Mode of nitric oxide action on the renal vasculature

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


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


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.


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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Mode of nitric oxide action on the renal vasculature