Endothelium-derived relaxing factors in the kidney of spontaneously hypertensive rats

Life Sciences, Vol. 56, No. 21 pp. PL 401-408, 1995 Copyright 0 19% Ekxier Science Lid Printed in the USA. All rights reserved w24-3205/95 $950 + .M)

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PHARMACOLOGY LETTERS Accelerated Communication

ENDOTHELIUM-DERIVED RELAXING FACTORS IN THE KIDNEY OF SPONTANEOUSLY HYPERTENSIVE RATS Hiroshi Hayakawa, Yasunobu Hirata **, Etsu Suzuki, Masao Kakoki, Kazuya Kikuchi*, Tetsuo Nagano*, Masaaki Hirobe* and Masao Omata The Second Department of Internal Medicine, Faculty of Medicine, University of Tokyo 7-3-l Hongo, Bunkyo-ku, Tokyo 113, Japan *Faculty of Pharmaceutical Sciences, University of Tokyo (Submitted January 11, 1995; accepted February 6, 1995; received in final form March 6, 1995)

Abstract: Acetylcholine (ACh)-induced vasodilation is mainly due to endothelium-derived nitric oxide (EDNO) and hyperpolarizing factor (EDHF). To explore the mechanisms underlying attenuated endothelium-dependent vasodilation in hypertensive arteries, we measured the EDNO released from isolated kidneys of spontaneously hypertensive rats (SHR) using a sensitive chemiluminescence assay system of NO. ACh-induced renal vasodilation was significantly smaller in SHR than in the normotensive control, Wistar-Kyoto rats (WKY). However, ACh-induced NO release did not differ between SHR and WKY (10m7M: SHR +37*2 [SE] vs. WKY +32+4 fmol/min/g kidney). Perfusion with a 20 mEqL high-K+ buffer, which is reported to inhibit action of EDHF, significantly reduced ACh-induced vasorelaxation in WKY but not in SHR, resulting in identical renal perfusion pressure in SHR and wKY under these conditions. These results indicate that attenuated ACh-induced vasorelaxation in the SHR kidney may be attributed to a decrease in EDHF rather than that in EDNO. Key Words: nitric oxide, hyperpolarizing factor, kidney, perfusion

It is well established that endothelial cells play an important role in the regulation of vascular tone and that vessels from hypertensive animals as well as humans show impaired endothelium-dependent vasodilation (1). Since vascular endothelial cells release multiple vasorelaxing and contracting factors, it remains unclear whether a change in endotheliumderived nitric oxide (EDNO) is the sole cause of altered endothelium-dependent vasodilation in hypertensive vessels. Moreover, because of limitations in the detectability of EDNO, the question of whether EDNO release in spontaneously hypertensive rats (SHR) is unchanged (2,3) or decreased (4, 5) has not been resolved. Endothelium-derived hyperpolarizing factor (EDHF) is another potent vasodilator which is released in response to acetylcholine (ACh) (6,7). Recent studies suggest that attenuated vasodilation in SHR in response to ACh may be due to an alteration in EDHF (8,9). Although the chemical structure of EDHF has remained **To whom correspondence should be addressed. FAX: +81-3-3814-0021

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elusive, EDHF causes membrane hyperpolarization by opening a K’ channel; as a consequence, the effect of EDHF can be suppressed by high concentration of extracellular K’ (10-12). Improved methods for measurement of NO release from hypertensive vessels will help clarify this issue. We have recently developed an assay system for NO which is highly sensitive and can be directly applied to physiological solutions. We have shown that this system can detect NO release in the venous effluent of isolated rat kidney and can be used to measure NO release during endothelium-dependent vasodilation as a function of renal perfusion pressure (RPP) (13, 14). In the present study, we applied this method to the study of isolated perfused kidneys from SHR, and have compared the effects of EDHF inhibition on ACh-induced vasodilation in SHR and the normotensive control, Wistar-Kyoto rats (WKY).

Methods The assay system for NO has been previously reported (13,14). The renal perfusate was introduced at 2 ml/min with a double plunger head pump into the rotating flow mixer. The chemiluminescence probe was concurrently pumped into the mixer at 0.5 ml/min. The mixture was then introduced into the chemiluminescence detector and the signal was continuously recorded. The EDNO concentration in the perfusate was calibrated using an authentic NO solution of known concentration determined by the HbO, method (13). The kidneys from SHR and WKY were isolated and perfused as previously reported (15). The right renal artery was cammlated via the superior mesenteric artery and perfused with a Krebs-Henseleit buffer with 95% 0, - 5% CO, at 37°C. To maintain the renal perfusion pressure at about 100 mmHg, the buffer contained 10” M phenylephrine instead of albumin and amino acids because these substances interfere with chemiluminescence detection. The chemiluminescence probe was composed of 10 mM H,O,, 18 pM recrystallized luminol, 2 mM potassium carbonate, and 150 mM desferrioxamine. The renal perfusion pressure (RPP) was simultaneously monitored through a pressure transducer connecting with the arterial cammla. The renal perfusion flow was maintained at 5 ml/min throughout the study. Following the equilibrium period, vehicle, lo-* M ACh, and 10e7 M ACh were infused sequentially. Finally, lOA M NG-monomethyl-L-arginine (L-NMMA) was added to 10e7 M ACh and infused. During the infusions, RPP and chemiluminescence were continuously monitored. SHR and WKY at 4 weeks of age were obtained from Japan Charles River Co. They were examined at 14 weeks of age. Systolic blood pressure (BP) was measured weekly by the tail cuff method. In order to assess the contribution of EDHF, the effects of high-K+ perfusion at 20 mEq/L on ACh-induced vasorelaxation were studied using a standard renal perfusion technique. The perfusate was a Krebs-Henseleit buffer which contained 6.7 g/d1 of fraction V bovine serum albumin, 20 essential amino acids and 100 mg/dl of glucose (15). Following the isolation of the kidney, 120 ml of the perfusate were recirculated at 37°C and continuously oxygenated with 95% O,-5% CO,. Renal perfusion flow was measured using an in-line flowmeter and perfusion pressure was monitored. After an equilibrium period, 100 pl of the ACh solution were added to the per&sate in a cumulative manner. The effects of ACh at lo-* M to lOa M on RPP were compared under both normal K’ concentration (4 mEq/L) and high K+ concentration (20 mEq/L). The perfusion pressure was initially set at 150 mmHg and the flow rate was maintained constant. In the high K’ perfusion study, the K’ concentration was adjusted by replacing NaCl with KCl. We have previously reported that ACh-induced vasodilation does not differ between the two perfusion methods, i.e., high flow with albumin and amino acids containing buffer and low flow with phenylephrine-containing buffer (3).

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Values are expressed as means&E. Effects of agents tested were assessed by ANOVA for repeated measures followed by the modified t-test. Differences between SHR and WKY were tested using the unpaired Student’s t-test. PcO.05 was considered statistically significant.

Results Table 1 shows the baseline variables of SHR and WKY rats. Although BP is higher in SHR than in WKY, RPP, kidney weight (KW) and baseline renal perfusion flow in both strains were comparable. TABLE 1 Blood Pressure (BP), Body Weight (BW), Kidney Weight (KW) and Baseline Values of Renal Perfusion in SHR and WKY Rats. n

BP mmHg

(A) NO measurement WKY 7 11427 SHR 7 212*9* (B) normal K’ perfusion WKY 6 11924 208*9* SHR (C) high K’ peusion WKY 7 128*6 SHR 6 221*5*

BW g

KW g

PRF ml/min

RPP mmHg

29126 27526

1.3420.04 1.22kO.04

5.0 5.0

11626 96k9

303211 333216

1.334.04 1.40&05

52.222.8 59.022.8

14822 148+1

26027 290*19

1.34*0.05 1.32kO.08

46.7k3.0 48.5k2.4

14823 14622

Values are expressed as means * SE. RPF: renal perfusion flow, RPP: renal perfusion pressure, * p
Figure 1 demonstrates the representative tracings of NO chemiluminescence and RPP during vehicle, ACh, L-NMMA, and L-arginine (L-Arg) administration in SHR and WKY. The NO chemiluminescence increased in response to ACh in a dose-dependent fashion, was diminished by L-NMMA and then restored by L-Arg. RPP changes mirrored changes in the NO signal. Figure 2 summarizes changes in the NO signal and RPP. Although the decrease in RPP in response to ACh was significantly smaller in SHR than in WKY, the baseline (WKY 26.825.2 vs. SHR 30.56.2 fmol/min/g kidney weight, ns) and stimulated NO signals in kidneys of SHR and WKY were indistinguishable. Figure 3 compares the effects of 4 mEq/L normal-K+ and 20 mEq/L high-K+ perfusate on ACh-induced vasorelaxation. The response to renal perfusion pressure to ACh was significantly greater at lo-’ M than at lo-’ M, however higher concentrations of ACh did not cause further vasodilation. This is consistent with results from previous reports (2, 3). The ACh-evoked decrease in RPP was smaller in SHR than in WKY at any concentration. High-K+ perfusion significantly reduced

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the response of RPP to ACh in WKY by about 50%. However, the reduction of the response in SHR was only lo%-20% and insignificant. Thus, the differences in ACh-induced vasodilation between SHR and WKY disappeared during the high K+ perfusion.

WKY

-

150

ap

100

&E

50 E

----A

_I,\

03

80-

SHR

60.

40.

20.

o-

Fig. 1 Representative tracings of nitric oxide (NO) release and renal perfusion pressure (RPP) in SHR and WKY. NO release and RPP in response to administration of acetylcholine (ACh), L-Ne-monomethyl arginine (L-NMMA) and L-arginine (L-Arg) are shown for (A) SHR and (B) WKY.

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+60

-60

a

-40 -50

+ lo-'M ACh

IO-% ACh

lo-'M ACh ,O-&M&IA

+p
-itp
Fig. 2 Effects of acetylcholine (ACh) and L-p-monomethyl arginine (L-NMMA) on renal perfusion pressure (RPP) and release rates of NO into the perfusate from the kidney of SHR and WKY. The mean and standard error of each value is shown.

This is the first report combining detection of NO release from SHR kidneys by the highly sensitive chemiluminescence method in the liquid phase with simultaneous measurement of

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SHR

WKY

-0-

normal

K+

-a-

high

K’

Fig. 3 Changes in renal perfusion pressure (RPP) induced by acetylcholine (ACh) in SHR and WKY with the perfusate containing normal-K+ (4 r&q/L) and highK’ (20 mEq/L) buffers. While in WKY there is a significant difference in RPP of normal and high K’ perfusate at all ACh concentrations, there is no difference in SHR at any level of ACh concentration. Moreover, while there are consistent differences in RPP of SHR and WKY in normal K’ perfusate, these differences are abolished with high K’ perfusate. The mean and standard error of each value is shown.

RPP. ACh-induced renal vasodilation was found to be attenuated in SHR. Numerous studies have reported similar results in hypertensive arteries (16-18); however the release rates of NO have remained controversial. Using a bioassay technique, L.iischer et al. (2) have shown that ACh released comparable amounts of relaxing factors from the aortas of SHR and WKY rats. However, the bioassay technique evaluates the integrated effects of endothelium-derived vasoactive substances and cannot discriminate the effect of EDNO from that of other EDRF including EDHF. Malinski et al. (5) have recently shown using their porphyrinic electrode that both cultured endothelial cells and vascular smooth muscle cells from stroke-prone SHR released less NO than those from WKY. They concluded that this is due to a genetic defect in NO synthase activity of stroke-prone SHR rather than secondary defects because the cells

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were passaged. Although the difference from our results may be due simply to the difference between SHR and stroke-prone SHR, one must be cautious in applying the results from the cultured cells to those in vivo. In the present study, the SHR and WKY kidneys released comparable amounts of EDNO, but the SHR showed attenuated vasodilation in response to ACh. This is compatible with our previous report that release rates of NO metabolites, NO,and NO,-, from SHR kidneys were similar to those from WKY kidneys (3). Therefore, it is likely that factors other than EDNO explain attenuation of ACh-induced vasorelaxation in SHR. Although the contribution of EDHF to ACh-induced vasodilation may differ among the vasculatures, it has been suggested that 20%-40% of the effect of ACh in the rat arteries is due to EDHF (10). The present study indicates that EDHF contributes significantly to renal endothelium-dependent vasodilation in WKY because inhibition of EDHF by high-K+ perfusion reduced the ACh-induced declines in RPP by 50%. Fujii and coworkers (6,7) have shown that ACh-induced hyperpolarization was significantly smaller in the SHR mesenteric artery than in WKY, and De Voorde et al. (19) have reported similar results in Goldblatt hypertensive rats. Potassium channel openers have been shown to be effective as antihypertensive agents. If EDHF is an endogenous K’ channel opener, decreases in EDHF may aggravate hypertension. We have previously demonstrated that NO release is markedly attenuated in DOCA-salt hypertensive rats (3,14,20). Such differential alterations in EDNO or EDHF in different experimental models may reflect underlying differences in the etiology of hypertension in these models. Further studies are required to confirm whether these changes are the result of hypertension or a change specific for SHR.

This study was partly supported by a Grant-in-Aid for Scientific Research on priority areas: “Vascular Endothelium-Smooth Muscle Coupling” and by Grant-in-Aid #06274209 and #06671132 from the Japanese Ministry of Education, Culture and Science, Japan.

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4. 5. 6. 7. 8. 9.

P.M. VANHOU’ITE, Hypertension l3 658-667 (1989). T.F. LUSCHER, J.C. ROMERO, and P.M. VANHOUTI’E, J. Hypertens. 4&uppl@ SSl-S83 (1986). H. HAYAKAWA, Y. HIRATA, E. SUZUKI, T. SUGIMOTO, H. MATSUOKA, K. KIKUCHI, T. NAGANO, M. HIROBE, and T. SUGIMOTO, Am. J. Physiol. 264 H1535-H1541 (1993). H. IKENAGA, H. SUZUKI, N. ISHIT, H. ITOH, and T. SARUTA, Kidney. Int. 43 205211 (1993). T. MALINSKI, M. KAPTURCZAK, J. DAYHARSH, and D. BOHR, B&hem. Biophys. Res. Commun. l$&l654-658 (1993). T.B. BOLTON, R.J. LANG, and T. TAKEWAKI, J. Physiol. (London) 3.5.l 549-572 (1984). G. CHEN, H. SUZUKI, and A.H. WESTON, Br. J. Pharmacol. 95 1165-1174 (1988). K. FUJII, M. TOMINAGA, S. OHMORI, K. KOBAYASHI, T. KOGA, Y. TAKATA, and M. FUJISHIMA, Circ. Res. 211 660-669 (1992). K. FUJII, S. OHMORI, M. TOMINAGA, I. ABE, Y. TAKATA, Y. OHYA, K. KOBAYASHI, and M. FUJISHIMA, Am. J. Physiol. 265 H509-H516 (1993).

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