Galanin is localized in sympathetic neurons of the dog liver
Thomas O.
Mundinger1,3,
C.
Bruce
Verchere1,3,
Denis G.
Baskin1,2,3,
Michael R.
Boyle1,3,
Stephan
Kowalyk1,3, and
Gerald J.
Taborsky Jr.1,3
1 Departments of Medicine and
2 Biological Structure, University
of Washington, Seattle 98195; and
3 Division of Endocrinology and
Metabolism, Veterans Affairs Puget Sound Health Care System, Seattle,
Washington 98108
 |
ABSTRACT |
Stimulation of canine hepatic nerves releases
the neuropeptide galanin from the liver; therefore, galanin may be a
sympathetic neurotransmitter in the dog liver. To test this hypothesis,
we used immunocytochemistry to determine if galanin is localized in
hepatic sympathetic nerves and we used hepatic sympathetic denervation
to verify such localization. Liver sections from dogs were
immunostained for both galanin and the sympathetic enzyme marker
tyrosine hydroxylase (TH). Galanin-like immunoreactivity (GALIR) was
colocalized with TH in many axons of nerve trunks as well as individual
nerve fibers located both in the stroma of hepatic blood vessels and in
the liver parenchyma. Neither galanin- nor TH-positive cell bodies were
observed. Intraportal 6-hydroxydopamine (6-OHDA) infusion, a treatment
that selectively destroys hepatic adrenergic nerve terminals, abolished
the GALIR staining in parenchymal neurons but only moderately
diminished the GALIR staining in the nerve fibers around blood vessels.
To confirm that 6-OHDA pretreatment proportionally depleted galanin and
norepinephrine in the liver, we measured both the liver content and the
hepatic nerve-stimulated spillover of galanin and norepinephrine from
the liver. Pretreatment with 6-OHDA reduced the content and spillover
of both galanin and norepinephrine by >90%. Together, these results
indicate that galanin in dog liver is primarily colocalized with
norepinephrine in sympathetic nerves and may therefore function as a
hepatic sympathetic neurotransmitter.
norepinephrine; hepatic glucose production; hepatic blood flow
 |
INTRODUCTION |
THE 29-AMINO ACID neuropeptide, galanin, is
found in both central and peripheral neurons of several species (4, 20,
26, 30). In the dog, galanin is found in intrinsic nerves of the gut
(14) and in postganglionic sympathetic nerves of the pancreas (8).
Galanin-like immunoreactivity (GALIR) (1) and galanin mRNA (29) have
been found in the majority of cell bodies of dog celiac ganglia, which
supply postganglionic sympathetic nerves not only to the pancreas but
also to the stomach, duodenum, and liver. Stimulation of hepatic
sympathetic nerves projecting from the celiac ganglia releases galanin
from the liver (17); therefore, galanin may be a sympathetic
neurotransmitter in the dog liver like it is in the pancreas (9).
Five criteria can be used to classify a peptide as a sympathetic
neurotransmitter or neuromodulator (2). First, the peptide is localized
in postganglionic sympathetic nerves within the organ. Second, the
peptide is released from the organ during sympathetic nerve
stimulation. Third, the biological effect of nerve stimulation is not
completely blocked by neutralizing classical neurotransmitter (i.e.,
norepinephrine) action. Fourth, specific peptide antagonism or
immunoneutralization abolishes the effect of sympathetic nerve stimulation that persists during adrenergic blockade. Fifth, the local
infusion of exogenous peptide is sympathomimetic. In the case of canine
hepatic galanin, only the second criterion has been clearly
demonstrated (17). However, preliminary data relevant to the fifth
criterion suggest that hepatic arterial infusions of galanin
potentiates norepinephrine's stimulation of hepatic glucose production
(HGP) (27).
The goal of the present study was to determine whether or not canine
hepatic galanin is localized in sympathetic nerves, thus addressing the
first criterion above. To demonstrate that galanin is colocalized with
norepinephrine in hepatic sympathetic nerves, we stained liver sections
of control dogs for both galanin and tyrosine hydroxylase (TH), an
enzyme marker of sympathetic nerves. We also looked for a parallel
decrease of GALIR and TH staining in dogs whose livers were
sympathectomized by a prior intraportal infusion of 6-hydroxydopamine
(6-OHDA). To verify that hepatic galanin is localized in sympathetic
nerves, we decreased both the liver content and the hepatic
nerve-stimulated release of norepinephrine with 6-OHDA pretreatment and
tested for parallel galanin reductions.
In addition to measuring sympathetic neurotransmitter release during
hepatic nerve stimulation (HNS), we compared the nerve-stimulated changes in HGP and hepatic arterial blood flow (HABF) in control vs.
6-OHDA-pretreated dogs. 6-OHDA pretreatment markedly decreased the HGP
response to 8-Hz nerve stimulation yet only modestly reduced the HABF
response. These observations, coupled with the staining data, are
consistent with greater sympathetic innervation of the vasculature than
the hepatocyte in the dog liver. To investigate the physiological
implications of such differential innervation, we electrically
stimulated the hepatic nerve at low frequency (1 Hz) in control dogs
and demonstrated its ability to elicit an HABF response in the absence
of an HGP response.
 |
MATERIALS AND METHODS |
Animals, surgical procedures, and 6-OHDA pretreatment.
For acute, terminal studies, adult male dogs (28-35 kg) of mixed
breed were fasted overnight and surgically prepared as described in
detail elsewhere (17). Briefly, anesthesia was induced with the ultra
short-acting barbiturate thiamylal sodium (Surital, Parke Davis, Morris
Plains, NJ; 30 mg/kg iv) and maintained with halothane (0.8%) in 100%
oxygen administered by positive pressure ventilation from a calibrated
vaporizer. Adequate levels of anesthesia during surgery and
experimentation were verified by maintenance of normal blood pressure
and heart rate and by an absence of a pedal and an eye reflex. A
midline laparotomy was performed to allow placement of sampling
catheters and blood flow probes (Transonic Systems, Ithaca, NY). Blood
sampling catheters were placed in the femoral artery, portal vein, and
hepatic vein, the latter being a Swan-Ganz catheter (Arrow
International, Reading, PA) introduced in the femoral vein and
fluoroscopically guided into a hepatic vein. The catheter tip was
advanced to a point where hepatic venous blood from several branches
flowed freely by the uninflated tip, and blood from the inferior vena
cava could not flow in a retrograde fashion around the inflated tip. An
infusion catheter was placed in the femoral vein for saline
administration. The nerve sheath surrounding the common hepatic artery,
the anterior hepatic plexus, was dissected free from the vessel midway
between the celiac ganglia and the branching of the gastroduodenal
artery. The anterior hepatic plexus was ligated and cut, and a
stimulation electrode was placed around the proximal end of the
anterior hepatic plexus at a distance of one half inch from the cut.
Transection of the anterior hepatic plexus ensured that no retrograde,
afferent stimulation resulted from HNS. Ultrasonic blood flow probes
were placed around the common hepatic artery at the point where the nerve sheath had been stripped from the artery and around the portal
vein as it enters the liver. After the completion of the surgery, a
60-min stabilization period preceded basal determinations.
Ten days before HNS studies, animals undergoing hepatic sympathectomy
received an intraportal infusion of 6-OHDA (Sigma, St. Louis, MO), a
treatment that markedly reduces the number of adrenergic nerve
terminals in the liver (3). Because extraction of catecholamines by the
liver is avid (17), intraportal 6-OHDA was expected to selectively
denervate the liver. The adrenal medulla, peripheral cholinergic
neurons, and central neurons are unaffected by intraportal 6-OHDA
infusion (13). To access the portal vein, a midline laparotomy was
performed under aseptic conditions, and a splenic vein was catheterized
for intraportal infusion of 6-OHDA (125 µg · kg
1 · min
1 × 20 min), as described recently (21). Fifteen minutes before the
intraportal infusion of 6-OHDA, the
-adrenergic antagonist esmolol
(500 µg/kg plus 300 µg · kg
1 · min
1
iv) was infused to reduce the potentially fatal arrhythmias induced by
large amounts of norepinephrine which are released from hepatic noradrenergic nerve terminals immediately after 6-OHDA treatment. Heart
rate and mean arterial blood pressure were monitored and kept in
acceptable ranges (heart rate <180 beats/min, mean arterial blood
pressure <160 mmHg) by further
- and
-adrenergic blockade (phentolamine, 1.5 mg iv bolus/injection; propranolol, 1.0 mg iv
bolus/injection, respectively) as needed. Femoral arterial and portal
venous catheters were removed, wounds were sutured, anesthesia was
discontinued, and dogs were allowed to recover from surgery only when
blood pressure and heart rate were stable in the absence of adrenergic
blockade.
All animals included in these studies were certified as healthy by the
Veterinary Medical Officer of the Veterans Affairs Puget Sound Health
Care System (VAPSHCS) and exhibited normal white blood cell counts,
hematocrit, temperature, food intake, urination, and defecation before
acute, terminal nerve stimulation studies. All research involving
animals was conducted in an American Association for Accreditation of
Laboratory Animal Care-accredited facility. All protocols were designed
to ensure appropriate ethical treatment of the animals and were
approved by the Institutional Animal Care and Use Committee of the
VAPSHCS.
HNS protocol, blood sampling, and assays.
Two separate HNSs were performed on each of four control and six
6-OHDA-pretreated dogs. HNS was performed by electrically stimulating
the sheath surrounding the hepatic artery using a model S-44 stimulator
(Grass Instruments, Quincy, MA). HNS was performed during hexamethonium
(1 mg/kg plus 0.7 µg · kg
1 · min
1
iv) and atropine (0.25 mg/kg plus 0.4 µg · kg
1 · min
1
iv) treatment to block the effect of any stimulated parasympathetic nerves that may run in the hepatic arterial sheath. HNS parameters were
as follows: frequency = 1, 4, or 8 Hz; current = 10 mA; pulse duration = 1 ms. Blood samples were taken from the femoral artery, portal vein, and hepatic vein, and both hepatic arterial and portal venous blood flows were monitored at each sampling time:
10,
5, and 0 min before; 2.5, 5, and 10 min during; and 15 and 25 min after HNS. The second HNS was performed 45 min after the first HNS.
Blood samples drawn for measurement of plasma GALIR concentration were
placed in tubes containing several proteolytic enzyme inhibitors (7).
Blood samples for norepinephrine analysis were drawn on a mixture (50 µl/2.5 ml blood) of ethylene glycol-bis(
-aminoethyl ether)-N,N,N',N'-tetraacetic
acid (0.09 mg/ml) and glutathione (0.06 mg/ml), and those for glucose
measurement were drawn on EDTA. All blood samples were kept on ice
until centrifugation (3,000 revolutions/min, 20 min, 2°C). The
centrifuged plasma was decanted and frozen at
20°C until
assayed. Unextracted plasma was assayed for galanin by radioimmunoassay
(RIA) (10) with a non-COOH-terminally directed antibody raised against
porcine galanin. Because the species variable portion of the galanin
molecule is limited to the COOH terminus (15, 24), this assay detects GALIR from all species yet examined. Synthetic porcine galanin was used
for assay standard, for canine galanin dilutes in parallel with porcine
standards (17). Plasma norepinephrine was measured in duplicate with a
sensitive and specific radioenzymatic assay (23). The intra- and
interassay coefficients of variation in this lab are 6% and 12%,
respectively. Plasma glucose was measured by the glucose oxidase method
(Beckman, Brea, CA).
Data analysis.
To assess the magnitude of galanin and norepinephrine release from the
liver during nerve stimulation, the hepatic spillovers of galanin
(HGALSO) and norepinephrine (HNESO) were calculated before and during
HNS, as previously described in detail (17) and recently validated
(21). Briefly, neurotransmitter spillover was calculated as the total
rate of neurotransmitter exiting the liver minus that portion that
enters the liver by the hepatic artery and portal vein and escapes
hepatic extraction. The fractional extraction of norepinephrine across
the liver was previously determined in this lab to be ~90% (17). To
calculate this hepatic norepinephrine extraction, tritiated
norepinephrine (NEN, Boston, MA; 1.5 µCi/min iv) was infused, and
after a stabilization period, blood samples were drawn from the femoral
artery, portal vein, and hepatic vein. Blood flows in the common
hepatic artery and portal vein were also measured. Tritiated
norepinephrine was extracted from plasma with alumina, radioactivity
was counted, and hepatic extraction of tritiated norepinephrine was
calculated by an arteriovenous difference equation (17).
To monitor the vascular and metabolic responses of the liver to nerve
stimulation, we measured blood flow in the common hepatic artery and
calculated HGP before and during HNS. HGP was calculated by the method
of Myers et al. (22), which incorporates plasma glucose concentrations
and blood flows in the common hepatic artery, portal vein, and hepatic
vein as well as conversion factors to convert plasma glucose
concentrations to blood glucose concentrations.
All data are expressed as means ± SE. Statistical comparison of
responses to HNS vs. basal were made using a single-tailed paired
t-test. Comparison of neurotransmitter
content between liver sections of control vs. 6-OHDA-treated dogs was
made using a two-sample t-test.
Correlation of the HGALSO and HNESO responses to HNS was performed
using the method of least squares.
Immediately after the completion of the HNS studies, dogs were
euthanized by an overdose of anesthesia and liver sections were
immediately excised for hepatic staining and extraction.
Immunocytochemistry.
The basic procedure for immunocytochemistry has been previously
described (11, 28). Pieces of liver (1-cm cubes) were placed in 4%
paraformaldehyde in 0.1 M phosphate buffer (pH = 7.2) for 2 h,
transferred to phosphate buffer overnight, dehydrated, and embedded in
paraffin. Sections (5 µm) were incubated overnight (4°C) with
antibodies raised in rabbits against porcine galanin (1:4,000) followed
by a 1-h, room temperature incubation with fluorescein isothiocyanate
(FITC)-labeled goat anti-rabbit immunoglobulin G. The sections were
then incubated overnight with monoclonal antibody to TH followed by a
1:20 dilution of Cy3-labeled anti-rabbit polyclonal antiserums for 1 h.
Sections were examined with a fluorescence microscope equipped with
filters for Cy3 and FITC microscopy. By switching filters, galanin and
TH stainings were viewed in the same liver section.
Acid extraction and chromatography.
To assess hepatic galanin and norepinephrine content, excised liver
sections were immediately snap-frozen on dry ice and stored at
70°C. Tissue (1-2 g) was homogenized and boiled in 1 N
acetic acid (10 ml/g tissue, 10 min). The homogenate was then
centrifuged twice (10,000 revolutions/min, 20 min), and the supernatant
was dried and reconstituted in 2 ml of galanin RIA buffer. The
reconstituted extract was stored at
20°C until assayed.
To characterize the molecular size of GALIR in canine liver, we
subjected liver extracts to gel-exclusion chromatography. Liver
extracts (1.5 ml) were applied to a 1 × 50-cm Sephadex G-50 column and eluted with galanin RIA buffer. Fifty 1-ml fractions were
collected and run in the galanin RIA. Synthetic porcine galanin (Bachem, Torrance, CA) and blue dextran were used as galanin-sized and
void volume-sized markers, respectively.
 |
RESULTS |
Hepatic galanin localization.
To determine if hepatic galanin is localized exclusively in hepatic
sympathetic nerves, we dual-immunostained sections of peripheral dog
liver (n = 5) for the presence of both
GALIR and TH. GALIR-positive staining was observed in nerve
trunks (Fig. 1B) and
in individual fibers both surround-ing blood vessels (Fig. 1D) and coursing through liver
parenchyma (Fig. 1F). Similar
localization was observed for TH (Fig. 1,
A, C,
and E). Indeed, most of the GALIR
staining was colocalized with TH. Neither GALIR-positive nor
TH-positive staining was observed in intrahepatic neuronal cell bodies
or other hepatic cell bodies.

View larger version (149K):
[in this window]
[in a new window]
|
Fig. 1.
Liver sections showing nerve trunks near the portal hilus
(A,
B) and individual fibers surrounding
blood vessels (C,
D) or coursing through parenchyma
(E,
F) immunostained for sympathetic
enzyme marker, tyrosine hydroxylase
(A,
C,
E) and galanin
(B,
D,
F). Dual immunostaining for galanin
and tyrosine hydroxylase was performed on same liver sections.
|
|
To verify that hepatic galanin is localized in noradrenergic nerves, we
stained liver sections of dogs that were previously subjected to local,
hepatic chemical sympathectomy induced by intraportal 6-OHDA infusion
(n = 5). The parenchymal staining of
GALIR fibers that was observed in control liver (Fig.
2C) was virtually absent in liver sections from 6-OHDA-treated dogs (Fig. 2D). Surprisingly, GALIR staining of
nerves surrounding blood vessels (Fig.
2A) was still prominent after 6-OHDA
treatment (Fig. 2B).

View larger version (114K):
[in this window]
[in a new window]
|
Fig. 2.
Liver sections showing blood vessels
(A,
B) and parenchyma
(C,
D) from control
(A,
C) and 6-hydroxydopamine (6-OHDA
Rx)-treated (B,
D) dogs immunostained for galanin.
Galanin-positive nerve fibers (arrows) are frequently observed in
stromal tissue surrounding blood vessels in liver from both control
(A) and 6-OHDA-treated dogs
(B ) as well as in liver parenchyma of control dogs
(C ). Liver parenchyma of 6-OHDA-treated dogs
(D ) did not have appreciable galanin-positive
staining. In all panels, bar = 40 µm.
|
|
Hepatic galanin content.
To quantitate the amount of hepatic galanin localized to noradrenergic
nerves, we measured the molar content of hepatic GALIR and
norepinephrine in liver sections of control and 6-OHDA-treated dogs.
The GALIR content of liver sections from control animal was 4.3 ± 1.0 pmol/g tissue (n = 5, Fig.
3A),
whereas that of animals pretreated with 6-OHDA was 0.58 ± 0.17 pmol/g tissue (n = 5). The
norepinephrine content of liver sections from control animals was 3,125 ± 422 pmol/g tissue (Fig. 3B),
whereas that of animals pretreated with 6-OHDA was 202 ± 75 pmol/g
tissue. Thus 6-OHDA pretreatment decreased the hepatic content of GALIR by 87% and decreased the hepatic content of norepinephrine by 94%.
To verify that the decrement of hepatic GALIR content after 6-OHDA
pretreatment was due to a reduction in the amount of a galanin-sized
molecule, acid extracts of peripheral liver tissue of control and
6-OHDA-pretreated dogs were subjected to size-exclusion gel
chromatography. Figure 4 depicts the
average gel filtration profiles of control
(n = 5) and 6-OHDA-pretreated
(n = 5) liver extracts. The majority
of GALIR present in liver extracts of control animals coeluted with
synthetic porcine galanin, and the amount of GALIR eluting at the
porcine galanin marker in 6-OHDA-pretreated animals was reduced by 77%
compared with controls. Therefore, GALIR appears to be predominantly
due to a galanin-sized peptide, and 6-OHDA treatment markedly reduces
the liver content of this peptide.

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 4.
Gel elution profile of GALIR in liver extracts of control
(n = 5) and 6-OHDA-pretreated
(n = 5) dogs. Blue dextran was used
for void volume marker (Vo), and
synthetic porcine galanin was used for galanin peptide marker (GAL).
Values are means ± SE.
|
|
Hepatic galanin release during nerve stimulation.
To demonstrate that 6-OHDA pretreatment depleted hepatic GALIR from a
releasable neuronal pool, we examined the effect of 6-OHDA pretreatment
on the hepatic spillover of GALIR and norepinephrine during 8-Hz HNS.
The calculated HGALSO in control dogs
(n = 8) increased from a
baseline of 0 ± 3 pmol/min to an average of 56 ± 17 pmol/min
(
= +56 ± 18 pmol/min, P < 0.01, Fig.
5A)
between 5 and 10 min of HNS, whereas HGALSO in 6-OHDA-pretreated dogs (n = 12) increased from a
baseline of 0 ± 0 pmol/min to only an average of 2 ± 1 pmol/min
(
= +2 ± 1 pmol/min, P < 0.05; Fig. 5A). Thus 6-OHDA
pretreatment decreased the HGALSO response to 8-Hz HNS by 97%.

View larger version (16K):
[in this window]
[in a new window]

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 5.
Change of hepatic galanin spillover (HGALSO;
A) or hepatic norepinepherine
spillover (HNESO; B) during 8-Hz
hepatic nerve stimulation (HNS) in control and 6-OHDA-pretreated dogs.
Values are means ± SE.
|
|
The concentration of GALIR in hepatic venous plasma, an alternative
index of hepatic neurotransmitter release (12), increased in control
dogs from a baseline of 105 ± 21 pM to an average of 360 ± 85 pM (
= +255 ± 80 pM, P < 0.01) between 5 and 10 min of 8-Hz HNS. In contrast, the concentration
of GALIR in hepatic venous plasma of 6-OHDA-pretreated dogs increased
from a baseline of 37 ± 5 pM to an average of 46 ± 5 pM
[
= +9 ± 9 pM, P = not significant (NS)]. 6-OHDA pretreatment thereby decreased the
increment of hepatic venous GALIR during HNS by 96%.
The HNS-induced increment of GALIR in femoral arterial plasma was
less than that seen in the hepatic vein. The concentration of
GALIR in femoral arterial plasma increased by 53 ± 24 pM
(P < 0.05) from a baseline of 100 ± 17 pM in control dogs but did not increase (
=
1 ± 5 pM, P = NS) from a baseline of
38 ± 4 pM in 6-OHDA-pretreated dogs. Thus 6-OHDA
pretreatment abolished the arterial GALIR increment during HNS.
The magnitude of the norepinephrine response to HNS was correlated to
that of the GALIR response, and 6-OHDA treatment reduced the magnitude
of each by the same percentage. The calculated HNESO in control dogs
increased from a baseline of 202 ± 73 pmol/min to an average of
1,994 ± 545 pmol/min (
= +1,792 ± 528 pmol/min, P < 0.01; Fig.
5B and Table
1). In control dogs, there was a significant positive correlation between the HNESO and HGALSO responses
to HNS (r = 0.95, P < 0.0005, n = 8). HNESO in 6-OHDA-pretreated dogs increased from a baseline of 77 ± 30 pmol/min to an average of
142 ± 65 pmol/min (
= + 65 ± 71 pmol/min,
P = NS, Fig.
5B and Table 1). Thus 6-OHDA
pretreatment produced a decrease of HNESO (96%) that was identical to
the decrease of HGALSO (97%).
The concentration of norepinephrine in hepatic venous plasma in control
dogs increased from a baseline of 94 ± 27 pg/ml to an average of
1,160 ± 304 pg/ml (
= + 1,066 ± 308 pg/ml,
P < 0.01) between 5 and 10 min of
HNS. In contrast, the concentration of norepinephrine in hepatic venous
plasma in 6-OHDA-pretreated dogs increased from a baseline of 33 ± 10 pg/ml to only 128 ± 61 pg/ml (
= + 94 ± 63 pg/ml,
P = NS). 6-OHDA pretreatment thereby decreased the increment of hepatic venous norepinephrine to HNS by
91%.
The increments of femoral arterial norepinephrine in response to
HNS were less than those of the hepatic vein in both control and
6-OHDA-pretreated dogs. Arterial plasma norepinephrine increased 159 ± 36 pg/ml (P < 0.005) from
a baseline of 90 ± 35 pg/ml in control dogs and increased 30 ± 15 pg/ml (P < 0.05) from a baseline of 27 ± 8 pg/ml in 6-OHDA-pretreated dogs. 6-OHDA
pretreatment thereby decreased the increment of femoral arterial
norepinephrine to HNS by 81%.
Metabolic and vascular responses during nerve stimulation.
To determine if 6-OHDA pretreatment produced an equivalent reduction of
the metabolic and vascular responses to HNS, we compared the HGP and
HABF responses to HNS in control vs. 6-OHDA-treated dogs. HGP in
control dogs increased from a baseline of 1.46 ± 0.50 mg · kg
1 · min
1
to an average of 4.97 ± 1.84 mg · kg
1 · min
1
(
= +3.51 ± 1.36 mg · kg
1 · min
1,
P < 0.025, Fig.
6 and Table 1) between 5 and 10 min of 8-Hz HNS. In contrast, HGP in 6-OHDA-pretreated dogs increased from a
baseline of 0.28 ± 0.25 mg · kg
1 · min
1
to 0.77 ± 0.39 mg · kg
1 · min
1
(
= +0.48 ± 0.25 mg · kg
1 · min
1,
P < 0.05) in response to HNS. Thus
6-OHDA pretreatment decreased the HGP response to 8-Hz HNS by 86%.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 6.
Change of hepatic glucose production (HGP) during 8-Hz HNS
in control and 6-OHDA-pretreated dogs. Values are means ± SE.
|
|
Arterial plasma glucose rose progressively during the 10 min of 8-Hz
HNS. Plasma glucose in control dogs rose from a baseline value of 93 ± 3 mg/dl to an average of 114 ± 5 mg/dl (
= +21 ± 6 mg/dl, P < 0.005) between 5 and 10 min of HNS, whereas plasma glucose in 6-OHDA-pretreated dogs rose 5 ± 2 mg/dl (P < 0.025) from a
baseline of 95 ± 2 mg/dl. Thus 6-OHDA pretreatment decreased the
arterial glucose response to 8-Hz HNS by 76%.
HABF in both control and 6-OHDA-pretreated animals decreased during
8-Hz HNS. HABF in control dogs decreased from a baseline of 120 ± 13 ml/min to an average of 64 ± 5 ml/min (
=
56 ± 6 ml/min, P < 0.0005; Fig. 5 and
Table 1) between 5 and 10 min of 8-Hz HNS. The decrement of HABF in
6-OHDA-pretreated dogs was 35 ± 7 ml/min
(P < 0.005; Fig.
7 and Table 1) from a baseline of 106 ± 21 ml/min. Thus 6-OHDA pretreatment decreased the HABF response to 8-Hz
HNS by only 38%, in marked contrast to the 70-100% reduction of
other parameters measured.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 7.
Change of common hepatic arterial blood flow (HABF) during 8-Hz HNS in
control and 6-OHDA-pretreated dogs. Values are means ± SE.
|
|
Low-frequency nerve stimulation.
Because 6-OHDA pretreatment decreased the HGP response more than the
HABF response during high-frequency (8-Hz) nerve stimulation, we sought
to determine if low-frequency nerve stimulation preferentially stimulated the vasculature over the hepatocyte. To simulate lower levels of neural activation in control dogs, we electrically stimulated the hepatic nerve at 1 and 4 Hz and measured HABF, HGP, and HNESO (see
Table 1). One-hertz HNS produced no significant change of either HGP or
HNESO, yet produced a significant decrease of HABF. Four-hertz HNS
increased HGP and HNESO and further decreased HABF. Therefore,
low-frequency (1-Hz) HNS in control dogs produced a disparity between
vascular and metabolic responses that was similar to that seen during
high-frequency (8-Hz) stimulation in 6-OHDA-treated dogs.
 |
DISCUSSION |
Galanin is released from the dog liver during HNS (17), and preliminary
data suggest that galanin potentiates norepinephrine-stimulated increases of HGP (27). Galanin may therefore be a sympathetic neurotransmitter in dog liver that aids in glucose mobilization. The
observation presented in this paper that GALIR is colocalized with TH
in liver nerves suggests that galanin is expressed by hepatic
sympathetic nerves. The colocalization of GALIR and TH in hepatic
nerves is consistent with the colocalization of GALIR peptide (1) and
galanin mRNA (29) with TH in roughly 90% of the cell bodies of the
celiac ganglia, the sympathetic ganglia which project the majority of
postganglionic nerves to the liver. Colocalization of GALIR with TH in
liver nerves is also consistent with the corelease of galanin and
norepinephrine from the liver during HNS (see below) (17). The lack of
GALIR staining in hepatic neuronal cell bodies or other cells in the
liver is evidence that galanin is not expressed by hepatocytes,
Kupffer cells, Ito cells, parasympathetic nerves, or sensory
nerves. Together these data support the hypothesis that galanin is a
sympathetic neuropeptide in the dog liver.
To independently confirm that galanin is localized in sympathetic
nerves of dog liver, we examined the effect of 6-OHDA on neural GALIR
and TH staining. As expected, staining of GALIR and TH in parenchymal
nerves was not observed in liver sections of dogs treated with 6-OHDA,
but, surprisingly, staining of GALIR nerves around blood vessels was
only modestly reduced in comparison to controls. The disproportionate
decrease of neural GALIR staining between the parenchyma and the
vasculature after 6-OHDA treatment could be due to a more dense
innervation of the canine hepatic vasculature compared with the
parenchyma, as suggested in other species by previous reviews (25, 31).
Equal degrees of parenchymal and vascular denervation by 6-OHDA
treatment might then account for the residual staining observed around
the vasculature. Alternatively, it is possible that the liver
parenchyma is perfused more completely than the vasculature with portal
venous blood. Therefore, the portal venous infusion of 6-OHDA could
preferentially denervate the parenchyma. Regardless of which
explanation is correct, because 6-OHDA selectively destroys
noradrenergic nerve terminals (16), the observation of a marked
reduction of GALIR in parenchymal nerves strongly supports the
hypothesis that galanin is a sympathetic neuropeptide in dog liver.
To further confirm that galanin is localized in sympathetic nerves, we
examined the effect of 6-OHDA treatment on GALIR content in acid
extracts of liver sections. The marked reduction of GALIR and
norepinephrine content after 6-OHDA treatment paralleled the marked
reduction of GALIR staining in the liver parenchyma. Again, because the
neurolytic action of 6-OHDA is specific to noradrenergic nerves, these
data again indicate that galanin is colocalized with norepinephrine in
hepatic sympathetic nerves.
The high correlation between changes in HNESO and HGALSO during HNS
suggests that galanin and norepinephrine are coreleased from the same
hepatic sympathetic nerve fibers. Furthermore, 6-OHDA pretreatment
produced a marked and parallel reduction of HGALSO and HNESO responses
to HNS. Interestingly, the molar ratio of galanin to norepinephrine
spillover (1:30) in control dogs during HNS was markedly different from
the molar ratio of galanin to norepinephrine content (1:750) in control
liver tissue. These data suggest either that there are significant
nonreleasable stores of hepatic norepinephrine or that there is
preferential release of galaninergic storage vesicles during the
high-frequency nerve stimulation employed in these experiments. The
latter explanation has been proposed for other neuropeptides (18, 19).
Nonetheless, the high correlation between the spillover of galanin and
norepinephrine in control dogs during HNS and the proportional decrease
of releasable galanin and norepinephrine in 6-OHDA-treated dogs
suggests that galanin is localized in hepatic sympathetic nerves.
The spillover of galanin and norepinephrine from the liver during HNS
was accompanied by an increase of HGP and arterial glucose. Although
norepinephrine is a known stimulator of HGP (6), preliminary data
suggest that coreleased peptides such as galanin may augment the
norepinephrine-stimulated HGP response (27). 6-OHDA pretreatment decreased the HGP response to HNS by an amount similar to the reduction
of nerve-stimulated galanin and norepinephrine spillover. Thus
sympathetic neurotransmitters mediate the HGP response during HNS, but
the relative roles of norepinephrine and galanin in glucose mobilization require clarification.
Although the HGP response to HNS was markedly reduced after 6-OHDA
pretreatment, the HABF response was only modestly reduced. Because the
majority of HABF is supplied to the liver, these data suggest that the
nerves of the liver that remain after 6-OHDA treatment predominantly
supply the vasculature. Alternatively, a sympathetic neurotransmitter
that is not colocalized with norepinephrine could modulate a
significant portion of the vasoconstrictor response to HNS. However,
this latter explanation is highly speculative, and the staining data in
this paper support the former explanation, since some GALIR-positive
fibers remain near the vasculature after 6-OHDA pretreatment. In
addition, GALIR staining of control liver sections show dense
innervation of the vasculature with more diffuse innervation of the
liver parenchyma, a pattern suggested by the available literature, as
summarized by Woods et al. (31) and Sawchenko and Friedman (25)
The possibility of differential innervation of hepatic vasculature vs.
hepatocytes is also indirectly supported by the effect of low-frequency
nerve stimulation on HABF and HGP in control dogs. For example, low
levels (1 Hz) of HNS in control dogs produced vasoconstriction without
increasing HGP (see Table 1), a pattern similar to that seen in
6-OHDA-pretreated dogs receiving high-frequency (8-Hz) HNS. Thus it
appears that the more highly innervated vasculature can respond to
degrees of neural activation that are insufficient to stimulate an HGP
response from the less densely innervated hepatocytes. These data do
not, however, rule out the alternative explanation that vascular
endothelium is more sensitive than the hepatocyte to low levels of
released norepinephrine.
In addition to increasing HGP, HNS produced an increase of arterial
GALIR. The arterial increment of GALIR was reduced by >90% by 6-OHDA
pretreatment, demonstrating that it was of hepatic origin. Because
small increments of arterial galanin can have significant physiological
effects (5), this finding raises the possibility that galanin released
from hepatic nerves may act as a neurohormone as well as a hepatic
neurotransmitter. One possible neuroendocrine effect of hepatic galanin
could be to decrease pancreatic insulin secretion (10). This potential
neuroendocrine effect may occur during stress, for several stresses
activate hepatic sympathetic nerves (21). If so, galanin released from the liver could aid in restraining glucose-stimulated insulin release,
thereby contributing to stress hyperglycemia.
Conclusion.
The finding that galanin is colocalized with TH in nerves of dog liver
argues that galanin resides in hepatic sympathetic nerves. The finding
that 6-OHDA treatment produces a parallel reduction of hepatic galanin
and norepinephrine content and spillover supports the hypothesis that
galanin is a sympathetic neurotransmitter in the dog liver. In
addition, the ability of HNS to significantly increase circulating
galanin levels, coupled with the potency of galanin to inhibit insulin
secretion (5), suggests a neuroendocrine role for hepatic galanin.
Finally, the hepatic vasculature is more responsive than parenchymal
hepatocytes to low levels of HNS, suggesting more dense sympathetic
innervation of the vascular smooth muscle, a hypothesis consistent with
the staining data from control and 6-OHDA-treated dogs.
 |
ACKNOWLEDGEMENTS |
This work was supported by the Medical Research Service of the
Department of Veterans Affairs and by National Institute of Diabetes
and Digestive and Kidney Diseases Grants DK-12829, DK-17047, and
DK-50154.
 |
FOOTNOTES |
Address for reprint requests: T. O. Mundinger, Division of
Endocrinology and Metabolism (151), Veterans Affairs Puget Sound Health
Care System, 1660 S. Columbian Way, Seattle, WA 98108.
Received 12 May 1997; accepted in final form 14 August 1997.
 |
REFERENCES |
1.
Ahren, B.,
G. Böttcher,
S. Kowalyk,
B. E. Dunning,
F. Sundler,
and
G. J. Taborsky, Jr.
Galanin is co-localized with noradrenaline and neuropeptide Y in dog pancreas and celiac ganglion.
Cell Tissue Res.
261:
49-58,
1990[Medline].
2.
Ahren, B.,
G. J. Taborsky, Jr.,
and
D. Porte, Jr.
Neuropeptidergic versus cholinergic and adrenergic regulation of islet hormone secretion.
Diabetologia
29:
827-836,
1986[Medline].
3.
Allman, F. D.,
E. L. Rogers,
D. A. Caniano,
D. M. Jacobowitz,
and
M. C. Rogers.
Selective chemical hepatic sympathectomy in the dog.
Crit. Care Med.
10:
100-103,
1982[Medline].
4.
Bishop, A. E.,
J. M. Polak,
F. E. Bauer,
N. D. Christofides,
F. Carlei,
and
S. R. Bloom.
Occurrence and distribution of a newly discovered peptide, galanin, in the mammalian enteric nervous system.
Gut
27:
849-857,
1986[Abstract].
5.
Boyle, M. R.,
C. B. Verchere,
G. McKnight,
S. Mathews,
K. Walker,
and
G. J. Taborsky, Jr.
Canine galanin: sequence, expression and pancreatic effects.
Regul. Pept.
50:
1-11,
1994[Medline].
6.
Connolly, C. C.,
K. E. Steiner,
R. W. Stevenson,
D. W. Neal,
P. E. Williams,
K. G. M. M. Alberti,
and
A. D. Cherrington.
Regulation of glucose metabolism by norepinephrine in conscious dogs.
Am. J. Physiol.
261 (Endocrinol. Metab. 24):
E764-E772,
1991[Abstract/Free Full Text].
7.
DeHaen, C. S.,
S. A. Little,
J. M. May,
and
R. H. Williams.
Characterization of proinsulin-insulin intermediates in human plasma.
J. Clin. Invest.
62:
727-737,
1978[Medline].
8.
Dunning, B. E.,
B. Ahren,
R. C. Veith,
G. Böttcher,
F. Sundler,
and
G. J. Taborsky, Jr.
Galanin: a novel pancreatic neuropeptide.
Am. J. Physiol.
251 (Endocrinol. Metab. 14):
E127-E133,
1986[Abstract/Free Full Text].
9.
Dunning, B. E.,
and
G. J. Taborsky, Jr.
Galanin
sympathetic neurotransmitter in endocrine pancreas?
Diabetes
37:
1157-1162,
1988[Abstract].
10.
Dunning, B. E.,
and
G. J. Taborsky, Jr.
Galanin release during pancreatic nerve stimulation is sufficient to influence islet function.
Am. J. Physiol.
256 (Endocrinol. Metab. 19):
E191-E198,
1989[Abstract/Free Full Text].
11.
Francis, B. H.,
D. G. Baskin,
D. R. Saunders,
and
J. W. Ensink.
Distribution of somatostatin-14 and somatostatin-28 gastrointestinal-pancreatic cells of rats and humans.
Gastroenterology
99:
1283-1291,
1990[Medline].
12.
Garceau, D.,
N. Yamaguchi,
R. Goyer,
and
F. Guitard.
Correlation between endogenous noradrenaline and glucose released from the liver upon hepatic sympathetic nerve stimulation in anesthetized dogs.
Can. J. Physiol. Pharmacol.
62:
1086-1091,
1984[Medline].
13.
Gilman, A. G.,
L. S. Goodman,
T. W. Rall,
and
F. Murad.
The Pharmalogical Basis of Therapeutics (7th ed.). New York: Macmillan, 1985, p. 210.
14.
Gonda, T.,
E. E. Daniel,
T. J. McDonald,
J. E. T. Fox,
B. D. Brooks,
and
M. Oki.
Distribution and function of enteric GAL-IR nerves in dogs: comparison with VIP.
Am. J. Physiol.
256 (Gastrointest. Liver Physiol. 19):
G884-G896,
1989[Abstract/Free Full Text].
15.
Kaplan, L. M.,
E. R. Spindel,
K. J. Isselbacher,
and
W. W. Chin.
Tissue-specific expression of the rat galanin gene.
Proc. Natl. Acad. Sci. USA
85:
1065-1069,
1988[Abstract].
16.
Kostrzewa, R. M.,
and
D. M. Jacobowitz.
Pharmacological actions of 6-hydroxydopamine.
Pharmacol. Rev.
26:
199-288,
1974[Medline].
17.
Kowalyk, S.,
R. Veith,
M. Boyle,
and
G. J. Taborsky, Jr.
Liver releases galanin during sympathetic nerve stimulation.
Am. J. Physiol.
262 (Endocrinol. Metab. 25):
E671-E678,
1992[Abstract/Free Full Text].
18.
Lundberg, J. M.,
J. Pernow,
and
J. S. Lacroix.
Neuropeptide Y: sympathetic cotransmitter and modulator?
News Physiol. Sci.
4:
13-17,
1989.[Abstract/Free Full Text]
19.
Lundberg, J. M.,
A. Rudehill,
A. Sollevi,
E. Theodorsson-Norheim,
and
B. Hamberger.
Frequency- and reserpine-dependent chemical coding of sympathetic transmission: differential release of noradrenaline and neuropeptide Y from pig spleen.
Neurosci. Lett.
63:
96-100,
1986[Medline].
20.
Melander, T.,
T. Hokfelt,
A. Rokaeus,
J. Fahrenkrug,
K. Tatemoto,
and
V. Mutt.
Distribution of galanin-like immunoreactivity in the gastro-intestinal tract of several mammalian species.
Cell Tissue Res.
239:
253-270,
1985[Medline].
21.
Mundinger, T. O.,
and
G. J. Taborsky, Jr.
Activation of hepatic sympathetic nerves during hypoxic, hypotensive and glucopenic stress.
J. Auton. Nerv. Syst.
63:
153-160,
1997[Medline].
22.
Myers, S.,
O. P. McGuinness,
D. W. Neal,
and
A. D. Cherrington.
Intraportal glucose delivery alters the relationship between net hepatic glucose uptake and the insulin concentration.
J. Clin. Invest.
87:
930-939,
1991[Medline].
23.
Peuler, J. D.,
and
G. A. Johnson.
Simultaneous single isotope radioenzymatic assay of plasma norepinephrine, epinephrine and dopamine.
Life Sci.
21:
625-636,
1977[Medline].
24.
Rokaeus, A.,
and
M. Carlquist.
Nucleotide sequence analysis of CDNAs encoding a bovine galanin precursor protein in the adrenal medulla and chemical isolation of bovine gut galanin.
FEBS Lett.
234:
400-406,
1988[Medline].
25.
Sawchenko, P. E.,
and
M. I. Friedman.
Sensory functions of the liver
a review.
Am. J. Physiol.
236 (Regulatory Integrative Comp. Physiol. 5):
R5-R20,
1979[Abstract/Free Full Text].
26.
Skofitsch, G.,
and
D. M. Jacobowitz.
Immunohistochemical mapping of galanin-like neurons in the rat central nervous system.
Peptides
6:
509-546,
1985[Medline].
27.
Taborsky, G. J., Jr., and T. O. Mundinger.
Differential actions of hepatic sympathetic neuropeptides.
Diabetes 46: 1997.
28.
Verchere, C. B.,
S. Kowalyk,
D. J. Koerker,
D. G. Baskin,
and
G. J. Taborsky, Jr.
Evidence that galanin is a parasympathetic, rather than sympathetic, neurotransmitter in the baboon pancreas.
Regul. Pept.
67:
93-101,
1996[Medline].
29.
Verchere, C. B.,
S. Kowalyk,
G. H. Shen,
M. R. Brown,
M. W. Schwartz,
D. G. Baskin,
and
G. J. Taborsky, Jr.
Major species variation in the expression of galanin messenger ribonucleic acid in mammalian celiac ganglion.
Endocrinology
135:
1052-1059,
1994[Abstract].
30.
Walker, L. C.,
N. E. Rance,
D. L. Price,
and
W. S. I. Young.
Galanin mRNA in the nucleus basalis of meynert complex of baboons and humans.
J. Comp. Neurol.
303:
113-120,
1990.
31.
Woods, S. C.,
G. J. Taborsky, Jr.,
and
D. J. Porte, Jr.
Central nervous system control of nutrient homeostasis.
In: Handbook of Physiology. The Nervous System. Intrinsic Regulatory Systems of the Brain. Bethesda, MD: Am. Physiol. Soc., 1986, sect. 1, vol. IV, chapt. 7, p. 365-412.
AJP Endocrinol Metab 273(6):E1194-E1202
0193-1849/97 $5.00
Copyright © 1997 the American Physiological Society