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Diabetologia (1999) 42: 978±986
Morphological evidence for the existence of nitric oxide and
carbon monoxide pathways in the rat islets of Langerhans:
An immunocytochemical and confocal microscopical study
P. A l m 1, P. Ekström2, R. Henningsson3, I. Lundquist3
1Department of Pathology, University of Lund, Sweden
2Department of Zoology, University of Lund, Sweden
3Department of Pharmacology, University of Lund, Sweden
ÓSpringer-Verlag 1999
Abstract
Aims/hypothesis. To map the cellular location of in-
ducible and constitutive nitric oxide synthase and
haem oxygenase in rat islets to clarify the morpholog-
ical background to putative nitric oxide and carbon
monoxide pathways.
Methods. Immunocytochemistry and confocal mi-
croscopy.
Results. After treatment with endotoxin, immunore-
activity for inducible nitric oxide synthase was ex-
pressed in a large number of islet cells, most of which
were insulin-immunoreactive beta cells and in single
glucagon-immunoreactive and pancreatic polypep-
tide-immunoreactive cells. Somatostatin-immunore-
active cells lacked immunoreactivity for inducible ni-
tric oxide synthase. In untreated rats, immunoreactiv-
ity for constitutive nitric oxide synthase occurred in
the majority of insulin-immunoreactive and gluca-
gon-immunoreactive cells, in most pancreatic
polypeptide-immunoreactive and somatostatin-im-
munoreactive cells and in islet nerves. Similarly, im-
munoreactivity for constitutive haem oxygenase was
detected in all four types of islet cells. Endotoxin
treatment did not change the pattern of immunoreac-
tivity for constitutive and inducible haem oxygenase.
After treatment with alloxan, insulin-immunoreactiv-
ity was observed only in single islet cells, being almost
devoid of immunoreactivity for constitutive nitric ox-
ide synthase and haem oxygenase.
Conclusion/interpretation. In vivo endotoxin-induced
expression of inducible nitric oxide synthase in insu-
lin-producing and in scattered glucagon-producing
and pancreatic polypeptide-producing cells strength-
ens previous suggestions of a pathophysiological role
for inducible nitric oxide synthase in the develop-
ment of insulin-dependent diabetes mellitus. The
presence of constitutive nitric oxide synthase and
haem oxygenase in all four types of islet cells, togeth-
er with recent functional data of ours support roles
for nitric oxide and carbon monoxide as intracellular,
paracrine or neurocrine modulators of islet hormone
secretion. [Diabetologia (1999) 42: 978±986]
Keywords Pancreatic islets, nitric oxide synthase,
haem oxygenase, imunocytochemistry, confocal mi-
croscopy.
Received: 19 November 1998 and in revised form: 22 March
1999
Corresponding author: P. Alm, MD, PhD, Department of Pa-
thology, University Hospital, S-22185 Lund, Sweden
Abbreviations: CO, Carbon monoxide; HO, haem oxygenase;
HO-1, inducible haem oxygenase; HO-2, constitutive haem ox-
ygenase; NO, nitric oxide; NOS, nitric oxide synthase; GLUC,
glucagon; IG, immunoglobulins; INS, insulin; IR, immunore-
active; LPS, lipopolysaccharide (endotoxin); iNOS, inducible
nitric oxide synthase; cNOS, constitutive nitric oxide synthase;
eNOS, endothelial nitric oxide synthase; nNOS, neuronal ni-
tric oxide synthase; PP, pancreatic polypeptide; SOM, soma-
tostatin; ZnPP, zinc protoporphyrin; FITC, fluorescein isothio-
cyanate conjugated.
Nitric oxide (NO) is a free radical gas that conveys
biological information in a way greatly differing
from that of the classical transmitters. In the nervous
system NO does not act on conventional receptors
but through effects on various regulatory processes,
intracellularly or in the membrane [1]. The formation
of NO is catalysed by the enzyme nitric oxide syn-
thase (NOS), in a reaction in which l-arginine and
oxygen are converted to NO and citrulline. There
are two major types of NOS enzymes; one inducible
isoform iNOS, originally described in macrophages
and also shown to be expressed in a variety of mam-
malian tissues among which are the islets of Langer-
hans [2, 3], and constitutive isoforms (cNOS) present
in neurons (nNOS) and endothelial cells (eNOS) [1].
We and others [4±8] have shown previously that the
pancreatic islets contain a constitutive NOS as deter-
mined by histochemical, immunocytochemical and
biochemical methods. Islet iNOS has been implicated
as an important factor in the pathogenesis of Type I
(insulin-dependent) diabetes mellitus [2, 3], whereas
islet cNOS has been suggested to be involved in the
physiological regulation of insulin and glucagon se-
cretion [4±15]. Thus, both cNOS and iNOS seem to
be of great physiological and pathophysiological im-
portance in the pancreatic islets. Constitutive nitric
oxide synthase was localised by the use of an antiser-
um to the neuronal isoform of cNOS.
There are diverging results as to the cellular loca-
tion of the NOS isoforms within the islets, i.e. whe-
ther only the insulin producing beta cells or, in addi-
tion, other endocrine cell types such as glucagon-pro-
ducing, somatostatin-producing, and pancreatic
polypeptide (PP)-producing cells also contain NOS
activity [4±8, 16±18]. Further, in this context, atten-
tion has also been drawn to another gaseous mole-
cule, carbon monoxide (CO), since a number of re-
cent studies have shown that CO may serve as a neu-
ronal messenger molecule similar to NO [19±21].
Carbon monoxide is produced by the action of haem
oxygenase (HO), at which haem from haemoglobin
is degraded to CO and biliverdin [19±22]. The latter
compound can then be converted to bilirubin, which
is an important antioxidant, the reaction being cataly-
sed by the enzyme biliverdin reductase. Similar to
NOS, HO consists of at least two isoenzymes, an in-
ducible (HO-1), and a constitutively expressed iso-
form (HO-2) [19±21]. Expression of HO-1 is induced
by various stress factors, e.g. fever, starvation, oxida-
tive injury. A cytokine-induced expression of a pro-
tein (presumably HO-1) has previously been ob-
served in islet tissue, and suggested to be a protective
mechanism against oxidative stress [2, 3, 23±25]. Con-
stitutive haem oxygenase is highly expressed in ner-
vous tissue, in which CO may have a transmitter-like
function [19, 20] and recent findings of ours suggest
a role for CO in the regulation of the release of islet
hormones in rats [26].
To further clarify the morphological background
to the putative NO and CO functional pathways in
the islets of Langerhans, the aim of this study was to
map the cellular location of iNOS, cNOS, HO-1 and
HO-2 in islets of normal and alloxan diabetic rats by
means of combined immunocytochemical and confo-
cal microscopical methods.
Materials and methods
Tissue handling. Female Sprague-Dawley rats (300±400 g body
weight, aged about 3±4 months) were purchased from B&K
Universal, Stockholm, Sweden. The animals had free access
to water and standard pellets and were used in different exper-
imental groups consisting of four to six animals. One group
consisted of rats that received no treatment. One group of
rats was given LPS (lipopolysaccharide endotoxin from salmo-
nella typhimurium, Sigma, St Louis, Mo., USA; 10 mg/kg i. p.,
dissolved in saline) and used after 6 h, at which time there is a
high expression of iNOS [34]. One group was treated with al-
loxan (Sigma; 60 mg/kg i.v., dissolved in saline with the addi-
tion of a drop of 0.1 N acetic acid to acidify the solution) and
killed after 5 days. Plasma glucose was determined by a glu-
cose oxidase method [18, 27] to ensure that the animals had be-
come diabetic. The concentrations of plasma glucose in freely
fed diabetic animals were 15.1±38.7 mmol/l (total range) com-
pared with 8.9±11.7 mmol/l in normal rats. The rats were an-
aesthesized with ketamine (100 mg/kg intramuscular; Ketalar,
Parke Davis, Barcelona, Spain) and xylazin (15 mg/kg intra-
muscular; Rompun, Bayer, Leverkusen, Germany) and perfus-
ed transcardially through the ascending aorta, first with 100 ml
of ice-cold calcium-free Krebs buffer (containing 0.5 g/l sodi-
um nitrite and 10.000 iU/l of heparin), and then with 300 ml of
an ice-cold, freshly prepared solution of 4% formaldehyde in
phosphate buffered saline (PBS, 0.1 mol/l, pH 7.4). The pan-
creatic glands were then rapidly dissected out and divided
into pieces, which were fixed in the same fixative for 4 hours.
After this they were rinsed in ice-cold 15 % sucrose in PBS
(three rinses during 48 h). The tissue specimens were frozen
in isopentane at ±40°C and then stored at ±70°C. Principles of
laboratory animal care (NIH publication No 85±23 1985)
were followed and the experimental design was also approved
by the animal ethics committee of the University of Lund,
Lund, Sweden.
Immunocytochemistry. Cryostat section were cut at a thickness
of 8 mm and thaw-mounted onto chrom alum-coated glass
slides and air dried for 30 min to 1 h. To show iNOS, nNOS,
HO-1 and HO-2, sections were pre-incubated in PBS with
0.2% Triton X-100 for about 2 h, and then incubated for
2 days in the presence of rabbit antisera to iNOS, nNOS, HO-
1 or HO-2. The antiserum to iNOS (1:500) was generated in
rabbits against a 25 amino acid peptide of a cloned inducible
NOS from a murine macrophage cell line [28, 29]. The antisera
to nNOS were generated in rabbits against a 15 amino acid se-
quence (nNOS-15, 1:1280) [30], or a 21 amino acid sequence
(nNOS-21, peptide 58; 1:2000) [31] from the C-terminal part
of a cloned rat cerebellar NOS [32]. The antiserum to HO-1
(1:500; code OSA 100, StressGen Biotechnol, Victoria, Cana-
da) was generated in rabbits against rat liver HO-1. The rabbit
HO-2 antiserum (1:1000, code OSA-200; StressGen) was gen-
erated in rabbits against rat testes HO-2. After rinsing in PBS
(three rinses during 10 min), the sections were incubated for
90 min with fluorescein isothiocyanate conjugated (FITC)
P.Alm et al.: Islet nitric oxide synthase and haem oxygenase 979
swine anti-rabbit immunoglobulins (IG) (1:80; Dakopatts,
Stockholm, Sweden) or Texas red-conjugated affinity purified
F(ab¢)2fragments of donkey anti-rabbit IG (1:80; code
711±076±132; Jackson Immuno Research, West Grove, Pa.,
USA). After rinsing, the sections were mounted in PBS/glycer-
ol with p-phenylenediamine to prevent fluorescence fading
[33].
To show two antigens simultaneously [34], sections were in-
cubated overnight with iNOS, nNOS, or HO-2 antisera (see
above), rinsed and then incubated overnight with antisera gen-
erated in guinea-pigs to insulin (1:16 000), glucagon (1:4000)
and pancreatic polypeptide (1:500) (the latter antisera pur-
chased from Linco Res, St Louis, Mo., USA), and a mouse
monoclonal antiserum to somatostatin (1:5 of a prediluted an-
tiserum; cat no 8330±0496, Biogenesis, Poole, England). After
rinsing, the sections were incubated for 90 min with FITC
goat anti-guinea-pig IG or goat anti-mouse IG (1:80; Sigma,
St Louis, Mo., USA), rinsed and then incubated with Texas
red conjugated affinity purified F(ab¢)2fragments of donkey
anti-rabbit IG (1:80; see above). The sections were rinsed and
mounted as described above. An Olympus 3 ´50 fluorescence
microscope (LRI Instrument AB, Lund, Sweden) equipped
with epi-illumination and appropriate filter settings for Texas
Red-immunofluorescence and FITC-immunofluorescence
was used for the examinations of the sections [35].
The primary and secondary antisera were diluted in PBS. In
control experiments no immunoreactivity could be detected in
sections incubated in the absence of the primary antisera or
with nNOS-15, HO-2, glucagon, somatostatin or pancreatic
polypeptide antisera absorbed with excess of the correspond-
ing immunizing antigen (100 mg/ml). No absorption controls
could be done with the iNOS or nNOS-21 antisera as antigenic
substances were not available. The characteristics of the iNOS
and the nNOS antisera have been presented previously
[29±31]. In control experiments iNOS-immunoreactivity was
only observed in LPS-induced tissues (macrophages in lung
and liver), in which no nNOS-immunoreactivity could be
seen. As cross reactions to antigens sharing similar amino acid
sequences cannot be completely excluded the structures shown
are referred to as iNOS-, nNOS-, HO-1-, HO-2-, insulin-(INS-),
glucagon-(GLUC-), pancreatic polypeptide-(PP-), or soma-
tostatin-(SOM-) immunoreactive (IR).
Confocal microscopy. To evaluate whether two immunoreac-
tivities were colocalized within the same cellular structures,
sections were analysed in a confocal laser scanning microscope
(Multiprobe 2001 TM CLSM; Molecular Dynamics) equipped
with an Ar/Kr laser and an inverted Nikon Diaphot TMD mi-
croscope as described elsewhere [35].
Results
No iNOS-immunoreactivity could be detected in is-
lets of untreated animals. After LPS treatment,
iNOS-immunoreactivity was expressed in a large
number of islet cells, which were diffusely spread
over the islets (Fig.1A, D, G, J). Double immuno-
staining showed that these cells were also INS-IR,
which was further verified by confocal microscopy
(Fig.1C).
Moreover, single iNOS-IR cells also displayed
GLUC-immunoreactivity and PP-immunoreactivity
(Fig.1F, I) but in most of the GLUC-IR and PP-IR
cells iNOS-immunoreactivity was lacking. No iNOS-
IR cells expressed SOM-immunoreactivity (Fig.1L).
In comparison, treatment with LPS did not seem to
change the nNOS-immunolabelling pattern with
both the nNOS antisera used.
Constitutive NOS expressed as nNOS-immunore-
activity could be detected in the cytoplasm of most is-
let cells of untreated animals (Fig.2A, D, G). The
number and distribution patterns of nNOS-IR cells
were similar with the two NOS antisera used, al-
though the intensity of the NOS-immunofluores-
cence was weaker with the nNOS-21 than with the
nNOS-15 antiserum. Vessels of capillary size, be-
tween the trabecula of islet cells (Fig.2G) and be-
tween exocrine acini, were accompanied by varicose
NOS-IR nerve terminals, which were also found
around arteries of various sizes. No nNOS-immu-
noreactivity could be detected in endothelial cells of
the islet microvasculature.
Double immunolabelling showed that nNOS-im-
munoreactivity was in most INS-IR cells (Fig.2A±C).
In the periphery of the islets there were, however,
several nNOS-IR cells which lacked INS-immunore-
activity. Most GLUC-IR cells, which were located
along the periphery of the islets, were also nNOS-IR
(Fig.2D±F), although single GLUC-IR cells were dis-
covered which lacked nNOS-immunoreactivity. Pan-
creatic polypeptide-IR and some SOM-IR cells,
which were also located in the periphery of the islets,
also expressed nNOS-immunoreactivity (data not
shown), which in the SOM-IR cells were as far as to
the ends of their long and gracile dendritic processes.
In the cytoplasm of almost all islet cells HO-2 im-
munoreactivity could be detected (Fig.3 A, D, G,
L). Double immunolabelling in combination with
confocal microscopy revealed that most INS-IR cells
also showed HO-2 immunoreactivity, although there
were HO-2 IR cell in the periphery of the islets that
lacked INS-immunoreactivity (Fig.3A±C). Further,
along the periphery of the islets there was a broad
ring of GLUC-IR, which also were HO-2 IR (Fig. 3
D±F) and dispersed PP-cells and SOM-IR cells with
extended processes. These also expressed HO-2 im-
munoreactivity (Fig.3G±K, L±N). No HO-1 immu-
noreactivity could be detected in any type of islet
cells.
After treatment with alloxan almost no specific
nNOS-immunoreactivity and HO-2 immunoreactivi-
ty was discovered in most of the damaged beta cells
and INS-immunoreactivity was only seen in single is-
let cells, which were vacuolized and enlarged. In com-
parison, nNOS-immunoreactivity and HO-2 immu-
noreactivity was well preserved only in non beta cells
(GLUC-IR cells and others) (data not shown).
P.Alm et al.: Islet nitric oxide synthase and haem oxygenase980
P.Alm et al.: Islet nitric oxide synthase and haem oxygenase 981
Fig. 1 A±L. Confocal microscopy of rat islets of Langerhans af-
ter treatment with LPS. Left panel: red fluorescence in A,D,G
and Jindicating expression of iNOS-immunoreactivity (Texas
red immunofluorescence). Middle panel: green fluorescence
(FITC-immunofluorescence) shows immunoreactivities for in-
sulin (B), glucagon (E), pancreatic polypeptide (H) and soma-
tostatin (K). Right panel: overlay picture of A+B (=C),
D+E(=F), G+H(=I) and J+K(=L). Cells showing yel-
lowish fluorescence (arrowheads) indicate colocalization of
iNOS/insulin (C), iNOS/glucagon (F), and iNOS/pancreatic
polypeptide (I). Bars with numerals indicate lengths (mm)
Discussion
A characteristic of Type I diabetes is a local inflam-
matory reaction in the pancreatic islets that are un-
dergoing autoimmune destruction [2, 3]. Nitric oxide
has been proposed as a possible mediator in the dam-
age process to the insulin producing beta cells and
there is ample in vitro evidence that IL-1 and other
cytokines are able to induce iNOS expression in islet
tissue [2, 3]. The pancreatic islet consists, however,
of a heterogeneous cell population, making it difficult
to localize the cellular source of iNOS expression and
NO production. It was recently shown that rat islets
exposed to cytokine in vitro expressed iNOS in their
insulin cells whereas the glucagon cells seemed unaf-
fected [7]. No data on the possible existence of iNOS
in somatostatin cells or PP-cells have so far appeared
in the literature.
Lipopolysaccharide (endotoxin) is known to stim-
ulate cytokine production [2, 3]. It is important, how-
ever, to note that apart from cytokines, other factors
also have been suggested to serve as direct or indirect
mediators of effects of LPS. Thus, it is known that
LPS may cause the synthesis of reactive oxygen spe-
cies such as superoxide and hydrogen peroxide, and
that NO can combine with superoxide to form the po-
tent oxidizing agent peroxynitrite [2, 3]. Hence, our
immunocytochemical data cannot be extrapolated to
answer questions on the intimate mechanisms of im-
mune destruction of the islet beta cells. Our results
show that after treatment with LPS in vivo iNOS is
expressed in most INS-IR cells and also, although to
a much lesser extent, in scattered GLUC-IR and PP-
IR cells. No iNOS was detected in cells immunostain-
ed for somatostatin. Our in vivo data of iNOS expres-
sion in INS-IR cells, agree with findings of previous
studies of islets exposed to cytokine in vitro [2, 3, 7].
The observation that iNOS expression can be elicited
in GLUC-IR cells and PP-IR cells has not been de-
scribed previously. This may be explained by differ-
ences between the in vitro and the in vivo situation
and the possible induction by LPS of unknown
iNOS stimulatory factors. On the other hand, since
the fraction of iNOS positive cells among the
GLUC-IR cell and PP-IR cells is much smaller than
among the INS-IR cells, it is not inconceivable that
refined techniques such as confocal microscopy are
required to observe these iNOS positive GLUC-IR
cell and PP-IR cells. It is difficult to explain why
SOM-IR cells are not influenced by LPS. It has
been shown [36, 37] that cells producing somatostatin
in contrast to those producing insulin, glucagon and
PP are not members of the group of endocrine cells
belonging to the amine precursor uptake and decar-
boxylation series. Whether the ability of endocrine
cells to store amine is coupled to that of expressing
iNOS remains to be explained. If NO finally turns
out, however, to be of pathophysiological importance
in the development of Type I diabetes, it is notable
that not only insulin cells but also GLUC-IR cells
and PP-IR cells are able to express iNOS. Whether
the number of these non-beta cells observed express-
ing iNOS is, however, enough to actually contribute
to damaging the beta cells remains to be explained.
It is also tempting to speculate that NO derived by
the action of iNOS in the glucagon cells is at least
partly responsible for the increased glucagon secre-
tion in the diabetic condition. Indirectly, such a
mechanism might also explain why there is an in-
creased somatostatin response to glucagon in diabe-
tes.
P.Alm et al.: Islet nitric oxide synthase and haem oxygenase982
Fig. 2 A±G. Confocal microscopy of islets of Langerhans of
untreated rats. Left panel: red fluorescence in Aand D(Texas
Red immunoflurescence) indicates nNOS-immunoreactivity.
Middle panel: green fluorescence (FITC-immunofluores-
cence) shows immunoreactivities for insulin (B) and glucagon
(E). Right panel: overlay picture of A+B(=C), and D+E
(=F). Yellowish fluorescent cells indicate colocalization of
nNOS/insulin (C) and nNOS/glucagon (F). Bars with numerals
indicate lengths (mm). G. Rat islet of Langerhans with nNOS-
IR varicose nerve terminals (arrowheads) running along ves-
sels of capillary size between endocrine cells. FITC-immuno-
fluorescence. Bar = 100 mm
P.Alm et al.: Islet nitric oxide synthase and haem oxygenase 983
Fig. 3 A±L. Confocal microscopy of islets of Langerhans of un-
treated rats. Left panel: red fluorescence in A,D,G and Jdis-
plays expression of HO-2 immunoreactivity (Texas red immu-
nofluorescence). Middle panel: green fluorescence (FITC-im-
munofluorescence) shows immunoreactivities for insulin (B),
glucagon (E), pancreatic polypeptide (H) and somatostatin
(K). Right panel: overlay picture of A+B (=C), D+E
(=F), G+H(=I) and J+K (=L). Cells showing yellowish
fluorescence indicate colocalization of HO-2/insulin (C), HO-
2/glucagon (F), HO-2/pancreatic polypeptide (I), and HO-2/
somatostatin (L). Bars with numerals indicate lengths (mm)
Our data show that all four endocrine cell types,
i.e. those containing insulin, glucagon, somatostatin
and PP showed immunoreactivity for two different
antisera of the neuronal isoform of constitutive
NOS. As previously discussed it has been known for
several years [2, 3] that rat pancreatic islets are able
to express the inducible NOS isoform after treatment
in vitro with different cytokines. The possibility of a
constitutive NOS isoform being present has, howev-
er, been a matter of debate. We and others [4, 5, 7, 8,
16, 38] have previously observed cNOS activity in rat
and mouse islet cells by both histochemical (NAD-
PH-diaphorase activity) and immunocytochemical
methods. In a recent study, nNOS immunoreactivity
was shown in rat islets with a weak intensity [38] that
was apparently lower than the findings in this study.
This is possibly due to differences in methodology
and nNOS antisera used but also to differences in
brightness of the fluorophores of the secondary anti-
bodies [39]. In comparison, other studies carried out
with other NOS antisera as well as with the NAD-
PH-diaphorase method were either unable to show
cNOS activity in rat islet cells or reported that only
cells containing somatostatin or islet nerves or both
were positive for NOS [40, 41, 42]. The reasons for
these discrepancies are not known. The existence of
different local isoforms, differences between antisera
in recognizing epitopes or differences in methodolo-
gy are possible explanations. Further, the recent ob-
servation that a constitutive eNOS is localized to glu-
cagon and somatostatin (but not to insulin cells) in rat
islets suggests the possibility that both eNOS and
nNOS are localized in the same cell type [5, 18]. In
the present study using two antisera of different
sources directed against neuronal NOS, we observed
that nNOS-immunoreactivity was located in all four
types of endocrine cells. Moreover, confocal micros-
copy disclosed coinciding profiles between cells ex-
pressing nNOS-immunoreactivity and immunoreac-
tivity for insulin, glucagon, pancreatic polypeptide
and somatostatin, respectively. Taken together with
the results from previous studies [4±8, 16] this data
strongly suggest that a constitutive nNOS resides in
all these four cell types and in nervous structures as
well. Moreover, results of most functional studies on
the influence of NOS-inhibitors and NO donors on
insulin and glucagon secretion favour NO as an im-
portant modulator of the secretory processes of these
hormones [4±6, 8±11, 13±15, 43]. Although some ear-
ly studies [8, 10] and a recent one (using islets from
newborn rats) [18] suggested that NO had a positive
effect on glucose-stimulated and l-arginine stimulat-
ed insulin release, we [4±6, 9, 14, 15, 44±46] and oth-
ers [13, 43, 47] have repeatedly shown that NO is
strongly inhibitory to insulin secretion induced by
these secretagogues. The inhibitory effect of NO on
insulin release induced by nutrients is probably due
to the formation of S-nitrosothiols [48], which impair
important regulatory thiol groups. Thiol groups have
long been shown to be essential for stimulus-secre-
tion coupling induced by glucose [49, 50]. In contrast,
NO is a positive modulator of glucagon secretion,
probably acting by stimulating the cyclic GMP system
[5, 6, 44±46].
It has previously been shown that cytotoxicity me-
diated by cytokine can induce expression of HO-1 in
cultured islets [23±25]. That we did not observe any
immunocytochemical evidence for HO-1 expression
in rats islets after injection with endotoxin does not
exclude that HO-1 could be detected by other more
sensitive methods but not by immunocytochemistry
which might be too insensitive in the present situa-
tion. We have very recently obtained evidence for
the presence of mechanisms mediated by HO-2 and
CO in the release of insulin and glucagon from rat is-
lets [26]. Thus, islet tissue was found to produce large
amounts of CO [26]. As this CO production was
strongly suppressed by the HO-inhibitor zinc proto-
porphyrin-IX (ZnPP-IX), and since both insulin and
glucagon secretion from intact islets could be sup-
pressed by ZnPP-IX and stimulated by the HO-sub-
strate haemin, we concluded that CO should be re-
cognized as a putative physiologic stimulator of insu-
lin and glucagon release [26]. The data in this study
strongly suggest that HO-2 resides in all four types
of endocrine cells in rat pancreatic islets and confocal
microscopy showed coinciding profiles between cells
expressing HO-2 immunoreactivity and immunoreac-
tivities for insulin and glucagon as well as for PP and
somatostatin. Hence, the CO-pathway could be of
functional significance as an intracellular modulator
system, not only for the release of insulin and gluca-
gon [26] but also for the secretion of PP and soma-
tostatin. Thus, from the most recent data and those
from this study it seems likely that NO as well as CO
formed within the islets of Langerhans are capable
of acting both within their cells of origin and also as
paracrine, neurocrine or even as endocrine media-
tors.
In conclusion, we have shown the existence of a
morphological substrate for a putative functional
role of iNOS, nNOS and HO-2 as important regulato-
ry enzymes in the physiology and pathophysiology of
hormone secretion from the islets of Langerhans.
Acknowledgements. The technical help of L. Thuresson and
the secretarial help of E. Björkbom is gratefully acknow-
ledged. This study was supported by the Swedish Medical Re-
search Council (12X-11205, 14X-4286), the foundations of
Crafoord, Magnus Bergvall, Albert Påhlsson, Thelma Zoga
and ke Wiberg, the Swedish Diabetes Association and the
Medical Faculty, University of Lund, Lund, Sweden. The gen-
erous supply of iNOS and nNOS-21 antisera by V. Riveros-
Moreno, Wellcome Research Laboratories, Beckenham,
England is gratefully appreciated.
P.Alm et al.: Islet nitric oxide synthase and haem oxygenase984
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