Characterization of nitric oxide synthase in the opossum esophagus

Characterization Esophagus JOSEPH

A. MURRAY

of Nitric Oxide Synthase in the Opossum

and EUGENE

D. CLARK

Department of Internal Medicine, University of Iowa College of Medicine and Department of Veterans Affairs Medical Center, Iowa City, Iowa

Bac~found/Aims: Nitric oxide mediates the nonadrenergic, noncholinergic neural control of esophageal motor function. The purpose of this study was to characterize the NO synthase found in the muscularis propria of the opossum esophagus and determine its distribution along the esophagus. Methods: Esophageal muscle was homogenized in HEPES buffer and ultracentrifuged. The supernatant was exposed to [3H]L-arginine. The [3H]Lcitrulline produced by NO synthase was separated from [3H]L-arginine with a Dowex AG 50 W-X8 column (Biorad, Hercules, CA). Assays were performed in the presence and absence of Ca*+ or reduced nicotinamide adenine dinucleotide phosphate (NADPH). The distribution of NO synthase activity along the esophagus was determined. Results: The apparent Michaelis constant and maximum velocity of NO synthase were 7.5 k 1.4 pmol/L L-aginine and 76.0 2 17.3 pmol.mg protein-‘. min-I, respectively. The enzyme required both Ca2’ and NADPH for activity. Smooth muscle tissue from the lower esophageal sphincter and the esophageal body l-2 cm or 5-6 cm above the lower esophageal sphincter differed little in enzymatic activity, ranging from 0.97 to 1.27 pmol .mg wet wt’. min-‘. Striated muscle had less activity with 0.40 pmol. mg wet wt-l. mini. Conclusions: These data indicate the presence of a constitutive NO synthase in the esophagus of the opossum.

N

itric oxide or an NO-containing ates several neuromuscular

gus that result from activating ergic

(NANC)

substance

functions

of the esopha-

nonadrenergic,

nerves. ‘-’ Normally,

medi-

noncholin-

activating

NANC

nerves produce relaxation of the lower esophageal sphincter (LES) and contraction muscle. This contraction

of circular

esophageal

is called the off response because

it occurs after stimulation

of the nerve has ended. The

time between the end of the stimulus of the contraction along the length

smooth

and the beginning

is called the latency. The latency varies of the esophagus

such that it describes

a gradient with the latency being shortest in the proximal smooth

muscle

LES.6 Inhibitors (L-NNA),

esophagus

and longest

just

above

the

of NO synthase, like W-nitro-L-arginine

antagonize

the nerve-induced

relaxation

of the

LES and the membrane

hyperpolarization

ies this relaxation. 2,3 They antagonize circular esophageal gradient,

smooth muscle, eliminate

and antagonize

underlying mimics

some events produced the esophagus contractions

nearly simultaneous This

suggests

events

exogenous NO

by nerve stimulation.

and the LES.le3

Inhibitors

of

of swallow-induced

such that the contractions

along the length

that differences

It

circular smooth mus-

NO synthase also change the timing peristaltic

the latency

the electrophysiological

these responses.lX2 In addition,

relaxes the LES and hyperpolarizes cle from

that accompan-

the off responses of

become

of the esophagus4

in NO

synthase

along the esophagus may underlie the gradient

activity

in latency.

There are several isoforms of the enzyme NO synthase. One type requires reduced P-nicotinamide cleotide phosphate activity

(NADPH),

Ca’+ , and calmodulin

and is a constitutive

other type is inducible dependent.8

adenine dinu-

and does not appear to be calcium-

Alterations

in NO

synthase

activity

play a role in physiological

and pathophysiological

cesses affecting

function.

esophageal

of the basic biochemical activity

processes.

synthase

use L-arginine

produce

NO

produced

All of the isoforms of NO

and L-citrulline

in a 1:l

is difficult

and they all

ratio.

because its half-life

is very short in biological is a stable

product

that can be assayed. This is performed arginine

as the substrate

the formation

The

NO

to assay directly

however,

citrulline

prerequi-

roles of NO in the

as their substrate,

by this reaction

pro-

of the enzyme

is an essential

site to further studies of the possible

may

An understanding

characteristics

found in the esophagus

pathophysiological

for

form of the enzyme.’ An-

systems;

of the reaction by using {3H]~-

for NO synthase and following

of [3H)L-citrulline.9-”

Abbreviations used in this paper: BCA, bicinchoninic acid; CHAPS, 3-[(3cholamidopropyl)dimethylammoniol]-l-propanesulfonate; DlT, dithriotheritol; ECW, 50% inhibitory concentration; EGTA, ethylene glycol bis(baminoethyl ether) N,N,N’,N’-tetraacetic acid; L-NNA, A’“-nitro+arginine; reduced NADPH, P_nicotinamide adenine dinucleo tide phosphate; NANC, nonadrenergic, noncholinergic; PMSF, phenylmethylsulfonyl fluoride; TLC, thin-layer chromatography; V,,., maximum initial velocity. This is a U.S. government work. There are no restrictions on its use. 0018-5085/94/$0.00

June 1994

ESOPHAGEAL

These studies were undertaken first was to show the presence within the muscularis propria

for three purposes.

The

of NO synthase activity of the esophagus. Such

studies must be performed as part of a rigorous proof that NO is a mediator of NANC neural control of esophageal neuromuscular

function.

found in the esophageal

lature.

to confirm

We were unable

that the gradient the length

in latency

results

along in

activity.

Products

obtained

was obtained

(Boston,

from Bio-Rad

from Du Pont NEN

MA). Dowex

Laboratories

chromatography

obtained

(Hillsboro,

from

Whatman

phenylmethylsulfonyl nonradioactive

fluoride

L-arginine,

diaminetetraacetic leupeptin,

(EDTA),

CA). LK6 silica HEPES

A,

L-NNA,

ethylene-

ethylene

glycol

acid (EGTA),

3-[(3-cholamidopropyI)dimethyl(CHAPS),

and P-NADPH

from Sigma Chemical

obtained

(DTT),

L-ornithine,

ammoniol-1-propanesulfonate were obtained

Louis, MO). Bicinchoninic

buffer,

dithiothreitol

ether) N,N,N’,N’-tetraacetic

pepstatin

was

(TLC) plates were

OR).

(PMSF),

L-citrulline,

acid

bis-(P-aminoethyl

AG 5OW-X8

(Hercules,

gel 20 X 20-cm thin-layer

2-mercaptoethanol,

acid (BCA) protein

from Pierce (Rockford,

tions of [‘H]L-arginine,

(0.75-2.9

@/incubate);

incubation

blank contained

assay kits were

IL).

and 0 or 1 mmol/L

Two hundred

petted

into a scintillation

liquid

scintillant

Prospect, rulline

was eluted

lengthwise,

at its length

through

with a cuff of stomach.

in situ in a dissection

intraperi-

a midline and pinned

removed

by sharp dissection.

by gross inspection.

strips

(including

the

circular,

plexus

measuring

1 cm in width

unseparated

stopped

LSGOOOIC liquid

Instruments,

incubate

Inc., Fullerton,

5-6

and

muscle

were investigated.

the supernatant

and 2 cm in length)

were

of the esophagus

cm above the LES, and the striated

muscle

scintillation

0 or 0.45 mmol/L

mL of ice-cold

homogenization Tenbroech

buffer per gram tissue with a

tissue grinder

(Corning

Inc., Corning,

tration

NADPH

ity (V,,)

These

maximum

incubation

tants with

with

L-arginine,

by incubating

(pH 7.5). The homogenate 100,000 X g at 8°C.

EDTA,

at

The contribumixture

L-NNA

muscle

superna-

0.45 mmol/L

Ca*+ with 0.5

for 15 minutes

with

was present

in the

of L-NNA. activity

the tissue was homogenized

as previously

in buffer A or buffer B. Homogenization composition

0.1; EGTA,

0.001; leupeptin,

was

on tissue

inhibitor

the smooth

if any NO synthase

had the following

from each

spectrophotometry

10 l.tmol/L L-arginine,

fraction,

EDTA

0.5 mmol/L

to the incubation

The effect of the NO synthase

To ascertain

for these

times were 3,

the supernatant

10 PmoliL

calcium

concentrations

particulate described

for 60 minutes

media

The incubation

using atomic absorption

was investigated

the

initial veloc-

Ca’+ with 0.5 mmol/L

by incubating

segment

homogenates.

concen-

(K,) values were determined

EDTA, and 0- 1.5 mmol/L Ca*+ for 15 minutes. measured

EDTA,

NO synthase

The effect of calcium on the NO synthase

activity was estimated esophageal

10 pmol/

time and L-arginine

The

0.45 mmol/L

4, 5, and 15 minutes.

with

data were used to calculate

constant

velocities.

contained

segments

The apparent

and Michaelis

the initial

by incubating

for 15 minutes.

of incubation

was studied.

NY). Homogenization buffer A contained 50 mmol/L HEPES, 0.1 mg/mL PMSF, 0.5 mmol/L EDTA, and 1 mmol/L DTT was centrifuged

on

(Beckman

Ca’+ in 0.5 mmol/L

varying

motor-driven

of the

cm above the LES, 5-6

buffer until

in 9

counter

This was performed

mmol/L EDTA, and 1 .O mmol/L NADPH

The tissue was homogenized

by 2

4-mL eluate

CA).

esophagus 12.5 - 13.5 cm above the LES. The tissue strips were blotted dry, weighed, and kept in the ice-cold homogenization homogenized.

followed

The radioactivity

from the LES, l-2

tion of endogenous

myenteric

was

(Na+ form). Cit-

and the eluate were counted

and l- 19.4 /.tmol/L L-arginine.

of

of the mixture

of NO synthase activity along the length the Ca” requirement of esophageal NO and the NADPH requirement of esophageal NO

synthase

flat

were

Solve

Corp., Mt.

The distribution of the esophagus,

studies

muscle

Transverse

longitudinal,

cut from the LES, the smooth muscle segment and

counting.

a Beckman

was

COz) Krebs’ solu-

The circular

the LES was identified

l-2

International

2OO+L aliquot

was taken for scintillation

from

bath. The bath contained

(95% OJ5%

tion (pH 7.4). The mucosa and most of the submucosa quickly

inci-

The esophagus

washed in Krebs’ solution,

room temperature-oxygenated

Products

was pi-

6 mL Budget

with 2 mL of stop solution

intramuscularly

opened

of this mixture

vial containing

mL of water. An aliquot (0.8 mL) of the combined

initial reaction velocities.

and 50 mg/kg sodium pentobarbital

An buffer

500 PL stop buffer 1pH 5.5]/1 mmol/L

onto 1 mL of Dowex AG 5OW-X8

activity as a function

sion and removed

microliters

IL). A separate

pipetted

and 0 or 1.0 mmol/L

was exposed

50 PL of homogenization

(Research

Adult opossums of either sex were anesthetized with 30 mg/kg ketamine and 0.3 mg/kg acepromazine administered The esophagus

L-arginine

NADPH.

in place of the supernatant.

L L-arginine,

toneally.

1.0, or 1.5

10, or 19.4 FmoliL

cm above the LES, and the striated

NO Synthase Assay

to yield

buffer of 0.5 mmol/

0, 0.05, 0.1, 0.3, 0.4, 0.45, 0.5, 0.75,

synthase,

Co. (St.

varying concentra-

Ca*+, and NADPH

in the homogenization 9.72,

assay.

to 60 l.tL of

at 37°C for 3, 4, 5, or

contained

L-arginine,

Ca’+; 1, 4.86,

citrulline).

Materials [2,3,-‘H]L-arginine

The reaction mixture

was added

The reaction was stopped by adding (10 mmol/L EDTA/lOO mmol/L HEPES

Materials and Methods

Research

This was incubated

mixture.

mmol/L

hypothesis

from a gradient

reaction

15 minutes.

1445

was saved for protein

of the supernatant

L EDTA;

muscu-

of the off contraction

of the esophagus

NO synthase

the third

of the supernatant

Fifty microliters

final concentrations

The second was to characterize

the type of NO synthase

An aliquot

NO SYNTHASE

(in mmol/L.):

0.1; 2-mercaptoethanol,

buffer B

Tris/HCL,

50;

12; pepstatin

A,

0.002; and PMSF, 1.0 (pH 7.4). This buffer

1446

MURRAY

is similar

AND

CLARK

GASTROENTEROLOGY

to that described

the homogenate supernatant

(soluble)

particulate

fraction

and resuspended vol) glycerol

by Mitchell

was centrifuged and a pellet was washed

and 0 or 20 mmol/L

was mixed

for

100,000

X g. The solublized

fraction.

homogenization

volume

of

X g to yield a

(particulate)

with

to the original

CHAPS

et al.‘* An aliquot

at 100,000

30 minutes

The pellet

and

L NADPH,

0.3-0.75

with

recentrifuged

at

fraction was used for incubations.

incubations stopped

Ca*+, and either

0.5 mmol/L

EDTA

and 0.1 mmol/L

EGTA.

were for 15 minutes

as previously

Protein pernatant

described. by serial dilution

using the BCA method One milliliter 0.4% NaOH,

the Pierce BCA protein

and 95% NaHCO,

or PMSF and centrifuged

until

in measured without

with or without

protein

DTT To an

preparation.

The

1: 10% with reagent A and stored

There

was no significant

concentrations

between

difference

samples

with or

These studies committee

were approved

of the University

by the animal

care and use

of Iowa.

TLC a pathway pathway ornithine,

can be formed from L-arginine

other than NO synthase.

via

The most likely alternate

is the urea cycle. In the urea cycle, arginase generates which is converted

carbamylase.

determined

To investigate

to citrulline this

by ornithine

possibility,

trans-

TLC was per-

formed on the eluate from the Dowex column

and an aliquot

of the unseparated stopped incubation mixture. Proteins were removed from the stopped incubate before chromatography by adding 3 vol of 10% trichloroacetic acid and centrifuging the mixture

for 10 minutes

tritiated

amino acids of interest,

at 2000

X g at 6°C. To locate the 0.6 pg/mL

of nonradioactive

L-arginine, L-ornithine, and L-citrulline were each added to the samples. These were subsequently located using ninhydrin. A sample was applied above the preabsorbent area, and the silica gel below it was removed to keep it from migrating. This was used as a reference value to calculate the percentages of arginine, ornithine, and citrulline. methanol, chloroform, water, above the preabsorbent

The plates were developed in NH40H (4:l:l:l) for 16 cm

layer. Those portions

of the silica gel

as mean

? SEM and n represents observations

significance.

(pmol/mg

protein)

V, is the initial velocity (pmol . mg protein-’ (pmol

and

a is the

mg protein-i.

nonlinear

(~mol/L).‘*

formed

both

using

t is time with

time

were determined

by

X S)/(K, + S), in

initial velocity and S is the L-arginine The

nonlinear

Quasi-Newton

analysis

and Simplex

available as part of the Systat statistical

age (Systat Inc., Evanston,

were

at each time,

mini),

in velocity

analysis of the model. V = (V,,

concentration methods

change

Km and V,,

min-‘).

which V is the calculated

to deter-

The initial velocities

analysis using the model P = (V,)t +

(a?), in which P is measured in minutes,

were made.

t test was used when appropriate

by nonlinear

was perestimation

software pack-

IL).

Results NO synthase

activity

was sought

in both the par-

ticulate and supernatant fractions. We were unable to detect any activity in the particulate fraction, although several types of homogenizations and incubations were used. To control for the possibility that there may be segregation

of necessary cofactors in the soluble fraction,

we compared

an uncentrifuged

sample with soluble frac-

tion and were unable to detect any increase in total activity. All of the subsequent results refer to the supernatant in buffer A unless noted.

NO activity

along the esophagus

was measured

in the

LES, at two levels in the smooth

muscle

in the striated

There was little differ-

ence in NO

In theory, citrulline

sections

vials and counted.

from which

mine levels of statistical

fraction

DTT and PMSF.

of animals

The paired Student’s

A of

DTT and PMSF were added to final

assayed.

Data are expressed the number

so-

buffer did not interfere

equal to those in the enzyme

samples were diluted

at -2O’C

(reagent

as in the enzyme preparation.

aliquot of the supernatant, protein

0.16%

assay kit) and frozen. To make sure

with the assay, tissue was homogenized

acid and the reference

Analysis of Data

was diluted

2% Na2C03.H20,

that the D’M’ in the homogenization

concentrations

of the su-

with bovine serum albumin

of the supernatant

10 mL of 1% BCA-Na,

dium tartrate,

The

at 37”C, and the reaction was

assays were performed

as standards.13 with

mmol/L

or 0.1 mmol/L

of amino

into scintillation

No. 6

The

with 0 or 10% (~011

CHAPS.

by bands

were scraped

buffer

To control for the possibility that the enzyme in the particulate fraction was separated from required cofactor(s) in the soluble fraction during centrifugation, uncentrifuged aliquots of homogenate with 0 or 10% (vol/vol) glycerol were also incubated. The incubate consisted of 50 PL of sample preparation and final concentrations of 0 or 10 PglmL calmodulin, 1.0 mmol/ EDTA

occupied

Vol. 106,

muscle esophagus. synthase

activity

among

esophagus, tissues

from

and the

LES and the smooth muscle portion of the esophagus. There was no significant gradient of NO synthase activity measured along the smooth muscle segment of the esophagus. NO synthase activity in the striated muscle segment (12.5-13.5 cm above the LES) was significantly less than that of the smooth muscle segments (n = 5; P I

0.04) (Table 1). No significant difference in activity of the synthase was found wherever it was measured in terms of total tissue (pmol/[mg wet wt X min]) or extractable material (pmol/[mg protein X min]) in the smooth muscle (data not shown). Inclusion of the NO synthase inhibitor L-NNA with the smooth muscle incubates significantly inhibited the production of E3H]citrulline; as pmol . mg wet wtt’ . min-‘, the LES (n = 4) decreased from 0.986 5 0.096 to 0.041 + 0.018. The l-cm segment (n = 3) decreased from 1.187 ? 0.108 to 0.076 + 0.016, and the S-cm segment (n = 4) decreased from 1.369 k 0.131 to 0.008 ?

ESOPHAGEAL NO SYNTHASE

June1994

0.005.

The

estimated

(I&) for L-NNA 0.437 2 0.034 0.33

+ 0.073

inhibitory

for the 5-cm

by L-NNA

being

exchange formed

contained

each of ornithine

0.8% 90.5%

trulline.

There

activity. that

blanks

0.8%

with stop solution

ornithine,

was no net formation

and 9.0%

activity

TLC of the Dowex eluate

of the counts

The ratio of amino nine,

that there is no arginase

measured

distance

0.53 for L-ornithine,

verified

distance

(cm) to sol-

(cm) was 0.35 for L-argi-

formation was not linear over time. There was a decrease in enzyme activity of 7.2% per minute for low L-arginine centration tested. The initial velocities

in NO synthase

activity

with time

and concentration

be

statistics.

7.5+

at the highest

1.4 pmol/L

estimated

to

from

L-arginine, be

76.0

+

K,,, was estimated and the apparent 17.3

pmol.mg

to V,, pro-

con-

Calcium and NADPH Dependence of NO Synthase The NO synthase activity was Ca*+-dependent. Omission of Ca2+ from the incubates resulted in no citrulline formation (Table 1). The Ca2+ dependence of smooth muscle NO synthase from the various segments on added Ca*+ was further investigated by incubating the homogenates

were calculated

The apparent

tein-* . min-‘.

time and L-arginine concentration 1). The time course of citrulline

and 4.4% per minute

nonlinear was

and 0.90 for L-citrulline.

Using the buffer A homogenate from the whole muscularis propria (LES to 6 cm), the change in enzy-

concentrations

Figure 1. Change of L-arginine.

that

Kinetics of NO Synthase Activity

matic activity with was assayed (Figure

minutes

were from citrulline.

acid migration

vent front migration

by

10

5

ci-

of ornithine

This suggests

present.

Thin-

with stop solution

the supernatant. 95%-97%

and not Thin-layer

and citrulline.

of the incubate

arginine,

the radioactivity

resin was citrulline

of incubation

layer chromatographs

(n = 2).

the measured

in the incubations.

chromatographs

yielded

that

is caused by enzyme

from the Dowex

segment

confirms

TLC was also used to confirm ornithine

concentrations

are 0.465+ 0.005pmol/L for the LES, pmol/L. for the l-cm segment, and

pmol/L

The inhibition [3H)citrulline

50%

1447

with

different

concentrations

of Ca2+

15

20

the time

course of citrulline formation at different L-arginine concentrations and plotted (Figure 2). The enzymatic activity appears to follow Michaelis-Menten ing neither

substrate

activation

kinetics,

nor substrate

show-

inhibition

with increasing L-arginine concentrations. The apparent Km and V,, values were estimated from the MichaelisMenten equation using Quasi-Newton and Simplex

Table1. Distribution of NO Synthase Activity Along the Esophagus

LES 1 cm above LES 5-6 cm above LES Striated muscle (12.5 cm from LES)

0.45 mmol/L Ca and 1 mmol/ L NADPH

0.45 mmol/L Ca and no NADPH

No Ca and 1 mmol/L NADPH

1.27 ? 0.08 0.97 * 0.15 0.98 -c 0.06

0.59 + 0.08 0.39 + 0.04’ 0.48 ? 0.05

0.00 2 0.01 -0.01 ? 0.01 0.00 2 0.01

1 0

0.41 2 0.05

NOTE. All values are expressed SEM. n = 5. “n = 4.

0.28 2 0.03

as pmol.mg

wet wt-‘.min?

0.00 + 0.01

5

10

micromolar

arginine

and mean 2

figure 2. The instantaneous velocities for NO synthase activity as it varies with concentration of L-arginine.

1446

MURRAY

AND

GASTROENTEROLOGY

CLARK

esophageal

motor

function.

No. 6

As part of a rigorous

that NO is in fact a mediator of the esophagus,

Vol. 106,

of neuromuscular

the enzymatic

proof

function

system for its biosynthe-

sis must be shown in the muscularis

propria of the esoph-

agus. These studies confirm the presence of NO synthase in the muscle layers of the esophagus, our previous contention

thereby supporting

that NO is a mediator

of NANC

motor events in the esophagus. There are several isoforms of the enzyme NO synthase. One general 1.5 1.0 -

class of enzyme

and calmodulin

for activity.

of the enzyme

that is known

somes from the cerebellum.

P

requires

0.0

0.5

1111

1.5

1.0

Total

Cs (mM)

to be present

rations

of the opossum

Figure 3. The effect that increasing concentrations of added calcium has on NO synthase activity in three different segments of the esophagus.

that

and the CaZf chelator

EDTA

The estimated

of total added Ca2+ were 0.22

0.024

E&(s)

mmol/L

(0.5

for the LES

mmol/L) (Figure

(n =

6), 0.166

mmol/L for 1 cm (n = 5), and 0.14

2

+

0.018

3). 2

0.029

mmol/L

for 5 cm (n = 6). Figure 3 shows a rapid increase in NO synthase activity

as added Ca2+ is increased from 0.2 to

0.3 mmol/L. The activity is increased

decreases slightly

to 1.5 mmol/L.

To estimate

EC& values, the endogenous

calcium

measured by atomic absorption obtained 0.07

(LES, 0.08

mmol/L)

EDTA

The

mmol/L; 1 cm, 0.09

apparent

was calculated

with

stability

bility constant

stability

constant

mmol/L; 5 cm,

the added calcium constant

of calcium

values of EDTA” of calcium

was calculated

EDTA.16

X lo-l2 NADPH

duced NO synthase activity

The free

The ECSo values

mol/L for LES,

mol/L for 1 cm, and 4.8 X lo-l2 exogenous

synthase

re-

to about one half of control

of NADPH

activity

X

mol/L for 5 cm.

and to about two

thirds of the control values for the striated segment NO

6.9

from the incubate

values for the smooth muscle segments ble 1). The absence

log

and the

from the apparent sta-

by the method of Segal.”

for free Ca2+ were 9.9 Omitting

was

The values

at pH 7.5 from the negative

constant

Ca’+ concentration

lo-l2

concentration

spectrometry.

were summed

of the dissociation absolute

free calcium

and then ECSo values for free Ca2+ were

concentrations calculated.

as the Ca’+

at each

significantly level

(Ta-

decreased

of the esophagus

(P < 0.005).

A number

of previous

on cal-

in the rat cerebellum. NO

synthesis

The

is found

fraction was included with the solu-

would suggest

that at least in our prepara-

tion, there does not seem to be any activity in the particulate fraction

(data not presented).

The dependence

of the esophageal

ity on Ca 2+ is similar the rat brain cerebellum,” celLI NO

activity

calcium

concentration

slightly

inhibits

In all of these systems,

increases

to a maximum

is increased.

NO synthase.

added Ca2+ are comparable as rat cerebellum

for

the bovine aortic endothelial

and the rat forebrain.20 synthase

NO synthase activ-

to what has been reported

Adding

as the

more Ca2+

The ECSo values for total

with other preparations

or bovine aortic endothelial

such

cells. How-

ever, the EC>, for free Ca2+, even with estimates of endogenous Ca2+ added, are considerably bellum or bovine aortic endothelial because

the esophageal

zyme activity

nute incubations

protein-‘.min-’

at 22°C).

When

values were multiplied

had much

protein-‘.min-’

at 34°C compared

with 960 nmol.mg bations

preparations

(35 pmol.mg

lower than rat cerecells. This is probably less en-

for 15-mi-

with rat cerebellum for 5-minute

the esophagus-free

incu-

Ca’+

EC,,

by the ratio of cerebellum/esopha-

gus specific activity,

a mean free Ca*+ EC5o value for the

esophagus

of 197 t

segments

which is almost identical bellum.

The rat cerebellum

mmol/L EDTA,

41 nmol/L was obtained,

to the 200 nmol/L for rat cerehomogenates

contained

which was twice the concentration

in our preparations.

Comparison

1.0 used

with bovine aorta endo-

thelial cell enzyme, which also used 1.0 mmol/L EDTA, is not possible because the CaZf also bathed the rat lung

Discussion plays an important

ble fraction

is dependent

it similar to the constitutive

no additional

when the particulate

from our crude prepa-

esophagus

found

does

and is found in mac-

activity

making

form of the enzyme observation

form

in synapto-

Another type, inducible,

The enzymatic

cium and NADPH,

Ca’+,

It is the constitutive

not appear to be calcium-dependent rophages8

NADPH,

studies

suggest

role in the neuromuscular

that NO control

of

fibroblast

(RFL-6)

5’-cyclic

adenosine

Our

inability

cells used to detect NO by stimulated monophosphate

to show NO

production.‘”

synthase

activity

in the

ESOPHAGEAL NO SYNTHASE

June 1994

particulate

fraction

differs from data reported

from the

Our identification ated

other investigators

NADPH-diaphorase-positive

seems more dependent

tors than the soluble

fraction.

on some cofac-

Forstermann

et a1.19 found

muscle

of NO synthase

rat anococcygeus21 and vascular endothelium. The NO synthase activity in the particulate fraction observed by

However, plexus

esophagus

it is consistent

of the striated

fraction

personal

communication,

of bovine aortic endothelial cells had a partial dependence on exogenous calmodulin. Mitchell et al.” found signifi-

aphorase

staining

for NO

synthase

cant activity

tional

that the potassium

chloride-washed

in the particulate

geus, which was increased added.

whereas

of the anococcy-

when tetrahydrobiopterin

cells and rat anococcy-

the anococcygeus

muscle

required

but not added calmodulin.

tion of calmodulin

to our preparation

did not enhance

(data not shown).

The enzyme esophagus

activity

appear

and show neither bition

added

The addi-

of the LES and smooth

to follow Michaelis-Menten substrate

activation

muscle kinetics

nor substrate

This is similar to that of the constitutive enzyme found in the crude enzyme preparation of cerebellar synaptosomes (8.4 Fmol/L inhibited

L-arginine).’

the NO synthase

was consistent antagonists

activity

with the assumption are altering

sic inhibition

of 5’-cyclic

in rat cerebella

that the NO synthase of the latency

guanosine

monophosphate

exposed to 100 pmol/L IC5o of about 6 nmol/L

with L-NNA

the cerebellum

and

were incubated with L-NNA range, a monophasic inhibi-

This is similar

cerebellum

to that observed

in purified

esophageal

muscle along the length of the esophagus may be differentially sensitive to NO or that NO may be compartmentalized in a way that allows it to generate a gradient.

is in

the constitutive it is highly a gradient

characterization NO

synthase.

enzyme

It

seen in

calcium-dependent.

in NO synthase activity

that would be a basis for the timing

physiology.

1. Du C, Murray J, Bates J, Conklin JL. Nitric oxide: mediator of

2.

3.

4.

5.

6. 7.

8.

length of the esophagus.* One possible explanation for the latency gradient and its abolition by antagonists of NO synthase might be a gradient in NO synthase activity along the length of the esophagus. These studies do not support this hypothesis because no gradient in NO synthase activity was seen. It is possible that the smooth

muscle

may be endothelial

References

seems to be important

in setting the timing of peristaltic contractions resulting from swallowing. Inhibitors of NO synthase make swallow-induced contractions nearly simultaneous along the

neurons.23X24 The funcin the striated

of esophageal circular muscle contraction. Work is underway to further characterize this enzyme and its role in

rat

homogenates.22

NO release from enteric neurons

et al.,

NADPH-di-

as a specific stain

NANC-elaborated

in that

along the esophagus

tion response was observed with ICso values of about 400 nmol/L.

(Fang

1993).

this is the first enzymatic

We could not identify

for-

N-methyl-D-aspartate with an and about 600 nmol/L. When

smooth muscle supernatants over a similar concentration

Some of the activity

In summary,

by a

East et al.” found a bipha-

slices incubated

in myenteric

of the esophageal

that L-NNA

along the esophagus

the gradient

direct effect on NO synthesis. mation

The finding

esophagus

January

of

in the myenteric

origin.

with respect to L-arginine.

geal activity

muscle

in the striobservation.

the demonstration

neurons

role of the NO synthase

unknown.

inhi-

The K,,, for the esophahad a value of 7.5 +- 1.4 pmol/L L-arginine.

with

has been proposed

most closely resembles

but not added tetrahydrobiopt-

tetrahydrobiopterin activity

was

fractions seem to have different The aortic endothelial cells re-

quire added calmodulin erin,

fraction

Bovine aortic endothelial

geus muscle particulate cofactor requirements.

particulate

activity

was a surprising

1449

9.

10.

11.

12.

nonadrenergic noncholinergic hyperpolarization of opossum esophageal muscle. Am J Physiol 1991;261:G1012-G1016. Murray J, Du C. Ledlow A, Bates J, Conklin JL. Nitric oxide: mediator of nonadrenegic noncholinergic responses of opossum esophageal muscle. Am J Physiol 1991; 261:G401-G406. Conklin JL. Du C, MurrayJ, Bates JN. Characterization and mediator of inhibitory junction potentials from opossum lower esophageal sphincter. Gastroenterology 1993; 104:1439-1444. Yamato S, Spechler SJ, Goyal RK. Role of nitric oxide in esophageal peristalsis in the opossum. Gastroenterology 1992; 103: 197-204. Tottrup A, Svane D, Forman A. Nitric oxide mediating NANC inhibition in opossum lower esophageal sphincter. Am J Physiol 1991;260:G385-G389. Weisbrodt NW, Christensen J. Gradients of contractions in the opossum esophagus. Gastroenterology 1972;62:1159-1166. Forstermann U, Schmidt HHHW, Pollock JS, Sheng H, Mitchell JA, Warner TD, Nakane M, Murad F. lsoforms of nitric oxide synthase: characterisation and purification from different cell types. Bio them Pharmacol 1991;42:1849-1857. Stuehr DJ, Griffith OW. Mammalian nitric oxide synthases. In: Meister A, ed. Advances in enzymology. New York: Wiley, 1992; 296:287-344. Knowles RG, Palacios M. Palmer RMJ, Moncada S. Formation of nitric oxide from L-arginine in the central nervous system: a transduction mechanism for stimulation of the soluble guanylate cyclase. Proc Natl Acad Sci USA 1989;86:5159-5162. Palmer RMJ, Moncada S. A novel citrulline-forming enzyme implicated in the formation of nitric oxide by vascular endothelial cells. Biochem Biophys Res Commun 1989; 158:348-352. Bredt DS, Snyder SH. Nitric oxide mediates glutamate-linked enhancement of cGMP levels in the cerebellum. Proc Natl Acad Sci USA 1989;86:9030-9033. Mitchell JA, Forstermann U, Warner TD, Pollock JS, Schmidt HHHW, Heller M. Murad F. Endothial cells have a particulate enzyme system responsible for EDRF formation: measurement by vascular relaxation. Biochem Biophys Res Commun 1991;176:1417-1422.

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MURRAY AND CLARK

13. Smith RK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD. Measurement of protein using bicinchoninic acid. Anal Biochem 1985;150:76-85. 14. Scrimgeour KG. Chemistry and control of enzyme reactions. London: Academic, 1977. 15. Mendham J, Cooper D. Analytical chemistry by open learning classical methods. Volume 2. Chichester, England: Wiley, 1987. 16. Cheng KL, Veno K, lmamura T, eds. CRC handbook of organic analytical reagents. Boca Raton: CRC, 1982. 17. Segal J. Cation chelators and their utilization in the preparation of low concentrations of calcium. Biotechnol Appl Biochem 1986;8:423-429. 18. Bredt D, Snyder SH. Isolation of nitric oxide synthase, a calmodulin-requiring enzyme. Proc Natl Acad Sci USA 1990;87:682-685. 19. Forstermann U, Pollock J, Schmidt HHHW, Heller M, Murad F. Calmodulindependent endotheliumderived relaxing factor/nitric oxide synthase activity is present in the particulate and cytosolic fractions of bovine aortic endothelial cells. Proc Natl Acad Sci USA 1991;88:1788-1792. 20. East SJ, Garthwaite J. Nanomolar @-Nitroarginine inhibits NMDAinduced cyclic GMP formation in rat cerebellum. Eur J Pharmacol 1990; 184:311-313.

21.

Mitchell JA, Sheng H, Forstermann U, Murad F. Characterization of nitric oxide synthase in non-adrenergic noncholinergic nerve containing tissue from the rat anococcygeus muscle. Br J Pharmacol 1991; 104:289-291. 22. Struehr DJ, Griffith OW. Mammalian nitric oxide synthases. In: Meister A, ed. Advances in enzymology. Volume 65. New York: Wiley, 1992:287-346. 23. Hope BT, Micheal GT, Knigge KM, Vincent SR. Neuronal NADPH diaphorase is a nitric oxide synthase. Proc Natl Acad Sci USA 1991; 88:2811-2814. 24. Dawson TM, Bredt DS, Fotuhi M, Hwang PM, Snyder SH. Nitric oxide synthase and neuronal NADPH diaphorase are identical in brain and peripheral tissues. Proc Natl Acad Sci USA 1991;88:7797-7801.

Received May 17, 1993. Accepted February 1, 1994. Address requests for reprints to: Joseph A. Murray, M.D., Department of Internal Medicine, University of Iowa Hospitals & Clinics, Iowa City, Iowa 52242. Dr. Murray is the recipient of a career development award from the Department of Veterans Affairs.