Intracellular pathways of α₁ adrenergic stimulation-evoked fluid secretion in isolated lacrimal gland ducts in mice

Abstract

Purpose : Recently we found that α₁ adrenergic agonist phenylephrine (PHE) resulted in a robust fluid secretion in isolated lacrimal gland (LG) ducts in mice. Earlier reports demonstrated the involvement of the NO/cGMP pathway in the PHE-induced protein secretion of LG acinar cells from rat, but the intracellular pathways used by α₁ adrenergic agonists to stimulate ductal fluid secretion is unknown. Therefore the aim of the present study was to investigate the intracellular mechanisms underlying α₁ adrenergic-stimulated LG ductal fluid secretion in mouse.

Methods : LG duct segments were isolated from mouse LG as we previously described. Ends of the ducts seal forming a closed intraluminal space during incubation. Fluid secretion as a swelling response can be quantified by video-microscopy. Effects of endothelial nitric oxide synthase (eNOS) inhibitor L-NAME, neuronal nitric oxide synthase (nNOS) inhibitor S-methyl-L-thiocitrulline, guanylate cyclase (GC) inhibitor ODQ and α_1D-adrenergic receptor inhibitor BMY-7378 were investigated in the PHE-induced ductal fluid secretion. Changes of intracellular Ca²⁺ concentration ([Ca²⁺]_i) after PHE stimulation was measured with microfluorometry using FURA 2AM. Data were expressed as the x-fold change of the PHE-induced luminal volume (LV) increase, which was standardized to 1.0. Data were presented as means ± SEM.

Results : PHE (10 µM) stimulation resulted in a significant LV change (1.64±0.28-fold increase, p=0.004). PHE-evoked ductal fluid secretion was dose-dependently reduced by L-NAME (max. inhibition at 10 µM: 73.06±10.5% decrease in LV, p=0.006) while inhibition of nNOS had no effect on the fluid secretory response (p=0.21). ODQ decreased PHE-induced LV increase (max. inhibition at 1 µM: 72.1± 12.1 % decrease in LV, p=0.005). BMY-7378 dose-dependently reduced ductal fluid secretion (max. inhibition at 1 µM: 80.06±12.7%. p=0.007). PHE stimulation resulted in a small, but statistically significant increase in [Ca2+]i (7.51± 1.07%, p=0.001) in isolated LG ducts.

Conclusions : LV increase induced by PHE was reduced by α1D adrenergic receptor blockage, or by inhibition of either eNOS or GC. These data suggest that α1-adrenergic agonists use the NO/cGMP pathway through α1D receptor stimulation to increase fluid secretion in isolated mouse LG ducts. The functional relevance of the PHE-induced small elevation in [Ca2+]i needs further investigation.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.