p38 Mitogen-activated protein kinase modulates exocrine secretion in rabbit lacrimal gland.

The lacrimal gland (LG) is an exocrine gland important for secretion of the tear film. The kinase p38 has important signal transduction functions, e.g. in gene transcription, but has previously not been known to modulate exocrine secretion. The aim of the current study was to investigate the role of p38 in carbachol (Cch)-induced LG secretion in LG acinar cells in vitro. Western blotting was used to determine the phosphorylation status of p38 and p42/44 and determine expression of p38 isoforms. To determine the effect of p38 inhibition on LG secretion, PD 169316, a general p38 inhibitor, and SB 239063, an inhibitor of p38α and ß, were added to the cells prior to secretion measurements. The results revealed activation of p38 mediated by Cch stimulation and inhibition of Cch-induced secretion as a result of p38 inhibition. The inhibition was observed with PD 169316 isoforms, but not with SB 239063. The p38δ isoform was shown to have robust expression both by Western blotting of acinar cells and immunofluorescence of the whole gland. In conclusion, p38 activation mediates secretion in cholinergic stimulation of rabbit LG cells.

Adenosine A2 receptor presence and synergy with cholinergic stimulation in rabbit lacrimal gland.

PURPOSE: Secretion from the lacrimal gland is an important part of the well-being of the eye, and a central part in the search for treatment of dry eye syndrome. Adenosine has stimulatory effects on the lacrimal gland, and can potentiate the effect of the cholinergic agonist carbachol (Cch). The aim of the present study is to investigate the presence of the adenosine A(2) receptor subtypes A(2A) and A(2B) in the rabbit lacrimal gland, and to characterize their role in regulated acinar cell secretion. METHODS: Expression of the receptors was investigated using reverse transcriptase-PCR (RT-PCR) and immunofluorescence, and secretion effects were studied using a secretion assay in isolated lacrimal gland acinar cells. RESULTS: Presence of both receptors was detected by RT-PCR and immunofluorescence. The secretion assay revealed a minor effect of stimulation of the A(2) receptors, and a strong synergistic effect with the cholinergic agonist Cch. The synergistic effect was significantly reduced by the A(2B) antagonist PSB 1115, but not by the A(2A) antagonist SCH 58261, indicating that A(2B) is the receptor responsible for this potentiation. CONCLUSIONS: The study reveals the presence of the adenosine A(2) receptor subtypes as well as a role for them in lacrimal gland secretion, and especially in the synergy with purinergic and cholinergic stimulation.

Functional expression of the adenosine A1 receptor in rabbit lacrimal gland.

It has become increasingly clear that purine compounds play a mediator role in exocrine secretion. Therefore, the present study was aimed at examining the presence of the adenosine A1 receptor in rabbit lacrimal gland and to evaluate the role of the A1 receptor in regulated secretion. The expression of the A1 receptor was investigated with reverse transcriptase PCR, cyto- and histochemistry as well as with pharmacological methods. Acinar cells were isolated, cultured in a serum-free medium for 2 days and thereafter treated with purinergic agonists/antagonists and/or carbachol and VIP. Secretory response was assessed by measuring secreted beta-hexosaminidase enzymatic activity. Microscopical evaluation of the immunocyto- and histochemistry specimens indicated that the adenosine A1 receptor is expressed in the rabbit lacrimal gland, which was also supported by reverse transcriptase PCR resulting in a sequence 100% identical with the previously published sequence for the rabbit A1 receptor gene. Incubation of acinar cells with adenosine and the A1 specific agonist N6-cyclopentyladenosine (CPA) resulted in a fourfold increase of secretion at the determined optimal concentrations. Incubation with carbachol alone resulted in a 10- to 15-fold increase. Carbachol combined with either adenosine or CPA increased the secretion 20-fold or more, demonstrating a synergistic effect. Our data provides evidence for the presence of adenosine A1 receptors in both tissue and cultured cells. Even though adenosine and CPA alone had only a moderate effect on secretion, the observed synergistic effect with carbachol suggests a modulatory role for the adenosine A1 receptor in the lacrimal gland.

Characterization of beta-hexosaminidase secretion in rabbit lacrimal gland.

The present study was aimed at validating the use of the lysosomal enzyme beta-hexosaminidase as a marker of secretory function in cultured rabbit lacrimal gland acinar cells. The secretory response and morphological characteristics of isolated acinar cells cultured in a serum-free medium supplemented with an extracellular matrix extract were monitored over time as part of optimization of our culturing protocol. Secreted beta-hexosaminidase activity was analyzed and compared with that of another lysosomal enzyme, cathepsin B, as well as protein secreted into the media, w or w/o the presence of secretagogues or protein kinase C activators and inhibitors. Lacrimal gland fluid was obtained from pilocarpine stimulated rabbits, and the activities of beta-hexosaminidase and cathepsin B were measured. A membrane fraction and a soluble fraction were obtained from isolated acinar cells and used for kinetic studies of beta-hexosaminidase in comparison with that released from cultured cells, in the lacrimal gland fluid and in serum. Optimal secretory response was obtained when the cells had been in culture for 2-3 days, coinciding with the formation of acinus-like structures. Stimulation of the cultured cells by carbachol or phorbol esters resulted in a more than 3-fold increase of beta-hexosaminidase release over basal, whereas no effect on cathepsin B release could be detected. Treatment with the protein kinase C inhibitor, chelerythrine chloride, significantly decreased the carbachol and phorbol ester-stimulated secretion. Cathepsin B could not be detected in rabbit lacrimal fluid, but beta-hexosaminidase was easily measured in quantities corresponding to as low as 0.4 microl of tear fluid. Using 4-methylumbelliferyl N-acetyl-beta-D-glucosaminide as a substrate for beta-hexosaminidase, the K(m) in lacrimal gland fluid (1.22+/-0.15 mM) was not significantly different from that of the membrane-associated fraction, the soluble fraction, rabbit serum or activity secreted from cultured cells. Beta-hexosaminidase is secreted by rabbit lacrimal gland, in vivo, and by acinar cells in primary culture, whereas cathepsin B is not secreted under the conditions described. Beta-hexosaminidase therefore provides a versatile marker for secretion in studies of tear production utilizing the rabbit as a model. Our results also indicate that PKC is an important regulator of rabbit lacrimal gland secretion.

Integrin adhesion in regulation of lacrimal gland acinar cell secretion.

The extracellular microenvironment regulates lacrimal gland acinar cell secretion. Culturing isolated rabbit lacrimal gland acinar cells on different extracellular matrix proteins revealed that laminin enhances carbachol-stimulated secretion to a greater extent than other extracellular matrix proteins investigated. Furthermore, immunofluorescence indicated that integrin subunits, potentially functioning as laminin receptors are present in acinar cells. Among these, the integrin alpha6 and beta1 subunit mRNA expression was also confirmed by RT-PCR and sequence analysis. Secretion assays, which measured beta-hexosaminidase activity released in the culture media, demonstrated that function-blocking integrin alpha6 and beta1 monoclonal antibodies (mAbs) induce a rapid, transient and dose-dependent secretory response in cultured cells. To determine the intracellular pathways by which integrin alpha6 and beta1 mAbs could induce secretion, selected second messenger molecules were inhibited. Although inhibitors of protein kinase C and IP(3)-induced Ca(2+) mobilization attenuated carbachol-stimulated secretion, no effect on integrin mAb-induced release was observed. In addition, protein tyrosine kinases do not appear to have a role in transducing signals arising from mAb interactions. Our data clearly demonstrate, though, that cell adhesion through integrins regulates secretion from lacrimal gland acinar cells. The fact that the integrin mAbs affect the cholinergic response differently and that the integrin beta1 mAb secretion, but not the alpha6, was attenuated by the phosphatase inhibitor, sodium orthovanadate, suggests that each subunit utilizes separate intracellular signaling pathways to induce exocytosis. The results also indicate that the secretory response triggered by the beta1 integrin mAb is generated through dephosphorylation events.

Sequencing, expression, and enzymatic characterization of beta-hexosaminidase in rabbit lacrimal gland and primary cultured acinar cells.

The lysosomal enzyme, beta-hexosaminidase, exists as two major isoforms; HexA and HexB. HexA is an alpha beta-subunit heterodimer and HexB a beta-subunit homodimer. Both isoforms can remove nonreducing beta-N-acetyl-D-glucosamine residues, whereas HexA hydrolyzes charged substrates as G(M2) gangliosides as well. beta-Hexosaminidase is present in both human and rabbit tear fluid and is secreted from rabbit lacrimal gland acinar cells in primary culture on stimulation with secretagogs. To further characterize the enzyme, the alpha- and beta-subunit mRNA expression was explored in rabbit lacrimal gland tissue as well as in cultured cells. Possible correlation between mRNA expression and HexA specific enzymatic activity was also investigated. Because existing beta-hexosaminidase antibodies are unable to recognize the rabbit enzyme, cloning and sequencing of the alpha- and beta-subunits were performed. Sequencing of the these subunits indicate that both are highly conserved between human, mouse, and rabbit. In contrast to the beta-subunit, showing an even mRNA expression between tissue and cultured cells, the level of alpha-subunit expression was higher in cultured acinar cells compared to tissue, with no alteration after cell stimulation. A minor but significant increase in total beta-hexosaminidase as well as HexA activity was observed in cultured cells compared to tissue. Enzymatic activity assays also revealed that HexA is the dominating isoform of beta-hexosaminidase in lacrimal gland and cultured acinar cells.