neuronal nitric oxide synthase in the neural pathways of the urinary bladder

J. Anat. (1999) 194, pp. 481–496, with 9 figures Printed in the United Kingdom 481

Review

Neuronal nitric oxide synthase in the neural pathways of the

urinary bladder

YUAN ZHOU1 AND ENG-ANG LING2

"Department of Experimental Surgery, Singapore General Hospital, and #Department of Anatomy,

National University of Singapore, Singapore

(Accepted 2 March 1999)

Nitric oxide (NO) is a unique biological messenger molecule. It serves, in part, as a neurotransmitter in the

central and peripheral nervous systems. Neurons containing NO have been identified histochemically by the

presence of nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) reactivity or

immunohistochemically by the antibody for neuronal NO synthase (n-NOS). Previous histochemical or

pharmacological studies have raised the possibility that NO may play an important role in the neural

pathways of the lower urinary tract. There is also considerable evidence to suggest that n-NOS is plastic and

could be upregulated following certain lesions in the lower urinary tract. The present review summarises the

distribution of n-NOS containing neurons innervating the urinary bladder and the changes of the enzyme

expression in some experimentally induced pathological conditions.

Key words : Autonomic nervous system; nitric oxide.

The urinary bladder and urethra serve 2 main

functions, namely, storage and evacuation of urine

(Lincoln & Burnstock, 1993), both being regulated

by a complex neural control that arises from the

lumbar and sacral levels of the spinal cord. At both

levels, the innervation has both afferent and efferent

components, with the latter being composed of

sympathetic, parasympathetic and somatic pathways

(Fletcher & Bradley, 1978). There is considerable

anatomical overlap and interaction between the

different components of the neural pathways of the

lower urinary tract with potential for integration or

modulation of activities. During urine storage and

micturition, the activities of the bladder, vesicoureteric

junction, internal sphincter and external sphincter are

all closely coordinated. It has been reported that the

coordination between the different neural pathways

could occur at the level of the spinal cord, within the

autonomic ganglia in the periphery, and at the

neuromuscular junction (Lincoln & Burnstock, 1993).

Correspondence to Professor E.-A. Ling, Department of Anatomy, National University of Singapore, 119260. Fax: 65-7787643; e-mail :

antlea!nus.edu.sg

Besides noradrenaline (NA) and acetylcholine (ACh),

numerous neurochemicals which have the potential

for transmitter and}or modulator activities have been

localised in neuronal cell bodies and terminal vari-

cosities in the neural pathways of the lower urinary

tract (Lincoln & Burnstock, 1993). The precise

mechanisms of action of these neurochemicals, how-

ever, are not fully understood. Along with the above

mentioned neurochemicals was the recent description

of nitric oxide (NO) as a nonadrenergic non-

cholinergic (NANC) inhibitory neurotransmitter at

various sites in the nervous system (Bredt & Snyder,

1992; Grozdanovic et al. 1994; Schuman & Madison,

1994). Its function has been documented in muscular

and sphincter relaxation in the cardiovascular (Palmer

et al. 1987; Lowenstein et al. 1994), digestive (Bult et

al. 1990; Sanders & Ward, 1992; Wang et al. 1996a)

and urogenital systems (Burnett et al. 1992; Mevorach

et al. 1994; Burnett et al. 1995). The possibility that

NO is involved in inhibitory transmission in the lower

urinary tract has recently been recognised in various

species (Dokita et al. 1991; Persson & Andersson,

1992; Persson et al. 1992; James et al. 1993).

Morphologically the neurons and fibres which express

NOS have been detected using neuronal nitric oxide

synthase (n-NOS) immunohistochemistry (Bredt et al.

1990) and nicotinamide adenine dinucleotide phos-

phate diaphorase (NADPH-d) histochemistry

(Grozdanovic et al. 1992; Vincent & Kimura, 1992).

Recent studies on NO have focused on its plasticity in

response to some pathological events (Solodkin et al.

1992; Zhang et al. 1993, 1994). As in other systems,

NADPH-d and NOS positive neurons or fibres have

been demonstrated in the neural pathways of the

lower urinary tract (Saffrey et al. 1994; Smet et al.

1996; Zhou et al. 1997a) and, furthermore, the

expression of n-NOS in such neural pathways has also

been shown to be plastic and could be altered

following some pathological lesions (Vizzard et al.

1995, 1996; Zhou & Ling, 1997a, b, c).

We summarise here the distribution of n-NOS and

the alteration of the enzyme expression following

certain pathological lesions in the neural pathways of

the urinary bladder. The pathways from the higher

centres in the brain to the spinal cord will not be

reviewed as they are beyond the scope of present

review.

As stated by Burnstock (1993), one of the major

advances in neuroscience since 1960 has been the

discovery of NANC nerves and recognition of a

multiplicity of neurotransmitter substances in auto-

nomic neuronal cell bodies and nerve fibres. These

substances include monoamines, purines, amino acids,

a variety of different peptides and more recently, nitric

oxide (Burnstock, 1993). Evidence of coexistence of

these neurotransmitters with NA and ACh in in-

dividual neurons or nerve terminals has been well

documented in the autonomic nervous system. The

concepts of cotransmission and neuromodulation are

now well established throughout the entire autonomic

nervous system (Lincoln & Burnstock, 1993).

In the urinary bladder, several transmitter sub-

stances such as 5-hydroxytryptamine (5-HT), his-

tamine, vasoactive intestinal peptide (VIP), somato-

statin (SOM), substance P (SP) and enkephalin (ENK)

have been proposed as NANC neurotransmitters.

These substances, however, do not mimic the neural

response since when their actions are modified by

pharmacological manipulation there are no parallel

effects on the response to nerve stimulation (Taira,

1972; Downie & Larsson, 1981; MacKenzie &

Burnstock, 1984). For example, VIP causes con-

traction of detrusor muscle in the guinea pig bladder,

but it is not likely to be an excitatory transmitter,

because agents which abolish the effects of electrical

field stimulation, such as atropine in combination

with α,β-methylene adenosine triphosphate (ATP),

have no effect on responses to exogenous VIP

(MacKenzie & Burnstock, 1984). Thus, it is suggested

that VIP may act as a neuromodulator in the urinary

bladder (Johns, 1979).

Among the various substances mentioned above,

ATP, a purinergic substance, seems to fulfil the role of

a NANC excitatory transmitter mediating smooth

muscle contraction in the urinary bladder (Crowe &

Burnstock, 1985; Hoyle, 1992). There is considerable

evidence to suggest that ATP may act as a neuro-

transmitter in the autonomic nervous system. It has

been shown that ATP is localised in both sympathetic

and parasympathetic neurons and is released in a

calcium-dependent manner following invasion of the

nerve terminal varicosity by an action potential (Hoyle

& Burnstock, 1993). After being released, ATP can act

on postjunctional receptors on the smooth muscle

membrane and evokes a biphasic contraction with a

primary phasic contraction followed by a relatively

slow secondary tonic contraction (Anderson, 1982). It

is likely that ATP acts as a transmitter along with NA,

ACh and other neuropeptides in the autonomic

nervous system, rather than existing separately in

distinct populations of purinergic nerves (Burnstock,

1990). Both ultrastructural and pharmacological

studies have shown that ATP cotransmits with ACh in

the intramural ganglia innervating the detrusor muscle

in the urinary bladder (Hoyes et al. 1975; MacKenzie

et al. 1982). Electrophysiologically, the biphasic

contractile response in the bladder to nerve stim-

ulation consists of an initial fast response that is

mediated by ATP and a second, slower response that

is mediated by ACh. ATP has been implicated as a

noncholinergic neuromuscular transmitter and as a

cotransmitter with ACh in parasympathetic nerves in

the urinary bladder (Hoyle et al. 1990).

Besides ATP, NO is another important NANC

inhibitory transmitter in the urethra and bladder neck

in various species (Dokita et al. 1991; Garcia-Pascual

et al. 1991; Persson & Andersson, 1992; Persson et al.

1992; Mevorach et al. 1994). NO, which is composed

of 1 atom each of nitrogen and oxygen, is an unusual

messenger (Lowenstein et al. 1994). When uncharged,

it can diffuse freely across membranes. With an

unpaired electron, it is highly reactive (having a half-

life of 2–30 s) and decaying into nitrite after trans-

mitting a signal spontaneously (Lowenstein et al.

482 Y. Zhou and E.-A. Ling

1994). As a neurotransmitter NO is highly unusual in

that it does not appear to be stored within nerve

terminals, but is synthesised and released as required.

The activity of conventional neurotransmitters is

terminated by either reuptake mechanisms or en-

zymatic degradation. The activity of NO terminates

when it reacts chemically with a substrate (Dawson &

Dawson, 1996). NO is generated intracellularly from

the semiessential amino acid -arginine by the action

of NOS, an NADPH-dependent enzyme (Grozdano-

vic et al. 1994). NADPH is a cosubstrate in the

production of NO (Bredt & Snyder, 1990). In the

conversion of arginine to citrulline, NADPH donates

electrons by the action of NOS which is thus an

NADPH-diaphorase. The latter is a term used to

define enzymes, which are able to oxidise NADPH;

on the other hand, not all NADPH-diaphorases can

produce NO (Tracey et al. 1993). NOS can be

inhibited by analogues of -arginine such as -NG-

nitroarginine (-NOARG) and -NG-nitroarginine

methyl ester (-NAME), and the inhibition of these

analogues can be reversed or prevented by additional

-arginine (Rand, 1992). The newly generated NO

probably diffuses out of the nerve terminal, rather

than being released from transmitter vesicles, and

diffuses into neighbouring cells, in this case smooth

muscle, where it activates cytosolic guanylate cyclase.

The activity of NO can therefore also be blocked by

inhibitors of guanylate cyclase, such as methylene

blue (Rand, 1992). Furthermore, NO can be prevented

from acting by adsorbing it with oxyhaemoglobin, or

deactivating it with a free radical scavenger (Rand,

1992).

NO has been shown to be synthesised in the urethra

and appears to mediate the smooth muscle relaxation

induced by nerve stimulation (Dokita et al. 1991;

James et al. 1993). Drugs that release NO mimic the

effect of nerve stimulation producing a relaxation of

urethral smooth muscle strips in vitro (Garcia-Pascual

et al. 1991; Persson & Andersson, 1992). Such effects

of nerve stimulation are blocked by NOS inhibitors in

rabbits (Dokita et al. 1991), pigs (Persson &

Andersson, 1992), rats (Persson et al. 1992) and sheep

(Garcia-Pascual et al. 1991) suggesting that NO plays

an important role in the relaxation of the sphincter

and detrusor muscle in the lower urinary tract

(Mevorach et al. 1994).

NO is synthesised by the enzyme NOS which has been

purified, sequenced and cloned (Bredt et al. 1991;

Lowenstein & Snyder, 1992; Marsden et al. 1992).

NOS is generally divided into 3 isoforms: neuronal

NOS (n-NOS), endothelial NOS (e-NOS) and in-

ducible NOS (i-NOS). The n-NOS and e-NOS share

many properties and are constitutive (Dawson &

Dawson, 1998). The n-NOS is a cytoplasmic enzyme

that is present in subpopulations of neurons in the

central and peripheral nervous systems (Fo$ rstermann

et al. 1991; Dun et al. 1993), whereas e-NOS is in the

endothelium where it is membrane bound (Lincoln et

al. 1992). i-NOS occurs in a variety of cell types,

including macrophages and vascular smooth muscle,

only after activation by cytokines (Lincoln et al.

1992). n-NOS was purified from rat cerebellum by

Bredt & Snyder (1990) and, furthermore, n-NOS

immunoreactivity was localised in the rat in which its

localisation pattern closely matched the distribution

of NADPH-d staining in the central nervous system

(Bredt et al. 1990; Dawson et al. 1991). NADPH-d

histochemistry has been used for over 30 y, but it was

not linked to any definitive functional significance

until recently (Grozdanovic et al. 1992; Vincent &

Kimura, 1992). The study by Hope et al. (1991) has

also shown that NADPH-d may be identical to n-

NOS. In addition, a one-to-one correlation between

n-NOS and NADPH-d has been found in the

myenteric neurons and plexus of the intestinal tract

(Young et al. 1992). These important findings suggest

that NADPH-d histochemistry may be adopted for

study of nitrergic neurons and fibres in both the

central and peripheral nervous systems (Bredt et al.

1990). However, some authors (Meller & Gebhart,

1993) have cautioned the interpretation of these

results since, as stated above, not all NADPH-

diaphorases are able to synthesise NO (Tracey et al.

1993). In fact, Matsumoto et al. (1993) reported a low

correlation between brain areas rich in NADPH-d

activity and n-NOS immunoreactivity in fresh brain

tissue, although in tissue treated with paraformal-

dehyde, they have shown a relatively greater cor-

relation between both enzymatic activities in cytosol.

These authors pointed out that the correlation

between brain NOS and NADPH-d in histochemical

studies may be coincidental. Valtschanoff et al.

(1992a) reported that neuronswith NADPH-d activity

that did not stain for n-NOS probably never produced

NO; the diaphorase activity was probably associated

with another enzyme that was not affected by fixation

(Traub et al. 1994). Other studies (Valtschanoff et al.

1992a ; Wang et al. 1995) have shown that the electron

dense reaction product of NADPH-d is associated

with the membrane components of endoplasmic

reticulum, Golgi apparatus and mitochondria (Wolf

et al. 1992; Tang et al. 1995; Wang et al. 1995), while

Neural nitric oxide synthase 483

Figs 1–6. For legends see opposite.


that of n-NOS is localised in the cytosol (Fo$ rstermann

et al. 1991). Furthermore, NADPH-d (Valtschanoff et

al. 1992a) and n-NOS (Dun et al. 1992; Valtschanoff

et al. 1992b) were found in similar regions of the

spinal cord but they did not appear to reside in similar

subcellular regions. All these results seem to suggest

that the localisation of neuronal NADPH-d may not

truly reflect the localisation of neurons that produce

NO (Meller & Gebhart, 1993) as was cautioned by

Matsumoto et al. (1993).

-d n-

n-NOS containing neurons have been identified in the

lower urinary tract of rat (McNeill et al. 1992), guinea

pig (Saffrey et al. 1994) and human (Ehren et al. 1994)

by using NADPH-d histochemical or n-NOS immuno-

histochemical methods. In tissue culture preparations

from neonatal guinea pigs, Saffrey et al. (1994) have

shown that 90% of the total neuronal population in

the intramural ganglia of the urinary bladder as

identified by labelling them with an antiserum to the

general neuronal marker protein gene product 9.5,

expressed NADPH-d reactivity. Almost all neurons

(99%) in culture stained for NADPH-d were also

found to be n-NOS immunoreactive. Just as in the

neonatal guinea pig urinary bladder (Saffrey et al.

1994), we have also shown that NADPH-d reactive

neurons were the most prevalent neuronal sub-

population in the urinary bladder of adult guinea pig

(Fig. 1) (Zhou et al. 1997). Besides NADPH-d

reactivity, n-NOS immunoreactivity was also localised

in the intramural ganglion cells in the wholemount

preparations of the urinary bladder of the guinea pig

by using immunofluorescence (Smet et al. 1996; Zhou

& Ling 1998) and DAB staining (Fig. 2) (Zhou et al.

Fig. 1. Photomicrograph showing a wholemount preparation of NADPH-d positive neurons in the intramural ganglia at the base of the

guinea pig urinary bladder. Note that some neurons are intensely stained (arrowheads) while others are moderately labelled (arrows) or

weakly stained (small arrows). Bar, 50 µm.

Fig. 2. Photomicrograph showing a wholemount preparation of n-NOS immunoreactive neurons (arrowheads) in the intramural ganglia at the

base of the guinea pig urinary bladder. Bundles of n-NOS positive processes (arrows) appear to connect ganglia of different sizes. Bar, 50 µm.

Fig. 3. Photomicrograph showing a wholemount preparation of NADPH-d positive intramural ganglion cells in the bladder base 24 h after

complete urethral obstruction in a guinea pig. The intramural ganglion cells appear to undergo degenerative changes as evidenced by the

fragmentation of the cytoplasm. A portion of the cytoplasm appears to have become detached from the soma of a neuron (arrows). Bar,

30 µm.

Fig. 4. Photomicrograph showing a wholemount preparation of NADPH-d positive intramural ganglia in the bladder base 48 h after complete

urethral obstruction in a guinea pig. Note the total disintegration and lysis of intensely stained NADPH-d neurons (arrows). The cellular

debris of disintegrated NADPH-d stained neurons (arrows) is strewn among normal-looking neurons which are weakly stained (arrowheads).

Bar, 30 µm.

Fig. 5. Electron micrograph showing a portion of an NADPH-d reactive DRG cell at L6 DRG of normal guinea pig. The NADPH-d

reaction product is deposited on the membrane of rough endoplasmic reticulum (rER), mitochondria (M) and some segments of the nuclear

envelope (arrows). Some Golgi saccules also appear to be stained (G), Bar, 1 µm.

Fig. 6. Electron micrograph showing a portion of an n-NOS reactive DRG cell at L6 DRG of normal guinea pig. n-NOS immunoprecipitate

(arrows) is patchily distributed throughout the cytoplasm except for the nucleus (N). Bar, 1 µm.

1997). Since the majority of the NADPH-d or n-NOS

positive neurons appear to be predominant in the

region of the bladder base, it was suggested that NO

might be involved in the relaxation activity in the base

during micturition (Saffrey et al. 1994; Smet et al.

1996; Zhou et al. 1997). This would be consistent with

recent pharmacological studies which demonstrated

that NO was a mediator for the neurogenic dilation of

the bladder neck during the micturition reflex in

different species (Dokita et al. 1991; Persson &

Andersson, 1992; Ehren et al. 1994).

The difference in cell population between NADPH-

d and n-NOS positive neurons has been shown in the

guinea pig bladder in vivo (Saffrey et al. 1994; Davies

et al. 1995; Smet et al. 1996; Zhou et al. 1997).

According to these studies, 70–90% of the intramural

ganglion cells in the bladder of the guinea pig were

NADPH-d positive, whereas 45–65% of them were n-

NOS immunoreactive (Saffrey et al. 1994; Davies et

al. 1995; Smet et al. 1996; Zhou et al. 1997). The lack

of total coincidence of the 2 enzymes has also been

observed by double labelling studies (Zhou et al.

1997; Zhou & Ling, 1998) suggesting that while

NADPH-d and n-NOS may be colocalised in the

majority of intramural ganglion cells, a one-to-one

correlation between these 2 enzymes does not always

exist in the urinary bladder.

n-

n-NOS and acetylcholinesterase (AChE )

Although anti-choline acetyltransferase (ChAT, the

enzyme responsible for the synthesis of ACh) has been

the antiserum of choice for localisation of ACh in the


central and peripheral nervous system (Furness et al.

1984; Wang et al. 1996b), the demonstration of ChAT

immunoreactivity in the urinary bladder had not been

successful (Vaalasti et al. 1986; Zhou & Ling 1998).

AChE histochemistry has, therefore, been adopted for

localisation of cholinergic neurons in the urinary

bladder (Burnstock et al. 1978; de Groat & Booth,

1984; Crowe et al. 1986; Gabella, 1990) and it has

been shown that the majority of the intramural

ganglion cells are stained for AChE (Burnstock et al.

1978; de Groat & Booth 1984; Gabella 1990).

Although the reliability of AChE as a cholinergic

marker has sometimes been questioned, it is none-

theless a useful marker for cholinergic neurons in the

autonomic ganglia (Lundberg et al. 1979). A recent

study by us (Zhou & Ling, 1998) has unequivocally

demonstrated the colocalisation of AChE and n-NOS

suggesting that the colocalisation of the 2 enzymes is

to complement each other for the neural control of the

micturition reflex. This result, along with previous

studies on the colocalisation of ChAT and n-NOS in

the sacral parasympathetic nucleus of rats (Barber et

al. 1984; Vizzard et al. 1994a), suggests that ACh and

NO may function in both the pre- and postganglionic

neurons in the parasympathetic nerves innervating the

urinary bladder. NO may contribute to the regulation

of pelvic visceral activities at the level of the

lumbosacral spinal cord (Burnett et al. 1995) as well as

in the intramural ganglia of target organs (Zhou &

Ling, 1998).

Tyrosine hydroxylase (TH ) and NADPH-d

TH is an enzyme responsible for the synthesis of

catecholamine (CA) (Smet et al. 1994), which is

known to be a sympathetic nerve mediator that serves

to maintain closure of the bladder outlet and internal

urethral sphincter (Lincoln & Burnstock, 1993). CA-

containing nerves have been detected in the bladder

base and some regions of the urethral smooth muscle

in a variety of species including man (Klu$ ck, 1980;

Crowe & Burnstock, 1989).

In the intramural ganglia of the guinea pig urinary

bladder, our study (Zhou & Ling, 1998) has dem-

onstrated the lack of colocalisation of TH and

NADPH-d. It is suggested that the TH positive

neurons in the urinary bladder represent a separate

neuronal population, which is not capable of pro-

ducing NO.

Vasoactive intestinal peptide (VIP) and NADPH-d

It has been reported that VIP acts as a cotransmitter

with ACh in parasympathetic nerves (Lundberg et al.

1979). In the urinary bladder, VIP may function as a

neuromodulator as it causes relexant responses to the

detrusor, trigone and neck (Larssen et al. 1981; Crowe

et al. 1985). VIP immunoreactivity is predominantly

located in the bladder base in guinea pigs, cats, pigs

and man (Alm et al. 1980; Mattiasson et al. 1985;

Crowe & Burnstock, 1989) and such immunoreactivity

is detected in 10–15% of the bladder ganglion cells

stained for AChE (Lundberg et al. 1979). It is

suggested that VIP is probably colocalised with ACh

in some bladder ganglion cells (Lundberg et al. 1979;

Kawatani et al. 1985).

VIP immunoreactive intramural neurons have been

demonstrated to coexpress NADPH-d reactivity

suggesting that VIP may be released together with NO

from the parasympathetic postganglionic neurons to

act as a neuromodulator coordinating the relaxation

of smooth muscle in the bladder base or urethra

(Zhou & Ling, 1998).

Calcitonin gene-related peptide (CGRP), substance P

(SP) and NADPH-d

Both CGRP and SP are known to exist in the afferent

cells and act as transmitters in sensory nerves (de

Groat, 1987; Maggi & Meli, 1988; Lincoln &

Burnstock, 1993). Furthermore, these peptidergic

substances are also involved in the initiation of central

autonomic reflexes as well as peripheral axon reflexes

which modulate smooth muscle activity, facilitate

transmission in autonomic ganglia and trigger local

inflammatory responses (de Groat, 1987). Their

immunoreactivities have been localised in the DRG

cells identified as supplying the bladder and urethra

by retrograde tracing technique (Papka, 1990).

CGRP and SP immunoreactivities have been

confined exclusively to nerve fibres (Ho$ kfelt et al.

1977; Crowe et al. 1986) and such fibres have recently

been demonstrated to associate closely with the

intramural ganglion cells stained for NADPH-d

(Zhou & Ling, 1998). It is postulated that through

such a unique configuration, NO may act as a

retrograde transmitter exerting its influence on the

sensory afferent pathways (Zhou & Ling, 1998). On

the other hand, the CGRP and SP immunoreactive

sensory afferents could also modulate activity in the

peripheral autonomic ganglia in an anterograde

manner (de Groat, 1987). This would be in line with

the view of the ‘sensory-motor nerve’ regulation

described by Burnstock (1993). Hence the close spatial

relationship between CGRP and SP positive nerve

fibres and NADPH-d positive neurons may be


involved in modulating the smooth muscle relaxation

in the intramural ganglia of the urinary bladder.

-d

Urethral obstruction is a common problem which

leads to many anatomical and physiological changes

in neural pathways to the urinary bladder (Speakman

et al. 1987; Steers et al. 1990). Partial urethral

obstruction due to enlargement of the prostate gland

in man, e.g. benign prostate hypertrophy (BPH) can

lead to increases in urethral resistance, smooth muscle

hypertrophy in the bladder and irritative bladder

conditions such as unstable contractions and

increased frequency of voiding (Andersen, 1976;

Chalfin & Bradley, 1982). Animal models have been

used for studying the pathophysiology of partial

outlet obstruction (Levin et al. 1990). These models

have been developed in a variety of species including

the rat (Mattiasson & Uvelius, 1982; Steers & de

Groat, 1988), rabbit (Levin et al. 1984; Kitada et al.

1989), cat (Kato et al. 1990; Radzinski et al. 1991),

guinea pig (Mostwin et al. 1991), pig (Sibley, 1985)

and dog (Rohner et al. 1978).

In the rat model, partial urethral obstruction causes

an initial rapid increase in bladder weight (30%

increase per day) during the first few days after

obstruction (Uvelius et al. 1984). Six weeks following

obstruction, the weight of the urinary bladder was

considerably increased, ranging from 8 to 11 times

that of controls ; the increase was attributed to smooth

muscle hypertrophy and hyperplasia (Uvelius et al.

1984). Cystometric analyses revealed that un-

anaesthetised animals with partial outlet obstruction

exhibited increased bladder compliance and capacity,

decreased voiding efficiency, increased voiding

pressures and unstable bladder contractions during

filling (Levin et al. 1990). Measurements of voiding

patterns in conscious animals showed increased

frequency of voiding with decreased voided volumes

and most of the changes were reversed following

removal of the obstruction (Malmgren et al. 1990).

Such changes including muscle hypertrophy and

increased frequency of voiding in the animal models

are similar to the clinical situation in man (Gabella &

Uvelius, 1993; Steers et al. 1996). Anatomical tracing

studies in partial urethral obstructed rats have revealed

the changes in the morphology of both afferent and

efferent pathways to the urinary bladder (Steers et al.

1990, 1991, 1996). In rats subjected to partial urethral

ligation for 6 wk, changes in the afferent pathways

included an increase (40%) in the average cross-

sectional area of labelled neuronal profiles in the

dorsal root ganglia (Steers et al. 1991). An increase in

the afferent projections to the spinal cord has been

demonstrated in the regions of the intermediolateral

nucleus (Steers et al. 1991) and the sacral para-

sympathetic nucleus (Steers et al. 1996). In efferent

pathways, the bladder postganglionic neurons in the

major pelvic ganglia of partially obstructed rats

exhibited a 90% increase in cross-sectional area when

compared with the controls (Steers et al. 1990). These

results suggest the remarkable plasticity of both

afferent and efferent pathways of the urinary bladder

in response to partial urethral obstruction. It was

speculated that the observed changes in the neural

pathways could have accounted for the enhancement

of a spinal micturition reflex.

Clinically, acute urinary retention is one of the most

common complications of BPH and it occurs in 47%

of patients (Christensen & Bruskewitz, 1990). Ex-

perimental studies on complete urethral obstruction

mimicking acute urinary retention have shown a 47%

reduction in muscarinic receptor density and 75%

decrease in the contractile responses to muscarinic

stimulation and field stimulation within 24 h following

urethral obstruction in rabbits (Kitada et al. 1989).

Following urethral obstruction, neuronal and axonal

degeneration and subsequent neuronal death in the

bladder (Speakman et al. 1987; Elbadawi et al. 1989)

and in the dorsal root ganglia (Zhou et al. 1998a) have

been reported in electron microscopic studies. These

changes are attributed to hypoxia as a result of

reduced blood flow as well as mechanical damage due

to high intravesical pressure (Kitada et al. 1989). The

presence of inflammation, ischaemic damage and

oedema in the overdistended bladder has been

implicated in facilitating the degeneration in the

bladder during the initial period of the urethral

obstruction (Kitada et al. 1989). It has been concluded

that the obstruction can produce partial denervation

of the bladder resulting in changes in the peptide

content in the bladder and alteration of responses of

the bladder smooth muscle to autonomic transmitters

(Andersson et al. 1988).

In our experimental model in guinea pigs with

complete urethral obstruction, increased NADPH-d

expression in the intramural ganglion cells was

detected which was followed subsequently by

neuronal degenerative changes and cell death (Figs 3,

4) (Zhou & Ling, 1997a). These results along with the

observations of NADPH-d reactivity in the intestinal

submucous ganglia (Wang et al. 1996c) and nodose


ganglion (Wang et al. 1996d ) of the guinea pig after

vagotomy suggest that the increased NADPH-d

expression may be a harbinger of consequent neuronal

degeneration and cell death as this might indicate

elevated n-NOS expression and, hence, greater NO

production.

-d n-

The neurons of the bladder afferents in the hypogastric

nerves (sympathetic) reside in dorsal root ganglia

(DRG) at L1 and L2 levels, while those in the pelvic

nerve (parasympathetic) are located in the DRG at L6

and S1 in rats (Hancock & Peveto, 1979; Nadelhaft &

Booth, 1984; Steers et al. 1991).

In guinea pigs, the majority of the bladder afferent

neurons are located in the DRG at L6–S2 levels with

fewer at L2–L3 suggesting that these cells provide the

main source of afferent innervation to the bladder

(Zhou & Ling, 1997b). A distinct bundle of tracer

labelled fibres has been detected in the lateral edge of

the dorsal horn extending from the Lissauer’s tract to

the region of the sacral parasympathetic nucleus

(SPN) of the spinal cord (Zhou & Ling, 1997b). This

feature parallels that in rats using wheat germ

agglutinin-HRP as the tracer (Steers et al. 1991).

NADPH-d reactivity and n-NOS immunoreactivity

have been demonstrated in the bladder afferent

neurons in the DRG using histochemical or immuno-

histochemical methods along with the retrograde

axonal tracing technique (Vizzard et al. 1993a). In

DRG at L6 and S1 segments of rats, 80.9% and

78.5%, respectively, of the bladder afferent neurons

labelled retrogradely with fluorescent dyes

(Flurogold or FB) were NADPH-d positive (Vizzard

et al. 1993a, 1994a). In the spinal cord of the same

species, NADPH-d reactivity was also identified in

neurons in the SPN at L6–S1 levels (Vizzard et al.

1993b). NADPH-d reactivity was also detected in

tracer labelled visceral afferent fibres after application

of retrograde tracer (horseradish peroxidase) to either

the pelvic nerves or urinary bladder (Vizzard et al.

1993b, 1994a). In guinea pigs, 30.9–42.1% of the

bladder afferent neurons expressed NADPH-d re-

activity (Zhou & Ling 1997b). These results (Vizzard

et al. 1993a ; Zhou & Ling, 1997b) suggest the

involvement of NO in the bladder afferent pathways.

However, n-NOS immunoreactivity is not paralleled

by NADPH-d reactivity in rats (Vizzard et al. 1995).

The discrepancy between NADPH-d reactivity and n-

NOS immunoreactivity in the bladder afferent

neurons is consistent with our observation in the

guinea pig whereby only 10.2–16.7% exhibited n-

NOS immunoreactivity compared with 30.9–42.1%

being NADPH-d reactive (Zhou & Ling, unpublished

observations). By electron microscopy, NADPH-d is a

membrane-associated protein with a widespread sub-

cellular distribution being localised chiefly in the

cisternae of rough endoplasmic reticulum, Golgi

saccules, mitochondria and nuclear envelope (Fig. 5).

On the other hand, n-NOS reaction product was not

specifically associated with any subcellular organelles

and plasma membrane (Fig. 6) (Zhou et al. 1998b). It

is possible that the actual number of neurons

expressing n-NOS neurons may have been over-

estimated by NADPH-d staining (Smet et al. 1996).

However, our triple labelling study has shown that

some bladder afferent neurons retrogradely labelled

with FB coexpressed n-NOS and NADPH-d (Fig. 7)

suggesting that the latter enzyme is still a reliable,

though not absolute, histochemical marker for NO-

producing DRG neurons.

Along with the above, it is relevant that NADPH-

d and n-NOS have been detected in the lateral edge of

the dorsal horn in a fibre bundle extending from

Lissauer’s tract to the region of the SPN (Vizzard et

al. 1993a, 1994a ; Zhou & Ling 1997b). These findings

have further strengthened the view of the involvement

of NO in the bladder afferent pathways.

-d n-

Being a small, reactive and gaseous molecule which is

able to pass through neuronal membranes easily, NO

may act as a ‘retrograde transmitter ’ in the sensory

pathways (Meller & Gebhart, 1993). However, it has

been shown that NO may not act as a ‘classic ’

transmitter in the nociceptive pathways because

antagonists of NOS do not alter baseline nociceptive

reflexes although they abolish facilitation of no-

ciceptive reflexes (Meller & Gebhart, 1993). Thus it

has been speculated that NO may play a pivotal role

in nociceptive processing in multisynaptic local circuits

of the spinal cord (Meller & Gebhart, 1993). This

concept has been supported by studies which showed

that nociceptive reflexes induced by mechanical

stimulation in the presence of inflammation could be

reduced by inhibitors of NOS (Meller et al. 1992,

1994). It has been shown that NADPH-d reactivity or

n-NOS immunoreactivity in the DRG and dorsal

horn of the spinal cord is plastic and could be

upregulated by pathological conditions (Solodkin et


Fig. 7. FB fluorescence (A), n-NOS immunofluorescence (B) and NADPH-d bright-field (C ) photomicrographs of the same section of L6

DRG in normal guinea pig. FB-labelled cells (A, arrows) also display n-NOS immunoreactivity (B, arrows) and NADPH-d reactivity (C,

arrows). Bar, 30 µm.

Fig. 8. FB fluorescence (A) and NADPH-d bright field (B) photomicrographs of the same sections showing NADPH-d staining (B, arrows)

in some FB-labelled bladder afferent neurons (A, arrows) in DRG at L6 segment of normal guinea pig receiving FB injection into the bladder

base. FB-fluorescence (C ) and NADPH-d bright-field (D) photomicrographs of the same sections showing increased NADPH-d staining (D,

arrows) in some FB-labelled bladder afferent neurons (C, arrows) in L6 DRG of the guinea pig, 48 h after complete urethral obstruction.

Bar, 30 µm.


Fig. 9. For legend see opposite.


al. 1992; Zhang et al. 1993, 1994). For example,

increased NADPH-d reactivity has been detected in

the afferent neurons in DRG in the models of hindpaw

inflammation (Traub et al. 1994) and neuropathic

pain (Steel et al. 1994).

In the pelvic organs, Vizzard et al. (1995) have

shown that expression of n-NOS in the afferent

neurons in DRG was increased following removal of

the major pelvic ganglia of rats. Upregulated n-NOS

immunoreactivity was also detected in the fibres along

the lateral edge of the dorsal horn extending from

Lissauer’s tract to the region of the SPN (Vizzard et

al. 1995). More recently Vizzard et al. (1996) further

showed that expression of n-NOS in the bladder

afferent pathways was increased following chronic

bladder irritation. It is suggested that NO does play a

role in the facilitation of the micturition reflex by

noxious chemical irritation of the bladder (Vizzard et

al. 1996). This notion is supported by pharmacological

studies which showed that bladder hyperreflexia

induced by either acetic acid (0.1%) (Kakizaki & de

Groat, 1996) or by turpentine (Rice, 1995) was

partially antagonised by intrathecal or topical spinal

cord administration of an NOS inhibitor. These

studies suggest that NO is involved in the facilitation

of the micturition reflex by nociceptive afferents at

spinal level (Rice, 1995; Vizzard et al. 1996).

Our study has shown increased NADPH-d re-

activity in the DRG and their projections in cor-

responding segments of the spinal cord following

urethral obstruction (Fig. 8) (Zhou & Ling, 1997b).

This raises the possibility that NO may act as a

neurotransmitter or neuromodulator involved in the

spinal nociceptive afferent input as a result of

overdistension of bladder and}or prolonged stimu-

lation of the mechanosensitive afferents in response to

the bladder outlet obstruction.

-d n-

Several studies have already dealt with the localisation

of n-NOS and NADPH-d in the lumbosacral spinal

cord in cats and rats (Aimi et al. 1991; Vizzard et al.

1994b). NADPH-d}NOS have been described in both

sympathetic and sacral parasympathetic preganglionic

neurons (Anderson, 1992; Blottner & Baumgarten,

Fig. 9. Photomicrographs showing NADPH-d reactivity and n-NOS immunoreactivity in the L6 segment of the spinal cord in the sham-

operated and complete urethral obstructed guinea pigs. (A) n-NOS immunoreactivity at L6 segment of the spinal cord in a sham-operated

animal. n-NOS positive neurons are distributed in the dorsal horn (DH) and the grey matter around the central canal (asterisk). n-NOS

positive neurons are barely detectable in the ventral horn (VH). (B) NADPH-d staining in the ventral horn neurons (arrows) at L5 segment

of the spinal cord in an animal killed 48 h after complete urethral ligation. (C) Induced n-NOS immunoreactivity in the ventral horn neurons

(arrows) at L6 segment of the spinal cord, 48 h after complete urethral ligation. Bar, 200 µm.

1992; Valtschanoff et al. 1992a) as well as in the

substantia gelatinosa of the dorsal horn, and around

the central canal (Dun et al. 1993; Vizzard et al.

1994b) ; sporadic positive cells were found in other

areas of the cord such as the ventral horn (Dun et al.

1992, 1993; Zhang et al. 1993; Vizzard et al. 1994b).

This is contrary to a recent report that a large number

of neurons in the somatic motor nuclei and Onuf’s

nucleus in the cat, monkey and man express intense

NOS immunoreactivity (Pullen et al. 1997).

-d n-

Several studies on NO have focused on its expression

in ventral horn motoneurons in the spinal cord after

peripheral nerve injury. It was reported that the spinal

motoneurons in new-born rats responded by ex-

pressing NADPH-d reactivity after axotomy

(Gonzalez et al. 1987; Lei et al. 1992; Hama & Sagen,

1994). NADPH-d and n-NOS are also induced in the

spinal motoneurons following spinal root avulsion in

rats (Wu & Li, 1993) and knee joint immobilisation in

guinea pigs (He et al. 1997). It has been suggested that

induction of n-NOS immunoreactivity in moto-

neurons following ventral root avulsion is involved in

the subsequent death of lesioned motoneurons be-

cause pretreatment with an NOS inhibitor significant-

ly increases the numbers of surviving motoneurons

(Wu & Li, 1993). All these studies suggest the plasticity

of n-NOS in motoneurons of the spinal cord.

In our experimental guinea pig model with complete

urethral obstruction, an increase in NADPH-d}n-

NOS reactivity was induced in ventral horn moto-

neurons in the lumbosacral spinal cord following

urethral obstruction (Fig. 9) (Zhou & Ling, 1997c). In

agreement with previous studies by others (see above),

this may have been induced by nerve lesions following

surgical ligation. It is speculated that the presumed

increase in NO production maybe involved in modu-

lating the threshold for triggering micturition (Zhou

& Ling 1997c). NO has been identified as a neuro-

transmitter in the NANC-induced urethral relaxation

in the micturition reflex (Persson et al. 1992;

Hashimoto et al. 1993; Mevorach et al. 1994). Ventral

horn motoneurons innervating the external urethral


sphincter have been located in the ventrolateral

division of the Onuf’s nucleus in the caudal

lumbosacral spinal cord by previous studies (de Groat,

1975; Morita et al. 1984). The upregulated NADPH-

d}n-NOS reactivity in ventral horn motoneurons

could also have been induced by the afferents from the

overdistended bladder in the animal model with

complete urethral obstruction (Zhou & Ling, 1997c).

The production of NO is probably to inhibit the

motoneurons and consequently relax the external

urethral sphincter in an attempt to overcome the

outlet resistance (Zhou & Ling, 1997c).

It is evident from the literature review that NO, as a

novel neurotransmitter, not only exists in the bladder

neural pathways including the intramural ganglia,

dorsal root ganglia and spinal cord, but also displays

remarkable plasticity following pathological lesions

such as pelvic nerve injury, chronic bladder irritation

and urethral obstruction.

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neuronal nitric oxide synthase in the neural pathways of the urinary bladder

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