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Autonomic Neuroscience: Basic and

Noradrenergic innervation of rabbit pancreatic ganglia

Eunyoung Yi, Tina G. Smith, Jeffrey A. Love*

Department of Pharmacology and Toxicology, University of Mississippi Medical Center, 2500 North State Street, Jackson, MS 39216-4505, United States

Received 14 May 2004; received in revised form 20 October 2004; accepted 16 November 2004

Abstract

Sympathetic nerve stimulation indirectly regulates pancreatic endocrine and exocrine secretion, in part, through actions on the cholinergic

parasympathetic innervation of the secretory tissues. Earlier work identified noradrenergic nerves in pancreatic ganglia and demonstrated the

effects of exogenous norepinephrine (NE) on synaptic transmission but no quantitative studies of ganglionic NE content and release exist.

Therefore, the distribution and density of catecholamine (CA)-containing nerves in rabbit pancreatic ganglia were studied using

paraformaldehyde/glutaraldehyde (FAGLU) staining and HPLC analysis of CA concentrations. Neural release of [3H]NE was measured in

ganglia isolated from the head/neck or body regions of the pancreas. CA-containing nerves densely innervated most ganglia (86%) from both

regions, while neural and non-neural CA-containing cell bodies were rarely found. Ganglia from the head/neck region contained significantly

higher concentrations of NE. Both 40 mM K+ and veratridine evoked Ca2+-dependent [3H]NE release and tetrodotoxin inhibited 80% of

veratridine-stimulated release. N-Conotoxin GVIA alone antagonized veratridine-stimulated release by 40% but the addition of nifedipine or

N-agatoxin IVA caused no further inhibition. There were no apparent regional differences in the Ca2+-dependence or toxin-sensitivity of NE

release. In conclusion, ganglia throughout the rabbit pancreas receive a dense, functional noradrenergic innervation and NE release is

dependent upon N- but not P/Q- or L-type voltage-dependent Ca2+ channels. These noradrenergic nerves may indirectly regulate pancreatic

secretion through actions on ganglionic transmission.

D 2004 Elsevier B.V. All rights reserved.

Keywords: Parasympathetic ganglia; Catecholamine fluorescence; Norepinephrine release; Na+ channels; Ca2+ channels; N-conotoxin GVIA; N-agatoxin IVA;

Nifedipine

1. Introduction

The pattern of sympathetic innervation of the pancreas,

where blood vessels, islets, ducts, and ganglia but not acini

receive direct innervation, suggests multiple direct and

indirect mechanisms of sympathetic regulation of pancreatic

endocrine and exocrine secretion (Anglade et al., 1987;

Cetin, 1992; Alm et al., 1967; Legg, 1968; Love and

Szebeni, 1999; Oomori et al., 1994). Indeed, activation of

the sympathetic nervous system, resulting in the local

release of norepinephrine (NE) from nerve terminals and

endocrine release of epinephrine (Epi) and NE from the

adrenal glands, has complex effects on exocrine and

1566-0702/$ - see front matter D 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.autneu.2004.11.004

* Corresponding author. Tel.: +1 601 984 1621; fax: +1 601 984 1637.

E-mail address: jlove@pharmacology.umsmed.edu (J.A. Love).

endocrine pancreatic secretion (Singer, 1993; Brunicardi

et al., 1995; Ahren, 2000).

Sympathetic regulation of endocrine pancreatic secretion

occurs primarily via direct actions of neurally released

norepinephrine (NE) on inhibitory a2 adrenoceptors on hcells and stimulatory a1 adrenoceptors on a cells of the

islets (Brunicardi et al., 1995; Ahren, 2000). Indirect

sympathetic regulation endocrine secretion also occurs via

a1 adrenoceptor-mediated constriction of the islet vascula-

ture (Lundgren, 1984). Pancreatic neurons, which provide

the cholinergic innervation of the islets (Brunicardi et al.,

1995; Ahren, 2000), regulate the oscillatory pattern of basal

endocrine secretion (Stagner and Samols, 1985), amplify

glucose-stimulated insulin secretion (Sha et al., 2001), and

increase pancreatic blood flow (Lundgren, 1984), also

receive a significant noradrenergic sympathetic innervation

(Alm et al., 1967; Legg, 1968; Love and Szebeni, 1999).

Clinical 117 (2005) 87–96

E. Yi et al. / Autonomic Neuroscience: Basic and Clinical 117 (2005) 87–9688

The effects of selectively activating this ganglionic sym-

pathetic pathway on endocrine pancreatic secretion are

currently unknown.

Conversely, sympathetic regulation of exocrine pancre-

atic secretion is primarily indirect since acini receive little or

no direct sympathetic innervation (Alm et al., 1967; Legg,

1968; Love and Szebeni, 1999; Love et al., 2000). Although

indirect sympathetic inhibition of exocrine secretion result-

ing from an a1 adrenoceptor-mediated vasoconstriction has

consistently been reported (Richins, 1953; Barlow et al.,

1974; Lenz et al., 1992), other indirect effects resulted from

actions on cholinergic nerves. Stimulation of sympathetic

nerves caused an atropine-sensitive increase in the volume

of exocrine secretion when vasoconstriction was blocked

with an a adrenoceptor antagonist (Babkin et al., 1939;

Kuntz and Richins, 1949; Barlow et al., 1974), and NE

indirectly stimulated amylase secretion by mouse and rabbit

pancreatic lobules via h adrenoceptors on the cholinergic

nerve terminals of pancreatic neurons (Berger and Laugier,

1986; Love et al., 2000).

In contrast, an NE re-uptake inhibitor (Nagain-Domaine

et al., 1999) and an a2 agonist (Love et al., 2000) inhibited

nerve-stimulated but not acetylcholine-stimulated enzyme

secretion in rats and rabbit pancreatic lobules, respectively.

Electrophysiological studies have demonstrated that exog-

enous a1 and a2 adrenoceptor agonists could, respectively,

facilitate and depress ganglionic transmission depending

upon the region of the pancreas studied (Yi and Love,

2003). These results all confirmed that pancreatic ganglia

and/or the terminals of cholinergic pancreatic neurons are

targets of the noradrenergic sympathetic nerves regulating

exocrine secretion.

Although considerable indirect evidence supports the

hypothesis that pancreatic ganglia are important sites of

sympathetic regulation of pancreatic endocrine and exocrine

secretions, there have been no specific quantitative studies of

the distribution, density, or release capability of noradrener-

gic nerves innervating pancreatic ganglia. Therefore, in the

present study, the frequency of catecholamine-containing

nerves, catecholamine content, and nerve-stimulated [3H]NE

release were measured and compared in ganglia from the

head/neck and body region regions of the rabbit pancreas. A

preliminary report of this work was published in abstract

form Yi et al. (2003).

2. Materials and methods

2.1. Materials

Ketamine and xylazine were purchased from Butler

(Columbus, OH) and EuthasolR was purchased from

Delmarva Labs (Midlothian, VA). SylgardR was purchased

from Dow Corning (Midland, MI). Scintillation cocktail

(Bio-Safe IIk) was purchased from RPI (Mount Prospect,

IL). [3H]NE was purchased from Perkin-Elmer (Boston,

MA). All other reagents were purchased from Sigma (St.

Louis, MO).

2.2. Animals

New Zealand White rabbits of either sex (1–1.5 Kg) were

anesthetized with an intramuscular injection of ketamine (40

mg/Kg) and xylazine (10 mg/Kg), and euthanized with an

intracardiac injection of pentobarbital sodium (50 mg/Kg) or

EuthasolR containing pentobarbital sodium (390 mg/ml)

plus phenytoin sodium (50 mg/ml). The Institutional Animal

Care and Use Committee at the University of Mississippi

Medical Center approved all the animal procedures used.

2.3. Isolation of rabbit pancreatic ganglia

The gross anatomy of the rabbit pancreas, unlike that of

other commonly used species (rat, mouse, guinea pig, cat,

dog), exhibits no clearly defined head, neck, body, and tail

regions. Therefore, it was divided into two anatomical

regions, head/neck and body, based on its relationship to the

adherent stomach, duodenum, and abdominal aorta. An

imaginary mid-line was visualized in the pancreas parallel to

the adjacent abdominal aorta. The ventral region, attached to

the stomach and initial segment of the duodenum and within

1.5–2 cm of their walls, was designated the head/neck. The

dorsal region, within 1.5–2 cm of the abdominal aorta, was

designated the body of the pancreas. Previous work

indicated that most pancreatic ganglia were found in these

two areas (Love and Szebeni, 1999), consistent with

observations in other species.

Pancreata with attached duodena were removed to a

dissecting dish containing an ice-cold Krebs solution

containing (mM): Na+, 137.4; K+, 5.9; Ca2+, 2.5; Mg2+,

1.2; Cl�, 134; HCO3�, 15.5; H2PO4

�, 1.2; glucose, 11.5 mM,

and bubbled with 95% O2/5% CO2. Pancreatic ganglia with

attached nerve bundles were dissected free of the paren-

chyma. For paraformaldehyde/glutaraldehyde (FAGLU)

staining experiments, connective tissue surrounding the

ganglia was left intact to allow pinning of the ganglia to

the floor of a tissue bath. For NE release experiments,

ganglia and their attached nerve trunks were dissected free

of all other tissue.

2.4. Paraformaldehyde/glutaraldehyde staining of

catecholamines

The methods of Furness et al. (1977) were used with slight

modifications. Pancreatic ganglia were pinned to the

SylgardR-coated bottom of a 35 mm culture dish. After

adding 3 ml of 0.5% glutaraldehyde + 4% paraformaldehyde

in 0.1 M phosphate buffer (pH 7.4), dishes were refrigerated

(4 8C) for at least 3, but no longer than 24 h. Under these

conditions, NE, Epi, and dopamine (DA) form a blue-green

flurophore (Furness et al., 1977; Wreford et al., 1982). Fixed

tissue was washed twice with phosphate buffered saline

E. Yi et al. / Autonomic Neuroscience: Basic and Clinical 117 (2005) 87–96 89

(PBS, pH 7.4) and examined using a microscope with a 40�water immersion lens equipped for epifluorescence (Olym-

pus BX50WI, exciter filter BP400-410, dichroic mirror

DM455, barrier filter BA455). Catecholamine (CA)-contain-

ing structures are identified under ultraviolet light. To delay

photo-bleaching of the fluorophores, 1,4-diazabicy-

clo[2.2.2]octane (DABCO, 100 mg/ml) was added to the

buffer during examination. The FAGLU staining method

alone could not differentiate among NE, Epi, and DA, but

simply demonstrated the presence, density, and regional

distribution of CA-containing nerves.

2.5. HPLC measurements

All isolated tissues were homogenized in 0.5 ml of 0.4 N

perchloric acid. Dihydroxy benzylamine (DHBA) was

added as an internal standard. The homogenates were

centrifuged at 13,000 �g for 5 min, and the supernatants

were passed through 0.45 Am syringe filter and stored at

�80 8C until HPLC analysis. The pellet was used for

determination of protein content.

For HPLC measurements, a buffer/methanol mixture

(85:15) was used as the mobile phase. The buffer contained

50 mM sodium acetate, 20 mM sodium citrate, 2 mM

sodium octyl sulfate, and 0.135 mM EDTA (pH 4.3, filtered

and degassed). The solution was delivered by a BAS PM-80

solvent delivery system (Bioanalytical Systems, West

Lafayette, IN) at a flow rate of 1 ml/min. One hundred

microliters of sample plus 100 Al of the extraction solution

(0.4 N percholoric acid in 0.1 M phosphate buffer) was

injected each time (loop volume 200 Al) and measurements

were repeated 2–4 times. The CAs were separated using a

microsorb-MV C18 reverse phase column (150�4.6 mm)

and detected using an amperometric electrochemical detec-

tor (Model LC-4C) equipped with a silver–silver chloride

reference electrode and a glassy carbon working electrode.

Detection potential was set at 0.7 V and the signal detection

range was set at 5 nA (filter 0.02 Hz) to maximize detected

peaks. Quantities of each monoamine in the sample were

determined by comparing integrated peak areas (HP 3390A

integrator) with those of standard solutions containing

known concentrations of NE, DA, Epi, and internal standard

dihydroxybenzylamine (DHBA).

2.6. Protein measurements

Themethod of Bradford (1976) was used. Briefly, 100mg

of Brilliant Blue G dyewas dissolved in 100ml of phosphoric

acid and 900ml of distilled water was slowly added to the dye

solution. After removing particulate matter by filtration

through filter paper (Whatman), the reagent was stored in a

dark bottle until used. To determine protein content, the tissue

pellets were dissolved in 0.5 N NaOH and 100 Al of eachsample was mixed with 1 ml of Bradford reagent. After 5 min

incubation at room temperature, 200 Al aliquots were

transferred to a 96-well plate and measured at an absorption

wavelength of 595 nm using a microplate reader (Thermo-

max, Molecular Devices). Bovine serum albumin dissolved

in 0.5 N NaOH (10–100 Ag/ml) was used as the protein

standard. All measurements were made in triplicate.

2.7. Tissue uptake and release of [3H]NE

The methods of Trendelenburg et al. (1999) were

modified and used for the NE release studies. Pancreatic

ganglia from the head/neck and the body were dissected free

and collected in separate microcentrifuge tubes. Ganglia

were then incubated with 100–150 nM NE (levo-[ring-

2,5,6-3H], specific activity, 40–80 Ci/mmol) in 0.5 ml

HEPES buffered Krebs containing 0.2 mM ascorbic acid for

1 h at 37 8C. At the end of incubation, the tubes were

centrifuged and the loading buffer was removed by

aspiration. Following three washes with 0.5 ml of fresh

[3H]NE-free, HEPES-buffered Krebs containing 1 AMdesipramine, the ganglia were incubated with a fresh 0.5

ml of the same medium. The incubation buffer was then

replaced every 10 min with 0.5 ml of fresh buffer.

The first samples of the experiments contained relatively

high basal levels of radioactivity, which precipitously

dropped to a stable level after 3–4 changes of medium.

Therefore, sampling was started after a 40 min stabilization

period. After collecting two 10 min samples to determine

basal release (HEPES buffered Krebs containing 1 AMdesipramine), the ganglia were incubated in buffer containing

stimulating agents. Following 40 mM K+ stimulation, the

release of [3H]NE immediately returned to basal levels.

Therefore, the basal release of radioactivity for high K+

stimulation was interpolated from the basal values of radio-

activity release immediately before and after the high K+

stimulation period. Unlike high K+ stimulation, the stimula-

tory effect of veratridine required an additional 30 min to

recover to the pre-stimulus level following washout. For this

reason the sample immediately preceding veratridine stim-

ulation was used as the measure of basal release. Three 10

min washes were allowed between veratridine stimulations

and up to 3 stimulations were attempted in each group of

ganglia. At the end of the experiments, tissues were collected

in separate tubes, mixed with 5 ml of scintillation cocktail

(Bio-Safe IIk, RPI), and radioactivity was measured for 15

min using a liquid scintillation analyzer (2500TR, Packard,

Meriden, CT). The released radioactivity was calculated as a

percent of total remaining radioactivity in the tissue at the

beginning of the respective collection period.

2.8. Statistical analysis

Statistical significance between groups was determined

using Sigma Stat (version 3.0). Briefly, when equal variance

and normality assumptions were met, paired or unpaired

t-tests and parametric variations of analysis of variance

(ANOVA) were used. The Student–Newman–Keuls test was

used as a post-hoc test. When either equal variance or

E. Yi et al. / Autonomic Neuroscience: Basic and Clinical 117 (2005) 87–9690

normality assumptions were not met, equivalent versions of

non-parametric tests were used. The specific statistical test

used in each case is indicated in the Results section. Data

are expressed as meanFstandard error unless otherwise

specified.

3. Results

3.1. Catecholamines in pancreatic ganglia

Paraformaldehyde/glutaraldehyde (FAGLU) staining pro-

vided a simple, effective alternative to immunohistochemis-

try for rapidly identifying the location of catecholamines

(CAs) in large numbers of pancreatic ganglia. Fluorescent

varicose nerves were observed in 37/43 ganglia (86%)

isolated from the head/neck region of 10 rabbits and in 67/78

ganglia (86%) isolated from the body region of 13 rabbits.

While, there was no regional difference in the frequency of

CA-containing nerves in pancreatic ganglia there was a

Fig. 1. Catecholamine-containing nerves in rabbit pancreatic ganglia.

Wholemounts of pancreatic ganglia were fixed in 4% paraformaldehyde

and 0.5% glutaraldehyde and observed under ultraviolet light. (A)

Catecholamine-containing varicose fibers travel along nerve trunk (far

lower right) and pass throughout the long, oval ganglion (upper left) from

the head/neck. (B) Catecholamine-containing varicose fibers are found in a

localized area of a ganglion from the body. Magnification 400�.

Fig. 2. Catecholamine-containing cells in pancreatic ganglia and nerve

trunks. Wholemounts of pancreatic ganglia fixed in 4% paraformaldehyde

and 0.5% glutaraldehyde and observed under ultraviolet light. (A) Two

groups of intensely fluorescent cell bodies without fluorescent processes in

a ganglion from the head/neck region. (B) Single fluorescent cell in a

ganglion from the body regions. (C) One large and two small (top)

fluorescent cells in a pancreatic nerve trunk. Magnification 400�.

qualitative regional difference in their density. CA-contain-

ing nerves were densely distributed throughout ganglia of the

head/neck region (Fig. 1A) but were of lower density and

appeared concentrated around particular neurons in ganglia

from the body (Fig. 1B). Thus, the overall density of CA-

containing fibers appeared noticeably higher in ganglia from

the head/neck compared to those of the body.

Fig. 3. High K+ stimulation of [3H]NE release from pancreatic ganglia.

Stimulation with 40 mM K+ significantly increased [3H]NE release from

ganglia in both regions. Lowering extracellular Ca2+ concentration to 0.1

mM and increasing extracellular Mg2+ concentration to 15 mM inhibited

release by 88F3% (n=7) in ganglia from the head/neck and by 90F4%

(n=7) in ganglia from the body. Each point represents release during a 10

min incubation period. *, **Significantly different from control ( pb0.05

and b0.01, paired t-test).

E. Yi et al. / Autonomic Neuroscience: Basic and Clinical 117 (2005) 87–96 91

Fluorescent cell bodies were rarely observed in the

ganglia (Fig. 2A,B) or in nerve bundles (Fig. 2C). CA-

containing cells were observed in only 1 of 43 ganglia from

the head/neck and 2 of 78 ganglia from the body. The larger

cells were oval with somal long axes of 20–40 Am(Fig. 2A,B) and usually occurred singly (Fig. 2B) although

multiple fluorescent cells were observed once (Fig. 2A).

One cell, found in an interganglionic nerve bundle, had a

long axis of 70 Am (Fig. 2C center). Viewed under normal

light, the larger cells had all the characteristics of pancreatic

neurons previously reported (Love and Szebeni, 1999).

Small non-neuronal cells (long axis 5–10 Am) with

fluorescent processes were occasionally observed in inter-

ganglionic nerve bundles (Fig. 2C top).

The apparent regional differences in the density of

ganglionic catecholamine-containing fibers were corrobo-

rated by the HPLC analysis of CA content. The concen-

tration of NE (1620F220 pmol/mg protein) in ganglia from

the head/neck region was approximately twofold greater

( pb0.05, unpaired t-test; n=5,4) than that in ganglia from

the body region (778F179 pmol/mg protein). Dopamine

concentration was also higher in the head/neck region

(587F131 vs. 232F62 pmol/mg protein), but this difference

did not reach statistical significance. Epi was not detected in

any of the ganglia studied.

3.2. Release of [3H]NE from pancreatic ganglia

HPLC analysis of samples of incubation medium from

groups of ganglia (25–60) stimulated with 40 mM K+ or 100

AM veratridine failed to detect the release of NE. Therefore,

the more sensitive method of [3H]NE release was used to

establish and characterize uptake, storage, and Ca2+-depend-

ent release of NE by pancreatic ganglia. An average of 33F1

and 40F1 ganglia per rabbit was collected from the head/

neck (59 rabbits) and body (53 rabbits) regions of the

pancreas, respectively. Ganglionic uptake of [3H]NE was

confirmed bymeasuring the amounts of radioactivity in tissue

extracts after a 1 h incubation with 100–150 nM [3H]NE

followed by 3 washes to remove extracellular NE. The mean

concentration of radioactivity in ganglia from the head/neck

region was greater ( pb0.01, Mann–Whitney test) than that in

ganglia from the body (1054F58 vs. 806F90 counts/min/

ganglion). Based on the specific activity of the [3H]NE (40–

80 Ci/mmol) used, these values are equivalent to 34F2 (head/

neck) and 26F3 (body) fmol of [3H]NE per ganglion. Thus,

isolated pancreatic ganglia transported and stored NE.

3.3. High K+-stimulated NE release

Initially, a release buffer containing 40 mM K+, whose

osmolarity was adjusted by an equimolar decrease in Na+

concentration, was used. In ganglia from the head/neck

region, 40 mM K+ increased [3H]NE release to 215% of

basal (Fig. 3). Release of [3H]NE immediately returned to

basal levels following washout of the 40 mM K+ solution.

The effect of a second stimulation of the same tissue (S2)

was not different from that of the first stimulation (S1) with

the ratio S2/S1 being 0.95F0.11 (n=7). The release of

[3H]NE by ganglia from the body was also increased to

263% of basal (Fig. 3) by 40 mM K+ stimulation and S2

was not different from S1 (S2/S1 ratio=1.11F0.32; n=7).

The magnitudes of release evoked by 40 mM K+ in ganglia

from the two regions were not different.

A solution containing 0.1 mM Ca2+ and 15 mM Mg2+

inhibited 40 mM K+-stimulated release by 88F3% in

ganglia from the head/neck and by 90F4% (Fig. 3) in

ganglia from the body. Similar results were observed

regardless of which stimulation (S1 or S2) was performed

in normal or low Ca2+ solution, indicating that decreased

release resulted from the relative lack of extracellular Ca2+

rather than depletion of [3H]NE stores. Thus, [3H]NE

release evoked by 40 mM K+ stimulation was consistent

with synaptic release from noradrenergic nerve terminals.

3.4. Veratridine-stimulated [3H]NE release

Veratridine, a plant alkaloid that selectively activates the

voltage-dependent Na+ channels found in nerve cell bodies

and fibers but not in SIF cells or glia (Ulbricht, 1998), was

used as a nerve-specific stimulus of ganglionic [3H]NE

release (Fig. 4 top). Unlike high K+ stimulation, the effects

of veratridine persisted after washout requiring an additional

30 min to recover to basal levels. Therefore, the time points

immediately preceding veratridine stimulations served as

the basal values used to calculate veratridine-stimulated

release. Veratridine evoked a concentration-dependent

release of [3H]NE in ganglia from both regions with no

regional difference in the magnitudes of release at each

concentration (Fig. 4 top).

Fig. 5. Calcium dependence of veratridine-stimulated [3H]NE release.

Stimulation with 50 AM veratridine significantly increased [3H]NE release

from ganglia of both regions. Lowering extracellular Ca2+ concentration to

0.1 mM and increasing extracellular Mg2+ concentration to 15 mM

inhibited release by 83F1% (n=6) in ganglia from the head/neck and by

84F2% (n=5) in ganglia from the body. Each point represents release

during a 10 min incubation period. ***Significantly different from control

( pb0.001, paired t-test).

Fig. 4. Veratridine-stimulated [3H]NE release from pancreatic ganglia.

(Top) Veratridine stimulated a concentration-dependent release of [3H]NE

from pancreatic ganglia of the head/neck (n=4, 5, and 7) and body (n=4, 4,

and 6,). (Bottom) Ganglia were stimulated with veratridine in the absence

and presence of 1–2 AM tetrodotoxin (TTX). In the presence of 1–2 AMTTX, [3H]NE release was antagonized by 87F3%, 81F1%, and 67F2%

(n=4, 5, and 4) in ganglia from the head/neck and by 85F3%, 76F7%, and

65F6% in ganglia from the body region (n=4). Each point represents

release during a 10 min incubation period.

Fig. 6. Effects of voltage-dependent Ca2+ channel blockers on nerve-

stimulated [3H]NE release. Ganglia were stimulated with 50 AM veratridine

in the absence and presence of various combinations of voltage-dependent

calcium channel (VDCC) blockers. Omega-Conotoxin GVIA (CTX, 1–3

AM), an N-type VDCC blocker, inhibited veratridine-stimulated release by

35F6% (n=6) in the head/neck and by 45F2% (n=5) in the body. Adding

agatoxin IVA (ATX, 1 AM), a P/Q-type VDCC blocker, or nifedipine (Nif,

10 AM), an L-type VDCC blocker, failed to significantly increase the

inhibition of release by CTX. Each point represents release during a 10 min

incubation period. *, **Significantly different from control ( pb0.05 and

b0.01, paired t-test).

E. Yi et al. / Autonomic Neuroscience: Basic and Clinical 117 (2005) 87–9692

To confirm that veratridine-stimulated release was due to

the activation of voltage-dependent Na+ channels, antago-

nism of its action with a selective blocker of voltage-

dependent Na+ channels, tetrodotoxin (TTX), was tested.

Adding TTX to the incubation buffer did not affect basal

release but significantly antagonized veratridine-stimulated

release. In ganglia from the head/neck, the release of [3H]NE

stimulated by 25, 50, and 100 AMveratridine was reduced by

87F3%, 81F1%, and 67F2%, respectively, in the presence

of 1–2 AM TTX (Fig. 4 bottom). A similar pattern of TTX

antagonism was observed in ganglia from the body. Release

of [3H]NE stimulated by 25, 50, and 100 AM veratridine was

reduced by 85F3%, 76F7%, and 65F6%, respectively, in

the presence of 1–2 AM TTX (Fig. 4 bottom). Thus, most of

the veratridine-stimulated release was mediated by stimula-

tion of noradrenergic nerves.

3.5. Voltage-dependent Ca2+ channels and NE release

The dependence of veratridine-stimulated release on

extracellular Ca2+ was tested next. For all remaining

experiments, 50 AM veratridine was used because it evoked

a robust [3H]NE release but allowed paired stimulations

without the depletion of labeled stores caused by 100 AMveratridine. The low Ca2+, high Mg2+ solution significantly

decreased veratridine-stimulated release by 83F1% and

84F2% in ganglia from the head/neck and body, respec-

tively (Fig. 5), consistent with release from noradrenergic

nerves.

Fig. 7. Toxin-resistant Ca2+ channels contribute to nerve-stimulated release.

Ganglia were stimulated with 50 AM veratridine in the absence and

presence of various voltage-dependent calcium channel blockers. Although

CTX, CTX+ATX, and CTX+Nif all significantly inhibited veratridine-

stimulated release, there was no significant difference in the degree of

blockade among these three groups. All combinations of channel blockers

caused significantly less inhibition of [3H]NE release than 0.1 mM Ca2+,

15 mM Mg2+ solution. ***Significantly different from CTX, CTX+ATX,

and CTX+Nif group ( pb0.001) using a one-way ANOVA followed by

Student–Newman–Keuls test.

E. Yi et al. / Autonomic Neuroscience: Basic and Clinical 117 (2005) 87–96 93

To complete the studies of nerve-stimulated [3H]NE

release, the voltage-dependent calcium channels (VDCCs)

mediating release were characterized using channel-selec-

tive blockers. VDCCs are classified into N, L, P/Q, R, and

T-types according to their pharmacological and kinetic

properties. The effect of N-conotoxin GVIA (CTX; 1–3

AM; IC50 of CTX 100 nM; Waterman, 2000), a selective

blocker of N-type VDCCs, on veratridine-stimulated

release was tested first. Two characteristics of CTX

necessitated modifications of our experimental protocol.

First, previous reports (Smith and Cunnane, 1997)

indicated that CTX effects were not readily reversed.

Therefore, the effect of veratridine alone was always tested

before CTX was added to the tissue. Second, CTX

required approximately 15 min to reach its maximal effect

(Eterovic et al., 1997) necessitating pre-incubation with

CTX for two 10 min periods prior to 10 min stimulation

with veratridine for a total of 30 min in CTX. CTX did

not change basal release but inhibited veratridine-stimu-

lated [3H]NE release by 35F6% and 45F2% in ganglia

from the head/neck and body, respectively (Fig. 6).

Increasing CTX concentration from 1 to 3 AM did not

further inhibit release. These results suggested that other

classes of VDCCs also participated in nerve-stimulated NE

release.

The effect of N-Agatoxin IVA (ATX), a blocker of P and

Q-type VDCCs, was tested on veratridine-stimulated

release. ATX blocks P-type VDCCs with an IC50 in the

low nM range and Q-type VDCCs with an IC50 in the high

nM range (Mintz et al., 1992; Waterman, 2000). Therefore,

1 AM ATX was used to block both P and Q-type VDCCs.

N-Agatoxin IVA, in the presence of 1 AM CTX, did not

further inhibit veratridine-stimulated release. In ganglia

from the head/neck and body, respectively (Fig. 6 top and

bottom), CTX+ATX decreased veratridine-stimulated

release by 51F7% and 39F4% of veratridine-stimulated3H release; values not significantly different from CTX

alone (Fig. 7). Thus, P/Q-type VDCCs did not appear to

play a significant role in nerve-stimulated NE release.

Lastly, the effect of a selective L-type VDCC inhibitor,

nifedipine (Nif; 10 AM; IC50~25 nM; Mery et al., 1996),

was tested on veratridine-stimulated release. Nifedipine plus

1 AM CTX (Fig. 6) inhibited veratridine-stimulated release

by 39F3% and 48F7% in ganglia from the head/neck and

body, respectively. This inhibition was not different from

that of CTX alone (Fig. 7). Thus, L-type VDCCs did not

appear to play a significant role in nerve-stimulated NE

release.

Taken together, in both regions, there were no statistical

differences in inhibition of release among the CTX,

CTX+ATX, and CTX+Nif groups. Thus, N-type but neither

P/Q nor L-type channels appeared to play a significant role

in veratridine-stimulated [3H]NE release from pancreatic

ganglia. Inhibition of veratridine-stimulated release was

significantly greater in 0.1 mM Ca2+ 15 mM Mg2+ solution

compared with specific VDCC blockers (Fig. 7) indicating

that toxin-resistant release was still dependent upon influx

of extracellular Ca2+.

4. Discussion

FAGLU staining demonstrated a dense innervation of

pancreatic ganglia from both regions by CA-containing

nerve fibers. The observation of blue-green fluorescence at

pH 7 indicated staining for CAs rather than 5-hydroxytrypt-

amine (5-HT), which emits a yellow fluorescence optimal at

pH 10 (Furness et al., 1977; Wreford et al., 1982). This

finding corroborated our previous reports of an absence of

5-HT immunoreactive nerves in rabbit pancreatic ganglia

and a lack of detectable 5-HT in rabbit ganglionic extracts

analyzed by HPLC (Love and Szebeni, 1999; Yi et al., 2003,

2004). These results were also consistent with previous

studies using fluorescent (Alm et al., 1967; Legg, 1968) or

immunohistochemical (Oomori et al., 1994; Love and

Szebeni, 1999) staining methods.

E. Yi et al. / Autonomic Neuroscience: Basic and Clinical 117 (2005) 87–9694

Catecholamine containing cell bodies were rare and

variable in size and shape. The larger of these cells, viewed

with standard illumination appeared to be pancreatic neurons

(Love and Szebeni, 1999). Previous studies have demon-

strated TH- and DBH-positive neurons in cultured neonatal

guinea pig pancreatic neurons (Liu et al., 1998) but not in

adult pancreatic neurons (Kirchgessner and Pintar, 1991;

Love and Szebeni, 1999). The CAs found in these cells

remain to be identified although the previously reported

absence of DBH-positive neurons and the low NE/DA ratios

observed in the present study suggest it may be DA. The low

concentrations of NE and DA (9F2 and 0.9F0.2 pmol/mg

protein, respectively) in pancreatic acini (Yi et al., 2004) and

the fact that ganglia were dissected free of the surrounding

parenchyma precluded a glandular source of the catechol-

amines. Both FAGLU staining and HPLC analysis of CA

concentrations indicated a higher density of noradrenergic

nerves in ganglia from the head/neck region. The average

amount of [3H]NE uptake per ganglion was also greater in

this region. Given that a ganglion is not a standard unit, these

observations are necessarily qualitative but are consistent

with the histochemical and chromatographical evidence as

well as recently reported regional differences in the

frequency and type of electrophysiological responses of

rabbit pancreatic neurons to NE and a adrenergic agonists

(Yi and Love, 2003).

Stored [3H]NE was readily released in response to

membrane depolarization with high K+ or veratridine

solutions and was highly dependent on influx of extrac-

ellular Ca2+. The fact that TTX inhibited the majority of

veratridine-stimulated release indicated release was predom-

inantly mediated by activation of voltage-dependent Na+

channels. These results were all consistent with active

neural transport, vesicular storage, and release of NE. High

intracellular Na+ concentrations, resulting from veratridine-

stimulation, can disrupt or reverse the Na+ gradient and

thereby cause transport of NE into the synapse (Gerevich

et al., 2001; Sumiya et al., 2001). In our experiments, the

incubation buffer contained 1 AM desipramine, which at a

concentration of 100 nM, inhibited both normal and reverse

transport by NE transporters (Smith and Levi, 1999). Thus,

while NE release due to reversal of the transporter might

have occurred in our experiments, its contribution was

probably small.

Antagonism of veratridine-stimulated release by TTX

decreased with increasing veratridine concentrations similar

to other tissues (Ulbricht, 1998). Sunami et al. (1993)

observed that 10 AM TTX inhibited 78% of 50 AMveratridine-evoked Na+ channel opening, comparable to

our results (81% block in the head/neck and 76% block in

the body) and an increase in the apparent Kd of TTX with

depolarization has been reported (Castillo et al., 1992).

Therefore, it is possible that the greater depolarizations

evoked by higher concentrations of veratridine reduced

TTX binding. Alternatively, TTX-resistant Na+ channels

might participate in veratridine-stimulated NE release in

pancreatic ganglia. Such channels have been found in

peripheral neurons from various species including rabbit

(Yoshida, 1994). The majority of veratridine-stimulated

release (N80%) was inhibited in 0.1 mM Ca2+ and 15 mM

Mg2+ solution indicating that veratridine-stimulated NE

release was dependent on extracellular Ca2+, consistent with

neural release of neurotransmitter. Complete block was not

achieved because the solution was not Ca2+-free and

probably allowed for a small Ca2+ influx and NE release

even in the presence of the high Mg2+ concentration.

Multiple VDCCs have been implicated in neurotrans-

mitter release from other peripheral autonomic nerves. The

relative importance of each channel type varied depending

on animal species, tissue, synapse, and the characteristics of

the stimuli used to evoke release (Smith and Cunnane,

1996a,b, 1997, 1999; Waterman, 2000). Only CTX exerted

significant inhibition (~40%) on veratridine-stimulated

release suggesting that N-type (but neither P/Q nor L-type)

VDCCs played a significant role in NE release from

pancreatic ganglia. The limited blocking effect of CTX

was not surprising because veratridine causes strong,

prolonged nerve stimulation. CTX-resistant neurotransmit-

ter release has been reported in various tissues, especially

when high frequency or prolonged stimulation was used to

evoke neurotransmitter release and P/Q- or L-type VDCCs

mediated CTX-resistant release in many of these studies

(Smith and Cunnane, 1996a,b, 1997; Waterman, 2000). The

lack of effect of ATX and Nif excluded a significant role of

P/Q or L-type VDCCs in veratridine-stimulated release.

The results suggest three possible mechanisms for the

CTX-resistant release. First, veratridine stimulation may acti-

vate R-type VDCCs like those demonstrated in superior

cervical ganglion neurons and adrenal chromaffin cells

(Martınez-Pinna et al., 2002; Albillos et al., 2000). Second,

T-type VDCCs contributed to nerve-stimulated release

similar to that observed in retinal bipolar cells (Pan et al.,

2001). Third, Ca2+-induced Ca2+ release, mediated by ryano-

dine receptors, contributed to veratridine-stimulated release.

Ryanodine receptors have been demonstrated in several

peripheral neurons (Turner et al., 2001; Hoesch et al., 2002;

Cifuentes et al., 2001), and ryanodine-sensitive excitatory

junction potentials have been reported in the guinea pig vas

deferens (Smith and Cunnane, 1996b). The existence of R or

T-type channels or ryanodine receptors in nerve terminals of

pancreatic ganglia remains to be demonstrated.

The regional differences in density of noradrenergic

innervation of the ganglia could result from specific

targeting of the head/neck region or simply because

substantial numbers of sympathetic fibers supplying the

gastrointestinal tract enter the pancreas in this region.

Indeed, a previous study in rat demonstrated regional

heterogeneity in the innervation of pancreatic ganglia by

enteric serotonergic nerves that primarily innervated ganglia

in the head/neck region (Kirchgessner and Gershon, 1990).

However, removal of the celiac and superior mesenteric

plexuses, whose nerves enter the immediately adjacent body

E. Yi et al. / Autonomic Neuroscience: Basic and Clinical 117 (2005) 87–96 95

of the pancreas, removed z90% of CA-containing nerves

(Anglade et al., 1987).

Regional differences in the enzyme content of pancreatic

acinar cells (Malaisse-Lagae et al., 1983), and the relative

numbers of pancreatic islet cells containing glucagon and

pancreatic polypeptide (Bishop and Polak, 1997; Malaisse-

Lagae et al., 1979, 1983; Orci et al., 1976) have also been

reported. The possible relationship between neural and

secretory heterogeneity remains to be explored.

The functional noradrenergic innervation reported here,

and earlier demonstrations that NE and a adrenergic

agonists modulate ganglionic transmission (Yi and Love,

2003) suggest pancreatic ganglia are likely sites of indirect

sympathetic regulation of secretion. While cholinergic

pancreatic neurons innervate all tissues of the exocrine

and endocrine pancreas (Singer, 1993; Ahren, 2000;

Brunicardi et al., 1995), the acini, which receive little if

any noradrenergic innervation (Love and Szebeni, 1999; Yi

et al., 2003, 2004), appear to be the most likely target for

indirect sympathetic regulation.

5. Conclusions

The results allow several important new conclusions

about the pancreatic innervation. First, functional noradre-

nergic nerves but rarely cell bodies are present in nearly all

ganglia from the head/neck and body regions of the

pancreas. Second, the density of noradrenergic innervation

of pancreatic ganglia and, presumably, the functional

importance of these sympathetic nerves is significantly

greater in ganglia from the head/neck region compared to

the body region. Third, nerve-stimulated NE release and the

VDCCs mediating it appeared the same in all ganglia.

Finally, pancreatic ganglia are possible sites of indirect

sympathetic regulation of pancreatic function.

Acknowledgements

The authors acknowledge Dr. Robert E. Kramer for

allowing us to use his equipment and lab space during

[3H]NE release experiments. This study was supported by

grant R06/CCR419466 from the Centers for Disease Control.

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