Secretomotor substances in the rat lacrimal gland: Immunocytochemical functional and ligand binding studies

Abstract

The rat lacrimal gland has a characteristic pattern of autonomic innervation which has important roles in the control of lacrimation and blood flow. Protein secretion is observed in the presence of cholinergic and adrenergic antagonists, implicating the involvement of a noncholinergic, nonadrenergic nervous pathway. Recent studies have demonstrated the presence of peptidergic nerves, and the neuropeptide VIP is established as a lacrimal secretagogue, however the
involvement ofother neurotransmitters in the control of lacrimal function remains unclear. Ageing has been suggested to be involved in one of the common malfunctions ofthis gland, that ofreduced tear production which may play a role in the development of the disease state keratconjunctivitis sicca (KCS). However, there is no evidence as to whether age-related changes occur in the innervation pattern ofthe lacrimal gland. This study employs histochemical and immunocytochemical
techniques to elucidate and compare the occurrence and precise distribution of nerve fibres and markers of mast cells in the lacrimal glands of 3-5, 14 and 24 month old rats. The glands of the three age groupswere innervated with acetycholinesterase (AChE), vasoactive intestinal polypeptide (VIP)-, neuropeptide Y (NPY)-, substance P (SP)-, calcitonin gene-related peptide (CGRP)-, tyrosine hydroxylase (TH)- and the phosphoprotein B-SO- immunoreactive (IR) nerve fibres. 13- 50-lit was distributed around acini, blood vessels and ducts, in a similar manner to VIP, SP and CGRP; though there was less VIP-IR nerves particularly associated with ducts and a generally sparse SP and CGRP innervation. NPY- and TH-IRnerves were apparent only around the vascular tissue. Mast cells, to date have lacked description and enumeration in the lacrimal gland. A high densityof5-HT and histamineimmunofluorescencewasobserved inclose oppositionto neurovascular
tissue, paralleling histochemical staining for mast cells.
In the 14 month, and to a greater extent, in 24 month old rats there were signs of chronic inflammation and patchy destruction of acinar, ductal and vascular tissue. The 3-5 and 14 month old rats displayed a similar pattern of innervation, however, by 24 months there was a reduction in the number and intensity of immunoreactive nerves. The loss of nerves was particularly associated with damage to the gland. In contrast to nerves, there was a large infiltration of mast cells in the aged tissue, indicative of inflammation and tissue degeneration. The decrease in innervation, chronic inflammation and mast cell infiltration observed in aged rats may be contributing factors to reduced tear output in ageing and KCS
This study investigates the role of VIP, NPY, SP, CORP and 5-HT in enzyme secretion and the presence of receptors for VIP and NPY on isolated rat acini. Protein secretion was measured from segments ofthe exorbital lacrimal gland and receptor binding studies were carried out on dispersed acini, separated on percoll density gradients, with WI-VIP or wINPY. All neuropeptides enhanced lacrimal secretion, but the net effect of all neuropeptides was dependent on dose. The response to VIP was abolished by the VIP receptor antagonist [4Cl-D-Phe 6, leu' 7} VIP, which reduced the response to NPY. In addition, when VIP was combined with NPY an attenuated response was observed. VIP and NPY binding sites were present on lacrimal acinar cells, but binding sites for NPY were far less numerous than those for VIP. Collectively, the results are
consistent with aphysiological role for VIP in the regulation ofsecretion in the lacrimal gland. NPY stimulates secretion, however, there is considerable interaction between these two peptides and their receptorsin both secretory and receptorbinding assays. Taken with the immunocytochemistry, a role for NPY in enzyme secretion is less clear.
In various tissues ofthe body, notably the gastrointestinal tract, nerves and mast cells are in close apposition suggesting a functional relationship. There are no reports available describing a neuroimmune connection in the lacrimal gland. Observations from this study have demonstrated peptidergic nerves in particular SP and CGRP, to be closely associated with mast cells. Pharmacological studies indicate that SP and CORP cause mast cell degranulation leading to 5-HT release. This data suggests an interaction of the nervous and immune systems in the control of lacrimation.
The morphological, biochemical and functional data presented provides new insights into the integration ofthe nervous control oflacrimal gland secretion. A better understanding ofthe normal lacrimal stimulus-secretion mechanisms will help to perceive the specific cellular mechanisms that are affected in diseases of the lacrimal gland or that break down during the aging process. Based on this knowledge it should be possible to develop scientifically based treatments for the decrease
in lacrimal gland secretion that occurs in KCS and related diseases ofaqueous tear film deficiency.