Structural and functional characterization of striated ducts isolated from the rabbit mandibular salivary gland

Abstract

SUMMARYDuctal elements within salivary glands are responsible for modifying the electrolyte composition of primary saliva secreted by the acini. To study the mechanism and regulation of the transport processes involved requires a suitable preparation of functional ducts. To this end we have isolated intralobular ducts from rabbit mandibular salivary glands using the technique of tissue dissociation and microdissection. Light and electron microscopy demonstrated that the ducts corresponded ultrastructurally to striated intralobular ducts of the intact gland. Ducts could be maintained in tissue culture on polycarbonate filter rafts for up to 36 h, during which time the ends of the ducts did not usually seal. The overall resting content of ductal adenosine 3',5'-cyclic monophosphate (cyclic AMP) was 160 + 3 0 fmol mm-' and increased dose dependently in response to stimulation with the /3-adrenoceptor agonist isoprenaline (10-9-10-4 M; concentration required to produce a half-maximal response, K0.5 = 2-1 X 10-6 M). The response to isoprenaline was blocked by the antagonist propranolol. Application of the calcium ionophore ionomycin induced a substantial maintained rise in[Ca2+]1. Taken together, these results indicate that isolated and cultured striated ducts (i) possess intact ,-adrenoceptors coupled to adenylate cyclase, putative receptors for prostaglandin E2 and muscarinic receptors, and (ii) represent a viable preparation for the study of the transport mechanisms involved in the ductal modification of salivary fluid composition.

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

“…However, recently a technique for the isolation of agonist-responding duct fragments from the submandibular salivary glands has been described (Dehaye & Turner, 1991) and used to characterize several aspects of signalling (Valdez & Turner, 1991;Dehaye et al 1993) and ion transport (Paulais, Cragoe & Turner, 1994;Xu, Zhao, Diaz & Muallem, 1995) respectively, lower than reported previously ( Valdez & Turner, 1991;Dehaye et al 1993;Dinudom et al 1993). Interestingly, the apparent affinity for isoprenaline in increasing [Ca2P]i of rat ducts (4-8 nm, present studies) is about 400-fold lower than that reported for increasing cellular cAMP in rabbit ducts (2-1 #m; Evans et al 1993). …”

“…However, in recent years techniques have been developed to examine these topics on the cellular level. Isolated ducts were used to show that isoprenaline stimulation increases cellular cAMP (Dehaye & Turner, 1991;Evans, Lau & Case, 1993 Dinudom, Porronnik, Allen, Young & Cook, 1993) and ,J-adrenergic (Dehaye, Valdez & Turner, 1993) Cholinergic and a-adrenergic agonists were proposed to mobilize [Ca2+]i from the IP3-sensitive pool, whereas the effect of the /J-adrenergic agonist isoprenaline on [Ca2+]i was attributed to Ca2P mobilization from an IP3-independent pool (Dehaye et al 1993).…”

Clcn2encodes the hyperpolarization-activated chloride channel in the ducts of mouse salivary glands

“…Figure 4A shows results from experiments with the muscarinic agonist CCh, whereas Fig. 4B is from experiments using carbachol with IPR to stimulate ␤-adrenergic receptors and raise the intracellular cAMP content of duct cells (12,16) and thus further enhance Cl Ϫ reabsorption through the activation of Cftr (25,38). As expected, the addition of IPR enhanced Cl Ϫ uptake from saliva by ϳ50% in wild-type glands, and to a comparable magnitude in Clcn2 null glands.…”

A rapid method for the preparation of single-cell suspensions from solid tissues

“…For example, to study DNA strand breaks using the microgel electrophoresis technique, an initial step is the dissociation of solid tissues into single cells. Various enzymatic (3,4,(7)(8)(9)(10)(11) and mechanical (1,5,6) methods have been used in the past to dissociate cells from solid tissues. They are all associated with higher cellular damage such as basal levels of DNA strand breaks, probably induced during the process.…”