Histamine-induced Cl- secretion in human nasal epithelium: responses of apical and basolateral membranes LANE

L. CLARKE,

ANTHONY

M. PARADISO,

AND RICHARD

C. BOUCHER

Department of Medicine and Cystic Fibrosis/Pulmonary Research Laboratories, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599 Clarke, Lane L., Anthony M. Paradiso, and Richard C. Boucher. Histamine-induced Cl- secretion in human nasal epithelium: responsesof apical and basolateralmembranes.Am. J. Physiol. 263 (Cell Physiol. 32): Cll90-Cll99, 1992.-The mechanismby which receptors coupled to phospholipaseC (PLC) induce Cl- secretion in amiloride-pretreated cultures of human nasal epithelial (HNE) cultures was investigated. Histamine (lo-* M, basolateraladministration) stimulated a rapid increase in equivalent short-circuit current, an index of Clsecretion, that returned to baselinewithin 5 min. Intracellular recordings with double-barreled Cl--selective microelectrodes showed that the apical and basolateral membranepotentials rapidly hyperpolarized, the fractional resistanceof the apical membrane increased, and the transepithelial resistance decreasedin responseto histamine. Intracellular Cl- activity remainedconstant. Equivalent circuit analysis revealed that the early portion (CO.9 min) of the Cl- secretory responsewas driven by an activation of a hyperpolarizing basolateralconductance, likely K+, whereasthe later (>0.9 min) phase of Clsecretion reflects activation of the apical membraneCl- conductance. Histamine raised intracellular Ca2+ (CaP+)measured by fura- in HNE with a potency similar to that observed for induction of Cl- secretion. Both intracellular release and plasmamembraneinflux pathways were identified, typical of receptor-mediated activation of PLC. The intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid (15 PM), coupled with reducedbathing solution Ca2+, blunted the rise in CaF+and the net transepithelial Cl- secretory responseto histamine. We conclude that 1) histamine induced Cl- secretion in HNE by a sequential mechanism: the rapid initial component reflects activation of the basolateral K+ conductance,and the later component reflects activation of an apical Cl- conductance; and 2) the level of Ca,‘+may participate in the activation of both the basolateral and apical conductances. intracellular calcium; potassiumconductance; chloride conductance; amiloride; fura-2; microspectrofluorimeter; ion-selective microelectrodes MECHANISMS that regulate the rate of transepithelial Cl- secretion are complex and appear to require the coordinated activities of basolateral and apical membrane ion conductances. The sequence of activation of apical and basolateral membrane ion conductances and the second messengers that activate these conductances may vary with the class of Cl- secretagogue. Electrophysiological studies of canine tracheal and human nasal epithelium indicate that P-adrenergic agonists, coupled via P-receptors to adenylate cyclase, initiate Cl- secretion by a sequence that involves an initial activation of the apical membrane Cl- conductance, likely by adenosine 3’,5’-cyclic monophosphate (CAMP)-dependent protein kinase (3, 9, 25). In canine tracheal epithelium, activation of the apical Cl- conductance is followed by a delayed activation of a basolateral membrane K+ conductance that sustains the Cl- secretory response (25). In contrast, activation of cell-surface

THE CELLULAR

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receptors that are coupled to phospholipase C (PLC), e.g., bradykinin and carbachol, may initiate Cl- secretion by a different mode. Although no electrophysiological intracellular studies have been performed, Ussing chamber and isotope efflux studies of canine tracheal epithelium and a human colonic carcinoma cell line (T84) indicate that activation of the basolateral K+ conductance may be the primary or sole initiating event generating Cl- secretion in response to bradykinin or carbachol, respectively (6, 14-16, 24, 31). In the present study, we investigated the mechanism of Cl- secretion triggered by cell surface receptors linked to PLC in human nasal epithelium (4). Histamine was selected for the study because 1) it has recently been shown to activate PLC in human airway epithelia (21), 2) it is relevant to the pathophysiology of allergic diseases that affect nasal epithelium (lo), and 3) its physiological effects have not been reported previously in human nasal epithelium. Amiloride was used to convert the tissue from its native Na+ absorptive state to a Clsecretory state (28, 29). Double-barreled Cl--selective microelectrodes were employed to characterize the dynamic effects of histamine on individual membrane potentials and resistances. We tested for histamine linkage to PLC in these studies by measuring the response of the intracellular Ca 2+ (Ca?+) levels to histamine using the fura- microspectrofluorimetric method. Both the effects of histamine on Caf+ metabolism and the possible relationship of Caf+ levels to the activation of ionic conductances involved in the cellular Cl- secretory response were also investigated. Because of greater availability and the quantitative correspondence of their transport activities to those of freshly excised tissues (33), cultured cells were employed for both aspects of these studies. METHODS Cell Culture

Cells were obtained from excised nasal specimensfrom patients undergoing elective rhinoplasty or nasal reconstructive surgery for sleepapnea. Tissueswere obtained from 12 males and 10 females(mean age 37 t 3 yr). All procedureswere approved by the University of North Carolina Committee for the Protection of the Rights of Human Subjects.Human nasalepithelium (HNE) cellswere harvested from polyps by enzymatic digestion [protease type XIV (Sigma) for 18-24 h at 4”C] as previously described (29). For bioelectric studies, cells were plated on collagenmembranesupports (CMS) with Ham’sF-12 hormone-supplementedmedia (mixed 1:1 with 3T3 fibroblast conditioned media after day 3) and studied 4-7 days after seeding (29). For single-cellmeasurementsof Ca”+,cellswereseeded on nitrocellulose-coatedcover slips, maintained in Ham’s F-12 hormone-supplementedmedia, and studied 24-48 h after plating (32). For unilateral perfusion studies of Caf+, cells were

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plated and grown to confluency on a Transwell collagen membrane (Costar, Cambridge, MA) affixed to a plastic wafer (17). Calcium studies were made on the polarized cell preparations 4-7 days after seeding. For CAMP studies, cells were plated and grown to confluency on a Transwell collagen membrane. Solutions

and Drugs

Histamine and amiloride were obtained from Sigma (St. Louis, MO). 1,2-Bis(2-aminophenoxy)ethane-N,N,N’,N’tetraacetic acid acetoxymethyl ester (BAPTA/AM) and furaacetoxymethyl ester (fura-2/AM) were obtained from Molecular Probes (Eugene, OR). 5’-(N-ethylcarboxamido)adenosine (NECA) was obtained from Research Biochemicals (Natick, MA). A standard Krebs-Ringer bicarbonate solution (KRB) was utilized for the bioelectric studies and microelectrode studies (29). The standard NaCl Ringer solution used for singlecell measurementsof Ca”+ contained (in mM) 150 NaCl, 2.5 K2HP04, 1.5 CaCl,, 1.5 MgCl,, 5 D-glucose,and 10 2v2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES). The Ca2+-freeRinger solution was identical except that CaCl, was replaced with ethylene glycol-bis(P-aminoethyl ether)N,N,N’JV’-tetraacetic acid (EGTA; 10msM). In experiments with La3+ (0.3 X 10B3 M), HP0,2- wasreplacedby Cl- to avoid precipitation of La3+ salts.External Ca2+calibration standards were prepared by utilizing the pentapotassiumsalt of fura-2. The calibration solutions contained 20 PM fura-2, 150 mM NaCl, 10 mM HEPES, and either 5 mM CaCl, or 2 mM EGTA. All HEPES-buffered solutions were adjusted to pH 7.4 (37°C) with either N-methyl-D-glucamine base(1 M) or NaOH (1 M). In experiments using polarized monolayers of fura-2-loaded HNE, solutions containing different concentrations of CaCl, were utilized (seeRESULTS). Bioelectric

Measurements

For measurementof changesin short-circuit current (I,,) in dose-effect studies, cells grown on CMS preparations were mounted in Ussingchambersthat were bathed bilaterally with KRB and short circuited (Physiologic Instruments, San Diego, CA). The preparationswere exposedto amiloride ( 10s4M, mucosal bath), and the effects of log-varying concentrations of histamine (range 10s7to 10q4 M) on Isc added to either the serosalor mucosalbathing medium were tested. For studies designedto explore the role of CaF+in the Cl- secretory response, tissues were mounted in reduced Ca2+ KRB (see RESULTS) and loadedwith BAPTA, and the responsesto histamine (10m4M, serosal)or NECA (10m4M, apical) were compared with those of tissuesmounted in standard KRB. Electrophysiology Microelectrode

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and the Cl-selective electrode signal ( VCJ were monitored on an oscilloscope(Iwatsu SS-5802) and a four-channel recorder (Linseis TY P-7045). The double-barreledCl--selective microelectrodeswere constructed asdescribedpreviously (7). The averageslope(S) of the Cl--selective microelectrode was -51 k 1 mV per decadeincreasein Cl- concentration (isosmoticsolutions, 23”C, n = 18). The resistanceof the reference barrel immersedin Ringer solution averaged134 MQ (n = 18). The responsetimes, selectivity coefficients, and contribution of interfering ions to the intracellular Cl- measurementshave been reported previously (29). Impalementswereperformed perpendicularto the apical surface of the cell cultures, and the criteria for cell penetration have been previously describedin detail (7, 30). After mounting in the chamber,cell cultures were allowedto stabilize in the bathing solutionsfor >l5 min, and basalelectrophysiologicalparameters were measured from a series of impalements (5-10 impalements/culture). After addition of amiloride (10B4M) to the apical bath (X0 min), studies of the electrophysiological responseto histamine were performed during continuous cell impalements. After a stable cell impalement was established (i.e., referenceand Cl-selective signalschangedby lessthan t2 mV for 15-20 s), the tissue cultures were exposedto 10e4M histamine in the basolateral bathing solution. For studies exploring the effects of intracellular Ca2+chelation on the histamine response,amiloride-pretreated tissues were exposed to BAPTA/AM and/or low external Ca2+ solutions as described above, and the responseto histamine (10s4M, basolateral)was monitored during continuous impalements. The transepithelial resistance (R,) and fractional apical membraneresistance(f&J were calculated from the changein Vt and Va in responseto constant current pulses R, = AV,lI

fR, = R,I(R,

I eq = ‘JR,

Intracellular Cl- activity (a,Cl) wascalculated as acl = acl

C

X

l()(AVCl-AVref)/S

0

where a,C1is the Cl- activity of the Ringer solution (89 mM). The electrochemical driving force for Cl- flow across the apical membrane(DF,Cl)was calculated from the equation DF,C’= (RTIF)

studies. The microelectrodeand transepithelial electrical setup hasbeen describedin detail previously (29). In brief, the CMS cups were placed horizontally in a modified mini-Ussing chamber. The luminal and basolateralsurfacesof the cultures were bathed with Ringer solutions that were warmed to 37°C and gassedwith 95% 02-5% Co,. The transepithelial potential difference (V& of the tissuewasmeasuredby calomelelectrodesconnectedto the half-chambersvia 3 M KC1 agar bridges.A secondpair of 3 M KC1 agar bridgesconnected a pair of Ag-AgCl electrodesto the bath, which permitted passageof 0.5-s current pulses (I, -40 PA/cm) every 6 s via a stimulator [World Precision Instruments (WPI) model 301-T] connected to a stimulus-isolation unit (WPI model 305). The reference and ion-selective microelectrode barrels were connected to high-impedanceamplifiers (WPI modelsN-707A and FD 223, respectively). The apical membranepotential (V,), i.e., the changein the referencebarrel electrodeupon impalement,

+ RJ = AV,/AV,

where R, and Rb are the resistancesof the apical and basolateral membranes,respectively. The equivalent short-circuit current (I,,) was calculated by the equation (9)

x

ln(aP/azl) - V,

where F is the Faraday constant, R is the gasconstant, and T is the absolutetemperature (OK). Sign conventions have been chosenso that both Vt and the basolateralmembranepotential ( Vb = Va - VJ are referenced to the basolateralbath and Va is referencedto the apical bath. The DF,C’is positive when the electrochemicaldriving force for Cl- movementacrossthe apical membraneis directed out of the cell. Equivalent-circuit analysis. The electrical properties of the epithelium were representedby a standard equivalent-circuit current model (23, 27). The method for determination of shunt resistance(R,) and absoluteconductive Cl- permeability of the apical membranehas been describedpreviously (27). The premisesfor application of this technique to HNE have been reviewed and include the following: 1) in amiloride-treated tissues, the apical membrane Cl- conductance is the sole conductancein the apical cell membrane;and 2) both the Cl-

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activity and apical membranepotential can be accurately obtained with intracellular microelectrodes(7, 27).

eredto the chamberby tubing that waskept asshort aspossible (-20 cm) to minimize heat loss.Solutions were changedusing two eight-port valves (Hamilton, Reno, NV). The half time for solution exchangewasco -L . ,m,l, , 0

1

E

O-

mm

mm

l

l

in single human nasal cells Ca?+1

mm

mm

0

Table 3. Summary of changes in Caf+

mm

mm

0

2

Fig. 4. Effects of histamine on intracellular Ca2+ (Ca’+) in a single fura-2-loaded HNE cell. A: in Ca2+-containing (1.5 x 10m4 M) solution, histamine (Hist.; 10m4 M) elicited a transient increase in Ca,2+,which was followed by an elevated plateau phase. B: in Ca2+-free (10s3 M EGTA) medium, histamine caused a rapid increase in Ca’+ that returned to control level. Each trace is representative of at least 10 separate experiments (3 or more individuals).

’

nM

Condition

mmmmm

Ca2+

Ca2+ free

(3

l

2

0

0.0

0.5

1.0 Time

1.5 (min)

2.0

2.5

Fig. 3. Data derived from equivalent circuit analysis of experimental data depicted in Fig. 2. A: equivalent short-circuit current (I&. B: resistance of basolateral membrane (Rb). C: electromotive force of basolateral membrane (Eb). D: resistance of apical membrane (R,). E: permeability of apical membrane to Cl- (cl).

Caa+ (post) = 190 t 13 nM; ACaf+ = 78 t 4 nM; n = 4 (3 individuals)]. This pattern parallels the data for Clsecretory currents (Fig. 1) and indicates that the preparation is polarized. Various protocols designed to reduce extracellular Ca2+ and blunt the ACaf+ response to histamine while preserving the integrity of the epithelial monolayer were explored. As shown in Fig. 7B, the maximal reductions of Ca2+ (300 PM mucosal; 510 PM serosal) that permitted preservation of an intact epithelial sheet did not blunt the initial rise in Caz+ in response to histamine (lo- 4 M, serosal) [ Caf+ (pre) = 102 t 17 nM; Caf+ (post) = 159 t 16 nM; ACaT+ = 57 t 6 nM; n = 4 (3 individuals)]. Therefore, to chelate Ca2+ released from intracellular stores, preparations were exposed to BAPTA/AM (15 ,uM) in the presence of low external Ca2+ (Fig. 7C). This combination routinely blunted the changes in CaF+ in response to histamine (10s4 M, sero-

Basal Histamine Peak Acaf+

Steady state La3+ + histamine Peak AcaP+

107t5” (6/62) 182+8t(4/14) 77t6

138+7t

89t6”

(4/28)

150+10~(3/10) 61+4$ 87&6$

176+9$ (4/9) 75t5

Steadv state 110+7§ Values are means t SE. Numbers in parentheses are number of individuals followed by total number of measurements for a given condition. Histamine and La3+ were used at 10m4 M and 0.3 X 10m3 M, respectively. Peak refers to maximum changes of intracellular Ca2+ (Ca”+) upon histamine addition. ACaf+ is difference between peak and basal values. Steady state refers to plateau value in presence of agonist. Statistical comparisons: * significant difference in Caf+ (P c 0.05) between cells bathed with Ca2+-containing (1.5 x lo-” M) and cells bathed with Ca2+-free (10B3 M EGTA) solutions. t These values of Caf+ were significantly higher (P < 0.01) than those measured in resting cells. $ Change of Ca”+ in Ca 2+-free solution was significantly lower (P < 0.05) than values obtained in Ca2+ -containing solutions. Q Steady-state Caf+ was not significantly higher (P > 0.05) than basal Ca’+ values.

sal) [Caf+ (pre) = 57 t 8 nM; Caf+ (post) = 76 t 9 nM; ACaf+ = 18 t 4 nM; n = 4 (3 individuals)]. The effects of loading preparations with BAPTA and reducing the Ca2+ in the external bathing media (300 PM mucosal; ~10 PM serosal) on the Cl- secretory responses to histamine were tested. The combination

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I

1

2 min

Ca2+

Fig. 5. Effects of cimetidine (HZ blocker) and diphenhydramine (H, blocker) on histamine-induced change of Ca*+ in a single fura-Z-loaded HNE cell. In Ca2+-containing (1.5 x lo-” M) Ringer solution, cimetidine (Cimet.; 10m4 M), histamine (Hist.; 10e4 M), and diphenhydramine (DPA; 10e4 M) were added as shown. Trace is representative of 6 separate experiments (3 individuals).

Iv’ 100

2 min

. M-Ca2+

300 PM/S-Ca2+ I

s 10 pM

M-Hist. S-Hist. I 1

M-C#+ 300 FM/S-Ca2+

1 -8

I -7

I -6 log [Hist]

I

I

1

-5

-4

-3

(M)

Fig. 6. Dose-effect relationship describing absolute change (peak-basal) of Caf+ in fura-Z-loaded HNE cells bathed with Ca2+-containing (1.5 x lo-” M) Ringer solution. Basal Caf+ values for cells in each group varied by not more than 15%. Each data point was determined from 5 or more cells (3 or more individuals). Each point represents mean t SE.

of reduced Ca2+ in the bathing media and BAPTA decreased the magnitude of the histamine-induced AIsc in amiloride-pretreated HNE as compared with control tissues (BAPTA/reduced Ca2+ A&, = -0.8 t 0.5 vs. control A&, = 4.1 t 0.6 pA/cm2, n = 7; preparations from 3 donors; Fig. 8). In contrast, the responses to a CAMP-mediated agonist, NECA (18), were similar in BAPTA/reduced Ca2+ solutions and standard KRB, indicating that the BAPTA/reduced Ca2+ protocol did not introduce nonspecific effects on the Cl- secretory apparatus (BAPTA/reduced Ca2+ Lv,, = -4.0 t 0.6 vs. control nr,C = -5.0 +- 1.7 pA/cm2, same culture preparations as above). Rt also did not differ significantly between the control (157 t 42 Qcm2) and the BAPTA/low Ca2+ (96 t 25 Qcm2) groups.

I

L 10 pM’

+

I

,

BAPTA

1 I S-Hist. Fig. 7. Effects of histamine on Caf+ in polarized monolayers of furaloaded HNE. A: monolayer was perfused bilaterally with Krebs-Ringer bicarbonate solution containing 1.5 x 10s3 M CaCl,. Histamine (Hist.; 10m4 M) was added to mucosal (M) and serosal (S) baths as indicated. B: protocol was same as in A except that extracellular Ca2+ was reduced to levels shown before adding histamine to either mucosal or serosal baths. C: same conditions as B except the monolayer of cells was pretreated with 1,2-bis(2-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid acetoxymethyl ester (BAPTA/AM; 15 x 10B6 M; 20 min) before adding histamine to serosal bath. Each trace is representative of 4 separate experiments (3 individuals).

We subsequently performed a series of intracellular Cl--selective microelectrode studies designed to test the effects of intracellular Ca2+ buffering (BAPTA/low Cl-) on individual membrane responses to histamine. Like the Ussing chamber experiments, little change in & was detected in response to histamine in six BAPTA/low Cl-treated tissues (& = -13.2 t 5.5 PA/cm2 pre; -13.6 t 4.5 PA/cm2 during histamine). Similarly, no significant changes were detected in Vt (-2.9 t 0.9 mV pre; -2.9 t 0.9 mV during), R, (237 t 59 Qcm2 pre; 237 t 59 &cm2 during), Va (-29.3 t 4.8 mV pre; -27.3 t 4.8 mV during), Vb (-32.1 t 5.5 mV pre; -30.2 t 5.5 mV during), or fR, (0.53 t 0.07 pre; 0.53 t 0.06 during). Calculation of equivalent circuit parameters revealed no significant changes in R, (1,760 t 985 Q. cm2 pre; 1,689 t 930 fl cm2 during), Rb (1,188 t 496 Q cm2 pre; 1,090 t 355 Q cm2 during), or PC1(9.3 + 3.6 X 10e6 cm/s pre; 9.8 t 4.0 X low6 cm/s during). l

l

l

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cl q

Control BAPTA/Low

HIS

Ca2+

NECA

Fig. 8. Sequential effects of histamine (HIS; low4 M, serosal) and 5’(N-ethylcarboxamido)adenosine (NECA; 10e4 M, mucosal) on absolute change (peak-basal) of I,, in HNE cultures bathed in standard KrebsRinger bicarbonate solution containing 1.2 x 10e3 M Ca2+ (control) or in HNE cultures pretreated with BAPTA/AM (15 x lob6 M; 20 min) and bathed in reduced Ca2+ Ringer (mucosal bath: 3 x 10m4 M Ca2+; serosal bath: 4.0 X low5 M Ca2+). All cultures were pretreated with amiloride floe4 M, mucosal) before HIS and NECA addition. Each bar represents mean t SE of 7 culture preparations from 3 individuals. * P < 0.01 vs. control.

Measurement of Intracellular in Response to Histamine

CAMP Content

Intracellular CAMP concentrations were measured before and during histamine exposure. At 10 min, basal CAMP and histamine-treated CAMP levels were 15 t 4 and 10 t 3 pmol cAMP/mg protein, respectively. Furthermore, CAMP concentrations measured 1, 5, 10, and 30 s after histamine treatment were not significantly different from control. DISCUSSION

The present studies establish that histamine can regulate ion transport activities of HNE. The receptors for histamine appear to be localized exclusively on the basolatera 1 membra ne and are predom inantly of the H1 subtype. The data linking histamine recepto r activation to increases in Caf+ support the thesis that the histamine receptor is linked to a PLC activity in HNE (5, 19). Because our goal was to study mechanisms of histamine-induced Cl- secretion, the HNE preparations were pretreated with amiloride, which converts the natively Na+-absorbing HNE into a Cl--secreting tissue (28, 29). Although pretreatment of tissues with amiloride permits studies of mechanisms for induction of Cl- secretion in HNE, it should be noted that the response of HNE in the native Na+ absorptive state to this class of agonists may be surprisingly different. In studies evaluating the pattern of ion transport response in HNE consequent to activation of other cell surface receptors linked to PLC, e.g., bradykinin, we found that the major response of the native tissue is to accelerate Na+ absorption rather than induce Cl- secretion (7). Thus the effects of histamine on Na+ transport rates in HNE will require investigation.

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Cl197

The transepithelial bioelectric responses to histamine in amiloride-pretreated HNE were transient, characterized by an initial rapid increase of Cl- secretion followed by a slow relaxation toward basal values over 5 min. Analysis of the intracellular microelectrode data revealed that at the time of maximum change in Cl- secretory rates (A&), both Va and Vb had hyperpolarized. Associated with increase in Cl- secretion was a major increase in the fR,. This observation, combined with the observation that R, decreased, indicates that a conductance in the basolateral membrane is activated. Activation of a basolateral membrane K+ conductance would be consistent with the pattern of change in fR, and the hyperpolarization of the intracellular potentials. Thus the data from the microelectrode analysis of preparations at the time of maximal Cl- secretory rates indicate that the major cellular process inducing increased Cl- secretion is activation of the basolateral membrane K+ conductance. This finding is consistent with measurements of K+ efflux in studies of canine trachea and T84 cells exposed to agonists linked to PLC (6, 11, 24). This response is qualitatively different from those observed in HNE exposed to ,&agonists where the Va depolarizes and fR, decreases, indicating that activation of an apical Cl- conductance is the major cellular process activated by the CAMP-dependent pathway (3, 9). Equivalent circuit analysis of the microelectrode data permit the continuous quantitation of the responses of the individual membrane resistances and electromotive forces in response to histamine. The estimates of changes in Rb and Eb calculated by the equivalent circuit technique are consistent with the notion that the activation of a basolateral K+ conductance is the major cellular ion conductance that initiates Cl- secretion in histamineexposed HNE. Indeed, until the time of the maximal change in current, it appears to be the only conductance participating in regulating the increase in Cl- secretion. The circuit analysis does reveal, however, a contribution of the apical membrane later in the Cl- secretory response. One minute into the histamine-induced response, a decrease in the resistance in the apical membrane and an increase in e1 can be detected (Fig. 3, Table 2). This response reached a maximum ~0.3 min later. The apical membrane response occurs during a period when the basolateral membrane potential is relaxing toward the basal value and indicates that the latter portion of the Clsecretory response is sustained by an activation of the apical membrane Cl- conductance (Fig. 3, Table 2). Thus it appears that the histamine-induced Cl- secretory response can be described as an initial component in which Cl- secretion is driven by the hyperpolarizing effect of activation of the basolateral membrane K+ conductance followed by a second component that reflects an activation of the apical membrane Cl- conductance that completes the response. In whole cell clamp studies of T84 cells exposed to acetylcholine, a sequential activation of a K+ conductance followed by a Cl- conductance has recently been observed (8). It should be noted that the response of the basolateral membrane may be more complex than activation of only a K+ conductance. After the rapid hyperpolarization, Eb

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appears to be relaxing toward the basal value despite the continuous monotonic fall in Rb (Fig. 3, Table 2). This pattern has been observed with activation of other cell surface receptors that interact with PLC, e.g., bradykinin (7). We speculate that a second conductance in the basolateral membrane is activated subsequent to activation of a K+ conductance. From the orientation of the voltage shift, a candidate is a basolateral Cl- conductance. This speculation is consistent with the recent detection of a small Cl- conductance in the basolateral membrane of HNE (29). In an attempt to analyze the signaling mechanisms that participate in the sequential activation of the basolateral membrane K+ conductance and the apical membrane Cl- conductance induced by histamine, we focused on Caz+ because elevations in intracellular Caf+ have been reported to activate apical membrane Cl- conductance in human (26) and canine airway epithelia (1) as well as activating a class of HNE basolateral K+ channels (6,24). The data shown in Fig. 4 and summarized in Table 3 demonstrate that histamine induces a pattern of changes in Caf+ that is typical of interactions of agonists with receptors that are linked with PLC and inositol trisphosphate (IP,) generation (22). Activation of the receptor leads to a rapid rise in Car+ that is independent of extracellular Ca2+, indicating release from intracellular stores by an IP,-dependent mechanism (5). The sustained or plateau phase, in contrast, is dependent on extracellular Ca2+, which indicates this phase of the response reflects activation of a plasma membrane Ca2+ permeability. The data showing that the plateau phase is blocked by La 3+ (Table 3) are consistent with other studies which show that La3+ can block Ca2+ influx from the extracellular solution to cytoplasm after receptor activation. It is difficult to assign to a CaH+ signaling mechanism a definitive role for sequential activation of basolateral and apical ion conductances. The dose-effect relationships relating histamine concentrations to Cl- secretory rates (Fig. 1) and changes in CaF+ (Fig. 6) are similar: K0 5 for Cl- secretion = 3 X 10s6 M; & 5 for ACaf+ = -3 X’10w6 M. The temporal pattern of changes in & (Fig. 2) and ACaF+ (Figs. 4 and 7) in response to equimolar concentrations of histamine are also similar. However, both of these associations, i.e., similar potencies and temporal relationships, may merely indicate that both phenomena are associated with receptor activation and not necessarily causally related to one another. The absence of change in intracellular CAMP levels in response to histamine activation indicates that CAMP is not generated by a direct or indirect mechanism consequent to histamine receptor activation, and CAMP is not a signaling system for histamine in HNE. The experiments in which Caf+ levels were clamped with a combination of the intracellular Ca2+ chelator BAPTA and a lowered bathing solution Ca2+ concentration (Fig. 7, A-C) provide evidence for a more definitive linkage between Cai2+ levels and regulation of rates of Clsecretion. A concentration of BAPTA and reduced bathing solution Ca2+ was developed that significantly blunted the changes in Caf+ induced by histamine with-

CHLORIDE

SECRETION

out greatly perturbing the basal Caf+. This same treatment blunted the Cl- secretory response to histamine in Ussing chambers (Fig. 8). The observation that responses to a CAMP-mediated agonist (NECA) were similar in KRB or BAPTA/low external Ca2+ solutions provides a control for the capacity of the Caz+-clamped preparation to develop a Cl- secretory response. Microelectrode studies of BAPTA/low Ca2+-treated preparations provided evidence that buffering Caf+ inhibited both activation of the basolateral membrane K+ conductance and the apical membrane Cl- conductance (El). Thus it would appear that Ca,“+ may be a signal that can coordinate both the initial activation of the basolatera1 K+ conductance, perhaps by direct effect of the Caz+ levels, and the subsequent activation of the apical membrane Cl- conductance. The temporal dissociation between activation of the basolateral membranes and activation of the apical membrane (-12-18 s) exceeds the time for Ca 2+ to diffuse from stores at the basolateral membrane to the apical membranes (estimated at -10 pm/s; airway epithelia -30 pm) (12). Thus interaction of Ca”+ with other biochemical paths is likely. Based on the work of Gardner and co-workers (20), interaction with Ca2+-calmodulin-dependent kinases may be one candidate pathway. In summary, HNE pretreated with amiloride exhibits a secretory Cl- response in response to histamine administration to the basolateral barrier. The interaction of the receptor appears to activate initially a basolateral K+ conductance that via cellular hyperpolarization increases the electrochemical driving force for Cl- exit across the apical membrane. Subsequently, as the basolateral membrane depolarizes, a primary activation of the apical Clconductance sustains the Cl- secretory response. Changes in Cai 2+levels triggered by the interaction of the receptor with the ligand may be a candidate second messenger for activating both conductances in a coordinated fashion. We thank Teresa Mace, Kelly Waicus, Elaine Cheng, and Angela Burnette for expert technical assistance and Lisa Brown and Aluoch Ooro for editorial assistance. This work was supported by National Heart, Lung, and Blood Institute Grants HL-34322 and HL-42384 and by Cystic Fibrosis Foundation Grant ROlS. Address for reprint requests: L. L. Clarke, Div. of Pulmonary Diseases, CB 7020,724 Burnett-Womack Bldg., Univ. of North Carolina at Chapel Hill, Chapel Hill, NC 27599. Received 4 December 1991; accepted in final form 29 June 1992. REFERENCES Al-Bazzaz, F. J., and T. Jayaram. Ion transport by canine tracheal mucosa: effect of elevation of cellular calcium. Exp. Lung Res. 2: 121-130, 1981. Boucher, R. C., E. H. M. J. Stutts, M. R. Knowles,

C. Cheng, A. and H. S. Earp.

M.

Paradiso,

Chloride secretory response of cystic fibrosis human airway epithelia: preservation of calcium but not protein kinase C- and A-dependent mechanisms. J. Clin. Invest. 84: 1424-1431, 1989. Boucher, R. C., C. U. Cotton, J. T. Gatzy, M. R. Knowles, and J. R. Yankaskas. Evidence for reduced Cl- and increased Na+ permeability in cystic fibrosis human primary cell cultures. J. Physiol. Lond. 405: 77-103, 1988. Boyer, J. L., J. R. Hepler, and K. T. Harden. Hormone and

growth factor receptor-mediated regulation activity. Trends PhurmacoZ. Sci. 10: 360-364,

of phospholipase 1989.

Downloaded from www.physiology.org/journal/ajpcell at Univ of Texas Dallas (129.110.242.050) on February 13, 2019.

C

HISTAMINE-INDUCED Brown, H. A., E. R. Lazarowski, R. C. Boucher, and T. K. Harden. Evidence that UTP and ATP regulate phospholipase C through a common extracellular Y-nucleotide receptor in human airway epithelial cells. 1Mol. PharmacoL. 40: 648-655, 1991. 6. Clancy, J. P., J. D. McCann, M. Li, and M. J. Welsh. Calcium-dependent regulation of airway epithelial chloride channels. Am. J. Physiol. 258 (Lung Cell. Mol. Physiol. 2): L25-L32, 1990. 7. Clarke, L. L., A. M. Paradiso, S. J. Mason, and R. C. Boucher. Effects of bradykinin on Na+ and Cl- transport in human nasal epithelium. Am. J. Physiol. 262 (Cell Physiol. 31):

CHLORIDE

C644-C655,

1992.

C1224-C1230, 12.

13.

14.

15. 16.

17.

131, 1990.

Lazarowski, E. R., S. J. Mason, L. Clarke, T. K. Harden, and R. C. Boucher. Characterization of adenosine receptors and relationship to chloride secretion in human airway epithelia. Br. J. Phurmucol. 106: 774-782, 1992. 19. Nakahata, N., and T. K. Harden. Regulation of inositol trisphosphate accumulation by muscarinic cholinergic and H1-hista18.

344, 1987.

20. Nishimoto, I., J. A. Wagner, H. Schulman, and P. Gardner. Regulation of Cl- channels by multifunctional CaM kinase. Neuron 6: 547-555, 1991. 21. Paradiso, A. M., E. H. C. Cheng, and R. C. Boucher. Effects of bradykinin on intracellular calcium regulation in human ciliated airway epithelium. Am. J. Physiol. 261 (Lung Cell. Mol. Physiol. 5): L63-L69, 1991. 22. Putney, J. W., Jr., H. Takemura, A. R. Hughes, D. A. Horstman, and 0. Thastrup. How do inositol phosphates regulate calcium signaling? FASEB J. 3: 1899-1905, 1989. 23. Schultz, S. G. Application of equivalent electrical circuit models to study of sodium transport across epithelial tissues. Federation Proc. 38: 2024-2029, 1979. 24. Smith, J. J., J. D. McCann, and M. J. Welsh. Bradykinin stimulates airway epithelial Cl- secretion via two second messenger pathways. Am. J. Physiol. 258 (Lung Cell. Mol. Physiol. 2): L369L377, 1990. 25. Welsh, M. J., P. L. Smith, and R. A. Frizzell. Chloride secretion by canine tracheal epithelium: the cellular electrical potential profile. J. Membr. Biol. 70: 227-238, 1982. 26. Willumsen, N. J., and R. C. Boucher. Activation of an apical Cl- conductance by Ca*+ ionophores in cystic fibrosis airway epithelia. Am. J. Physiol. 256 (Cell Physiol. 25): C226-C233,

1989.

Dissing, S., B. Nauntofte, and 0. Sten-Knudsen. Spatial distribution of intracellular, free Ca*+ in isolated rat parotid acini. Pfluegers Arch. 417: 1-12, 1990. Grynkiewicz, G., M. Poenie, and R. Y. Tsien. A new generation of Ca indicators with greatly improved fluorescence properties. J. Biol. Chem. 260: 3440-3450, 1985. Kunzelmann, K., H. Pavenstaedt, C. Beck, 0. Uenal, P. Emmrich, H. J. Arndt, and R. Greger. Characterization of potassium channels in respiratory cells. I. General properties. Pfluegers Arch. 414: 291-296, 1989. Kunzelmann, K., H. Pavenstaedt, and R. Greger. Characterization of potassium channels in respiratory cells. II. Inhibitors and regulation. Pfluegers Arch. 414: 297-303, 1989. Kunzelmann, K., H. Pavenstaedt, and R. Greger. Properties and regulation of chloride channels in cystic fibrosis and normal airway cells. Pfluegers Arch. 415: 172-182, 1989. Larsen, E. H., I. Novak, and P. S. Pedersen. Cation transport by sweat ducts in primary culture. Ionic mechanism of cholinergically evoked current oscillations. J. Physiol. Lond. 424: 109-

Cl199

mine receptors on human astrocytoma cells. Biochem. J. 241: 337-

5.

8. Cliff, W. H., and R. A. Frizzell. Separate Cl- conductances epithelial cells. activated by CAMP and Ca*+ in Cl-secreting Proc. Nutl. Ad. Sci. USA 87: 4956-4960, 1990. 9. Cotton, C. U., M. J. Stutts, M. R. Knowles, J. T. Gatzy, and R. C. Boucher. Abnormal apical cell membrane in cystic fibrosis respiratory epithelium. An in vitro electrophysiologic analysis. J. Clin. Invest. 79: 80-85, 1987. 10. Davies, R. J., and J. L. Devalia. Histamine levels in nasal secretions: effect of methacholine and allergen. In: Allergic and Vasomotor Rhinitis: Puthophysiologicul Aspects, edited by N. Mygind and U. Pipkorn. Copenhagen: Munksgaard, 1987, p. 179-188. 11. Dharmsathaphorn, K., J. Cohn, and G. Beuerlein. Multiple calcium-mediated effector mechanisms regulate chloride secretory responses in T84 cells. Am. J. Physiol. 256 (Cell Physiol. 25):

SECRETION

1989.

Willumsen, N. J., and R. C. Boucher. Shunt resistance and ion permeabilities in normal and cystic fibrosis airway epithelium. Am. J. Physiol. 256 (Cell Physiol. 25): C1054-C1063, 1989. 28. Willumsen, N. J., and R. C. Boucher. Sodium transport and intracellular sodium activity in cultured human nasal epithelium. Am. J. Physiol. 261 (Cell Physiol. 30): C319-C331, 1991. 29. Willumsen, N. J., C. W. Davis, and R. C. Boucher. Intracellular Cl- activity and cellular Cl- pathways in cultured human airway epithelium. Am. J. Physiol. 256 (Cell Physiol. 25): C103327.

C1044,

1989.

30. Willumsen, N. J., C. W. Davis, and R. C. Boucher. Cellular Cl- transport in cultured cystic fibrosis airway epithelium. Am. J. Physiol. 256 (Cell Physiol. 25): C1045-C1053, 1989. 31. Wong, S. M. E., R. P. Lindeman, S. Parangi, and H. S. Chase, Jr. Role of calcium in mediating action of carbachol in T84 cells. Am. J. Physiol. 257 (Cell Physiol. 26): C976-C985, 1989.

32. Wu, R., J. Yankaskas, E. Cheng, M. R. Knowles, and R. Boucher. Growth and differentiation of human nasal epithelial cells in culture. Serum-free, hormone-supplemented medium and proteoglycan synthesis. Am. Rev. Respir. Dis. 132: 311-320, 1985.

33. Yankaskas, J. R., C. U. Cotton, M. R. Knowles, J. T. Gatzy, and R. C. Boucher. Culture of human nasal epithelial cells on collagen matrix supports. Am. Rev. Respir. Dis. 132: 1281-1287,

1985.

Downloaded from www.physiology.org/journal/ajpcell at Univ of Texas Dallas (129.110.242.050) on February 13, 2019.