Salivary and serum biomarkers of inflammation in a man with metastatic medullary thyroid carcinoma and hyperreactive gingiva: a fourteen year odyssey.

A peripheral (gingival) fibroma, a gingival cyst and hyperplastic gingivitis occurred simultaneously in a man with metastatic medullary thyroid carcinoma (MCT). The gingival growths and hyperplasia appeared to be related to poor oral hygiene rather than to the MTC. Despite the patient's improved oral hygiene, the hyperplastic gingivitis and peripheral fibroma recurred, and a new peripheral fibroma and gingival cyst developed, which prompted reconsideration of a link with the MTC. MTC cells secrete calcitonin (CT), procalcitonin (ProCT) and growth factors; the patient's serum CT and ProCT were several fold higher than normal. The patient's salivary CT and ProCT also were elevated, but α-amylase and epidermal growth factor (EGF) were not, compared to three healthy controls. A possible link between the MTC and gingival hyper-reactivity due to CT and/or ProCT promoting inflammatory cytokines, and the utility of salivary ProCT as an indicator of periodontitis in this patient were explored further. Unstimulated whole saliva and serum were collected from the patient followed by a standard periodontal examination before periodontal treatment, and 3 weeks and 3 months after treatment. This cycle was repeated 7 months after the previous periodontal treatment. The saliva was assayed for ProCT and the serum was assayed for ProCT, high sensitivity C-reactive protein (CRP), interleukin-6 (IL-6) and proadrenomedullin (ProADM). The results were analyzed for correlations among the severity of periodontitis and the biomarkers/cytokines. Only the salivary ProCT was correlated with the severity of periodontitis, i.e. it was higher just before and lower at 3 weeks and 3 months after each periodontal treatment. The patient's salivary ProCT content also was much higher than reported elsewhere. The other biomarkers/cytokines were within normal ranges. Our findings indicate that salivary ProCT is independent of serum ProCT and therefore may be a useful marker for moderate to severe periodontitis in patients with MTC. The greatly elevated salivary and serum CT and ProCT, and a trend toward correlation between the serum CRP and ProCT suggest a pro-inflammatory link between the MTC and the hyperreactive gingiva in this patient. Further studies are warranted to determine whether hyperplastic gingivitis and gingival growths, such as cysts and fibromas, occur with unusual frequency in patients with MTC.

Botryoid odontogenic cyst. Exploration of proliferative activity, apoptosis and expression of TP53 and BCL2 compared to the histologically identical lateral periodontal and gingival cysts.

The botryoid odontogenic cyst (BOC) is a rare, locally more aggressive variant of the usually indolent lateral periodontal cyst (LPC) and gingival cyst (GC). A recent case of BOC provided an opportunity for an exploratory study on the causes of its more aggressive behavior. The limited objective was to see if the BOC was sufficiently different from the other cysts to warrant an investment in a large study. Sections of neutral buffered formalin fixed, paraffin-embedded tissues from the BOC and archival specimens of four GCs, four LPCs and three odontogenic keratocysts (OKCs) were stained using immunohistochemistry for Ki-67, a marker of proliferating cells, caspase-3, a marker of cells undergoing apoptosis, tumor suppressor p53, and the apoptosis inhibitor BCL2. The mean labeling index (LI) of immunoreactive cyst epithelial cells was computed for each antibody and type of cyst. Compared to the LPCs and GCs, the BOC exhibited a moderately larger Ki-67/caspase-3 LI difference, which indicates that the BOC had a net higher rate of growth. We found a much higher level of LI, therefore likely dysregulation of p53. We also found a much higher LI of BCL2. The LIs of p53 and BCL2 in the BOC were similar to and more than twice that of the OKCs, respectively. Although meaningful statistical analysis was precluded by our use of only one case of BOC and a small number of the other cysts, the high p53 and very high BCL2 labeling indices of the BOC offer a potential explanation for its reportedly more aggressive behavior that clearly is worthy of further investigation.

Salivary and serum procalcitonin and C-reactive protein as biomarkers of periodontitis in United States veterans with osteoarthritis or rheumatoid arthritis.

Serum procalcitonin (ProCT) is elevated in response to bacterial infections, whereas high sensitivity C-reactive protein (hsCRP) is a nonspecific inflammatory marker that is increased by excess adipose tissue. We examined the efficacy of ProCT and hsCRP as biomarkers of periodontitis in the saliva and serum of patients with arthritis, which is characterized by variable levels of systemic inflammation that potentially can confound the interpretation of inflammatory biomarkers. Blood and unstimulated whole saliva were collected from 33 patients with rheumatoid arthritis (RA) and 50 with osteoarthritis (OA). Periodontal status was assessed by full mouth examination and patients were categorized as having no/mild, moderate or severe periodontitis by standard parameters. Salivary and serum ProCT and hsCRP concentrations were compared. BMI, diabetes, anti-inflammatory medications and smoking status were ascertained from the patient records. Differences between OA and RA in proportionate numbers of patients were compared for race, gender, diabetes, adiposity and smoking status. Serum ProCT was significantly higher in arthritis patients with moderate to severe and severe periodontitis compared with no/mild periodontitis patients. There were no significant differences in salivary ProCT or salivary or serum hsCRP in RA patients related to periodontitis category. Most of the OA and RA patients were middle aged or older, 28.9% were diabetic, 78.3% were overweight or obese, and slightly more than half were either current or past smokers. The OA and RA groups differed by race, but not gender; blacks and males were predominant in both groups. The OA and RA groups did not differ in terms of controlled or uncontrolled diabetes, smoking status or BMI. The RA patients had been prescribed more anti-inflammatory medication than the OA patients. Our results demonstrate that circulating ProCT is a more discriminative biomarker for periodontitis than serum hsCRP in patients with underlying arthritis. Any elevation in salivary and serum hsCRP due to periodontitis apparently was overshadowed by differences among these patients in factors that influence CRP, such as the extent of inflammation between RA and OA, the extent of adipose tissue, the use of anti- inflammatory medications and smoking status. Although our study showed no differences in salivary ProCT related to severity of periodontitis, this biomarker also may be useful with further refinement.

Effects of double ligation of Stensen's duct on the rabbit parotid gland.

Salivary gland duct ligation is an alternative to gland excision for treating sialorrhea or reducing salivary gland size prior to tumor excision. Duct ligation also is used as an approach to study salivary gland aging, regeneration, radiotherapy, sialolithiasis and sialadenitis. Reports conflict about the contribution of each salivary cell population to gland size reduction after ductal ligation. Certain cell populations, especially acini, reportedly undergo atrophy, apoptosis and proliferation during reduction of gland size. Acini also have been reported to de-differentiate into ducts. These contradictory results have been attributed to different animal or salivary gland models, or to methods of ligation. We report here a bilateral double ligature technique for rabbit parotid glands with histologic observations at 1, 7, 14, 30, 60 days after ligation. A large battery of special stains and immunohistochemical procedures was employed to define the cell populations. Four stages with overlapping features were observed that led to progressive shutdown of gland activities: 1) marked atrophy of the acinar cells occurred by 14 days, 2) response to and removal of the secretory material trapped in the acinar and ductal lumens mainly between 30 and 60 days, 3) reduction in the number of parenchymal (mostly acinar) cells by apoptosis that occurred mainly between 14-30 days, and 4) maintenance of steady-state at 60 days with a low rate of fluid, protein, and glycoprotein secretion, which greatly decreased the number of leukocytes engaged in the removal of the luminal contents. The main post- ligation characteristics were dilation of ductal and acinar lumens, massive transient infiltration of mostly heterophils (rabbit polymorphonuclear leukocytes), acinar atrophy, and apoptosis of both acinar and ductal cells. Proliferation was uncommon except in the larger ducts. By 30 days, the distribution of myoepithelial cells had spread from exclusively investing the intercalated ducts pre-ligation to surrounding a majority of the residual duct-like structures, many of which clearly were atrophic acini. Thus, both atrophy and apoptosis made major contributions to the post-ligation reduction in gland size. Structures also occurred with both ductal and acinar markers that suggested acini differentiating into ducts. Overall, the reaction to duct ligation proceeded at a considerably slower pace in the rabbit parotid glands than has been reported for the salivary glands of the rat.

Dispersed donor salivary gland cells are widely distributed in the recipient gland when infused up the ductal tree.

The salivary glands often are severely and permanently damaged by therapeutic irradiation for cancer of the head and neck. The markedly reduced quantity and quality of saliva results in greatly increased susceptibility to dental caries and infection of the oral mucosa and alveolar bone. Recently, subcapsular injection of cultured mouse salivary gland cells has achieved a significant degree of regeneration in a previously irradiated mouse salivary gland; however, the recovery was limited to one lobule. We describe here a method for delivering donor rat salivary gland cells via the main duct that distributes several thousand cells throughout the recipient rat's salivary gland. The donated cells exhibited the cytodifferentiation of the structures in which they lodged, i.e., acini, granular convoluted tubules, and the several types of ducts. This method may facilitate the simultaneous functional recovery of almost all of the lobules of irradiated rat salivary glands.

Exogenous thyroid hormone affects myoepithelium and proliferation in the developing rat parotid gland.

In the mature rat parotid gland, myoepithelial cells (MEC) invest intercalated ducts, but not acini. During postnatal development, however, these cells differentiate around both intercalated ducts and acini, then translocate to only intercalated ducts during weaning. Previously, we found that thyroxine (T(4)) accelerates translocation of cells with small secretory granules from acini into intercalated ducts and the number of apoptotic cells increased tremendously with high doses. We present here additional analysis of the effects of T(4) on developing rat parotid gland, namely, the distribution of MEC and the proliferation of parenchymal cells. Beginning at age four days, pups were given daily subcutaneous injections of low, medium, and high doses of T(4) or vehicle or no injection. At ages 4, 7, 10, and 15 days, glands were excised and processed for light microscopy. Sections were double-immunostained with antibodies against proliferating cell nuclear antigen (PCNA) and actin, and counterstained with hematoxylin. Proliferative activity was assessed via PCNA histochemistry and MEC were identified using actin histochemistry. MEC in the T(4) groups invested mostly acini at 15 days in vehicle/normal glands and mostly intercalated ducts after 10 days in the T(4) groups. The proliferative activity of acinar cells and MEC in vehicle/normal glands declined progressively with age and T(4) increased the rate of this decline in the MEC in a dose-dependent manner. We conclude that T(4) accelerates the translocation of MEC from acini to intercalated ducts and that an important mechanism is the more rapid decline in the proliferative activity of MEC than in acinar cells in the T(4) groups. Some of the decline in the proliferative activity of all cells in the high and medium dose T(4) groups after seven days may have been due to dose-related thyroxine toxicity.

On approaches to the functional restoration of salivary glands damaged by radiation therapy for head and neck cancer, with a review of related aspects of salivary gland morphology and development.

Radiation therapy for cancer of the head and neck can devastate the salivary glands and partially devitalize the mandible and maxilla. As a result, saliva production is drastically reduced and its quality adversely altered. Without diligent home and professional care, the teeth are subject to rapid destruction by caries, necessitating extractions with attendant high risk of necrosis of the supporting bone. Innovative techniques in delivery of radiation therapy and administration of drugs that selectively protect normal tissues can reduce significantly the radiation effects on salivary glands. Nonetheless, many patients still suffer severe oral dryness. I review here the functional morphology and development of salivary glands as these relate to approaches to preventing and restoring radiation-induced loss of salivary function. The acinar cells are responsible for most of the fluid and organic material in saliva, while the larger ducts influence the inorganic content. A central theme of this review is the extent to which the several types of epithelial cells in salivary glands may be pluripotential and the circumstances that may influence their ability to replace cells that have been lost or functionally inactivated due to the effects of radiation. The evidence suggests that the highly differentiated cells of the acini and large ducts of mature glands can replace themselves except when the respective pools of available cells are greatly diminished via apoptosis or necrosis owing to severely stressful events. Under the latter circumstances, relatively undifferentiated cells in the intercalated ducts proliferate and redifferentiate as may be required to replenish the depleted pools. It is likely that some, if not many, acinar cells may de-differentiate into intercalated duct-like cells and thus add to the pool of progenitor cells in such situations. If the stress is heavy doses of radiation, however, the result is not only the death of acinar cells, but also a marked decline in functional differentiation and proliferative capacity of all of the surviving cells, including those with progenitor capability. Restoration of gland function, therefore, seems to require increasing the secretory capacity of the surviving cells, or replacing the acinar cells and their progenitors either in the existing gland remnants or with artificial glands.

Primary culture of polarized human salivary epithelial cells for use in developing an artificial salivary gland.

Therapeutic irradiation for head and neck cancer, and the autoimmune disease Sjogren's syndrome, lead to loss of salivary parenchyma. They are the two main causes of irreversible salivary gland hypofunction. Such patients cannot produce adequate levels of saliva, leading to considerable morbidity. We are working to develop an artificial salivary gland for such patients. A major problem in this endeavor has been the difficulty in obtaining a suitable autologous cellular component. This article describes a method of culturing and expanding primary salivary cells obtained from human submandibular glands (huSMGs) that is serum free and yields cells that are epithelial in nature. These include morphological (light and transmission electron microscopy [TEM]), protein expression (immunologically positive for ZO-1, claudin-1, and E-cadherin), and functional evidence. Under confocal microscopy, huSMG cells show polarization and appropriately localize tight junction proteins. TEM micrographs show an absence of dense core granules, but confirm the presence of tight and intermediate junctions and desmosomes between the cells. Functional assays showed that huSMG cells have high transepithelial electrical resistance and low rates of paracellular fluid movement. Additionally, huSMG cells show a normal karyotype without any morphological or numerical abnormalities, and most closely resemble striated and excretory duct cells in appearance. We conclude that this culture method for obtaining autologous human salivary cells should be useful in developing an artificial salivary gland.

Tissue compatibility of two biodegradable tubular scaffolds implanted adjacent to skin or buccal mucosa in mice.

Radiation therapy for cancer in the head and neck region leads to a marked loss of salivary gland parenchyma, resulting in a severe reduction of salivary secretions. Currently, there is no satisfactory treatment for these patients. To address this problem, we are using both tissue engineering and gene transfer principles to develop an orally implantable, artificial fluid-secreting device. In the present study, we examined the tissue compatibility of two biodegradable substrata potentially useful in fabricating such a device. We implanted in Balb/c mice tubular scaffolds of poly-L-lactic acid (PLLA), poly-glycolic acid coated with PLLA (PGA/PLLA), or nothing (sham-operated controls) either beneath the skin on the back, a site widely used in earlier toxicity and biocompatibility studies, or adjacent to the buccal mucosa, a site quite different functionally and immunologically. At 1, 3, 7, 14, and 28 days postimplantation, implant sites were examined histologically, and systemic responses were assessed by conventional clinical chemistry and hematology analyses. Inflammatory responses in the connective tissue were similar regardless of site or type of polymer implant used. However, inflammatory reactions were shorter and without epithelioid and giant cells in sham-operated controls. Also, biodegradation proceeded more slowly with the PLLA tubules than with the PGA/PLLA tubules. No significant changes in clinical chemistry and hematology were seen due to the implantation of tubular scaffolds. These results indicate that the tissue responses to PLLA and PGA/PLLA scaffolds are generally similar in areas subjacent to skin in the back and oral cavity. However, these studies also identified several potentially significant concerns that must be addressed prior to initiating any clinical applications of this device.

Cationic liposome-mediated gene transfer to rat salivary epithelial cells in vitro and in vivo.

BACKGROUND: Previously we have shown that gene transfer to salivary gland epithelial cells readily occurs via recombinant adenoviruses, although the response is short-lived and results in a potent host immune response. The aim of the present study was to assess the feasibility of using cationic liposomes to mediate gene transfer to rat salivary cells in vitro and in vivo. METHODS: Initially, for transfection in vitro, we used two cationic liposome formulations (GAP-DLRIE/DOPE and DOSPA/DOPE) complexed with plasmid encoding human growth hormone (hGH) as a reporter gene. Thereafter, using GAP-DLRIE/DOPE, plasmids were transferred to rat salivary glands in vivo, and hGH levels measured in saliva, serum and gland extracts. RESULTS: Under optimal conditions, transfection of rat submandibular glands (SMGs) was consistently observed. Approximately 95% of the cells transfected with a plasmid encoding beta-galactosidase were acinar cells. Maximal hGH expression was obtained during the first 48 h post-transfection using a plasmid encoding the hGH cDNA and complexed with GAP-DLRIE/DOPE. hGH was detected in gland extracts and saliva, and occasionally in serum. No systemic or local gland pathology was consistently or significantly observed. CONCLUSIONS: The levels of the reporter gene product, hGH, obtained after GAP-DLRIE/DOPE-mediated gene transfer are considerably lower (<0.5%) than those achieved with adenoviral vectors (10(8) PFU). Nonetheless, cationic liposome-mediated gene transfer to salivary glands may be useful for potential therapeutic applications.

Enzyme histochemical localization of Na(+),K(+)-ATPase and NADH-DE in the developing rat parotid gland.

Information on ductal differentiation in the developing rat parotid gland is sparse. Striated and excretory ducts are rich in a number of enzymes related to ion movement. The objective of this investigation was to delineate histochemically the chronology of two of these, ouabain-sensitive Na(+),K(+)-ATPase and NADH-DE, in the developing rat parotid gland. Parotid glands were excised from rats at representative ages from 20 days in utero to 42 days. Enzyme histochemistry was performed on air-dried frozen sections. For Na(+), K(+)-ATPase, some sections also were fixed in phosphate-buffered formalin. Ouabain blocked Na(+),K(+)-ATPase activity, and neither enzyme reacted without substrate. Weak Na(+),K(+)-ATPase reactions were initially seen in unfixed sections at 1 day, and increased steadily to the adult pattern of strong (concentrated basolaterally) in striated ducts and excretory ducts, respectively, and weak to modest (diffuse) in acini and intercalated ducts at 28 days. In fixed sections, localization was sharper but the reaction was somewhat reduced. NADH-DE was modest in terminal buds and ducts before birth, then progressively changed to the adult pattern of weak in acini and intercalated ducts and strong (concentrated basally and luminally) in striated and excretory ducts at 28 days. As demonstrated by enzyme histochemistry of Na(+),K(+)-ATPase and NADH-DE, differentiation of rat parotid striated ducts and excretory ducts occurs mainly between birth and 28 days. Anat Rec 256:72-77, 1999. Published 1999 Wiley-Liss, Inc.

Oral hairy leukoplakia: an ultrastructural study and review of the literature.

A 39-year-old, homosexual, Caucasian man with a 9-month history of acquired immunodeficiency syndrome by reduced CD4 lymphocyte count alone was found to have extensive, asymptomatic, nonremovable, corrugated, white patches on the lateral borders and ventral aspects of the tongue typical of oral hairy leukoplakia (OHL). Histologically, irregular hyperparakeratosis, acanthosis, and clusters of ballooned keratinocytes in the stratum spinosum were present in the biopsied lateral tongue. Some of the superficial ballooned keratinocytes had peripherally beaded nuclei, whereas others had ground glass intranuclear inclusions. Ultrastructurally, the ballooned keratinocytes had three important findings of diagnostic significance. First, frequent herpesvirus nucleocapsids were largely confined to superficial ballooned keratinocytes having marginated and condensed chromatin. In searching for herpesvirus nucleocapsids, the marginated and condensed chromatin was an invaluable marker for cells harboring the virions. Second, the marginated and condensed chromatin frequently had a distinctive punched-out or cribriform appearance. Third, the ground glass intranuclear inclusion bodies consisted of central, medium electron-dense, finely granular material containing frequent herpesvirus nucleocapsids and partially surrounded or capped by prominent, clumped chromatin. The patient died with progressive multifocal leukoencephalopathy 24 months after OHL was diagnosed.

Radiation-induced progressive decrease in fluid secretion in rat submandibular glands is related to decreased acinar volume and not impaired calcium signaling.

The mechanism(s) of radiation-induced salivary gland dysfunction is poorly understood. In the present study, we have assessed the secretory function (muscarinic agonist-stimulated saliva flow, intracellular calcium mobilization, Na+/K+/2Cl- cotransport activity) in rat submandibular glands 12 months postirradiation (single dose, 10 Gy). The morphological status of glands from control and irradiated rats was also determined. Pilocarpine-stimulated salivary flow was decreased by 67% at 12 months (but not at 3 months) after irradiation. This was associated with a 47% decrease in the wet weight of the irradiated glands. Histological and morphometric analysis demonstrated that acinar cells were smaller and occupied relatively less volume and convoluted granular tubules were smaller but occupied the same relative volume, while intercalated and striated ducts maintained their size but occupied a greater relative volume in submandibular glands from irradiated compared to control animals. In addition, no inflammation or fibrosis was observed in the irradiated tissues. Carbachol- or thapsigargin-stimulated mobilization of Ca2+ was similar in dispersed submandibular gland cells from control and irradiated animals. Further, [Ca2+]i imaging of individual ducts and acini from control and irradiated groups showed, for the first time, that mobilization of Ca2+ in either cell type was not altered by the radiation treatment. The carbachol-stimulated, bumetanide-sensitive component of the Na+/K+/ 2Cl- cotransport activity was also similar in submandibular gland cells from control and irradiated animals. These data demonstrate that a single dose of gamma radiation induces a progressive loss of submandibular gland tissue and function. This loss of salivary flow is not due to chronic inflammation or fibrosis of the gland or an alteration in the neurotransmitter signaling mechanism in the acinar or ductal cells. The radiation-induced decrease in fluid secretion appears to be related to a change in either the water-handling capacity of the acini or the number of acinar cells in the gland.

Development and characterization of immortalized rat parotid and submandibular acinar cell lines.

The purpose of this investigation was to develop well-differentiated rat parotid and submandibular acinar cell lines. Acinar cells dissociated from rat parotid and submandibular glands were grown on Mitomycin C-treated 3T3 fibroblasts or Matrigel in primary culture and transfected by CaPO4 precipitation using a plasmid containing a replication-defective simian virus (SV40) genome. Cytokeratin analysis via indirect immunofluorescence and receptor mediated changes in intracellular calcium and cyclic AMP were assessed and used for the identification and selection of immortalized epithelial cells. Of the more than 60 clonal cell lines, four retained moderate to high levels of acinar differentiation through >60 passages. Ultrastructurally, there were tripartate junctional complexes and moderate amounts of rough endoplasmic reticulum and secretory granules. Functional studies indicated that beta-adrenoceptors, vasoactive intestinal peptide, and prostaglandin E1 were effective activators of intracellular cyclic AMP production in all cell lines. Alpha-adrenoceptors, muscarinic cholinoceptors, and P2U-purinoceptor agonists were effective in increasing intracellular inositol phosphate production and free calcium levels whereas substance P was ineffective. These data document the utility of the SV40 plasmid in immortalizing rat parotid and submandibular acinar cells that retain most of the features of acinar differentiation.

In vivo use of adenoviral vectors: effects on salivary gland structure.

Recently there has been considerable progress in the development of in vivo gene transfer technology. By this means, new genetic information may be introduced directly to cells, while the cells remain in their natural milieu. The ability to express exogenous proteins makes it possible to explore the functions of native or altered proteins and thereby develop new insights into cell function and dysfunction. We have demonstrated that the major salivary glands are efficiently infected by recombinant adenovirus vectors. These vectors are capable of expressing transgenes in both acinar and ductal cell types. Recently, we have developed vectors that contain cell-specific promoters so that proteins may be expressed in selected subpopulations of salivary cells. Early generations of adenoviral vectors elicited potent immune responses in vivo. However, modified vectors and adjunctive measures have improved the safety of gene transfer to salivary glands. Future studies will aim to increase the duration of adenovirus-based gene expression and to produce vector systems that are not toxic to the host.

A rat parotid gland cell line, Par-C10, exhibits neurotransmitter-regulated transepithelial anion secretion.

Because of the lack of salivary gland cell lines suitable for Ussing chamber studies, a recently established rat parotid acinar cell line, Par-C10, was grown on permeable supports and evaluated for development of transcellular resistance, polarization, and changes in short-circuit current (Isc) in response to relevant receptor agonists. Par-C10 cultures reached confluence in 3-4 days and developed transcellular resistance values of >/=2,000 Omega . cm2. Morphological examination revealed that Par-C10 cells grew as polarized monolayers exhibiting tripartite junctional complexes and the acinar cell-specific characteristic of secretory canaliculi. Par-C10 Isc was increased in response to muscarinic cholinergic and alpha- and beta-adrenergic agonists on the basolateral aspect of the cultures and to ATP and UTP (through P2Y2 nucleotide receptors) applied apically. Ion replacement and inhibitor studies indicated that anion secretion was the primary factor in agonist-stimulated Isc. RT-PCR, which confirmed the presence of P2Y2 nucleotide receptor mRNA in Par-C10 cells, also revealed the presence of mRNA for the cystic fibrosis transmembrane conductance regulator and ClC-2 Cl- channel proteins. These findings establish Par-C10 cells as the first cell line of salivary gland origin useful in transcellular ion secretion studies in Ussing chambers.

Mutual occlusion of P2X ATP receptors and nicotinic receptors on sympathetic neurons of the guinea-pig.

1. The interaction of ion channels activated by nicotinic receptor agonists with ion channels gated by extracellular ATP (i.e. P2X receptors) was studied on sympathetic neurons acutely dissociated from coeliac ganglia of the guinea-pig. Patch clamp methods were used to measure the inward current generated through these non-selective cationic channels under voltage clamp. 2. At the whole cell level, the specific nicotinic receptor agonists nicotine (5-100 microM) or cytisine (50-75 microM) and the P2X receptor agonists ATP (0.1-7 microM) or alpha,beta-methylene ATP (6 microM) were examined separately and in the presence of the other receptor activator. When a nicotinic and P2X receptor agonist were applied together, mutually occlusive effects were generally observed. This occurred even with concentrations of agonists that in themselves generated little to no inward current. 3. The occlusive effects of nicotinic agonists on ATP-gated currents were blocked by the nicotinic receptor/ion channel blocker hexamethonium (150 microM). The occlusive effects of ATP analogues on inward currents generated by nicotinic agonists were blocked by the P2X receptor antagonist suramin (100 microM). 4. Mutual occlusion of the effects of nicotinic agonists and ATP analogues were also observed when currents through single channels were studied in excised (outside-out) patches. 5. The results suggest that nicotinic receptors and P2X ATP receptors do not act independently in these sympathetic neurons.

Development and characterization of SV40 immortalized rat parotid acinar cell lines.

Rat parotid salivary gland acinar cells were transfected by CaPO4 precipitation using a plasmid containing a replication-defective simian virus (SV40) genome. Out of 30 clonal cell lines, 2 were shown to have moderate to high levels of cytodifferentiation and salivary gland acinar cell function. Functional studies with the two cell lines indicated that the beta-adrenergic agonist (isoproterenol), vasoactive intestinal peptide prostaglandin E1, and forskolin were effective activators of intracellular cyclic adenosine 3':5'-cyclic monophosphate production. Phenylephrine, carbamylcholine, and UTP were effective in increasing inositol phosphate production and intracellular free calcium levels, whereas substance P was without affect. Utilizing indirect immunofluorescence analysis, both cell lines were shown to express the SV40 large T antigen. Electron microscopic evaluation documented moderate to high levels of cytodifferentiation with the maintenance of tripartite junctional complexes, cellular polarization, and presence of moderate amounts of secretory granules and rough endoplasmic reticulum. The two cell lines had doubling times of 22 and 36 h, respectively.