extracellular calcium and camp: second messengers as ''third … · 2016. 11. 7. ·...

doi: 10.1152/physiol.00019.200722:320-327, 2007. ;Physiology

Aldebaran M. Hofer and Konstantinos Lefkimmiatis''Third Messengers''?Extracellular Calcium and cAMP: Second Messengers as

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It seems that yet another domain within the organismtouched by Ca2+ and cAMP is the extracellular space. Ithas long been known that intracellular signaling eventsare associated with changes in second messenger con-centrations outside the cell. Can these fluctuations beregarded as “signals” in their own right? This article willaddress how cAMP and Ca2+ move across the cell mem-brane, the potential mechanisms for sensing theseextracellular changes in messenger concentration, andthe physiological outcomes of these signaling events.

Export of cAMP from Stimulated Cells

In 1963, just 5 years after the seminal descriptions ofthe second messenger function of cAMP, Davoren andSutherland reported the existence of a probenecid-sensitive mechanism for cAMP extrusion in nucleatedpigeon erythrocytes (18). Over the years, numerousreports have appeared demonstrating that cAMP canbe expelled following agonist stimulation from a widevariety of cell types, including adipocytes (57), hepato-cytes, renal epithelial cells (56), neuronal cells (53),fibroblasts, T lymphocytes (67), and skeletal muscle(27). In some earlier studies, investigators even useddeterminations of external [cAMP] as a surrogate formeasurements of hormone-dependent cAMP accu-mulation inside the cell (8). Nevertheless, this abilityof cells to eject the second messenger is apparentlycell-type specific, since certain other cell types do notappear to extrude cAMP whatsoever.

cAMP export can proceed across a concentrationgradient, is temperature dependent, and requires ener-gy in the form of ATP (FIGURE 1). This process is sus-ceptible to inhibitors of organic anion transport, suchas probenecid and sulfinpyrazone (56). In a coloniccarcinoma model (CC531mdr+), the efflux of cAMP wasfound to be exquisitely sensitive to cell swelling (28).Certain multi-drug resistance proteins in the MRP fam-ily have now been identified as significant pathways forcAMP egress (39, 69). These proteins belong to thesuperfamily of ATP binding cassette (ABC) proteins,specifically the ABCC subfamily. Most of these areknown to function as active membrane transporters fororganic anions and various drugs [e.g., for bile acidsand chemotherapeutic agents, including nucleoside

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The foundations of the second messenger concept wereestablished nearly 50 years ago when Earl Sutherlandand Ted Rall identified a heat stable factor that mediat-ed the intracellular actions of the hormones glucagonand epinephrine on glycogen metabolism in the liver(49). That factor was, of course, cyclic AMP, a discoverythat earned Sutherland a Nobel Prize in 1971 (58).

The next signaling molecule to be officially consid-ered as a second messenger was the calcium ion (12).The importance of Ca2+ as a trigger of muscle contrac-tion was known since the 19th century from the pio-neering work of London-based physiologist SydneyRinger. However, acceptance of the general signaltransduction function of Ca2+, as originally proposedby Lewis Victor Heilbrunn in the 1940s, initially metwith resistance (43). Recognition of the validity of thistheory slowly built following the identification of theCa2+ ATPases, intracellular Ca2+ release channels, andprotein targets for the Ca2+ signal throughout the 1960sand 1970s. Around this same time, several laboratoriesmanaged to make direct measurements of agonist-stimulated intracellular Ca2+ transients using the lumi-nescent jellyfish photoprotein aequorin. Together,these findings firmly vaulted Ca2+ to the status of abona fide second messenger.

Ca2+ and cAMP are now recognized as universal reg-ulators of cell function. Between them, these two clas-sic textbook second messengers impact nearly everyaspect of cellular life in diverse organisms rangingfrom amoebae to plants to human beings. A recurrenttheme that has emerged since the early descriptions ofthese fundamental messengers is the importance oflocalized signaling events within the cell (6, 16, 60, 70).By confining Ca2+ or cAMP to precise subcellulardomains (e.g., plasma membrane, organelles), thesemolecules can selectively activate a subset of targets,thereby expanding the repertoire and range of the sig-nal. Discovery of this property was greatly facilitatedby the parallel development of fluorescent probes anddigital microscopic imaging techniques, methods ini-tially applied for the visualization of Ca2+ signalingevents (71). More recently, FRET-based indicators forimaging cAMP in single cells have been used to con-firm that the metabolism and disposition of cAMP canbe regulated independently in different parts of thecell (25, 42, 46).

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Extracellular Calcium and cAMP: SecondMessengers as “Third Messengers”?

Aldebaran M. Hofer andKonstantinos Lefkimmiatis

Department of Surgery, VA Boston Healthcare System andBrigham & Women’s Hospital, Harvard Medical School,

West Roxbury, [email protected]

Calcium and cyclic AMP are familiar second messengers that typically become ele-

vated inside cells on activation of cell surface receptors. This article will explore

emerging evidence that transport of these signaling molecules across the plasma

membrane allows them to be recycled as “third messengers,” extending their abili-

ty to convey information in a domain outside the cell.

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analogs; see Kruh and Belinsky for review (39)]. In par-ticular, MRP4, MRP5, and MRP8 are established trans-porters of cyclic nucleotides (cAMP and cGMP) (69).These drug efflux pumps often assume polarized distri-butions in epithelial cells (e.g., MRP4 is found in theapical membrane of kidney tubule cells and the baso-lateral membrane of prostate glandular cells) (39).

Quantitatively, the amount of cAMP expelled by cer-tain cell types can be quite significant. For instance,human CD4+ T lymphocytes stimulated with choleratoxin release more than 50% of the total cAMP pro-duced over a 24-h period to the extracellular space(67). Strewler found an even more vigorous degree ofprobenecid-sensitive export in polarized LLC-PK1

renal epithelial cells; 40 min after stimulation witharginine vasopressin, the external cAMP concentra-tion in the apical bath was more than twice thatretained inside the cells (56). In humans, the concen-tration of cAMP in plasma and urine can become dra-matically elevated under a variety of physiologicalconditions and also following infusion of exogenouscAMP-elevating hormones (particularly epinephrine,parathyroid hormone, and glucagon). For example,Hendy and colleagues showed many years ago thatinfusion of human subjects with a large bolus ofglucagon causes massive release of cAMP from theliver, elevating plasma levels from low nanomolar lev-els to over 450 nM within 10 min (31). This couldtranslate into dramatic local accumulations of cyclicnucleotides in the diffusion-restricted spaces adjacentto cells. Direct measurements using microdialysistechniques have further demonstrated that significantfluctuations in extracellular cAMP can occur in intactbrain tissue during agonist treatment (11).

The cAMP extrusion mechanism has been largelydismissed as a means of regulating the intracellularcAMP signal, since the cyclic nucleotide phosphodi-esterases (PDEs: the enzymes that degrade cAMP andcGMP) already provide powerful and rapid control ofintracellular cAMP (3, 39). Furthermore, it has beenargued that this strategy is too energetically unfavor-able to be a viable means of regulating the cyclicnucleotide levels within the cell, with the furtheradverse consequence of depleting the cellular purinepool (38). As seen in FIGURE 2, however, acute inhibi-tion of this efflux pathway with probenecid can result inmeasurable increases in resting intracellular [cAMP] asmeasured using sensitive FRET-based reporters. Thissuggests that in some cells this molecular device maybe capable of altering resting cAMP levels and, poten-tially, the profile of the intracellular cAMP signal (27,56), in addition to altering extracellular levels of cAMP.

Actions and Targets of Extracellular cAMP

In the social amoeba Dictyostelium discoideum, extra-cellular cAMP serves as a chemical alarm bell that

signals single-celled individuals to aggregate duringstress into a multicellular “slug” or pseudoplasmodi-um. This fascinating organism, long used as a modelfor cellular migration and chemotaxis, is known toexpress four G-protein-coupled receptors (GPCRs)used to detect secreted cAMP (cAR1, cAR2, cAR3,cAR4; not to be confused with the Ca2+-sensing recep-tor “CaR” described below) (5). These receptors haveaffinities for cAMP ranging from sub-micromolar tomicromolar.

To date, no mammalian homologs of this well char-acterized cAMP receptor have been reported. It isnoteworthy, however, that the Dictyostelium GPCRsshare some limited sequence similarity to the secretinfamily of GPCRs expressed in mammalian cells,which includes receptors for parathyroid hormoneand calcitonin. It has been speculated that the latterclass of GPCRs may mediate the actions of extracellu-lar cAMP in vertebrates (5). Extracellular cAMP isknown to have many actions on diverse organ

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FIGURE 1. “Third messenger” activity of extracellular cAMPIntracellular cAMP is typically generated via adenylyl cyclases (AC) fol-lowing hormonal activation of G-protein-coupled receptors (GPCRs)linked to the heterotrimeric G-protein, G�s. Once formed, the secondmessenger can be actively transported to the extracellular space via aprobencid- and sulfinpyrazone-sensitive efflux mechanism belonging tothe ATP-binding cassette (ABC) transporter family. Extracellular cAMP ishypothesized to have direct actions on putative receptor proteins (as ofyet unidentified) that are expressed on neighboring cells. Alternatively,it is well established that extracellular cAMP can be sequentially metab-olized, first by ecto-phosphodiesterase to adenosine monophosphate(AMP), and then by ecto-5’-nucleotidase to adenosine. Adenosine canthen act as a paracrine or autocrine messenger to activate other signaltransduction cascades via one of four subtypes of adenosine receptors(A1, A2A, A2B, A3). In addition, since cAMP is relatively stable in blood,circulating cAMP can be converted by ecto-enzymes at a distant site,effectively rendering cAMP as a prohormone for adenosine (which has afleeting half-life in the circulation).

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cell model (22). Again, intracellular cAMP and extra-cellular cAMP had opposing actions on PGHS-2expression, causing upregulation and downregulationof the enzyme, respectively.

Paracrine Action of cAMP on theRenal System: The ExtracellularcAMP-Adenosine Pathway

The hormone glucagon, which is released from thepancreas directly into the portal blood flow, causesintracellular cAMP signaling in hepatocytes, and, assuggested above, this leads to substantial efflux ofcAMP into the general circulation. It appears that animportant target of this circulating cAMP is the renalsystem, particularly the proximal tubule. Glucagon iswell known to cause a marked enhancement of renalsodium and phosphate excretion in vivo, althoughspecific receptors for glucagon have never been iden-tified in the kidney. This prompted Bankir and colleagues (5) and others to propose that cAMPreleased from the liver might be acting as a circulating factor mediating the renal actions ofglucagon. A sequence of studies by Ahloulay et al.showed that cAMP infusion alone reproduced theactions of glucagon on renal Na+ and PO4

–2 handling(2). This phenomenon has been named the “pancreato-hepatorenal cascade.”

A comprehensive series of animal experiments car-ried out by Jackson and coworkers (36, 37) have givena further twist on this general theme. These investiga-tors showed that the cAMP entering the general circulation from the liver is able to undergo enzymaticconversion to adenosine once it reaches the kidney (FIGURE 1). Adenosine has a short half-life in the cir-culation (~1 s); therefore, cAMP (which is stable inplasma) may be regarded as a sort of prohormone foradenosine. Once produced (either locally or at a dis-tant site), adenosine can activate one of four differentreceptor subtypes (A1, A2A, A2B, and A3). Complex sce-narios can be envisioned considering that adenosinereceptor subtypes A1 and A3 interact with G�i/G�o toreduce intracellular cAMP levels in target cells, where-as the A2A and A2B subtypes serve to increase cAMP viaG�s. Therefore, cAMP released from one cell type couldconceivably initiate cAMP signaling in a neighboringcell or suppress cAMP signaling depending on the par-ticular adenosine receptor subtype expression pattern.

As described above, the cAMP-adenosine pathwayis prominent in the kidney, but substantial evidencefor this phenomenon also exists in the central nervoussystem (11, 21, 38). Moreover, the cAMP-adenosinepathway has been speculated to be important in thecardiovascular system and also for systemic metabolichomeostasis (57). The presence of the pathway doesnot preclude the possibility that cAMP may exertdirect actions on cells in addition to indirect effects viaadenosine production.

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systems, including renal, hepatic, and central nervoussystem. This subject has recently been reviewed in depthby Bankir et al. (5) and Jackson and Raghvendra Dubey(38). As described below, some of these actions may bethe indirect result of the metabolism of cAMP to adeno-sine in the extracellular space (the “extracellular cAMP-adenosine pathway”), although some effects appear tobe direct. For example, Sorbera and Morad (55) showedin 1991 that 50 �M extracellular cAMP rapidly (~50 ms)inhibited a sodium current in ventricular myocytesderived from several vertebrate species. This effect wassensitive to pertussis toxin, suggesting a GPCR-basedmechanism dependent on G�i or G�o proteins (55). Asanother example, secreted cAMP (but not adenosine)derived from stimulated human CD4+ T lymphocyteswas recently shown to exert significant growth effects onneighboring T cells in a co-culture system (67).

Detrick and colleagues demonstrated that nanomo-lar concentrations of extracellular cAMP and cGMP(but not adenosine or guanosine) enhanced colonyformation in myeloid progenitor cells. Interestingly,membrane permeant forms of the cyclic nucleotidesused at high micromolar concentrations had theopposite effect on the proliferation of the cells, imply-ing that an intracellular elevation of cAMP or cGMPcould antagonize the action of extracellular secondmessenger (20). Elalamy and colleagues have provid-ed compelling evidence for the involvement of anecto-PKA (protein kinase A) in mediating the actionsof extracellular cAMP (used at a concentration of 5�M) on expression of prostaglandin H synthase(PGHS-2) in a pulmonary microvascular endothelial

REVIEWS

FIGURE 2. Blockade of cAMP extrusion with probenecid altersintracellular free [cAMP] Intracellular cAMP was imaged using a FRET-based biosensor (46) in sin-gle HEK293 cells as described previously (25). This sensor (courtesy of Dr.Kees Jalink) is based on the cAMP binding protein Epac, which has beenlabeled with CFP and YFP. cAMP-dependent conformational changes ofthe Epac protein result in changes in FRET, providing a measure of free[cAMP]. Acute treatment of cells with 1 mM probenecid caused a smallbut significant change in the resting FRET signal (the 480:535 nm emis-sion ratio), consistent with an increase in intracellular [cAMP]. These datasuggest that the cAMP export process can contribute to intracellularcAMP homeostasis, in addition to mediating the elevation in extracellularcAMP. Shown for comparison is the action of the direct adenylyl cyclaseactivator forskolin (50 �M).

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Extracellular Calcium as a ThirdMessenger

Just as it has long been known that intracellular cAMPsignaling events are associated with extracellularaccumulation of the second messenger, so has it beenlong appreciated that Ca2+ can fluctuate outside cells,owing to activation of influx and efflux pathways forthe cation during Ca2+ signaling events (41). As withcAMP, early measurements of hormone-stimulatedCa2+ signals frequently relied on determinations ofCa2+ in the external media. Because diffusion is great-ly limited in the interstitial spaces (which occupy onlya fraction of the tissue volume; e.g., ~20% in brain tissue) (66) and the buffering capacity for Ca2+ insidethe cell is so much greater than outside, these fluxescan lead to significant alterations in free [Ca2+] in theextracellular milieu.

Fluctuations in Extracellular Ca2+

As indicated in FIGURE 3, agonist-stimulated Ca2+

signaling events involve 1) the release of Ca2+ frominternal storage compartments into the cytoplasmvia intracellular release channels (i.e., the InsP3

receptor); 2) the extrusion of Ca2+ into the extracellu-lar space by plasma membrane Ca2+ ATPases (PMCA)or other export mechanisms (e.g., Na+/Ca2+ exchang-ers); and 3) the activation of Ca2+ entry through store-operated channels (SOCs), such as the recently identified Ca2+ release-activated pathways known asthe Orai proteins (47).

Tepikin and colleagues have provided directdemonstrations of the significant quantitative impactof the Ca2+ extrusion process on extracellular Ca2+

levels adjacent to stimulated cells (61–63). One studyemployed simultaneous real-time measurements ofintracellular and extracellular [Ca2+] in single pancre-atic acinar cells suspended in a small droplet (40–90times the volume of the cell; FIGURE 4). By measuringthe extracellular [Ca2+] in the droplet with a Ca2+-sen-sitive dye, it was estimated that the total intracellularcalcium content was reduced by 0.7 mM duringcholinergic stimulation, owing to active transport ofthe cation by the PMCA.

Temporal and spatial separation of Ca2+ entry andefflux across the plasma membrane can give rise tophysiologically significant excursions in extracellular[Ca2+] (13, 35), particularly in polarized epithelial cellsand other functionally polarized cells such as neurons(33). For example, Caroppo and colleagues showedthat [Ca2+] in the luminal micro-compartment of theintact gastric gland increases by 200–500 �M followingcholinergic stimulation, owing to an abundance ofPMCA on the apical membrane of the gastric epithelial cells (13). At the same time, a comparabledepletion of Ca2+ was recorded in the basolateral inter-stitium of the intact mucosa as a result of Ca2+ influx

via pathways located predominantly at the basal cellside. As described below, these extracellular [Ca2+]fluctuations have been shown to have functional con-sequences.

It is also well established that specific elements of theCa2+-handling machinery (as well as certain Ca2+ sen-sors) can be confined in cell surface microdomains,such as caveolae (17, 26), potentially giving rise to localgradients of Ca2+ in the caveolar nanospaces. Other fac-tors can influence free [Ca2+] in the external milieu,including dilution and concentration of ionic species,owing to cellular water transport. In addition, the


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FIGURE 3. “Third messenger” activity of extracellular Ca2+

Local extracellular [Ca2+] can fluctuate as a consequence of agonist-induced intracellular Ca2+ signaling events. In the typical scenario, acti-vation of a G�q/11-coupled receptor by a Ca2+-mobilizing agonist resultsin inositol 1,4,5-trisphosphate (InsP3) production, giving rise to the lib-eration of stored Ca2+ via InsP3 receptor release channels in intracellularCa2+ pools. A substantial fraction of Ca2+ released into the cytoplasm israpidly extruded by plasma membrane Ca2+ ATPases (PMCAs), poten-tially resulting in significant local elevation of the extracellular [Ca2+].Store emptying also triggers Ca2+ influx via store-operated Ca2+ chan-nels (SOCs) in the plasma membrane, leading to transient depletion ofCa2+ in the volume-limited interstitial spaces. The ensuing local fluctua-tions in Ca2+ can influence a variety of Ca2+-sensing proteins on adja-cent cells or on the same cell. Examples include HERG K+ channels,several types of nonselective cation channels, and a host of G-protein-coupled receptors modulated by extracellular Ca2+ (e.g., the extracellu-lar Ca2+-sensing receptor, the GPRC6A orphan receptor, ormetabotropic glutamtate receptors). Gap-junction hemichannels andthe transmembrane protein notch are also susceptible to alterations inextracellular [Ca2+] (32).

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Extracellular Ca2+ Sensors

Although specific GPCRs for cAMP have not beenidentified in mammalian cells, cell-surface receptorsfor Ca2+ are known to exist, and some of these havebeen well characterized. Without question, the bestknown of these is the extracellular calcium-sensingreceptor (CaR), which was originally cloned frombovine parathyroid gland in 1993 by Brown and col-leagues (9). The structural and functional propertiesof this widely expressed divalent cation receptor havebeen reviewed extensively elsewhere (10, 33) and willnot be addressed in detail here. The CaR (of whichonly a single isoform appears to exist) is indispensa-ble for life in mammals, acting as the Ca2+ sensor thatcontrols systemic Ca2+ and Mg2+ homeostasis via PTHsecretion. An emerging literature describes numer-ous physiological functions of this receptor through-out the body and in diverse vertebrate species,including birds and fish. Deletion of CaR is lethal, butthe developments of viable “rescued” CaR knockoutmouse models that maintain normal parathyroidfunction are being used increasingly to examine thisreceptor’s physiological role in other organ systems(1, 45).

CaR is a member of family C of the GPCR superfam-ily, which also includes three taste receptors (T1–T3),the GABAB receptors, eight metabotropic glutamatereceptors (mGluR1–mGluR8), and six orphan recep-tors, including the recently characterized GPRC6A (7,68). These receptors appear to share an evolutionarythread with CaR based on their common functionalorigins as nutrient/salinity sensors (15, 30). The CaR isallosterically modulated by extracellular amino acids(15). Conversely, other members of this family that areregarded as amino acid sensors, such as certainmGluRs, GABAB receptors, and GPRC6A, are modulat-ed by extracellular Ca2+ (44). GPRC6A has 34% aminoacid sequence identity with CaR (68) and is activatedby relatively high extracellular [Ca2+] (5–10 mM) (44).This receptor has been suggested to serve as a sensorfor Ca2+ in bone (44), a tissue where local extremes inexternal [Ca2+] are believed to occur during the boneremodeling process.

Many other cell surface proteins are susceptible tophysiological fluctuations in external [Ca2+] (recentlyreviewed in Ref. 32). These include gap-junctionhemichannels (64), which can open in response to amodest (~200 �M) decrease in external [Ca2+], and thereceptor Notch, which may sense external [Ca2+] todrive the establishment of right-left symmetry duringembryogenesis (50, 51). A distinct Ca2+-sensing recep-tor known as CAS has been recently described inplants (59). In addition, a number of ion channels altertheir open probability depending on the local extracel-lular Ca2+, including the proton-gated cation channelsASIC1a/ASIC1b, HERG K+ channels, and other nonse-lective channels found in neuronal tissue (32).


transport of Ca2+ buffers (e.g., HCO3–, PO42–) would also

be expected to influence the free [Ca2+] in the intersti-tium. Ca2+ taken up into endocytic vesicles could con-ceivably impact the local extracellular [Ca2+] (23).Finally, secretory vesicles are known to contain highconcentrations of Ca2+ and other divalent cations (Zn2+,Mg2+), and synchronous secretory activity could inprinciple lead to rapid increases in extracellular diva-lents (29, 48). Gray et al. recently proposed that libera-tion of these metals from vesicles of insulin-secretingcells may constitute a means of communicationbetween cells (29) via sensors for extracellular Ca2+, asdescribed in the following section.

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FIGURE 4. Extracellular [Ca2+] becomes elevated adjacent tostimulated cells due to Ca2+ exportDirect measurements of Ca2+ extrusion from pancreatic acinar cells per-formed by Tepikin and colleagues (62) using the “droplet technique”demonstrates that large quantities of Ca2+ are exported from stimulatedcells. The total drop in cellular Ca2+ following acetylcholine (ACh) treat-ment was estimated to be about 0.7 mM over 2–5 min (modified withpermission from Ref. 62). A: photomicrograph of fluid micro-droplet con-taining a single mouse pancreatic acinar cell. The cell was loaded with theCa2+ indicator fura-2, whereas the droplet contained a second Ca2+ dye,fluo-3. At right is seen the pipette tip, used for iontophoretic delivery ofagonists. B: simultaneous measurement of free intracellular Ca2+ concen-tration ([Ca2+]i) in a single acinar cell and extracellular Ca2+ ([Ca2+]o) in thedroplet following challenge with 20 nM ACh.

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Intercellular Communication Via Ca2+

Our laboratory demonstrated some years ago in aproof-of-concept study using a co-culture model sys-tem that it is possible for CaR to detect extracellularfluctuations in [Ca2+] that occur secondary to intracel-lular Ca2+ signaling events (34, 65). This opened up theprospect that Ca2+ might function as a paracrine messenger, used, for example, to communicate infor-mation about the signaling status of a neighboring cellor to integrate or reinforce signals in multicellularensembles. We further provided evidence for a varia-tion on this theme, whereby exported Ca2+ can activateCaR expressed on the same cell in an autocrine fash-ion (19). Caroppo et al. (13) later showed a physiolog-ical role of this third messenger signaling system in theintact gastric mucosa. These investigators took advan-tage of information gained from their previous extra-cellular microelectrode studies aimed at measuringthe profile of the extracellular Ca2+ “signal” in the apical and basolateral microdomains followingcholinergic stimulation (see above) (13). Remarkably,reproducing this physiological pattern of extracellular[Ca2+] variation was able to elicit changes in pepsino-gen and alkaline secretion from the tissue (14), andmore recently this third messenger activity has beenlinked to changes in water transport (24) in the samemodel system. The CaR, which is expressed apically inthe amphibian oxyntic cell, is involved in detecting theextracellular [Ca2+] elevation that occurs in the lumi-nal compartment of the gastric gland, although itappears that another entity may be responsible forsensing the basolateral decrease in [Ca2+] (14). Theseintriguing data are suggestive of a novel mode of Ca2+

signaling that takes advantage of extracellular, ratherthan intracellular, changes in [Ca2+], but it remains tobe seen whether this process occurs in other tissues.

Other Second Messengers as “ThirdMessengers”?

Are there other hydrophilic signaling molecules thatare exported by cells to inform neighboring cells oftheir signaling or metabolic status? Cyclic GMP, thesecond messenger generated by either atrial natri-uretic peptide or nitric oxide gas via guanylatecyclases, is vigorously exported from many cell typesin quantities that surpass that of cAMP (4, 54). Thiswidespread phenomenon is mediated by many of thesame MRP family members (e.g., MRP4, MRP5,MRP8) known to transport cAMP, as well as theorganic anion transporter OAT1 (69). Diverse biologi-cal actions of extracellular cGMP have been describedin brain and kidney [recently reviewed by Sager (54)],but as is the case for extracellular cAMP, specificmolecular receptors for cGMP in mammalian cellshave yet to be identified.

Isolated reports of extracellular accumulation ofinositol 1,4,5 trisphosphate (InsP3) following choliner-gic stimulation as measured using microdialysis tech-niques in brain have also appeared (40, 52). Roberts etal. found that several inositol phosphate metabolitesof InsP3 appeared (in addition to InsP3) in the intersti-tial space under these conditions, although it is uncer-tain whether the appearance of the additional inositolderivatives reflects metabolism of extracellular InsP3

or a separate transport process (52). It is not knownwhether InsP3 egress is a widespread phenomenon orwhether it has any functional significance.

Conclusions

Second messengers are the cellular currency of infor-mation transfer. However, the generation of cAMP fromATP and the energy required to maintain the gradientsthat permit Ca2+ signaling to take place come at a cer-tain energetic cost. Thus it is attractive to imagine thatmulticellular organisms might capitalize on fluctua-tions in extracellular second messengers to expand theinformational content of the intracellular signal trans-duction process. The concept of the interstitialmicrodomain as a specialized signaling compartmentis in its infancy, however. There is still much to learnabout how and when the local concentrations of cAMPand Ca2+ change in this space and what the physiologi-cal consequences of these fluctuations are. The devel-opment of practical methods for probing the profile ofsuch extracellular “signals” will be an important firststep to understanding whether this constitutes a gener-alized device to extend the scope and range of secondmessenger molecules to a domain outside the cell.

We are grateful to Dr. Kees Jalink of the NetherlandsCancer Institute for graciously providing us with the Epac-based FRET sensor used for imaging intracellular cAMPand to our colleague Prof. Silvana Curci for reading themanuscript.

Support for the work conducted in the Hofer laboratorywas provided by the Medical Research Service of theDepartment of Veteran’s Affairs and the Brigham SurgicalGroup.

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