestinal motility independently of their effects on gastric emptying.13,18 Moreover, if WIN did not exert an independent action on the intestine, the radiographic motility curves for the small intestine, caecum and colorectum would have simply been displaced to the right but would have kept approximately the same shape/slope as in controls, as after treatment with other drugs that mainly delay gastric emptying.33 Instead, in rats treated with high-WIN, the curve for the small intestine was wider (Fig. 3A), and the slopes of those for caecum (Fig. 4A) and colorectum (Fig. 5A) were slower than in saline- or vehicle-treated rats. In addition, although our method does not allow for a quantitative analysis of peristaltic activity, peristaltic waves in the small intestine were less apparent after WIN administration (compare Fig. 3D,F with Fig. 6F), further supporting the existence of a direct action in the small intestine.

Our results also indicate that the intestine might be more sensitive than the stomach to the depressive effect of cannabinoids, which is in disagreement with other studies reporting similar sensitivity for both regions.13,15,16,18 In those studies, analyses were made within 1 h after drug administration using invasive techniques. In our study, alterations in motility curves of all GI regions were best detected 2–8 h after drug administration, but 1 h after high-WIN, gastric emptying tended to be slower, and motility in the small intestine was already significantly uncoupled.

Central and CV acute effects of WIN were evaluated at least 20 min and no later than 2 h after drug administration, while gastric emptying was still uncoupled. As expected, WIN induced the typical central effects.4,5,36 On the other hand, even though most cannabinoids induce both bradycardic and hypotensive effects in the rat,8,41 we did not detect any remaining effect 1 h after WIN administration. Differences in the time point chosen for recording and/or the methodology used might explain the discrepancy with other reports. In fact, we have observed that intraperitoneal WIN administration provokes a transient decrease in MBP (about 20 mmHg) and HR (about 30 beats min−1) in the rat, starting 5 min and lasting for 30–40 min after administration (unpublished observations).

Interestingly, these results suggest that different systems respond differently to the same cannabinoid treatment. In both GI tract and CNS, whose functions were acutely depressed by WIN, cannabinoids are known to exert their actions through a similar mechanism, the presynaptic inhibition of neurotransmitter release.17,42 In contrast, in the CV system, cannabinoids act on both nerve fibres innervating the tissue and the tissue itself.11

Effects of chronic administration of WIN

No alterations in MBP or HR were found after chronic administration of WIN. In contrast, the last administration of WIN still induced some effects on GI motility and CNS, whereas other cannabinoid-induced alterations were less intense or even disappeared.

In agreement with previous reports,30,43–46 some central effects were more resistant to attenuation after chronic treatment than others. Thus, tolerance was complete to hypothermia and hypolocomotion and partial to catalepsy, whereas antinociception remained unchanged, which might be a useful feature if cannabinoid agonists were used therapeutically.

Most studies of GI signs of tolerance have been carried out in vitro.17,31,32 Nevertheless, an early report showed that mice pretreated with THC at 10 mg kg−1 orally once daily for 2–4 days developed long-lasting tolerance to the inhibitory effect of this cannabinoid on the passage of a charcoal meal.30 In our study, tolerance to reduced intestinal transit (measured as arrival and filling of the caecum) developed after daily treatment for 14 days, but tolerance to gastric emptying delay did not develop to the same extent, and 4–8 h after the last high-WIN administration, a significant amount of barium was still present in the stomach. In agreement with previous reports testing WIN and other cannabinoid agonists,47–49 daily food intake and body weight gain were significantly lower in rats receiving high-WIN each day. Although anxiogenic effects50 and some depressed locomotor activity could have contributed, the fact that each administration delayed gastric emptying for about 2 h might have also influenced feeding and body weight gain.

It is likely that treatments longer than 14 days are needed to fully attenuate WIN-induced central and GI effects. In any case, our results support the idea that development of tolerance may be dependent on the particular cannabinoid effect and the anatomical region acted upon.46 Furthermore, it might involve different mechanisms for hypothermia, catalepsy, and reduction of intestinal transit on one hand, and for antinociception and gastric emptying delay, on the other.

As reported for other WIN chronic effects, like anxiogenic responses,50 1 week after the last dose, no residual GI or central effects were apparent. This is not surprising as the half life of WIN is 24–36 h.47,50 Thus, those cannabinoid effects that are resistant to tolerance are also relatively short-lasting. As acute or daily WIN administration did not produce any permanent MBP or HR changes, it seems that chronic administration of cannabinoids, even at high doses, might be relatively safe.

CB1 receptor involvement in GI motor function

Although cannabinoids may modulate GI function through central receptors,21,51 they are known to primarily decrease GI contractility and peristalsis by a direct action on the myenteric neurons.23,52–55 CB1 receptors, located in these neurons,25,26,56 are responsible for the effects on GI motility in vitro and in vivo.14,16 Although we have not totally excluded the involvement of CB2 receptors, which are now known to exist in the brain and, perhaps, in the peripheral nervous system, and have been implicated in emesis57 and changes in motility induced by lipopolysaccharide,58 the remaining GI effect (namely, gastric emptying delay) after daily WIN treatment was completely blocked by the CB1 antagonist AM 251, further confirming that CB1 receptors are important modulators of GI motor function, even after chronic cannabinoid treatment.

The CB1 antagonist showed some accelerating effect in most GI regions when administered alone, which is in agreement with previous reports suggesting that CB1 antagonists, including AM 251,59 might exert an inverse agonist action or unmask an endogenous cannabinoid inhibitory tone, in the GI tract.14,21,23,52,60,61 However, whereas in previous reports in which invasive methods were used, the CB1 antagonist rimonabant did not alter gastric emptying per se in fasted rats,15,18 we did find a slight transient accelerating effect on this parameter. Differences in methodology and/or the drug used might account for this discrepancy.

Concluding remarks

The therapeutic potential of chronic cannabinoid administration might be counteracted by the presence of persistent adverse effects. Our data indicate that CV function might remain well preserved but that some central and GI signs might still occur during chronic treatment. Thus, under similar experimental conditions, the different systems show differential sensitivity to the effects of cannabinoids and to the development of tolerance. Although chronic cannabinoid treatment, even at high doses, might be relatively safe, delayed gastric emptying seems particularly resistant to attenuation, and this could have some influence on feeding and weight gain. Therefore, our data suggest that monitoring GI motor function could improve safety of chronic cannabinoid treatment.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Competing interests
  8. Conflict of Interest
  9. References

This work was supported by Ministerio de Educación y Ciencia (SAF2006-13391-C03-01), Universidad Rey Juan Carlos – Comunidad de Madrid (URJC-CM-2006-BIO-0604), and Comunidad de Madrid (S-SAL/0261/2006). The authors wish to thank Óscar Gutiérrez (Servicio de Radiología, Clínica Universitaria, Universidad Rey Juan Carlos) for technical radiological advice.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Competing interests
  8. Conflict of Interest
  9. References