Integrative Physiology |
From the Institut für Pharmakologie und Toxikologie der TU München (M.S., V.V., C.H., A.P., T.K., P.R., F.H.), München, Germany; Institut für Physiologie der Universität Rostock (R.S.), Rostock, Germany; and Abteilung Pharmakologie für Pharmazeuten, Universitätskrankenhaus Eppendorf (M.K.), Hamburg, Germany.
Correspondence to Franz Hofmann, Institut für Pharmakologie und Toxikologie der TU München, Biedersteiner Str 29, 80802 München, Germany. E-mail pharma{at}ipt.med.tu-muenchen.de \ © 2000 American Heart Association, Inc.
| Abstract |
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Key Words: cGMP-dependent protein kinase I arteries K+ channels
| Introduction |
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The important effector of cGMP, cGKI, is highly expressed in smooth muscle. Deletion of the gene for cGKI can led to multiple phenotypes, including high blood pressure in young mice.17 Interestingly, and in contrast to previous suggestions,13 adenosine A2 receptor- and cAMP-dependent relaxation of aortic rings was not affected by the lack of cGKI.17 Older cGKI-deficient (cGKI/) mice, many of which have multiple infections, have normal or only slightly elevated blood pressure,17 suggesting that cGKI is not absolutely required to lower vascular tone and can be bypassed. A potential alternative pathway could be an increased production of endothelium-derived hyperpolarizing factors (EDHF).18 Activation of large and small KCa channels, calcium-activated chloride channels, or voltage-dependent K+ channels by EDHF or NO would hyperpolarize the membrane, close calcium channels, and reduce cytosolic calcium concentrations ([Ca2+]i).14 18
Thus far, the in vitro analysis of hypertensive knockout mice has been performed on isolated aortic segments. In our studies, we noticed that aortic segments can be relaxed by high concentrations of NO in the absence of cGKI. To investigate whether this is also valid for other vessels, we used a small artery (arteria tibialis) that may behave similar to resistance vessels and develop a spontaneous myogenic tone in wild-type (wt) and cGKI/ mice. In this study, we show that high concentrations of NO relax vascular smooth muscle independent of the activation of Ca2+-activated K+ (BKCa) channels by cGMP-dependent cross-activation of cAMP kinase (cAK). This pathway may be important in situations where high concentrations of NO prevail, such as in endotoxin shock.19 20
| Materials and Methods |
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Measurement of Contractility
Arteria Tibialis
The arteria tibialis, a second order branching from
arteria femoralis, was dissected and transferred to the experimental
chamber containing ice-cold physiological buffer. The chamber was
perfused at 37°C and a rate of 2
mL/min.21 A
permanent intravascular pressure of 80 mm Hg was applied at nonflow
conditions, resulting in an inner vessel diameter of 65% to 85% of
the diameter obtained in Ca2+-free solution.
The diameters of fully relaxed wt and
cGKI/ vessels were 78±6 µm (n=6) and
68±5 µm (n=7), respectively. These values are not significantly
different. All compounds were applied to the adventitial side. Final
concentrations are always reported. Diclofenac (1 µmol/L) was present
in all buffers. To block endothelial NOS, 30 µmol/L L-NOARG was added
60 minutes before DEA-NO.
Aorta
Aortic rings from male mice were prepared as
described
elsewhere.17 For
other experimental details, see our previous
study.17
Analysis of BKCa Channel
Activity
Aortic smooth muscle cells were isolated as described
elsewhere.22
Membrane currents were recorded in whole-cell configurations. The bath
solution contained (in mmol/L) NaCl 140, KCl 5.6,
CaCl2 1.8, MgCl2 1,
glucose 10, and HEPES 10 (pH 7.4). The pipette solution contained KCl
40, potassium aspartate 100, NaCl 10, MgATP 3, glucose 10, HEPES 10,
and 300 nmol/L free calcium (pH 7.4). The holding potential was -20
mV. Test pulses lasted 500 ms to potentials from -30 to +80 mV.
Amplitudes of the final 50 ms of depolarizing test pulses were averaged
from 3 to 5 consecutive trials.
Determination of Cyclic Nucleotide
Levels
Aortic rings were incubated for 10 minutes with
norepinephrine (NE) followed by vehicle (100 µmol/L NaOH) or
100 nmol/L or 100 µmol/L DEA-NO for 3 minutes. Aortic rings were
transferred into liquid nitrogen and pulverized together with 400 µL
10% trichloracetic acid (TCA) under liquid nitrogen. After thawing and
centrifugation, the supernatant was extracted 5 times with 5 volumes of
ether. The ether was evaporated at 70°C for 10 minutes. cGMP and cAMP
were determined by specific enzyme immunoassays (Cayman
Chemical).
| Results |
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Acetylcholine (1 nmol/L to 1 µmol/L) that stimulates the
endothelial NO synthesis induced a concentration-dependent relaxation
only in small arteries of wt but not
cGKI/ mice
(Figures 1A
and 1B
), suggesting that cGKI is essential for
endothelium-dependent relaxation. Endothelial dysfunction has been
reported in hypertensive animals and
humans.24 To rule
out that the defect in relaxation was caused by a dysfunction of the
endothelium, the NO donor DEA-NO was applied directly to the arteries
(Figure 1C
). NO relaxed the wt vessels
concentration-dependently without affecting the tone of the
cGKI/ vessels up to a concentration of 1
µmol/L. However, high concentrations of DEA-NO (10 µmol/L) relaxed
partially cGKI/ vessels, suggesting
regulation of vascular tone independent of cGKI. The cAMP-dependent
regulation of the small vessel contractility was not disrupted in
cGKI/ mice
(Figure 1D
). Adenosine acting via cAMP/cAK elicited a similar
relaxation in wt and cGKI/ vessels,
confirming that cAMP relaxes arteries independent of
cGKI.17
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BKCa Channels Are
Involved in Acetylcholine-Induced Relaxation
The above results suggest that high concentrations of
NO relaxed the vessels by mechanisms independent of cGKI. Previously,
it was shown that NO can affect smooth muscle contractility by
hyperpolarizing the membrane potential through direct or indirect
activation of BKCa channels, which are widely
distributed in vascular smooth
muscle.14 We tested
the contribution of BKCa channels to the
cGMP/cGKI-mediated relaxation in small vessels using the specific
BKCa channel blocker IBTX. Relaxation of wt
small arteries induced by 100 nmol/L ACh was significantly reduced by
200 nmol/L IBTX (n=6, P<0.05;
Figure 2A
), indicating that ACh-induced relaxation was at
least in part mediated by BKCa channels in
murine small vessels. However, the inhibitory effect of IBTX was no
longer observed when the concentration of ACh was raised to 1 µmol/L
(Figure 2A
). Similar results were obtained in the presence of
DEA-NO. IBTX partly inhibited vasorelaxation induced by 1 µmol/L
DEA-NO but had no effect on the relaxation induced by 10 µmol/L
DEA-NO
(Figure 2B
). These results confirm that high concentrations
of NO can relax murine arteries independent of
BKCa channels and cGKI.
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cGKI Is Required to Activate
BKCa Channels
A direct activation of BKCa
channels by NO has been
reported.11 An
electrophysiological analysis of the BKCa
channels in isolated murine smooth muscle cells from small vessels was
not feasible because of the extremely low amount of cells obtained.
Others have used isolated aortic smooth muscle cells to investigate the
regulation of BKCa
channels.11
Therefore, we tested whether the relaxation of murine aortic rings was
also affected by inhibition of BKCa channels
(Figure 2C
). ACh-induced (10 µmol/L) relaxation of aortic
rings was attenuated by 50% in the presence of 200 nmol/L
IBTX.
This finding encouraged us to analyze the regulation of
BKCa channels in isolated aortic wt and
cGKI/ smooth muscle cells
(Figures 3A
and 3B
). In wt cells, BKCa
currents were increased by 250 nmol/L and 5 µmol/L DEA-NO
(Figure 3A
), and the DEA-NOincreased current was inhibited
by 100 nmol/L IBTX. The current increase was significant at a membrane
potential of +20 mV
(Figure 3A
, inset). In contrast, DEA-NO up to a concentration
of 50 µmol/L had no effect on the activity of
BKCa channels in
cGKI/ smooth muscle cells
(Figure 3B
and inset). In agreement with other
reports,25 26 27 28
this finding indicates that NO regulates murine
BKCa channels only in the presence of
cGKI.
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NO-Dependent Relaxation of Aortic Rings
To gain more insight into the molecular mechanism of
NO-dependent relaxation in cGKI/vessels,
rings from wt and cGKI/ thoracic aorta
were precontracted with NE and then incubated with increasing
concentrations of DEA-NO. DEA-NO relaxed the wt and
cGKI/ aorta with
EC50 values of 35 and 850 nmol/L, respectively
(Figures 4A
and 4B
). These results are in agreement with those
obtained from small arteries and show that NO at higher concentrations
induced vascular relaxation by an unidentified mechanism in small and
large vessels.
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In the next series of experiments, we investigated whether
the relaxing effect of NO depended on the synthesis of cGMP. WT and
cGKI/ aortic rings were preincubated
with the competitive inhibitor of soluble guanylyl cyclase, ODQ (3
µmol/L). ODQ shifted the dose-response curves in each genotype to
higher concentrations of NO with EC50 values of
3 and 60 µmol/L
(Figures 4A
and 4B
), proving that cGMP synthesis was required
for NO-induced relaxation of cGKI/
aortic rings. Because cAMP/cAK-dependent relaxation was not impaired in
the cGKI/ vessels
(Figure 1D
; see also Figures 2B
and 2C
in reference 17), it
was possible that cGMP relaxed the aortic rings by cross-activation of
the cAMP-signaling pathway. Addition of Rp-8-Br-cAMPS, a competitive
antagonist of cAMP at the cAMP-binding sites of cAK, did not affect the
dose-response curve for DEA-NO in wt aortas. Rp-8-Br-cAMPS inhibited
effectively relaxation of aortic rings by the
adenosine-A2 receptor agonist CGS21680
(Figure 4C
). The A2 receptor has been
shown to relax murine aortic rings through activation of cAK and
independent of
cGKI.17 29
Therefore, these results support the notion that low concentrations of
NO relaxed vascular smooth muscle exclusively via cGKI and not via cAK.
In contrast, preincubation of the cGKI/
aortic rings with Rp-8-Br-cAMPS increased the
EC50 of DEA-NO 10-fold, from 0.85 µmol/L to
9.4 µmol/L
(Figure 4B
). This 10-fold shift supported the hypothesis that
cGMP relaxed cGKI/ aorta by
cross-activation of cAK.
Cyclic Nucleotide Levels in WT and
cGKI/ Aorta
The experiments carried out so far are compatible with
the hypothesis that NO-increased cGMP levels can affect vascular tone
by activation of the cAMP/cAK signaling pathway. However, the above
finding did not determine whether cGMP inhibited the activity of a
cAMP-hydrolyzing phosphodiesterase and thereby increased the cellular
cAMP concentration or activated directly the cAK. Direct activation of
cAK by cGMP requires cGMP concentrations around 10
µmol/L,30 whereas
phosphodiesterase III is inhibited by cGMP with an
IC50 value of 0.13
µmol/L.31 Using
the difference in required cGMP concentration as criterion should allow
differentiation between a direct and indirect activation mechanism by
measuring NO-stimulated cGMP and cAMP levels. The cyclic nucleotide
levels were measured in aortic rings of wt and
cGKI/ mice before and after the addition
of a low (100 nmol/L) and high (100 µmol/L) concentration of DEA-NO
under the same conditions used for the relaxation experiments
(Figure 5
). DEA-NO at a concentration of 100 nmol/L increased
cGMP levels 2-fold in both genotypes but had no significant effect on
the cAMP levels. These cGMP levels activate cGKI in wt
vessels.32 The high
concentration of DEA-NO stimulated cGMP level over 80-fold in both
tissues. cAMP levels were not increased significantly. Assuming a 60%
water content of the aortic rings, the final cGMP concentration of 30
pmol/mg wet weight yields a cytosolic cGMP concentration of 50
µmol/L. This cGMP concentration exceeds 5-fold the
Ka values for activation of
cAK.30 These values
strengthen additionally the conclusion that high concentrations of
DEA-NO relaxed small and large vessels in the absence of cGKI by
cross-activation of cAK.
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| Discussion |
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The molecular mechanisms involved in NO-mediated regulation
of BKCa channels presently are not clear. It was
reported that the activity of these channels is regulated directly by
NO11 by
cGKI-dependent phosphorylation of the channel
protein25 26 27
or of a protein
phosphatase.28 33 34
NO-dependent activation of BKCa channels was
only observed in the presence of cGKI. Even a concentration of 50
µmol/L DEA-NO did not activate BKCa channels
in mice lacking cGKI
(Figure 3
). In agreement with these results, IBTX did not
prevent relaxation induced by high NO concentrations. These findings
establish firmly that NO activates BKCa channels
in murine vascular smooth muscle via cGKI. This implies that
BKCa channels or closely associated proteins are
in vivo targets for cGKI.
ACh induced relaxation in the presence of IBTX. Similar experiments led to the detection of EDHF, a family of endothelial-derived hyperpolarizing factors.18 It is possible that ACh induced relaxation by stimulating the production of EDHF and not by that of NO. However, IBTX-independent relaxation was also observed when NO was applied directly to the vessels. NO has not been reported to stimulate the production of EDHF, suggesting that relaxation in the presence of IBTX was caused through a different pathway involving cGMP. Possible targets of cGKI that regulate vascular tone could be IRAG, a protein modulating calcium release from IP3-sensitive stores,35 and myosin phosphatase.36 Phosphorylation of these proteins could be involved in the IBTX-insensitive relaxation of wt vessels.
Numerous cGMP-independent effects of NO have been described.37 Experiments designed to test alternative pathways suggested that NO effects were mediated by cGMP even in the cGKI/ mice. The inhibition of the soluble guanylyl cyclase by the competitive inhibitor ODQ shifted the dose relaxation curves to higher concentrations of NO. Activation of cAK by cGMP was identified as the most likely pathway leading to relaxation. cGMP-dependent activation of cAK has been observed previously in isolated smooth muscle cells after downregulation of cGKI.10 The mechanism by which cAK relaxes vascular tone is not known. It is unlikely that cAK activated BKCa channels, because relaxation of cGKI/ vessels occurred in the presence of IBTX.
The cGKI/ mice develop hypertension that could be caused by endothelial dysfunction.24 A change in endothelial function associated with a reduced but not abolished vascular responsiveness to ACh as well as vascular remodeling has been proposed as a common phenomenon in hypertension. The wall thickness as potential index of vascular remodeling23 was not altered in cGKI/ small arteries, demonstrating no compensatory effects in the young mutant mice. The data presented here do not exclude an impaired endothelium-dependent relaxation in older cGKI/ mice. The evidence presented in this study and a previous one17 supports the hypothesis that the lack of smooth muscle cGKI caused hypertension. The relaxation induced by DEA-NO or 8-Br-cGMP did not depend on a functional endothelium.17 Hence, it is very likely that the cGKI deficiency and not endothelial dysfunction led to the impaired ACh/NO-dependent vasorelaxation.
There is clear evidence from this and a previous
study17 that the
NO/cGMP and adenosine/cAMP cascades regulate vascular tone in mice
independent of each other under physiological conditions.
cGMP-dependent activation of cAK was responsible for the relaxation
obtained at high concentrations of NO. This pathway is not operative
under physiological conditions, as shown in
Figure 4A
. However, under pathophysiological circumstances,
for example, during endotoxin shock when NO production is high,
activation of cAK by cGMP may be an additional regulatory mechanism
that produces generalized
hypotension.19 20
| Acknowledgments |
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This work was supported by grants from Deutsche Forschungsgemeinschaft, Thyssen-Stiftung (grant 9.26/91), and Fond der Chemie. We thank M. Wöckner, S. Kamm, and A. Salusky for excellent technical assistance.
Received February 23, 2000; revision received August 23, 2000; accepted August 29, 2000.
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