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Departments of * Clinical Pharmacology, Edited by Salvador Moncada, University of London, London, United
Kingdom, and approved December 15, 1999 (received for review September
30, 1999)
The generation of nitric oxide (NO) in penile erectile tissue and
the subsequent elevation of cyclic GMP (cGMP) levels are important for
normal penile erection. Current treatments of erectile dysfunction
elevate either cGMP levels by blocking cGMP degrading phosphodiesterase
5 or cyclic AMP (cAMP) levels by intrapenile injection of prostaglandin
E1. The molecular target or targets of cGMP in erectile tissue and the
role of cAMP for normal penile erection are not known. Herein, we
report that mice lacking cGMP-dependent kinase I (cGKI) have a very low
ability to reproduce and that their corpora cavernosa fail to relax on
activation of the NO/cGMP signaling cascade. Elevation of cAMP by
forskolin, however, induces similar relaxation in normal and cGKI-null
corpus cavernosum. In addition, sperm derived from cGKI-null mice is
normal, can undergo acrosomal reactions, and can efficiently fertilize
eggs. Altogether, these data identify cGKI as the downstream target of
cGMP in erectile tissue and provide evidence that cAMP signaling cannot
compensate for the absence of the cGMP/cGKI signaling cascade in vivo.
Penile erection is produced
by an increased blood flow to the corpus cavernosum (CC), made possible
by opening of penile resistance vessels (helicine arteries), relaxation
of the CC cells, and occlusion of the venous outflow (1). The erectile
response in several animal models depends on nitric oxide (NO),
produced by nerves as well as vascular endothelium (1, 2-4). NO
activates soluble guanylate cyclase, which leads to the production of
cyclic GMP (cGMP). cGMP signals via three different receptors in
eukaryotic cells, including ion channels, phosphodiesterases, and
protein kinases (5). At present, however, the molecular targets that are activated by cGMP and finally execute the relaxation of penile smooth muscle are not known. In addition, two different cGMP-dependent protein kinases (cGKI and cGKII) have been identified in mammals (6,
7). cGKII is expressed in the small intestine, brain, and cartilage
(8-10), whereas high levels of cGKI are found in vascular and
intestinal smooth muscle, platelets, Purkinje cells of the cerebellum,
and CC cells (11, 12). Inactivation of cGKI in mice abolished both
NO/cGMP-dependent relaxation of vascular and intestinal smooth muscle
and inhibition of platelet aggregation, causing hypertension,
intestinal dysmotility, and abnormal hemostasis (13). In the present
study, we investigated the function of cGKI in erectile tissue and the
capability of cGKI-deficient mice to reproduce. Furthermore, we
analyzed whether a cross-talk exists between the cGMP and cyclic AMP
(cAMP) signaling cascades in smooth muscle (5, 14), i.e., whether cAMP
can cause relaxation via cGKI.
Drugs and Chemicals.
The following drugs were used: l-noradrenaline (NA; Aldrich),
carbachol,
N Animals and Tissues.
Mice (cGKI+/+ and cGKI Immunohistochemistry.
Tissue sections were preincubated in PBS with 0.2% Triton X-100 for
2 h and then incubated overnight at +4°C with rabbit antisera against protein gene product 9.5 (1:2,000; Ultraclone, Wellow, Isle of
Wight, U.K.), cGKI (1:1,000), tyrosine hydroxylase (1:2,000; Pel-Freez
Biologicals), or vesicular acetylcholine transporter (VAChT; 1:2,400;
Euro-Diagnostica, Malmö, Sweden); or incubated with guinea pig
antiserum to vasoactive intestinal polypeptide (VIP, 1:640;
Euro-Diagnostica); or incubated with sheep antiserum to neuronal NO
synthase (nNOS, 1:6,000; kind gift of P. Emson, Department of
Neurobiology, Babraham Institute, Cambridge, U.K.). After rinsing in
PBS, the sections were incubated for 90 min with FITC-conjugated swine
anti-rabbit IgG (1:80; Dakopatts, Stockholm, Sweden), FITC-conjugated
donkey anti-sheep IgG (1:80; Sigma), FITC-conjugated goat anti-guinea
pig IgG (1:80; Sigma), Texas Red-conjugated donkey anti-rabbit IgG
(1:125; code 711-075-152, Jackson ImmunoResearch), Texas Red-conjugated
donkey anti-sheep IgG (1:125; code 713-075-147, Jackson
ImmunoResearch), or Texas Red-conjugated donkey anti-guinea pig IgG
(1:125; code 706-075-148, Jackson ImmunoResearch). The immunoreactive
structures were evaluated as described (15).
Functional Experiments.
The tunica albuginea was carefully opened from its proximal extremity
of the CC toward the penile shaft, and the erectile tissue within the
CC was microsurgically excised. One preparation (0.3 × 0.3 × 3 mm) was obtained from each CC. All preparations were used
immediately after removal. Strip preparations were prepared and mounted
in thermostatically controlled organ baths (5 ml; 37°C) containing
Krebs solution bubbled with a mixture of 95% O2
and 5% CO2 (pH 7.4). Isometric tension was
recorded, and electrical field stimulation (EFS) was performed as
described (15).
Physiology
Erectile dysfunction in cyclic GMP-dependent kinase
I-deficient mice
,
,,, and
Experimental
Pathology, and § Pathology, University of Lund, S-221 85 Lund, Sweden; Laboratory of Genetics, The Salk
Institute, La Jolla, CA 92037; and ¶ Institut für
Pharmakologie und Toxikologie, Technische Universität, D-80802
München, Germany
Abstract
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Materials and Methods
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
-nitro-L-arginine
(L-NNA), forskolin (Sigma), and
1H-[1,2,4]-oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; Tocris Cookson,
Bristol, U.K.). NO was freshly prepared for each experiment. An
airtight glass beaker containing 20 ml of distilled water was
deoxygenated for 1 h with helium gas. The beaker was then bubbled
with medical NO gas (purity > 99.5%) for 15 min until saturated
solutions were obtained (NO; 3 × 10
3 M).
/; ref. 13) were killed by carbon
monoxide asphyxia followed by exsanguination. The penis was removed from each animal by cutting the crura CC at the point of adhesion to
the lower pubic bone, and the CC were then excised and processed for
immunocytochemistry or used for functional experiments as described
(15).
/;
n = 9) were obtained. To verify the contractile ability
of the preparations, a K+ solution (124 mM) was added to the organ
baths at the end of the equilibration period. The mean contractile
response to K+ amounted to 0.92 ± 0.07 mN (n = 11) and 0.69 ± 0.04 mN (n = 9) in cGKI+/+ and
cGKI/ mice, respectively. The NA concentration used (3 × 106 M) corresponded to the approximate
EC70 value and produced stable and reproducible
contractions. The effects of NO, carbachol, and forskolin were
investigated in NA-contracted preparations.
4
M) or the guanylate cyclase inhibitor ODQ (106
M). Effects of EFS were then investigated as described above.
A Krebs solution of the following composition was used (in mM): NaCl
119, KCl 4.6, CaCl2 1.5, MgCl2 1.2, NaHCO3 15, NaH2PO4 1.2, and glucose
5.5. In the high K+ solution (124 mM), the NaCl in the normal Krebs
solution was replaced by equimolar amounts of KCl.
In Vitro Fertilization (IVF) and Embryo Culture. The experiments were performed as described (16). Spermatozoa were obtained from the cauda epididymidis of 7- to 8-week-old wild-type and cGKI-deficient male mice. The sperm cells were capacitated in IVF medium (Whittingham's medium supplemented with 30 mg/ml BSA) for 1.5 h at 37°C. At 13.5 h after human chorion gonadotropin, cumulus-enclosed oocytes were inseminated with 106 sperm cells per ml. The mixture of eggs and sperm was incubated in IVF medium for 4 h at 37°C, and thereafter eggs were transferred into pregassed M16 medium supplemented with 4 mg/ml BSA. Fertilization was assessed 24 h after insemination by counting two- and four-cell embryos. Morphologically normal two/four-cell embryos were cultured further in M16 medium until the blastocyst stage.
To estimate sperm motility, a small amount of sperm was removed from IVF medium after 1-h capacitation and investigated under a cover slide by using phase-contrast microscopy. Cells with hyperactivity were considered as progressively motile. Assessment of spontaneous acrosome reactions was done by the Coomassie brilliant blue staining method as described (17).Calculations.
Student's paired or unpaired two-tailed t tests were used
for statistical comparison of two means. ANOVA with Bonferroni
correction was used for multiple comparisons. A probability of
P < 0.05 was accepted as significant. When
appropriate, results are given as mean values ± SEM. n
denotes the number of animals and strip preparations. The log
IC50 values (the logarithm of the drug
concentrations producing 50% relaxation of the induced response) were
determined graphically for each curve by linear interpolation.
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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There were no obvious differences in sexual behavior between
wild-type and cGKI/ mice. However, when 15 cGKI-null male mice were crossed with wild-type female mice, we observed a single pregnancy
resulting in eight offspring. In contrast, wild-type male mice
(n = 15; cGKI+/+) produced more than 500 offspring. cGKI-null females were able to reproduce normally when they were mated
with wild-type males. These data clearly show that cGKI-null males, in
contrast to females, have a reduced fecundity.
Immunohistochemistry.
To unravel the cause of the infertility in cGKI-null males, we first
tested whether the integrity of and the nerve population in the
erectile tissue were altered by the lack of cGKI activity. In normal
penile tissue, intense immunoreactivity against cGKI was seen in the
smooth muscle of the walls of the central and helicine arteries and in
the smooth muscle of the trabecular septa surrounding the cavernous
spaces (Fig. 1A). In
connective tissue and striated muscle outside the CC tissue, no
specific immunoreactivity was observed. In cGKI-deficient mice, both
central and helicine arteries as well as trabecular septa are normally
developed, and as expected, no cGKI-specific immunoreactivity could be
detected in cGKI/ mice (Fig. 1B). In addition, no
difference in number and distribution patterns of nerve populations
believed to be of importance for erectile responses could be detected
between cGKI+/+ and cGKI/ mice. In the trabecular tissue
surrounding the central and helicine arteries, nerves were observed
showing immunoreactivity to protein gene product 9.5 (18), a
nonspecific nerve marker (Fig. 1 C and D),
tyrosine hydroxylase (Fig. 1 E and F), VAChT
(specific for cholinergic nerves; Fig. 2
A and B), VIP (Fig. 2 C and
D), and NOS (Fig. 2 E and F). The
central and helicine arteries were supplied by nerves rich in protein
gene product 9.5-, NOS-, VAChT-, VIP-, and tyrosine
hydroxylase-immunoreactive terminals. Double immunolabeling showed that
in varicosities and intervaricose segments, VAChT- and VIP-, VAChT- and
NOS-, and NOS- and VIP-immunoreactive terminals were colocalized.
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IVF.
Next, we tested whether the reduced fertilization capability of
cGKI/ males is a consequence of sperm dysfunction. Motility, spontaneous acrosome reactivity, and IVF efficiency were compared between sperm derived from normal and cGKI-null mice. After a 1-h
capacitation, cGKI+/+ (n = 2) males and cGKI/
(n = 2) males showed 82 ± 6% and 79 ± 8%,
respectively, of progressively motile spermatozoa. The percentages of
acrosome-reacted sperm were 27 ± 4% for normal and 29 ± 5% for cGKI/ mice. IVF of normal mouse oocytes with sperm
obtained from wild-type and cGKI/ males showed no differences in
success rate (Table 1).
|
Smooth Muscle Contraction.
To test whether the distorted fertility of cGKI-null mice is due to
erectile dysfunction, we isolated CC tissue from normal and mutant mice
and investigated its ability to contract in vitro. Spontaneous contractile activity was observed neither in normal nor in
cGKI-deficient CC preparations. The contractile capacity, as tested by
NA (106 M), was similar in CC tissue from
cGKI+/+ and cGKI/ mice and amounted to 0.45 ± 0.03 mN
(n = 11) and 0.39 ± 0.03 mN (n = 9), respectively.
6 M). Carbachol relaxed preparations from
cGKI+/+ mice by 59 ± 6% (n = 11) but had
almost no effect (1 ± 1%; n = 8;
P < 0.001) in preparations from cGKI/ mice (Fig.
3). Blockade of NO synthesis in CC tissue
from cGKI+/+ mice by pretreatment with L-NNA
(104 M) reduced the carbachol-induced
relaxation to 1 ± 1% (n = 5; P < 0.001; Fig. 3). Inhibition of soluble guanylyl cyclase by addition
of ODQ (106 M) reduced the carbachol-induced
relaxation to 2 ± 1% (n = 6; P < 0.001; Fig. 3).
|
log
IC50 value was 5.85 ± 0.07, and complete relaxation was obtained at NO concentrations of 3 × 105 to 104 M. Pretreatment of the CC tissues with ODQ markedly inhibited the
NO-induced relaxations, shifting the concentration-response curve for
NO to the right (log IC50 = 4.88 ± 0.09).
The NO concentration-response curve of normal tissues in the presence
of ODQ (104 M) was almost identical to the NO
concentration-response curve obtained in CC from cGKI-null mice (log
IC50 = 4.66 ± 0.11), indicating that cGKI
mediates the cGMP effects in erectile tissue. However, at high
concentrations, NO induced relaxations independently of cGKI.
|
4 M L-NNA (n = 5) or
106 M ODQ (n = 5) were observed
at frequencies above 2 Hz (Figs. 5 and
6).
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log
IC50 = 7.04 ± 0.11) and cGKI/ mice
(n = 6; log IC50 = 6.73 ± 0.11), demonstrating that the cAMP signaling is not affected by the
lack of cGKI (Fig. 7). However, at
106 M, the highest forskolin concentration
used, a statistically significant (P < 0.05)
difference in relaxation amplitude was found (72 ± 5% for
cGKI/ and 90 ± 2% for cGKI+/+ mice).
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Discussion |
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Abstract
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Materials and Methods
Results
Discussion
References
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An important role for neuronally and endothelially produced NO and for the NO/cGMP pathway in penile erection is widely accepted (1, 4). Initially, it was thought that nNOS is the major source for NO in CC tissue (2, 3). A recent report, however, showed that nNOS-deficient mice have normal mating behavior and a normal erectile response to electrical stimulation of the cavernous nerves (19, 20). In addition, isolated CC tissue has normal relaxant responses to electrical nerve stimulation (20), and it was suggested that endothelial NOS is essential for erection, not only in nNOS-deleted mice, but also in normal mice. At present, the role of nNOS in this setting is still debated, because thorough analyses revealed that an alternatively spliced mRNA of the nNOS gene is still expressed in nNOS mutant mice (21). The majority of NO effects are mediated by cGMP (22), and a number of reports have underlined the importance of cGMP in erectile tissue (1-4, 23). Damage to the penile nerves and/or endothelium, as can be found in, e.g., diabetes mellitus, can lead to a decreased production of cGMP in the CC tissue and consequently to erectile dysfunction. Additional support for a central role of cGMP in erection comes from the impressive clinical results with sildenafil (24), which selectively inhibits the activity of phosphodiesterase 5 (which breaks down cGMP) and consequently increases intracellular cGMP levels. A few patients with erectile dysfunction, however, do not respond to sildenafil, suggesting that they may have a functional disturbance of the NO/cGMP signaling cascade distal to the production/breakdown of cGMP.
There is presently no consensus as to whether transmitters other than NO play an important role in the erectile process. The murine penile tissue is well supplied with nerves containing VIP, which has also been shown for humans and other species (1, 25). VIP stimulates adenylyl cyclase and subsequently elevates intracellular concentration of cAMP, which in turn activates cAMP-dependent protein kinase. VIP has a well documented, pronounced, inhibitory, and relaxation-producing effect on strips of CC tissue and cavernosal vessels in vitro (1). Therefore, it has been speculated that the cAMP pathway should be involved in the control of penile smooth muscle tone (26) and in normal erection. The most compelling evidence comes from the effective treatment of erectile dysfunction in humans by intracavernosal injection of prostaglandin E1, which acts through the adenyl cyclase/cAMP signaling pathway (27). We also show in this study that forskolin can activate the adenylyl cyclase and elevate cAMP levels both in normal and cGKI-deficient CC tissue. Nevertheless, cGKI-null mice have a very low ability to reproduce, and their CC tissue shows no relaxation in response to nerve stimulation, suggesting that putative transmitters released from nerves and acting through the cAMP pathway, such as VIP and VIP-related peptides (25), play a minor role in normal erection. Furthermore, under normal physiological situations, signaling via cGKI seems critical in obtaining penile erections. The relaxing effects of NO at high concentrations in the absence of cGKI could be due to a cGMP-independent action of NO (e.g., activation of large conductance calcium-activated potassium channels) and/or an increase of cellular cGMP to such high concentrations that the cAMP-dependent protein kinase is activated. Analyses of isolated cGKI-deficient vascular smooth muscle clearly favor the latter notion (unpublished work).
Our results provide convincing evidence that the highly reduced fecundity of mice lacking cGKI is due to erectile dysfunction and show that NO/cGMP mediates its effect through cGKI. These mice will be a useful model for testing drugs that act downstream of cGKI and stimulate penile erection.
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Acknowledgements |
|---|
This work was supported by Swedish Medical Research Council Grants 6837, 11205, and 12531 and the Medical Faculty of Lund University.
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Abbreviations |
|---|
cGMP, cyclic GMP;
cAMP, cyclic AMP;
cGKI and cGKII, cGMP-dependent kinase I and II;
CC, corpus cavernosum;
NA, l-noradrenaline;
L-NNA, N-nitro-L-arginine;
ODQ, 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxalin-1-one;
VAChT, vesicular
acetylcholine transporter;
VIP, vasoactive intestinal polypeptide;
NOS, nitric-oxide synthase;
nNOS, neuronal NOS;
EFS, electrical field
stimulation;
IVF, in vitro fertilization.
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Footnotes |
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To whom reprint requests should be addressed.
E-mail: Karl-Erik.Andersson{at}klinfarm.lu.se.
This paper was submitted directly (Track II) to the PNAS office.
Article published online before print: Proc. Natl. Acad. Sci. USA, 10.1073/pnas.030419997.
Article and publication date are at www.pnas.org/cgi/doi/10.1073/pnas.030419997
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References
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M. Craven, G. P. Sergeant, M. A. Hollywood, N. G. McHale, and K. D. Thornbury Modulation of spontaneous Ca2+-activated Cl- currents in the rabbit corpus cavernosum by the nitric oxide-cGMP pathway J. Physiol., April 15, 2004; 556(2): 495 - 506. [Abstract] [Full Text] [PDF]
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 J. Bonnevier, R. Fassler, A. P. Somlyo, A. V. Somlyo, and A. Arner Modulation of Ca2+ Sensitivity by Cyclic Nucleotides in Smooth Muscle from Protein Kinase G-deficient Mice J. Biol. Chem., February 13, 2004; 279(7): 5146 - 5151. [Abstract] [Full Text] [PDF]
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 M. J. Fitzpatrick and M. B. Sokolowski In Search of Food: Exploring the Evolutionary Link Between cGMP-Dependent Protein Kinase (PKG) and Behaviour Integr. Comp. Biol., February 1, 2004; 44(1): 28 - 36. [Abstract] [Full Text] [PDF]
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 D. R. Kaiser, K. Billups, C. Mason, R. Wetterling, J. L. Lundberg, and A. J. Bank Impaired brachial artery endothelium-dependent and -independent vasodilation in men with erectile dysfunction and no other clinical cardiovascular disease J. Am. Coll. Cardiol., January 21, 2004; 43(2): 179 - 184. [Abstract] [Full Text] [PDF]
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 R. Feil, S. M. Lohmann, H. de Jonge, U. Walter, and F. Hofmann Cyclic GMP-Dependent Protein Kinases and the Cardiovascular System: Insights From Genetically Modified Mice Circ. Res., November 14, 2003; 93(10): 907 - 916. [Abstract] [Full Text] [PDF]
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 T. Munzel, R. Feil, A. Mulsch, S. M. Lohmann, F. Hofmann, and U. Walter Physiology and Pathophysiology of Vascular Signaling Controlled by Cyclic Guanosine 3',5'-Cyclic Monophosphate-Dependent Protein Kinase Circulation, November 4, 2003; 108(18): 2172 - 2183. [Full Text] [PDF]
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 M. E. DiSanto Corpus Cavernosum Smooth Muscle Physiology: A Role for Sex Hormones? J Androl, November 1, 2003; 24(6_suppl): S6 - S16. [Full Text] [PDF]
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T. J. Bivalacqua, M. F. Usta, H. C. Champion, P. J. Kadowitz, and W. J. G. Hellstrom Endothelial Dysfunction in Erectile Dysfunction: Role of the Endothelium in Erectile Physiology and Disease J Androl, November 1, 2003; 24(6_suppl): S17 - S37. [Full Text] [PDF]
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M. A. Palese, J. K. Crone, and A. L. Burnett A Castrated Mouse Model of Erectile Dysfunction J Androl, September 1, 2003; 24(5): 699 - 703. [Abstract] [Full Text] [PDF]
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