Volume 12 number 6 1993, 539-551

Current Eye Research

Carbachol-induced increase of Na+/H antiport and recruitment of Na+,K-ATPase in rabbit lacrimal acini

Ross W.Lambert, Carol A.Maves and Austin K.Mircheff

Departments of Physiology and Biophysics, and Ophthalmology, University of Southern California School of Medicine, Los Angeles, CA 90033, USA

ABSTRACT Parallel arrays of Na'/H' and CI-/HC03- antiporters are believed to catalyze the first step of transepithelial electrolyte secretion in lacrimal glands by coupling Na' and C1- influxes across acinar cell basolateral membranes. Tracer uptake methods were used to confirm the presence of Na'/H' antiport activity in membrane vesicles isolated from rabbit lacrimal gland fragments. Outwardly-directed H' gradients accelerated "Na' uptake, and amiloride inhibited 96% of the Ht gradient-dependent "Na' flux. Amiloride-sensitive TINa' influx was half-maximal at an extravesicular Na' concentration of 14 mM. Zn vitro stimulation of isolated lacrimal acini with 10 pM carbachol for 30 min increased Na'/H' antiport activity of a subsequently isolated basolateral membrane sample 2.5- fold, but it did not significantly affect Na'/H+ antiport activity measured in intracellular membrane samples. The same treatment increased basolateral membrane Na',K'- ATPase activity 1.4-fold; this increase could be accounted for by decreases in the Na',K'-ATPase activities of intracellular membranes. Thus, it appears that cholinergic stimulation causes recruitment of additional Na',K'- ATPase pump units to the acinar cell basolateral plasma membrane. The mechanistic basis of the increase in basolateral membrane Nat/H' antiport activity remains unclear.

INTRODUCTION

The lacrimal glands produce an approximately isotonic

fluid which represents the major component of the

aqueous layer of the precorneal tear film. This fluid is

essential for the health of the ocular surface and the

quality of the visual image, and insufficient lacrimal fluid

production is a major cause of painful and vision-

threatening dry eye conditions. The lacrimal glands are

typical tubulo-acinar exocrine glands. The acini secrete an

isotonic, NaC1-rich fluid, while the ducts secrete a KCl-rich

fluid. Fluid secretion in both segments is believed to be

the osmotic consequence of transepithelial electrolyte

secretion. Ductal mechanisms have received little

attention, but specific transport processes underlying

acinar electrolyte secretion have been addressed in studies

using intact cells and subcellular membrane preparations

isolated from rat and mouse exorbital lacrimal glands.

The mechanisms elucidated to date are consistent with the

general principles of the model for epithelial electrolyte

secretion proposed by SiIva et af. (1). As has been

reviewed elsewhere (2), C1- channels are expressed in lacrimal acinar cells and are believed to be present in the

apical membranes. Na',K'-ATPase pumps, K' channels,

Na'/H' antiporters, and Cl-/HCO,- antiporters are

expressed in the basolateral plasma membranes. It has

been proposed that the antiporter array drives the first

step of transcellular C1- secretion by using the energy of

the transmembrane Na' electrochemical potential

gradient, established and maintained by Na',K'-ATPase,

to accumulate C1- at an intracellular electrochemical

potential greater than in either the interstitium or the

luminal fluid (3).

Cholinergic stimulation is known to trigger several

events in rodent lacrimal acinar cells. These include

opening of the apical C1- channels and basolateral K'

channels, acceleration of Na' influx (3, 4), cytoplasmic

alkalinization (9, exocytic release of secretory products,

retrieval and recycling of secretory vesicle membrane

constituents (6, 7), acceleration of basolateral membrane

recycling traffic (7), and net translocation of Na+,K'-

Received on December 22, 1992; accepted on May 14, 1993

0 Oxford University Press 539

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ATPase pump units from intracellular pools to the

basolateral membranes (8, 9). Amiloride blocks both

alkalinization of the cytoplasmic and acceleration of Na'

influx (3 - 5) , suggesting that both phenomena result from

an increase in plasma membrane Nat/Ht antiport activity.

This conclusion has remained provisional, however,

because amiloride is known to also inhibit the binding of

ligands to exocrine acinar cell muscarinic cholinergic

receptors (3, 10).

The present study was undertaken on the premise

that if basolateral membrane Na'M' antiport activity

increases in response to cholinergic stimulation, it should

be possible to detect such an increase in plasma

membrane vesicles isolated from lacrimal acini which have

been stimulated in vitro. Because the absolute amounts

of membrane material that can be isolated from rat

exorbital gland acinar preparations are too small for such

an experiment, we have used two new experimental

preparations, fragments and isolated acini from the rabbit

lacrimal gland (11). Rabbit acinar preparations have

recently been shown to retain the ability to respond to

cholinergic stimulation by releasing protein and

accelerating Na' influx ( l l ) , apparently in the context of

an overall acceleration of recycling traffic between the

basolateral plasma membrane and endocytic

compartments (12). However, despite the greater size of

the rabbit lacrimal gland and better yield of viable acini,

the amounts of material in rabbit lacrimal acinar

preparations are still relatively limited. Therefore, we first

used fragment preparations to obtain membrane samples

for experiments confirming that Nat/H' antiporters with

typical kinetic characteristics are present in the rabbit

lacrimal gland. We then used isolated acini to measure

the effects of stimulation on the subcellular distribution of

Nat/Ht antiport activity. These analyses confirm that

basolateral membrane-associated Na'lH' antiporter

activity increases following cholinergic stimulation. The

same analyses show that, as in rat lacrimal acinar cells,

~ ~ ~~ ~ ~

stimulation also causes additional Na',Kt-ATPase pumps

to be recruited to the basolateral membranes.

MATERIALS AND METHODS

Materials

N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid

(HEPES), 2[N-morpholino]ethanesulfonic acid (MES),

carbamylcholine chloride (carbachol), amiloride, and

valinomycin were from Sigma (St. Louis, MO). Ham's F-

12 medium was from Irvine Scientific (Irvine, CA).

nNaCl was from Amersham (Arlington Heights, IL). Nitrocellulose filters were from Schleicher and Schuell

(Keene, NH). Filtron X was from National Diagnostics

(Highland Park, NJ). All other chemicals were reagent

grade and were obtained from standard suppliers.

Premration of lacrimal tissue

Subcellular fractionation analyses performed with the goal

of isolating large membrane samples were done with

lacrimal gland fragment preparations. Analyses

performed with the goal of characterizing the effects of

stimulation on Nat/Kt-ATPase and Na'/H' antiport

subcellular distributions were done with isolated lacrimal

acini. Male New Zealand white rabbits (2.0 - 2.5 kg, Irish

Farms, Norco, CA) were used in all experiments, which

were performed in accord with the Guiding Principles in

the Care and Use of Animals. Fragment preparations

were obtained from lacrimal glands of 6 rabbits, while

glands from 12 rabbits were required for each acinar

preparation. Rabbits were anesthetized with

intramuscular injections of 40 mgkg ketamine and 10

mgkg xylazine and were sacrificed by an overdose of Na-

pentobarbitol (60 mgkg).

Lacrimal acini were isolated as described by Bradley

et al. (11). These were washed, suspended in Ham's

medium (pH 7.6), and placed in a 250 ml polypropylene

Erlen-Meyer flask in a shaking (100 osclmin) water bath

at 37°C for 30 min. Viability of cells in intact acini as

assessed by Trypan Blue exclusion after 5 min exposure

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was consistently greater than 90%. After equilibration in

Ham’s medium, acini were collected by centrifugation,

washed once, resuspended in Ham’s medium, and divided

into two equal samples. These were incubated for an

additional 30 min in Ham’s medium with or without 10

pM carbachol. Acini were then pelleted and washed once

with fresh 37” Ham’s medium with and without carbachol.

Each pellet was suspended in 6 ml ice-cold isolation

buffer (8, 13 - 15). All remaining steps were performed at

4”.

Subcellular fractionation of eland fragments and intact

- acini

Fragments were preincubated for 45 min in a modified

Krebs-Ringer bicarbonate solution (8, 9) constantly gassed

with 95% 0, / 5% CO,. Subsequent homogenization in

isolation buffer and separations by differential

sedimentation and density gradient centrifugation in a

Beckman 2-60 zonal rotor were performed exactly as

described previously for analyses of rat exorbital lacrimal

gland fragments (8, 13 - 15). Swinging bucket rotors were

used for triplicate analyses of resting and carbachol-

treated acinar samples according to the methods described

by Bradley et al. (11). In both zonal rotor and swinging

bucket procedures, membranes were harvested from the

density gradient fractions by dilution and high-speed

centrifugation.

Tracer uutake

Nat/Ht antiport activity was assessed by the rapid

filtration methods used in previous studies (16).

Membrane fractions were rapidly thawed, and samples

were diluted with loading buffer, which contained, in mM: sorbitol, 200; K-gluconate, 50; Mg-gluconate, 1; MES, 30;

Tris, 5; and HEPES, 10; pH 6.0. Membranes were

pelleted at 250,000 g for 75 min, suspended in loading

buffer using a 25 gauge syringe, and incubated at 23°C for

60 minutes. Valinomycin (5 pg/ml) was added to all

membrane vesicle suspensions in order to minimize the

effects of outwardly-directed Ht gradients on the

transmembrane voltages. Uptake buffer at pH 7.5 was

composed of (in mM): sorbitol, 200, Na-gluconate, 1; K- gluconate, 50; Mg-gluconate, 1; Tris, 20; HEPES, 20; and

MES, 5; pH 7.5. Uptake buffer at pH 6.0 was identical to

the loading buffer except for the addition of 1 mM Na- gluconate. 2Nat was used at a concentration of 13

pCi/ml. Uptake reactions were performed at room

temperature and were initiated by mixing 5 p1 vesicle

suspension with 20 p1 uptake buffer. Tracer uptake was

terminated by dilution with 1 ml ice-cold stop solution

(uptake buffer without tracer). Vesicles were rapidly

collected on 0.45 pm nitrocellulose filters, which were then

rinsed twice with 4 ml aliquots of stop solution. Filters

were dissolved in 5 ml Filtron X, and radioactivity was

counted in a Beckman LS8000 liquid scintillation counter.

Analytical methods The Kt-dependent p-nitrophenylphosphatase reaction of

Nat,Kt-ATPase was measured as described previously

(17). The density gradient distribution of K+-pNPPase

activity closely parallels that of ouabain-sensitive, (Nat + Kt)-dependent ATP hydrolysis (14). GalactosyltransferM

was determined as described by Bradley ef al. (15).

Protein in subcellular fractions was determined with the

BioRad assay kit (BioRad, Richmond, CA). Other

biochemical markers were determined as described

previously (15, 17). Marker cumulative enrichment factors

were calculated by dividing the percent recovered marker

activity in a fraction by the percent recovered protein in

that fraction.

When the marker enzyme and Nat/Ht antiport

activities of resting and carbachol-stimulated samples were

to be compared, the activity measured in each fraction

was normalized to the total amount of protein recovered

in the pelleted density gradient fractions. This calculation

made it possible to compile results from replicate

experiments in which acinar yields differed. It also makes

it possible to evaluate whether observed changes resulted

from redistributions between subcellular compartments or

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from selective activation or inhibition. Statistical analyses

were performed using Student's T-test for paired samples.

Results were considered significant when P-values were

membrane constituents, and in earlier studies it was

assumed to have been derived from the basolateral

membrane itself (13, 16).

less than 0.05. Figure 1 presents the density gradient distributions of

RESULTS Subcellular fractionation analvsis of lacrimal eland

fraements

As has been reviewed recently (14), density gradient

analyses of lacrimal acinar cell preparations appear to

separate three types of membrane sample that contain

Na+,K+-ATPase and other basolateral membrane

constituents: A sample believed to be derived from the

basolateral membrane, a . mixture of samples believed to

be derived from endosomal compartments, and a series of

Golgi-derived samples. The presumptive endosomal

membrane samples represent a major pool of basolateral

several marker enzyme activities from a rabbit lacrimal

gland fragment preparation. The major features,

including the peaks of Naf,Kt-ATPase in the regions of

the density gradient designated density windows I , I& and

N and the overlap between Nat,Kt-ATPase and

galactosyltransferase in windows II through V, are similar

to features noted in previous analyses of rat exorbital

lacrimal gland fragments (8, 13 - IS), rat lacrimal acini (9,

18), and rabbit lacrimal acini (1 1). There are, however,

several quantitative differences between the marker

distribution patterns from rabbit fragment and acinar

preparations. These result, in part, from differences in

12

c .- >

a U

Q) > 0

.- z 4 L O

4

0

I II Ill IV v VI 1 1 I I I I I]

20

10

0

I ' 1 I I I

n

5 10 15 20

12

0

4

0

Density window

I II 111 IV v VI

Acid phosphatase I 1 I I I I - 5 1 0 15 2 0

I II Ill IV v VI I ' ' T 1 1 I I ]

Galactosyltransferase

A 5 10 15 20

Fraction

Figure 1. Subcellular fractionation analysis of lacrimal gland fragment preparation. Density distributions of marker enzyme activities and protein, assayed in duplicate after density gradient centrifugation in a 2-60 zonal rotor. Values presented are percentages of the total activities recovered in the pellets generated by high-speed centrifugation of the density gradient fractions. Similar

542

0 4u3 0 5 10 15 20

I I1 111 IV v V I I 1 I 1 I I 1

Protein 12

4 LA 5 10 15 20

0

distribution patterns, with minor differences in the positions of some of the sharper peaks, were noted in analysis of a second preparation. Density gradient fractions were pooled into 6 density windows on the basis of salient features of the marker distribution patterns prior to assays for NADPH-cytochrome c reductase and Nat/Ht antiport activity.

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the resolution achieved with zonal and swinging bucket

rotors and, in part, from changes in subcellular

organization which occur during isolation and in vitro

incubation of the acini (18). There is also a noteworthy

difference between rabbit and rat preparations: In rat

lacrimal gland fragments and acini, Nat,Kt-ATPase

activity is divided almost equally between the peaks

centered in windows N and W, while in rabbit lacrimal

gland the peak in window N accounts for a much smaller

fraction of the total Nat,Kt-ATPase.

Density window I, which contains the basolateral

membrane sample, contained the least protein of any

window and was characterized by the largest enrichment

factors for the typical plasma membrane markers, Nat,Kt-

ATPase (9.0) and alkaline phosphatase (10.0). It also

contained a clearly defined peak of acid phosphatase

activity while having the lowest enrichment factors for

succinate dehydrogenase and NADPH cytochrome c

reductase. Its enrichment for galactosyltransferase was

1.9. Density window 11, which contains a mixture of

endosomal membrane samples and, perhaps, also an

apical membrane sample, contained nearly 4-times more

protein than density window I ; its Na+,K+-ATPase, alkaline

phosphatase, and galactosyltransferase enrichment factors

were 7.8, 8.9 and 3.7, respectively. Density windows IIZ

through V contained most of the Golgi-derived membrane

samples, marked by galactosyltransferase. These were

overlapped by endoplasmic reticulum membranes, marked

by NADPH-cytochrome c reductase, and mitochondria,

marked by succinate dehydrogenase (not shown).

Nat/H+ antiporter activitv in isolated membrane vesicles

Figure 2 depicts the time-courses of '*Na+ uptake by a

density window II sample in the presence and absence of

an outwardly directed H+ gradient. As seen in Figure 24,

"Nat uptake was linear for at least 5 sec, and the

outwardly-directed Ht gradient increased the initial rate

more than 5-fold. Amiloride at a concentration of 1 mM

inhibited 96% of the pH gradient-dependent Na+ influx.

Steady-state levels of nNat uptake were similar fbr

vesicles that had been incubated in the presence and

absence of Ht gradients (Figure 2B). Since all transport

experiments were performed in the presence of the Kt

ionophore, valinomycin, and 50 mEqL Kt in the intra-

and extravesicular media, it is unlikely that Ht diffusion

potentials could have contributed enough to the trans-

membrane voltage difference to drive significant w a t

influx via conductance pathways. Therefore, the results

A 2.0 1 A

0 ' 0 1 2 3 4 5

Time (sec)

5.0

.E 4.0

3.0

B

- 3 2

= c s

m

+m ; 2.0 z - 1.0

0 0 30 60 90 120

Time ( m i d

Figure 2. A. Effect of amiloride on the initial rates of Ht gradient-dependent and -independent Nat uptake. Values are means & S.D. of triplicate determinations. Uptake reactions shorter than 10 seconds were timed with a metronome. Membranes from density window II (Figure 1) were prepared for transport as described in MATERIALS AND METHODS. The uptake reactions were initiated by mixing 5 p1 vesicles (pH, = 6.0, 0 Nat) with 20 pl uptake buffer. Closed circles, pHo = 7.5; closed triangles, pHo = 7.5, plus 1 mM amiloride; open circles, pHo = 6.0; open triangle, pH, = 6.0, plus 1 mM amiloride. B. Effect of an outwardly-directed Ht gradient on the time course of zNa+ uptake into isolated membrane vesicles. Open circles, pH, = 6.0; closed circles, pHo = 7.5. Similar results were obtained with a second density window II preparation.

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summarized in Figure 2 indicate the presence of Na+/H'

antiporters in the isolated membrane sample.

The measurements summarized in Figure 3A indicate

that the amiloride-sensitive component of Na' uptake

became saturated at a sodium concentration of

approximately 50 mM. The Lineweaver-Burk

transformation in Figure 3B yields a K, of approximately

14 mM and a maximal velocity (J-) of 120 nmol/mg

protein min.

Effects of carbachol on Na+/H' antiport and Na+.K+-

ATPase

Figures 4 and 5 present the density gradient distributions

of enzymatic marker and Na+/HC antiport activities from

resting and stimulated acini. As summarized in Table 1,

stimulation had no significant effect on the manner in

which marker enzymes were distributed among the

fractions defined by differential sedimentation. However,

it significantly ( P c 0.05) decreased the fraction of the

recovered protein present in the initial low-speed pellet,

Po, and significantly increased the fraction of the

recovered protein present in XP, the series of pellets

obtained by high-speed sedimentation of the density

gradient fractions. Altogether, stimulated preparations

contained 18% & 4% (P < 0.05) less total protein than

resting preparations. This loss can likely be attributed to

the stimulation of protein secretion during the 30 min in

vifro incubation (11). If intact secretory vesicles were

preferentially enriched in Po, secretory protein release and

translocation of retrieved secretory membrane constituents

to the Golgi complex could plausibly account for the

observed change in the distribution of the remaining

protein. Release of secretory proteins from the cell

probably also accounts for the slight increases in marker

enzyme specific activities observed after stimulation.

As reported previously ( l l ) , density window I samples

were characterized by the largest cumulative enrichment

factors for Na',K+-ATPase and alkaline phosphatase (18.9

zr: 4.1 and 16.1 2 2.4, respectively, from resting

preparations), but these enrichment factors were larger

A 1601

N d concentration (mEq/l)

-0.2 -- 0 0.2 0.4 0.6 0.8 1.0

1 I N $

Figure 3. A. Dependence of nNat influx on external Na' concentration. Membrane vesicles were from density window II of a zonal rotor analysis, with pHi = 6.0. Initial rates of Na' uptake from a medium of pH = 7.5 were calculated from triplicate measurements at 2 and 5 sec. Influx was measured in the presence (closed circles) and absence (open circles) of 1 mM amiloride. Uptake buffers contained concentrations of Na'-gluconate from 1 to 50 mM and were constructed by replacing 100 mM sorbitol in the loading buffer described in MATERIALS AND METHODS with Na-gluconate and TMA-gluconate. Values are means 2 S.D. B. Lineweaver-Burk transformation of antiporter-mediated Na' influxes estimated from differences between total influxes and amiloride-insensitive influxes in Figure 3A.

than those noted for the corresponding samples from

fragment preparations. Cholinergic stimulation increased

the Na',K'-ATPase activity of density window I by 42% ( P

< 0.05), and it significantly decreased the Na',K+-ATPase

activities of windows II and N. The combined decreases

of activity in density windows II and N were greater than

the increase of activity in window I, and stimulation had

the net effect of decreasing the total Na+,K'-ATPase

activity recovered in XP, by 7% (P c 0.05). Cholinergic

stimulation had no significant effect on the acid and

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l ; : q p *

Resting

160 r

120

80

4 0

0'

- -

-

-

Density window

1.6

1.2

0.8

0.4

0 -

Stimulated

- -

- - -

120 '""1

120 1

40

0

o-6 r 0.4

0.2

0

Figure 4. Subcellular fractionation analysis of resting and stimulated acini. Density gradient centrifugation was performed in the SW-28 swinging bucket rotor. As discussed in the text, differences from the distributions depicted in Figure 1 result both from differences in resolution and from changes in subcellular organization that occur with acinar isolation. Activities of Nat,Kt- ATPase and alkaline phosphatase were first calculated in nrnoles/hr per density window. Acid phosphatase activity

I I1 111 IV Density window

Stimulation-induced change

1 0 2ol -20 - 1 : / T

0.2 1

-0.2 - O . l I

- 0.10 - 0.06 -

-0.05 - -0.10 - - L

I I1 111 IV Density window

was calculated in pmoleshr per density window. The total activity in each density window was then divided by the total protein (in mg) in all four density windows. The parallel calculation for the protein content in each densiry window yields a dimensionless value. Values presented are means & S.E.M. from 4 separate acinar preparations. The stimulation-induced change is the mean difference between the resting and stimulated samples from each preparation; * indicates P < 0.05.

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0.08

f+f -0.08

Resting

:if&+=+ 0.3

e

f: 0.2 .-

? $. 0.1

z 0

I II 111 IV

03

0.2

0.1

0

Density window Density window Density window

Figure 5. Density distribution of Nat/Ht antiport activities from resting and stimulated acini. Nat/Ht antiport activity was first calculated in nmoles/min per denSity window from quadruplicate determinations at 2 and 5 sec either in the presence or absence of outward Ht gradients or in the presence of Ht gradients with and without 1 mM amiloride. The activity in each density

window was divided by the total mg protein in all four density windows. Values presented for density windows I and II are means f S.E.M. from 3 separate acinar preparations; * indicates P c 0.05. Values for density windows ZII and N are means f range for 2 acinar preparations.

Table 1. Fractionation analysis of isolated acini. Distribution of markers after differential sedimentation steps.

Marker Initial Specific Percent Recovered Activity

Activity Po CPi CSi

Nat ,Kt -ATPase Resting 95 f 16 27.9 2 6.4 67.1 f 6.8 5.1 2 1.0 Stimulated 99 f 30 31.3 2 9.5 67.7 f 10.9 1.0 f 2.3

Resting 204 f 63 18.4 f 3.5 58.2 f 2.6 23.3 f 2.1 Stimulated 211 f 38 23.0 f 9.0 54.7 f 6.1 22.3 f 3.4

Resting 1.74 f 0.30 18.6 f 5.3 58.4 f 4.6 23.0 f 1.5 Stimulated 1.92 2 0.49 19.7 f 6.6 58.9 f 5.8 21.4 f 2.2

Alkaline Phosphatase

Acid Phosphatase

Protein Resting Stimulated

- 14.8 ? 5.2 19.3 f 1.1 65.9 2 5.0 - 11.6 f 1.2 23.4 f 1.9 65.1 f 1.3

Initial activities are nmoles/mg protein hr for Nat,Kt-ATPase and alkaline phosphatase and pmoles/mg protein the initial low-speed centrifugation steps. cPi and cSi are the series of pellets and supernatants generated by high-speed centrifugation of the density gradient fractions. All values are means f S.E.M.

hr for acid phosphatase. Po denotes the pellet generated by

alkaline phosphatase activities in density window I, but it

significantly decreased the alkaline phosphatase activities

of windows II and 111. Because stimulation increased the

fraction of total protein recovered in window I by 36%, it

was accompanied by a slight increase in the Nat,Kt-

ATPase specific activity and by significant decreases in the

alkaline and acid phosphatase specific activities in this

density window.

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The density gradient distribution of Na'/H' antiport

activity from resting acini, depicted in Figure 5, was

qualitatively similar to the distributions of Na+,K'-ATPase

and alkaline phosphatase (Figure 4), indicating that, like

the other plasma membrane constituents, the antiporters

are present both in surface-expressed and in intracellular

pools. Cholinergic stimulation significantly increased the

Na'/H' antiport activity of the densiry window I

membrane samples from 0.037 f .010 to 0.094 f .026

nmole/min . mg total protein (mean k S.E.M., n = 3, P < 0.05). The increase of Na'/H+ antiport activity in

density window I appeared to be accompanied by a small

decrease of antiport activity in density window IZ, but the

change in window II was not statistically signifcant.

Na'/H+ antiport activity was measured in window III and

window N samples from two of the acinar preparations.

These measurements gave no indication of a stimulation-

associated decrease which could have accounted for the

increase of antiport activity in density window I .

Dependence of basolateral membrane Na'/H' antiport on

intravesicular DH While it is clear that the increase in basolateral membrane

Na+,K'-ATPase activity results from the recruitment of

additional pump units from an endosomal compartment,

the mechanistic basis of the increase in antiport activity is

more difficult to discern. In other cell types which have

been examined, rapid increases in plasma membrane

Na'/H' antiport have been attributed to decreases in the

K, for intravesicular H' (19 - 22). It was not practical to

compare the pH vs rate relationships of window I samples

from resting and stimulated acinar preparations because

of the small amounts of material available in these

preparations. However, we found it instructive to

determine how intravesicular pH influenced amiloride-

sensitive Na' flux into density window I samples from

lacrimal gland fragment preparations. The experiment

depicted in Figure 6 was performed with an extravesicular

pH of 8.0 in order to increase antiporter-mediated influx

at the higher intravesicular pH values. Under these

0 0.5 1 1.5 2 2.5 3 3.5

H+ concentration (pEq / L)

Figure 6. Effect of intravesicular Ht concentration on Na'/Ht antiporter-mediated Nat influx. Membrane vesicles from the density window I sample from a fragment preparation (Figure 1) were divided into four groups and prepared for transport using loading buffers at pH = 5.5, 6.0, 6.5, and 7.0. Initial rates of Na' uptake from a medium of pH = 8.0 were calculated from the amiloride- sensitive uptake at 2 and 5 sec. Values presented are means f S.E.M. from triplicate determinations. The value predicted for a 2.5-fold increase in influx at pHi 6.0, i.e., an increase of the magnitude caused by cholinergic stimulation (Figure 5), is indicated by an X.

conditions, amiloride-sensitive PNa' influx was detectible

at pHi = 7.0, and it increased more than 10-fold as pHi

decreased to 5.5. A second experiment, with pH, 7.5, yielded qualitatively similar results, but in this case no

significant amiloride-sensitive "Na' influx was detectible

at pHi 7.0. These results indicate that Na+M+ antiport

decreases to a small fraction of the antiporter's J,. at pHi

values above the cytoplasmic pH set-point of 7.1 (5, 23).

Moreover, the H' concentration-flux data from both

experiments suggest a possible sigmoid relationship .in

which small decreases of pH, below 7.0 would produce

relatively large increases in Na' influx. Antiporter-

mediated Nat influx appeared to approach its maximum

as pHi decreased below 6.0.

DISCUSSION This study has provided evidence that rabbit lacrimal

acinar cells express both Na'/Ht antiporters and Na',K+-

ATPase pumps in their basolateral plasma membranes.

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As in acinar cells of the rat exorbital lacrimal gland, both

transporters also exist in substantial intracellular pools.

As in the rat lacrimal gland, cholinergic stimulation causes

a portion of the intracellular Na',K'-ATPase to be

translocated to the basolateral membranes. Cholinergic

stimulation also increases basolateral plasma membrane

Na'/H' antiport activity.

The functional characteristics of the rabbit lacrimal

Na'/H' antiporter, to the extent that it has been feasible

to describe them, are generally similar to those of other

cells. The K, of 14 mM for Na' measured in a sample of

isolated endocytic membrane vesicles enriched in

basolateral membrane constituents (Figure 3) falls within

the range of values reported for Na'/H' antiporters in

other epithelial cell types, including Necfurur gallbladder

(24), MDCK cells (25), and acinar cells of rat exorbital

lacrimal gland (16). Since endocytic membranes generally

retain their orientation upon cell disruption, this value

probably reflects the affinity of the antiporter's

cytoplasmfacing Na' binding site. The orientation of the

basolateral membrane vesicles has not yet been

established. However, it is interesting to note that, as in other systems (9, 21, 22), antiporter-mediated Na' influx is

relatively modest at intravesicular pH values near the

cytoplasmic pH of unstimulated cells, and it increases

markedly with increasing intravesicular H' concentration

(Figure 6). This behavior would allow the antiporter to

function efficiently to dissipate intracellular acid loads

( 19)- The 2.5-fold acceleration of Na'/H' antiporter-

mediated Na' flux into isolated basolateral membrane

vesicles observed after 30 min stimulation with carbachol

(Figure 5 ) is remarkably similar to the acceleration of Na'

flux into intact acini which occurs after cholinergic

stimulation (3, 11). We believe this observation lends

substantial support to the hypothesis that a parallel array

of Na'/H+ and Cl-/HCO,- antiporters mediates the influx

step of secretagogue-induced trans-cellular C1- secretion

(2, 3). It is possible that acinar cells also express

secretagogue-sensitive Na'-Cl- or Nat-Kt-2Cl- symporters

which couple C1- influx to the Na+ gradient across the

basolateral membranes, but that these are inactivated

during cell disruption and membrane isolation (16).

However, there is little reason to postulate their presence.

The increase in Na'/H+ antiporter activity must be

related in some way to the cascade of second messengers

triggered by agonist binding to the cholinergic receptor.

Evidence is accumulating that muscarinic receptors in

lacrimal acinar cells are coupled to the same intracellular

mediators as in other epithelial cells (26), i.e., that

phospholipase C is activated, hydrolyzing

phosphatidylinositol-bis-phosphate to inositol-tris-

phosphate and diacylglycerol (DAG). DAG activates

protein kinase c, and the functional significance of this

process is indicated by the fact that phorbol esters

stimulate lacrimal peroxidase secretion (27, 28).

The evidence that cholinergic receptor activation

triggers protein kinase c activation in lacrimal acinar cells

is of particular interest because this kinase mediates

Na'/H' antiporter activation in other cell types (21, 22).

Therefore, it is plausible to suggest that protein kinase c

also mediates the increase in lacrimal acinar cell

basolateral membrane Na'/H' antiporter activity. In

lymphocytes protein kinase c-mediated phosphorylation is

believed to increase the affinity of an intracellular H'-

binding regulatory site on the antiporter (21, 22). The

available data certainly do not exclude the possibililty that

such an increase in intravesicular H'-binding affinity

occurs in lacrimal acinar cells, where it be manifest in the

format of Figure 6 as a left-ward shift in the Ht concentration - flux relationship. However, the

relationship in Figure 6 suggests that an affinity change

may not be sufficient to produce the 2.5-fold increase that

was observed after cholinergic stimulation (Figure 5) .

One possibility is that stimulation caused additional

antiporters to be recruited to the basolateral membranes

but that the removal of antiporters from the endosomal

compartment was masked by kinetic changes in the

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~ ~ ~ ~~

remaining antiporters. Another possibility worthy of

future attention is that stimulation specifically increased

the turnover rates of the pool of antiporters residing in

the basolateral membranes.

The recruitment of Na',K'-ATPase pump units which

accompanies the overall increase of basolateral membrane

Na'/H' antiport should help the cell to compensate for

the acceleration of Na' influx (3, 11). It may be noted

that the increase in basolateral membrane Na',K'-

ATPase does not correspondend in a 1:l fashion with the

measured increase in Na'/H' antiport (Figures 4 and 5 ) .

This relationship may reflect the interplay of events that

occur in the intact cell but are not detected by our

reductionist analytical approach. The data available on

mouse lacrimal acinar cells suggest that one such event is

a significant increase of cytosolic Na' activity (29). Such

an elevation of "a'], would contribute to an overall

increase of the Na',K'-ATPase pump rate by moving the

system toward the right on a sigmoid Na' concentration - rate curve (30), the I,,,, of which has been increased by

the recruitment of additional pump units.

An increase of "a'], could also, plausibly, function as

the signal for recruitment of Na',KC-ATPase to the

basolateral membrane. However, it also seems possible

that the observed recruitment phenomenon is more

directly coupled to the intracellular signal transduction

mechanisms triggered by receptor activation. The latter

hypothesis is supported by the existence of

servomechanisms which modulate Na' pump rates

independently of changes in cytosolic Na' activity in

several other Na'-transporting epithelia (31).

While the intracellular messenger systems triggered by

receptor activation lead directly to increases in the

basolateral membrane Na'/H' antiport and, perhaps,

Na',K'-ATPase rates, the net influx of C1- via Cl-/HCO,-

antiporters appears to increase as the result of altered ion

driving forces and of pH,-mediated changes in the anion

exchanger's kinetic charactertistics. C1- flux into resting

acinar cells primarily represents Cl-/Cl- self-exchange (3).

The opening of apical C1- channels leads cytoplasmic C1-

activity to decrease. The cytoplasmic alkalinization that

follows an increase in Nat/H' antiport activity leads the

cytoplasmic HC0,- activity to increase. The result of

these changes is an increase in the intracellular [HCO,-] :

[Cl-] ratio, which should have the consequence of

increasing the fraction of anion exchanger-mediated C1-

influx occurring as Cl-/HCO,- exchange and, therefore, as

net influx across the basolateral membrane. Cytoplasmic

alkalinization also increases the turnover rate of the anion

exchanger regardless of whether the intracellular substrate

in a particular cycle is C1- or HCO,- (3).

The secretagogue-induced increases in Nat,Kt-

ATPase and Nat/Ht antiport activities amount to a

functional remodeling of the acinar cell basolateral

membrane. The remodeling process occurs in the context

of an overall acceleration of recycling traffic between the

basolateral membrane and endocytic compartments (6, 7,

12). It is possible that the accelerated recycling traffic

also accuunts for the slight but statisticaily significant

decrease in the total membrane-associated Nat,Kt-

ATPase activity and the marked decrease in the total

membrane-associated alkaline phosphatase activity which

occur after 30 min stimulation (Figure 4). It may also

account for the decrease in total muscarinic receptor

content which occurs when rat exorbital gland fragments

are stimulated (15). One pIausibIe working hypothesis is

that a constant fraction of the membrane constituents

internalized in each cycle are targeted to lysosomes, so that acceleration of basolateral membrane recycling traffic

is accompanied by accelerated degradation of the

recycling membrane constituents. In this context it is of

interest to note preliminary experiments in which acinar

cells were cultured overnight in the presence and absence of carbachol. The results suggest that sustained

cholinergic stimulation profoundly depletes the

intracellular pool of every basolateral membrane

constituent which has been measured (32).

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ACKNOWLEDGEMENTS

This work was supported by NIH grant EY 05801. R.W. Lambert was the recipient of a Grant-in-Aid of Research

from Sigma Xi, the Scientific Research Society.

CORRESPONDING AUTHOR

Austin K. Mircheff, Ph.D., Department of Physiology and

Biophysics, University of Southern California School of

Medicine, 1333 San Pablo Street, Los Angeles, CA 90033.

REFERENCES 1. Silva, P., Stoff, J., Field, M., Fine, L., Forrest, J.N. and

Epstein, F.H. (1977) Mechanism of active chloride secretion by shark rectal gland: role of Na-K-ATPase in chloride transport. Am. J. Physiol. 233, F29&F306.

2. Mircheff, A.K. (1989) Lacrimal fluid and electrolyte secretion: a review. Curr. Eye Res. 8, 607-617.

3. Lambert, R.W., Bradley, M.E. and Mircheff, A.K. (1991) pH sensitive-anion exchanger in rat lacrimal acinar cells. Am. J. Physiol. 260, G517-G523.

permeability coupling in rat lacrimal gland. Am. J. Physiol. 239, G106-G113.

Intracellular pH regulation in the mouse lacrimal gland acinar cells. J. Membrane Biol. 101, 73-81.

6. Herzog, V. and Farqhuar, M.G. (1977) Luminal membrane retrieved after exocytosis reaches most Golgi cisternae in secretory cells. Proc. Natl. Acad. Sci. (USA), 74, 5073-5077.

7. Oliver, C.A. (1982) Endocytic pathways at the lateral and basal cell surfaces of exocrine acinar cells. J. Cell Biol. 95, 154-161.

8. Yiu, S.C., Lambert, R.W., Bradley, M.E., Ingham, C.E., Hales, K.L., Wood, R.L. and Mircheff, A.K. (1988) Stimulation-associated redistribution of Nat,Kt-ATPase in rat lacrimal gland. J. Membrane Biol. && 185-194.

9. Yiu, S.C., Lambert, R.W., Tortoriello, P.J. and Mircheff, A.K. (1991) Secretagogue-induced Na,K- ATPase redistributions in rat lacrimal acini. Invest. Ophthalmol. Vis. Sci. Z, 2976-2984.

10. Kuijpers, G.A.J., De Pont, J.J.H.H.M., Van Nooy, I.G.P., Fleuren-Jakobs, A.M.M., Bonting, S.L. and Rodrigues de Miranda, J.F. (1984) Amiloride is a cholinergic antagonist in the rabbit pancreas. Biochim. Biophys. Acta, 804, 237-244.

11. Bradley, M.E., Lambert, R.W., Larnbert, R.W., Lee, L.M. and Mircheff, A.K. (1992) Isolation and subcellular fractionation analysis of acini from rabbit lacrimal glands. Invest. Ophthalmol. Vis. Sci. 33,

12. Lambert, R.W., Maves, C.A., Gierow, J.P., Wood,

4. Parod, R.J. and Putney, J.W. (1980) Stimulus-

5. Saito, Y., Ozawa, T. and Nishiyama, A. (1988)

2951-2965.

R.L. and Mircheff, A.K. (1993) Plasma membrane

internalization and recycling in rabbit lacrimal acinar cells. Invest. Ophthalmol. Vis. Sci. 34, 305-316.

13. Mircheff, A.K. and Lu, C.C. (1984) A map of membrane populations isolated from rat exorbital gland. Am. J. Physiol. 247, G651-G661.

14. Bradley, M.E., Larnbert, R.W. and Mircheff, A.K. (1993) Isolation and identification of plasma membrane populations. Meth. Enzymol., in press.

15. Bradley, M.E., Peters, C.L., Lambert, R.W., Yiu, S.C. and Mircheff, A.K. (1990) Subcellular distribution of muscarinic acetylcholine receptors in rat exorbital lacrimal gland. Invest. Ophthalmol. Vis. Sci. 3 l , 191- 200.

16. Mircheff, A.K., Ingham, C.E., Lambert, R.W., Hales, K.L., Hensley, C.B. and Yiu, S.C. (1987) Nat/Ht antiporter in lacrimal acinar cell basal-lateral membranes. Invest. Ophthalmol. Vis. Sci. 28, 1726- 1729.

17. Mircheff, A.K. (1989) Isolation of plasma membranes from polar cells and tissues: Apicalbasolateral separation, purity, function. Meth. Enzymol. l72, 18- 34.

Analytic subcellular fractionation of acini from rat lacrimal gland. Invest. Ophthalmol. Vis. Sci. 3 l , 2437- 2447.

Modifier role of internal H t in activating the Nat-Ht exchange activity in renal mircovillus membrane vesi- cles. Nature, 299, 161-163.

induced activation of Nat/Ht exchange in rat parotid acinar cells. J. Membrane Biol. Jll, 191-198.

21. Grinstein, S., Cohen, S., Goetz, J.D., Rothstein, A. and Gelfand, E.W. (1985) Characterization of the activation of Na+/Ht exchange in lymphocytes by phorbol esters. Change in cytoplasmic pH dependence of the antiport. Proc. Natl. Acad. Sci. (USA), 82,

22. Grinstein, S . and Rothstein, A. (1986) Mechanisms of regulation of the Na/H exchanger. J. Membrane Biol.

23. golchini, K., Lambert, R.W., Ghadishah, E.,

18. Yiu, S.C., Wood, R.L. and Mircheff, A.K. (1990)

19. Aronson, P.S., Nee, J. and Suhm, M.A. (1982)

20. Manganel, M. and Turner, R.J. (1989) Agonist-

1429-1433.

90, 1-12.

Yasharpour, F., Gierow, J.P. and Mircheff, A.K. (1991) Sodium-proton exchange in rabbit lacrimal acinar cells characterized with a pHi-sensitive dye. Invest. Ophthalmol. Vis. Sci. 32 (Suppl.), 726.

24. Weinman, S.A. and Reuss, L. (1982) Nat/Ht exchange at the apical membrane of Necturus gallbladder. J. Gen. Physiol. 80, 299-321.

25. Goldfarb, D. and Nord, E.P. (1987) Asymmetric affinity of Nat/Ht antiporter for Nat at the cytoplasmic versus external site. Am. J. Physiol. 253, F959-F968.

26. Dartt, D.A. (1989) Signal transduction and control of lacrimal gland protein secretion: a review. Curr. Eye Res. 8, 619-636.

27. Dartt, D.A., Ronco, L.V., Murphy, S.A. and Unser,

Cur

r E

ye R

es D

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oade

d fr

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onal

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Current

Research Eye

M.F. (1988) Effect of phorboI ester on rat lacrimal gland protein secretion. Invest. Ophthalmol. Vis. Sci. 29, 1726-1731.

28. godges, R.R. and Dartt, D.A. (1989) Cholinergic and alpha-adrenergic agonist stimulation translocates lacrimal gland protein kinase c. Invest. Ophthalmol. Vis. Sci. 30 (Suppl.), 473.

29. Saito, Y., Ozawa, T. and Nishiyama, A. (1987) Acetylcholine-induced Na+ influx in the mouse lacrimal gland acinar cells: Demonstration of multiple Na+ transport mechanisms by intracellular Na' activity measurements. J. Membrane Biol. 98, 135- 144.

30. Lewis, S.A. and Wills, N.K. (1983) Apical membrane permeability and kinetic properties of the sodium pump in rabbit urinary baldder. J. Physiol. (London), 341, 169-184.

31. Schultz, S.G. (1981) Homocellular regulatory mechanisms in sodium-transporting epithelia: Avoidance of extinction by "flush-through." Am. J. Physiol. 24l, F579-F590.

32. Maves, C.A., Rismondo, V. and Mircheff, A.K. (1992) Chronic stimulation of rabbit lacrimal acinar cells decreases intracellular pools of Na,K-ATPase and other surface enzymes. Invest. Ophthalmol. Vis. Sci. 33 fsuppl.), 1289.

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