Agonist-dependent cannabinoid receptor signalling in human trabecular meshwork cells

Abstract

Background and purpose: Trabecular meshwork (TM) is an ocular tissue involved in the regulation of aqueous humour outflow and intraocular pressure (IOP). CB1 receptors (CB1) are present in TM and cannabinoid administration decreases IOP. CB1 signalling was investigated in a cell line derived from human TM (hTM).

Experimental approach: CB1 signalling was investigated using ratiometric Ca2+ imaging, western blotting and infrared In-Cell Western analysis.

Key results: WIN55212-2, a synthetic aminoalkylindole cannabinoid receptor agonist (10-100 microM) increased intracellular Ca2+ in hTM cells. WIN55,212-2-mediated Ca2+ increases were blocked by AM251, a CB1 antagonist, but were unaffected by the CB2 antagonist, AM630. The WIN55,212-2-mediated increase in [Ca2+]i was pertussis toxin (PTX)-insensitive, therefore, independent of Gi/o coupling, but was attenuated by a dominant negative Galpha(q/11) subunit, implicating a Gq/11 signalling pathway. The increase in [Ca2+]i was dependent upon PLC activation and mobilization of intracellular Ca2+ stores. A PTX-sensitive increase in extracellular signal-regulated kinase (ERK1/2) phosphorylation was also observed in response to WIN55,212-2, indicative of a Gi/o signalling pathway. CB1-Gq/11 coupling to activate PLC-dependent increases in Ca2+ appeared to be specific to WIN55,212-2 and were not observed with other CB1 agonists, including CP55,940 and methanandamide. CP55940 produced PTX-sensitive increases in [Ca2+]i at concentrations>or=15 microM, and PTX-sensitive increases in ERK1/2 phosphorylation.

Conclusions and implications: This study demonstrates that endogenous CB1 couples to both Gq/11 and Gi/o in hTM cells in an agonist-dependent manner. Cannabinoid activation of multiple CB1 signalling pathways in TM tissue could lead to differential changes in aqueous humour outflow and IOP.

Figure 1

CB₁ receptor is present in hTM cells. (a) hTM cells labelled with anti-human CB₁ rabbit polyclonal IgG (−BP), and hTM cells treated with 1:1 mixture of anti-CB₁ rabbit polyclonal IgG with blocking peptide (+BP). AlexaFluor 546 goat anti-rabbit IgG was used as the secondary antibody. (b) CB₁ was detected using western blot analysis. Four prominent bands are present, ranging from 97 to 45 kDa (−BP), three of which were substantially reduced following pre-absorption with a blocking peptide (+BP). CB₁ was detected at the expected size for this antibody (66–64 kDa). α-Tubulin was used as a loading control. (c) Reverse transcription-PCR was performed using normal hTM cells and hTM(rCB₁) cells, which stably overexpress human CB₁. β-Actin was used as a loading control. htm, human trabecular meshwork; IgG, immunoglobulin G.

Figure 2

WIN55212-2 and CP55940 induce an increase in [Ca²⁺]_i in hTM cells. (a) Ratiometric calcium imaging of hTM cells in the absence and presence of 10 μM WIN55,212-2. The representative image was captured at the peak increase in [Ca²⁺]_i (18 min). (b) hTM cells were treated with 10 μM WIN55,212-2 for 5 min. WIN55,212-2 induces an increase in [Ca²⁺]_i, represented as an increase in the fura-2 ratio (340/380 nm), that is sustained for 17.2±1.4 min. (c) Mean Ca²⁺ increases at concentrations of 0.1–100 μM WIN55,212-2, CP55940 and methananadamide. (d) The WIN55,212-2-induced increase in [Ca²⁺]_i is mediated by the CB₁ receptor. Graph shows the mean peak amplitude of the Ca²⁺ increase for 10 μM WIN55,212-2 in the absence or presence of AM251 (1 μM, n=24; 10 μM, n=20), a specific CB₁ receptor antagonist, or AM630 (1 μM, n=26; 10 μM, n=18), a specific CB₂ receptor antagonist. ^*P<0.05 and ^**P<0.01 in comparison with 10 μM WIN55,212-2 control (no inhibitor).

Figure 3

[Ca²⁺]_i increase involves phospholipase C and mobilization of Ca²⁺ stores. (a) The mean peak amplitude of the WIN55,212-2-induced [Ca²⁺]_i increase following pretreatment with the sarco/endoplasmic reticulum calcium ATPase inhibitors thapsigargin (TG) (n=23) and cyclopiazonic acid (CPA) (n=29). (b) The mean peak amplitude of the WIN55,212-2-induced [Ca²⁺]_i increase in the presence of 0.1 μM (n=22) and 1 μM (n=22) U73122, a phospholipase C inhibitor, and 1 μM U73343 (n=18), the inactive analogue of U73122. ^**P<0.01 and ^***P<0.001 in comparison with 10 μM WIN55,212-2 control (no inhibitor).

Figure 4

WIN55,212-2(WIN)-mediated [Ca²⁺]_i increase is a result of CB₁-G_q/11 signal transduction. (a) The peak amplitude of the WIN and CP55940 (CP)-induced [Ca²⁺]_i increases in hTM cells that had been incubated in 500 ng ml⁻¹ of pertussis toxin for 20–24 h (+PTX) in comparison to control cells (−PTX). WIN: −PTX, n=28; +PTX, n=23. CP: −PTX, n=38; +PTX, n=25. (b) The peak amplitudes of the WIN-induced [Ca²⁺]_i increases in hTM cells receiving a mock transfection (n=12), cells receiving the empty vector (n=3) and cells expressing a dominant-negative G_q/11 α-subunit (DNG_αq) protein (n=6). ^*P< 0.05 in comparison with pIRES2-enhanced green fluorescent protein expressing cells.

Figure 5

WIN55,212-2 activation of MAP kinase pathway is a result of CB₁-G_i/o signal transduction. Figures show the increase in phosphorylation of extracellular signal-regulated kinase 2 (pERK2) following a 5 min exposure to WIN55,212-2 or CP55940 using Western blot (a and b) and infrared In-Cell Western analysis (c–e). pERK2 levels were normalized to total ERK2 protein and expressed as relative pERK. (a) Western blot showing concentration effect of WIN55,212-2 treatment (0.25–5 μM). (b) Relative pERK2 following densitometric quantification of western blot. (c) Increase in pERK with 5–10 μM WIN55,212-2 in the presence and absence of 10 μM AM251 (n=16). (d) Increase in pERK with 5–10 μM WIN55,212-2 with and without PTX pretreatment (n=8). (e) Increase in pERK with 5–10 μM CP55940 with and without PTX pretreatment (n=8). Results are normalized to 0.1% DMSO control treatments. ^*P<0.05, ^**P<0.01 and ^***P<0.001. PTX, pertussis toxin; DMSO, dimethylsulphoxide.