The cannabinoid agonist WIN55,212-2 increases intracellular calcium via CB1 receptor coupling to Gq/11 G proteins

Abstract

Central nervous system responses to cannabis are primarily mediated by CB(1) receptors, which couple preferentially to G(i/o) G proteins. Here, we used calcium photometry to monitor the effect of CB(1) activation on intracellular calcium concentration. Perfusion with 5 microM CB(1) aminoalkylindole agonist, WIN55,212-2 (WIN), increased intracellular calcium by several hundred nanomolar in human embryonic kidney 293 cells stably expressing CB(1) and in cultured hippocampal neurons. The increase was blocked by coincubation with the CB(1) antagonist, SR141716A, and was absent in nontransfected human embryonic kidney 293 cells. The calcium rise was WIN-specific, being essentially absent in cells treated with other classes of cannabinoid agonists, including Delta(9)-tetrahydrocannabinol, HU-210, CP55,940, 2-arachidonoylglycerol, methanandamide, and cannabidiol. The increase in calcium elicited by WIN was independent of G(i/o), because it was present in pertussis toxin-treated cells. Indeed, pertussis toxin pretreatment enhanced the potency and efficacy of WIN to increase intracellular calcium. The calcium increases appeared to be mediated by G(q) G proteins and phospholipase C, because they were markedly attenuated in cells expressing dominant-negative G(q) or treated with the phospholipase C inhibitors U73122 and ET-18-OCH(3) and were accompanied by an increase in inositol phosphates. The calcium increase was blocked by the sarco/endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin, the inositol trisphosphate receptor inhibitor xestospongin D, and the ryanodine receptor inhibitors dantrolene and 1,1'-diheptyl-4,4'-bipyridinium dibromide, but not by removal of extracellular calcium, showing that WIN releases calcium from intracellular stores. In summary, these results suggest that WIN stabilizes CB(1) receptors in a conformation that enables G(q) signaling, thus shifting the G protein specificity of the receptor.

Fig. 1.

Activation of CB₁ by WIN increased [Ca²⁺]_i to a variable extent and with varied kinetics. (A) The time course of changes in [Ca²⁺]_i in HEK293 cells loaded with fura-2 and perfused with 5 μM WIN. The lines above indicate drug application. The solid line indicates the WIN-induced response in CB₁-HEK293 (n = 8). A subset of CB₁-HEK293 were coperfused with 1 μM of the CB₁ antagonist SR (dashed line, n = 5). The change in [Ca²⁺]_i in response to WIN in nontransfected HEK293 cells is also shown (dotted line, n = 8). (B) Calcium elevations with WIN recorded in four different CB₁-HEK293 cells, tested on 4 different days. (C)CB₁-HEK293 were perfused with 5 μM WIN (solid line), and HEK293 cells transiently expressing M₁R were perfused with 10 μM Oxo-M (dashed line). These traces show the change in calcium in representative cells. (D) The black bar shows the 10-90% rise time for the calcium caused by 5 μM WIN in CB₁-HEK293 (n = 6). Open bar indicates the calcium rise time caused by a 40-s application of 10 μM Oxo-M in HEK293 cells transiently expressing M₁R (n = 7). The rise times were significantly different (P < 0.01, t test).

Fig. 2.

Only the AAI agonist WIN caused a large increase in [Ca²⁺]_i in CB₁-HEK293. The WIN-induced change in [Ca²⁺]_i (black bar, n = 5) is significantly greater than that of other agonists (P < 0.05, ANOVA). Agonists tested: the classical cannabinoids, THC (10 μM, n = 9), HU-210 (1 μM, n = 4), the phytocannabinoid, cannabidiol (CBD, 3 μM, n = 4), the nonclassical cannabinoid, CP (n = 6), the endocannabinoid, 2-AG (n = 5), and the anandamide analog, AM356 (n = 9). All agonists were perfused for 150 s.

Fig. 3.

PTX pretreatment augmented the WIN-induced [Ca²⁺]_i increase in CB₁-HEK293. Black bars indicate the response in untreated cells, and open bars indicate overnight treatment with 500 ng/ml PTX. (A) Pretreatment with PTX enhanced the rise in [Ca²⁺]_i at all WIN concentrations. n = 5 for all conditions. (B) PTX pretreatment had no effect on the 10-90% rise time for the calcium rise following perfusion of 3 or 5 μM WIN.

Fig. 4.

G_q/11 is involved in the WIN-induced increase in [Ca²⁺]_i.(A) The rise in [Ca²⁺]_i during perfusion of either 5 μM WIN (left three bars) or 10 μM Oxo-M (right two bars) is plotted. Control cells were not treated with PTX and did not express dominant negative G_αq (black bars, n = 10 for WIN control, n = 4 for Oxo-M control). The rise in [Ca²⁺]_i in HEK293 cells expressing DN G_αq was less than control (P < 0.001 for WIN-treated and P < 0.05 for Oxo-M treated, t test). PTX pretreatment (open bar, n = 5) augments the rise in calcium (P < 0.05, t test). (B) The change in calcium upon WIN application in a representative cell for each treatment. Solid line indicates WIN only, dashed line is WIN+DN G_αq, and dotted line is WIN+PTX.

Fig. 5.

PLC mediates the increase in [Ca²⁺]_i by WIN. (A) CB₁-HEK293s were either untreated (black bar, n = 12) or pretreated for 160 s with either 3 μM of the PLC inhibitor U73122 (n = 5) or 3 μM of its inactive analog U73343 (n = 4) followed immediately by perfusion of 5 μM WIN for 150 s. U73122 strongly suppressed the calcium rise (P < 0.01, t test), whereas U73343 had no significant effect. (B) Neither 3 μM U73122 (solid line) nor 3 μM U73343 (dashed line) increased [Ca²⁺]_i on its own.

Fig. 6.

WIN increases IP levels. CB₁-HEK293 transiently transfected with M₁R (solid bars, n = 9) and nontransfected HEK293 cells (open bars, n = 6) were stimulated with agonist for 10 min and then assayed for IP production. IP levels increased in CB₁-HEK293 treated with either 3 or 5 μM WIN compared with basal levels or levels in nontransfected controls. In cells expressing M₁R, 10 μM Oxo-M robustly increased IP accumulation. *, comparison with basal control; †, comparison with nontransfected HEK293 cells treated with the same agonist concentration. * and †, P < 0.05; ** and ††, P < 0.01.

Fig. 7.

WIN releases calcium from IP₃- and Ry-sensitive intracellular stores. In CB₁-HEK293, the 5 μM WIN-induced rise in [Ca²⁺]_i did not require extracellular calcium (“0 Ca,” n = 12 vs. “Control,” black bar, n = 16). One micromolar TG (n = 13), the IP₃R inhibitor XeD, 1 μM, n = 10), the RyR inhibitors dantrolene (Dan, 10 μM, n = 11) and 1,1′-diheptyl-4,4′-bipyridinium dibromide (DHBP) (50 μM, n = 6) all reduced calcium rises in response to WIN (P < 0.05, ANOVA), as did coapplied XeD and Dan (n = 4). The reversible SERCA pump inhibitor 2,5-di-t-butyl-1,4-benzohydroquinone (BHQ) in the absence of WIN, modestly increased calcium (100 μM, n = 5, P < 0.05, ANOVA). (Inset) Removal of extracellular calcium (“0 Ca,” open bar, n = 12) affected neither the latency nor rise time of the calcium transient (“Control,” black bar, n = 16). Latency was calculated as the time from start of drug application until the [Ca²⁺]_i rose to 10% of the maximum value.