Vol. 83, No. 4, 1978

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS Pages

August 29,1978

1595-1601

THE INFLUENCEOF CYSTICFIBROSIS SERUM ANDCALCIUM ON SECRETIONIN THE RABBIT TRACHEAL MUCOCILIARY APPARATUS

J.H. Conover and E.J. Conod Denartment of Pediatrics St. Vincent's Hospital and Medical Center of New York and New York University Medical School New York, New York Received

July

25,1978

SUMM4RY: Cystic Fibrosis serum or its isolated component IgG fraction and calcium ionophore A23187 all produced a quantitatively greater increase of mucus glyconrotein secretion in the rabbit tracheal epithelium than did control serum or its isolated component IgG fraction. These values were determined by dry weight secretion per gram of tissue and on subsequent sialic acid content of secretions. This demonstrable increase in mucus production represents a measurable difference in the functioning of the cultured mucociliarv apparatus due to the influence of cystic fibrosis serum. Cystic fibrosis the ciliary cultured

(CF) sera has been shown to adversely affect

and mucous components of the microscopically rabbit

tracheal

ciliated

epithelium

(1,2).

both

observed

Hhile the dual

nature of this CF serum-induced response is well known, much of the attention

has previously

hence the term ciliary

been focused on the prominent ciliary dyskinesia factor

shown that both secretory

and ciliarv

(CDF) (3,4).

asnects of

the

vector--

He have recently mucociliary

res-

ponse are induced by CF sera and are mediated by calcium ions (1,5,(i). In these instances, specific the total

chelation of calcium in CF sera eliminated

CDF response and control

cium ionophore A23187 generated of which was distinctly Both light

identified

typical

CDF resoonse--each

complete

by electronmicroscopy

and electron microscopic cellular

gested that mucoussecretion lium after

a

sera supplemented bv 2 X 1W4M cal-

observations have sug-

is increased in the rabbit

exposure to CF affected

and carrier

(6).

tracheal epithe-

sera or their

purified

IgG

0006-291X/78/0834-1595$01.00/0 1595

Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 83, No. 4, 1978

fractions tions,

as compared together

clinically sera

with

with

the

control

led

mucous

mucous

substances.

production

do control

or in

This

and heterozygotes

report

stimulate sera.

purified

serum-derived

sociated

substance

in viscositv

could

whether

that

production were

IgG fractions,

further

in the pathophysiology

mucus seen

in the

rate

of one or more specific

demonstrate

Such increases

ohserva-

CF and CF carrier

an increase

the production

greater

These

of nulmonarv

potentiate

will

RESEARCH COMMJNKATIONS

or IgG fractions.

us to investigate

IgC fractions,

of total

AND BIOPHYSICAL

sera

increase

in CF subjects

or their

than

BIOCHEMICAL

sera

of r&hit similarly

CF patients

tracheal

extended

imolicating

of this

from

mucus

to individual

TgG and/or

some as-

disorder.

Rabbit tracheal tissue was prepared according to our orevious renort separated luminal epithelial mucosa (4), except that the mechanically (tracheal sheath) was used as a whole intact unft without mincing into smaller pieces. These tracheal sheaths are composed of both submucosa and mucosa as defined bv electronmicroscopy (6) and in general contain a large abundance of mucous-producing epithelial gohlet cells, and a variable degree of the predominantlv mucous-producjng tubule-alveolar glands. Four intact tracheal sheaths nrepared in this fashion were immediately placed in a common Petri dish (35 X 10 mm) containing 2.5 ml of either CF, CF obligate heterozygote CF-H or normal control sera. Replicate Petri dishes for each test sera were incubated at 37OC for different intervals (1,2,4,6,8 and 24 hours). At the end of each incubation period, the test serum of each dish was collected and the respective tracheal sheaths removed and gently agitated with forceps in another Petri dish containing 5 ml of Hanks' Ralanced Salt Solution without phenol red indicator at pi! 7.0. In each instance, the wash, containing apparent mucus-like insoluble material, was combined with its respective test serum and centrifuged at 2fWO mm for lo minutes to isolate the insoluble mucus-like material. Tn every instance, the re suiting pellet was washed twice with 5 ml distilled water and resuspended reisolated bv centrifugation and finally in 0.5 ml of distilled water, placed in a pre-weighed glass ampule where it was lyophilized for 24 hours. Each of the washed intact tracheal sheaths was blotted once on filter paper and also ulaced in pre-weighed glass amnules for lyophilization. The resulting lyophilized secretions and tissues were then weighed on a ?fettler H 20 T balance and secretions expressed as dry sialic acid (measured as weight per gram tissue. Subsequently, N-acetylneuraminic acid) by the thiobarbituric acid method of Warren (7) and DNA measured by the method of Schneider (8) were performed on each test sample secretion. Figure as control evidenced: It

1 demonstrates sera

in this

dry weight

can be seen that

DNA content

a typical

of this

test

system.

of secretion, after system

time-response

2 hours rises

All

three

sialic

acid

of incubation sharply.

1596

This

curve test

parameters

(NANA) with

for

CF as well are

and DNA.

any sera,

that

mav be due to a noted

the

BIOCHEMICAL

Vol. 83, No. 4, 1978

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

180 170 160 I!%l1

.-g. -I c m140

l

2 mz IlO-. i=

l

Em F goz 2, 00t; 70-

’ is E"

l

0 l

0. ‘W! :. 0

60-

50403020-

‘,’

INCUBATION

igure

1.

Time-resvonse curve sera. Open circles represent CF sera.

Figure

2.

Tracheal secretory response heterozygotes and controls.

microscopic

appearance

which

serves

earlier

than

the

NANA and mucous appreciably Figure

lower

2 depicts

tracheal

tissue,

The vertical grout:

CF=76.6+5.4

control=30.4+2.6 between

line

one-hour

production, than

the

2 hour

Vhile

also

(S.E.Y.)

used

of the wejehts

mr:

me secretion. and control

1597

for

substances for

this

and 15 normal

was

study. aer

gram

control

the mean value

subjects. for

each

CF-F-69.73.6

mp: secretion;

t-test,

the mean difference

Ry Student's subject

intervals

of secretion

indicates

secretion;

wash solution,

seemed effective

interval

each column

CF patients,

time

of these

13 CF, 21 V-1'

emnloying across

value

from

fn the hanks'

damage.

the quantity

the results

CF homozygotes

to sera

cells

of tissue

listed

CONTROL

of tracheal explants to CF and control represent control sera; closed circles

of epithelial

as an index

CF

02

AT 37” c (SCOURS)

is

significant

(p