THC Regulates Tearing via Cannabinoid CB1 Receptors

Abstract

Purpose: Aqueous deficiency dry eye (ADDE) is a chronic condition affecting millions, with symptoms ranging from a dry itchiness to blurred vision and accompanied by an increased risk of eye infections. ADDE typically arises from disorders of the lacrimal gland that produces tears necessary for eye lubrication. Cannabis users frequently report dry eye, but the basis for this is unknown. If the effects occur via the endogenous cannabinoid signaling system, then this may represent a novel mechanism for the regulation of tearing.

Methods: We examined expression of cannabinoid CB1 receptors in the lacrimal gland using immunohistochemistry, Western blotting, and PCR and tested tetrahydrocannabinol (THC) regulation of tearing in wild-type and CB1-null mice.

Results: We now report that CB1 receptors are expressed in the axons of cholinergic neurons innervating the lacrimal gland. Little if any staining is seen in lacrimal gland epithelial cells (acinar and ductal) or myoepithelial cells (MECs). Activation of CB1 receptors by THC or the cannabinoid agonist CP55940 reduces tearing in male mice. In female mice, THC has no effect, but CP55940 increases tearing. In both sexes, the effect of CP55940 is absent in CB1 knockout mice. CB1 mRNA and protein levels are approximately four- to fivefold higher in males than females. In male knockouts, THC increases tearing, suggesting that THC also acts through different receptors.

Conclusions: Our results suggest a novel, albeit sex-dependent, physiologic basis for the dry eye symptoms experienced by cannabis users: activation of neuronal CB1 receptors in the lacrimal gland reduces tearing.

Figure 1.

CB1 receptor protein is detected in the lacrimal gland. (A) Immunohistochemical staining in murine lacrimal gland slice shows CB1 protein expression in WT tissue. (B) CB1 staining is absent in CB1^–/– tissue. Scale bars: 50 µm.

Figure 2.

CB1 receptors are expressed in extra-acinar processes and cells. (A, B) Lacrimal CB1 protein expression (green) is seen outside acini, including small extra-acinar cell somata. Expression is not seen in green fluorescent protein (GFP)-expressing myoepithelial cells (“SMA”). (C, D) In a higher magnification image, CB1 is also visible in extra-acinar processes suggestive of neuronal axons. Scale bars: (A, B) 50 µm; (C, D) 30 µm.

Figure 3.

CB1 is expressed on neuronal axons. (A) CB1 (green) colocalizes with axonal marker neurofilament H (NFH; red). (B) Triple stain for CB1 (green), β3-tubulin (red), and β-actin (cyan) shows colocalization with β3-tubulin. (C) Staining for CB1 (green), tyrosine hydroxylase (TH; red), and phalloidin shows closely associated but discrete TH- and CB1-positive neuronal processes. (D) CB1 (green) and substance P (red) do not colocalize. (E) CB1 (green) colocalizes with cholinergic marker choline acetyl transferase (ChAT; red).

Figure 4.

THC differentially alters basal tear volume in mice in a sex-dependent manner via activation of cannabinoid CB1 receptors. (A) In male wild-type mice, THC (4 mg/kg, IP) and the CB1 receptor agonist CP55940 (CP; 0.5 mg/kg, IP) lowered tear volume while the CB1 receptor antagonist SR141716 (SR1; 4 mg/kg, IP) did not. In CB1 knockouts, THC did not reduce tearing but instead increased tearing while CP55940 was without effect. *P < 0.05, **P < 0.01, one-way ANOVA with Dunnett's post hoc test versus control. (B) Female mice saw no effects from THC treatment (4 mg/kg, IP), but an increase in tearing after treatment with CP55940 (0.5 mg/kg, IP). SR141716 (4 mg/kg, IP) did not alter tearing. In CB1 knockouts, neither THC nor CP55950 affected tearing. ***P < 0.005, one-way ANOVA with Dunnett's post hoc test versus control. (C) Pilocarpine (7 mg/kg, subcutaneously) elicited tearing in male mice as expected, but no difference in tearing was seen between animals injected with vehicle versus CP55940 (0.5 mg/kg) an hour prior to pilocarpine. NS, unpaired t-test.

Figure 5.

CB1 mRNA and protein are expressed at higher levels in male lacrimal gland. (A) Quantitative RT-PCR shows that message for CB1 receptors is approximately five times more abundant in male than in female mice. n = 3, 4, **P < 0.01 by unpaired t-test. (B) Western blot for CB1 protein shows CB1 expression in lacrimal glands of four male and four female mice. Ponceau S staining is shown in inset, bottom right. (C) Quantification of CB1 protein expression (shown in C) in males and females (CB1 protein level relative to whole protein). n = 4, ***P < 0.0001 by unpaired t-test. (D) RT-PCR shows the presence of mRNA for CB1 in lacrimal gland (LG) and cerebellum (Crb) of a male mouse, using two different probe sequences (p1, p2 in figure). Glutaraldehyde 3-phosphate dehydrogenase (GAPDH) is included as a control.

Figure 6.

CB1 activation does not alter distribution of acinar vesicles in the lacrimal gland. (A) Staining for VAMP8 (green) is seen in acinar cells. Staining is polarized within acini, associated with intra-acinar ducts labeled with phalloidin (red), a marker for F-actin. (B, C) Higher magnification image of untreated lacrimal gland has similar staining pattern to that of a CP55940-treated (0.1 mg/kg, IP, 1 hour) animal. Lacrimal glands are from male mice. Scale bars: 10 µm.