Histamine activates phospholipase C in human airway epithelial cells via a phorbol ester-sensitive pathway

Histamine epithelial

activates phospholipase C in human airway cells via a phorbol ester-sensitive pathway

M. RUGOLO, F. BARZANTI, D. C. GRUENERT, AND S. HRELIA Dipartmento di Biologia Ev.Sp. and Dipartmento di Biochimica G. Moruzzi, Universitb di Bologna, 40126 Bologna, Italy; Cardiovascular Research Institute, Gene Therapy Care Center, and Department of Laboratory Medicine, University of California, San Francisco, California 94143 Rugolo, M., F. Barzanti, D. C. Gruenert, and S. Hrelia. Histamine activates phospholipase C in human airway epithelial cells via a phorbol ester-sensitive pathway. Am. J. PhysioZ. 271 (Lung CeZZ. 2MoZ. Physiol. 15): L665-L671, 1996.-In human airway epithelial cell lines SHTEo- and CFNPESo-, histamine causes a transient elevation of intracellular free calcium concentration ( [Ca2 +1;) detected by fura 2 fluorescence, which is due to both release from intracellular stores and extracellular Ca2+ entry. The effect of histamine is abolished by the Ca 2--ATPase inhibitor thapsigargin. Histamine also stimulates inositol phosphate accumulation. Changes in [Ca’+]; and inositol phosphate production exhibit a similar dose-response relationship for histamine (maximal effect at 10Y4 M), with both phenomena being blocked by the H1 antagonist mepyramine and being insensitive to pertussis toxin treatment. The effects of histamine on phosphoinositide metabolism and [Ca”-]i are abolished by a short-term preincubation with phorbol ester, and this effect is reversed by staurosporine and calphostin C, suggesting a feedback regulation by protein kinase C. The results indicate that human airway epithelial cells contain H1 receptors coupled to phospholipase C through a pertussis toxin-insensitive G protein. H1 receptors; cystic fibrosis

calcium

fluxes;

fura

2; inositol

phosphates;

IT IS WELL ESTABLISHED that several hormones, neurotransmitters, and growth factors bind to specific membrane receptors, which in turn activate phospholipase C (PLC) and stimulate phosphoinositide hydrolysis, to produce diacylglycerol and inositol trisphosphate (IP& Diacylglycerol is a well-known activator of protein kinase C (PKC), whereas IP3 releases Ca2+ from intracellular stores (1). The increase in intracellular calcium concentration ([Ca’+]i) plays a pivotal role in many intracellular events, including activation of membrane K+ and Cl- currents. In particular, the Ca2+-regulated Cl- conductance is potentially very important in airway epithelial cells from patients with cystic fibrosis (CF) (28), sirice the pharmacological amplification of Ca2+-mediated Cl- secretion has been suggested as a mean to bypass the adenosine 3’,5’-cyclic monophosphate (CAMP&dependent Cl secretory pathway defect in CF (24,27,28). Clarke et al. (8) have previously reported that histamine induced Cl- secretion in primary cultures of human nasal epithelial cells through elevation of [Ca2+];. This [Ca2-]i elevation, in turn, causes activation of basolateral K+ conductance and apical Cl conductance. Futhermore, Harris and Hanrahan (14) demonstrated that histamine stimulated a biphasic calcium response in a CF tracheal cell line, suggesting the 1040-0605/96

$5.00

Copyright

o 1996

involvement of both IP3-sensitive and IPs-insensitive intracellular Ca2+ pools. Histamine is released from mast cells and basophils, and its action is also relevant to the physiopathology of allergic responses in the airway epithelium. The broad range of biological effects of histamine is mediated by its binding to specific membrane receptors, classified into H1 (17)- and H2-type receptors (3). This classification is based on their sensitivity to different antagonists. Another class of receptors, the H3, has recently been identified and localized exclusively in the nervous system (15). Both H1- and H2-type histamine receptors have been implicated in the airway reactivity; however, the Ca2+ response has been shown to be associated with stimulation of Hi receptors (8,14). In the present study, our attention was focused on the signaling mechanism involved in the response to histamine of two human airway epithelial cell lines: SHTEo-, from tracheal epithelium of a normal individual (12), and CFNPEgo-, from nasal epithelium of a CF patient (10). We therefore investigated whether the effect of histamine was due to binding to a classical HI-type receptor, associated with PLC, leading to stimulation of IP3 accumulation and Ca2’ mobilization. We also compared the effect of histamine in both cell lines. Furthermore, it was determined whether the histamine response was modulated by PKC. MElTHODS

Materials. Histamine, cimetidine, mepyramine, sulfinpyrazone, phorbol 12-myristate 13-acetate (PMA), and pertussis toxin were from Sigma, St. Louis, MO. 4a-PMA and staurosporine were from BIOMOL Research Laboratories, Plymouth Meeting, PA. Thapsigargin and calphostin C were from Calbiochem, San Diego, CA. Fura 2-acetoxymethyl ester (AM) was from Molecular Probes, Eugene, OR. [2-3H]myoinositol was from Amersham International (Amersham, Bucks, UK). Dowex 1X8 (100-200 mesh formate form) was from Bio-Rad Laboratories, Richmond, CA. All other chemicals were of analytical grade. Cell cultures. SHTEoand CFNPESocell lines were derived from tracheal epithelium of a normal individual and from nasal epithelium of a CF patient, respectively (10, 12). Cells were grown in Dulbecco’s modified Eagle medium (DMEM) supplemented with 4 mM L-glutamine, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 10% fetal calf serum. Measurements of [Ca2+]i and Mn2+ influx. Loading with 4 uM fura 2- AM was performed in trypsinized cells (4 X lo61 ml) for 30 min at 37°C with continuous stirring in DMEM growth medium (pH 7.4) supplemented with 2 mg/ml bovine serum albumin (BSA) and 0.2 mM sulfinpyrazone. Cells were washed and resuspended in the same growth medium and left at 15-17°C until use. Before each experiment, an aliquot of 3 X 10” cell s was centrifuged and resuspended in a Ca2+-free the American

Physiological

Society

L665

L666

HISTAMINE

H1 RECEPTORS

IN HUMAN

saline solution containing (in mM) 125 NaCl, 5 KCl, 1 MgSO,, 1 KHzP04, 5.5 D-ghCOSe, 20 Na-N-Z-hydroxyethylpiperazineN’-2-ethanesulfonic acid, 1 mg/ml BSA, and 0.2 sulfinpyrazone (pH 7.4). Fura 2 fluorescence measurements were carried

out

in

a water-jacketed

cuvette

(37°C)

with

dual

wavelength

excitation

system

and

Kd

is

the

apparent

equilibrium

CELL

significant (NS)]. IP,, accumulation is expressed as percent increase over total [2-3H]myoinositol incorporation. Statistical anaZysis. Data are expressed as means t SD. Statistical analysis was performed using the Student’s t-test.

emission

RESULTS

at 510

nm. The ratio of the fluorescence signals at 340 and 380 nm (R) was measured to calculate the [Ca2+]i according to the equation ( 13 >

[Ca” *Ii = & x CR - R,,i,)/(R,,,,

EPITHELIAL

continuous

stirring. Fluorescence was monitored with a Multiscanspectrofluorimeter (AMKO-LTI) equipped with an alternating

AIRWAY

- R) x Sf380/Sb380 (1) dissociation

constant

for

of CFNPESo- cells with

Stimulation

histamine

(10~~

M) caused an immediate increase in [Ca2+]; from a resting value of 35 2 15 nM (n = 19) to a peak value of 430 t 34 nM (n = 4). This increase is followed by a

rapid decrease to 265 ? 15 nM and a subsequent slower decrease to a plateau phase characterized by a value of [Ca2+li

slightly

h’ lg h er than that

before stimulation

association of fura 2 with Ca2+ (225 nM>, Rmin and R,,, were determined in an aliquot of cells lysed with 0.1% Triton X-100, after addition of 1 mM ethylene glycol-bis(@aminoethyl ether)-N,N,N’ ,N’-tetraacetic acid (EGTA) plus 15 mM tris(hydroxymethyl)aminomethane base, pH 8.8 (R,i,) and 4 mM CaC12 (R,,,,), respectively. Sf3&Sb380 was the ratio of the fluorescence values for CaZT-free and Ca2+-bound dye measured at 380 nm. Autofluorescence values, determined in an aliquot of cells not loaded with fura 2-AM, were subtracted

indicates that, although the initial spike is primarily of intracellular origin, there may be a component that is

from

due to extracellular

all

of the

experimental

measurements

traces at both wavelengths. Mn2- influx was determined fluorescence

excitation

and

in fura 2-loaded

signal

at 360

nm,

and

calibration

cells, from the emission

at 510

nm, as described (18). Because the affinity of fura 2 for Mn2+ is very high, all Mn2f ions entering fura 2-loaded cells are trapped as fura 2-Mn2+ complexes. These complexes are nonfluorescent at all wavelengths. Thus the rate of quenching of fura 2 fluorescence at the excitation wavelength of 360 nm (isobestic point) provides an estimate of the rate of Mn2+ influx

into

cells,

irrespective

of the

magnitude

of

end

of the

recordings

by permeabilization

0.1% Triton X-100. Measurements of phosphoinositide cultures

were

harvested

of cells

turnover.

by trypsinization

and

Confluent were

with

at

5-6 x lo5 cells/well in 36-mm plastic culture dishes (Nunc, Denmark). At confluence, cells were preincubated in inositolfree medium containing [2-3H]myoinositol (1 pCi/ml) for 24 h. The labeling medium was aspirated, and cells were then rinsed and incubated with 2 ml of a buffer containing (in mM) 130 NaCl, 4.7 KCl, 1.3 CaC12, 20 NaHCOs, 0.44 NaH2P04, 1.1 MgC12, 10 D-glucose, and 10 LiCl (pH 7.4). After 30 min, histamine and/or other drugs were added, as described, and cells were stimulated for 10 min. The reaction was stopped by rinsing

the

cells

three

times

with

ice-cold

buffer,

followed

by

addition of ice-cold methanol-HCl (1OO:l vol./vol). Total inosito1 metabolites (IP + IP2 + IP3 + IP, = IP,,) were measured in the

water-soluble

phase

after

lipid

extraction

(2, 4, 5). The

aqueous phase was diluted five times with distilled water and applied to disposable columns containing 1 ml AG 1X8 resin in

the

formate

form

(Bio-Rad).

[3H]inositol

through with 20 ml of water followed glycerophosphoinositol with 10 ml of 5 mM rate plus 30 mM sodium formate. IP, were of 0.1 M formic acid plus 1.5 M ammonium (1 ml) were collected, and radioactivity

was

observed in Ca2+-containing n = 8, P < 0.001, Fig. lB),

medium (240 t 30 nM, and the subsequent slow

declining phase and the plateau were lost, This result

Ca 2+. In the cell line SHTEo-, the

addition of histamine induced similar elevations in [Ca2+]; in the presence and absence of extracellular Ca2+ (405 -+ 70 nM, n = 4, and 214 f- 35 nM, ,V = 4,

respectively; not statistically obtained in CFNPSo- cells). Thapsigargin

different

is known to inhibit

from the values the Ca2+-ATPase

associated with the endoplasmic reticulum, thereby causing irreversible depletion of intracellular Ca2+ cells incubated in Ca2+-free, EGTA-containing caused a slow elevation of [Ca2+]i that

medium reached a

maximal value of 120 _t 45 nM (n = 4) and persisted fur

cell

seeded

histamine was decreased in magnitude relative to that

stores (25). Addition of lOA7 M thapsigargin to CFNPSo-

[Ca”]i

changes that may occur simultaneously. Maximal Mn2+ quenching values (100%) were estimated in each preparation at the

(95 t 21, n = 4, Fig. 1A). In Ca2+-free medium containing 0.1 mM EGTA, the initial [Ca2+]; spike caused by

washed

by the elution of disodium tetraboeluted with 10 ml formate. Fractions was determined.

Elution volumes needed to separate the appropriate standards were determined in preliminary experiments. No significant difference was observed in [2-3H] myoinositol incorporation between basal and stimulated conditions [basal = 678,535 t 83,504 disintegrations per minute (dpm)/mg protein, n = 4; stimulated = 658,158 t 65,126 dpm/mg protein, n = 4, not

4-6 min (Fig. 1C). Subsequent addition of histamine did not affect [Ca2+]i (Fig. lC>, suggesting that thapsigargin emptied the intracellular Ca2+ stores available to histamine. When thapsigargin was added after histamine, a small but significant Ca2+ release was still detectable (Fig. ID), possibly due to intracellular stores that have been partially refilled after initial histamineinduced depletion. A similar behavior was also observed in SHTEo- cells (not shown). A lo-min incubation in the presence of lOA M thapsigargin failed to induce IP,, accumulation in SHTEo- cells (control, 5.76 ? 0.4%, n = 3, over total inositol incorporation; thapsigargin, 5.90 ? 0.3%, n = 3). In CFNPESocells, the values were control, 6.14 ? 0.3%, n = 3; thapsigargin, 6.2 t 0.25%, n = 3. It has been reported that in chromaffin cells histamine stimulates Ca2+ release from both IP,-sensitive and ryanodine-sensitive stores (23). We have therefore investigated whether the two airway cell lines have ryanodine-sensitive

contribute However,

Ca2+-release channels that could

to the histamine-induced Ca2+ response. addition of the ryanodine-receptor agonist

(10M2 M) to both airway epithelial cell lines failed to induce any [Ca2+]i elevation (not shown), caffeine

indicating

that

these

cells do not have

ryanodine

recep-

tors. In another experiment, cells were treated with rvanodine (lo+ M) for 3 min and then with low2 M

HISTAMINE

Hi RECEPTORS

IN HUMAN

AIRWAY

EPITHELIAL

L667

CELL

285 B [Ca*+li nM

1

Iz

351 I

t

HIST

1 min

4

t

t

275 D

1

1 min

HIST

1

145 1

Fig. 1. Effect of histamine and thapsigargin on intracellular calcium concentration ( [Ca2+];> in airway epithelial cells. Fura 2-loaded CFNPESo cells were incubated in Ca2+-containing saline solution (A) or in Ca2i -free saline solution containing 0.1 mM EGTA (B0). Where indicated, lop4 M histamine (HIST) and 10e7 M thapsigargin (TG) were added. In this and the following figures, [Ca2+]; in nM, is reported in the Left. Results are shown from a representative experiment.

85

AC

43-

t

t TG

;min’

HIST

t

t HIST

caffeine for 3 min to trigger the use-dependent activation of the ryanodine-sensitive channel (9). This treatment also had no effect, as the subsequent response to histamine was indistinguishable from control in both airway cell lines (not shown). To test whether the histamine-induced Ca2+ spike is dependent on Ca2+ entry from the medium, Ca2+ entry was monitored as the rate of Mn2+ quenching of the fura 2 signal at 360 nm (18). Mn2+ (lob4 M) was added to the cells either in the absence (control) or presence of histamine and thapsigargin. Figure 2 shows that exposure of the cells to 10e4 M histamine 3 min before Mn2+ addition induced a significant increase in the rate of fura 2 quenching (2.4 2 0.2-fold over basal rate, n = 4, significantly different from control, P < 0.01). A similar stimulation of quenching (2.3 2 0.3-fold, n = 4, significantly different from control, P < 0.01) was observed by addition of 10e7 M thapsigargin (Fig. 2). In both cases, this stimulation was completely prevented by the addition of La3+ (50 uM) to the incubation medium. Quenching of unstimulated cells was unaffected by La3+ (not shown). Figure 3 shows that [Ca2+]i elevation and IP, accumulation increased in parallel as functions of histamine concentration. In both cases, the maximal effect was observed at concentrations >10e4 M histamine, but a significant effect was observed at concentrations as low as 10e6 M. At 10ee4M h ist amine, the increase in IP,, accumulation was 2.49-fold over control (P < 0.001) in SHTEo- cells and 2.50-fold (P < 0.001) in CFNPEgo-. When expressed as percent of total labeled phosphoinositide, the control cells released 6.06 2 0.5% (n = 10, 9HTEo) and 6.34 t 0.36% (n = 5, CFNPESo-) of the total inositol incorporated, whereas the histaminetreated cells released 15.00 t 0.44 (n = 4, SHTEo-) and 15.35 t 0.8% (n = 8, CFNPEgo-).

‘-? 1 mln

TG

Figure 4A shows that elevation of [Ca2+]; induced by histamine was abolished by the H1 receptor-antagonist mepyramine, at concentrations as low as lop6 M. The H2 receptor-antagonist cimetidine had no effect at concentrations up to lop4 M. At the concentrations tested, mepyramine or cimetidine alone had no effect on [Ca2+]i (not shown). Figure 4B shows that IP, production by 10m4 M histamine was almost completely inhibMn*’

L 0

HIST

TG c

1 min Fig. 2. Mn2+ quench of fura 2 fluorescence in airway epithelial cells. Fura 2-loaded CFNPESocells were incubated in Ca2’-free saline solution without EGTA. Where indicated, 10h4 M HIST, lop7 M TG and/or 10e4 M MnC12 (Mn2+) were added. Left: vertical bar specifies extent of %Mn2quench, determined as described in METHODS. Fluorescence value before Mn 2+ addition was 65,000 + 1,500 (n = 8) arbitrary units. Data shown are a representative experiment. Resting and stimulated [Ca2+]i values were similar to those reported in Fig. 1, B and C.

L668

HISTAMINE

50

Hi RECEPTORS

IN HUMAN

AIRWAY

EPITHELIAL

CELL

the adenosine receptor agonist 2-chloroadenosine (22). As shown in Fig. 5, right, pertussis toxin almost completely abolished the normal cellular response (22). The effect of PMA on the Ca2+ response caused by histamine was measured over a wide range of PMA concentrations. Histamine-induced [Ca2+]i elevation was completely abolished by a 2-min preincubation with 5 x 1O-8 M PMA (F’lg. 6A). Conversely, the inactive phorbol ester 4~PMA was without effect (Fig. 6A). After a 2-min preincubation with PMA, the addition of staurosporine (2 x lop8 M), an inhibitor of PKC, completely restored the histamine-induced Ca2+ response (285 t 15 nM, n = 4, after addition of histamine alone;

-

0 -8

-7

-6

log 7

16

I

.-0 w 0

-4

-5

[HISTAMINE], I

I

-3

id I

I

K

100

5 CL E

80

ol+

60

5

c

.-c

B .-E

40

g E

20

N 0 -9

-8

-7

log 0

-8

-7

log

-6

[HISTAMINE],

-5

-4

---.I

-

-6

-5

[antagonist],

-4

-.I

M

18,

M

Fig. 3. Dose response for histamine-induced [Ca2 -1; elevation and total inositol phosphate (IP,,) production in airway epithelial cells. A: fura 2-loaded CFNPESo cells were incubated in CagT-free saline solution containing 0.1 mM EGTA. Data are expressed as absolute change of [Ca” ’ ]i (JnM) over the resting value. Each data point is mean k SD from 3 to 4 independent experiments. A, significant difference from control (P < 0.001). B: IP,, production was measured as described in METHODS. Radioactivity of IP,, fraction is expressed as %total incorporation of [2-3H]myoinositol measured in each dish. No significant difference was observed in [2-3H]myoinositol incorporation between basal and stimulated conditions (see METHODS). Results are shown as means + SD for 4 to 10 determinations. IP,, formation in absence of histamine was 6.5 f 0.3% (n = 4) of total inositol incorporation. A, Significant difference from control (P < 0.001).

i c .-0 w 2 u 0

& c

a. -

0u ‘:: .-c 0

+0 0 8?

ited by 10e6 M mepyramine, whereas cimetidine had no significant effect. Preincubation of CFNPESo-- or SHTEo- cells with pertussis toxin (400 rig/ml, 4 h) had no significant effect on the [Ca” ‘Ii elevation caused by histamine (Fig. 5). The [Ca”-1; peak values after histamine were 300 t 18 nM (n = 3) and 285 t 10 nM (n = 3, NS) in the presence and absence of pertussis toxin treatment, respectively. Furthermore, stimulation of IP, accumulation by histamine is similar in the absence or presence of pertussis toxin treatment (2.53 t O.l-fold, n = 3, and 2.75 5 O.l-fold stimulation, n = 3, respectively, not shown). As a control, the pertussis toxin treatment was tested against the rise in [Cazi ]i normally elicited by

10

HISTAM. CIMET. MEPYR.

8 6

-

+ -

+ + -

II + -+

Fig. 4. Effect of cimetidine and mepyramine on histamine-induced [Ca2 +]i change s and inositol phosphate production in airway epithelial cells. A: fura 2-loaded 9HTEo cells were incubated with 10 4 M histamine, as described in Fig. 1B. Cimetidine (0) or mepyramine (0) was added 1 min before histamine. Maximal Ca2 response (100%) was 195 ? 15 anM increase over the resting value. Data are means + SD of 3 independent experiments. A, significant difference from control (P < 0.01). B: 9HTEo cells were stimulated with 10e4 M histamine for 10 min, as described in METHODS. Where indicated, 10 m4 M cimetidine or lo- 6 M mepyramine was added 1 min before histamine. Data (means + SD of 4 independent experiments) are presented as in Fig. 3B. A, Significant difference from control (P < 0.001) . l

HISTAMINE

Hi RECEPTORS

IN HUMAN

310-

[Ca’+li nM

40 I-MT

2-CAD0 2 mln

Fig. 5. Effect of pertussis toxin on histamine-induced [Ca2+]; elevation. CFNPESocells were preincubated for 4 h with 400 rig/ml of pertussis toxin. Cells were then loaded with fura 2 as described in METHODS. Fura 2-loaded cells were incubated in Ca2’-free saline solution containing 0.1 mM EGTA. HIST, lop4 M histamine; 2-CADO, 2 x 1O-5 M 2 -c hl oroadenosine. Results shown are a representative experiment.

AIRWAY

EPITHELIAL

L669

CELL

HI-receptor antagonist mepyramine, and the ineffectiveness of the H2 antagonist, cimetidine, supports this conclusion. Recently, Yamashita et al. (29) have reported the DNA sequence of H1 receptors from bovine adrenal cells. These authors indicate that the H1-receptor gene codes for a single polypeptide chain with seven predicted transmembrane domains that includes a- sequence for G protein binding. The finding that pertussis toxin did not affect both [Ca2+]; elevation and IP, production induced by histamine in airway cell lines suggests that this G protein is pertussis toxin insensitive. This finding is supported by the results in CF/T43 cells that IP, production by histamine was not modified by pertussis toxin treatment, but IP, accumulation induced by stimulation of the Vnucleotide receptor

A

120\ ii

100

'

t

290 2 20 nM, n = 4, after PMA, staurosporine, and histamine treatment). Similarly, IP, production induced by histamine was significantly attenuated by PMA (Fig. 7). Histamine-induced IP, accumulation was only -1.3-fold over basal levels, compared with the 2.6%fold increase observed in the absence of PMA pretreatment. The addition of staurosporine after PMA preincubation completely restored histamine-induced IP,, accumulation (2.83-fold over control). Staurosporine alone had no effect on resting and stimulated levels of [Ca”+]; or on basal IP,, accumulation (not shown). Calphostin C, another inhibitor of PKC, at the concentration 100 nM also significan .tly restored histamineinduced IP,, act umul ation and Ca2+ mobili zation (not shown). DISCUSSION

The present study greatly expands previous data reporting the presence of histamine receptors in human airway epithelial cell lines (6, 8, 14) and demonstrates that binding of histamine to its receptors causes changes in [Ca2+]; that are dependent on stimulation of phosphoinositide turnover. In particular, we showed that both [Ca2+]i changes and IP, production were affected in parallel by histamine, with a maximal effect at 10e4 M. This finding is consistent with data from another human airway epithelial cell line, CF/T43, that show both stimulation of IP, accumulation (6) and Ca2+ mobilization (8, 14) by histamine. It is noteworthy that the extent of histamine-induced activation of both [Ca2’l; elevation and IP, accumulation was similar in SHTEo- and CFNPES o- cell lines, in agreement with previous data indicating that Ca2+ signaling is intact in CF epithelial cells (27, 28) and confirming previous studies on the CFNPESo- cells (10, 22). Furthermore, these responses in human airway epithelial cells to histamine appear to be mediated by binding to H1 receptors. The finding that IP, accumulation and Ca2+ mobilization were almost completely abolished by the

Et

80

E

B 2900 [Ca2+l i nM

QMA

HIS1

PMA t

STAU

HIST

I

2 min Fig. 6. Effect of phorbol 12-myristate 13-acetate (PMA), 4a-PMA, and staurosporine (STAU) on histamine-induced [Ca2+]i elevation. A: fura 2-loaded CFNPESocells were incubated with 1O---4 M histamine, as described in Fig. 1B. PMA (0) or 4tx-PMA (0) was added 2 min before histamine. Maximal Ca2+ response (100%) was 280 2 13 AnM increase over resting value. Data are means + SD of 4 experiments. A, Significant difference from control (P < 0.001). B: fura 2-loaded cells were incubated in Ca2+-free saline solution. Where indicated, lop7 M PMAwas added and followed 2 min later by 1O-4 M histamine (HIST, Left); 10e7 M PMA was added and followed 2 min later by 2 X lops M STAU and after 5 min by lop4 M HIST (right>. Results are representative of 4-6 experiments.

L670

HISTAMINE

r‘\ c

Hi RECEPTORS

IN HUMAN

20

.-0 + F 0 cl

16

.-k z0 03 .-c -0 0L Q

-E .--+ :

8 6

PMA

PMA STAU

Fig. 7. Effect of PMA and staurosporine on the histamine-induced total inositol phosphate (IP,, > accumulation. CFNPESo cells were stimulated in absence (open bars, control) and presence (hatched bars) of 10 4 M histamine for 10 min. Where indicated, 10 7 M PMA was added 2 min before histamine; 2 X 10 8 M staurosporine (STAU) was added after PMA, followed after 5 min by 1O-4 M histamine. Data are means 5 SD of 3-7 experiments. A, Significant difference from control; P < 0.001.

was reduced by 30-40% (6). In CFNPEgoand SHTEocells, we have previously reported a similar inhibition (30%) by pertussis toxin of the [Ca”+]i response induced by ATP (22). It follows that the different immortalized cell lines respond to the indicated agonists in a qualitative, reproducible manner. The data reported in Fig. lA are partially in agreement with those reported by Harris and Hanrahan (14) in the CF/T43 cell line, showing a biphasic Ca2+ response to histamine, consisting of a rapid spike followed by a second peak. Although we were unable to distinguish two distinct peaks, a biphasic behavior is clearly apparent in CFNPESoand SHTEo- cells incubated in the presence of extracellular Ca2+. However, in the absence of extracellular Ca2+, only one peak was clearly present in either cell line, in contrast to the two peaks that were detectable in CF/T43 cells (14). It is possible that single cell [Ca’+] i measurements utilized in the previous studies were more accurate than our measurements performed with populations of cells. Elevation of [Ca”+]; caused by histamine seems to be due to both release from intracellular stores and Ca2+ entry from the extracellular medium. It is well established that depletion of the intracellular Ca2’ pool acts as a signal for Ca2+ entry, as originally proposed by Putney (20) in the “capacitative Ca2+ entry model.” Strong support for this model stems from the findings that depletion of IP,-sensitive stores by thapsigargin (25) in the absence of significant IP, production is sufficient to activate the Ca2+ influx pathway (21). However, there is other evidence that suggests that the capacitative Ca2+ entry model may not be the only mechanism controlling Ca2+ entry during PLC-linked receptor activation. In several different cellular systems, it has been reported that there is a component of

AIRWAY

EPITHELIAL

CELL

receptor-mediated Ca2+- entry that does not mimic the simple depletion of intracellular Ca2+ stores (7, 9, 11). The data of Mn2+ influx reported in Fig. 2 support the capacitative model, because depletion of the intracellular Ca2+ stores by thapsigargin completely mimics H1 receptor-activated Ca2+- entry. These data, however, do not allow us to define whether thapsigargin and histamine activate the same or different Ca2+ channels. In the CF/T43 cell, it has been shown that Ca2+ influx from the extracellular medium makes a negligible contribution to overall [Ca2+]i elevation induced by histamine (14). The reason for this difference is not known, but it might be due to differences in cell type or to differences in cell culture conditions. Another possible explanation is that CF/T43 cells and those used in this study might express different types of Ca2+ channels. The lack of response to caffeine and the failure of ryanodine/caffeine treatment to reduce the [Ca2+] peak induced by histamine suggest that the calcium response in the cell lines used did not involve ryanodinesensitive Ca2+- stores. This finding is also supported by the lack of response to histamine after treatment with thapsigargin, which is thought to release Ca2+ from IP,-sensitive stores only (25). Desensitization of several receptors coupled to phosphoinositide hydrolysis is caused by short-term treatment with phorbol esters, presumably caused by activation of PKC. For the al-receptor in cultured smooth muscle cells, this desensitization appears to be associated with phosphorylation of the receptor itself (16). In cultured airway smooth muscle cells, it was demonstrated that phorbol ester inhibition of histamineinduced IP,, production is caused, in part, by a postreceptor site of action of PKC, possibly via a direct effect on phosphoinositide-specific PLC (19). Conversely, in phorbol ester-treated endothelial cells, it was shown that desensitization of IP, production after histamine stimulation is not mediated by PKC (26). In our experiments, a 2-min preincubation of cells with PMA caused total desensitization of both the histamine-stimulated IP, accumulation and elevation of [Ca”+]i. If PMA specifically activates PKC, as the lack of effect of 4~PMA would indicate, then it follows that PKC is likely to be involved in Hi histamine-receptor desensitization in airway epithelial cells. This was confirmed by the use of the PKC inhibitors staurosporine and calphostin C that were both able to reverse PMAinhibition. The fact that PMA blocks both Ca2’- release and IP,, accumulation suggests that this PKC-mediated inhibition is not confined to the Ca2+ release mechanism but involves PLC. In conclusion, in this study we have reported that the airway cell lines SHTEoand CFNPSopossess Hi histamine receptors coupled to PLC through a pertussis toxin-insensitive G protein, leading to intracellular Ca2+ mobilization. Furthermore, we have demonstrated a negative feedback effect by PKC activation on the histamine-mediated response. Finally, the histamine signaling is similar in normal and CF-derived cells. This work was supported by a grant from Progetto Finalizzato Ingegneria Genetica, Consiglio Nazionale delle Ricerche, Rome

HISTAMINE

H1 RECEPTORS

IN HUMAN

(to M, Rugolo) and by National Institutes of Health Grants DK47766, HL-41928, and DK-46002 (to D. C. Gruenert). Address for reprint requests: M. Rugolo, Dip. di Biologia E. S. Univ. di Bologna, Via Irnerio 42,40126 Bologna, Italy. Received

16 November

1995;

accepted

in final

form

26April

2.

M. J. Inositol trisphosphate Nature Land. 361: 315-3251993. Berridge, M. J., C. P. Downes, and

16. Leeb-Lundberg, Caron,

1996.

amplifies

agonist-dependent

phosphatidylinositol

brain and salivary glands. Biochem. 3. Black,

Biol.

responses

in

J. 206: 587-595, 1982. C. J. Durant, C. R. Ganellin,

J. W., W. A. M. Duncan, E. M. Parsons. Definition and antagonism of histamine H2 receptor. Nature Land. 236: 385-390,1972. Bligh, E. G., and W. J. Dyer. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37: 911-

918,1959. 5. Bordoni, A., P. L. Biagi, C. A. Rossi, and S. Hrelia. Dynamic of alfa-1-adrenoceptor mediated degradation of membrane phospholipids in cultured rat cardiomyocytes. Biochem. Biophys. Res. 6.

Commun. Brown, Harden.

198:

R. C. Boucher,

and

T. K.

Evidence that UTP and ATP regulate phospholipase C through a common extracellular 5’-nucleotide receptor in human airway epithelial cells. Mol. Pharmacol. 40: 648-655, 1991. 7. Byron, K. L., G. Babbnigg, and M. L. Villereal. Bradykinininduced CaZ+ entry, release, and refilling of intracellular Ca2+ stores. J. BioZ. Chem. 267: 108-118, 1992. 8. Clarke, L. L., A. M. Paradiso, and R. C. Boucher. Histamineinduced Cl secretion in human nasal epithelium: responses of apical and basolateral membranes. Am. J. Physiol. 263 (Cell Physiol. 32): C1190-Cll99,1992. 9. Clementi, E., H. Scheer, D. Zacchetti, C. Fasolato, T. Pozzan, and J. Meldolesi. Receptor-activated Ca2+ influx. Two independently

regulated

neurosecretory 10. Cozens, Finkbeiner, tion

mechanisms

of influx

stimulation

in

267: 2164-2172, 1992. A. L., M. J. Yezzi, L. Chin, E. L. M. Simon, W. E. J. A. Wagner, and D. C. Gruenert. Characteriza-

of immortal

cystic

fibrosis

11. Ely, J. A., C. Ambroz,

12.

coexist

PC12 cells. J. Biol. Chem.

tracheobronchial

gland

cells. Proc. Natl. Acad. Sci. USA 89: 5171-5175, A. J. Baukal,

T.

Gruenert,

D.

C.,

and

C. B. Basbaum,

M.

J. Welsh,

M.

Li,

W. E.

Characterization of human cells transformed by an origin-defective simJ. A.

Nadel.

tracheal epithelial ian virus 40. Proc. NatZ. Acad. Sci. USA 85: 5951~5955,1988. 13. Grynkiewicz, G., M. Poenie, and R. Y. Tsien. A new generation of Ca2 ’ indicators with greatly improved fluorescence properties. J. BioZ. Chem. 260: 3440-3450, 1985. 14.

Harris,

R. A.,

and

J.

W. Hanrahan.

Histamine

stimulates

a

biphasic calcium response in the human tracheal epithelial cell line CF/T43. Am. J. Physiol. 265 (CeZZ PhysioZ. 34): C781-C791, 1993.

Oxford:

G.

receptor

Pergamon,

antihistamines. and

Therapeutics,

1973,

In: International edited by M.

p. 127435.

1522-X27,1989.

19. Murray,

R. K.,

induced

Am.

C.

F. Bennet,

Mechanism

Kotlikoff. I&

formation

J. Physiol.

S. J.

Fluharty,

and

of phorbol ester inhibition in

cultured

257 (Lung

airway

CeZZ. Mol.

M.

I.

of histamine-

smooth

muscle

cells.

1): L209-L216,

Physiol.

1989. 20. Putney,

calcium entry.

21.

revisited.

22.

J. W., Jr. A model for receptor-regulated Cell Calcium 7: l-12, 1986. Putney, J. W., Jr. Capacitative calcium entry Calcium 11: 611-624,199O. Rugolo,

M.,

T. Mastrocola,

C.

Wohrle,

A.

Rasola,

CeZZ D.

C.

ATP and Ai adenosine receptor agonists mobilize intracellular calcium and activate K+ and Cl- currents in normal and cystic fibrosis airway epithelial cells. J. Biol. Chem. 268: 24779-24784, 1993. 23. Stauderman, K. A., and M. M. Murawsky. The inositol 1,4,5-trisphosphate-forming agonist histamine activates a ryanomechanism in bovine adrenal chrodine-sensitive Ca2+ release maffin cells. J. BioZ. Chem. 266: 19150-19153, 1991. 24. Stutts, M. J., T. C. Chine& S. J. Mason, J. H. Fullton, L. L. Clarke, and R. G. Boucher. Regulation of Cl- channels in normal and cystic fibrosis airway epithelial cells by exkacellular ATP. Proc. NatZ. Acad. Sci. USA 89: 1621-1625, 1992. 25. Takemura, H., A. R. Hughes, 0. Thastraup, and J. W. Gruenert,

Putney.

epithelial

Balla, and K. J. Catt. Relationship between agonist- and thapsigargin-sensitive calcium pools in adrenal glomerulosa cells. Thapsigargin-induced Ca2 ’ mobilization and entry. J. Biol. Chem. 266: 18635-18641,199l.

Finkbeiner,

M.

of adrenergic

Chem.

Schachter.

G. Romeo,

Activation

thapsigargin 12271,1989.

1992.

S. B. Christensen,

A. DeBlasi,

Regulation

18. Merrit, J. E., R. Jacob, and T. J. Hallam. Use of manganese to discriminate between calcium influx and mobilization from internal stores in stimulated human neutrophils. J. BioZ. Chem. 264:

366-371,1994.

H. A., E. R. Lazarowski,

IF., S. Cotecchia,

R. J. Lefkowitz.

262: 309%3105,1987. K. I. Histamine and Encyclopedia of Pharmacology

and

4.

L. M.

and

17. Melville,

Lithium

R. Hanley.

L671

CELL

function by phosphorylation. Agonist-promoted desensitization and phosphorylation of ai-adrenergic receptor coupled to inositol phospholipid metabolism in DDTi MF-2 smooth muscle cells. J.

and calcium signalling. M.

EPITHELIAL

15. Ishikawa, S., and N. Sperelakis. A new class (Ha) of histamine receptors on perivascular nerve terminals. Nature Lox!. 327: 158-160,1987.

REFERENCES

1. Berridge,

AIRWAY

and

of

calcium

L. J.

V. Galietta.

entry

by

the

tumor

in parotid acinar cells. J. Biol. Chem.

promoter

264:

12266-

26. Thorglirsson, G. Desensitization of inositol phosphate production after agonist stimulation of endothelial cells is not mediakd by protein kinase C. Biochem. Biophys. Res. Commun. 161: 1064-1069,1989. 27.

Wagner,

J.A.,A.

L. Cozens,

H. Schulman,

D. C. Gruenert,

L.

Activation of chloride channels in normal and cystic fibrosis airway epithelial cells by multifunctional calcium/calmodulin-dependent protein kinase. Nature Stryer,

Land.

and

P. Gardner.

349: 793-796,1991.

28. Welsh, M. J. Abnormal regulation of ion channels in cystic fibrosis epithelia. FASEB J. 4: 2718-2725, 1990. 29. Yamashita, M., H. Fukui, K. Sugama, Y. Horio, S. Ito, H. Mizuguchi, and H. Wada. Expression cloning of a cDNA encoding the bovine histamine HI receptor. Proc. NatZ. Acad. Sci. USA 88: 11515-11519,199l.