NADPH-Diaphorase activity in neurons of the mammalian pancreas: Coexpression with vasoactive intestinal polypeptide

1993;105:999-1008

GASTROENTEROLOGY

NADPH-Diaphorase Activity in Neurons of the Mammalian Pancreas: Coexpression With Vasoactive Intestinal Polypeptide TOORU SHIMOSEGAWA, TAKASHI ABE, AKIHIKO MASARU KOIZUMI, and TAKAYOSHI TOYOTA Third Department

icotinamide

adenine

dinucleotide

phosphate-

diaphorase (NADPH-d) is an enzyme that catalyzes the chemical reaction of converting soluble ni-

troblue tetrazolium salt to insoluble blue dye formazan in the presence of NADPH.’ By histochemistry for NADPH-d, a specific group of neurons in the brain have been shown to possess the activity of this enzyme, although

REISHI

ABE,

YOSHIFUMI

KIKUCHI,

of Internal Medicine, Tohoku University School of Medicine, Sendai, Japan

Background: To provide a morphological basis for understanding the role of nitric oxide in the pancreas, the present study was designed to clarify the localization and distribution of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) activity, a marker of NO synthase, in the pancreas of several mammalian species, including humans. Methods: NADPH-d activity was examined in the rat, guinea pig, dog, and human pancreas by histochemistry. In addition, the possibility of coproduction of NO and vasoactive intestinal polypeptide (VIP) was investigated by a combined use of histochemistry and immunohistochemistry. Results: In the pancreas, NADPH-d activity was localized in nerve fibers, nerve cell bodies, and the vascular endothelium. Nerve fibers with the enzyme activity were chiefly distributed in the exocrine pancreas and showed species differences in the distribution. Nerve fibers stained for NADPH-d were also observed in the endocrine pancreas, but the enzyme activity was not detected in the islet cells. Part of the nerve fibers and nerve cell bodies coexpressed NADPH-d activity and VIP-immunoreactivity. _Conclusions: These results suggest that NO may act as a neuronal mediator and an endothelium-derived relaxing factor and may physiologically interact with VIP in the mammalian pancreas.

N

SATOH,

its role has remained

unknown.’

Recently,

however, strong evidence has been presented that NADPH-d corresponds to neuronal nitric oxide synthase (NOS), which forms nitric oxide by conversion of L-arginine to L-citrulline.3-5 NO is a free radical gas, recently identified as an endothelium-derived relaxing factor,6s7 and may be a candidate for nonadrenergic, noncholinergic neurotransmitters in both the central and peripheral nerves.*-” It- is well known that pancreatic functions are under

the strong influence of autonomic nerves.” Besides the classical neurotransmitters like acetylcholine and noradrenaline, garded

various

small

peptides

as neurotransmitters

pancreatic ported

nerve

systems.13

that NADPH-d

detected result

in intrinsic

strongly

However,

activity, neurons

suggests

pancreatic

study,

and distribution because

of

we recently

a marker

re-

of NOS, was

of the rat pancreas.14

This

to classi-

neurotransmitters, should neuronal mediator regu-

we compared

of NADPH-d pigs,

dogs,

the colocalization

intestinal

re-

functions.

In the present of rats, guinea

been

that NO, in addition

cal and putative peptidergic be considered as a putative lating

have

or neuromodulators

the localization

activity and

humans.

of NOS-like

polypeptide-like

in the pancreas In addition, and vasoactive

immunoreactivity

(VIP-

LI) has recently been shown in guinea pig enteric neurons, l5 the possibility of coexpression was examined in the intrapancreatic

neurons.

Materials and Methods Materials ley rats (200-250

Pancreatic

specimens from five male Sprague-Dawg body wt), five male Hartley guinea pigs

(250-300

wt),

g body

five male

body wt), and five humans Two

fresh

from

the

operations, autopsy

specimens segments

Necropsy

patients

specimens

All pancreatic tine histological ducted

of human of resected

and three pancreatic

from

mongrel

dogs

(lo-15

were used in the present pancreas materials specimens

were

obtained

during

surgical

were obtained

who died of nonpancreatic were fixed within

specimens

with the consent

3 hours

The present

of the Ethics

at

diseases. after death.

were judged to be normal

examinations.

kg

study.

by rou-

study was con-

Committee

for Use of

Experimental Animals and Research Use of Human rials of the Tohoku University School of Medicine.

Mate-

Abbreviations used in this paper: NADPH-d, nicotinamide adenine dinucleotide phosphate-diaphorase; NOS, nitric oxide synthase; PFA, paraformaldehyde solution; VIP-L& vasoactive intestinal polypeptide-like immunoreactivity. Q 1993 by the American Gastroenterological Association 0016-5085/93/$3.00

1000

GASTROENTEROLOGY Vol. 105. No. 4

SHIMOSEGAWA ET AL.

Figure 1. NADPH-d actmty in the pancreas. (A)Nerve fibers around acini in the human pancreas (original magnification x500). (8) Nerve fibers around intralobular ducts in the guinea pig pancreas (original magnification X485). (C) Nerve fibers around a capillary in the rat pancreas (original magnification x500). (D) Nerve plexus around a small artery in the rat pancreas (original magnification X410). (E) An interlobular nerve bundle containing numerous nerve fibers in the dog pancreas (original magnification X3 10). (F)Nerve fibers and terminals in an islet of the rat pancreas (original magnification x525).

Fixation and Tissue Preparation While under deep anesthesia with sodium pentobarbital (60 mg/kg Nembutal;

Tokyo-Kasei,

Tokyo, Japan),

rats and guinea pigs were perfused with 60 mL of ice-cold saline through the ascending aorta, followed by perfusion with 360 mL of ice-cold (PFA)

in phosphate

2% paraformaldehyde

solution

buffer (pH 7.4, 0.1 mol/L)

solution.

The pancreas was quickly removed and divided into the head, body, and tail segments. The tissue pieces were then immersed in 2% PFA for 12-24

hours at 4°C. Dogs were

laparotomized under deep anesthesia with sodium pentobarbital (60 mg/kg), and specimens were excised from the head, body, and tail segment of the pancreas. The tissue pieces were immersed in cold 2% PFA for at least 48 hours at 4°C. Specimens of the human pancreas were quickly immersed in cold 2% PFA and kept there for at least 48 hours at 4°C. After fixation, all pancreatic specimens were kept in cold phosphate-buffered saline (PBS; pH 7.2, 0.01 mol/L) containing 10% sucrose for 24 hours at 4°C for cryoprotection. Tissue sections were cut to a thickness of 12 pm on a cryostat (Leitz 1720 digital cryostat; Ernst Leitz Wetzlar,

Table 1. Distribution of NADPH-d-Stained

Nerve Fibers in

the Pancreas Tissues

Rat

Exocrine pancreas Aclni Duct systems Large-caliber ducts Small-caliber ducts Vascular systems Capillaries Arterioles Arteries Venules Large-caliber veins Nerve fibers In nerve bundles Endocrine pancreas Islet. cells t. few; t+,

Guinea pig

Human

Dog

+++

++

+t

++ +

+ +++

++ +

+ ++

+ +++

+++ ++

++ +++

++ ++

t

+

+

+

+

+

t

+

++

+

+

+

t-t+

mixture

reflected

light

455 were used as exciter

t+t+

dak Tri-X

-t

moderate; +++.

abundant;

+++t,

Germany).

All sections

VIP-Antiserum

alum gelatin-coated taining Osaka,

0.3%

by a method

VIP-L1

Japan)

X-100

at 4°C

until

(Wako

anti-VIP

+

A, calcitonin Y, porcine

(Oriental

Yeast

nitroblue (pH

Co. Ltd.,

tetrazolium

8) containing

stained

for VIP

glycerol

tissue

(Wako)

X-100

was stopped

sections

were

pus BHS microscope

Tris-HCl

by rinsing

buffer at

the sections

in

a mixture

of

with

examined

(Olympus

field

for 60 minutes

covered

and PBS (2:1, vol/vol),

P-NADPH

Optical

Co. Ltd.,

the indirect

immunofluorescence

the anti-VIP

serum

Inc., Belmont, 24 hours

7161N;

According

methionine

for 60 minutes

1:700 for the anti-VIP

secondary

antibody.

The

One hundred

the anti-VIP

substance

tissues

serum were

for

rat, guinea P, secretin,

somatostatin,

fourth

specimens

section

nerve

cell bodies

with

only VIP-L1 activity,

pancreas,

and

error of deviation)

cell body was calculated

cut

species. described (1)

activity,

(2)

(3) nerve

cell

and (4) nerve cell bodactivity.

pig, dog,

These percentof the rat, guinea

the mean

percentage

of the respective

in each animal

type

species.

Results

The dilutions 1: 160 for the in a PBS-

animal

activity,

for every specimen

of nerve

Within Nerve

and NADPH-d

and NADPH-d

human

photomi-

were determined:

ages were determined and

by

for VIP immu-

as previously

VIP-L1

+ standard

bright-

evaluated

were consecutively

was processed

with

(mean

Previously

under

of the four

cell bodies

Scientific Inc., Nasections in a moist

mounted

staining. was

of the following

VIP-L1

were

and the same

bright-field

nerve

activatingpolyG conjugated

and

locations

rephotographed

to 200 sections

all pancreatic

and

by the

stainings.

ies without

at room temperature.

used were

with

precise

Percentage of Colocalization Cell Bodies from

fibers

photographed removed,

the corresponding

for NADPH-d

bodies with only NADPH-d

with fluorescein isothiocyanate (Miles perville, IL) was applied to the tissue

and

first immuno-

photomicrographs

chamber

27, glucagon,

pepto the

Nerve

Colocalization

Laboratories

endothelin-1, and pituitary adenylatecyclase peptide 38. Goat anti-rabbit immunoglobulin

chamber

by

were

the dark-field with

were

for NADPH-d

fields

in a moist

with

releasing

P, neu-

of VIP-LI.

were

and their

Peninsula

with porcine,

VIP, but none

histidine

Incubation

to the supplier,

100% cross-reactivity

pig, and human peptide

method.

CA) was performed

at 4°C.

serum shows

(RAS

was performed

VIP-L1

were then

nostainings crographs

Every for VIP

gastrin

described.

microscope,

and the percentages

lmmunohistochemistry

porcine

sections

The coverslips visual

comparing

Tokyo,

lmmunohistochemistry

with

illumination.3s4

using an Olym-

Japan) with a green filter, and photographed with Fuji Neopan F-film (lso 32; Fuji Photo Film Co., Tokyo, Japan).

tissue

were processed

recorded

and 0.2 mmol/L

in 0.1 mol/L

peptide,

of NADPH-d Activity

as previously

cell bodies

sections

by incubating

1 mmol/L Japan)

0.2% Triton

3i”‘C4 The reaction PBS. The

Tokyo,

porcine

of substance

or Met-enkephalin-Arg6-Gly’-Leu*

The pancreatic

Co.,

for histochemistry

was performed with

species exam-

of 100 pg/mL

galanin,

abol-

VIP to the

in PBS con-

recorded.

sections

of porcine

did not affect the strength

fluorescence

tissue

was completely

gene-related

Colocalization VIP-LI

Histochemistry slide--mounted

in tissues were per-

1:700) in any animal

the addition

bombesin,

nerve

staining

Ko-

Co., Roches-

reported.16

of the pancreas

serum (dilution,

ropeptide

and/Nor immunohistochemistry.

NADPH-d

Kodak

of VIP-L1

more than 0.2 pg/mL

ined. However,

tide,

Pharmaceutical

processed

filter.

with

Specificity

previously

in nerves

ished by adding

up on chrome

glass slides and immersed

Triton

and EY-

as a barrier

were photographed

Tests of the specificity formed

very dense.

were picked

filters, and O-515

sections

BHS-RFC

BP-490

ter, NY).

++t+

+

with an Olympus microscope.

pan film (Iso 400; Eastman

neurokinin +t+

and examined fluorescence

The immunostained

+

++

glycerol

antiserum

Wetzler,

1001

NADPH-D ACTIVITY IN THE PANCREAS

October 1993

tivity

Localization Activity

and Distribution

In all animal

species

shown

by blue-colored

of NADPH-d

examined,

NADPH-d

staining

was localized

acin

1002

SHIMOSEGAWA

ET AL.

GASTROENTEROLOGY

Vol. 105,

No. 4

NADPH-D ACTIVITY IN THE PANCREAS

October 1993

nerve

fibers,

thelium cies

nerve

cell bodies,

of the pancreas. differences

NADPH-d

in

activity

tween

body,

spe-

in nerve

of

rat and

In

within

they were not different

be-

and

distribution

neural

and tail segments

of the pan-

creas. NADPH-d

activity

the .parenchyma whereas

pancreas.

In the human

gicall specimens

than

In general, single dles with NADPH-d in the exocrine

pancreas,

in autopsy

nerve

fibers

observed

with in sur-

specimens.

close

to acini

guinea

showed

(Figure

2F and H).

that NADPH-d

staining

terminals

surrounded

(Figure

1E). NADPH-d

ure lF),

terminals

nerve bundistributed (Figure

lA),

but it was not detected

of any animal The density

species

pancreas,

slightly

man pancreas,

of NADPH-d

moderately

in

the islets (Fig-

in the endocrine positive

arteries

nerve

around

NADPH-d lium

density

in the walls

of thicker

ducts,

species activity

examined, were

fine nerve

fibers

acini

and

observed

moderate

whereas

with

moderately

or

in

in the

the enzyme abundantly

around arterioles, where they formed nerve plexuses. Species differences were remarkable in the innervation of islets; in the rat islets, many dotlike nerve terminals

pig and hu(Fig-

activity

veins (Fig-

of endothelial

and the individual staining

(Figure

was not detected or small

cells were 3B and C).

in the endothe-

veins.

C).

fibers

The

distribution

was essentially

However,

nerve

similar

of VIP-L1

to that

in the dog and human

of VIP-L1 nerve fibers that of NADPH-d-positive In the rat, guinea

appeared nerve

animal

much fibers.

that

higher

pancreas,

than

many of ganstrong

VIP (Figure 2A, C, and G). pancreas, most nerve cell bodimmunoreactivity for VIP (Figviews, VIP-L1 within perikarya

in the appearance.

surrounded

species.

the density

the nerve cell bodies within the intrapancreatic glia showed weak to moderate and occasionally

nals

nerve

of NADPH-d-

pancreas

pig, and human

was granular was

pattern

fibers of the respective

in the periphery

of the islets.

of VIP-LI in

As has been shown previously,‘“21 VIP-L1 was localized in nerve elements of the pancreas (Figure 4,4

with NADPH-d activity were observed, whereas in other animal species they were only occasionally seen activity

activity

and large-caliber

by strong

immunoreactivity for Meanwhile, in the dog ies showed very strong ure 2E). In magnified

Nerve cell bodies. NADPH-d

that

in the dog

of arterioles,

Localization and Distribution Pancreatic Nerves

in

they were present ducts. In all animal

fibers

in the guinea

The cytoplasm

pale blue,

of capillaries

positive

guinea pig and human pancreas, more densely around small caliber

activity

were dense

in the endothelium

3A andB),

bordered

rat and dog pancreas,

were

showed

and only sparse in the rat pancreas

(Figure

cells stained

and

fibers

views

and homogeneous

NADPH-d

less dense

also observed

capillaries of the guinea pig, dog, and human pancreas, whereas they were less dense in the rat pancreas. In the nerve

High-power

cells

animals is summarized in NADPH-d activity were

or abundantly

of this en-

Other tissue components. NADPH-d was

examined.

the pancreata of the various Table 1. Nerve fibers with observed

tissues

was also localized within

cell bodies

ure 2I3, D, F, and H).

connective

activity

fibers and nerve

nerve activity

was diffuse

cell bodies

clearly

nerve

most strong

with

ganglion

the

interlobular

of in-

in the perikarya.

ure 3C) in the pancreas.

and

terminals

2B, 13, F, and H). In the

pig pancreas,

around ducts (Figure lB), capillaries (Figure lC), arterioles and arteries (Figure lo), and in nerve bundles in intralobular

and nerve

(Figure

zyme (Figure 2B and D), whereas fewer nerve cell bodies stained for NADPH-d in the human and dog pan-

Nerve

in the rat

nerve trunks and thin activity were chiefly

pancreas

in

and human

less abundant

were more clearly

fibers

distributed

pig, dog,

they were

activity

of nerve

were diffusely

of the guinea

pancreas, NADPH-d

abundance

cell bodies

ganglia

the ganglia

creas

Nerve fibers. An with

shown

trapancreatic

elements.

density

however,

endo-

were considerable

in pancreatic

the same species, the head,

and the vascular

There

1003

nerve

Dotlike

cell bodies

nerve

termi-

also showed

Figure 2. Colocalizahon of NADPH-d actlvrty and VIP-L1 In intrapancreatic ganglion nerve cell bodies. Photomicrographs A. C, E, and G show VIP-LI, and B, D, F, and H show NADPH-d activity. (A and 8) Rat pancreas. All nerve cell bodies in this ganglion show both VIP-LI and NADPH-d activity (original magnification X390). (C and D) Guinea pig pancreas. Most nerve cell bodies in this ganglion show weak to moderate strength of VIP-L1 (C). and most show strong activity for NADPH-d (D) (original magnrfication x415). (E and F) Dog pancreas. Most nerve cell bodies show strong immunoreactivity for VIP (E), but only a few stain for NADPH-d (original magnification x390). (G and H) Human pancreas. Some nerve cell bodies show both VIP-LI and NADPH-d activity, but some show only VIP-L1 (original magnrfication x280).

1004

SHIMOSEGAWA ET AL.

GASTROENTEROLOGY Vol. 105, No. 4

for VIP.

immunoreactivity

nerve

terminals

within

The

density

of VIP-L1

intrapancreatic

ganglia

was

high in the guinea pig, dog, and human pancreas but low in the rat pancreas (Figure 2A, C, E, and G).

Colocalization VIP-LI Nerve fibers.

of NADPH-d Activity

In the rat and guinea pig pan-

creas, many nerve fibers around arterioles ducts showed

and

both VIP-L1

and small

and NADPH-d

activity

(Figure 4A and B), but some showed only VIP-L1 NADPH-d

activity.

In the dog and human pancreas,

numerous nerve fibers showed VIP-L& showed NADPH-d

or

but only a few

activity (Figure 4C and 0). VIP-L1 and NADPH-d

Nerve cell bodies. ity were also colocalized

activ-

in some of the nerve cell bod-

ies within the intrapancreatic

ganglia (Figure U-H).

In the rat and guinea pig pancreas, many of the nerve cell bodies

(Figure

2A-D)

NADPH-d

activity,

whereas in the human pancreas

coexpressed

slightly fewer did the same (Figure contrast,

in the dog pancreas,

VIP-L1

and

2G and H). By

most nerve cell bodies

showed VIP-L1 but only a small part of them simultaneously showed NADPH-d activity (Figure 2E and F). In the dog pancreas, it seemed that nerve cell bodies with a weak or negative

immunoreactivity

were stained strongly for NADPH-d

for VIP

(Figure 2E and

0 To determine which

VIP-L1

calized, 92-196

the percentage of nerve cell bodies in and NADPH-d intrapancreatic

ined in each specimen.

activity

were colo-

neurons

were exam-

The results are summarized in

Table 2. In the rat and guinea pig pancreas,

VIP-L1

and NADPH-d activities were colocalized in 86% and 78% of nerve cell bodies, respectively. In these animals, more than 80% of intrapancreatic ies showed VIP-L1 man

and

activities

Figure 3. NADPH-d activrty in the vascular endothelium of the pancreas. (A) Endothelium of an artery stained for NADPH-d in the human pancreas. The asterisk indicates the arterial lumen (original magnification X195). (6) Endothelial cells showing strong starning for NADPH-d in an artery of the guinea pig pancreas. The asterisk indicates the arterial lumen (original magnification X590). (C) A sheet of an endothelial layer of a large-caliber vein in the guinea prg pancreas. The cytoplasm of endothelial cells stains weakly for NADPH-d. and the individual cells are delineated by strong staining for NADPH-d (original magnification X350).

dog

or NADPH-d

pancreas,

were colocalized

VIP-L1

nerve cell bod-

activity. In the huand

NADPH-d

in 53% and only 16% of

nerve cell bodies, respectively. 80% and 75% of total nerve

However, more than cell bodies examined

showed VIP-L1 in the dog and human pancreas, respectively. No particular differences were noted in the strength of VIP-L1 or NADPH-d activity within the nerve cell bodies or in the percentage of nerve cell bodies with VIP-L1 and/or NADPH-d activity between surgical and autopsy specimens of the human pancreas. Nerve cell bodies that showed neither VIPLI nor NADPH-d activity were
October 1993

Figure 4. Colocalization of NADPH-d activity and VIP-L1 in nerve fibers of the pancreas. (A and 13)The same visual field of a section of the rat pancreas, immunostarned for VIP (A) and stained for NADPH-d (B). Most nerve fibers around an arteriole show both VIP-L1 and NADPH-d activity, but some show only one type of activity. The strong fluorescence in A is an artifact of staining (original magnification X660). (C and D) Nerve fibers around capillaries of the dog pancreas, immunostained for VIP (C) and stained for NADPH-d (D). Thin nerve bundles containing numerous VIP-LI nerve fibers surround capillaries, whereas only a few nerve fibers show NADPH-d activity. It is also noted that VIP-LI and NADPH-d activity are colocalized In some nerve fibers (original magnification x600).

Discussion

atic nerve

In this study, we showed that the presence of NADPH-d activity in neurons and vascular endothelium was a finding common to the rat, guinea pig, dog, and human colocalized

pancreas and that NADPH-d activity was with VIP-L1 within a part of intrapancre-

Table 2. Percentage of Nerve Cell Bodies With NADPH-d Activity and/or VIP-LI in the Pancreas Nerve cell type NADF’H-d+, NADPH-d-, NADF’H-d+, NADPH-d-,

VIP+ VIP+ VIPVIP-

Rat

Guinea pig

Dog

Human

86 + 2 a&i 61+1 0.2 i 0.2

78 t 4 a&2 a*3 3-+1

16 + 6 66 f 5 lOk3 a+ i

53 t 2 23? 1 19?2 3fl

NOTE. The numbers shown for each type of neuron are percentages (mean + SE) of the total nerve cell bodies examined. +, positive; -, negatrve.

fibers and nerve

cell bodies.

Recent

studies

have presented strong evidence that NADPH-d corresponds to neuronal NOS. 3,4 Our present histochemical results, together with the evidence, suggest that the nerves and vascular endothelium of the pancreas may produce NO, which may work as a neuronal mediator and an endothelium-derived relaxing factor in the mammalian pancreas. The presence of several isoforms of NOS has been clarified.22-24 By biochemical characteristics, these isoforms have been classified into two major categories. One is designated as the constitutive NOS, present in neurons and vascular endothelium, and the other as the inducible NOS, present in macrophages, neutrophils, pancreatic islet cells, or hepatocytes.23-25 Recent reports suggest that the constitutive NOS is localized in the rat islet cells or in the cultured rat insulinoma cells and that NO produced in islet cells may be the

1006

SHIMOSEGAWA

mediator

of arginine-stimulated

Careful tion

observations

activity

of guanylate of cyclic

cyclase

guanosine

and the resultant

the only known

numerous

activity

in the rat islets,

obtained

of other

cells agrees with but

NADPH-d

activity

previous

disagrees

cells.25*26 There

animal

activity

in

ancy, but the possibility

pancreatic

should

endocrine

as a neuronal

activity parenchyma,

activity

ganglion

cells,

re-

cells by the activation

of

and it has been

suggested

pancreatic

acinar

are increased

cells,

intracellular

by acetylcholine,

levels

providing in acinar

cells. 36-38 However,

ported

that elevation

(at least

troprusside

release.39 As shown and nerve in the

It is, therefore,

have

nerve from been

with

NADPH-d

vascular

by nitrosourea

on Ca2+ fluxes

the neurotransmitter

could be roughly

grouped

walls of the mouse.28

they suggested a general capable of producing NO

the intracellular

signal

into the enteric

and parasym-

pathetic postganglionic nerves. Our findings indicate that intrinsic neurons of the mammalian pancreas should also be added to the nerve systems that may produce NO. Nerve bundles

in intralobular

and interlobular

nective tissues of all animal species examined tained numerous nonvaricose nerve fibers NADPH-d activity, suggesting that extrapancreatic

con-

neurons

rat and guinea were

coexpressed

pig pancreas, coexpressed

or neuromo-

transduction

by NO and the biological pancreas.

and VIP-LI, suggesting that these duce both NO and VIP as neuronal VIP-L1

nerve fibers not only in

it is important action

pancreatic

is localized in nerve cell bodies pharynx, bladder walls, pelvic

or ni-

or amylase

study, were distributed

role of NO in the pancreas,

induced exocrine

re-

but also in ducts and acini in the pan-

creas. To establish clarify

it has been

in the p resent

activity

systems

dulator

of cGMP

has no effect

NADPH-d activity within the tongue,

and the intestinal

bom-

cyclase

The present study of colocalization and nerve cell bodies confirmed that

Based on these observations, rule that peripheral neurons

of cGMP

cholecystokinin,

regarded as the parasympathetic postganglionic. Recently, it has been shown by Grozdanovic et al. that

nerve plexuses,

that gua-

nylate cyclase-dependent mechanisms are involved in the insulin secretion from islet B cells.35 In dispersed

phore,

were diffusely

which

path-

that NO

endocrine

conceivable that at least a part of intrapancreatic fibers stained for NADPH-d may have originated the intrapancreatic

muscle

confirmed

and A231 87, a calcium ionoproof of the presence of guanylate

were shown

examined.

cyclase

signal transduction

are

physalaemin,

mediator.

in the pancreatic

guanylate

smooth

produc-

(cGMP)

besin,

the pancreatic

fibers with NADPH-d

of all the animals

on rat

laxes arterial

intracellular

No. 4

showing

be considered

that NO regulates

cell bodies with NADPH-d

Our

for this discrep-

in the rat pancreas) functions

species.

in pancreatic

others

is no clear explanation

ob-

was not

observations

with rat

termi-

were

this finding

of no NADPH-d

pancreas2’

nerve

for NADPH-d

although

in the pancreas

observation

dotlike

Vol. 105,

monophosphate

way for NO. 30-34 It has been

a strong

pancreas

tion

species

with

Nerve

vation

but no

nals

within

the localiza-

islet cells,

By contrast,

distributed

secretion.25’26

was seen in the islet cells of any animal

examined. served

insulin

were made to clarify

of NADPH-d

staining

acinar

GASTROENTEROLOGY

ET AL.

to

pathway

of NO on the in nerve fibers a part of intra-

NADPH-d

activity

neurons may promediators. In the

NADPH-d in a large

activity

and

proportion

of

nerve cell bodies within the ganglia (about 80%). However, in the dog and human pancreas, only about 16% and 53% of ganglion cell bodies, respectively, coexpressed them, although in both animal species more than 75% of nerve cell bodies showed VIP-LI. These observations strongly suggest that VIP-containing rons in the mammalian pancreas could be divided

neuinto

conwith ori-

two groups: neurons capable of synthesizing both VIP and NO and neurons synthesizing only VIP. Most

gins with the enzyme activity may also contribute to the innervation of the pancreas. In rats, NADPH-d activity has been shown in the nerve cell bodies of the sensory ganglia,‘” but not in the nerve cell bodies of the sympathetic ganglia. ‘* It is possible, therefore, that nerve fibers with sensory origins may constitute a part of the NO-synthesizing nerve system in the mammalian pancreas. Species differences were evident in the distribution of NADPH-d-positive nerve fibers in the pancreas, suggesting that NO may exert different modes of action on the pancreas from species to species. The acti-

nerve cell bodies in the rat and guinea pig pancreas correspond with the first type, whereas in the dogpancress, the latter type neurons are dominant. Because the variation of the ratio of the two types of VIP neurons was relatively small in the respective animal species, the ratio may be genetically determined from species to species. The importance of VIP in the mammalian pancreas was suggested by the presence of VIP-L1 in most neurons in the pancreas, irrespective of animal species. Meanwhile, the existence of NADPH-d activity in only a part of VIP-L1 neurons in the dog pancreas suggests that NO may exert a supple-

October 1993

ment-ary

NADPH-D ACTIVITY IN THE PANCREAS

role for VIP or may bear some specialized

in the pancreas of at least the dog species. By immunohistochemistry, it has been that

NADPH-d

choline

is colocalized

acetyltransferase

in certain

in the brain.40T4’ Recently, LI and VIP-L1

has been

teric neurons,15

mdicating

sion and coproduction to th.e intrinsic but instead

systems

needed

to

meaning

neurons

clarify

suggested

neuropeptide groups

of neurons

the colocalization shown

Y or

of NOS-

in the guinea

that the possible

pig encoexpres-

of NO and VIP are not specific

represent

nervous

with

role

of the mammalian a general

of digestive

pancreas,

rule for the intrinsic

organs.

the

physiological

of the possible

coproduction

Further

study

and

biological

is

fiber types innervating the taenia of the guinea-pig caecum. Cell Tissue Res 1992;270: 125- 137. 16. ShimosegawaT, Moriizumi S, Koizumi M, Kashimura J, Yanaihara N, Toyota T. lmmunohistochemical demonstration of galaninlike immunoreactive nerves in the human pancreas. Gastroenterology 1992; 102~263-27 1. 17. Larsson LI. Innervation of the pancreas by substance P. enkephalin. vasoactive intestinal polypeptide and gastrin/CCK immunoreactive nerves. J Histochem Cytochem 1979;27: 1283- 1284. 18. Bishop AE, Polak JM, Green IC, Bryant MG, Bloom SR. The location of VIP in the pancreas of man and rat. Diabetologia 1980; 18:73-78. 19. DeGiorgio R, Sternini C, Anderson K, Brecha N, Go VLW. Tissue distribution and innervation pattern of peptide immunoreactivities in the rat pancreas. Peptides 1992;13:91-98. 20.

of NO and VIP.

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Received January 13, 1993. Accepted June 3, 1993. Address requests for reprints to: Tooru Shimosegawa, M.D., Third Department of Internal Medicine, Tohoku University School of Medicine, l-l Seiryo-machi, Aoba-ku, Sendai, Miyagi 980, Japan. The autopsy specimens of the human pancreas used in this study were supplied by the Department of Pathology and the surgical specimens by the First Department of Surgery, Tohoku University School of Medicine, Sendai, Japan.