Involvement of REG Ialpha protein in the regeneration of ductal epithelial cells in the minor salivary glands of patients with Sjögren's syndrome.

The regenerating gene (Reg) was originally isolated from regenerating rat pancreatic islets and revealed recently to constitute a multi-gene family in humans. REG Ialpha protein is known to be overexpressed not only in various human inflammatory diseases but also in various experimental models of inflammation in animal tissues. However, its involvement in pathophysiology of the minor salivary gland (MSG) is not clear. We investigated REG Ialpha expression in the MSG of patients with primary Sjögren's syndrome (SS) and assessed its role in ductal epithelial cell proliferation in such tissues. Lip biopsy specimens were obtained from 40 patients with primary SS and examined using immunohistochemistry for REG Ialpha protein, Ki67 and single-strand DNA (ssDNA). The relationships among clinicopathological factors and expression of REG Ialpha protein, Ki67 and ssDNA in the MSG were then analysed. REG Ialpha protein was expressed rarely in ductal epithelial cells of the normal MSG but was apparently overexpressed in those of patients with SS. The labelling indices for both Ki67 and ssDNA in the ductal cells of the MSGs were significantly higher in SS patients than in controls. Moreover, these labelling indices were significantly higher in REG Ialpha-positive than in negative SS patients. REG Ialpha protein may play a role in the regeneration of ductal epithelial cells in the MSGs of patients with SS.

Multiple functions of Na/K-ATPase in dopamine-induced salivation of the Blacklegged tick, Ixodes scapularis.

Control of salivary secretion in ticks involves autocrine dopamine activating two dopamine receptors: D1 and Invertebrate-specific D1-like dopamine receptors. In this study, we investigated Na/K-ATPase as an important component of the secretory process. Immunoreactivity for Na/K-ATPase revealed basal infolding of lamellate cells in type-I, abluminal interstitial (epithelial) cells in type-II, and labyrinth-like infolding structures opening towards the lumen in type-III acini. Ouabain (10 µmol l(-1)), a specific inhibitor of Na/K-ATPase, abolished dopamine-induced salivary secretion by suppressing fluid transport in type III acini. At 1 µmol l(-1), ouabain, the secreted saliva was hyperosmotic. This suggests that ouabain also inhibits an ion resorptive function of Na/K-ATPase in the type I acini. Dopamine/ouabain were not involved in activation of protein secretion, while dopamine-induced saliva contained constitutively basal level of protein. We hypothesize that the dopamine-dependent primary saliva formation, mediated by Na/K-ATPase in type III and type II acini, is followed by a dopamine-independent resorptive function of Na/K-ATPase in type I acini located in the proximal end of the salivary duct.

Biological behavior of myoepithelial cells in the regeneration of rat atrophied sublingual glands following release from duct ligation.

The present study aimed to clarify how myoepithelial cells behave during regeneration of an atrophied sublingual gland by investigating cell proliferation and ultrastructure. Atrophy of rat sublingual glands was induced by unilateral ligation of the excretory duct near the hilum with metal clips, which were then removed after one week of ligation for regeneration. The sublingual glands 0-14 days after unligation were examined with single immunohistochemistry for actin as a marker of myoepithelial cells, double immunohistochemistry for actin and proliferating cell nuclear antigen (PCNA) as a marker of proliferating cells, and transmission electron microscopy (TEM). The single immunohistochemistry and TEM showed that myoepithelial cells surrounded residual ducts in the atrophied glands and immature and mature acini in the regenerating glands. Although PCNA-positive myoepithelial cells were identified during regeneration, PCNA labeling indices of myoepithelial cells were low at all time points except at day 7. Ultrastructurally, myoepithelial cells showing bizarre shaped structures in the atrophy changed with maturation of differentiating acinar cells and appeared normal in the regenerated glands. There was no differentiation of the remaining duct cells to myoepithelial cells. These observations suggest that proliferation of myoepithelial cells and differentiation to myoepithelial cells do not commonly participate in the regeneration of atrophied sublingual glands and that the bizarre shaped myoepithelial cells in the atrophied sublingual glands recover the original shapes with acinar cell regeneration.

Active participation of apoptosis and mitosis in sublingual gland regeneration of the rat following release from duct ligation.

This study was designed to establish how mitotic cell proliferation and apoptotic cell death participate in the regeneration of atrophied rat sublingual glands. To induce atrophy to the sublingual gland of rats, the excretory duct was ligated unilaterally near the hilum, and after 1 week of ligation (day 0) the duct ligation was released to enable gland regeneration. The regenerating glands were examined with routine histology, immunohistochemistry for proliferating cell nuclear antigen (PCNA) as a marker of proliferating cells, terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) as a marker of apoptotic cells, and transmission electron microscopy. At day 0, a few acini and many ducts remained in the atrophic sublingual glands, and newly formed immature acini were observed at day 3. Thereafter acinar cells progressively matured and increased in number, although the number of ducts decreased. Many PCNA- and some TUNEL-positive cells were seen in acini and ducts during regeneration. The labeling indices for both cell types were statistically significantly different from that of the control at several time points of the regeneration. Apoptotic and mitotic cells were also confirmed to be present in the experimental sublingual glands by electron microscopy. These observations suggest that apoptosis as well as mitosis of duct and acinar cells actively participate in and play important roles in sublingual gland regeneration.

Dopamine and serotonin receptors mediating contractions of the snail, Helix pomatia, salivary duct.

The combination of high performance liquid chromatography, bioassay and immunocytochemistry was applied to study the regulation of the salivary duct muscle of the snail, Helix pomatia. The major function of the duct appears to be to propel the saliva toward the buccal cavity during feeding. It has been established that serotonin and dopamine applied exogenously mimic the effect on the duct exerted by the stimulation of the salivary nerve. Immunohistochemistry revealed the presence of serotonin, but not dopaminergic nerve elements in the nerve and along the duct surface. However, both serotonin (14.9-15.5 pmol/mg) and dopamine (0.38-0.58 pmol/mg), as well as the synthesizing enzymes (tyrozine hydroxylase 0.28 pmol/mg tissue/h and DOPA 0.32 nmol synthesized DA/mg tissue/h) could regularly be assayed in the salivary duct by high performance liquid chromatography. When released following the stimulation of the salivary nerve, both monoamines were shown to interact with distinct membrane receptors. Dopamine elicited a sustained increase of the muscle tone in concentration-dependent manner (K(d)=1.5 microM). Mammalian D(1) receptor antagonist flupenthixol and fluphenazine attenuated, whereas the D(1) receptor agonist SKF-38393 mimicked the effect elicited by exogenous dopamine. Serotonin had a double effect on the salivary duct: a relaxing and a contracting one with different K(d) values 76 nM and 2.4 microM, respectively. 5-HT(2) receptor antagonist ritanserin and ketanserin attenuated the serotonin-induced relaxation. In contrast 5-HT(3) antagonist metoclopramide and MDL2222 decreased and 5-HT(3) receptor agonist 1-(m-chlorophenyl)-biguanide mimicked the serotonin-induced contraction, suggesting that serotonin exerted its action on two different receptor subtypes. The release of radiolabeled serotonin and dopamine upon nerve stimulation was found to be Ca-dependent. Furthermore, the increase in serotonin concentration induced a decrease of the potency of dopamine to elicit sustained contraction. These results provide evidence for the transmitter role of serotonin and dopamine in salivary duct. It is concluded that receptors reveal a pharmacological profile related to vertebrate D(1), 5-HT(2) and 5-HT(3) receptor subtypes. Moreover, it was found that the process of conveying the saliva is modulated by an interaction of dopamine and serotonin.

A morphogenetic concept of salivary duct regeneration and metaplasia.

The exact mechanisms of physiological regeneration and of metaplastic processes of the salivary duct have not been definitely established, although regeneration from a putative uncommitted stem cell population has long been favored. In the present study, double immunohistochemical labeling for Ki 67 and alpha-actin or different cytokeratin subtypes, respectively, made possible an exact localization and quantification of cellular proliferation in the regular salivary duct and in different types of metaplasia. Our data demonstrate a baseline proliferative capacity in all five cell types of the salivary duct. Luminal secretory cells of the acinus and intercalated duct regenerate independently from myoepithelial or basal cells. In contrast, the renewal of oxyphilic cells in the striated and excretory duct is maintained by proliferation and differentiation of basal cells. The great majority of metaplasias develops from uncommitted, Bcl-2 positive basal cells of striated/excretory ducts which possess an enormous capacity for pluridirectional morphogenetic differentiation. Despite this important role of basal cells, our findings demonstrate that all cell types principally have to be considered as potential progenitor cells for salivary gland tumors. The improved insight into regenerative and metaplastic processes of the salivary duct may contribute to a better understanding of the complex formal carcinogenesis.

Morphological and functional characteristics of acinar atrophy and recovery in the duct-ligated parotid gland of the rat.

Although acinar atrophy occurs frequently in salivary diseases, the relationship between structural changes and functional decrements is not well-established, and the potential for recovery of histological and functional integrity has not been wholly quantified. We aimed, therefore, to develop further our understanding of pathological acinar atrophy. Stensen's duct was ligated for periods up to six weeks and, in separate experiments, was de-ligated after two weeks and allowed to recover for up to two weeks. Qualitative and quantitative histological analyses were carried out. Additionally, the ability of enzymatically dispersed cells from ligated and de-ligated glands to respond to neurohormonal stimuli was also measured. The results confirmed that totally obstructed glands undergo a rapid, progressive severe atrophy amounting to absolute losses of over 85% of acinar tissue by two weeks. Acinar shrinkage and cell losses through apoptosis accounted for the glandular atrophy. Remaining intralobular epithelia consisted of extremely atrophic acini and numerous duct-like structures with intermediate forms. Dispersed cells from atrophic glands exhibited agonist-induced release of chloride similar to normal. Together, these structural-functional results confirm the persistent viability of acinar-like cells in the obstructed gland and suggest that the duct-like structures are derived from surviving atrophic acini. De-ligated glands exhibited a near-normal recovery of structure by two weeks. Their enzymatically dispersed cells responded normally to agonist stimulation. The results support the view that pathological atrophy is largely similar to physiological atrophy, providing a mechanism for acinar cell survival under adverse conditions, with the possibility of eventual recovery.

Sour taste stimulation facilitates reflex swallowing from the pharynx and larynx in the rat.

Chemical stimulation of the pharynx and larynx is effective in eliciting reflex swallowing. A sour taste bolus facilitates the onset of swallowing in patients with neurogenic dysphagia, but the mechanism of the facilitation has not been clarified. We investigated the effect of sour solutions on the elicitation of reflex swallowing in anesthetized rats. The main ducts of salivary glands were ligated to avoid the effect of saliva. A small amount of water, sour solutions, and other taste solutions were applied to the mucosa of the pharyngolaryngeal region. Acetic acid and citric acid, which provide a sour taste, had a stronger effect on evoking reflex swallowing as compared with other taste solutions. The effectiveness of these acids increased with increasing concentrations. We also examined the contribution of the superior laryngeal nerve (SLN) and the pharyngeal branch of the glossopharyngeal nerve (GPNph) to reflex swallowing. Acetic acid was greatly effective in evoking swallowing in both the region innervated by the SLN and the GPNph. On the other hand, water was effective in the SLN region but only slightly effective in the GPNph region. The results indicate that stimulation of the pharyngolaryngeal region with sour solutions facilitates reflex swallowing, suggesting that the facilitation may be due to increases of sensory inputs via the SLN and GPNph.

Membrane stretch and salivary glands - facts and theories.

Cell shape in salivary glands is affected by mechanical forces. In the acini and ducts cell shape is modified by the contractions of the myoepithelial cells in both the secretory and ductal portions of the glands. At the organ level shape changes are due to muscle contraction during mastication, food intake and speech. All these factors may cause some degree of stretching of salivary cell membranes. Recent studies suggest that physical forces influence signal transduction, gene expression, secretory function, cell differentiation and proliferation. Here we overview membrane stretch-activated cellular events. Evidence from a variety of tissues suggests that mechanical forces may alter the properties of acinar cells leading to cytoskeletal reorganisation, changes in ion fluxes, modulation of secretory activity and subsequent release of transmitters such as ATP. Transmitters released from acinar cells may modulate the secretory activity of salivary tissue, and interact with classical regulatory pathways.

Oral focal acantholytic dyskeratosis induced in sialoadenectomized rats painted orally with the carcinogen 4-nitroquinoline N-oxide.

This study examines the effects of xerostomia on the development of mucosal changes proceeding carcinoma in rats painted orally with 4-nitroquinoline N-oxide (4NQO). Sialoadenectomy enhanced the incidence of focal acantholytic dyskeratosis (FAD) on the palate and tongue of rats painted on the palate with 0.5% 4NQO three times weekly. At one month, no changes were observed. After two months of painting with 4NQO, sialoadenectomized rats, but not controls, had FAD. After three months, FAD was seen in both test and control groups, but the incidence of FAD was higher in the test group and seen more on the palate than on the tongue of xerostomic animals. Neither test nor control animals developed carcinoma within this study period.

Study of human adult parotid duct in the area of penetration through buccinator muscle and their functional relationship as a sphincter.

The adult human parotid duct is roughly 6-8 cm long. From the parotid gland, parotid duct traverses through masseter muscle, penetrates through buccinator muscle, and opens into the oral cavity. This unique form of the parotid duct is likely correlated with the function of the duct, directly affected by the movement of the buccinator muscle during mastication and swallowing. Histological structure of the duct is known to be different in each region, and details of smooth muscle present in the parotid duct are mostly unclear. In this study, we conducted SEM and histological observations of the area where the parotid duct penetrates the buccinator muscle, and an observation of smooth muscle to investigate its existence using alpha-smooth muscle antibody. We confirmed the presence of an abundance of skeletal muscle bundles likely originating from the buccinator muscle under the epithelium of the parotid duct wall in the region where it penetrates the buccinator muscle. We also observed that some of the muscle fibers were completely attached to the epithelium. We observed a lack of smooth muscle in this region of the duct wall. From these results, we suggest that the area of the duct penetrating buccinator muscle plays a role in regulating the salivary passage through the contraction of the surrounding buccinator muscle fibers.

Cannulation of the mouse submandibular salivary gland via the Wharton's duct.

Severe salivary gland hypofunction is frequently found in patients with Sjögren's syndrome and those who receiving therapeutic irradiation in their head and neck regions for cancer treatment. Both groups of patients experience symptoms such as xerostomia (dry mouth), dysphagia (impaired chewing and swallowing), severe dental caries, altered taste, oro-pharyngeal infections (candidiasis), mucositis, pain and discomfort. One innovative approach of regenerative medicine for the treatment of salivary gland hypo-function is speculated in RS Redman, E Mezey et al. 2009: stem cells can be directly deposited by cannulation into the gland as a potent method in reviving the functions of the impaired organ. Presumably, the migrated foreign stem cells will differentiate into glandular cells to function as part of the host salivary gland. Also, this cannulation technique is an expedient and effective delivery method for clinical gene transfer application. Here we illustrate the steps involved in performing the cannulation procedure on the mouse submandibular salivary gland via the Wharton's duct (Fig 1). C3H mice (Charles River, Montreal, QC, Canada) are used for this experiment, which have been kept under clean conventional conditions at the McGill University animal resource center. All experiments have been approved by the University Animal Care Committee and were in accordance with the guidelines of the Canadian Council on Animal Care. For this experiment, a trypan blue solution is infused into the gland through the opening of the Wharton's duct using a insulin syringe with a 29-gauge needle encased inside a polyethylene tube. Subsequently, the mouse is dissected to show that the infusions migrated into the gland successfully.

Salivary gland regeneration.

The ability of animal salivary glands to recover from an experimentally-induced atrophic state offers hope that human salivary glands may be regenerated following injury. Examination of the mechanisms of regeneration in animal models has revealed processes which resemble the embryonic formation of salivary glands. Secretory proteins present in regenerated acinar and ductal cells are the same as found in the perinatal salivary glands. The use of microarrays to reveal global gene changes has, in combination with bioinformatic techniques, identified some of the important signalling cascades operating in the early stages of glandular regeneration. The role of stem cells is also considered and would fit in with current ideas of glandular regeneration, however the isolation and subsequent differentiation of stem cells into a normal reflexly secreting gland still requires considerable research.

Spontaneous tissue regeneration of the Stensen duct in dogs.

OBJECTIVE: There is a high incidence of wounds in the excretion duct of the salivary parotid gland owing to facial trauma that causes a loss of duct tissue. The aim of this study is to evaluate the ability of the salivary tissue to spontaneously self-regenerate, which is here widely demonstrated. STUDY DESIGN: This work was carried out on 20 adult dogs. A biomaterial catheter was inserted using microsurgical procedures to regenerate the lost duct tissue. This catheter served as an indicator for regenerating tissue growth. The results were assessed through clinical and radiographic exams by means of salivary charts and a histologic study. RESULTS: The results obtained demonstrate the complete regeneration of the Stensen duct in all cases. The self-expulsion of the intraductal guide was satisfactory, albeit unexpected, such that subsequent management of the patient was unnecessary. CONCLUSION: Spontaneous tissue regeneration of salivary ducts is possible using a biomaterial guide.

Abnormal basement membrane type IV collagen alpha-chain composition in labial salivary glands in Sjögren's syndrome.

OBJECTIVE: Sjögren's syndrome (SS) is characterized by atrophy and malfunction of the acinar cells. The aim of this study was to investigate whether type IV collagen alpha-chain composition of acinar cell compartments could be abnormal in diseased glands. METHODS: Messenger RNA (mRNA) from human submandibular gland (HSG) cells, cultured with or without growth factor-depleted Matrigel, was analyzed using quantitative reverse transcription-polymerase chain reaction (RT-PCR). Labial salivary glands were analyzed using quantitative RT-PCR and immunohistochemistry. RESULTS: HSG cells of both the ductal and acinar phenotypes synthesized all alpha-chain mRNA, in particular those of the alpha1 and alpha2 chains. Labial salivary glands (LSGs) contained alpha1/2 chains but also contained mRNA of all the other alpha-chains, although the mRNA copy numbers for the alpha3 and alpha4 chains were low, and the corresponding proteins were absent. Type IV collagen alpha1/2-chains were observed in all tubuloalveolar basement membranes. In healthy glands, alpha5 and alpha6 chains were continuous around ducts but discontinuous around acini. In SS glands, these chains were absent or patchy around the ducts and absent around the acini. CONCLUSION: Ductal and acinar epithelial cells are able to locally produce mRNA for all 6 different alpha-chains. Type IV collagen alpha1/2-chains seem to form the backbone in the tubuloalveolar basement membrane in salivary glands. Type IV collagen alpha3 and alpha4 chain mRNA were found in cultured salivary epithelial cells and LSG explants but were not translated to the corresponding alpha-chains in LSGs. Both alpha5 and alpha6 mRNA were observed in salivary epithelial cells and glands. In healthy glands, immunolabeling always disclosed corresponding alpha-chains around ducts, but their synthesis and/or degradation seemed to be locally regulated around acinar cells.

Topographical distribution of cells in the rat submandibular gland duct system with special reference to dark cells and tuft cells.

The duct system of the rat submandibular gland consists of the intercalated duct, the granular convoluted tubule, the striated duct, the excretory duct, the main excretory duct, and the salivary bladder. The duct system contains special cell types, such as dark cells and tuft cells, in addition to principal cells. However, little is known about cell distribution in the duct system. The purpose of the present study was to examine cell distribution and to perform a morphometric analysis of the duct system. Transmission and scanning electron microscopy were used to examine the duct system of the rat submandibular gland. Six regions in the duct system, the striated duct, the interlobular excretory duct, the 5-mm proximal excretory duct from the hilus, the main excretory duct at the hilus, the 10-mm distal main excretory duct from the hilus, and the salivary bladder, were investigated. Morphometric and statistical analyses of the data were then performed. The epithelium of the duct system consisted of a heterogeneous cell population. Dark cells and tuft cells were present throughout the duct system. The principal, dark, and tuft cells were distinguished by their different microvilli by using a scanning electron microscope. The frequency of these cells in the total epithelial cell population was as follows: The percentage of principal cells in the six regions of the duct system varied from 87.5% to 94.4%, that of dark cells varied from 4.1% to 7.2%, and that of tuft cells varied from 1.8% to 7.2%. The number of principal and tuft cells was significantly different between the striated duct and the main excretory duct at the hilus (P < 0.01). However, no significant difference in number of dark cells throughout the duct system was observed (P > 0.05). The abundance of the principal, dark, and tuft cells in the duct system of the rat submandibular gland was determined. Few tuft cells were distributed in the striated duct, and most were found at the hilus. Dark cells were distributed equally throughout the duct system.

An unusual parotid gland in the tent-building bat, Uroderma bilobatum: possible correlation of interspecific ultrastructural differences with differences in salivary pH and buffering capacity.

The tent-building bat, Uroderma bilobatum, is a small, frugivorous phyllostomid bat with a broad neotropical distribution. Generally found in humid forest, this bat lives in small groups that create daytime "roosts" from large leaves of a variety of tropical plants. Fruit eating engenders a variety of ecological and physiological challenges for bats, some of which could require adaptive features in their salivary glands. The parotid salivary glands of Uroderma bilobatum were prepared for transmission electron microscopy by using methods that have become standard for field work. The parotid gland is extremely unusual in structure. Although the secretory endpieces still produce serous granules with a complex substructure, they are modified into quasi striated ducts. Their basal folds, which are extensive, occasionally harbor some vertically oriented mitochondria, imparting a resemblance to striated ducts. Other evidence for the endpiece origin of these parenchymal components is a well-developed system of intercellular canaliculi, structures that never occur in bona fide striated ducts. The long but sparse intercalated ducts consist of two types of cells, each of which elaborates a modest number of secretory granules of differing substructure. Striated ducts are of conventional morphology, except that a few dark cells shaped like wine glasses are present in their walls. The striated duct cells produce no secretory granules, but their apical cytoplasm may contain some small, empty vesicles. Capillaries lie in longitudinal grooves in the base of the duct cells, an arrangement that might enhance electrolyte exchange. Excretory ducts consist of simple cuboidal epithelium composed of cytologically unspecialized cells that sometimes includes a dark cell. It was concluded that salivary glands could have a major role in adapting species to acquire nutrients from marginal sources, such as tropical fruits, which have a low protein and sodium content. The unusual parotid acinar cells in Uroderma bilobatum are discussed in the context of salivary pH and buffering capacity. Comparisons are made with four other bat species, including an insectivorous species with a salivary pH > 8.0 and a very high buffering capacity, an intermediate species, and a fruit bat with acidic-stimulated saliva and very low buffering capability. Such interspecific comparisons provide a foundation for hypothesizing that ultrastructural features of the acinar cell basolateral membranes and intercellular canaliculi correlate with differences involving Na/H+ exchangers and release of HCO3- and, thus, are associated with the species differences that are important to diet and nutrient acquisition.

Nedd4 mediates control of an epithelial Na+ channel in salivary duct cells by cytosolic Na+.

Epithelial Na+ channels are expressed widely in absorptive epithelia such as the renal collecting duct and the colon and play a critical role in fluid and electrolyte homeostasis. Recent studies have shown that these channels interact via PY motifs in the C terminals of their alpha, beta, and gamma subunits with the WW domains of the ubiquitin-protein ligase Nedd4. Mutation or deletion of these PY motifs (as occurs, for example, in the heritable form of hypertension known as Liddle's syndrome) leads to increased Na+ channel activity. Thus, binding of Nedd4 by the PY motifs would appear to be part of a physiological control system for down-regulation of Na+ channel activity. The nature of this control system is, however, unknown. In the present paper, we show that Nedd4 mediates the ubiquitin-dependent down-regulation of Na+ channel activity in response to increased intracellular Na+. We further show that Nedd4 operates downstream of Go in this feedback pathway. We find, however, that Nedd4 is not involved in the feedback control of Na+ channels by intracellular anions. Finally, we show that Nedd4 has no influence on Na+ channel activity when the Na+ and anion feedback systems are inactive. We conclude that Nedd4 normally mediates feedback control of epithelial Na+ channels by intracellular Na+, and we suggest that the increased Na+ channel activity observed in Liddle's syndrome is attributable to the loss of this regulatory feedback system.

Autonomic nervous control of myoepithelial cells and secretion in submandibular gland of anaesthetized dogs.

In dog submandibular gland, the activity of myoepithelial cells was assessed by simultaneous measurement of intraductal pressure (P(du)) and subcapsular pressure (P(ca)) using catheter-tip pressure transducers; their resting values were 2.5 +/- 0.21 and 3.0 +/- 0.19 mmHg, respectively (n = 40). Retrograde infusion of saliva (collected from preceding parasympathetic nerve stimulation) increased P(du) (coefficient of 50 mmHg ml(-1) for rates < 1 ml min(-1) and 85 mmHg ml(-1) for higher rates) and P(ca) (coefficient of 0.47 mmHg ml(-1) for all rates). Blood flow changes did not affect P(du) but increased P(ca) (coefficient of 0.04 mmHg ml(-1)). Parasympathetic nerve stimulation increased P(du) but decreased P(ca) abruptly; the response threshold was 0.1 Hz, with maximal responses at 16 Hz. The coefficients for P(du) and P(ca) on salivary secretion to parasympathetic nerve stimulation in glands with spontaneous blood flow (5.3 x 10(-3) and 4.87 x 10(-2) ml min(-1) g(-1) mmHg(-1)) were close to their values in glands with constant-flow vascular perfusion (4.9 x 10(-3) and 3.68 x 10(-2) ml min(-1) g(-1) mmHg(-1)). The finding that P(ca) fell despite concomitant increased blood flow suggests contraction of myoepithelial cells. Additional ductal occlusion further increased P(du) and enhanced the fall in P(ca), suggesting that the myoepithelial cells can contract when distended. Atropine blocked salivary secretion and responses of P(du) and P(ca) to parasympathetic nerve stimulation. ACh elicited responses similar to that of parasympathetic nerve stimulation. VIP caused very scanty salivary secretion and gradual slight increases in P(du) and P(ca); the change in P(ca) was abolished in glands with constant-flow vascular perfusion. Hence, contraction of myoepithelial cells to parasympathetic nerve stimulation is via muscarinic receptors. Sympathetic nerve stimulation increased P(du) and decreased P(ca) abruptly; the response threshold was 0.1 Hz, with maximal responses at 16 Hz. The coefficients for P(du) and P(ca) on salivary secretion to sympathetic nerve stimulation in glands with spontaneous blood flow (3.0 x 10(-3) and 3.2 x 10(-3) ml min(-1) g(-1) mmHg(-1)) were similar to their values in glands with constant-flow vascular perfusion (3.2 x 10(-3) and 3.1 x 10(-3) ml min(-1) g(-1) mmHg(-1)). The finding that P(ca) fell even in glands with constant-flow vascular perfusion suggests contraction of myoepithelial cells. Superimposed sympathetic nerve stimulation immediately enhanced the pressure changes and secretory response to parasympathetic nerve stimulation, indicating that the two autonomic nerves act synergistically to evoke myoepithelial cell contraction. Phentolamine and prazosin but not propranolol and yohimbine blocked the sympathetic enhancement. The finding that phenylephrine, but not clonidine and isoproterenol, abruptly decreased P(ca) in glands with constant-flow vascular perfusion suggests that the sympathetic activation of myoepithelial cells is via the alpha(1)-adrenoceptors.

Characterization, localization and axial distribution of Ca2+ signalling receptors in the rat submandibular salivary gland ducts.

1. To characterize [Ca2+]i signalling in salivary duct cells a procedure was developed for the rapid preparation and isolation of intralobular ducts, some of which had attached intercalated ducts. The isolated ducts retained agonist-induced Ca2+ signalling after permeabilization with streptolysin O (SLO). 2. The improved cell preparation technique was reflected in the repertoire and intensity of agonist responsiveness of the cells. Measurements of [Ca2+]i in intact cells showed that all agonists previously reported to affect electrolyte transport by the submandibular salivary gland (adrenaline, carbachol, isoprenaline and forskolin) mobilized Ca2+ from internal stores and increased Ca2+ influx across the plasma membrane. 3. The use of the SLO-permeabilized ducts showed that all agonists, including isoprenaline and forskolin, mobilized Ca2+ exclusively from the inositol 1,4,5 trisphosphate (IP3)-sensitive pool. However, in granular ducts only adrenaline mobilized the entire IP3-sensitive pool whereas all other agonists mobilized only part of the pool. 4. All regions of the duct responded to substance P and the luminally secreted agonist ATP. Interestingly, the intercalated duct was most responsive to ATP and demonstrated only a minimal response to all other agonists. The granular region of the same duct and the extralobular duct always responded best to stimulation by adrenaline. 5. The perfused extralobular duct was used to show that adrenaline and carbachol stimulated the duct through the basolateral membrane whereas the receptors for ATP were localized in the luminal membrane of the duct. This suggests the presence of an ATP-dependent positive feedback loop in salivary duct with decreased activity along the ductal tree.