Our confocal microscopy observations showed that there was extensive α
1-IR and β
1-IR in the mouse LG, suggesting the presence of both α
1- and β
1-mediated pathways. Although both α
1-IR and β
1-IR were in close association with acinar cells, they exhibited different distribution patterns in the interstitial areas. Extensive α
1-IR was in the interstitial areas, in association with blood vessels and ducts, and less was in contact with acinar cells. This suggests that the α
1 pathway, in addition to regulating protein secretion from acinar and ductal cells, also plays a role in modulating blood flow within the gland. Vasoconstriction in the LG is mediated by α-receptors,
16 and in cat and rabbit, vascular tone in the LG correlates with tear flow.
2 17 Other studies demonstrated that blood flow in the submandibular gland influences secretory function.
18 19 In contrast, our study showed that most of the β
1-IR is in association with acinar cells rather than with blood vessels and ducts, suggesting that the β
1 pathway may be involved only in the secretory process at the acinar cell level.
Even in the acinar cells, α1-IR and β1-IR appeared to have different distribution patterns. α1-IR was relatively evenly distributed on the surfaces and inside the cytoplasm, whereas much of the β1-IR appeared to concentrate in the central areas of cytoplasm, with only some labeling on the membrane.
Lacrimal acinar cells perform constitutive vesicle-mediated transport between their basolateral plasma membranes and an endomembrane system that includes recycling, transcytotic, and degradative compartments.
20 There is ongoing recycling of β-receptors between a large intracellular pool (recycling endosome) and a small surface-expressed pool,
21 and their residence time at the basal-lateral membranes is quite brief,
22 consistent with the distribution pattern of β
1-receptors that we observed. The unique expression pattern of β
1-receptors may explain the gland’s low
relative responsiveness to the agonist at low concentrations and robust
absolute responsiveness at higher concentrations (i.e., more β
1-receptors are transported to the membrane when the cells were stimulated with agonist at higher concentration, whereas only a small number of receptors were on the membrane when the agonist concentration was low). The LG’s robust response to phenylephrine at low concentration may be explained by the abundance of α
1-receptors on the membranes, although acinar cells also have a large intracellular pool of α
1-receptors.
The internalized β-receptors in the early endosome may be sorted to the lysosome for degradation or to the recycling endosome for return to the plasma membrane,
23 24 through the highly coordinated intracellular trafficking that is regulated by many factors at distinct organelles.
25 26 27 This possibility suggests that enhanced lacrimal protein secretion, as a therapeutic approach for lacrimal dysfunction, may be achieved by facilitating the recycling of internalized β-receptors through modifying components of the vesicular transport machinery.
Protein secretion studies using α
1- and β
1-adrenergic agonists suggested that activation of either of these pathways could stimulate protein secretion. Our dose-response curves of carbachol, norepinephrine, and phenylephrine were very similar to those in rat LG, which measured either newly synthesized protein
28 or peroxidase secretion.
29 30 31 Between 10
−6 and 10
−4 M, norepinephrine and phenylephrine induced similar protein secretion to that of carbachol, suggesting that adrenergic stimulation was as effective as the cholinergic activation. Although carbachol, norepinephrine and phenylephrine all induced similar amounts of protein secretion between 10
−6 and 10
−4 M, carbachol was more effective at lower concentrations (<10
−6 M). In fact, the threshold of carbachol-induced protein secretion was approximately 10 times lower than that for norepinephrine and phenylephrine, and at least 100 times lower than that for isoproterenol. By comparing each EC
50, the order of potency of these agonists can be estimated: carbachol (EC
50 = 3 × 10
−7 M) > phenylephrine (EC
50 = 5 × 10
−7 M) > norepinephrine (EC
50 = 10
−6 M) > isoproterenol (EC
50 = 3 × 10
−5 M).
The dose-response curve for isoproterenol indicates that although isoproterenol failed to induce protein secretion at concentrations <10
−5 M, it significantly increased protein secretion at concentrations >10
−5 M. The isoproterenol-induced protein secretion increased almost linearly with the increase of agonist concentration. It appeared that isoproterenol had not yet achieved its maximum effect at 10
−4 M, which may be higher than that of norepinephrine, phenylephrine, or carbachol. However, the EC
50 of isoproterenol was approximately 100 times that of carbachol and 60 times that of phenylephrine. These results are in contrast to those from rabbit LG
3 and rat salivary gland.
32 In rabbit LG, the threshold for isoproterenol-induced protein secretion (3 × 10
−8 M) was 100 times lower than that for carbachol (3 × 10
−6 M).
3 In rat isolated submandibular cells, isoproterenol induced IgA secretion at a lower threshold and showed a larger increase of secretion at higher concentrations compared with that elicited with carbachol.
32
Phentolamine inhibited phenylephrine-induced protein secretion in a dose-dependent manner. Although the inhibitory effect of phentolamine appeared incomplete (maximum inhibition was 80% at 10
−5 M phentolamine), protein secretion was decreased by 50% at 10
−8 M, the lowest concentration used. In rat lacrimal acini, maximum inhibition by phentolamine on phenylephrine (10
−4 M)-induced peroxidase secretion was 90%, which was considered to be complete inhibition, whereas the half inhibition was achieved at 10
−6 M.
33 34 Our data suggest an α
1-mediated protein secretion pathway in the mouse LG, and the immunofluorescence observations suggest that this pathway may be of the α
1 type.
Propranolol also inhibited isoproterenol-induced protein secretion in a dose-dependent manner, although the inhibition was only partial. Maximum inhibition of 52% was achieved at 10
−5 M and with 20% inhibition at 10
−8 M. In the rat, propranolol achieves complete inhibition of isoproterenol-induced protein secretion.
35 Our data demonstrated the presence of the β-mediated protein secretory pathway in the mouse LG, and the observation of β
1-IR in the gland suggests that the β pathway may be of the β
1 type.
Both immunofluorescence and protein secretion findings suggest that there is an α
1-adrenergic pathway in the mouse LG. Confocal microscopy demonstrated that extensive α
1-IR was present on the surface and inside the acinar cells and that α
1-IR was found in the interstitial areas. Phenylephrine, the specific α
1 agonist, induced a significant amount of protein secretion from the gland fragments. The inhibitory effects of phentolamine on phenylephrine-induced protein secretion provide further evidence of the presence of an α-mediated protein secretory pathway. The findings are in agreement with a previous report that α
1-receptors are present in the mouse LG.
36
In rat LG, α-receptors were shown to be located on the acinar cells. Activation of these cells by phenylephrine increases potassium permeability and release, and this effect was almost completely blocked by phentolamine.
4 Phenylephrine has been reported to stimulate peroxidase secretion from rat lacrimocytes and this effect was suppressed by phentolamine, even though radioligand-binding assays have indicated that the gland did not possess a substantial number of α
1- and α
2-receptors.
33
Isoproterenol-induced protein secretion suggests the presence of a β-mediated secretory pathway in the mouse LG, a conclusion that is further supported by the inhibitory effect of propranolol on the isoproterenol-induced response. It is known that isoproterenol markedly enhances ATP-induced inward and outward currents, which are blocked by propranolol.
5 These findings are in agreement with our results. However, other studies in mouse LG have indicated that the mouse LG contains only the α
1-receptor,
36 and intracellular recordings from acinar cells failed to detect any isoproterenol-induced membrane potential and resistance changes.
6 Data supporting the β-receptor-mediated modulation of LG function has been reported in the rat: isoproterenol stimulated peroxidase secretion
35 37 38 and increased potassium permeability.
4 However, other reports claimed that isoproterenol had no effect on inducing radiolabeled protein
39 and peroxidase
14 secretions. In rabbit, in vitro studies showed the existence of β-receptors and their association with protein secretion.
3
Taken together, the results of the present study showed three lines of evidence in support of the involvement of the β-adrenergic system in the regulation of protein secretion in the mouse LG: (1) Isoproterenol stimulated protein secretion from the gland fragments; (2) the isoproterenol-induced secretion was specifically inhibited by propranolol, the nonselective β antagonist; and (3) β1-IR was observed in close association with acinar cells and interstitial areas. The microscopy findings further suggested that the β-pathway in the mouse LG was of the β1 type.
From the dose-response curves, phenylephrine, at concentrations of 10
−6 to 10
−4 M, appeared to act similarly to norepinephrine in eliciting protein secretion. Phenylephrine specifically activates an α
1 pathway, to stimulate lacrimal protein secretion.
3 At 10
−5 M, our data showed that protein secretion induced by phenylephrine was approximately 90% of that induced by norepinephrine and 97% of that at 10
−4 M. At 10
−6 M, phenylephrine-induced secretion was 1.6 times of that of norepinephrine, which was a significant difference. The adrenergic neurotransmitter norepinephrine can activate both α- and β-receptors. Our data indicate that phentolamine decreased norepinephrine-induced protein secretion by 67%, and propranolol achieved 47% inhibition. These data suggest that most of the adrenergic activation in the mouse LG is mediated by α-receptors and that β activation plays a less important role. Our data are in agreement with those obtained in the rat,
37 but are contrary to the data from rabbit LG, in which phentolamine reduced norepinephrine-evoked protein secretion by 47%, whereas propranolol achieved 71% inhibition.
3
When the LG fragments were exposed to both phenylephrine and isoproterenol, the protein secretion rate was higher than that evoked by either agonist. However, the rate was significantly lower (
P < 0.05) than the theoretical addition of the rates resulting from exposure to each agonist separately. Based on the definition of synergism,
15 37 it appears that there is no synergistic effect between the α
1 and β activation in the mouse LG. Agonist binding to the α
1-receptors partially activates the Ca
2+/diacylglycerol (DAG)/cGMP-dependent pathway, whereas binding to β-receptors activates the cAMP-dependent pathway.
12 31 40 A report of experiments in the rat described synergism between α
1 and β stimulation
37 ; and, in the rabbit, synergism between the cholinergic and β-adrenergic systems has also been reported.
3
Adrenergic innervation in the mouse LG, as reflected by markers such as TH, is sparse and far less extensive than is parasympathetic innervation. However, the fact that there is a high number of adrenergic receptors in the lacrimal gland and that systemic catecholamines circulating in the blood stream can enter the gland suggests that the adrenergic pathways is an important regulatory pathway.
41 42