Nitric oxide and cyclic GMP formation upon electrical field stimulation cause relaxation of corpus cavernosum smooth muscle

Vol. 170, No. 2, 1990

BIOCHEMICALANDBIOPHYSICALRESEARCH

COMMUNICATIONS

July 31, 1990

Pages

NITRIC

OXIDE AND CYCLIC GNP FORMATION UPON ELECTRICAL FIELD STIMULATION CAUSE RELAXATION OF CORPUS CAVERNOSUM SMOOTH MUSCLE

Louis

J.

Department

Received

843-850

Ignarro,

Peggy A. Bush, Georgette M. Buga, Jon M. Fukuto and Jacob Rajfer*

Keith

S. Wood

of Pharmacology and Division of Urology, *Department of University of California, Los Angeles, California 90024

June

18,

Surgery

1990

SUMMARY: In the presence of functional adrenergic and cholinergic blockade, electrical field stimulation relaxes corpus cavernosum smooth muscle by unknown mechanisms. We report here that electrical field stimulation of isolated strips of rabbit corpus cavernosum promotes the endogenous formation and release of nitric oxide (NO), nitrite, and cyclic GMP. Corporal smooth muscle relaxation in response to electrical field stimulation, in the presence of guanethidine and atropine, was abolished by tetrodotoxin and potassium-induced depolarization, and was markedly inhibited by NG-nitro-L-arginine, NG-amino-L-arginine, oxyhemoglobin, and methylene blue, but was unaffected by indomethacin. The inhibitory effects of NG-substituted analogs of L-arginine were nearly completely reversed by addition of excess L-arginine but not D-arginine. Corporal smooth muscle relaxation elicited by electrical field stimulation was accompanied by rapid and marked increases in tissue levels of nitrite and cyclic GMP, and all responses were nearly abolished by NG-nitro-L-arginine. These observations indicate that penile erection may be mediated by NO generated in response to nonadrenergic-noncholinergic neurotransmission. 01990 Academic Press, Inc.

by

In the

human

least

three

at

and rabbit,

adrenergic,

cholinergic,

Adrenergic

neurotransmission

which

causes

adrenoceptors

fibers

In the atropine marked

corporal

tested neither

smooth

attenuated

of

(a muscarinic in

the

muscle

muscarinic

both

of

smooth

found

of

penile

or

NANC

corporal

receptors

smooth

whereas

relaxation of

innervated

muscle

erection,

by activation

only

muscle

and both

tone: (1,2). muscle,

cholinergic

penile

erection.

prejunctional

innervated

be without

mechanism

(an adrenergic

blocker), the

electrical

NANC neurons

relaxation

that

Many classes

(1,2). to

guanethidine

receptor

excitation

tetrodotoxin and

contraction

is

smooth

alpha,-

by interneuronal

(l-3).

presence

results

cavernosum

control

nonadrenergic-noncholinergic mediates

and prejunctional

cholinergic

blocker

is

corpus that

or diminished

promotes

function

trabecular pathways

and

detumescence

neurotransmission Adrenergic

the

neuroeffector

of

effect

field

and the is abolished

of

on this smooth

corporal

neuronal

pharmacological relaxation muscle

blocker)

and

stimulation

response

is

by the

sodium

agents response relaxation

(EFS) rapid

channel have

(3).

been Thus,

nor 0006-291X/90

843

and

the $1.50

Copyright 0 1990 by Academic Press. Inc. All rights of reproduction in any form reserved.

Vol.

170,

No.

2,

1990

neurotransmitter study corpus

was to

BIOCHEMICAL

released elucidate

cavernosum

smooth

AND

by NANC neurons the

mechanism

BIOPHYSICAL

is known.

by which

RESEARCH

COMMUNICATIONS

The objective

EFS causes

relaxation

of the of

present rabbit

muscle.

MATERIALS

AND METHODS

Chemicals and solutions: L-Arginine, D-arginine, indomethacin, phenylephrine HCl, acetylcholine chloride, atropine sulfate, guanethidine sulfate, methylene blue, hemoglobin (human), NG-nitro-L-arginine, and reagents for the diazotization The water soluble reaction were from Sigma Chemical Co. (St. Louis, MO). crystalline HCl salt of authentic NG-nitro-L-arginine was prepared by standard chemical procedures and used in this study. S-Nitroso-N-acetylpenicillamine (SNAP) was prepared, stored, and used as described (4). Oxyhemoglobin solutions Krebs-bicarbonate solution consisted of were prepared as described (5). (millimolar): NaCl, 118; KCl, 4.7; CaCl , 1.5; NaHCO,, 25; MgSO,, 1.2; KH,PO,, 11; and disodium ethylenec&aminetetraacetic acid, 0.023. 1.2; glucose, Preparation of rabbit corpus cavernosum for relaxation measurements: Corpus cavernosum excised from the oenis of New Zealand White rabbits (3 kq) was cut into 4 or 5 strips (6.3 x-b.3 x dissected free from the tunica albuginea, 0.7 cm), and mounted under 2 g resting tension in organ chambers containing Krebs-bicarbonate solution at 37'C gassed with 95% 0, - 5% CO . Resting tensions of 2 g for strips of corpus cavernosum prepared from 5 kg rabbits were predetermined to produce optimal length-tension relationships (potassium-induced contractions) for experiments on smooth muscle relaxation. Changes in isometric force were measured and recorded (5), and EFS was accomplished with the aid of two parallel platinum electrodes, on either side of the strips, connected to a current amplifier and stimulator (SD9 Grass Stimulator). EFS was conducted at 10 volts for 10 set at frequencies of 2, 4, 8, and 16 Hz in the form of square wave pulses (0.2 msec). Bathing media contained 5 PM guanethidine and 1 PM atropine (atropine was omitted when acetylcholine was tested). Strips were allowed to equilibrate under tension for 60 min. Corporal smooth muscle was precontracted submaximally (60-70% of maximal) by addition of 10 PM phenylephrine, and the smooth muscle developed 3-4 g of tension that was remarkably well-maintained for at least 60 min in between washing the strips. In general, EFS was applied to precontracted strips at varying frequencies, allowing tissues to recover completely from each relaxation response prior to the next EFS. Sequential frequencies of 4, 2, 4, 8, and 16 Hz were used in most protocols in order to obtain frequency-dependent relaxant responses and to assess the reproducibility of the responses at a given frequency (see Fig. 1). Cyclic GMP determination: Cyclic GMP levels were measured in corpus cavernosum strips that had been equilibrated under tension and subjected to precontraction as was performed with all other strips, and tone was monitored until the time of quick-freezing with the aid of brass clamps precooled in liquid nitrogen. Samples (25-40 mg) were extracted and assayed for cyclic GMP by radioimmunoassay as described (5). Inorqanic nitrite determination: Nitrite levels were measured in corpus cavernosum strips that had been equilibrated under tension. orecontracted. and quick frozen as'described above. 'Each strip (25-40 rngj was homogenized in 0.5 ml of ice-cold absolute methanol, allowed to stand at 4 C for 18-20 hr to ensure complete extraction of nitrite, and samples were centrifuged at 10,000 x g for 10 min. Aliquots (300 ~1) of clear supernatant were assayed for nitrite by a modification of procedures described previously (6). Briefly, 400 ~1 of 1% w/v sulfanilic acid in 4 N HCl were added to each sample. Samples were mixed and 100 ~1 of cont. HCl were added to clarify samples that had turned slightly cloudy. After 10 min, 300 ~1 of 1% w/v N-(1-naphthyl)-ethylenediamine in methanol were added and optical densities were measured at 548 nm. 844

Vol.

170,

No.

2,

1990

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

RESULTS Inhibition that

of EFS-induced

interfere

dependent, smooth

with rapidly

muscle

biological

and actions

2

0

1

transient

of

8

frequency

of at

all

corporal

channel

blocker,

frequencies of

agent

was inhibited

bv aqents

a frequency-

precontracted sodium

an inhibitor

Relaxation

muscle

EFS caused

a neuronal

an NO sequestering

NO (7,8).

4

relaxation

blue,

smooth

NO:

relaxation

by methylene

oxyhemoglobin,

of

(1 PM),

EFS-induced

was inhibited (7),

cavernosum

or formation

Tetrodotoxin

abolished

Relaxation cyclase

ofcorous

actions

developing,

(Fig.1).

consistently

relaxation

the

tested.

cytosolic that also

guanylate inhibits

the

by NG-nitro-L-

100 16

0

(Hz,

2

2

4

8

16

frequency ulz)

Fia. 1. Relaxation of corpus cavernosum smooth muscle by EFS is inhibited Strips of by agents that interfere with the biosynthesis or actions of NO. mounted under 2 g of tension, and rabbit corpus cavernosum were prepared, equilibrated for 60 min. EFS was conducted at 10 volts for 10 set at frequencies Bathing of 2, 4, 8, and 16 Hz in the form of square wave pulses (0.2 msec). The inset is a typical media contained 5 PM guanethidine and 1 PM atropine. tracing illustrating relaxation of phenylephrine (PE; 10.'M)-precontracted strips in response to EFS at various frequencies (Hz). W signifies washing of strips. After obtaining control responses (0) to EFS, NG-nitro-L-arginine (A; 30 PM) or NG-amino-L-arginine (A; 30 PM) was added 15 min prior to a second round of EFS. Oxyhemoglobin (m; 10 PM) was added 60 min and methylene blue (0; 10 PM) was added 30 min prior to a second round of EFS. Each data point represents the mean f S.E.M. of 12-18 strips from 3-5 rabbits. Values obtained in the presence of each inhibitor are significantly different (kO.01) from corresponding control values (Student's t test for paired values). Fia. 2. Reversal by L-arginine of the inhibitory effect of NG-nitro-Larginine and NG-amino-L-arginine on EFS-elicited relaxation of corpus cavernosum smooth muscle. Experiments were performed as described in Fig. 1. After obtaining control responses (0) to EFS, NG-nitro-L-arginine (A,A; 30 PM) or NGamino-IL-arginine (m,0; 30 PM) was added 15 min prior to a second round of EFS. L-Arginine ( A,[7; 300 PM) was then added to baths containing inhibitor, and Each data point represents the mean responses to EFS were obtained 15 min later. Values obtained in the presence of f S.E.M. of 14-18 strips from 3-5 rabbits. L-arginine are significantly different (kO.01) from corresponding values obtained in the presence of inhibitor without L-arginine (Student's t test for paired values).

845

Vol.

170,

No.

arginine inhibit

2,

and the

unaffected

of is not

the

inhibitory

illustrated

here

two

endogenous

responses

Influence

of

relaxation

of to

cornus be

to

an inhibitor of

by their to

COMMUNICATIONS

of

L-arginine

NO (9,lO).

of prostaglandin

of D-arginine EFS and caused

(not little

shown).

smooth

endothelium-dependent

endothelial

muscle:

whereas

on

SNAP,

inactive, EFS-induced

caused a labile

ACh +m:;H;

JPE

4 Ini”

-fpE SNAP

ACll -m

j7i6cs;#:5

-fPE

4 min

-I

PE

ACC

SNAP

+p:65

xi.!5

t KCI

1mmM

120mM

Fiq. 3. Influence of membrane depolarization (120 mM KCl) and apparent endothelial damage (-E) on relaxant responses of corpus cavernosum smooth muscle to EFS, acetylcholine (ACh), and S-nitroso-N-acetylpenicillamine (SNAP). As described in the text, corporal tissue could not be mechanically denuded and -E signifies tissue that was unresponsive to ACh as shown. Experiments were performed as described in Fig. 1. Strips were precontracted by phenylephrine (PE; 10.sM) or 120 mM KC1 as indicated. ACh and SNAP were added in cumulatively increasing concentrations (1O“M to 10-5M). W/E signifies washing and equilibration for 30 min. The tracings shown are representative of 12-16 strips from 3-4 rabbits for each test condition.

846

did

relaxation. were

Acetylcholine

relaxation,

(Fig.

L-Arginine

or no direct

damaqe

The

NG-amino-L-

of L-arginine

and NG-amino-L-arginine and

was

formation. and

upon addition

that

Relaxation

NG-nitro-L-arginine

reversal

deDolarization cavernosum

RESEARCH

analogs

L-arginine

of NG-nitro-L-arginine membrane

BIOPHYSICAL

structural

actions

concentrations

control

The D-enantiomers

appeared

of

equivalent

enhance

AND

NG-amino-L-arginine,

by 10 PM indomethacin,

arginine not

BIOCHEMICAL

conversion

selectivity 2) but

1990

what S-

Vol.

170,

No.

2,

nitrosothiol

1990

that

inhibited observations

(ll),

in

The elicited

smooth

question relaxation,

be

answered

attempting

to

cavernous

spaces

approximately to cause

5% of the

cyclic

formation

for

because the

NG-nitro-L-arginine

response

(Figs.

1 and 2).

with

did

not

relaxant to EFS was actions

same as their

actions

in

is

of

technical

endothelium

problems lining

trabecular

corporal

on

Moreover,

4),

that

muscle, which

relaxation

for

EFS-

(12),

could

encountered blood

in

sinuses

and

It was observed acetylcholine

normal

GMP in corpus

smooth

(Fig.

the

network.

however,

and cyclic

obligatory

relaxation

EFS and SNAP caused

of nitrite

by

the

line

but

Similarly, response

in

endothelium

studied,

whereas

GMP accumulation

In

pharmacological

acetylcholine-elicited

in a complex

inhibited

agents

all

inhibited

the

are

vascular

preparations

any relaxation,

EFS-induced

is

selectively

arranged

to

shown).

3).

Thus,

cavernosum

the

unequivocally damage

(Fig.

preparations.

whether as it

(not

(Fig. relaxation

strips.

muscle

of

blue

depolarization

to acetylcholine diminished but

and NO on corpus

relaxation

NO, and responses

and methylene

potassium-induced

potassium-depolarized

vascular

endothelium-independent

by authentic

by oxyhemoglobin

acetylcholine

not

also

relaxant responses to SNAP were only

abolished other

NO, caused

was caused

past

of

liberates

Relaxation

3). were

abolish responses

BlOCHEMlCALANDBlOPHYSlCALRESEARCHCOMMUNlCATlONS

failed

relaxation

(Fig.

cavernosum: and

inhibited

and cyclic

this also

in 3).

EFS caused response the

was

relaxant

GMP accumulation

were

I Cyclic CUP

Nitrite

T*

10 0 i

c-

cm EFS 16 Hz

EiS 16 Hz

Stimulation of cyclic GMP and nitrite formation in corpus cavernosum 4. as described in Fig. response to EFS. Experiments were performed essentially 1. Untreated strips (hatched bars) or strips treated with 100 hM NG-nitro-Larginine for 15 min (solid bars) were quick frozen at 10 set after onset of EFS (16 Hz). Control strips (C) were mounted under tension, equilibrated for 60-90 Samples (25-40 mg) were extracted and assayed for min, and then quick frozen. cyclic GMP. Nitrite determinations were performed on samples that were extracted with absolute methanol. Each data point represents the mean f S.E.M. of lo-12 strips from 2-3 rabbits. *Significantly different (Pt0.01) from control strips Fia. in

and

NG-nitro-L-arginine-treated

strips

(Student's

847

t test

for

unpaired

values).

Vol.

170,

No.

2,

accompanied

1990

BIOCHEMICAL

by tissue

oxidation

product

arginine

(Fig.

of

AND

BIOPHYSICAL

RESEARCH

accumulation

of nitrite

(NO,-),

NO, and this

response

was also

the

COMMUNICATIONS

principal

spontaneous

inhibited

by NG-nitro-L-

4).

DISCUSSION The

mechanism

by which

EFS was

studied. cholinergic

performed

blockade

in

order

pathways

of neurotransmission.

are:

EFS

1,

abolished blue,

caused

oxyhemoglobin, the

were

by addition

of

excess

by increased

were did

not relaxed

and cyclic EFS

study

is

due

demonstrates to

evidence

for

formation

or target

Moreover,

the

which reversed

by

cell

These analog,

between

of

but

of

reversed relaxation GMP, both

and

5,

and

appeared

EFS-induced to

be

unresponsive

was assessed

spontaneous

not

analogs

and were

were

was

by methylene

and cyclic

that

to

by monitoring

oxidation

relaxation

and

of nitrite

product

of NO,

similar

GMP formation

potassium-induced

in

and cavernosum

action

tissues,

support

via

cholinergic are mediated 848

inhibit

the

relaxation.

nearly

completely selective

with both NO is

heme-dependent

coupled the view The

EFS-induced

experimental

to

findings tissues

Accordingly,

cavernosum. of

were

of endogenous

GMP formation (17,18).

The

known

EFS-induced

previous

GMP formation,

corpus

depolarization

adrenergic in corpus

cell

elicited

an enantiomerically

made in other

cyclic cyclic

NO.

(9,10),

in to

relaxation

and NG-amnio-L-arginine,

NO formation

target

in the

of agents

inhibited

L-arginine

are

cyclase

muscle

of NG-nitro-L-arginine

excess

intracellular

smooth

chemical

NG-methyl-L-arginine,

guanylate

accumulation

responses

that

EFS-induced

nitrite

formation

that

endogenous

One major

of

cytosolic

functional

L-isomer

tissue

of NO markedly

effects

observations

(13-16).

stimulation

cyclic

communication

substituted 4,

of

corporal

is

actions

addition

arginine

that

hypothesis

inhibitory

manner.

of

the

NO formation

NANC neuron-dependent

this

are known to inhibit

in viva

for

preparations the

parasympathetic

NG-amino-L-arginine, NG-nitro

D-arginine;

to EFS.

and

relaxation

NG-nitro-L-arginine;

of nitrite,

adrenergic

was inhibited

potassium-depolarized

normally

formation

was

in this

muscle

the

formation by

and

cavernosum

GMP.

This by

not

tissue

because

simultaneous

of

selective but

functional

observations

and

effects

corpus

sympathetic

relaxation

in

endothelium-independent acetylcholine

the

2, EFS-induced

abolished occur

of

smooth

L-arginine

of

presence

transient

enantiomerically

nearly

the

exclude

inhibitory

of

relaxation

to

but

was accompanied which

the

NG-nitro-L-arginine,

3,

L-arginine

in

relaxation

The principal

rapid

by tetrodotoxin;

indomethacin;

EFS causes

abolition relaxation

the with that

close

a related in

vitro

likely

and to

be

activation association

the simultaneous EFS causes NO and by tetrodotoxin in the presence

blockade indicates that via the NANC neuronal

EFS-induced pathway.

or of

Vol.

170,

No.

2,

1990

Mammalian cavities

BIOCHEMICAL

corpus

arranged

squamous

cavernosum cells

it

is

tends

preparations

but

findings

for

from

of

NANC neurons, It

response such

the

muscle

that

to

EFS was much more rapid and

documented

(l-3),

well

whereas

by

rational

basis

for

rate

of than

arteries

(19).

The present

investigation

in

of the

for

is

smooth

5% of

of

(12),

the

muscle

EFS-elicited

may be of

erection

penile

and therapy

in

preparations

in other

that

study.

relaxation

observations penile

these

current

is

well

vascular erection

NANC neurotransmission

etiology

detergent

by EFS derives

under

in arterial

vasodilation to

endothelial or

NO generated

These

observations

response

anatomical

endothelium

observed

regulation

with

to relax in response to NO and EFS. Assuming that

the

that

lined

the

be obligatory

corporal

neuronally-mediated

NO generated

remove

or endothelium

as neuronal

established.

mediated

the

pulmonary

significance

Whether

is

this

collagenase

a functioning

cells,

sinusoidal

Approximately

failed to

may not

muscle.

of

that of

or in

as well.

response

endothelium

smooth

damage

reasons, requires

muscle

tissue

muscle in

noteworthy

as cerebral

not

unknown normally

smooth

to

COMMUNICATIONS

consists

Because

of

smooth

is

physiological is

that

corporal

smooth

endothelium.

relaxation

indicate

relaxation

the

RESEARCH

tissue

of

difficult

relaxed

acetylcholine-elicited

erectile

Incubation

damage

studied,

acetylcholine

or

means.

to

BIOPHYSICAL

network

technically

by mechanical

solutions

or

a trabecular

epithelial

arrangement, cells

in

AND

beds may be

provides of

a

impotence.

ACKNOWLEDGMENTS This and a grant

work from

was supported the

Laubisch

in part Fund for

by U.S.P.H.S. Cardiovascular

grants

HL35014

and HL40922,

Research.

REFERENCES

1.

Saenz

de Tejada,

Kim, N., Lagan, I., Krane, R.J. and Goldstein, I. 1117-1121. Saenz de Tejada, I., Blanco, R., Goldstein, I., Azadzoi, K., de las Morenas, A., Krane, R.J. and Cohen, R.A. (1988) Am J. Phvsiol. 254: H459H467. Saenz de Tejada, I., Goldstein, I., Azadzoi, K., Krane, R.J. and Cohen, R.A. (1989) N. Enql. J. Med. 320: 1025-1030. Ignarro, L.J., Lippton, H., Edwards, J.C., Baricos, W.H., Hyman, A.L., Kadowitz, P.J. and Gruetter, C.A. (1981) J. Pharmacol. EXD. Ther. 218: 739749. Ignarro, L.J., Byrns, R.E. and Wood, K.S. (1987) Circ. Res. 60: 82-92. Ignarro, L.J., Buga, G.M., Wood, K.S., Byrns, R.E. and Chaudhuri, G. (1987) Proc. Natl. Acad. Sci. USA 84: 9265-9269. S. and Kimura, H. (1978) Murad, F., Mittal, C.K., Arnold, W.P., Katsuki, Adv. Cvclic Nucleotide Res. 9: 145-158. Gruetter, C.A., Barry, B.K., McNamara, D.B., Gruetter, D.Y., Kadowitz, P.J. and Ignarro, L.J. (1979) J. Cvclic Nucleotide Res. 5: 211-224. Moore, P.K., al-Swayeh, O.A., Chong, N.W.S., Evans, R.A. and Gibson, A. (1990) Br. J. Pharmacol. 99: 408-412. Fukuto, J.M., Wood, K.S., Byrns, R.E. and Ignarro, L.J. (1990) Biochem. &hvs. Res. Commun. 168: 458-465.

(1989) J. Urol.

2. 3. 4. 5. 6. 7. 8. 9. 10.

I., 142:

849

Vol.

11. 12. 13. 14. 15. 16. 17. 18. 19.

170,

No.

2,

1990

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Chand, N. and Altura, B.M. (1981) Science 213: 1376-1379. Furchgott, R.F. and Zawadzki, J.V. (1980) Nature (Land.) 288: 373-376. D.S. and Moncada, S. (1988) Siochem. Palmer, R.M.J., Rees, D.D., Ashton, Bionhvs. Res. Commun. 153: 1251-1256, 1988. Nathan, C. and Levi, R. (1988) Proc. Sakuma, I., Stuehr, D., Gross, S.S., Natl. Acad. Sci. USA 85: 8664-8667. R.M.J. and Moncada, S. (1989) Proc. Natl. Acad. Sci. Rees, D.D., Palmer, USA 86: 3375-3378. Aisaka, K., Gross, S.S., Griffith, O.W. and Levi, R. (1989) Biochem. Bionhvs. Res. Commun. 160: 881-886. Ignarro, L.J., Byrns, R.E., Buga, G.M. and Wood, K.S. (1987) Circ. Res. 61: 866-879. Ignarro, L.J. (1989) Circ. Res. 65: 1-21. Frank, G.W. and Bevan, J.A. (1983) Am. J. Phvsiol. 244: H793-H798.

850