mode of nitric oxide action on the renal vasculature
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Mode of nitric oxide action on the renal vasculature
A . K U R T Z , K .- H . G OÈ T Z , M . H A M A N N and P . S A N D N E R
Institut fuÈr Physiologie der UniversitaÈt Regensburg, Regensburg, Germany
ABSTRACT
Our study aimed to characterize the essential cellular pathways along which nitric oxide (NO) exerts
its well-known vasodilatatory properties in the kidney. Using the isolated perfused rat kidney model
we examined the roles of potassium channels, cGMP-protein kinase activity and cAMP-phosphodi-
esterases (PDE) in the effect of NO on renovascular resistance. We found that neither potassium
channel activity nor G-kinase activity was essential for the vasodilatatory effect of NO. The effect of
NO, however, was essentially mimicked by pharmacological inhibition of PDE-3, which is a cGMP-
inhibitable PDE. As PDE-3 is strongly expressed in renal preglomerular vessels and NO stimulates
cGMP formation in renal vessels, it appears likely that inhibition of cAMP degradation and
consequently the cAMP pathway are crucially involved in mediating the effects of NO on renal
vascular resistance.
Received 30 September 1999, accepted 6 October 1999
It is well established that nitric oxide is a prominent
vasodilator in the renal vasculature (Navar et al. 1996,
Kone & Baylis 1997). By doing that it does not
essentially interfere with the autoregulation of renal
blood ¯ow, but it ampli®es renal blood ¯ow at any
given renal perfusion pressure (Navar et al. 1996, Kone
& Baylis 1997). How does NO exert these vasodilatory
properties in the renal preglomerular renal vessels at the
level of vascular smooth muscle cells is less understood.
By extrapolating ®ndings on the effects of NO in
other vascular beds, one could speculate that NO might
hyperpolarize arterial smooth muscle cells by the acti-
vation of potassium channels such as apamin- (Garcia-
Pascual et al. 1995), charybdotoxin- (Khan et al. 1993,
Archer et al. 1994, Bolotina et al. 1994) or iberiotoxin-
(Khan et al. 1993, Hohn et al. 1996, Mistry & Garland
1997) sensitive calcium-activated potassium channels or
such as voltage-gated (Yuan et al. 1996) or ATP-
regulated potassium channels (Miyoshi et al. 1994,
Murphy & Brayden 1995).
Another possibility is that NO via formation of
cyclic GMP (cGMP) leads to activation of cGMP-
dependent protein kinase, which is known to be
expressed at a rather high level in the renal vasculature
(Joyce et al. 1986) and which relaxes smooth muscle
cells either by activating potassium channels (Robertson
et al. 1993), by directly interfering with the intracellular
calcium handling (Cornwell & Lincoln 1989, Ishikawa
et al. 1993, Liu et al. 1997) or by inhibiting protein
kinase C (Kumar et al. 1997).
Finally, it is also conceivable that NO acts via the
cAMP pathway by interfering with cAMP degradation
through cGMP-regulated cAMP phosphodiesterase
activity (Beavo 1995). Cyclic AMP (cAMP) is known as
a potent vasodilator signal molecule in the renal
vasculature and cGMP-regulated cAMP phosphodi-
esterases have been demonstrated in the renal vascu-
lature (Reinhardt et al. 1995).
Using the isolated perfused rat kidney model, in
which the potent vasodilatatory effect of endogenous
and exogenous NO is still well preserved we were
interested in characterizing the contribution of each of
the three above mentioned pathways to the vasodila-
tory properties of NO in the renal vasculature.
Speci®cally we examined the effects of NO on renal
vascular resistance in the presence of potassium
channel blockers, inhibitors of cGMP-dependent
protein kinase activity and cAMP-phosphodiesterase
inhibitors.
MATERIALS AND METHODS
Isolated perfused rat kidney
Male Sprague±Dawley rats (250±300 g body weight)
having free access to commercial pellet chow and tap
Correspondence: Armin Kurtz MD, Institut fuÈr Physiologie, UniversitaÈt Regensburg, D-93040 Regensburg, Germany.
Acta Physiol Scand 2000, 168, 41±45
Ó 2000 Scandinavian Physiological Society 41
water were obtained from the local animal house and
used throughout. Kidney perfusion was performed in a
recycling system (Scholz & Kurtz 1992). In brief, the
animals were anaesthetized with 150 mg kg±1 of 5-ethyl-
(1¢-methyl-propyl)-2-thiobarbituric acid (Inactin, Byk
Gulden, Konstanz, FRG). Volume loss during the
preparation was substituted by intermittent injections
of physiological saline via a catheter inserted into the
jugular vein. After opening of the abdominal cavity by a
mid-line incision, the right kidney was exposed and
placed in a thermoregulated metal chamber. The right
ureter was cannulated with a small polypropylene tube
(PP-10) which was connected to a larger polyethylene
catheter (PE-50). After intravenous heparin injection
(2 U g±1) the aorta was clamped distal to the right renal
artery and the large vessels branching off the abdominal
aorta were ligated. A double-barrelled cannula was
inserted into the abdominal aorta and placed close to
the origin of the right renal artery. After ligation of the
aorta proximal to the right renal artery, the aortic clamp
was quickly removed and perfusion was started in situ
with an initial ¯ow rate of 8 mL min±1. The right
kidney was excised and perfusion at constant pressure
(100 mmHg) was established. To this end the renal
artery pressure was monitored through the inner part of
the perfusion cannula (Statham Transducer P
10 EZ) and the pressure signal was used for feedback
control of a peristaltic pump. The perfusion circuit was
closed by draining the venous ef¯uent via a metal
cannula back into a reservoir (200±220 mL). The basic
perfusion medium, which was taken from a thermo-
stated (37 °C) reservoir, consisted of a modi®ed
Krebs±Henseleit solution containing (mmol L±1): all
physiological amino acids in concentrations between
0.2 and 2.0 mmol L±1, 8.7 glucose, 0.3 pyruvate, 2.0 L
lactate, 1.0 a-ketoglutarate, 1.0 L malate and 6.0 urea.
The perfusate was supplemented with 60 g L±1 bovine
serum albumin, 10 mU L±1 vasopressin 8-lysine, and
with freshly washed human red blood cells (10%
haematocrit). Ampicillin 3 mg 100 mL±1 and ¯oxacillin
3 mg 100 mL±1 were added to inhibit possible bacterial
growth in the medium. To improve the functional
preservation of the preparation, the perfusate was
continuously dialysed against a 25-fold volume of the
same composition but lacking erythrocytes and
albumin. For oxygenation of the perfusion medium the
dialysate was gassed with a 95% oxygen, 5% carbon
dioxide mixture. Perfusate ¯ow rates were obtained
from the revolutions of the peristaltic pump which was
calibrated before and after each experiment. Renal ¯ow
rate and perfusion pressure were continuously moni-
tored by a potentiometric recorder. After establishing
the reperfusion loop, perfusate ¯ow rates usually
stabilized within 15 min. Stock solutions of the drugs
to be tested were dissolved in freshly prepared perfu-
sate and infused into the arterial limb of the perfusion
circuit directly before the kidneys at 3% of the rate of
perfusate ¯ow.
RESULTS
Role of potassium channels for the effect of NO
on renal vascular resistance
To investigate the role of potassium channels, in
particular the activation of those channels, in the vaso-
dilatory effect of NO in the kidney, we examined the
effect of the NO-donor sodium nitroprusside (SNP) on
perfusate ¯ow in the presence of different established
blockers of potassium channels, such as 4-aminopyri-
dine, barium, charybdotoxin and apamin. (Fig. 1). All of
these drugs caused signi®cant reductions of perfusate
¯ow at constant perfusion pressure, indicating an
Figure 1 Effect of sodium nitroprusside (10 lM) on perfusate ¯ow at
constant perfusion pressure of 100 mmHg through isolated rat
kidneys in the presence of different potassium channel blockers,
namely, 4-aminopyridine 1 mM (panel a) and barium chloride 100 lM
(panel b), apamin 200 nM (panel c) and charybdotoxin (panel d). Data
are mean � SEM of ®ve kidneys in each protocol.
Mode of nitric oxide action � A Kurtz et al. Acta Physiol Scand 2000, 168, 41±45
42 Ó 2000 Scandinavian Physiological Society
increase of renovascular resistance (Fig. 1). The increase
of renovascular resistance achieved by these drugs ranged
from 20 to 40%. The increase of renovascular resistance
induced by any of these drugs was rapidly and completely
reversed by the SNP as indicated by the restoration of
normal ¯ow rates (Fig. 1).
Role of G-kinase for the effect of the NO
on renal vascular resistance
To assess the relevance of the cGMP-G-kinase pathway
for the renal vasodilatation induced by NO, the effect
of the NO-donor SNP was examined in the presence of
the established G-kinase inhibitor Rp-8-pCPT-cGMP
(Fig. 2). Rp-8-pCPT-cGMP itself lowered ¯ow rates
indicating an increase of renovascular resistance by
inhibition of G-kinase activity (Fig. 2). The decline of
perfusate ¯ow induced by the G-kinase inhibitor was
completely reversed by the NO donor SNP (Fig. 2).
Role of cAMP-phosphodiesterases for the effect of NO
on renal vascular resistance
To assess a possible involvement of cAMP-
phosphodiesterases in the effect of NO on renal
vascular resistance, we examined the effects of endo-
genous and of exogenous NO on kidney perfusate ¯ow
at elevated intracellular cAMP levels. Those were
achieved with receptor-induced activation of cAMP
formation (isoproterenol, Fig. 3a) or with inhibition of
cAMP degradation (Fig. 3b, c). For the latter purpose
we used more selective inhibitors of the different
cAMP-PDE subclasses. Isoproterenol at a lower
concentration of 3 nmol L±1 moderately increased
blood ¯ow (Fig. 3a). Inhibition of endogenous NO
formation by L-NAME substantially decreased ¯ow in
the presence of isoproterenol. The reduction of
perfusate ¯ow induced by L-NAME was completely
reversed by the NO-donor SNP (Fig. 3a).
8-MM-IBMX an inhibitor of the PDE-1 family
signi®cantly increased perfusate ¯ow (Fig. 3b). Inhibi-
tion of endogenous NO formation by L-NAME
markedly decreased ¯ow in the presence of 8-MM-
IBMX and this reduction of perfusate ¯ow was
completely reversed by the NO-donor SNP (Fig. 3b).
Similarly, rolipram, an inhibitor of the PDE-4
family, signi®cantly increased perfusate ¯ow (Fig. 3c).
Inhibition of endogenous NO formation by L-NAME
markedly decreased ¯ow in the presence of rolipram
and this reduction of perfusate ¯ow was completely
reversed by the NO-donor SNP (Fig. 3c).
Quite different results were obtained with milrinone,
which is an established inhibitor of the PDE-3 family.
Milrinone also increased perfusate ¯ow (Fig. 3d). But in
Figure 2 Effect of sodium nitroprusside (10 lM) on perfusate ¯ow at
constant perfusion pressure of 100 mmHg through isolated rat
kidneys in the presence of the cGMP-protein kinase inhibitor Rp-8-
pCPT-cGMP. Data are mean � SEM of ®ve kidneys.
Figure 3 Effects of the NO-synthase inhibitor L-NAME (1 mM) and
of the NO donor SNP (10 lM) on perfusate ¯ow at constant
perfusion pressure of 100 mmHg through isolated rat kidneys in the
presence of isoproterenol 3 nM (panel a) and of different cAMP-PDE
inhibitors, namely 8-MM-IBMX 20 lM (panel b), rolipram 20 lM
(panel c) and milrinone 20 lM (panel d). Data are mean � SEM of
®ve kidneys in each protocol.
Ó 2000 Scandinavian Physiological Society 43
Acta Physiol Scand 2000, 168, 41±45 A Kurtz et al. � Mode of nitric oxide action
the presence of milrinone, neither inhibition of
endogenous NO formation by L-NAME nor adminis-
tration of exogenous NO by SNP had a further effect
on perfusate ¯ow (Fig. 3d). Rather similar results were
obtained with trequinsin, another PDE-3 inhibitor (not
shown). Apparently, inhibition of PDE-3 activity
essentially mimicked the effect of NO on renovascular
resistance.
DISCUSSION
Our study aimed to dissect the cellular pathways along
NO causes vasodilatation in the kidney. In this context
we focused on the participation of potassium channels,
in particular, on opening of potassium channels, on the
involvement of cGMP-dependent protein kinase
activity and on the involvement of cAMP-phosphodi-
esterases.
The results obtained show that various well-
established potassium channel blockers, among those
known to preferentially block calcium activated potas-
sium channels, such as apamin or charybdotoxin per se
increased vascular resistance suggesting that they were
effective in blocking potassium channels and therefore
caused depolarization of the vascular smooth muscle
cells in the kidney. However, none of these potassium
channel blockers attenuated the renal vasodilatation
induced by exogenous NO in the isolated perfused rat
kidney (Fig. 1). From these results we would infer that
the activation of potassium channels by NO is of minor
relevance for the effect of NO on renovascular resist-
ance.
The G-kinase inhibitor Rp-8-pCPT-cGMP also
increased renovascular resistance suggesting that the
basal G-kinase activity in the renal vasculature provides
a vasodilatatory effect. This inference ®ts with the
observation of a relatively high expression of G-kinase
in the renal vasculature (Joyce et al. 19861 ) and with our
observation that G-kinase activators are potent
vasoldilators in the isolated perfused rat kidney (Kurtz
et al. 1998). As the vasoconstriction induced by the G-
kinase inhibitor could be reversed by the NO donor,
we would assume that also G-kinase activation is not
essential for mediating the vasodilatory effect of NO in
the renal vasculature.
Our observation that several different inhibitors of
cAMP phosphodiesterases signi®cantly decreased
renovascular resistance con®rms the concept that
cAMP is a potent vasodilatory signal in the kidney and,
moreover, suggests a high turnover rate of cAMP in the
renal vasculature. This is indicated by the observation
that inhibition of cAMP degradation without additional
stimulation of cAMP formation already markedly
affects renovascular resistance (Fig. 3). Most effective
in this context was inhibition of PDE-3 activity, which
corroborates the ®nding that PDE-3 is the main
cAMP-PDE expressed in renal preglomerular vessels
(Reinhardt et al. 1995, Sandner et al. 1999). PDE-3
activity is naturally inhibited by cGMP (Beavo 1995) the
formation of which is known to be stimulated by NO.
The observation that pharmacological inhibition of
PDE-3 selectively mimicked the effect of NO on
renovascular resistance and prevented further effects of
NO therefore strongly suggests that inhibition of PDE-3
activity and in consequence the cAMP pathway is
importantly involved in the action of NO on renal
vascular resistance.
The authors' work is ®nancially supported by grants of the Deutsche
Forschungs-gemeinschaft.
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