S Ito,, L A Juncos, O A Carretero. Find the latest version: J Clin Invest. 1993;91(5):

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1 ndothelium-derived relaxing fator/nitri oxide modulates angiotensin II ation in the isolated miroperfused rabbit afferent but not efferent arteriole. S Ito,, L A Junos, O A arretero J lin Invest. 1993;91(5): Researh Artile It has been reported that sensitivity to angiotensin II (Ang II) is higher in efferent (f) than afferent (Af) arterioles (Arts). We tested the hypothesis that this is due to arteriolar differenes in the interation between Ang II and endothelium-derived relaxing fator/nitri oxide (DNO). Rabbit Af-Arts with glomerulus intat were miroperfused in vitro at a onstant pressure. f-arts were perfused from the distal end of either the Af-Art (orthograde perfusion) or the f-art (retrograde perfusion) to eliminate influenes of the Af-Art or glomerulus, respetively. Ang II did not alter Af-Art luminal diameter until the onentration reahed 1(-9) M, whih dereased the diameter by 11 +/- 2.6% (n = 11; P <.2). In ontrast, f-arts beame signifiantly onstrited at onentrations as low as 1(-11) M with either perfusion. Surprisingly, the derease in f-art diameter at 1(-1), 1(-9), and 1(-8) M was signifiantly greater with retrograde perfusion (44 +/- 6.9%, 7 +/- 5.6%, and 74 +/- 4.1%, respetively; n = 5) than with orthograde perfusion (16 +/- 4.2%, 25 +/- 2.9%, and 35 +/- 3.5%; n = 9). NDO synthesis inhibition with 1(-4) M nitro-l-arginine methyl ester (L- NAM) dereased the diameter to a greater extent in Af-Arts (22 +/- 3.%; n = 11) ompared to f-arts with either orthograde (9.5 +/- 2.3%; n = 8) or retrograde perfusion (1.2 +/- 2.1%; [ ] Find the latest version: Pdf

2 ndothelium-derived Relaxing Fator/Nitri Oxide Modulates Angiotensin 11 Ation in the Isolated Miroperfused Rabbit Afferent but Not fferent Arteriole Sadayoshi Ito, Shuji Arima, Yi Lin Ren, Luis A. Junos, and Osar A. arretero Hypertension and Vasular Researh Division, Department ofmediine and Heart and Vasular Institute, Henry Ford Hospital, Detroit, Mihigan 4822 Abstrat It has been reported that sensitivity to angiotensin II (Ang II) is higher in efferent (f) than afferent (Af) arterioles (Arts). We tested the hypothesis that this is due to arteriolar differenes in the interation between Ang II and endothelium-derived relaxing fator/nitri oxide (DNO). Rabbit Af-Arts with glomerulus intat were miroperfused in vitro at a onstant pressure. f-arts were perfused from the distal end of either the Af-Art (orthograde perfusion) or the f-art (retrograde perfusion) to eliminate influenes of the Af-Art or glomerulus, respetively. Ang II did not alter Af-Art luminal diameter until the onentration reahed 1-9 M, whih dereased the diameter by 11±2.6% (n = 11; P <.2). In ontrast, f-arts beame signifiantly onstrited at onentrations as low as 1-11 M with either perfusion. Surprisingly, the derease in f-art diameter at 1-1, 1-', and 1-8 M was signifiantly greater with retrograde perfusion (44±6.9%, 7±5.6%, and 74±4.1%, respetively; n = 5) than with orthograde perfusion (16±4.2%, 25±2.9%, and 35±3.5%; n = 9). NDO synthesis inhibition with 1-4 M nitro-l-arginine methyl ester (L-NAM) dereased the diameter to a greater extent in Af-Arts (22±3.%; n = 11 ) ompared to f-arts with either orthograde (9.5±2.3%; n = 8) or retrograde perfusion (1.2±2.1%; n = 6). With L- NAM pretreatment, Af-Art onstrition indued by 1-1 M (14±4.%, n = 9) and 1-9 M Ang 11 (38±3.9%) was signifiantly greater ompared to nontreated Af-Arts. In ontrast, L- NAM pretreatment had no effet on Ang II-indued onstrition in f-arts with either perfusion. In onlusion, this study demonstrates higher sensitivity of f-arts to Ang II, partiularly with retrograde perfusion. Our results suggest that DNO signifiantly modulates the vasoonstritor ation of Ang II in Af-Arts II but not f-arts, ontributing to the differential sensitivity to Ang II. (J. lin. Invest : ) Key words: glomerular hemodynamis * nitro-l-arginine methyl ester * renal miroirulation * sensitivity Introdution The renin-angiotensin system plays an important role in the regulation of blood pressure, homeostasis of fluid volume and eletrolytes, and pathogenesis of various forms of hyperten- Address reprint requests to Dr. Sadayoshi Ito, Hypertension and Vasular Researh Division, Henry Ford Hospital, 2799 West Grand Boulevard, Detroit, MI Reeivedfor publiation 8 September 1992 and in revisedform 18 Deember J. lin. Invest. The Amerian Soiety for linial Investigation, In /93/5/212/8 $2. Volume 91, May 1993, sion. A number of studies have examined the role of angiotensin II (Ang II), ' the physiologially ative omponent of the system, in the ontrol of renal hemodynamis as well as its sites ofation within the kidney ( 1 ). Infusion ofang II or inhibition of its ation in the kidney has been shown to result in an inreased or dereased filtration fration, respetively (2-5). Thus it has been postulated that sensitivity to Ang II is higher in the postglomerular efferent (f) than in the preglomerular afferent (Af) arteriole (Art). Although reent studies using isolated mirovessels have demonstrated higher sensitivity of the f-art (6, 7), the mehanism(s) responsible for this differene in sensitivity is (are) not well understood. We have reently shown that in isolated miroperfused rabbit Af-Arts, inhibition of endothelium-derived relaxing fator/ nitri oxide (DNO) not only redues basal luminal diameter but also augments the vasoonstritor ation of Ang II (8). This suggests that DNO, whih is produed loally in the Af-Art, is an important autaoid hormone that ontrols vasular reativity. Although studies have shown that endogenous DNO plays an important role in the ontrol ofrenal hemodynamis (9), it is not known whether either ativity ofdno or its interation with other vasoative substanes differs between the Af-Art and f-art. Sine the Af-Art and f-art are ruial vasular segments that ontrol glomerular hemodynamis (and hene renal exretory funtion), it would be important to understand the ation of DNO in these vessels. In the present study, we tested the hypothesis that endogenous DNO modulates Ang II ation to a greater extent in the Af-Art than in the f-art, ontributing to the differene in sensitivity to Ang II. For this, we mirodisseted and perfused rabbit Af-Arts and f-arts in vitro and studied whether (a) response to DNO inhibition or (b) DNO modulation of Ang II ation is different between the two vessels. Methods Isolation and miroperfusion ofthe rabbit Af- and f-art We used a method similar to that desribed previously to isolate and miroperfuse Af-Arts (8, 1). Briefly, young male New Zealand white rabbits ( kg), fed standard rabbit how (Ralston Purina o., St. Louis, MO) and tap water ad lib., were anesthetized with intravenous sodium pentobarbital (4 mg/kg) and given an intravenous injetion of heparin (5 U). The kidneys were removed and slied along the ortiomedullary axis. Slies were plaed in ie-old MM (Gibo Laboratories, Grand Island, NY) ontaining 5% BSA (Sigma hemial o., St. Louis, MO) and disseted under a stereomirosope (model SZH; Olympus orp., Tokyo, Japan) as desribed previously. From 1. Abbreviations used in this paper: Af-Art and f-art, afferent and efferent arteriole, respetively; Ang II, angiotensin II; DNO, endothelium-derived relaxing fator/nitri oxide; L-NAM, nitro-l-arginine methyl ester; N, norepinephrine. 212 Ito et al.

3 -. zj4jjua ::... ':.._ni' R...s., a': a '3.'-, g... ::.:-:<:l Kim Aft ::. XY- S eah rabbit, a single superfiial Af-Art with its glomerulus intat was mirodisseted. Using a miropipette, the Af-Art was transferred to a temperature-regulated hamber mounted on an inverted mirosope (model IMT-2; Olympus orp.) with Hoffman modulation. The Af- Art was then annulated with an array of glass pipettes as desribed previously (8, 1). Intraluminal pressure was measured by Landis' tehnique, using a fine pipette introdued into the arteriole through the perfusion pipette. This tehnique was used beause it has been reported that pressure measurement by a servo-null system is not aurate with our arrangement of pipettes. The arteriole was perfused with oxygenated medium 199 ontaining 5% BSA, and intraluminal pressure was maintained at 6 mmhg throughout the experiment. We employed two approahes to study f-art, namely orthograde and retrograde perfusion. For orthograde perfusion, a short (- 5 Mm) Af-Art with its glomerulus intat and a segment of the attahed f-art (25-3 Mm) were mirodisseted. The Af-Art was annulated as desribed above, exept that the perfusion pipette was advaned to the end of the Af-Art (Fig. 1, top). The tip of the pressure pipette was plaed just beyond the distal end of the Af-Art, and intraluminal pressure at this point was maintained at 5 mmhg throughout the experiment so as to eliminate the hemodynami influenes of the Af-Art. In five experiments, we measured pressure in the f-art. The f-art was drawn into a holding pipette (i.d., 2gm) whih had a slightly negative internal pressure (-2 mmhg). A pressure pipette (o.d., 2 Mm) was inserted into the f-art from the distal end and advaned until its tip reahed a point 5gm distal to the glomerulus. In that only a 2-am - pressure pipette was inserted into the f-art with a luminal diameter of 15 Mm (thus the pipette oupied only 2% of the ross-setional Orthograde Perfusion s>..; Per~f~o.n -~~~~~ ~ ~ far Prem.PIpl Retrograde Perfusion in :S 3.;f,_6A''<+, W..-re.i: w w Figure 1. fferent arteriole (f-art) perfused in orthograde (top) and retrograde diretion (bottom). Perf-Pip, perfusion pipette; Pre-Pip, pressure pipette; Af-Art, afferent arteriole. area), perfusate flowed freely into the holding pipette. The pressure measured at this point varied between 32 and 41 mmhg, with an average of 36.4±1.3 mmhg. For retrograde perfusion, most of the Af-Art was removed and the f-art ( 1-15 Mm) perfused from its distal end at 35 mmhg throughout the experiment (Fig. 1, bottom). In this preparation, there are no influenes ofeither hemodynami hanges indued by the glomerulus or vasoative substanes produed by the glomerulus/af-art. The bath, whih was idential to the arteriolar perfusate exept that it ontained.1% BSA, was exhanged ontinuously. Mirodissetion and annulation ofthe arteriole were ompleted within 9 minat 8, after whih the bath was gradually warmed to 37 for the rest of the experiment. One the temperature was stable, a 3-min equilibration period was allowed before taking any measurements. Images of the arteriole were displayed at magnifiations up to X 1,98 and reorded with a video system onsisting of a amera (model DX-755; Sony, Tokyo, Japan), monitor (model PVM1942Q; Sony) and video reorder (model DV-95; Sony). The diameter at the most onstrited point (whih was seen at a segment within 5 Mm from the glomerulus) was measured with an image analysis system (Fryer, arpentersville, IL). xperimental protools 1. Response to Ang II. After the 3-min equilibration period, inreasing doses of Ang 11 ( 1-" to 1-8 M; Sigma hemial o.) were added to the bath. Luminal diameter was measured immediately before adding Ang II and observed for at least 3 min at eah dose. 2. Response to norepinephrine (N). To examine whether differenes in sensitivity between the Af- and f-art are ommon to all vasoonstritors, we ompared the effet of N on luminal diameter of Af- and f-arts. After the equilibration period, inreasing doses of N (1-8 to 1-6 M; Sigma hemial o.) were added to the bath. Luminal diameter was measured immediately before adding N and observed for at least 3 min at eah dose. 3. Inhibition ofdno synthesis with nitro-l-arginine methyl ester (L-NAM). After the equilibration period, inreasing doses of L- NAM ( l-7 to l-4 M; Sigma hemial o.), a ompound that inhibits DNO synthesis ( I I ), were added to the lumen. Luminal diameter was measured immediately before adding L-NAM and observed for at least 15 min at eah dose. 4. ffet ofl- or D-arginine on L-NAM-indued onstrition. We examined whether Af-Art vasoonstrition indued by L-NAM is speifially due to inhibition of DNO synthesis from L-arginine ( 12). We first inhibited DNO synthesis with L-NAM at l-4 M, after whih either L-arginine or its inative isomer D-arginine at 1-' M (Sigma hemial o.) was added to the lumen together with L-NAM. 5. ffet ofl-nam on Ang II-indued vasoonstrition. After the equilibration period, l-4 M L-NAM was added to the arteriolar perfusate. Fifteen minutes later, the effet of Ang II was examined as in protool 1. We have previously shown that L-NAM at this onentration bloks aetylholine-indued vasodilation in Af-Arts preonstrited with N (8). We have also onfirmed that L-NAM is just as effetive in the f-art. The f-arts were preonstrited with N (5 x 1-7 M) to 82±3.6% of basal diameter in orthograde perfusion (n = 5) and 46±6.5% in retrograde perfusion (n = 5), while intraluminal addition of aetylholine ( I-5 M) reversed the diameter to 15±7.1% and 16±5.6% of basal values, respetively. When N was given to f-arts pretreated with L-NAM, the diameter dereased to 79±4.7% of baseline in orthograde perfusion (n = 6) and 63±12% in retrograde perfusion (n = 4), whih remained unhanged after addition of aetylholine (8±4.6% and 64±12%, respetively). (See Results and Disussion for weaker responses to N in orthograde than in retrograde perfusion.) 6. ffet ofl-nam on N-indued vasoonstrition. We examined whether L-NAM-indued enhanement of vasoonstritor ation is ommon to all vasoonstritors. The experimental design was the same as in protool 5, exept that N (I-8 to 1-6 M) was used instead of Ang II. Nitri Oxide in Afferent and fferent Arteriole 213

4 7. ffet ofn pretreatment on Ang II-indued vasoonstrition. Sine L-NAM not only augmented Ang II-indued vasoonstrition in Af-Arts but also redued basal diameter (see Results), we tested whether L-NAM affeted the ation ofang II by inreasing basal tone. The experimental design was the same as in protool 5, exept that we used only Af-Arts and redued basal diameter with I-7 M N instead of 1-4 M L-NAM. Statistis Values were expressed as mean±sm, and all statistial analyses were done using absolute values. A paired t test was used to examine whether the diameter at a given onentration was different from the ontrol value. Analysis of ovariane (ANOVA) was used to examine whether the hange in diameter at a given onentration was different between groups. For both analyses, P <.125 (.5/4; Bonferroni adjustment) was onsidered signifiant. Results 1. Response to Ang II. Basal luminal diameter of the Af-Arts was 16.1±.9 Mm (n = 11), while that of the f-arts was 15.7±.6,um with orthograde perfusion (n = 9) and 16.3±1.3,Mm with retrograde perfusion (n = 5). As shown in Fig. 2, Ang II had no effet on Af-Arts until the onentration reahed 1-9 M, whih dereased luminal diameter by 2.±.45 Mm or 11±2.6% (P<.2); at 1-8 M, the derease was 6.7±1.1 zm or 4±5.3% (P <.1). In ontrast, in f-arts perfused in either diretion, Ang II began to ause signifiant onstrition at onentrations as low as 1"-I M. In orthograde perfusion, the derease in diameter indued by Ang II was 1.8±.55,um or 11±3.8% at 1-1" M (P <.1), 2.4±.61,um or 16±4.2% at 1-1 M (P <.5), 3.9±.39 Mum or 25±2.9% at 1-9 M (P <.1), and 5.5±.57,um or 35±3.5% at 1-8 M (P <.1); in retrograde perfusion, the orresponding values were 1.6±.25 Mm or 1±2.1% (P <.5), 7.1±1.11 Mm or 44±6.9% (P <.3), 11.2±.68,m or 7±5.7% (P <.1), and 11.8±.76 Mm or 74±4.1% (P <.1). ompared to the Af-Arts, the derease in f-art diameter was signifiantly greater at doses of 1-1 and 1-9 in orthograde perfusion and at all doses examined in retrograde perfusion. In addition, the derease in f-art diameter indued by 11 to :8. -j S " I -14 L * Af-Art (n-11) O f-art OP (n-8) A& f-art.rp (n-5) O Figure 2. hange in luminal diameter indued by Ang II in miroper- fused afferent arterioles (Af-Art) and efferent arterioles studied with orthograde (f- Art. OP) or retrograde perfusion (f-art RP). \ *.tlt *.**.***p <.12, I z ** t.3, and.1, re- I i spetively, ompared with Af-Art. +++P <.3 and.1, re- -8 spetively, ompared Angiotensin 11 (log M 1) with f-art * OP M Ang II was signifiantly greater in retrograde than in orthograde perfusion (Fig. 2). 2. Response to N. Basal luminal diameter of the Af-Arts was 16.2±.9 Atm (n = 7), while that of the f-arts was 14.8±.5 Am with orthograde perfusion (n = 9) and 14.5±1.4 Am with retrograde perfusion (n = 5). In both the Af-Arts and the f-arts with retrograde perfusion, N dereased luminal diameter in a similar dose-dependent manner, with signifiant onstrition ourring at l-' M (Fig. 3). In the f-arts studied with orthograde perfusion, N at 1-8 and l-7 M indued similar responses ompared to either Af-Arts or f-arts with retrograde perfusion; however, N at higher onentrations indued signifiantly weaker onstrition. 3. Inhibition ofdno synthesis with L-NAM. Basal luminal diameter ofthe Af-Arts was 17.4±.8Mm (n = 11 ), while that of f-arts was 14.7±.7,m with orthograde perfusion (n = 8) and 15.2±.72 Am with retrograde perfusion (n = 6). As shown in Fig. 4, L-NAM dereased Af-Art diameter in a dosedependent manner, with the derease beoming 2.6±.76 Mm or 14±4.3% at 1-5 M (P <.1) and 3.9±.58 Am or 22±3.% at 1-4 M (P <.1). With orthograde perfusion, l-4 M L-NAM aused a statistially signifiant derease in f-art diameter (1.4±.33 Am or 9.5±2.3%; P <.1); however, it tended to be smaller than in the Af-Arts (P =.5). Moreover, with retrograde perfusion, L-NAM had no effet on f-arts at any dose, and the derease in diameter at l-4 M was signifiantly different ompared to the Af-Arts (P <.5). 4. ffet ofl- or D-arginine on L-NAM-indued onstrition. L-NAM pretreatment dereased Af-Art diameter from 18.5±.7 to 14.8±.7 (n = 6), while addition of L-arginine at l-3 M reversed the diameter to ontrol levels ( 19.4±1.1 Am). On the other hand, D-arginine had no effet on the L-NAMindued derease in diameter; with L-NAM, the diameter dereased from 18.7±.7 to 15.±.8 Mm (n = 4) and remained Mm). unhanged after addition of D-arginine ( 14.4± ffet ofl-nam on Ang II-indued vasoonstrition. hanges in diameter indued by Ang II in L-NAM-treated and nontreated Af-Arts are depited in Fig. 5, while an example of the responses observed at 1-' M is shown in Fig. 6. After L-NAM pretreatment, basal diameter dereased signifiantly by 22±5.6%, falling from 17.3±1. to 13.2±.8 Mm (n = 9). When Af-Arts were pretreated with L-NAM, Ang II at 1-1 M, whih had no effet on nontreated Af-Arts, dereased the diameter signifiantly by 2.±.62 Mm or 14±4.% (P <.12); at 1-" M, the diameter dereased by 1.9±.66 Mm or 13±4.2%, but it did not reah statistial signifiane (P =.2). When ompared to non-treated Af-Arts, the Ang IIindued derease in diameter was greater in the L-NAM-pretreated Af-Arts at doses of 1-", 1-1, and 1-' but not at 1-8 M. In ontrast, L-NAM pretreatment had no effet on f-arts whether they were perfused in an orthograde or retrograde diretion (Fig. 7). Thus, with L-NAM pretreatment, Ang IT-indued onstrition beame no different in Af-Arts and f-arts with orthograde perfusion. In the ase of f-arts with retrograde perfusion, onstrition at 11`, 1-9, and 1 8 M seemed to be greater ompared to Af-Arts even with L-NAM pretreatment; however, the differenes was not statistially signifiant (P >.4). 6. ffet of L-NAM on N-indued vasoonstrition. L- NAM pretreatment dereased luminal diameter by 25±3.4%, 214 Ito et al.

5 :k I-, q._ ' J " Z. Lr F -1 F -12 k -14 L * Af-Art (n-7) o f-art-op (n-9) AL f-art-rp (n-5) Norepinephrine (log M) Figure 3. hange in luminal diameter indued by N in miroperfused afferent arterioles (Af Art) and efferent arterioles studied with orthograde (f-art- OP) or retrograde perfusion (f-art.rp). *,**P <.7 and.5, respetively, ompared with either Af-Art or f-art RP. :x._. a -J " - -2 k L -1 o Nontreated (n-1) * L-NAM (n-9) Angiotensin 11 (log M) Figure 5. hange in luminal diameter indued by Ang II in nontreated afferent arterioles (Nontreated) and afferent arterioles pretreated with L-NAM. * **P <.2 and.1, respetively, for nontreated versus L-NAM. from 2.5±1. to 15.7±1.1,um (n = 6). However, it did not affet N-indued onstrition in Af-Art (Fig. 8), in marked ontrast to the augmented ation of Ang II (Figs. 5 and 6). 7. ffet of N pretreatment on Ang II-indued vasoonstrition. Pretreatment with N redued basal diameter by 16±2.8%, from 17.3±.8 to 14.6±.9 ztm (n = 1), whih was no different from that indued by L-NAM. However, it did not affet the vasoonstritor ation of Ang II (Fig. 9). Disussion Differential sensitivity of the pre- and postglomerular arterial vessels to Ang II has been of great interest to many investigators. Using various experimental models, researhers have reported that sensitivity to Ang II is higher (3, 7, 13, 14) or exlusively present in the postglomerular vessels (2, 6, 15, 16), or similar in both vessels ( 17-2). To avoid neurohormonal and systemi hemodynami influenes, dwards (6) developed an in vitro preparation onsisting of an isolated rabbit zk J a a -2 F -4-6 L * Af-Art (n-11) o f-art-op (n-8) A& f-art-rp (n-6) 1 A I L-NAM (log M) I Figure 4. hange in luminal diameter indued by L-NAM, whih inhibits synthesis of DNO, in miroperfused afferent arterioles (Af-Art) and efferent arterioles studied with orthograde (f- Art- OP) or retrograde perfusion (f-art- RP). *P <.5 ompared with Af-Art. renal mirovasular segment with one end annulated and the other oluded. He reported that, although Ang II aused dosedependent vasoonstrition in the f-art, the Af-Art was ompletely nonresponsive to Ang II even at 1-6 M. Using a similar tehnique in rats, Yuan et al. (7) reported that Ang II auses vasoonstrition in both Af-Art and f-art, but with a lower 5 in f-art. Our results are qualitatively onsistent with these in vitro studies, even though there was a lear differene in the vasular responses of the f-arts with orthograde versus retrograde perfusion. Nevertheless, our observations, taken together with previous studies, learly demonstrate that sensitivity to Ang II is higher in f-arts than in Af-Arts. We found striking differenes in the vasular reativity of f-arts with orthograde versus retrograde perfusion. In retrograde perfusion, both Ang II- and N-indued vasoonstrition were very strong, and the dose-response urves obtained were almost idential to those reported by dwards (6) and Yuan et al. (7) in isolated but nonperfused f-arts. On the other hand, vasoonstrition was muh weaker in orthograde perfusion. Thus, when Ang II ation on Af-Arts and f-arts was ompared, the differene was less marked (albeit signifiant) with orthograde than with retrograde perfusion of the f-arts. The reason for the differenes in f-art reativity is not lear. It has been reported that f-art response to Ang II but not N is pressure dependent (21 ). However, we observed that not only the response to Ang II but also that to N (whih is pressure independent) were learly different with orthograde versus retrograde perfusion. Furthermore, when we measured pressure in f-arts studied with orthograde perfusion, it was found to be similar to that used for retrograde perfusion. The fat that L-NAM did not affet the differenes in f-art reativity suggests that fators other than DNO may be involved. It may be that in orthograde perfusion, the release of other vasoative substanes (suh as prostaglandins) by the glomerulus (22) ould influene the f-art response. The differenes may also be related to shear stress. Due to ultrafiltration at the glomerulus, protein onentration in the f-art (and hene shear stress) would be higher in orthograde than retrograde perfusion. This differene as well as the diretion and rate of flow may have affeted synthesis of various vasoative substanes by the endothelium (23, 24). Nitri Oxide in Afferent and fferent Arteriole 215

6 ,...;,,>.tf... : *' 5.lm Figure 6. Vasoonstrition indued by i- L-NAM (right). Note that L-NAM not only redued basal diameter but augmented the vasoonstritor ation of angiotensin II. M angiotensin II (AngIl) in a nontreated afferent arteriole (left) and afferent arteriole pretreated with In ontrast to the differene in sensitivity to Ang II, N has been reported to have a similar effet on isolated Af-Arts and f-arts (6, 7). onsistent with these studies, we found no differene in N-indued vasoonstrition between Af-Arts and f-arts with retrograde perfusion. However, in the f-arts studied with orthograde perfusion, the dereases in diameter indued by higher onentrations (5 x 1'- and 1-6 M) were signifiantly less ompared to either Af-Arts or f-arts with retrograde perfusion. Weaker responses off-arts than Af-Arts to N or eletrial stimulation of renal nerves have also been observed in rat juxtamedullary nephrons (P. K. armines, personal ommuniation) and the isolated perfused hydronephroti kidney (25). The fat that N-indued onstrition of f- Arts is weaker or at best similar ompared to Af-Arts suggests that the f-art's higher sensitivity to Ang II is not due to either strutural or physial fators, suh as its thinner arteriolar wall or lower perfusion pressure. In fat, Yuan et al. (7) have shown that reduing intraluminal pressure ofthe Af-Art from 9 to 3 mmhg did not inrease sensitivity to Ang II. They also reported that indomethain had no effet on the Af-Art, suggesting that prostaglandins are not involved in the differene in sensitivity. We have reently presented evidene that endogenous prodution ofdno is important in the ontrol ofaf-art reativity (8). We have shown that Ang II at l-7 M auses only transient onstrition of miroperfused rabbit Af-Arts, whih beomes stronger and more persistent when they are pretreated with L-NAM. These effets ofl-nam are most likely due to inhibition of DNO, sine the present study has shown that L-arginine (the preursor of DNO) but not D-arginine (an inative isomer) reverses L-NAM-indued onstrition ofthe Af-Art. We have now extended our previous findings, showing that L-NAM pretreatment inreases sensitivity of the Af-Art to Ang II at more physiologial onentrations. It is unlikely that this enhanement is due to the hange in basal tone, sine a similar redution in basal diameter with N had no effet on Ang II-indued vasoonstrition. In addition, L-NAM pretreatment did not alter Af-Art responses to N, showing that enhanement of vasoonstrition by L-NAM is not ommon to all vasoonstritors. On the other hand, L-NAM pretreatment had no effet on f-art response to Ang II whether it was studied with orthograde or retrograde perfusion. Furthermore, we found that the L-NAM-indued derease in basal luminal diameter is muh smaller in f-arts than in Af-Arts. These results suggest that in the Af-Art, endogenous DNO not only ontrols basal tone but also ounterats the vasoonstritor ation of Ang II (but not N), whereas it plays a minimal role in the f-art. Thus the differene in sensitivity to Ang II may be 216 Ito et al.

7 S.. e11 -A S e ORTHOGRAD PRFUSION 2 O Nontreoted (n-u) * L-NAM (n-7) A ontrol (n-5) A L-NAM (n-6) a Anglotenaln 11 (log Id) Figure 7. hange in luminal diameter indued by Ang II in nontreated efferent arterioles (Nontreated) and efferent arterioles pretreated with L-NAM. Note that L-NAM had no effet in either orthograde or retrograde perfusion. partially due to its signifiant interation with DNO in the Af-Art, ompared to little, if any, interation in the f-art. The reason why L-NAM had less effet on the f-art than on the Af-Art is not lear. It is not likely to be due to insuffiient blokade of DNO synthesis, sine L-NAM ompletely bloked aetylholine-indued vasodilation in the f-art. Neither is it likely to be due to arteriolar differenes in the rate of diffusion of DNO into underlying vasular smooth musle ells nor their response to DNO, sine aetylholine indues similar vasodilation in both Af-Arts and f-arts (26). However, there are several possibilities that ould explain our observation. First, it may be that the basal (unstimulated) rate of DNO synthesis was somehow higher and/or degradation lower in Af-Arts than in f-arts. Seond, there is experimental evidene that vasular smooth musle ells of the Af-Art but not f-art may possess voltage-dependent alium hannels (27, 28), and that NDO ould diretly hyperpolarize smooth musle ells of some vessels independently of a putative endothelium-derived hyperpolarizing fator (29). Thus, inhibition of DNO with L-NAM may have altered membrane poten-,. -J ~.) L F -6 F -8 F -1 F -12 I -14 L Anglotensin 11 (log M) o Nontreated (n-7) * L-NAM (n-b) Norepinephrine (log M) RMlOGRAD PRFUSION Figure 8. hange in luminal diameter indued by norepinephrine in nontreated afferent arterioles (Nontreated) and afferent arterioles pretreated with L- NAM. Note that L- NAM had no effet on N-indued vasoonstrition, in marked ontrast to enhaned Ang II ation (Figs. 5 and 6). -%._._. -J ' a, r -2-4 F -6 F -8 L o Nontreated (nil1) A N (n-l) O Angiotensin 11 (log M) Figure 9. hange in luminal diameter indued by Ang II in nontreated (Nontreated) and norepinephrine-pretreated afferent arterioles (N). Note that N had no effet on Ang II-indued vasoonstrition despite a similar derease in basal diameter. tial, preferentially affeting the Af-Art. Third, DNO has been shown to modulate myogeni ontration (3). Therefore, L- NAM may have potentiated myogeni tone in the Af-Art, but not in the f-art whih does not exhibit a myogeni response (6, 7). Finally, DNO have been shown to influene endothelial synthesis of vasoonstritors suh as endothelin (31). Thus, it may be possible that L-NAM inreased the level of vasoonstritors signifiantly in the Af-Arts but not f-arts. In our preparation of Af-Arts, the ation ofboth Ang II and endothelin (32) but not N is augmented by L-NAM. However, the reason for suh disparate effets of L-NAM is not lear. It has been proposed that yli GMP is involved in the intraellular mehanism of DNO ation (33). It is possible that in the Af-Art, the mehanism ofthe vasoonstritor ation of both Ang II and endothelin is more suseptible to modulation by intraellular yli GMP than that of N, so that a L-NAM-indued derease in the basal level of yli GMP resulted in enhaned ations of Ang II and endothelin but not N. It may also be that Ang II and endothelin stimulated DNO synthesis more than N under our experimental onditions. In addition, a reent preliminary report suggests that DNO ould displae bound endothelin from its reeptors (34), raising the possibility that DNO may have differential effets on Ang II and endothelin reeptors versus N reeptors. Several studies have reently examined the interation between Ang II and DNO in the systemi and renal irulation. De Niola et al. (35) and Sigmon et al. (36) reported that in anesthetized rats intravenous L-NAM inreased systemi blood pressure as well as systemi and renal vasular resistane. The inrease in renal vasular resistane was greatly attenuated by either an Ang II antagonist or a onverting-enzyme inhibitor (I), whereas systemi vasular resistane remained elevated, suggesting a unique interation between Ang II and DNO within the kidney. The study of De Niola et al. (35) has further shown that the interation takes plae not only in the glomerular arterioles but also at the glomerular tuft and proximal tubule. In addition, an interation between Ang II and DNO has been shown to our in the absene of systemi hemodynami hanges in onsious dogs (37). These in vivo findings, taken together with our study strongly suggest that intrarenal DNO plays an important role in the regulation of renal funtion by modulating the ations of Ang II. Nitri Oxide in Afferent and fferent Arteriole 217

8 learane studies in the whole animal showed that aute inhibition of DNO resulted in dereased renal blood flow with little hange in the GFR, suggesting preferential onstrition of the postglomerular vessel (f-art) (38, 39). onsistent with this observation, a miropunture study by Zatz and de Nui (4) showed that intravenous administration of L- NAM, whih raised mean systemi blood pressure by 3 mmhg, dereased single-nephron plasma flow but had no effet on single-nephron GFR. These hanges were assoiated with marked glomerular hypertension (73 mmhg in L- NAM-treated rats versus 5 mmhg in vehile-treated rats), and alulation of regional vasular resistane indiated a preferential effet on the f-art. Although these in vivo studies show the overall effets of DNO inhibition, various hanges in systemi hemodynamis, hormones and sympatheti nerve ativity ould have influened the renal hemodynami response. In order to avoid systemi neurohormonal and hemodynami influenes, Radermaher et al. (41 ) employed a preparation of isolated rat kidney perfused with a syntheti solution at onstant pressure. In this preparation, L-NAM dereased the GFR and inreased the filtration fration signifiantly, suggesting onstrition of both Af-Arts and f-arts. However, when red blood ells (whih ontain hemoglobin that savenges DNO) were added to the kidney perfusate, L-NAM aused a similar derease in the GFR but no hange in the filtration fration, suggesting a predominant effet on the Af-Art. These authors speulated that when the kidneys were perfused with the hemoglobin-free solution, DNO, whih was produed only in the Af-Art and/or glomerulus, reahed the f-art through the lumen, whereas addition of red blood ells prevented DNO from reahing the f-art but not from ating on the luminal side in the Af-Art and/or glomerulus. This would explain why L-NAM inreased vasular resistane in both Afand f-arts in the syntheti solution, whereas it had no effet on f-arts in the solution ontaining red blood ells. These observations are onsistent with our study that L-NAM indued a small but signifiant onstrition of the f-art with orthograde perfusion, but had no effet when possible influenes of the Af-Art and/or glomerulus was eliminated by perfusing the f-art in a retrograde diretion. In ontrast to our results showing the predominant effets of L-NAM on the Af-Art, L-NAM has been shown to produe similar dereases in the luminal diameter of both Af-Arts and f-arts in rat juxtamedullary nephrons perfused with blood in vitro (42). The dereases were markedly attenuated by pretreatment with either a onverting-enzyme inhibitor or Ang II antagonist, suggesting interation between endogenous Ang II and DNO. However, L-NAM did not augment the vasoonstrition indued by exogenous Ang II, while the sensitivity of the Af-Art and f-art to Ang II was the same in this preparation ( 17-19). The reason for the disrepanies between these studies and ours is not lear, but may be related to the perfusion solution (syntheti solution versus blood) and/or the presene or absene of maula densa-mediated tubuloglomerular feedbak. It may also be possible that in the juxtamedullary preparation, DNO produed in the Af-Art and/or glomerulus somehow reahed the f-art at onentrations high enough to exert its ation. These and previous studies provide evidene that endogenous DNO regulates Af-Art resistane by itself as well as by modulating the ations of both Ang II and endothelin (8, 32, 35, 37, 4-42). Sine the Af-Art is a site ofboth myogeni and tubuloglomerular feedbak responses as well as renin synthesis and seretion (43), alteration of DNO in the Af-Art would have profound effets on glomerular and systemi hemodynamis, either diretly or indiretly by altering renin release (9). Indeed, reent experimental evidene suggests that DNO may be involved (whether primarily or seondarily) in the altered renal hemodynamis seen in various pathologial onditions suh as hypertension and ishemi aute renal failure (44). In spontaneously hypertensive rats at a phase of established but unompliated hypertension, inhibition of DNO has been shown to ause exaggerated hypertension ompared with their normotensive ontrols, Wistar-Kyoto rats (45). We have previously shown that L-NAM dereases the luminal diameter ofmiroperfused Af-Arts to a greater degree in spontaneously hypertensive rats than in Wistar-Kyoto rats, suggesting that endogenous DNO may be high (or intat) in SHR Af- Arts (46). This ativity of DNO may help maintain glomerular filtration in the fae of various strutural and funtional alterations that favor Af-Art ontration (47, 48). On the other hand, DNO appears to be low in ishemi aute renal failure (49), whih is haraterized by profound diminution of the GFR that is disproportionate to the observed derease in renal blood flow (5). It is oneivable that low DNO may ause a preferential inrease in Af-Art resistane by itself as well as by augmenting the ations of various vasoonstritors, suh as Ang II and endothelin whih are elevated in ishemi aute renal failure. In onlusion, our observations learly demonstrate that sensitivity to Ang II is higher in the isolated miroperfused rabbit f-art than in the Af-Art, whereas L-NAM, an inhibitor of DNO synthesis, auses stronger vasoonstrition in the Af-Art than in the f-art. Furthermore, L-NAM pretreatment augments the vasoonstritor ation of Ang II in the Af- Art but not the f-art. Thus endogenous DNO would appear to modulate Ang II ation in the Af-Art but not in the f-art, whih may ontribute to the differene in sensitivity to Ang II. Aknowledgments This study was supported by National Institutes of Health grants HL28982 and HL Referenes 1. Ihikawa, I., and R.. Harris Angiotensin ations in the kidney: renewed insight into the old hormone. Kidney Int. 4: Davalos, M., N. S. Frega, B. Saker, and A. Leaf ffet of exogenous and endogenous angiotensin II in the isolated perfused rat kidney. Am. J. Physiol. 235:F65-F Ihikawa, I., J. F. Miele, and B. M. Brenner Reversal ofrenal ortial ations of angiotensin II by verapamil and manganese. Kidney Int. 16: Kastner, P. R., J.. Hall, and A.. Guyton ontrol of glomerular filtration rate: role of intrarenally formed angiotensin I. Am. J. Physiol. 246:F897-F Ihikawa, I., J. M. Pfeffer, M. A. Pfeffer, T. H. Hostetter, and B. M. Brenner Role of angiotensin II in the altered renal funtion ofongestive heart failure. ir. Res. 55: dwards, R. M Segmental effets of norepinephrine and angiotensin II on isolated renal mirovessels. Am. J. Physiol. 255:F526-F Yuan, B. H., J. B. Robinette, and J. 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