Daily treatment with cannabinoids results in tolerance to many, but not all, of their behavioral and physiological effects. The present studies investigated the effects of 7-day exposure to 10 mg/kg/day Δ9-tetrahydrocannabinol (THC) on the diuretic and antinociceptive effects of THC and the synthetic cannabinoid AM4054. Comparison studies determined diuretic responses to the κ-opioid agonist U50,488 and furosemide. Following determination of control dose-response functions, mice received 10 mg/kg/day THC for 7 days, and dose-response functions were redetermined 24h, 7 days, or 14 days later. THC and AM4054 had biphasic diuretic effects under control conditions with maximum effects of 30 and 35 ml/kg urine, respectively. In contrast, antinociceptive effects of both drugs increased monotonically with dose to >90% MPE. Treatment with THC produced 9- and 3-fold rightward shifts of the diuresis and antinociception dose-response curves for THC and, respectively, 7- and 3-fold rightward shifts in the AM4054 dose-response functions. U50,488 and furosemide increased urine output to >35 ml/kg under control conditions. The effects of U50,488 were attenuated following 7-day treatment with THC, whereas the effects of furosemide were unaltered. Diuretic effects of THC and AM4054 recovered to near-baseline levels within 14 days after stopping daily THC injections, while tolerance to the antinociceptive effects persisted longer than 14 days. The tolerance induced by 7-day treatment with THC was accompanied by a 55% decrease in the Bmax value for cannabinoid CB1 receptors. These data indicate that repeated exposure to THC produces similar rightward shifts in the ascending and descending limbs of cannabinoid diuresis dose-effect curves, and to antinociceptive effects, while resulting in a flattening of the U50,488 diuresis dose-effect function.
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In contrast to the effects of the cannabinoid agonists, the cannabinoid antagonist rimonabant had neither diuretic nor hypothermic effects ( Fig. 2 ). Rimonabant also did not have antidiuretic effects in rats that had been preloaded with 10.0–30.0 ml/kg water ( Table 3 ). Although diuresis was slightly increased after 0.3 mg/kg rimonabant in rats prehydrated with 30.0 ml/kg water, this effect was not dose-related and further increases in dose to 1.0 and 3.0 mg/kg did not reveal any significant increase in diuresis. Among other cannabinergic drugs that were evaluated for diuretic effects, only methanandamide increased voided urine and, these effects were observed only with a single dose, 10 mg/kg. In contrast to other CB1 agonists displaying intermediate effects, a 3-fold increase in dose, to 30 mg/kg of methanandamide, did not correspondingly increase urine output but, instead, reduced diuresis to control levels. The remaining drugs, including the CB2 selective cannabinoid agonist AM1241 and the endocannabinoid transport inhibitor AM404, were without effect on urine output ( Table 4 ).
After initial exposure to handling procedures, thermal probes (YSI, No. 401) were inserted rectally to a depth of 7 cm, 30 minutes before drug administration. The probes were secured to the tails with porous tape, and the animals were placed in individual chambers measuring 30 × 12 × 12 cm. Two baseline temperature readings were recorded before drug injection, and temperature was recorded every 30–60 minutes for 6 hours after injection. The change in temperature was determined for each rat by subtracting temperature readings from the mean of the 2 baseline measures.
Diuresis and hypothermia after 0.1 or 0.3 mg/kg AM4054 were compared with saline and 10 mg/kg furosemide at different times over a 6-hour period. AM4054 had a very quick onset to action, and relative to saline, AM4054 significantly increased urine loss within 30 minutes after injection, with maximum values obtained 60–120 minutes after injection. In contrast, significant decreases in body temperature occurred later, with peak effects occurring more than 2 hours after injection ( Fig. 3 ). To determine whether fluid loss contributes, at least in part, to the later onset of hypothermic effects, the diuretic and hypothermic effects of pentobarbital given alone and in combination with 10.0 mg/kg furosemide were determined. Pentobarbital, at 10.0–30.0 mg/kg, did not produce diuresis ( Table 4 ) but did decrease temperature (F3,20 = 9.13; P < 0.05); peak hypothermic effects of 30.0 mg/kg pentobarbital, a decrease in temperature of 5.2±1.0°C, occurred 2.5–3 hours after injection with a recovery toward baseline temperatures within 6 hours. The dose of 10.0 mg/kg furosemide alone had no effects on body temperature but did produce significant diuresis within 30 minutes after injection (see Fig. 3 ). Coadministration of 30.0 mg/kg pentobarbital and 10.0 mg/kg furosemide did not have additive effect in either measure. Thus, rats that received both pentobarbital and furosemide voided 27.2±5.1 g/kg urine and had peak hypothermic effects of 5.3±0.6°C that recovered toward baseline values within 6 hours after injection.
Effects of Water-Loading and Noncannabinioid Diuretics.
Many synthetic cannabinoid ligands have been developed in the past 20 years, including compounds such as CP55,940 and WIN55,212 (Devane et al., 1988; Compton et al., 1992); however, potential diuretic effects of these drugs have not been previously evaluated. The present studies were undertaken to compare the net diuretic effects of systemic administration of cannabinoid agonists in conscious rats. Results show that CB1 agonists dose-dependently increase diuresis concomitantly with hypothermic effects, and further studies with the nonselective agonist AM4054 (Thakur et al., 2012) reveal that these effects are antagonized by rimonabant. Overall, our data indicate that diuresis is a CB1-mediated effect that may serve as a reliable and objective physiologic measure of cannabinoid action in rats; the circumstances under which these results represent a potential therapeutic benefit or potential liability of cannabinoids remain to be determined.
Adult female Sprague-Dawley rats (Charles River, Wilmington, MA) weighing 200–350 g and adult male Sprague-Dawley rats weighing 530–770 g were used. Animals were group-housed in a climate-controlled vivarium with a 12-hour light/dark cycle (lights on at 7 AM ). Subjects had unrestricted access to food and water outside experimental sessions. All studies were approved by the McLean Hospital and Northeastern University Institutional Animal Care and Use Committees.
(A) Diuretic effects after injection of vehicle, 0.1 mg/kg AM4054, or 0.1 mg/kg AM4054 after a 15-minute pretreatment with 10 mg/kg capsazepine. (B) Diuretic effects after injection of vehicle; 3.0 mg/kg WIN 55,212; or 3.0 mg/kg WIN 55,212 after a 30-minute pretreatment with 10 mg/kg AM630; other details as in Fig 2 .
Voided urine after injection with listed drugs in female rats (n = 6 except saline, n = 6–12)