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Journal of Steroid Biochemistry & Molecular Biology 108 (2008) 310–317

Rapid responses to aldosterone in the kidney and colon�

Brian J. Harvey ∗, Rodrigo Alzamora, Adam K. Stubbs, Mustapha Irnaten,Victoria McEneaney, Warren Thomas

Department of Molecular Medicine, Royal College of Surgeons in Ireland,Beaumont Hospital, Dublin 9, Ireland

bstract

Aldosterone is a crucial modulator of ion transport across high resistance epithelia and regulates whole body electrolyte balance throughts effects on the kidney and colon. The net consequence of aldosterone release is to promote salt conservation. The genomic mechanism ofldosterone action is relatively well characterized and the role of the classical mineralocorticoid receptor as a ligand-dependent transcription

actor is well established. The rapid effects of aldosterone on target tissues are less well understood and there is still controversy over thedentity of the aldosterone non-genomic receptor. Greater understanding of the physiological consequences of aldosterone’s rapid responsesn the kidney and colon has been achieved through the identification of definite and putative membrane targets and their signaling regulators.

2007 Elsevier Ltd. All rights reserved.

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eywords: Aldosterone; Rapid responses; Kidney; Colon

. Introduction

The kidney and distal colon are the principal target organsor aldosterone regulation of whole body electrolyte balance.he classical mechanism for aldosterone to elicit its effects is

o increase the expression of membrane transporters in targetpithelia through the action of the classical mineralocorti-oid receptor (MR) as a ligand dependent transcription factor1,2]. Rapid physiological responses to aldosterone may alsoe observed well in advance of transcriptional changes, how-ver it is the genomic effects of aldosterone that have beenost intensively studied. The earliest investigation of rapid

esponses to aldosterone almost 50 years ago, described theormone’s effects on Na+ and K+ excretion into the urineithin 5 min following its intra-arterial administration [3].he critical role for aldosterone in the regulation of Na+, K+

nd H+ fluxes across high resistance epithelia both rapidlynd over extended periods of time has subsequently been

onfirmed by many investigators [4]. In general terms, aldos-erone promotes Na+ absorption and net K+ secretion in the

� Presented at the ‘12th International Congress on Hormonal Steroids andormones & Cancer’ (Athens, Greece, 13–16 September 2006).∗ Corresponding author. Tel.: +353 1 809 3817; fax: +353 1 809 3778.

E-mail address: bjpharvey@rcsi.ie (B.J. Harvey).

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960-0760/$ – see front matter © 2007 Elsevier Ltd. All rights reserved.oi:10.1016/j.jsbmb.2007.09.005

istal nephron and distal colon, additionally since water fol-ows Na+ by osmosis the net effect of aldosterone releases to increase extracellular fluid volume and to raise bloodressure [5]. Consequently, the effects of aldosterone on theidney and colon not only modulate whole body, homeostatic,lectrolyte balance but can also contribute to pathophysiolog-cal effects on tissues including the kidney and vasculaturehrough the development of hypertension [6,7].

. Kidney

In the kidney, aldosterone exerts its most pronouncedhysiological effects on the epithelial cells of the distal, reab-orbative region of the nephron. The aldosterone sensitiveistal nephron is comprised of the thick ascending limb (TAL)f the loop of Henle; the distal convoluted tubule; the connect-ng tubule and the cortical collecting duct (CCD). This regionf the nephron is the principal site for regulating the rate ofa+ efflux from the body. Na+ is reabsorbed at the apical sur-

ace of the principal epithelial cells from the renal ultrafiltrate

hrough the epithelial sodium channel (ENaC). Na+ is trans-orted out of the epithelium at the basolateral membrane byhe Na+/K+ ATPase pump and into the blood, which in turnaintains a gradient for apical Na+ uptake. The Na+ mem-

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B.J. Harvey et al. / Journal of Steroid Bioch

rane transporters and others such as the Na+/H+ exchangersNHE) are regulated by aldosterone at the transcriptionalevel over a number of hours after treatment and some arelso activated rapidly, within a few minutes through the stim-lation of cell signaling cascades that modulate the activitynd subcellular distribution of pre-expressed transporters.

.1. Membrane targets

.1.1. NHEThe eight known mammalian NHE sub-types mediate

he electroneutral exchange of Na+ for H+ across the cellembrane. In polarized epithelia NHE1 is located at the baso-

ateral membrane and contributes to homeostatic processesuch as cell volume regulation and cytoplasmic pH modu-ation. NHE3 is located at the apical surface of cells fromhe proximal nephron, the distal nephron and the colorectalpithelium where it mediates NaHCO3 and NaCl reabsorp-ion. Trans-membrane Na+/H+ exchange was identified asesponding rapidly to aldosterone in the MDCK canine, renalell line [8]. The rise in cytoplasmic pH associated with thisctivation was preceded by, and dependent upon, a rise inCa2+]i within 1 min of aldosterone treatment [9]. NHE3 acti-ation in proximal tubule cells was sensitive to the classicalR antagonist spironolactone [10] and was dependent on

RK1/2 MAP kinase activation [11]. Aldosterone stimulatedHE1 activity in the M1-CCD cells was also preceded bycalcium influx, and was PKC and ERK1/2-dependent, butas spironolactone-insensitive [12]. It has recently been pro-osed that aldosterone inhibits the activity of apical NHE3 inhe medullary TAL through a non-genomic, MR-independent

echanism to block HCO3− reabsorption [13,14]. This effect

s contrary to aldosterone’s stimulatory effect on NHE3 activ-ty in the proximal tubule [10]. The stimulation of NHE1y aldosterone may mediate the hormone’s transcriptionalffects through the activation of pH sensitive signaling pro-esses while NHE3 activity contributes to the aldosteroneensitive absorption of HCO3

− from the renal ultrafiltrate.he pH shift generated by NHE1 stimulation contributes to

he activation of other membrane transporters that promote+ recycling in the distal tubule such as K+

ATP channels15].

.1.2. K+ channelsActive transport of Na+ across the basolateral membrane

equires activation of Na+/K+ ATPase to export Na+ inxchange for K+. The ATP sensitive K+ channels facilitate+ recycling across basolateral membrane. Stimulation of+

ATP channel activity was detected within 2 min of aldos-erone treatment of A6 amphibian renal principal cells [15].his effect was blocked by amiloride, an inhibitor of NHE

ctivity and could be mimicked by a cytosolic pH shiftrom pH 7.15 to 7.4 [15]. More recent work has shown thathe aldosterone induced up-regulation of the Kir1.1/ROMK

+ channel in murine TAL cells relies upon cystic fibrosis

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& Molecular Biology 108 (2008) 310–317 311

rans-membrane conductance regulator (CFTR) Cl− channelctivation [16] and is also dependent on ENaC expression17]. Complex regulatory mechanisms therefore integratehe different aldosterone responsive membrane transporters.FTR is regulated through multiple potential phosphoryla-

ion target sites for PKA and PKC. The coupling of CFTRctivation to Kir1.1 confers ATP sensitivity on Kir1.1 andFTR may act as a PKA-dependent switch for the regu-

ation of K+ secretion by the distal nephron [16]. RapidKC but not PKA activation has been identified as a con-equence of aldosterone treatment in the M1-CCD cell line18]. However, increased cAMP production has been detectedn isolated inner medullary collecting duct cells within 4 minf aldosterone treatment that could potentiate PKA activ-ty [19]. It may prove to be that the increase in cytosolicH stemming from increased NHE activity is the switchhat initially engages K+ recycling but other factors latereed into the regulation once the expression of transportersuch as ENaC and CFTR in the cell membrane has beendjusted.

.1.3. Na+/K+ ATPaseThe basolateral Na+/K+ ATPase pump by reducing

ntracellular Na+ and raising K+ provides the main electro-hemical driving force for the luminal influx of Na+ and theasolateral efflux of K+ in the distal nephron. The activa-ion of Na+/K+ ATPase is generally regarded to occur inwo phases early (1–4 h) where pre-existing pump subunitsre recruited to the cell membrane and late (+4 h) whenhere is a detectable change in Na+/K+ ATPase expressionhrough the transcriptional effects of activated MR. Aldos-erone also stimulates Na+/K+ ATPase activity in isolatedCD tubules within 30 min of treatment [20]. The serumnd glucocorticoid-induced kinase 1 (SGK-1) is the onlyinase identified which directly regulates Na+/K+ ATPasen response to aldosterone in the kidney [21]. The promi-ent role of SGK-1 as an intermediate in aldosterone’s earlyffects (1–2 h) on Na+ transport is well established. Thisinase is however regulated at the transcriptional level and itsffects on Na+/K+ ATPase activity are only observed after anxtended period [21]. The Na+/K+ ATPase also has a PKChosphorylation site on the � subunit, residue Ser-23 [22]nd mutation of this site compromises its capacity to trans-ort Na+ [23]. Phosphorylation of Na+/K+ ATPase by PKAas been observed at Ser-943 and phosphorylation at this siteotentiates activation by PKC [24]. Many investigators havelso observed the pH sensitivity of Na+/K+ ATPase activity inifferent experimental systems including renal cells [25,26].his has been attributed to the effect of intracellular pH on

on binding specificity of the transporter [27]. Since PKC

n the rapid responses stimulated by aldosterone in the kid-ey, these may contribute to the early phase in aldosteronenduced Na+/K+ ATPase activity in advance of changes inGK-1 expression.

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12 B.J. Harvey et al. / Journal of Steroid Bioch

.1.4. ENaCEpithelial sodium channel (ENaC) activity is the rate-

imiting factor in Na+ reabsorption by the principal cells ofhe distal nephron. The aldosterone induced increase in cellurface ENaC expression is regulated in two stages by: (1)romoting the trafficking of pre-expressed ENaC subunitso the cell membrane and (2) through the MR-dependentegulation of ENaC subunit gene transcription. ENaC chan-el density increases in the membrane following aldosteronereatment through the phosphorylation and inactivation ofhe E3 ubiquitin-protein ligase Nedd4-2 by SGK-1 [28,29].KA regulates Nedd4-2 in response to vasopressin to pro-ote ENaC activation 5–15 min after treatment [30]. PKC

as been implicated in the phosphorylation of each of theNaC subunits and to increase channel activity in response

o insulin in A6 cells [31]. The activation of a negativeeedback pathway involving PKC and ERK1/2 to suppressNaC activity through enhanced proteasome degradation of

he ENaC� subunit has been described in A6 cells treatedith phorbol ester for >1 h [32]. The same group described a

rosstalk in ERK1/2 and PI3-K signaling following aldos-erone treatment, where PI3-K activation promotes ENaCctivity through SGK-1 and ERK1/2 activation has a sup-ressive effect on ENaC activity [33]. Here the activationf ERK1/2 is through increased expression of its upstreamegulator, the small G-protein K-RasA 1 h after aldosteronereatment rather than a rapid response within minutes ofreatment [33]. K-RasA is also methylated in response toldosterone in A6 cells and activation of ENaC is proteinethylation sensitive [34,35]. The transcriptional regulation

f signaling intermediates cannot account for the most rapidtimulation of ENaC activity by aldosterone. An increasen the amiloride sensitive ENaC current within 2 min fol-owing 100 nM aldosterone treatment has been observed insolated rabbit principal collecting duct cells that was notetected in equivalent rat cells [36]. The rapid signaling pro-ess that leads to ENaC activation in principal cells has yeto be elucidated, however ENaC activation has been reportedo be sensitive to an inhibitor of protein methylation and wasTP-dependent [36].

.1.5. H+-ATPaseThe kidneys contribute to homeostatic acid–base regula-

ion through the reabsorption of bicarbonate and the release of+ into the renal ultrafiltrate in the distal nephron. The releasef H+ takes place in the CCD and outer medullary collectinguct (MCD) at the apical surface of type A intercalated cellshrough the vacuolar H+-ATPase pump. H+-ATPase activ-ty can be regulated through systemic electrolyte balance,ngiotensin II release and by aldosterone. In the CCD, aldos-erone stimulates H+ secretion directly through increasednsertion of H+-ATPase pumps into the apical membrane

nd indirectly through increased absorption of Na+ largelyhrough ENaC in the principal cells, which renders the tubuleumen electro-negative and so promotes H+ diffusion. In theuter MCD aldosterone stimulates H+-ATPase directly in

bcom

& Molecular Biology 108 (2008) 310–317

he absence of Na+ reabsorption. The aldosterone inducedncrease in H+-ATPase activity resulting in urine acidifica-ion has been observed over prolonged exposure, however

rapid activation of H+-ATPase has also been described.ldosterone stimulated a 2–3-fold increase in H+-ATPase

ctivity in isolated outer MCD after 15 min treatment [37].his activity was spironolactone sensitive but insensitive to

nhibition of transcription or translation. The increase in H+-TPase activity was blocked by disruption of the subcellularicrotubule network using colchicine and also sensitive toKC inhibition. The rapid activation of H+-ATPase activity

n the intercalated cells of the outer MCD is therefore depen-ent on the trafficking of already expressed H+-ATPase to thepical membrane.

.2. MR and rapid responses to aldosterone in theidney

The identity of the receptor responsible for initiatinghe rapid responses to aldosterone has been the subject ofebate. One school of thought holds that there is a distinct,embrane-associated receptor for aldosterone that exclu-

ively initiates the different rapid “non-genomic” responses.he contention is in part supported by evidence that some of

he rapid responses are detected in MR knockout mice [38]r are not blocked by specific pharmacological antagonistsf the classical MR. The MR antagonists spironolactone andU26752 do not block ERK1/2 activation in the M1-CCDell line [12,39]. The conclusions presented in the latter reportuggest that RU28318 and canrenone also have no effect how-ver the data presented appear to show some inhibition ofhe ERK1/2 activation. The ERK1/2 dependent activation ofHE1 [12] and the transient rise in intracellular calcium [40]

n aldosterone treated M1-CCD cells were also spironolac-one insensitive. A novel membrane-associated aldosteroneeceptor has been proposed, based on impeded ligand experi-ents. Aldosterone-BSA stimulated a rapid increase in PKC

ctivity 10 min after treatment in rat RCCD2 cells [41] with aoncurrent increase in MR phosphorylation which was sen-itive to PKC� inhibition.

The alternative view is that the classical MR mediatesll the non-genomic responses either directly or throughncillary signaling molecules. This appears to be the caset least in cardiac myocytes [42,43]. Intracellular MR is pro-ected from activation by glucocorticoids through the activityf 11� hydroxy-steroid dehydrogenase (11�HSD2) whichonverts cortisol to cortisone [44]. Inhibition of 11�HSD2ith carbenoloxone allowed the activation NHE by corti-

ol, mimicking the effect of aldosterone [44]. This suggestshat the classical receptor, rather than a distinct membraneeceptor is responsible for this response. MR inhibitors

locked aldosterone-stimulated effects with different poten-ies. For example, RU28318 is a more effective inhibitorf aldosterone-induced alkalinization in vascular smoothuscle compared to spironolactone [44]. Difference in the

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fficacy of these inhibitors for MR mediated rapid responseso aldosterone in particular experimental systems may reflectonformational changes in the aldosterone-binding site ofR [45]. MR resides in a complex with other proteins and

he effect of aldosterone binding is to change MR confor-ation and to modulate the interaction of MR with the other

roteins in the complex, one major consequence being the dis-lacement of Hsp90 [46]. The release of Hsp84, the murineomolog of Hsp90 has been implicated in the activation of-Src by aldosterone in M1-CCD cells [47]. It has also beenroposed that the differential effects of MR antagonists isvidence for the existence of an aldosterone receptor distinctrom MR that regulates some of the non-genomic effects ofhe hormone [48]. A plasmid expressing MR conferred rapidRK1/2 and JNK responses but not Ca2+ responsiveness inHO cells treated with aldosterone [49]. It may thus prove toe that distinct receptors including MR regulate the differentapid responses to aldosterone.

.3. Activation of signaling intermediates

.3.1. Protein kinasesAldosterone stimulates a rapid change in the cellular

ignaling background that supplements and modulates thelassical transcriptional effects of the hormone. These sig-aling responses are focused on the activation of membraneransporters through phosphorylation, free Ca2+ availabilityr changes in intracellular pH. The activation of the PKCamily of serine/threonine protein kinases is an importantomponent in the rapid responses to aldosterone in the kid-ey and was first described in isolated renal cells [50]. Theirect activation of the Ca2+ dependent PKC isoform PKC�y aldosterone in a cell free system has been proposed [18]kin to the direct activation of PKC achieved by other lipidsuch as arachidonic acid [51]. The stimulation of NHE activ-ty by aldosterone in the M1-CCD cells could be blocked byKC� inhibition [18] and the activation of PKC� in RCCD2ells is detectable within 5 min of treatment and peaks at0–15 min [52]. Activation of other PKC isoforms has noteen demonstrated in response to aldosterone in renal cellsowever PKC� is emerging as a probable regulator of Na+/K+

TPase activity in aldosterone treated cardiomyocytes53,54].

The rapid activation of ERK1/2 mitogen activated pro-ein kinase (MAPK) in aldosterone treated M-1 CCD cellss PKC dependent [12]. The activation of the PKC/MAPKathway culminates in enhanced activity of the NHE1 incid loaded cells [9,12]. The coupling of MR activation toownstream signaling pathways is now being elucidated.ldosterone stimulates the activation of c-Src tyrosine kinaseithin 2 min of treatment [47]. Aldosterone not only stim-lates EGFR expression at the level of transcription [55],

ut also rapidly promotes its c-Src dependent phosphory-ation [56]. Additionally there is a synergism in the rapidctivation of ERK1/2 between aldosterone and EGF stim-lated pathways which confirms EGFR family receptor

aart

& Molecular Biology 108 (2008) 310–317 313

yrosine kinases as critical signaling integrators in steroidnduced rapid responses [56]. A physiological relevance isent to EGFR activation in response to aldosterone by thebservation that inhibition of EGFR by tyrphostin AG1478ompletely blocked the activation of NHE3 by the hormone10,57].

.3.2. CalciumA role for aldosterone in regulating calcium homeostasis

s uncertain. Clearance studies showed either an increase inalciuria [58] or an absence of significant changes in Ca2+

xcretion [59,60] after aldosterone administration. A rise inCa2+]i as a component of aldosterone induced rapid sig-aling is more established. Sequestration of free Ca2+ inrganelle stores stably maintains low [Ca2+]i. Release of Ca2+

rom these stores or the activation of Ca2+ channels in theell membrane results in a transient rise in [Ca2+]i whichctivates Ca2+-dependent protein kinases such as PKC� orembrane transporters such as Ca2+-dependent K+ channels.transient, 3-fold rise in [Ca2+]i has been observed in M1-

CD cells within 2 min of treatment with 1 nM aldosterone40]. PKC inhibition with chelerythrine blocked this effectuggesting a possible role for Ca2+-independent PKC acti-ation in the response. The rise in [Ca2+]i stimulated byldosterone in MDCK cells was sensitive to inhibition ofGFR and ERK1/2 [9,57]. The extracellular or store sourcef the Ca2+ rise observed in these experiments has not yeteen established experimentally. The activation of inositolignaling pathways has been observed in other experimentalystems including the A6 cell line [61], which may result inhe activation of IP3 sensitive Ca2+ channels in the endoplas-

ic reticulum or mitochondria.

. Colon

The rapid effects of aldosterone on electrolyte absorptionn the colon have been subjected to less intensive investiga-ion than its effects in the kidney. The mammalian colon is a

ajor target for aldosterone, and the level of mineralocorti-oid receptor gene expression is higher in the distal colon thann any other target tissue including the kidney [62,63]. In theistal colon, aldosterone causes a switch from electroneutralaCl absorption to stimulated electrogenic Na+ absorptiony inducing expression of apical ENaC and basolateral Na+,+-ATPase [64,65]. In parallel, net K+ absorption is con-erted to net K+ secretion by induction of apical K+ channels66]. This process has been well characterized, however, sev-ral studies have demonstrated that changes in electrolyteransport precede the increase in induction of these proteins.he current model for rapid aldosterone action in the colon

s that the hormone stimulates the activation of PKC�, which

ctivates Na+/H+ exchange resulting in rapid cytoplasmiclkalinization. This pHi change negatively affects potassiumecycling, which is the driving force for chloride secretion inhe distal colon.

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.1. Regulation of ion transport

Early studies in the 1970s reported rapid effects of aldos-erone on ion transport in rabbit distal colon [67]. Aldosteronenduced a rapid (<30 min) increase in the short-circuit cur-ent (Isc), an equivalent increase in the rate of active Na+

bsorption (JNa net) and a decline in tissue resistance (Rt).ldosterone produced no change in Na+/K+-ATPase pro-

ein abundance or activity within the time frame of thesetudies. Aldosterone also had no effect on the bi-directionalr net transepithelial movements of K+ under short-circuitonditions, suggesting that the enhanced secretion of K+

bserved in vivo is the result of increased diffusion of K+

rom plasma to lumen via paracellular pathways in responseo an increased transepithelial electrical potential differencelumen negative).

In early studies from our laboratory [68], aldosteronehowed rapid (<1 min) activation of Na+/H+ exchange and+ recycling in rat colonic epithelium that was coupled

o cyclooxygenase signaling. This effect was mediated byncreases in [Ca2+]i, in response to aldosterone-inducedKC activation [69]. It is notable that these kinases studiesere performed in isolated cytosolic fractions, suggesting no

nvolvement of a putative membrane receptor. Further studiesrom our laboratory using the human T84 colonic epithe-ial cell line [70] showed that aldosterone fludrocortisone,nd DOCA were equivalently effective in inducing a rapidncrease in [Ca2+]i within 2 min, independently of intracel-ular calcium stores and not mimicked by cortisol. Althoughecorded basal [Ca2+]i levels varied substantially betweenndividual cells, the extent of the stimulation (>10-fold) inntracellular calcium was of the same order as that for thelevation of PKC in the rat tissue [69]. Very similar find-ngs were seen in normal human colon cells from surgicalpecimens [71].

Basolateral K+ channels in colonic epithelia are veryensitive to changes in intracellular pH within the phys-ological range [72]. In the rat distal colon, aldosteronenduces rapid activation of NHE that leads to intracellu-ar alkalinization which is also coupled to cyclooxygenaseignaling [73]. This change in intracellular pH has a sig-ificant impact on basolateral K+ conductances producingctivation of K+

ATP channels and inhibition of a calcium-ependent potassium channel (K+

Ca) [72,15]. Previous studiesave demonstrated that KATP channels are involved in elec-rogenic Na+ absorption in distal colon [72,74,75]. Theseesults suggest that aldosterone’s non-genomic action wouldead to a rapid increase in Na+ absorption. In contrast to rab-it tissue, there are no reports of rapid activation of ENaCn rat distal colon; this is consistent with previous studieshat reported lack of rapid ENaC activation in mice and ratidney cells [36]. Therefore, it appears that the activation of

+ATP channels will not increase Na+ absorption by itself butill counteract membrane depolarisation produced by Na+

ntry into the cell during the genomic phase of aldosteronection.

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& Molecular Biology 108 (2008) 310–317

Finally, Bowley et al. [76] explored the rapid effectsf aldosterone (EC50 < 1 nM) on intermediate conductanceasolateral K+ channels in human colonoscopy specimenssing patch clamp techniques. Aldosterone appeared botho decrease individual channel activity (in single channelatches) and to decrease the apparent number of channelser patch, suggesting that it may stimulate channel recyclingnto cytoplasmic vesicles as well as having a direct inhibitoryffect. The 27 pS K+ channel was provisionally identified asnhibited by aldosterone on the basis of the detection of theognate KCNN4 mRNA by RT-PCR. This channel is knowno be calcium sensitive and is a possible candidate for the

Ca channel we have previously identified to be inhibited byldosterone.

.2. Activation of signaling intermediates

.2.1. Protein kinasesPysiological concentrations of aldosterone stimulated

ytosolic and membrane associated PKC activity in isolatedemale rat distal colonic epithelium (<5 min) [69]. Simi-ar observations were subsequently made in isolated humanistal colonic epithelium [71]. A tolbutamide-sensitive K+

urrent in human distal colonic epithelia was rapidly stim-lated following aldosterone treatment (0.1 nM) in humanistal colonic epithelium, which was sensitive to the PKCnhibition [72]. The calcium dependency of the PKC activitybserved in these studies suggested that classical isoforms ofKC were implicated [69,71,72]. Aldosterone rapidly stimu-

ated PKC-dependent NHE activity in rat distal colonic crypts73]. Further experiments confirmed the involvement of clas-ical PKCs when rapid cytoplasmic alkalinization in rat distalolonic crypts was found to be sensitive to the specific PKC�nhibitor HBDDE [18].

.2.2. CalciumAldosterone rapidly increases [Ca2+]i in isolated rat dis-

al colonic crypts and T84 cells through a mechanism thatas dependent on extracellular Ca2+ availability and PKC

ctivation [69,70]. Aldosterone did not potentiate the releasef Ca2+ from intracellular stores by thapsigargin (an endo-lasmic reticulum Ca2+-ATPase inhibitor) in cells incubatedn Ca2+ free conditions, but did increase [Ca2+]i follow-ng thapsigargin-induced calcium release [70]. These datandicate that aldosterone stimulates the influx of calciumrom the extracellular medium. In contrast to the immedi-te increase in [Ca2+]i following aldosterone treatment in thebsence of thapsigargin, when intracellular Ca2+ stores areepleted, the aldosterone induced increase in [Ca2+]i is muchlower. This suggests that aldosterone stimulated Ca2+ entrynto T84 cells, which in turn resulted in Ca2+ release fromntracellular stores [70]. Aldosterone (0.1 nM) significantly

ncreased [Ca2+]i along the whole length of isolated humanolonic crypts by a PKC and extracellular Ca2+-dependentechanism [71]. L-type Ca2+ channels were implicated,

s the pre-treatment of human colonic crypts with vera-

B.J. Harvey et al. / Journal of Steroid Biochemistry

Fig. 1. Membrane transporters that respond rapidly to aldosterone. Aldos-terone regulates ion transporters that facilitate the flux of electrolytes acrossthe high resistance epithelia of the kidney and the colon. In the principal cellsof the distal nephron rapid activation of these transporters has been observedwithin minutes of aldosterone treatment. Intracellular calcium levels risethrough the activation of Ca2+ channels in intracellular stores such as themitochondria (M) or endoplasmic reticulum or at the cell membrane within1 min. Changes in cytoplasmic pH are detectable within 1 min through acti-vation of NHE1. Apical sodium absorption through ENaC and basolateralK+ recycling through KATP channel activity is also detectable within 2 min.Pumping of Na+ into the blood at the basolateral membrane in exchange forK+ through the Na+/K+ ATPase is increased within 30 min. In the intercalatedcells increased H+ ATPase activity at the apical membrane results in urineafli

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cidification within 15 min. The rapid activation of KATP channels resultingrom increased NHE3 activity has also been reported in the colonic epithe-ium within 1 min of aldosterone treatment to facilitate the K+ recycling thats permissive for Na+ absorption in the colon.

amil abolished the aldosterone induced increase in [Ca2+]i72].

. Conclusion

Aldosterone rapidly stimulates multiple signaling path-ays, which modulate the earliest stages in its physiological

esponses in target tissues. The regulation of salt reabsorptioncross the renal and colonic epithelia requires the integratedctivation or suppression of membrane transport targets at thepical and basolateral surface of the epithelial cells (Fig. 1).t is at this initial, synchronizing stage of transport andignaling activity where the rapid responses to aldosteroneay have their greatest physiological relevance, effectively

reparing the cells for changes in gene expression. The acti-ation of signaling pathways such as ERK1/2 also have theotential to directly modulate gene expression through thehosphorylation of transcription factors and provide a linkor genomic/non-genomic crosstalk.

cknowledgements

The authors are supported by program grant60809/Z/00 from the Wellcome Trust and by

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& Molecular Biology 108 (2008) 310–317 315

he Higher Education Authority of Ireland PRTLIycle 3.

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