Vertebrate salt glands: Short- and long-term regulation of function

JOURNAL OF EXPERIMENTAL ZOOLOGY 283:689–701 (1999)

© 1999 WILEY-LISS, INC.

Vertebrate Salt Glands: Short- and Long-TermRegulation of Function

TREVOR J. SHUTTLEWORTH1* AND JAN-PETER HILDEBRANDT2

1Department of Pharmacology and Physiology, University of RochesterSchool of Medicine and Dentistry, Rochester, New York 14642

2Physiologisches Institut, Medizinische Fakultät, Universität desSaarlandes, D-66421 Homburg/Saar, Germany

ABSTRACT Excess salt loads in most non-mammalian vertebrates are dealt with by a varietyof extra-renal salt-secreting structures collectively described as salt glands. The best studied ofthese are the supra-orbital nasal salt glands of birds. Two distinct types of response to osmoregu-latory disturbances are shown by this structure: a progressive adaptive response on initial expo-sure to a salt load that results in the induction and enhancement of the secretory performance orcapabilities of the gland; and the rapid activation of existing osmoregulatory mechanisms in theadapted gland in response to immediate osmoregulatory imbalance. Not only is the time-frame ofthese two types of response very different, but the responses usually involve fundamentally differ-ent processes: e.g., the growth and differentiation of osmoregulatory structures and their compo-nents in the former case, compared with the rapid activation of ion channels, pumps etc. in thelatter. Despite marked differences in the nature and time-frame of these responses, they both areapparently triggered by neuronally released acetylcholine, which acts at muscarinic receptors onthe secretory cells to induce an inositol phosphate-dependent increase in cytosolic-free calciumconcentrations ([Ca2+]i). Therefore, the question arises as to how the cells produce the appropriatedistinct response using a single common signal (i.e., an increase in [Ca2+]i). Examination of thefeatures of this signaling pathway in the two conditions described, reveals that they each areuniquely tuned to generate a response with the characteristics appropriate for the cells’ require-ments. This tuning of the signal involves often rather subtle changes in the overall signalingpathway that are part of the adaptive differentiation process. J. Exp. Zool. 283:689–701, 1999.© 1999 Wiley-Liss, Inc.

Animals respond to environmental challengesby activation of a variety of homeostatic mecha-nisms that are instrumental in keeping the inter-nal conditions in the animal constant, or at leastclose to given “set-points.” While the consequencesof acute environmental stress on body functionsare generally balanced by immediate changes inanimal behavior, or functional changes at the levelof organs or individual cells, prolonged environ-mental alterations often induce long-term changesin body functions. These changes are associatedwith structural rearrangements of cells, cell com-ponents, or entire organs. Since these processesoccur within genetically defined limits in indi-vidual organisms, they are characterized as physi-ologic or phenotypic adaptations (Bennett, ’95).Within the realm of osmoregulatory systems, ex-cellent examples of these processes are seen inthe responses of non-mammalian vertebrates toexcess salt loads. Because the kidneys of such non-mammalian vertebrates possess only a limited

ability, at best, to effectively excrete hypertonicurine, excess salt loads must be excreted by non-renal means. The tissues involved vary from thebranchial and opercular “chloride cells” of marineteleosts, through various specialized salt-secret-ing glands in other groups (such as the rectalgland of elasmobranchs, modified nasal, lachry-mal, and salivary glands in reptiles, and supra-orbital nasal glands of birds). In addition to theregular response associated with the routinemodulation of ion secretion, many of these tissuesdemonstrate marked adaptive responses to pro-longed stimulation or in response to the first ex-posure of the animal to an excess salt load. Thisreview discusses these “immediate” and “long-

Grant sponsor: National Institutes of Health; Grant number:GM40457; Grant sponsor: Deutsche Forschungsgemeinschaft; Grantnumber: Hi 448.

*Correspondence to: Trevor J. Shuttleworth, Ph.D., Department ofPharmacology and Physiology, Box 711, University of Rochester Medi-cal Center, 601 Elmwood Avenue, Rochester, NY 14642. E-mail:[email protected]

690 T.J. SHUTTLEWORTH AND J-P. HILDEBRANDT

term” responses in the exocrine avian nasal gland,the specific extrarenal salt-secreting organ of ma-rine or potentially marine birds. Depending on theprior osmotic experience of the individual bird,this structure is capable of showing both a pro-found adaptive differentiation response, and aneffective immediate secretory response to excesssalt loads. It is the cellular basis of each of thesetypes of response, and the signaling mechanismsinvolved, that form the focus of this review.

STRUCTURE OF THE NASAL GLANDKnut Schmidt-Nielsen and his coworkers

(Schmidt-Nielsen, ’60) first showed that marinebirds are able to excrete highly concentratedsalt solutions from paired structures located inshallow depressions in the skull above the eyes,the supraorbital nasal glands. The internalstructure of the nasal gland, its blood supplyand innervation are well documented (Hosslerand Olson, ’90; Butler et al., ’91; Gerstbergerand Gray, ’93), so will not be detailed here. Thefunctional unit of the salt gland is the secre-tory tubule. Non-secretory, poorly differentiated,peripheral cells line the tubules close to theirblind endings. However, the majority of the tu-bule is lined by more differentiated secretorycells that show the characteristic features ofion-transporting epithelial cells, e.g., infoldingsof the basolateral plasma membrane and a highdensity of mitochondria. Many secretory tubulesform a secretory lobule and open into a com-mon central ductule. Several of these ductulesempty into primary ducts, then into two mainducts that carry the secreted fluid to the nasalcavity (Butler et al., ’91). The fluid is removedvia the nostrils by shaking movements of thehead or by passively dripping from the tip ofthe beak (Blösch, ’66). In some species, the con-centration of secreted sodium chloride can reach10 times the plasma concentration (Peaker andLinzell, ’75) and the glands are capable of re-moving more than 20% of the sodium chloridedelivered by the blood (Kaul et al., ’83). Clearly,the avian nasal salt gland has to be consideredone of the most efficient ion-transporting organsin the animal kingdom.

MECHANISM OF SALT GLANDSECRETION

When birds are osmotically stressed, the nasalglands produce a hypertonic solution comprisedprincipally of sodium chloride solution with onlyminor contributions from other ions. Although

other models for the secretory mechanism havebeen proposed (as discussed by Shuttleworth, ’95),it is generally accepted that the key process is asecondary active secretion of chloride (Frizzell etal., ’79) involving a basolateral Na/Cl/K-cotrans-port mechanism (Ernst and Van Rossum, ’82;Torchia et al., ’92) and apical chloride and baso-lateral potassium channels (Richards et al., ’89;Martin and Shuttleworth, ’94a; Martin et al., ’94)(see Fig. 1). The entire process is ultimately en-ergized by the ion gradients generated by abasolaterally localized electrogenic Na/K-ATPase(Hopkins et al., ’76; Ernst and Mills, ’77; Boldyrevet al., ’95). The critical role of the Na/K-ATPaseand the Na/Cl/K-cotransporter in the secretoryprocess has led to the widespread use of determi-nations of changes in tissue oxygen consumption,particularly that component inhibitable by eitherouabain or bumetanide, respectively, as an indi-rect measure of secretory activity (Borut andSchmidt-Nielsen, ’63; Ernst and Van Rossum, ’82).The evidence for the presence of such an ion-trans-port mechanism in the secretory cells of the aviannasal salt gland is extensive (see Shuttleworth,’95, for review), and it seems likely that an es-sentially identical mechanism operates in most ofthe other salt-secreting tissues of non-mammalianvertebrates. Considerable evidence in support ofthis proposition exists for the teleost chloride cell,the elasmobranch rectal gland, and the turtle lach-

Fig. 1. Diagram illustrating the mechanism of secretionin the avian nasal gland. Increases in cytosolic Ca2+ activatesecretion by increasing the opening of basolateral Ca2+-acti-vated K+ channels and apical Ca2+-activated Cl– channels.(Adapted from Lowy et al., ’89; Shuttleworth et al., ’97).

VERTEBRATE SALT GLANDS 691

rymal salt gland. An important exception seemsto be the potassium-secreting nasal salt gland ofcertain desert lizard species (e.g., Sauromalus).Here the production of a secretion with potassiumconcentrations in excess of 1,000 mM, and the vir-tual absence of any sodium, obviously suggests arather unique secretory mechanism (Shuttleworthet al., ’87), the details of which are far from clear.

Although the underlying mechanism for ion se-cretion in the avian nasal gland seems clear, theprocess that determines the final concentrationof the secreted fluid, which is generally fairly con-stant in a given bird species even under differentconditions of salt stress, is not well understood.It has been suggested that sodium chloride is se-creted isotonically by the salt gland cells, and fluidcomposition is secondarily altered during the pas-sage of the fluid through the ductal system(Marshall et al., ’85, ’87). However, the structuralfeatures required by such a model (e.g., a loopedexcretory duct and some form of countercurrentmultiplier system) are not seen in the avian na-sal gland. The most likely mechanism, therefore,is that the secretion is produced as a hypertonicfluid in the secretory tubules, without majorchanges in the overall concentration during pas-sage along the duct.

SIGNALS CONTROLLING THESECRETORY RESPONSE

Early in the history of the investigation of aviannasal gland function, Schmidt-Nielsen (’60) proposedthat the control of nasal gland function involvedcentral osmoreceptors. This was subsequently sup-ported by experimental results obtained some 25years later by Gerstberger et al. (’84a,b). Extra-cerebral osmoreceptors, however, may provide ad-ditional information on the osmotic status of theanimal (Hanwell et al., ’72). A modulatory role ofchanges in extracellular volume, often associatedwith osmotic stress, seems likely (Hammel et al.,’80). Output from these various receptors is con-veyed to the gland via cholinergic fibers in thesecretory nerve (Fänge et al., ’58; Hanwell et al.,’71). Hormonal effects on nasal gland function can-not be ruled out, but may be confined to modula-tory actions (Shuttleworth, ’95; Shuttleworth etal., ’97). The secretory nerve contains parasym-pathetic fibers whose endings carry two types oftransmitter-filled vesicles (Kühnel, ’72): one con-taining acetylcholine (ACh) (Hootman and Ernst,’81), and the other that contains vasoactive intes-tinal peptide (VIP) (Lowy et al., ’87; Gerstberger,’88). Both types of vesicles are released on elec-

trical stimulation of the secretory nerve, indicat-ing that both transmitters are likely involved inthe control of nasal gland function. Since at leastsome of the signaling cascades induced by thesetransmitters may be involved in secretory andadaptive growth and differentiation responses ofthe gland, they are briefly reviewed in the follow-ing paragraphs.

Acetylcholine receptors and signalsLigand binding assays indicate that nasal gland

cells possess ACh-receptors of the muscarinic type(mAChR), and that these receptors are respon-sible for the control of secretion (Hootman andErnst, ’80, ’81, ’82; Hossler and Sarras, ’80;Shuttleworth and Thompson, ’89). However, at-tempts to fit the nasal gland receptors into exist-ing schemes of muscarinic receptor subtypes haveproved difficult. Binding studies indicated thatonly one type of receptor is expressed in duck na-sal gland cells and that the receptor showed char-acteristics similar to the mammalian M3 receptor.However, Northern hybridization studies, usingDNA-probes derived from the sequences of mam-malian receptors, showed hybridization only withthe m1-specific probe (Hildebrandt and Shuttle-worth, ’94). This apparent contradiction may reflectsubtle differences in the avian vs. mammalian re-ceptors, or simply the inadequacy of current phar-macological-based receptor subtype classificationschemes.

Binding of an appropriate agonist to the mAChRcouples, via a member of the Gq-family of G pro-teins (Hildebrandt and Shuttleworth, ’93), to theintracellular effector phospholipase C (PLC), result-ing in cleavage of the phospholipid phosphatidylino-sitol 4,5-bisphosphate (PIP2) to release the twosecond messengers: inositol 1,4,5-trisphosphate(InsP3), and diacylglycerol (Santiago-Calvo et al., ’64;Fisher et al., ’83; Snider et al., ’86; Shuttleworthand Thompson, ’89; Hildebrandt and Shuttleworth,’91, ’92). Diacylglycerol induces the translocationand activation of protein kinase C, while InsP3 bindsto specific receptors on intracellular calcium stores,releasing calcium ions into the cytoplasm (Shuttle-worth and Thompson, ’89; Stuenkel and Ernst, ’90;Shuttleworth, ’95; Shuttleworth et al., ’97). As inmany cell types, this initial calcium peak due torelease of calcium from intracellular stores is fol-lowed, at least at high agonist concentrations, bya sustained plateau of elevated cytosolic calciumconcentration ([Ca2+]i) (Fig. 2A). This plateau de-pends on calcium influx from the extracellularmedium and is activated by an unidentified


mechanism after the depletion of intracellular cal-cium stores. Since the rate of calcium influx seemsto depend on the amount of calcium released fromthe stores, this process was named capacitativecalcium influx (Putney, ’86; Putney and Bird, ’93;Berridge, ’95).

A rather different calcium signal is produced atlower, presumably more physiologically relevant,agonist concentrations. Here, regular oscillationsin [Ca2+]i are seen (Fig. 2B), with frequency de-pendent on agonist concentration (Crawford et al.,’91). The basic mechanism that generates theseoscillations in [Ca2+]i involves the unique biphasicresponse to cytosolic Ca2+ levels shown by theInsP3 receptors on the intracellular stores (seeBezprozvanny and Ehrlich, ’95 for details). How-

ever, it recently became clear that receptor-acti-vated Ca2+ entry also plays a role in the genera-tion and frequency of the oscillations (Martin andShuttleworth, ’94a; Shuttleworth and Thompson,’96a). Evidence shows that the entry of Ca2+ acts,along with the low levels of InsP3 generated atthe relevant agonist concentrations, to drive thecyclical release of [Ca2+]i from the stores by act-ing as a co-agonist at the InsP3 receptors (Shuttle-worth and Thompson, ’96a). Interestingly, itappears that the nature of the Ca2+ entry acti-vated under these conditions is distinct from thecapacitative entry described above (Shuttleworthand Thompson, ’96b). Instead, Ca2+ entry duringoscillatory [Ca2+]i signals has been shown to re-sult from the receptor-activation of a cytosolicphospholipase A2 with the subsequent generationof arachidonic acid. This apparently opens a Ca2+-permeable ion channel in the plasma membrane(Shuttleworth, ’96).

In the secretory response, the mAChR receptor-induced elevations in [Ca2+]i open Ca2+-activated po-tassium channels in the basolateral membrane ofthe secretory cells (Richards et al., ’89; Martin andShuttleworth, ’94a) and Ca2+-activated chloridechannels in the apical membrane (Martin andShuttleworth, ’94a) (Fig. 1). Loss of these ions fromthe cells presumably then stimulates the activitiesof the Na/Cl/K-cotransporter and the Na/K-ATPase,resulting in continuous chloride secretion via themechanism discussed above.

In parallel with the activation of the inositolphosphate/calcium-signaling cascade, phospholi-pase C activity also results in the accumulationof diacylglycerol (DG) in appropriately stimulatedcells. This signaling molecule remains in the in-ner leaflet of the plasma membrane, where it ac-tivates certain isoforms of protein kinase C (PKC)(Nishizuka, ’88). Once activated, PKC is then ableto phosphorylate selected target proteins in thecells on serine and threonine residues, therebychanging their functional properties. In the aviannasal salt gland, DG production or PKC activityhave never been directly measured, but indirectevidence indicates potentially important functionsfor this signaling pathway in the gland cells. Forexample, in vitro studies indicate that PKC canphosphorylate the Na/K-ATPase from avian na-sal gland cells (Lowndes et al., ’90). The Na/Cl/K-cotransporter in the gland may also be a substratefor PKC, as muscarinic receptor activation leadsto phosphorylation of a 170 kDa-protein that hasbeen immunologically identified as the Na/Cl/K-cotransporter (Torchia et al., ’91; Torchia et al.,

Fig. 2. Cytosolic calcium signals in isolated nasal glandcells loaded with a Ca2+-sensitive fluorescent probe (indo-1).A: Typical sustained “plateau-type” [Ca2+]i response to a maxi-mal concentration of an appropriate agonist (added at arrow).B: An oscillatory [Ca2+]i response such as typically seen fol-lowing stimulation with a submaximal concentration of anappropriate agonist (added at arrow).


’92). As yet, it is unknown whether phosphoryla-tion of the Na/K-ATPase or the cotransporter oc-curs in vivo, or how phosphorylation may affectthe biological activities of these proteins.

VIP-receptors and signalsAs noted above, evidence indicates a co-release

of VIP with ACh from the secretory nerve end-ings in the gland. The implication that VIP playsa role in the control of salt gland secretion wasconfirmed by the identification of specific bindingsites for VIP in the salt gland parenchyma (Gerst-berger, ’88); by the findings that VIP applied toconfluent cell layers of cultured nasal gland cellsstimulated ion transport (measured as short-cir-cuit current) (Lowy et al., ’87); and by the obser-vation that intracarotid infusion of VIP inducedboth secretion from the gland and increased bloodflow into the gland (Gerstberger et al., ’88).

Generally, VIP receptors are coupled to a Gs-typeG protein that activates adenylyl cyclase to formcyclic adenosine 3′,5′-monophosphate (cAMP). Thiscan then activate a specific cAMP-dependent pro-tein kinase (protein kinase A, PKA) to transfer phos-phate groups to serine and threonine residues incellular proteins, thereby changing their function.Gs proteins are present in salt gland tissue (Hilde-brandt and Shuttleworth, ’93). Treatment of ducknasal gland tissue slices with forskolin, an agentthat directly activates adenylyl cyclases, or a mem-brane-permeable analogue of cAMP, 8-cpt-cAMP,results in the stimulation of secretory activitymeasured as bumetanide-sensitive oxygen con-sumption (Shuttleworth and Thompson, ’87). Fur-thermore, in patch-clamp experiments, Martinand Shuttleworth (’94b) showed that treatmentof cells with VIP, cAMP-analogs, or forskolin re-sulted in the activation of a plasma membranechloride conductance distinct from the calcium-activated chloride conductance activated by cal-cium-mobilizing agonists. The properties of thisconductance were consistent with those of the so-called cystic fibrosis transmembrane conductanceregulator (CFTR), and the presence of this pro-tein at the apical plasma membrane has beendemonstrated (Ernst et al., ’94). Since cAMP-el-evating agents do not activate basolateral potas-sium channels in nasal gland cells (Martin andShuttleworth, ’94b; Martin et al., ’94), it seemsunlikely that cAMP is able to sustain secretionfrom the gland by itself. However, it does potenti-ate the secretory response to calcium-mobilizingagonists (Martin et al., ’94). In addition to theCFTR-channel, cAMP may have other target pro-

teins in avian nasal gland cells. For example, aPKA-mediated protein phosphorylation of the Na/Cl/K-cotransporter was observed in vitro (Torchiaet al., ’92), although the regulatory significanceof this response in vivo is, as yet, unknown.

Other plasma membrane receptorsSeveral studies have indicated the presence of

other plasma membrane receptors in nasal glandcells that may have functions in the modulationof secretion or other cellular functions. β-Adren-ergic agonists that elevate cytosolic cAMP-levelshave been shown to mediate secretory responses(Lowy et al., ’85; Lowy and Ernst, ’87; Martin etal., ’94), although these effects may be limited tocertain developmental stages of the nasal gland.Adenosine also induces an increase in intracellu-lar cAMP, and a marked stimulation of secretoryactivity in duck nasal gland cells (Shuttleworth,’95). A purinergic receptor of the P2U-type has beenidentified in duck nasal gland cells. This receptorbinds nucleotides (ATP) with high affinity, mobi-lizes calcium from intracellular stores, and acti-vates calcium-sensitive ion channels (Martin andShuttleworth, ’95). In addition, the naturally oc-curring phospholipid lysophosphatidic acid (LPA)activates inositol phosphate production and cal-cium-mobilization in duck nasal gland cells (Hilde-brandt, ’95). Binding sites for atrial natriureticfactor (ANF) have been identified in nasal glandparenchyma (Schütz and Gerstberger, ’90) andANF has been shown to induce the cellular accu-mulation of cyclic guanosine 3′,5′-monophosphate(cGMP) (Hübschle et al., ’96), possibly modulat-ing ongoing salt secretion from the gland (Schützand Gerstberger, ’90). The physiological relevanceof these multiple receptor systems however, hasnot been fully elucidated and may well lie in cel-lular processes not directly related to salt and fluidsecretion.

ADAPTIVE CHANGES IN NASAL GLANDSTRUCTURE AND FUNCTION

With few exceptions, the nasal gland is presentin all bird species, but in terrestrial birds it issmall and poorly developed (Technau, ’36). How-ever, as originally observed by Heinroth andHeinroth (’28), even in marine, or potentially ma-rine birds, the size of the gland depends onwhether animals ingest excess salt or not. Thenasal glands of birds not previously exposed toan excess salt stress are small and secrete little,if any, fluid. Initial exposure to an osmotic stress,however, initiates rapid and profound hyperpla-


sia and hypertrophy responses in the gland thatresult, within 1 to 7 days, in a greatly enhancedsalt-secretory capacity. The most obvious changeduring this adaptive response to an initial saltstress is an increase in gland size and glandweight (Ellis et al., ’63). Three major factors con-tribute to this: (1) an increase in cell number dueto accelerated proliferation of peripheral cells lin-ing the blind endings of the secretory tubules(Holmes and Stewart, ’68; Knight and Peaker, ’79;Hossler, ’82); (2) an increase in cell volume of in-dividual secretory cells resulting from elevationsin protein and lipid synthesis rates (Holmes, ’72);and (3) an increased fluid content associated witha marked vasodilation occurring at the onset ofsalt-secretion (Ballantyne and Wood, ’69; Hanwellet al., ’71; Hossler and Olson, ’90; Kaul et al., ’83).

The hyperplasic response is reflected by an in-creased DNA content in the salt glands (Holmesand Stewart, ’68; Hanwell and Peaker, ’75; Knightand Peaker, ’79; Hossler, ’82; Hildebrandt andShuttleworth, ’91), the precise timing of which var-ies with the age at which the initial salt stress isexperienced. As originally proposed by Ellis et al.(’63), the cells involved in DNA synthesis andadaptive cell proliferation are the peripheral cellsof the secretory tubules (Hossler, ’82). These cellsclearly are less differentiated than the principalcells in the mid-section of the secretory tubules(Ernst and Ellis, ’69) and their proliferation inresponse to osmotic stress results in the elonga-tion of the secretory tubule.

In parallel with this, processes related to theadaptive differentiation of the principal secretorycells are observed. These processes include an ap-proximate doubling of cell size (Hootman andErnst, ’80) preceded by a prolonged period of ac-celerated protein and lipid synthesis (Holmes andStewart, ’68; Stewart and Holmes, ’70; Karlssonet al., ’71; Levine et al., ’72; Stewart et al., ’76;Lingham et al., ’80; Sarras et al., ’85). This re-sults in amplification of the plasma membranearea, especially at the basolateral surface of thesecretory cells, and in an increase in the numberof mitochondria per cell (Ernst and Ellis, ’69; Mer-chant et al., ’85). These morphological changes areaccompanied by increases in the activities of gly-colytic and mitochondrial enzymes (McFarland etal., ’65; Spannhof and Jürss, ’67; Stainer et al.,’70), and an increased abundance and activity ofthe Na/K-ATPase (Fletcher et al., ’67; Stewart etal., ’76; Ernst and Mills, ’77; Hossler et al., ’78;Lingham et al., ’80; Barrnett et al., ’83, Sarras etal., ’85; Mazurkiewicz and Barrnett, ’85; Hilde-

brandt, ’97a) (Fig. 3A). Recently, an analysis ofthe latter response indicated that inhibition of thedegradation of Na/K-ATPase subunits might bethe initial mechanism of elevating Na/K-ATPaseabundance and activity within the first day of saltstress (Hildebrandt, ’97). This is followed by ac-celerated protein synthesis in both subunits of theNa/K-ATPase during the subsequent four to sixdays, resulting in a final three- to four-fold eleva-tion in the activity of the enzyme (Barrnett et al.,’83). Another transport-related protein that seemsto be upregulated during salt stress in ducks isthe CFTR-like chloride channel located in the api-cal membrane of the secretory cells (Ernst et al.,’94). The glycosylation state of the CFTR-like pro-tein (Ernst et al., ’94), as well as that of the β-subunit of the Na/K-ATPase (Hildebrandt, ’97) isincreased following salt stress. Whether thesemodifications have any function in ensuring a cor-rect membrane insertion or stabilization of theproteins in the plasma membrane is unknown.

As noted above, after a few days of salt stressthese structural and biochemical changes in thenasal gland transform the organ into a fully dif-ferentiated salt gland whose salt-excretory capac-ity is, at least in the duck, two- to three-fold higherthan that seen in animals not previously exposedto salt stress. This is judged from differences inthe maximal rate of sodium secretion (Stewart etal., ’76) and the bumetanide-sensitive portion ofoxygen consumption in stimulated salt gland cells(Hildebrandt and Shuttleworth, ’91) (Fig. 3B).

Fig. 3. Changes in (A) Na/K-ATPase activity, and (B)unstimulated (open columns), and carbachol-stimulatedbumetanide-sensitive oxygen consumption associated with theadaptive response to salt stress in the avian nasal gland. Mea-surements were made in cells isolated from ducklings eitherdrinking tapwater (naive cells) or 48 hours after changing to1% NaCl solution as drinking water. Data taken fromHildebrandt and Shuttleworth (’91) and unpublished data.


Interestingly, evidence indicates that at leastsome of these dramatic adaptive changes are re-versible on removal of the salt stress. Salt glandsof female Eider ducks, which do not drink salt-water while incubating their eggs, undergo a sub-stantial reduction in size and weight (McArthurand Gorman, ’78). Similar observations have beenmade on gulls (Komnick and Kniprath, ’70) andducks (Hossler et al., ’78) on changing from a sa-line drinking water to freshwater. The reductionin gland size during de-adaptation is associatedwith proportional reductions in plasma membranesurface area, protein content, and Na/K-ATPaseactivity in the secretory cells (Bonting et al., ’64;Komnick and Kniprath, ’70; Stewart and Holmes,’70; Hossler et al., ’78; McArthur and Gorman, ’78;Hossler, ’82; Merchant et al., ’85). During the de-adaptation process, the specific activities of phos-phatases and proteases in the salt gland cells areupregulated (Hossler et al., ’78). An accumulationof membranous material has been described in in-tracellular vesicles of the cells during de-adapta-tion (Barrnett et al., ’83). These observationsindicate that the removal of plasma membranecomponents during de-adaptation can be accom-plished by endocytosis and degradation of proteinsand lipids within cellular organelles. During suchresponses, however, the number of secretory cellsper gland seems to stay constant, as the totalamount of DNA per gland does not change (Holmesand Stewart, ’68; Hossler, ’82). This suggests thatadaptive hyperplasia in the nasal gland is essen-tially an irreversible process occurring only duringthe initial period of osmotic stress.

SIGNALS CONTROLLING ADAPTIVECELL PROLIFERATION AND

DIFFERENTIATIONIt is clear that the cholinergic innervation to the

gland plays a critical role in triggering the adap-tive changes described above. For example, unilat-eral postganglionic denervation of one salt gland,followed by salt-loading of the animal, abolishes theadaptive processes in the gland on the operated sidewhile the opposite gland grows and differentiates(Hanwell and Peaker, ’73, ’75; Pittard and Hally,’73). Furthermore, infusion of atropine into the cir-culation of simultaneously salt-loaded geese sup-pressed the adaptive hypertrophy in the glands(Hanwell and Peaker, ’75). Nevertheless, such datado not necessarily exclude additional hormonal ef-fects, for example, as potential modulators or per-missive factors for the induction of adaptive cellgrowth and differentiation.

c-Fos expression and the adaptive responseRecent studies have indicated that the adap-

tive differentiation response described above in-volves activation of the protooncogene c-fos(Hildebrandt et al., ’98). c-Fos is known to beactivated by a variety of extracellular stimulithat use different intracellular signaling path-ways to increase the transcription rate of thegene. Its protein product, Fos, dimerizes withoften constitutively expressed Jun proteins toform the transcription factor AP-1 that binds withhigh affinity to AP-1 sites in the promoter regionsof late genes whose transcription rates are there-by altered. Many of these genes under the controlof AP-1 are important mediators of developmentalprocesses, including cell proliferation and cell dif-ferentiation (Angel and Karin, ’91).

Osmotic stress induces a transient increase inthe transcription rate of the protooncogene c-fos,as well as an increase in Fos protein abundancein the nasal gland cells of ducklings. Maximumlevels of Fos expression are achieved between 4and 6 hr after the beginning of oral salt intake(Hildebrandt et al., ’98). In contrast, the c-Jun pro-tein is constitutively expressed and does notchange during osmotic stress (Hildebrandt et al.,’98). Adaptive cell proliferation is considered tobe restricted to peripheral cells, while differen-tiation is thought to occur in all parts of the glandexcept the peripheral cells (Ernst and Ellis, ’69;Hossler, ’82). In the above experiments, labelingwith Fos-specific antibodies was seen mainly inthe nuclei of the secretory (principal) cells liningthe distal portions of the secretory tubules in thegland. Although, in some experiments, nuclei ofperipheral cells were also stained. Therefore, itremains unclear at this stage which of these spe-cific adaptive responses is triggered by Fos accu-mulation. Additional studies revealed that theactivation of muscarinic receptors was the neces-sary stimulus that resulted in increases in Fosexpression, which appeared to be under transcrip-tional regulation (Hildebrandt et al., ’98). It is im-portant to note that these changes in Fos proteinexpression are unlikely to be related to the secre-tory response, as they occur only after secretionis fully activated. However, such a time-scale isconsistent with a role in initiating the adaptiveresponses seen in the gland.

As to the signals involved in the mAChR-medi-ated induction of c-fos, it appears that a sustainedelevation in the cytosolic concentration of free cal-cium ions is the critical signaling step (Hilde-


brandt et al., ’98), as it can be mimicked by theSERCA-pump inhibitor thapsigargin and is blockedby chelation of extracellular calcium by addingEGTA to the culture medium. Protein kinase C didnot appear to be involved (Hildebrandt et al., ’98).Calcium-mediated induction of c-fos has been de-scribed by several authors in various cell types. Insome cases, elevations in cytosolic calcium concen-trations activate Ras and the mitogen-activated pro-tein kinases Erk1 and Erk2, which results in thephosphorylation of ternary complex factors boundat the serum response element (SRE) in the c-fosgene (Finkbeiner and Greenberg, ’96). In othercases, calcium activates calcium/calmodulin-de-pendent protein kinases that phosphorylate thecAMP-response element binding protein (CREB).Phosphorylated CREB, in turn, may activate c-fos transcription (Sheng et al., ’91). However, noneof the above kinase cascades seem to be involvedin c-fos induction in nasal gland tissue. Instead,the possible involvement of p38 MAP kinase wasindicated. Inhibition of this kinase completelyblocked carbachol- or thapsigargin-induced Fos ac-cumulation, and an accumulation of phosphorylatedp38 occurred within minutes after mAChR-activa-tion, along with an increase in p38-specific pro-tein kinase activity (Hildebrandt et al., ’98).

RESPONSE DISCRIMINATIONFrom the above information, it can be seen that

muscarinic receptor activation serves as the sig-nal for both the adaptive growth and differentia-tion responses in salt gland cells from birds notpreviously exposed to a salt stress (“naive” cells)and for the sustained salt secretion in the fullydifferentiated gland. Furthermore, in both casesthe critical intracellular signal appears to be thesame, i.e., an increase in intracellular calcium con-centration. This raises the obvious question as toexactly how the nasal gland cells are able to in-terpret this common signal in order to induce theappropriate response at the relevant time.

Some indication of how this may be achievedcan be obtained by comparing the nature of thegeneration of the specific Ca2+ signals in the twosituations and their respective sensitivities to ago-nist concentrations. Hildebrandt and Shuttleworth(’91) made the surprising observation that maxi-mal mAChR activation resulted in a three- to four-fold higher rate of InsP3 accumulation in the naivecells compared with the cells from ducklings af-ter only 48 hours of mild salt stress (Fig. 4A).These differences could be entirely accounted forby a marked difference in the densities of musca-

rinic receptors in naive and stressed salt glandcells (Hildebrandt and Shuttleworth, ’91, ’94).While the binding affinities for muscarinic ligandswere the same in both cell types (Fig. 4B), themaximum binding level of the mAChR-antagonistquinuclidinyl benzilate in isolated naive salt glandcells was more than three times higher than thatin stressed cells (Fig. 4C). The implication of thesedata is that the same degree of extracellularstimulation results in a much higher second mes-senger response in the cytosol of naive cells com-pared with stressed cells. Despite this, the higheraccumulation rate of InsP3 in maximally activatednaive cells did not induce faster or higher calciumsignals than those observed in stressed cells(Hildebrandt and Shuttleworth, ’91). This was notdue to a difference in the sensitivity of the cal-cium release mechanism in both cell types, as in-dicated by the fact that dose response curves forthe release of stored calcium by externally added

Fig. 4. Changes in (A) unstimulated (open columns) andcarbachol-stimulated cellular InsP3 levels, (B) mAChR affin-ity, and (C) mAChR number, associated with the adaptiveresponse to salt stress in the avian nasal gland. Determina-tions of mAChR affinity and number are based on bindingstudies using the muscarinic agonist quinuclidinyl benzilate.Other details as in Fig. 3. Data taken from Hildebrandt andShuttleworth (’91).


doses of InsP3 in permeabilized naive or stressedcells overlapped perfectly (Shuttleworth, ’95;Shuttleworth and Thompson, unpublished data)(Fig. 5).

Consideration of these findings reveals that wehave a situation where the relationship betweenagonist concentration and InsP3 generation ismuch steeper in the naive cells as compared to thestressed cells, but the sensitivity of the “responsesystem” (i.e., InsP3-induced Ca2+ release) is un-changed. What is this likely to mean to the cell? Inthe stressed cells, it is known that an oscillatory[Ca2+]i signal is only generated at low agonist con-centrations where significantly submaximal levelsof InsP3 are generated (Fig. 6A). Given the identi-cal sensitivity of the InsP3-induced Ca2+ releasemechanism in the two cell types, examination ofthe relative agonist concentration vs. InsP3 genera-tion curves for stressed and naive cells would indi-cate that such submaximal levels of InsP3 are onlyproduced in a very narrow, restricted range of ago-nist concentrations. In other words, the response ofthe naive cells switches from no response to a maxi-mal response (stores fully depleted and a sustained“plateau-like” [Ca2+]i signal) over a very small rangeof agonist concentrations. In the stressed cells, how-ever, a graded response represented by increasingfrequency of [Ca2+]i oscillations is seen over a fairlywide range of agonist concentrations until a maxi-mal response is obtained (Fig. 6B). The implicationsof this are that the naive cell responds to agonistsin an “on-off” manner to generate a sustained [Ca2+]i

signal, while the stressed cell produces a variable,oscillatory [Ca2+]i signal whose frequency is relatedto the degree of agonist stimulation, much like a“dimmer switch.” It can be argued that these dis-tinct characteristics of the agonist-induced signalare precisely what might be required to initiate theappropriate response. For example, the activationof c-fos and the initiation of gene expression associ-ated with the adaptive differentiation response islikely to require the generation of a signal that ismaintained for an extended period, but which is asimple reflection of the presence or absence of ad-equate agonist stimulation. On the other hand, theinitiation of secretion involves the rapid activation

Fig. 5. Concentration-response curves for InsP3-inducedCa2+ release in saponin-permeabilized naive and adaptedavian nasal gland cells (Shuttleworth and Thompson, unpub-lished data).

Fig. 6. (A) Theoretical relationship between agonist (car-bachol) concentration, stimulated InsP3 levels, and predictedtype of [Ca2+]i response induced in naive and adapted nasalgland cells based on the data presented in Figs. 4 and 5. (B)Linear transformation of part of the graph depicted in (A)showing the rapid switch in agonist-induced [Ca2+]i signalsfrom no response to a maximal (plateau-type) response inthe naive cells, compared to a graded [Ca2+]i response (re-flected as an increasing frequency of [Ca2+]i oscillations) inthe adaptive cells. See text for details.


of ion channels, therefore requiring only a repeti-tive transient signal. However, for the accurate re-sponse to varying salt loads, such a signal must becapable of being modulated to closely reflect the par-ticular degree of agonist stimulation existing at thetime. This is appropriately achieved by a frequency-modulated oscillatory signal. In this way, the samebasic intracellular messenger (changes in cytosolicCa2+ concentration) can be used to deliver eitherthe adaptive response or the secretory response, asappropriate. Interestingly, it would appear that thecritical component in determining the switch be-tween these two types of response, namely thedownregulation of mAChR number, is itself partof the adaptive differentiation response (Fig. 7).

Of course, it is likely that the above is a majoroversimplification of a full story that likely in-volves multiple interacting signaling pathways.For example, it is entirely possible that the highrate of PIP2 hydrolysis in naive cells serves to gen-erate some other component or components of theoverall mAChR signaling cascade with criticalroles in the initiation of adaptive growth and/ordifferentiation processes in the nasal glands ofanimals exposed to a salt stress. For example, themuscarinic activation of nasal gland cells resultsin the generation of equimolar amounts of diacyl-glycerol (DG) along with InsP3. Although the rateof DG accumulation in avian nasal gland cells hasnot been measured, it is likely that muscarinicstimulation of naive cells results in a higher DGgeneration rate compared with similar stimula-tion in stressed cells. Since DG is generally in-volved in recruitment and activation of proteinkinase C, there is a clear possibility that this sig-naling moiety also plays a role in the adaptive

differentiation response. One such role may in-volve changes in the regulation of intracellular pH(pHi) in the salt gland cells following stimulation.Metabolic activation of cells often results in anincrease in the cytosolic proton concentration (de-crease in pHi) that is generally balanced by sev-eral pH-homeostatic mechanisms. In duck nasalgland cells, bicarbonate-dependent and bicarbon-ate-independent pHi-regulatory mechanisms havebeen identified (Shuttleworth and Wood, ’92) thatcontribute to the regulation of pHi in the restingstate of the cells and after activation of ion secre-tion by muscarinic agonists. Stimulation of iso-lated cells from fully differentiated salt glandsresults in a transient acidification of the cytosol,with a subsequent restoration of pHi to control lev-els. The major component of this recovery processshows properties consistent with the activation ofa sodium/proton-exchanger (NHE) (Shuttleworthand Wood, ’92). In contrast, in the cells from birdsnot previously salt-stressed the initial cytosolicacidification is absent (Bentz and Hildebrandt, ’95),presumably reflecting the less pronounced metabolicactivation and lower rate of acid production in thesenaive cells. Instead, a more slowly developing in-crease in pHi occurs that reaches a sustained pla-teau at approximately 0.1 pH-units above thecontrol level (Bentz and Hildebrandt, ’95). Again,the properties of this pHi-shift are consistent withthe activation of an NHE and appear to involve aprotein kinase C-mediated phosphorylation (Bentzand Hildebrandt, ’95). These findings raise thequestion of whether sustained changes in pHi instimulated naive cells have any regulatory role inthe initiation of adaptive cell proliferation or differ-entiation. Experiments examining this have shownthat increasing pHi by 0.1 units accelerates the rateof DNA-synthesis, while cytosolic acidification low-ers the DNA-synthesis rate (Bentz et al., unpub-lished data). Furthermore amiloride, presumably bycausing an acidification in mAChR-activated cells,suppressed the onset of DNA-synthesis both in vivoand in vitro (Bentz et al., unpublished data), point-ing to the involvement of the NHE in these pro-cesses. These data indicate that changes in pHi mayplay a role as a permissive factor or a cofactor ofmitogenic signaling in the nasal gland and suggestthat mAChR-induced cytosolic alkalinisation maybe a factor in the signaling cascade that initiatesadaptive cell proliferation in the nasal gland.

SUMMARYAs noted earlier, broadly similar combinations

of immediate and adaptive responses are seen in

Fig. 7. Diagram illustrating the proposed mechanism,whereby specific changes in [Ca2+]i signal either an adaptivedifferentiation (and proliferation) response, or a secretory re-sponse, as appropriate. See text for details.


the various salt-secreting tissues of other non-mammalian vertebrate groups. For example, theproliferation of branchial chloride cells in teleo-sts is a well-documented response to increased saltloads. Similarly, the potassium-secreting nasalsalt glands of the desert lizard Sauromalus dem-onstrate profound changes in Na/K-ATPase lev-els, in response to salt loading, that mimic thosedescribed above for the avian nasal gland (Shuttle-worth et al., ’87). Interestingly, and in contrast tothe avian gland, the changes seen in the lizardgland, which is enclosed in a bony nasal capsule,are not associated with any overall increase ingland size. Clearly then, the extrarenal salt-se-creting tissues of non-mammalian vertebrateswould seem to be excellent model systems for thestudy of the underlying cellular mechanisms re-sponsible for the profound proliferative and cellu-lar reorganization responses associated withphysiologic or phenotypic adaptations to environ-mental challenges. The mechanisms we have pro-posed above for the avian nasal gland are clearlyjust part of an overall response that undoubtedlyinvolves multiple interacting pathways. However,they do serve to illustrate what is likely to be awidespread, although often overlooked, phenom-enon—even apparently relatively simple signals,like an increase in cytosolic calcium ion concen-tration, are capable of encoding and delivering amultitude of complex, diverse messages within thecell. The critical components of such messages,and the cellular machinery required for their ac-curate read-out, are exciting and important areasfor current and future research in cell signaling;areas in which vertebrate salt glands can usefullyplay a significant role.

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Vertebrate salt glands: Short- and long-term regulation of function