Nicotinic receptor signaling in nonexcitable cells

Nicotinic Receptor Signaling in Nonexcitable Cells

Geeta Sharma, Sukumar Vijayaraghavan

Department of Physiology and Biophysics, and the Neuroscience Program, University of ColoradoHealth Sciences Center, 4200 E. 9th Avenue, Denver, Colorado 80262

Received 16 May 2002; accepted 25 June 2002

ABSTRACT: The finding that neuronal nicotinicacetylcholine receptors (nAChRs) are present in non-neuronal cells both within and outside the nervous sys-tem raises some interesting issues. The mechanismsunderlying receptor signaling and its downstream con-sequences in these cells remain to be elucidated. Factorscontrolling the release of acetylcholine and the extent ofits diffusion are likely to be different for these cells thanfor traditional neuronal synapses. Recent advances onthe physiologic functions of some of these cell types have

provided a better insight into possible functional rolesfor nAChRs in nonexcitable cells. The presence ofnAChRs on these cells also implies a broader scope forthe actions of nicotine that needs to be considered froma clinical viewpoint. Revealing the potential physiologicroles for nAChRs on nonexcitable cells is likely to pro-vide a more complete understanding of cholinergic sig-naling. © 2002 Wiley Periodicals, Inc. J Neurobiol 53: 524–534, 2002

Keywords: non-neuronal; acetylcholine; glia; calciumsignaling

INTRODUCTION

The past decade has seen a dramatic expansion in ourknowledge of nAChR composition, pharmacology,and channel properties (for review, see Role and Berg,1996; Colquhoun and Patrick, 1997; Clementi et al.,2000). The additional information obtained on recep-tor properties, however, has not resulted in a similarincrease in our understanding of their physiologicrole. From a number of studies, one consensus thatemerges is that some traditional views of ligand-gatedion channel function do not necessarily apply tonAChRs. A few possible instances of nicotinic trans-mission have been demonstrated in central nervoussystem (CNS) neurons, for example, in the ferret

visual cortex (Roerig et al., 1997) and in the rathippocampus (Frazier et al., 1998). Current evidencesuggests that a major role for nAChRs, at least in themammalian CNS, is not as mediators of rapid chem-ical transmission at synapses but rather as modulatorsof synaptic and cellular signaling.

Once one accepts this nontraditional view of li-gand-gated ion channel function, the observation thatnAChRs are located at unusual loci on neurons, andon nonexcitable cells, should come as no surprise. Theissues raised in terms of transmitter availability, sig-naling mechanisms, and functional relevance, how-ever, are different for non-neuronal cells. ExaminingnAChR function in these cells, therefore, becomes aprerequisite for our understanding of both cholinergicsignaling and potential consequences of nicotine ex-posure.

nAChRs in Non-neuronal Cells

In this section we provide an overview of what isknown about nAChR expression in selected non-neu-ronal cells both within, and outside of, the nervoussystem. The cell types and tissues discussed here thatexpress nAChRs are representative and do not purport

Correspondence to: S. Vijayayaraghavan ([email protected]).

Contract grant sponsor: National Institutes of Health; contractgrant number: DA 10266 (to S.V.).

Contract grant sponsor: the American Heart Association, Moun-tain Affiliate (fellowship to G.S.), and the National Science Foun-dation (POWRE grant to G.S.).© 2002 Wiley Periodicals, Inc.Published online in Wiley InterScience (www.interscience.wiley.com).DOI 10.1002/neu.10114

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to be comprehensive. Where available, the reader isreferred to reviews on nAChRs in specific non-neu-ronal cell types.

Outside the Nervous System. Human keratinocytesboth in situ and in primary culture express �3 and �7nAChR subunits (Zia et al., 2000) in addition to �2and �4 subunits (Grando et al., 1995). Using poly-clonal antibodies to C-terminal peptides of �7 and �3subunits, it was shown in keratinocytes, that the twogene products are differentially distributed. (Table 1).The �3 protein was seen in small cells, presumablyearly in development. In cultures, they were localizedto membranes forming cell–cell junctions. On theother hand, the �7 protein was present in large, im-motile, presumably fully differentiated cells (Zia etal., 2000). In the lung, bronchial epithelial cells alsoexpress a wide array of nAChR subunits and showsignificant [3H]-epibatidine and [125I]-�-Bungaro-toxin (�Bgt) binding (Table 1). Application of nico-tine and ACh elicits measurable inward currents inthese cells further strengthening the evidence for func-tional nAChRs (Maus et al., 1998; Wang et al., 2001).

Polymorphonuclear (PMN) cells and lymphocytesisolated from human blood show the presence ofnAChRs. RT-PCR and Western blotting have beenused to demonstrate the presence of �3, �4, �7, �2,

and �4 in these cells (Table 1). Coimmunoprecipita-tion experiments suggest that the predominant nAChRsubtype in PMN cells is the �3�4 subtype with minorpopulations of other combinations, while the recep-tors in lymphocytes are likely to be partially com-posed of the �7, �4�2, and �3�4 subtypes (Benham-mou et al., 2000). In addition, PMN cells havesignificant [3H]-nicotine binding that appears to beupregulated in smokers as has been reported for thebrain receptors (Benhammou et al., 2000), suggestingregulatable expression of nAChRs in these cells.

Nervous System. Among developing precursor cellsin the nervous system, the O2A-oligodendrocyte pre-cursor cells (O2A/OPCs) are, perhaps, the best char-acterized. These cells, upon differentiation, give riseto oligodendrocytes. A recent study has shown thatO2A/OPCs express a number of nAChR subunits asdemonstrated by RT-PCR and immunofluorescencetechniques (Rogers et al., 2001). The presence ofthese subunits was not detectable once these cellswere induced to differentiate, suggesting that receptorexpression is developmentally controlled.

In contrast, cultured hippocampal astrocytes pos-sess functional nAChR responses with pharmacologyconsistent with the expression of low levels of func-tional �7-containing receptors (Sharma and Vija-

Table 1 nAChRs in Non-Excitable Cells

Cell Type Genes Detection MethodLigandBinding Function Ref.

Lymphocytes (Human) �4, �4, �3,�7, �2

RT-PCR, Southernblot, Western blot

ND ND 1

Vascular EndothelialCells (Human)

�3, �5, �2,�4, �7

in situ Hybridization,RT-PCR,

3H-Epi Electro-physiology 2

Polymorphon uclearcells (Human)

�3, �4, �7,�2, �4

RT-PCR, Southernblot Western blot

3H-Nicotine ND 1

Lymphocytes (Murine) RT-PCR 125I-�Bgt ND 3Bronchial Epithelium

(Human, Rat)�3, �5, �4,

�2, �7RT-PCR, Immuno-

fluorescence, insitu Hybridization

3H-Epi,125I-�Bgt

Electro-physiology 4

Keratinocytes (Human) �3, �7 Immunofluorescence ND Calcium Imaging 5O2A Progenitors (Rat) �3, �4, �5,

�7, �2,�4

RT-PCR, Immuno-fluorescence

ND Calcium Imaging 6

Astrocytes (Rat) �7 (?) Immunofluorescence(mAb W6)

3H-nicotine, Calcium Imaging,Electro-physiology

7

Glial cells (Lymnaeastagnalis)

�7-like’ Fluorescence �Bgtbinding, Westernblots, Northernblots

125I-�Bgt Electro-physiology 8

A listing of nAChRs detected in non-excitable cells. Data taken from 1. Benhammou et al., 2000. 2. Macklin et al., 1998; Wang et al., 2001.3. Toyabe et al., 1997. 4. Maus et al., 1998; Wang et al., 2001. 5. Zia et al., 2000. 6. Rogers et al., 2001 7. Hosli and Hosli, 1988; Hosli etal., 2001; Sharma and Vijayaraghavan, 2001. 8. Smit et al., 2001.

Nicotinic Receptor Signaling in Nonexcitable Cells 525

yaraghavan, 2001). Using W 6, a monoclonal anti-body raised against the Torpedo AChR and shown tocompete for �-Bgt binding to that receptor (Conti-Tronconi et al., 1990), Hosli and coworkers providedevidence for AChRs in cultured cortical astrocytes aswell as their colocalization with androgen and estro-gen receptors on these cells (Hosli et al., 2001).

nAChR currents, blocked by �Bgt, have beenshown in glial cells of the mollusc Lymnaea stagnalis.In addition these cells secrete a truncated version ofnAChR in response to receptor activation that acts asa released ACh binding protein (AChBP). The se-quence of the AChBP reveals homology to the �7class of nAChRs. From binding studies, the AChBPhas a high affinity for both �Bgt and nicotine (Smit etal., 2001).

A general consensus that has emerged over the lastfew years is that nAChRs are not restricted to neuronsor even the nervous system in their distribution. Abetter understanding of the mechanisms involved innAChR activation and signaling would be an impor-tant first step towards elucidating their physiologicalrole in nonexcitable cells.

Calcium as a Possible Mediator ofnAChR Signaling in Nonexcitable Cells

In nonexcitable cells, once one excludes the unlikelypossibility of direct metabotropic actions of nAChRs,one is left with signaling mechanisms that are basedon specific ion fluxes upon receptor activation.Among these, calcium is likely to play a major role innAChR signaling.

In human keratinocytes, application of high dosesof nicotine causes a transient and a small increase inintracellular free calcium concentrations ([Ca]i). Thischange in [Ca]i was inhibited when nicotine wasapplied in the presence of high doses of mecamyl-amine, a nonspecific nAChR antagonist (Zia et al.,2000). In the human nasal epithelium, nicotine causesa large increase in [Ca]i that is sensitive to nanomolarlevels of methyllycaconitine (MLA), suggesting acti-vation of functional �7-containing nAChRs (Blank etal., 1997). In T-lymphocytes, although a direct dem-onstration of nicotine-induced calcium transients hasbeen lacking, long-term exposure to nicotine de-creases the calcium rises seen upon T-cell receptoractivation (Singh et al., 2000).

O2A/OPCs respond to nicotine application with alarge rise in [Ca]i. In a subset of these cells, nicotinealso initiated calcium oscillations that are relativelylong lasting and occur in the continued presence ofnicotine (Rogers et al., 2001). As both AMPA andhigh potassium only elicit transient calcium rises in

these cells, the oscillations appear to be nAChR-specific. The nicotine-mediated increases in [Ca]i areabolished by dihydro-�-erythroidine (DH�E), sug-gesting an obligatory involvement of the �4�2 sub-type of nAChRs. It will be interesting to determine thecellular mechanisms that underlie the nAChR-medi-ated calcium oscillations and their functional conse-quences in these cells.

Similarly, in primary cultured astrocytes from therat hippocampus, both nicotine and ACh induce arapidly desensitizing current. This is accompanied bya robust increase in [Ca]i (Sharma and Vijayaragha-van, 2001). Unlike the O2A/OPCs, both currents andthe calcium signals in astrocytes were inhibited byMLA and �Bgt, suggesting that these signals arosefrom the activation of �7-containing nAChRs.

Mechanisms of nAChR-MediatedIncreases in [Ca]iLigand-gated ion channels commonly increase [Ca]i

by three possible mechanisms: (1) calcium fluxthrough the ion channel itself. In this regard, theNMDA class of glutamate receptors (NMDARs) are aclassic example where calcium flux through the ionchannel can have important physiologic functions(Ghosh and Greenberg, 1995). (2) The most prevalentmechanism for raising global [Ca]i upon ligand-gatedion channel activation in neurons is depolarization-mediated activation of voltage-gated calcium chan-nels (VGCCs). (3) Calcium influx upon ligand-gatedion channel activation can be further amplified byrelease from endoplasmic reticulum calcium stores.As calcium signaling is spatially and temporallycoded, each of these sources of calcium could poten-tially control a separate set of downstream eventsthat affect cellular functions. Upon examination ofnAChR-mediated calcium rises in a number of sys-tems, it appears that these receptors might employ allthree mechanisms.

Calcium Flux Through nAChRs. A noteworthyproperty of nAChRs is their high relative permeabilityto calcium. The PCa/PNa ratios calculated for nAChRsrange from about 1 for ganglionic nAChRs (Fieberand Adams, 1991; Rathouz and Berg, 1994) to about20 for heterologously expressed �7 gene product(Seguela et al., 1993). The �Bgt-sensitive currentfrom cultured hippocampal neurons exhibit a PCa/PNa

ratio of 5 (Castro and Albuquerque, 1995). Thesefindings suggest that the nAChRs show higher relativepermeability to calcium than their muscle counterpart(Vernino et al., 1992) and can, in some cases, like thatfor the �7-containing nAChRs, be comparable to that

526 Sharma and Vijayaraghavan

seen for the NMDARs, the current benchmark for acalcium-permeable receptor. From direct measure-ments of calcium flux using fluorescence calciumindicators, it is estimated that calcium contributes toabout 4–5% of the total current carried by �Bgt-insensitive nAChRs (Vernino et al., 1994; Rogers andDani, 1995). Such measurements have not been per-formed on nAChRs containing the �7 subunit, but inneurons, flux through these nAChR subtypes might besufficient to modulate neurotransmitter release (Grayet al., 1996).

In nonexcitable cells, ion flux through nAChRchannels is likely to be especially significant, becauseVGCCs are present at low densities or completelyabsent. Also, in cells like astrocytes, which have asignificantly lower resting membrane potential thanneurons, flux through ligand-gated ion channels couldrepresent a major influx pathway for raising [Ca]i bothlocally to modulate calcium-activated channels (Hosliet al., 1988) and globally to activate a variety of calcium-dependent signaling mechanisms (Zia et al., 2000).

Calcium Flux through VGCCs. This is clearly adominant mechanism in neurons. In chick ciliary gan-glion neurons, a major portion of nAChR-inducedchanges in bulk calcium is due to influx throughVGCCs (Vijayaraghavan et al., 1992; Rathouz andBerg, 1994). In cultured hippocampal neurons, about85% of nAChR-mediated bulk calcium changes wereblocked by cadmium, suggesting that most of the cal-cium flux was through VGCCs (Barrantes et al., 1995).

Functional VGCCs have been described in endo-thelial cells (Vinet and Vargas, 1999) and lympho-cytes (Ricci et al., 1996), although their physiologicsignificance is not well understood. In O2A/OPCs,nAChR-mediated changes in [Ca]i are inhibited bynifedipine, consistent with a role for L-type calciumchannels (Rogers et al., 2001) although these channelshave yet to be characterized in these cells. In primaryastrocytes cultured from the rat hippocampus, largecalcium rises are seen in response to nAChR activa-tion, which are not affected by VGCC blockersdemonstrating that in these cells, VGCCs do not par-ticipate in nAChR signaling (Sharma and Vija-yaraghavan, 2001).

Downstream Activation Mechanisms. One possiblecalcium signaling pathway triggered by nAChR acti-vation that has received less attention is the down-stream amplification of incoming calcium signals byrelease from intracellular stores. This form of release,known as calcium-induced calcium release (CICR), islikely to play an important role in nAChR-mediatedcalcium signaling events. A number of recent studies

have implicated store calcium release in nAChR sig-naling in neurons. In the substantia nigra nAChR-mediated increases in [Ca]i inhibited by dantroline(Tsuneki et al., 2000). Local nAChR-mediated cal-cium changes at local nerve terminal varicosities inthe vas deferens are almost completely due to CICR(Brain et al., 2001), suggesting a role for CICR inmodulating local calcium-dependent events. RapidnAChR-mediated calcium transients on somaticspines of ciliary ganglion neurons are not store-de-pendent, while a portion of global calcium signals areamplified by release from stores (Shoop et al., 2001).These studies raise the possibility that in addition toamplification, CICR might allow a single nAChRsubtype to activate more than one temporally andspatially distinct downstream signaling mechanisms.For example, in the hair cell efferent synapses of themammalian cochlea, nAChRs suppress sound-evokedactivity of the auditory nerve on a millisecond timescale, mediated by activation of calcium-activatedpotassium channels by calcium flux through nAChRchannels. In addition, there is a slower inhibition overtens of seconds, which is mediated by CICR uponnAChR activation (Sridhar et al., 1997). Thus, thisdownstream calcium signaling mechanism allowsregulation of two temporally distinct processes by theactivation of nAChRs.

In non-neuronal cells, perhaps the extreme exam-ple of amplification, allowing for complex spatial andtemporal calcium dynamics, comes from our studieson cultured hippocampal astrocytes (Sharma and Vi-jayaraghavan, 2001). In these cells, activation ofnAChRs leads to a rapid and large calcium transient.As expected for signaling via a ligand-gated ion chan-nel, this rise in [Ca]i is entirely dependent on externalcalcium demonstrating an obligatory requirement forcalcium influx. Blocking VGCCs with cadmium doesnot affect these transients. Depleting intracellular cal-cium stores with thapsigargin (TG), a microsomalCa/ATPase inhibitor, or high concentration of ryano-dine to block CICR, more or less completely blocksnAChR-mediated calcium transients [Fig. 1(A) and(B)]. The complex nature of this calcium signalingcascade is further illustrated by the finding that thereis a delayed release component from IP3-sensitivestores [Fig. 1(C)]. These findings imply that in cul-tured astrocytes there is a three-step process for theelevation of [Ca]i-influx through nAChRs followed byCICR from ryanodine-sensitive stores and subsequentrelease from IP3-sensitive stores. It is our belief thatsuch an elaborate cascade allows for a complex spatialand temporal control of calcium signaling in astro-cytes that have potential implications in their role asmodulators of neuronal function (see below).


The effective range for calcium diffusion in cells isthought to be small. For example, in bovine adrenalchromaffin cells, this range has been calculated to beabout 5 �m in the presence of mobile calcium buffersand 0.1 �m in their absence (Zhou and Neher, 1993).The argument is that because of limited mobility ofcalcium in cells, for a calcium signal to be transmittedacross large distances, for example, from the termi-nals of one process of an astrocyte to the other (whichis on an average �100 �m), it has to be regenerative.By strategic placement of stores and by using down-stream second messengers like IP3, activation ofnAChRs on astrocytes can be spread in the form of anintracellular calcium wave across the length of anastrocyte. An example of such an intracellular cal-cium wave is illustrated in Figure 2(A). Calciumsignals in response to the activation of nAChRs weremeasured across space and time within an astrocyte.A single narrow, nonstellate astrocyte (selected toapproximate linear propagation) was challenged with100 �M ACh in the presence of 500 nM atropine.Calcium rises were measured in 1 �m optical slices

spaced 1 �m apart, along the length of the glial cell.The origin of calcium rise was discrete and propa-gated along the length of the cell as a calcium wavewith measurable delays in onset [Fig. 2(A), left panel]at the rate of 12 �m/s. In addition, as the wavepropagated, the rate of fluorescence rise [Fig. 2(A)] aswell as the amplitude of the calcium signals increased,likely due to store amplification. In a stellate astro-cyte, in situ, such a mechanism could provide the cellwith an ability to modulate direction and distance ofpropagation as well as allowing the cells to integratespatially discrete inputs.

In addition, local activation of nAChRs on astro-cytes using a patch pipette and a diagonally placedsuction pipette (restricting diffusion of agonist to acircle with a diameter of less than 20 �m), caused thespread of calcium signals in the form of intercellularcalcium waves [Fig. 2(B)] that propagate, on an av-erage, at a rate of 25 �m/s. Intercellular waves inthese cultured astrocytes are not blocked by blockersof gap junctions but are inhibited by suramin, a gen-eral blocker of ATP receptors (Rizolli, Sharma, and

Figure 1 nAChR-mediated calcium rise in cultured hippocampal astrocytes. Hippocampal astro-cytes were cultured at low density and were loaded with 10 �M fluo 3-AM. The cells were thenchallenged with a 2-s application of 100 �M ACh in the presence of 500 nM atropine using apiezo-driven rapid application system. Calcium transients were reported as a rise in fluo 3 fluores-cence. Values on the Y-axis represent fold change in fluorescence in a cell normalized to themanganese-saturated fluorescence for the same cell. (A) Astrocytes were pretreated with 1 �M TGfor 30 min to deplete ER calcium stores. Depletion of stores was confirmed by the application of 20mM caffeine, which induced a change in calcium much smaller than that seen under controlconditions. An application of ACh 5 min after the caffeine application showed no increase in fluo3 fluorescence (Compare with Control response in Panel C), suggesting that depletion of storecalcium abolished the nAChR-mediated calcium transient in astrocytes. (B) A second test for storeinvolvement was then carried out. Cells were preincubated with 100 �M Ryanodine to block CICRchannels. An application of caffeine was used to confirm block of store release. Application of AChagain resulted in very little change in fluo 3 fluorescence (see Control Response in panel C). Thisimplied that most of the calcium rise seen upon nAChR activation in these cells came from CICRfrom ryanodine-sensitive stores. (C) The involvement of IP3 receptors (IP3Rs) in nAChR calciumtransients was estimated using cells pretreated with 20 �M Xestospongin C (Xe-C), a noncompet-itive blocker of IP3Rs. In the presence of Xe-C, nAChR calcium transients were similar in amplitudeto the control transients (Control) but the decay was much faster. Taken together, our results suggesta three-step process for nAChR-mediated calcium rise in astrocytes. Influx of calcium throughnAChRs causes a rapid release of calcium from intracellular stores via CICR. This is followed byfurther amplification due to release from IP3-sensitive stores (Sharma and Vijayaraghavan, 2001).


Vijayaraghavan, unpublished data). Thus, calciumrises in one astrocyte appears to be communicated toothers by extracellular mechanisms involving ATPrelease and the recruitment of purinergic receptors.

These results indicate that nonexcitable cells canemploy complex calcium signaling mechanisms toboth transduce input signals within themselves, aswell as possibly communicate to other cells. Thus,these nonexcitable cells might have a form of excit-ability that is based not on electrical propagation buton calcium signaling mechanisms. Signaling bynAChRs on astrocytes would thus be one means ofmodulating this excitability.

Possible Sources of ACh for nAChRActivation in Nonexcitable Cells

The presence of ligand-gated ion channels at nontra-ditional sites also implies a nonconventional mode ofactivation by the ligand. Assuming that ACh is theendogenous ligand for nAChRs (see elsewhere in thisissue for other possible ligands), the presence of the

receptors in non-neuronal cells also calls for the ex-istence of nonsynaptic sources of ACh as well.

In the nervous system, presence of nAChRs onnon-neuronal cells and possibly on nonsynaptic siteson neurons, raises the possibility of activation viaACh diffusion. Much of what we know about AChdiffusion comes from studies at the neuromuscularjunction where the radius of diffusion based receptoractivation is at best about 300 nm (Land et al., 1981).If activation of nAChRs in the nervous system canarise by diffusion of ACh across the extracellularspace, then this warrants a more careful examinationof the acetylcholine esterase (AChE) concentrationsas well as the location and geometry of release sites.

It is interesting and a further testimony to therelevance of non-neuronal nAChRs that presence ofthe receptors is in many cases accompanied by aproximal source of ACh. The parasympathetic vagalefferents form a neuronal source of ACh, and vagalcontrols of many potentially nAChR-mediated path-ways has been shown, for example, in airways (Rog-ers, 2001) and in the immune system (Borovikova et

Figure 2 Spread of nAChR-mediated calcium signals in astrocytes. (A) Intracellular calcium wavein fluo 3-loaded astrocytes. Rapid imaging (average acquisition at 17 Hz) of nAChR-mediatedcalcium transient was performed on an isolated astrocytes challenged with ACh in the presence ofatropine using a rapid application system. Long and flat cells were chosen to approximate linearpropagation. Fluorescence was measured at 2-�m intervals along the length of the cell. Upon agonistapplication calcium rise originated at a single locus in this cell and spread at the rate of about 12�m/s. The rate of calcium rise was amplified as the signal progressed presumably due to releasefrom stores. Numbers next to each transient represents distance (in �m) from the point of origin (0).(B) Intercellular spread of nAChR-mediated calcium signals. Agonist (100 �M ACh � 500 nMatropine) was applied to a single astrocyte in a confluent culture using pressure application from apatch pipette. Spread was further limited using a large (5-�m diameter tip) suction pipette placeddiagonally across from the application pipette. Activation of nAChRs caused a rapid calciumtransient (t � 0) in a couple of cells that spread across the field at an average rate of 22 �m/scompletely spanning the field in less than 10 s (t � 10). Fluorescence traces from astrocytes selectedat specific distances from the point of origin (numbers next to each transient is the center-to-centerdistance in �m). Activation of nAChRs triggered an intercellular calcium wave in astrocytes. Thediameter of the field shown in the images is 250 �m.


al., 2000; Tracey et al., 2001). Further, at least insome cell types, a likely mechanism for nAChR acti-vation is by paracrine or autocrine release of ACh. Inhuman airways, both basal and ciliated epithelial cellsproduce ACh as detected by HPLC measurementsfrom acutely isolated cells (Klapproth et al., 1997).This represents a possible source of ligand fornAChRs present on bronchial epithelial cells (Wanget al., 2001). Vascular endothelial cells themselvessynthesize acetylcholine suggesting a source of theligand for autocrine activation of nAChRs (Ka-washima et al., 1990). In keratinocytes, there is aspatial pattern of distribution of both choline acetyl-transferase (ChAT) and AChE. ChAT is distributedthroughout the epidermal layer while AChE is re-stricted to cells in the basal half (Nguyen et al., 2001).Low levels of ACh have also been detected in bloodcells. In the blood, ACh is present in submicromolarlevels but likely exists in specific blood cells at amuch higher concentration. The presence of ACh,ChAT mRNA, and protein, has been reported fromT-lymphocytes (Kawashima and Fujii, 2000).

ACh has been shown to be synthesized by culturedglial cells. Astrocytes cultured from neonatal mousebrain, contain significant levels of ACh (about 80pmol/106 cells) as well as activity of ChAT, suggest-ing the endogenous production of ACh in these cells(Wessler et al., 1997). In addition, a recent reportindicates that synaptically released ACh in hippocam-pal slices can activate muscarinic receptors on astro-cytes (Araque et al., 2002). Coupled with evidencethat glial cells also express nAChRs (Rogers et al.,2001; Sharma and Vijayaraghavan, 2001), there ap-pears to be a complete cholinergic signaling comple-ment in these cells.

Based on these findings, one could speculate thatcholinergic signaling in many non-neuronal cells ex-ists as a complete signaling machinery responding tolocal stimuli and resulting in local effects. Identifica-tion of stimuli for ACh release from these cells as wellas convincing demonstrations of autocrine/paracrineactivation of nAChRs by released ACh would be thenext step in validating the physiological relevance ofthese systems.

Possible Physiologic Roles for nAChRson Nonexcitable Cells

It is tempting to try to arrive at a single universalphysiologic role for nAChRs, but this is unlikely to bethe case. If, as evidence suggests, calcium is the majormediator of nAChR effects, then consequences ofnAChR activation are likely to be as diverse as thecell types in which they are expressed. Calcium sig-

naling in cells is under stringent spatial and temporalcontrol, and a whole host of factors, for example,sources of calcium, buffering mechanisms, distribu-tion of downstream signaling proteins, etc., are likelydetermining factors for the end physiologic conse-quences. Hence, consequences of nAChR activationare likely to be both cell-type and context specific. Itis, therefore, not surprising that signaling by nAChRscan have contradictory effects. For example, nAChRactivation can be protective for differentiated hip-pocampal neurons (Dajas-Bailador et al., 2000), whileit is cytotoxic to developing hippocampal progenitorcells (Berger et al., 1998). Current evidence suggeststhat nAChRs on nonexcitable cells might have roles toplay in cellular functions like cell death and cellmigration in addition to the modulation of cellularsignaling.

Development, Cell Death, and Migration. Expres-sion of nAChRs is clearly controlled during develop-ment. In vertebrate muscle, transcripts for �4, �5, �7,and �4 have been detected, and their levels are reg-ulated with embryonic age, suggesting a role fornAChRs during skeletal muscle development (Cor-riveau et al., 1995). The nAChR �7 gene is expressedin the chick tendon and periosteum during develop-ment (Romano et al., 1997). Neuronal stem cells andprogenitors express functional nAChRs ( Komourianand Quik, 1996; Berger et al., 1998; Atluri et al.,2001). Activation of nAChRs by low concentrationsof nicotine in undifferentiated hippocampal progeni-tors causes calcium-dependent apoptosis, specificallyin undifferentiated progenitor cells. This suggests apossible mechanism for eliminating excess progenitorcells during development (Berger et al., 1998). Func-tional nAChRs have been shown to be present inO2A/OPC progenitors, but the expression is lost oncethey differentiate into oligodendrocytes (Rogers et al.,2001). Interestingly, �7 expression in these cells wasrestricted to dying cells, suggesting the possibility thatthese receptors might have a similar role in the elim-ination of developing glial progenitors. Combinedwith studies on nAChRs in nonexcitable cells outsidethe nervous system, these results suggest a commonrole for nAChRs during development and differenti-ation.

nAChRs might have a role to play in the formationof new blood vessels. Once again, what we knowcomes from studies on the effects of nicotine. Nico-tine increases proliferation of vascular endothelialcells via the activation of nAChRs (Villablanca,1998). In addition, nicotine stimulates expression ofspecific genes in endothelial cells (Zhang et al.,2001a, 2001b), chief among them being the endothe-


lial nitric oxide synthase (Tonnessen et al., 2000).Recently a direct demonstration of nAChR activation-mediated angiogenesis has been demonstrated (Hee-schen et al., 2001). Although these studies have im-plicated nAChRs in atherosclerosis and in tumorpromotion (by increasing tumor vascularization), it isnot clear whether angiogenesis represents a physio-logic function for nAChRs. As endothelial cells them-selves can produce ACh (Kawashima et al., 1990),this could potentially be an autocrine mechanism forblood vessel production as well as regulation of theirtonicity.

In keratinocytes, exposure to nicotine causes areduction in cell migration. The extent of this inhibi-tion is inversely related to increases in [Ca]i

.. Resultssuggest that in these cells, calcium rise upon nAChRactivation modulates cell adhesion, detrimentally af-fecting migration and, by extension, the wound-heal-ing process. Furthermore, it has been shown thatsimultaneous activation of nAChRs and their musca-rinic counterpart (mAChRs) on keratinocytes leads tosecretion of filaggrin followed by apoptotic cell death(Nguyen et al., 2001). As keratinocytes also synthe-size and secrete ACh (Grando et al., 1993; Nguyen etal., 2001), it appears that in the human epidermis,ACh, via nAChRs, controls migration and cornifica-tion of skin cells in a autocrine/paracrine manner. Theunequal distribution of AChE in the different layers ofepidermis (see above) suggests that there is a gradientin the levels of ACh. ACh-mediated apoptotic celldeath is mainly observed in the terminally differenti-ated keratinocytes on the surface layer. Thus, it ap-pears that this gradient of ACh and perhaps the in-duction of specific nAChR subtypes in the terminallydifferentiated keratinocytes allow for specific target-ing of surface epithelial cells for elimination (Maus etal., 1998).

Recent studies have demonstrated a role for glialcells in synaptic development and function (Haydon,2001). Retinal ganglion cells grown in the absence ofastrocytes show dramatically decreased number ofsynapses as well as a large decrease in the pool offunctional synapses (Ullian et al., 2001). The presenceof nAChRs on glial cells suggests a potential role forthe receptors in formation of functional synapses.Another feature of the developing retina is the exis-tence of waves of spontaneous, correlated neural ac-tivity and calcium transients. These waves are thoughtto be required for the sculpting of synaptic connec-tions between the retina and the lateral geniculatenucleus (Shatz, 1996). Nicotinic receptor activationhas been shown to modulate the spatiotemporal pat-terns of these spontaneous waves (Feller et al., 1996;Sernagor et al., 2000). At present, there is no evidence

for glial (and by extension glial nAChR) participationin modulation of spontaneous activity in the develop-ing retina but this remains an intriguing possibility.Interestingly, mechanical stimulation of a single as-trocyte leads to a propagating calcium wave involvingboth astrocytes and Muller cells in the intact adultretina. These glial waves modulate the firing rate ofretinal neurons, presumably by release of glutamatefrom the glial cells (Newman and Zahs, 1998).

Role in Signaling. A major consequence of nAChRactivation is the modulation of cellular signaling. Thisis certainly true for neurons, and is likely to be validfor nonexcitable cells as well.

It has been postulated that nAChR activation mighthave adverse consequences on immune function (So-pori and Kozak, 1998). These receptors are expressedin peripheral blood cells where chronic nicotine ex-posure upregulates nAChR number just as it does onCNS neurons (Benhammou et al., 2000). Acute expo-sure of rats to nicotine causes a significant inhibitionin concanavalin A-induced T-cell proliferation, aneffect reversed by mecamylamine. Chronic exposureto nicotine has an additional effect in that it causesT-cell anergy, coupled with the abolishment of T-cellreceptor–mediated calcium rise in lymphocytes(Singh et al., 2000). Activation of nAChRs inhibitscytokine responses by alveolar macrophages (Mat-sunaga et al., 2001). Although endogenous regula-tion by ACh for immune cells has not been con-vincingly demonstrated, it does indicate a potentialphysiologic role for nAChRs in the control of im-mune function.

To appreciate the potential significance of nAChRsin astrocytes and other glial cells in situ, one needs tounderstand the importance of glial cells in nervoussystem function. Perisynaptic Schwann cells in theperiphery and astrocytes in the CNS are now viewedas integral modulatory elements of tripartite synapses.In the CNS, astrocytic processes are intimately asso-ciated with synapses, and a single astrocyte can en-velop many synapses. Thus, these cells are ideallysituated to integrate neuronal activity (Araque et al.,2001). The theory is that astrocytes can detect synap-tic activity and transmit this information to othersynapses in an as yet unappreciated form of bidirec-tional signaling. Three requirements need to be satis-fied for this theory to be valid: (1) astrocytes need tobe able to detect activity at synapses; (2) local signalsneed to be propagated across astrocytes; (3) astrocytesneed to be able to communicate back to neurons andthus modulate synaptic activity.

The presence of a number of neurotransmitter re-ceptors (Araque et al., 2001) and the localization of


some of these receptors to astrocytic terminals inclose proximity to synapses (Aoki, 1992) stronglysuggest that these cells are in a position to detectsynaptic activity. The presence of elaborate calciumsignaling mechanisms in astrocytes (discussed above)provides a mechanism for transmission of local infor-mation at one astrocytic terminal to the ends of otherterminals. In addition, increasing [Ca]i in astrocytescauses a large and transient increase in glutamatergicand GABAergic spontaneous synaptic currents fromadjoining hippocampal neurons in culture (Araque etal., 1998) presumably by releasing glutamate (Araqueet al., 2000) and activating presynaptic NMDA recep-tors. Taken together, these studies provide strong ev-idence for an active role for astrocytes in signaling. Inwhat might emerge as a computational nightmare forthose trying to understand neural networks, astrocyteswould play a role monitoring and integrating synapticactivity across diverse populations of synapses in thebrain. From our current understanding of the mecha-nisms involved, such an integration would occur at amuch slower time course (limited by calcium dynamicsand diffusion in the cells). The consequences of suchmodulation for network activity could be profound.

It is thus possible that activation of nAChRs onastrocytes would similarly modulate network activityin the brain. It remains to be seen whether nAChRs onastrocytes have a role to play in the demonstratedactions of nicotine on synaptic transmission.

Perhaps the most unusual potential role for glialnAChRs comes from a recent study on synaptic trans-mission in CNS neurons of the snail Lymnaea stag-nalis (Smit et al., 2001). Glial cells in the snail syn-thesize and exocytose nAChR fragments containingthe ligand binding domains, assembled in a pentamer.This ACh binding protein (AChBP) released fromglial cells is most homologous to the �7-subtype ofnAChRs. The release of AChBP is induced by pre-synaptic ACh release and activation of nAChRs onthe glial plasma membrane and depresses transmis-sion in these neurons presumably by buffering ACh inthe synaptic cleft. This would imply a scavengingfunction for the AChBP, similar to the role played bythe esterase. Another, perhaps even more interesting,interpretation of these findings is that the AChBP actsas a carrier protein allowing for long-distance diffu-sion of the transmitter. In this context binding of AChto AChBP would increase its lifetime, protecting itfrom surrounding esterases. The relevance of thisunusual finding, and whether such novel roles for glialnAChRs are more widespread in the animal kingdom,remains to be seen.

CONCLUSIONS

Work done thus far clearly demonstrates the existenceof all the essential elements of a nicotinic signalingpathway in nonexcitable cells. Activation of nAChRsin these cells might control a number of processes likecell migration, survival, and differentiation, in addi-tion to some of the cell type-specific roles described inthis review. Current research provides an interestingpicture of the evolution of an important signalingsystem—from simple autocrine forms of signaling tocomplex neurocrine mechanisms.

There are a number of reasons why neurobiologistsshould be interested in nAChRs on nonexcitable cells.The obvious reason comes from the recent recognitionthat glial cells in the brain have important roles inboth the development of neurons and integration ofsynaptic signals, and that glial cells express nAChRs.From a more general perspective, it is very likely thatmechanisms of nAChR signal transduction in nonex-citable cells share many common features with thoseoperational in neurons. Hence, many of the rolesascribed to the receptors in non-neuronal cells mightbe applicable for neurons as well. Calcium signalingproperties in these cells are likely to be valid fornAChR-mediated neuronal signaling. Issues like theimportance of calcium flux through nAChR channelsare perhaps more easily addressable in nonexcitablecells. Understanding diffusion-based mechanisms foractivation of nAChRs by ACh in nonexcitable cellswill provide insights into how receptors present onnonsynaptic locations on neurons might be activatedin the brain. For these reasons, and others, futuredevelopments in the area of nAChR signaling in non-excitable cells will likely be followed with consider-able interest by neurobiologists.

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Nicotinic receptor signaling in nonexcitable cells