In most cells, the ubiquitously expressed Na+/H+exchanger isoform 1 (NHE1) is thought to be a primary regulator of pH homeostasis, cell volume regulation, and the proliferative response to growth factor stimulation. To study the function of NHE1 during embryogenesis when these cellular processes are very active, we targeted the Nhe1 gene by replacing the sequence encoding transmembrane domains 6 and 7 with the neomycin resistance gene. NHE activity assays on isolated acinar cells indicated that the targeted allele is functionally null. Although the absence of NHE1 is compatible with embryogenesis, Nhe1 homozygous mutants (−/−) exhibit a decreased rate of postnatal growth that is first evident at 2 wk of age. At this time, Nhe1 −/− animals also begin to exhibit ataxia and epileptic-like seizures. Approximately 67% of the −/− mutants die before weaning. Postmortem examinations frequently revealed an accumulation of a waxy particulate material inside the ears, around the eyes and chin, and on the ventral surface of the paws. Histological analysis of adult tissues revealed a thickening of the lamina propria and a slightly atrophic glandular mucosa in the stomach.

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Multiple Na؉ /H ؉ exchangers (NHEs) are expressed in salivary gland cells; however, their functions in the secretion of saliva by acinar cells and the subsequent modification of the ionic composition of this fluid by the ducts are unclear. Mice with targeted disruptions of the Nhe1, Nhe2, and Nhe3 genes were used to study the in vivo functions of these exchangers in parotid glands. Immunohistochemistry indicated that NHE1 was localized to the basolateral and NHE2 to apical membranes of both acinar and duct cells, whereas NHE3 was restricted to the apical region of duct cells. Na ؉ /H ؉ exchange was reduced more than 95% in acinar cells and greater than 80% in duct cells of NHE1-deficient mice (Nhe1 ؊/؊ ). Salivation in response to pilocarpine stimulation was reduced significantly in both Nhe1 ؊/؊ and Nhe2 ؊/؊ mice, particularly during prolonged stimulation, whereas the loss of NHE3 had no effect on secretion. Expression of Na ؉ /K ؉ /2Cl ؊ cotransporter mRNA increased dramatically in Nhe1 ؊/؊ parotid glands but not in those of Nhe2 ؊/؊ or Nhe3 ؊/؊ mice, suggesting that compensation occurs for the loss of NHE1. The sodium content, chloride activity and osmolality of saliva in Nhe2 ؊/؊ or Nhe3 ؊/؊ mice were comparable with those of wild-type mice. In contrast, Nhe1 ؊/؊ mice displayed impaired NaCl absorption. These results suggest that in parotid duct cells apical NHE2 and NHE3 do not play a major role in Na ؉ absorption. These results also demonstrate that basolateral NHE1 and apical NHE2 modulate saliva secretion in vivo, especially during sustained stimulation when secretion depends less on Na ؉ /K ؉ /2Cl ؊ cotransporter activity.

SUMMARY1. We have studied the effects of acetylcholine (ACh) on fluid secretion and intracellular messengers in interlobular ducts isolated from the rat pancreas and maintained in short-term tissue culture.2. Ductal fluid secretion was measured using micropuncture techniques.Intracellular free calcium ([Ca2+]1) and cyclic AMP concentrations were measured in single ducts using fura-2 microspectrofluorimetry and radioimmunoassay techniques respectively. Changes in the levels of these intracellular messengers were correlated with fluid secretion.3. ACh stimulated ductal fluid secretion. The dose required for a half-maximal response was about 0 4 ,/M and maximal secretion was achieved with 10 /LM ACh.These effects of ACh were blocked by atropine and by removal of extracellular Ca2". 4. ACh was about four orders of magnitude less potent as an activator of ductal fluid transport than the hormone secretin; however, the maximal rates of fluid secretion evoked by these two agonists were similar.5. ACh caused a dose-dependent rise in duct cell [Ca2+]i, but had no effect on cyclic AMP. In contrast, secretin increased duct cell cyclic AMP, but had no effect on [Ca2+]1. tTo whom reprint request should be sent.

The onset of salivary gland fluid secretion in response to muscarinic stimulation is accompanied by up-regulation of Na ؉ /H ؉ exchanger (NHE) activity. Although multiple NHE isoforms (NHE1, NHE2, and NHE3) have been identified in salivary glands, little is known about their specific function(s) in resting and secreting acinar cells. Mice with targeted disruptions of the Nhe1, Nhe2, and Nhe3 genes were used to investigate the contribution of these proteins to the stimulation-induced up-regulation of NHE activity in mouse parotid acinar cells. The lack of NHE1, but not NHE2 or NHE3, prevented intracellular pH recovery from an acid load in resting acinar cells, in acini stimulated to secrete with the muscarinic agonist carbachol, and in acini shrunken by hypertonic addition of sucrose. In HCO 3 ؊ -containing solution, the rate of intracellular pH recovery from a muscarinic agoniststimulated acid load was significantly inhibited in acinar cells from mice lacking NHE1, but not in cells from NHE2-or NHE3-deficient mice. These data demonstrate that NHE1 is the major regulator of intracellular pH in both resting and muscarinic agonist-stimulated acinar cells and suggest that up-regulation of NHE1 activity has an important role in modulating saliva production in vivo.

1. The effects of fluid secretory stimuli on the Na+-K+-2Cl-cotransporter in rat parotid acini were investigated. Cotransporter activity was measured using NH4+ as a K+ surrogate and following cotransporter-mediated NH4+ fluxes by monitoring intracellular pH. 2. A dramatic upregulation (15-to 20-fold) of acinar Na+-K+-2Cl-cotransporter activity was induced by muscarinic, a1-adrenergic and peptidergic stimuli. A half-maximal effect of the muscarinic agonist carbachol was observed at -0 5 /M.3. Our results indicate that the rise in intracellular calcium concentration ([Ca2P]1) which accompanies these stimuli is both a necessary and a sufficient condition for this effect; but it is not a consequence of the KCI loss and concomitant isotonic shrinkage caused by increased [Ca2+]i as it persists when these effects are prevented. 4. The effect of muscarinic stimulation on the cotransporter can, however, be blocked by inhibitors of phospholipase A2 (4-bromophenacylbromide and manoalide), by a general inhibitor of arachidonic acid metabolism (5,8,11,14-eicosatetraynoic acid) and by specific inhibitors of the cytochrome P450 pathway (methoxsalen and ketoconazole). 5. These latter results argue strongly for the involvement of a product of the cytochrome P450 pathway of arachidonic acid metabolism in upregulation of the salivary Na+-K+-2ClF cotransporter.6. Owing to the complexity of the arachidonic acid cascade a wide variety of agents could potentially interfere with this upregulation of the cotransporter, and thereby result in decreased salivary fluid production. We suggest that such an effect could underlie the dry mouth (xerostomia) that occurs as an unexplained side-effect of many commonly prescribed medications.

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.

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