An organotypic culture system of Merkel cells using isolated epidermal sheets.

BACKGROUND: Merkel cells (MCs) exist in the epidermal basal layer, in contact with keratinocytes. This direct contact seems critical for maintaining MCs in vitro. OBJECTIVES: To estimate the effects of nerve cells on the maintenance of MCs within epidermal sheets in a new organotypic culture system of MCs. METHODS: We developed a new organotypic culture system of MCs, using MC-containing epidermal sheets embedded in collagen gel. To estimate the effects of nerve cells on the maintenance of MCs within the epidermal sheets, we cocultured nerve cells and MC-containing epidermal sheets. In these culture assemblies, cellular behaviour was analysed by histochemistry, immunohistochemistry, electron microscopy and enzyme-linked immunosorbent assay. RESULTS: This culture, even in the absence of neurotrophin (NT)-3 and nerve growth factor (NGF) (which are crucial for MC biology), retained cytokeratin (CK)-20-positive and neuroendocrine granule-containing MCs within the sheets for over 2 weeks. Coculture of MCs with PC-12 nerve cells significantly increased the number of MCs within the epidermal sheets, and the keratinocytes had almost identical expression levels of CK1, CK10, CK14 and the progenitor marker p63 to those produced by keratinocytes in vivo. Uptake of the growth marker bromodeoxyuridine by MCs and levels of NT-3 and NGF in the culture supernatants were undetectable in this system, regardless of the presence or absence of PC-12. CONCLUSIONS: The data suggest, first, that direct contact between MCs and keratinocytes may be critical for retaining MCs in vitro; second, that nerve cell-affected maintenance of keratinocyte differentiation, but not NT-3 and NGF, may contribute to MC maintenance; and third, that MCs are not able to grow, at least in our system. Our method would be useful for studying MC biology.

Localization of neuronal-constitutive nitric oxide synthase and secretory regulation by nitric oxide in the rat submandibular and sublingual glands.

The distribution of neuronal-constitutive nitric oxide synthase (ncNOs)-positive nerve fibres was compared immunohistochemically, and the effect of NOs inhibitor and NO scavenger on the secretory response was compared functionally, in the two glands. Numerous ncNOs-positive fibres were distributed around acini in the submandibular gland but scarcely any around acini in the sublingual gland. Within the submandibular ganglion (parasympathetic), the nerve-cell bodies were strongly positive. Within the superior cervical ganglion (sympathetic), the nerve-cell bodies were negative, although some positive nerve fibres were observed. The secretory responses to the electrical stimulation of the chorda were significantly reduced by the NOs inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME, 10(-9)-10(-3) M) in a dose-dependent manner. The NO scavenger, 2-(4-carboxyphenyl)4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (carboxy-PTIO) also reduced the chorda-evoked secretion (10(-9)-10(-6) M). The submandibular secretions evoked by stimulation of the superior cervical ganglion were not affected by L-NAME or carboxy-PTIO. In the sublingual gland, neither L-NAME nor carboxy-PTIO affected chorda-evoked salivary secretion. The histochemical and functional results both suggest that NO plays an excitatory role in the regulation of parasympathetic nerve-induced salivary secretion in the rat submandibular gland, but not in the sublingual gland.

Regulated permeability of tight junctions by E-cadherin in rat submandibular gland acini.

We examined the relationship between E-cadherin (E-CD), protein kinase C (PKC) and assembly of the tight junction (Tj) in rat submandibular gland acinar cells. The junctional complex of the acinar cells was double-labeled with anti-ZO-1 antibody and anti-E-CD antibody. When Ringer's solution was intraductally injected into the main duct, ZO-1 labels were highly concentrated at the Tj zone, and the adherens junction (Aj) was exclusively labeled by E-CD. In addition, the Tj was impermeable to microperoxidase. Neither intraductal injection of anti-E-CD antibody solution nor infusion of carbachol produced labels for ZO-1 at the Tj zone, although these proteins were occasionally intermixed at the Aj zone. In these cases, the Tj was permeable to microperoxidase. Intraductal injection of anti-E-CD antibody solution with PKC agonist, resulted in a reduction of E-CD labels in the Aj zone, while the ZO-1 was labeled exclusively in the Tj zone. In this case, Tj was impermeable to microperoxidase. These results suggest that E-CD plays a major role in mediating intercellular physical adhesion, and that PKC may be active in signaling the pathway activated by E-CD-mediated cell-cell adhesion.

Chorda-evoked opening of tight junctions in rat submandibular salivary acini demonstrated by microperoxidase.

The permeability of these junctions from the interstitium to the lumen was examined by using an ultrastructural tracer, microperoxidase, in conjunction with electron microscopy. In the resting gland, the reaction product of microperoxidase was seen in the interstitial and intercellular spaces, but not within acinar lumina; thus the tight junction was impermeable to microperoxidase (junction closed). Intraductal injection of hypertonic sucrose solution (1000 mOsm; 30 microliters) caused a sustained elevation of the luminal pressure, indicating osmotic water flow into the lumen due to the presence of a hypertonic solution. In this gland no opening of the tight junctions was observed. In the chorda-stimulated gland, microperoxidase entered the lumen through the tight junctions, that is, they became permeable to microperoxidase (junction open). These findings suggest that chorda stimulation opens the acinar tight junctions and that the paracellular secretory pathway may be involved in the secretion of small molecules and water from the submandibular acini.

Distribution of anionic sites during increasing tight junctional permeability in the rat submandibular gland.

We sought to determine the effect of substance P salivary stimulation on both electrical charges and cellular permeability in the tight junctions of rat submandibular gland cells. Microperoxidase (1,900 daltons) was used as a tracer. It was administered by close-arterial infusion via the glandular arteries, and secretory routes of acinar cells in the gland were determined cytochemically. In the resting gland, microperoxidase reaction product filled the lateral intercellular spaces up to the tight junctions, but did not penetrate them. In the substance P-stimulated gland, microperoxidase reaction product was present within tight junctions and the lumen. Both distribution and mobility of anionic sites on the surface of the submandibular gland cells were studied utilizing multivalent ligand, ruthenium red and cationized ferritin as probes. In the resting gland, ruthenium red deposits were located uniformly in all areas of the basal membrane and intercellular spaces except for the tight junctional region of acinar and ductal cells. In substance P-stimulated gland, ruthenium red deposits were present in the tight junctional region and, to a lesser extent, in the intercellular spaces. Electrical charges of the tight junctions area of the lateral plasma membrane were studied using intraductal injection of cationized ferritin. In the resting gland, cationized ferritin probe was present in the intercellular spaces and was bound weakly in the tight junctional region. In the substance P-stimulated gland, cationized ferritin was firmly adherent to the tight junctional region.

Degranulation of acinar cells in von Ebner's gland of the rat.

We investigated the effect of sympathetic agonists, parasympathetic muscarinic agonists and substance P on depletion of secretory granules in acinar cells of rat von Ebner's gland. Drugs were injected intraperitoneally at several different concentrations. Antagonists were given 15 minutes before injection of the agonist, and the extent of depletion of secretory granules in glandular acini was calculated using a computerized color image analyzer. The specific alpha 2-sympathetic agonist clonidine and the beta 1-sympathetic agonist dobutamine produced a depletion of secretory granules. When combined with injections of the alpha 2-sympathetic antagonist yohimbine and the beta 1-sympathetic antagonist acebutolol, depletion of secretory granules was blocked. The parasympathetic muscarinic agonist carbachol also produced a depletion of secretory granules. QNB blocked the depletion caused by carbachol, while atropine partially inhibited depletion. The specific M1-muscarinic agonist McN-A-343 caused some depletion, although there was no significant differences between it and the control. Complete depletion of the secretory granules was achieved by carbachol stimulation superimposed on substance P stimulation. We concluded that the activation of the sympathetic alpha 2- and beta 1-receptors, as well as the M2 (M2 beta)-muscarinic and substance P receptors, results in degranulation of acinar cells in von Ebner's gland of the rat.

Permeability increase in rat submandibular gland by glandular substance P.

When the chorda tympani was stimulated at 20 Hz, infused horseradish peroxidase (HRP) entered the saliva through the tight junctions in the rat submandibular gland. This response was not inhibited by atropine but was reduced by a substance P(SP)-antagonist. The increase in junctional permeability for HRP never occurred in the gland that was stimulated at 5 Hz or in the SP-depleted gland. Our data suggest that the effect of parasympathetic stimulation on the increase in junctional permeability for macromolecules was due to the release of SP in the gland.

Sialographic damage in rat submandibular gland.

The damage caused to rat submandibular glands by intraductal injection was examined. When a small volume of isotonic saline was injected, even if salivation was not influenced, salivary composition was greatly affected because the intercellular junctions of the duct cells were damaged. Intraductal injections of hyperosmotic solutions or highly viscous liquids were more effective in damaging intercellular junctions. When interpreting the significance of a salivary composition in clinical diagnosis, it is necessary to consider the sialographic damage which might influence the salivary composition.