Interactive modulation of renal myogenic autoregulation by nitric oxide ...

Am J Physiol Regul Integr Comp Physiol 292: R354 –R361, 2007. First published September 21, 2006; doi:10.1152/ajpregu.00440.2006.

Interactive modulation of renal myogenic autoregulation by nitric oxide and endothelin acting through ET-B receptors Ying Shi,1,2 Catherine Lau,2,3 and William A. Cupples2,3 1

Biology Department, Concordia University, Montreál, Que´bec; 2Lady Davis Institute for Medical Research, SMBD-Jewish General Hospital, Montreál, Que´bec; and 3Centre for Biomedical Research & Biology Department, University of Victoria, Victoria, British Columbia, Canada Submitted 27 June 2006; accepted in final form 15 September 2006

Shi Y, Lau C, Cupples WA. Interactive modulation of renal myogenic autoregulation by nitric oxide and endothelin acting through ET-B receptors. Am J Physiol Regul Integr Comp Physiol 292: R354 –R361, 2007. First published September 21, 2006; doi:10.1152/ajpregu.00440.2006.—In rats, nitric oxide modulates renal autoregulation in steady-state experiments and the myogenic mechanism in dynamic studies. Interactive modulation of autoregulation by nitric oxide and endothelin-1, predominantly involving endothelin B receptors, has been reported although it remains unclear whether the interaction is synergistic or obligatory or whether it affects the myogenic component of autoregulation. Nonselective inhibition of nitric oxide synthase (L␻-nitro-L-arginine methyl-ester; L-NAME) with endothelin A and B selective receptor antagonists BQ-123 and BQ-788, all infused into the renal artery, plus time series analysis were used to test the interactive actions of nitric oxide and endothelin on renal vascular conductance and on autoregulation. Nonselective endothelin receptor antagonism blunted the constrictor response to subsequent L-NAME but had no effect on previously established L-NAME-induced vasoconstriction. BQ-123 did not affect conductance and caused only minor reduction in myogenic autoregulatory efficiency. Responses to BQ-123 and L-NAME were additive and not interactive. BQ-788 and L-NAME each caused strong vasoconstriction alone and in the presence of the other, indicating that coupling between nitric oxide- and endothelin B-mediated events is not obligatory. L-NAME augmented myogenic autoregulation, and subsequent BQ-788 did not alter this response. However, BQ-788 infused alone also enhanced myogenic autoregulation but resulted in significant impairment of myogenic autoregulation by subsequent L-NAME. Thus the interaction between nitric oxide and endothelin is clearly nonadditive and, because it is asymmetrical, cannot be explained simply by convergence on a common signal pathway. Instead one must postulate some degree of hierarchical organization and that nitric oxide acts downstream to endothelin B activation.

synthases (NOS) by N␻-nitro-Larginine derivatives causes an important and long-lasting rise in blood pressure accompanied by profound reduction of renal blood flow (RBF) (e.g., see Ref. 3) so that typically renal vascular conductance is reduced by 50 to 60%. It has been known for some time that nitric oxide is a strong modulator of renal autoregulation in rats. In classic steady-state pressure ramps, this appears as a left shift of the lower limit of autoregulation after NOS inhibition (24, 25, 32, 38). When single pressure steps were used to examine the kinetics of autoregulation, NOS inhibition enhanced the faster component

of autoregulation (i.e., the myogenic mechanism) (17). In dynamic studies the signature of NOS inhibition includes reduction of coherence at frequencies ⬍ 0.08 Hz, increased slope of gain reduction by the myogenic mechanism plus increased amplitude of the associated phase peak, and usually, increased gain reduction in the frequency band from 0.05 to 0.28 Hz (8, 14, 17, 18, 40, 41). Although the left shift of the lower limit could result from the marked rise in efferent resistance (23, 40), other results are not susceptible to this explanation (5, 11, 12, 33). Together, these studies demonstrate that nitric oxide modulates the myogenic component of renal autoregulation, and they show that the modulation can be detected by several different experimental designs. It is tacitly assumed that the left shift seen in steady-state experiments during NOS inhibition reflects the increased autoregulatory efficiency displayed by the myogenic mechanism in dynamic studies. It is also presumed that the absence of significant change in gain in the steady-state experiment (e.g., see Refs. 2, 3, and 27) reflects the difficulty of demonstrating positive modulation of the already highly efficient autoregulation. It has been reported that vascular responses to NOS inhibition are mediated by endothelin-1 (1, 31). Certainly endothelin subtype B (ET-B) receptors on endothelial cells are coupled to endothelial NOS (37), although renal vascular smooth muscle has a substantial population of ET-B receptors that mediate vasoconstriction (20). Interactive modulation of steady-state renal autoregulation by endothelin-1, mediated by the ET-B receptor and by nitric oxide has been reported. Using the steady-state pressure ramp experiment in vivo, Kramp et al. (25) explored potential interactions between endothelin-1 and nitric oxide with NOS inhibition plus ET-A and ET-B selective receptor blockers. As expected, the lower limit of autoregulation was left-shifted by N␻-nitro-L-arginine methyl ester (L-NAME). This response was abrogated by the nonselective endothelin antagonist bosentan but was not affected by blockade of ET-A alone. Blockade of ET-B receptors by BQ-788 had no effect on either the lower limit of autoregulation or on the efficiency of autoregulation at higher renal perfusion pressure. However, when L-NAME was administered in the presence of BQ-788, the efficiency of autoregulation was impaired and, in those animals in which a lower limit could be discerned, it was significantly right shifted. These results indicate a rather complex interaction between endothelin and nitric oxide, although they do not provide information concerning the location or mechanism of the interaction.

Address for reprint requests and other correspondence: W. A. Cupples, Centre for Biomedical Research, Univ. of Victoria, PO Box 3020, STN CSC, Victoria, BC, V8W 3N5 Canada (e-mail: [email protected]).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

myogenic mechanism; dynamics; L-NAME; BQ-788

INHIBITION OF NITRIC OXIDE

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0363-6119/07 $8.00 Copyright © 2007 the American Physiological Society

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NITRIC OXIDE-ENDOTHELIN INTERACTION

Both endothelin-1 and NOS inhibition (17, 18, 33, 40, 41) augment myogenic autoregulation in the renal circulation under circumstances in which the other agonist would not be expected to contribute substantially. In the hydronephrotic kidney, in vitro endothelin-1 has been demonstrated to augment myogenic autoregulation at concentrations below those required to cause vasoconstriction (22), although most studies show little effect of endogenous nitric oxide in this preparation (33, 36). In addition, there is recent evidence that neuronal NOS at the macula densa accounts for a substantial portion of the modulation of autoregulation by nitric oxide (14, 17, 33), and available evidence suggests that the macula densa is not a target of endothelin (35). Thus it is not clear how nitric oxide and endothelin-1 interactively modulate autoregulation. Oddly, most previous studies of endothelin-nitric oxide interactions have compared NOS inhibition alone with NOS inhibition in the presence of endothelin receptor blockade. This limits the conclusions that can be drawn from these studies because, if endothelin were to mediate the effects of nitric oxide, prior endothelin receptor blockade should prevent responses to subsequent NOS inhibition. The present study used receptor subtype selective antagonists with bidirectional design and assessment of RBF dynamics to test the role of endothelin in renal vascular responses to nonselective NOS inhibition. METHODS

This study was performed at the SMBD-Jewish General Hospital in Montreál and at the University of Victoria and was approved by the Animal Care Committees at both institutions, both operating under the guidelines promulgated by the Canadian Council on Animal Care. Procedures. All experiments were performed in 10- to 14-wk-old male Wistar rats obtained from Charles River (Canada). Rats had free access to water and food at all times prior to experiments. Twenty minutes prior to anesthesia, each rat received buprenorphine (Temgesic, 0.01 mg/kg ip; Reckitt and Colman Pharmaceuticals, Wayne, NJ). Anesthesia was induced by 5% isoflurane in inspired gas (30% O2-70% air). After induction, the anesthetic concentration was reduced to ⯝2%. The animal was transferred to a servocontrolled, heated table to maintain body temperature at 37°C, intubated, and ventilated by a respirator (model RSP 1002; Kent Scientific, Litchfield, CT). During the 1-h postsurgical equilibration period, inspired anesthetic concentration was titrated to the minimum concentration that precluded a blood pressure response when the tail was pinched (⯝1%). Cannulas were placed in the right femoral artery (PE-90 with narrowed tip) and vein (PE-50). A constant infusion delivered 1% body wt/h throughout the experiment and contained 2% charcoalwashed bovine serum albumin in normal saline. A fine Teflon cannula was placed in the left femoral artery and advanced into the abdominal aorta. The left kidney was approached by a flank incision, immobilized in a plastic cup, and covered with plastic wrap. The distal end of the Teflon cannula was manipulated into the renal artery after the artery was stripped from hilus to aorta. The flow probe (model 1PRB; driven by a Transonic Systems model T401 flowmeter) was placed around the renal artery; it was fixed in place, and acoustic coupling was assured by application of a gel based on surgical lubricant and NALCO 1181 as recommended (35a). Femoral arterial pressure was measured by a Kent pressure transducer (model TRN050) driven by a model TRN005 amplifier. In autoregulation experiments, a motorized clamp was placed on the aorta between the right and left renal arteries and was used to force blood pressure. The motor was driven by a program implemented in DT-VEE (Data Translation), which operates as a negative-feedback controller to maintain downstream pressure 15–20% below the sponAJP-Regul Integr Comp Physiol • VOL

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taneous level of blood pressure. It iterates at 2 Hz, and at each iteration the target pressure is randomly changed within a predefined range, typically ⫾ 5% (42). Experimental drugs were infused into the renal artery using the mixing pump reported by Parekh (30). Pilot experiments established that placing the cannula tip into the renal artery or operating the mixing pump had no discernible effect on spontaneous blood pressure or RBF. Inhibition of NOS was achieved by L-NAME (Sigma) delivered at 10 ␮g/min ⫻ 20 min, then at 3 ␮g/min for the remainder of the experiment (33). The nonselective endothelin antagonist PD-145,065 (Sigma) was infused at a nominal concentration (assuming RBF ⫽ 5 ml/min and hematocrit ⫽ 0.4) of 75 nM (9), except as noted below. The endothelin subtype A receptor (ET-A) selective antagonist BQ123 and the ET-B selective antagonist BQ-788 (Alexis Biochemicals) were each delivered at a nominal concentration of 1 ␮M (15, 16, 21, 29, 39). Preliminary experiments (n ⫽ 8) showed that intrarenal infusion of endothelin-1 (Sigma) at 5 ng 䡠 kg⫺1 䡠 min⫺1 reduced RBF by 52 ⫾ 7% and that this constriction was effectively blocked by PD-145,065 (⌬RBF ⫽ ⫺3 ⫾ 3%) and by combined infusion of BQ-123 and BQ-788 (⌬RBF ⫽ ⫹1 ⫾ 5%). Experiments. If the effects of two compounds are additive, then the response to combined application will be independent of the order in which they are applied. Dependence of the final result on the sequence of application defines an interaction between the two (6). Consequently, in the three experiments described below, the initial control period was followed in some rats first by intrarenal infusion of L-NAME and then by L-NAME plus endothelin receptor antagonist, while in other rats the endothelin receptor antagonist was infused first and then L-NAME was added. Experiment 1 tested whether the renal vasoconstrictor actions of nonselective endothelin inhibition and nonselective NOS inhibition are interactive. Because endothelin-1 dissociates slowly from its receptors, PD-145,065 was infused for at least 80 min before measurements were made. Eight rats received first L-NAME alone, then L-NAME plus PD-145,065 (75 nM). Eight rats received first PD145,065, after which L-NAME was added to the infusate. RBF was measured at the end of each interval. In four rats PD-145,065 was infused at a nominal concentration of 75 nM in renal arterial blood and in four rats at 225 nM. No differences were apparent, and the results were pooled. Experiment 2 examined the responses of RBF and of RBF dynamics to single and combined inhibition of NOS and of ET-A receptors. In 10 rats, the control period (saline vehicle) was followed sequentially by L-NAME and then by L-NAME plus BQ-123. In another seven rats, the control period was followed sequentially by BQ-123 and then by BQ-123 plus L-NAME. In each period, drugs were infused for 30 min and renal perfusion pressure was then forced for 25 min during continued infusion. Experiment 3 examined the responses of renal vascular conductance and of RBF dynamics to single and combined inhibition of NOS and of ET-B receptors. In seven rats, the control period (saline vehicle) was followed by L-NAME and then by L-NAME plus BQ788; in another six rats, the control period was followed by BQ-788 and then by BQ-788 plus L-NAME. In each period, drugs were infused for 30 min and renal perfusion pressure was then forced for 25 min. Data acquisition and analysis. The blood pressure and RBF signals were low-pass filtered at 20 Hz and sampled at 100 Hz online. Data segments of 1311 s were band-pass filtered (0.004 and 1 Hz) by using a fast-Fourier transform (FFT)-inverse FFT filter with a rectangular window and subsampled to 3.125 Hz. Power spectra, transfer functions, and squared coherences based on the FFT were computed by using standard algorithms as described previously (33, 42), using 1024-point segments shaped by the Hann window. Coherence is an index of the linear relationship between the input and output; if coherence ⫽ 1, all variation in the output variable can be explained as 292 • JANUARY 2007 •

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a linear function of variation of the input, while coherence ⫽ 0 suggests that the signals are unrelated. Gain in decibels (dB) was calculated as 20 ⫻ log (admittance magnitude/conductance). When gain ⬎ 0 pressure fluctuations are amplified into blood flow as expected from passive, compliant vessels; flow is being stabilized when gain ⬍ 0. The slope of gain reduction by a control system and the height of the associated phase peak provide information about the internal dynamics of that system. A system that responds only to the level of the input variable will typically have a slope ⯝20 dB/decade and a phase peak of ␲/4 radians (rad) at the corner frequency, whereas a system that responds to both the level and the rate of change of the input variable will have a slope ⯝40 dB/decade and a phase peak of ␲/2 rad (18, 28). Slope was determined by least squares fitting of the linear region of gain reduction, determined by inspection; the phase peak was estimated as the average phase within the same region of the spectrum. Under control conditions in normotensive rats, the slope of gain reduction is typically in the range of 20 –30 dB/decade (40). In rats anesthetized by isoflurane, the myogenic system operates at ⯝0.2 Hz, while tubuloglomerular feedback operates at 0.03– 0.05 Hz (7, 41, 42). To assess the contribution of the myogenic system to stabilization of RBF, we determined fractional compensation that is calculated from gain: fractional compensation ⫽ 1 ⫺ [10(gain/20)]. Fractional compensation ⫽ 1 implies complete autoregulation and fractional compensation ⫽ 0 indicates that autoregulation is ineffective in the frequency interval from 0.05 to 0.08 Hz that was used to minimize corruption by tubuloglomerular feedback (⬍ 0.05 Hz) and by myogenic transients (⬎ 0.08 Hz) (42). Treatment effects were assessed by two-way, repeated-measures ANOVA (Statistica, version 5.5) and completed by planned contrasts comparing consecutive periods within an experimental sequence and comparing between coincident periods in the two sequences within each experiment. The rationale for this testing procedure was twofold. First, we wished to compare responses to an agent in the absence vs. the presence of the other agent. Second, we wished to determine whether the start (control) and end (both agents) points differed between the two experimental sequences. P ⬍ 0.05 was considered to be significant. Results are presented as means ⫾ SE.

conductance. Renal perfusion pressure was not altered significantly in either sequence by L-NAME or by BQ-123. L-NAME caused profound renal vasoconstriction when infused alone and very similar vasoconstriction when infused during BQ-123 infusion. In contrast, BQ-123 did not significantly alter renal conductance when infused alone or when infused during L-NAME infusion. Figure 1 presents the RBF dynamics acquired in the BQ-123 experiments. On the left (Fig. 1, A–D) are shown the data acquired when L-NAME was infused first and BQ-123 was added; data acquired when BQ-123 was infused first and L-NAME added are shown on the right (Fig. 1, E–H). Figure 1, A and E show the power spectra of renal perfusion pressure, which did not differ among periods and were very similar in the two sequences. The discrete high-frequency peaks reflect respiratory signals from animals that breathed slowly. Coherences between renal perfusion pressure and RBF are shown in Fig. 1, B and F; coherence was high in control, was reduced in the low frequency band from 0.01 to 0.08 Hz by L-NAME in the absence (P ⫽ 1 ⫻ 10⫺5) or presence of BQ-123 (P ⫽ 0.008), and was unaffected by BQ-123 in the absence or presence of L-NAME. Admittance gains are shown in Fig. 1, C and G, and admittance phases are shown in Figs. 1, D and H. Variables extracted from these transfer functions (fractional compensation, slope of gain reduction, phase peak) are reported in Table 2. L-NAME alone (Fig. 1, C and D) increased the slope of gain reduction (shown by the dashed lines; P ⫽ 5 ⫻ 10⫺4) and the corresponding phase peak (P ⫽ 5⫻10⫺5), while L-NAME during BQ-123 infusion (Fig. 1, G and H) caused comparable increases in the slope (P ⫽ 0.003) and the phase peak (P ⫽ 6⫻10⫺7). L-NAME also increased the fractional compensation achieved by the myogenic mechanism, both alone (P ⫽ 2 ⫻ 10⫺5) and during BQ-123 infusion (P ⫽ 8⫻10⫺4). BQ-123 had lesser effects on the transfer function. When infused alone, it reduced fractional compensation (P ⫽ 0.029) but did not significantly alter the slope of gain reduction or the phase peak (both P ⬎ 0.3). When infused during L-NAME infusion, BQ-123 reduced fractional compensation (P ⫽ 0.029) but did not significantly alter the slope of gain reduction (P ⬎ 0.4) or the associated phase peak (P ⬎ 0.2). Inspection of Fig. 1 and Table 2 indicates that the effects of L-NAME and BQ-123 on RBF dynamics are independent and additive. Renal vascular responses to infusion of L-NAME and BQ-788 are presented in Table 3. Renal perfusion pressure was stable in the forward experiments (control to L-NAME to BQ-788 plus L-NAME), but in the reverse sequence, infusion of BQ-788 alone was associated with a rise of renal perfusion pressure that was maintained during subsequent L-NAME infusion. Both L-NAME

RESULTS

Table 1 shows the effects of intrarenal arterial infusion of and PD-145,065 on blood pressure, RBF, and renal vascular conductance. L-NAME was associated with a significant rise of blood pressure and induced profound vasoconstriction, although the changes in RBF and blood pressure were uncorrelated (r2 ⫽ 0.026). Subsequent prolonged infusion of PD-145,065 had no effect on blood pressure, on RBF, or on renal vascular conductance. When infused alone, PD-145,065 did not affect renal hemodynamics. Subsequent infusion of L-NAME substantially reduced RBF and conductance, although to a significantly lesser extent than when L-NAME was infused alone. Table 2 shows the effects of intrarenal arterial infusion of L-NAME and BQ-123 on renal perfusion pressure, RBF, and L-NAME

Table 1. Responses of blood pressure and conductance to nonselective endothelin receptor blockade and inhibition of nitric oxide synthesis

Blood pressure, mmHg Renal blood flow, ml/min Renal vascular conductance, ml䡠min⫺1䡠mmHg⫺1

Control

L-NAME

PD-145065 During L-NAME

Control

PD-145065

L-NAME During PD-145065

121⫾4 8.40⫾0.56 0.070⫾0.005

135⫾3* 3.84⫾0.39† 0.029⫾0.003†

134⫾2 3.98⫾0.42 0.030⫾0.003

110⫾4 8.94⫾0.89 0.083⫾0.009

115⫾4a 8.55⫾0.63c 0.075⫾0.006c

114⫾4b 5.70⫾0.52†a 0.050⫾0.005b†

Data are reported as means ⫾ SE. L-NAME, Nw-nitro-L-arginine methyl ester. Superscripts denote statistical significance: comparison with previous period in the same sequence: *P ⬍ 0.01, †P ⬍ 0.001; comparison with the same period in the reverse protocol: a P ⬍ 0.05, b P ⬍ 0.01, c P ⬍ 0.001. AJP-Regul Integr Comp Physiol • VOL

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Table 2. Responses of renal perfusion pressure, conductance, and variables extracted from the pressure-flow transfer function to selective endothelin A receptor blockade and inhibition of nitric oxide synthesis

Renal perfusion pressure, mmHg Renal blood flow, ml/min Renal vascular conductance, ml䡠min⫺1䡠mmHg⫺1 Fractional compensation, % Slope of gain reduction, dB/dec Phase peak, rad

Control

L-NAME

BQ-123 During L-NAME

Control

BQ-123

L-NAME During BQ-123

105⫾3 6.81⫾0.98 0.064⫾0.009 18⫾7 22.0⫾2.5 0.82⫾0.09

106⫾2 3.27⫾0.45‡ 0.031⫾0.004‡ 51⫾6‡ 37.8⫾2.9‡ 1.47⫾0.12‡

103⫾2 3.31⫾0.51 0.032⫾0.005 38⫾9* 35.3⫾4.9 1.33⫾0.13†

102⫾3 5.75⫾0.63 0.057⫾0.008 6⫾8 17.1⫾3.5 0.76⫾0.17

96⫾3 6.38⫾0.75a 0.068⫾0.009b ⫺9⫾8*b 14.1⫾3.4b 0.60⫾0.12b

98⫾4 3.32⫾0.57‡ 0.035⫾0.007‡ 17⫾11‡ 27.3⫾5.3† 1.09⫾0.16‡

Data are reported as means ⫾ SE. Ten rats received L-NAME before BQ-123; 7 rats received BQ-123 first. dB/dec, decibles per decade. Superscripts denote statistical significance: comparison with previous period in the same sequence: *P ⬍ 0.05, †P ⬍ 0.01, ‡P ⬍ 0.001; comparison with the same period in the reverse sequence: a P ⬍ 0.01, b P ⬍ 0.001.

and BQ-788 caused profound renal vasoconstriction, whether acting alone or in the presence of the other antagonist. Figure 2 presents the RBF dynamics acquired in the BQ-788 experiments. On the left (Fig. 2, A–D) are shown the data acquired when L-NAME was infused first and BQ-788 was added; data acquired when BQ-788 was infused first and L-NAME added are shown on the right (Fig. 2, E–H). The power spectra of renal perfusion pressure were similar among periods and between the two sequences (Fig. 2, A and E). Coherences, shown in Fig. 2, B

and F, were high in control and reduced in the low- frequency band from 0.01 to 0.08 Hz by L-NAME in the absence of BQ-788 (P ⫽ 0.001); coherence was unaffected by BQ-788 in the absence or presence of L-NAME. Admittance gains are shown in Figs. 2, C and G, and admittance phases are shown in Figs. 2, D and H. Variables extracted from these transfer functions are reported in Table 3. In the forward experiment (L-NAME first), the slope of gain reduction by the myogenic system (shown by the dashed lines) was high during the control period and consequently was

Fig. 1. RBF dynamics acquired in experiment 2 during control and intrarenal infusion of L␻-nitro-L-arginine methyl-ester (L-NAME) and BQ-123. The control record is in black, the second record is in red (L-NAME on left, BQ-123 on right), and the third record (L-NAME plus BQ-123) is in blue. A–D: data acquired when L-NAME was infused first. E–H: data acquired when BQ-123 was infused first. A and E: power spectra of renal perfusion pressure that were similar in both sequences and all periods. B and F: coherence approached unity at ⬎ 0.1 Hz under all conditions. At lower frequencies, coherence was reduced by L-NAME, but not by BQ-123. C and G: admittance gain spectra. In both cases the slope of gain reduction by the myogenic mechanism (the dashed lines) was significantly increased by L-NAME and little affected by BQ-123. Admittance phase spectra are shown in D and H. The phase peak of the myogenic mechanism was increased by L-NAME and reduced by subsequent BQ123 (D). In the reverse sequence, the phase peak was not altered by BQ-123 alone and was increased by subsequent L-NAME.

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Table 3. Responses of renal perfusion pressure, conductance, and variables extracted from the pressure-flow transfer function to selective endothelin B receptor blockade and inhibition of nitric oxide synthesis

Renal perfusion pressure, mmHg Renal blood flow, ml/min Renal vascular conductance, ml䡠min⫺1䡠mmHg⫺1 Fractional compensation, % Slope of gain reduction, dB/dec Phase peak, rad

Control

L-NAME

BQ-788 during L-NAME

Control

BQ-788

L-NAME during BQ-788

97⫾3 6.66⫾0.65 0.068⫾0.006 36⫾6 43.9⫾3.5 1.36⫾0.09

99⫾4 3.14⫾0.44‡ 0.032⫾0.005‡ 56⫾6 47.1⫾3.8 1.77⫾0.08†

101⫾3 2.19⫾0.28† 0.022⫾0.003† 61⫾4 49.6⫾6.1 1.76⫾0.12

102⫾5 7.78⫾0.58 0.076⫾0.004 34⫾9 30.1⫾3.8a 1.03⫾0.12a

108⫾3* 4.04⫾0.67‡ 0.037⫾0.006‡ 57⫾4 41.8⫾4.9 1.36⫾0.06*b

110⫾3 2.08⫾0.36‡ 0.019⫾0.003‡ 38⫾4*c 25.8⫾2.3*b 1.26⫾0.05b

Data are reported as means ⫾ SE. Seven rats received L-NAME before BQ-788 and 6 rats received BQ-788 first. Superscripts denote statistical significance: comparison with previous period in the same sequence: *P ⬍ 0.05, †P ⬍ 0.01, ‡P ⬍ 0.001; comparison with the same period in the reverse sequence: a P ⬍ 0.05, b P ⬍ 0.01, c P ⬍ 0.001.

not affected by L-NAME or by subsequent BQ-788 (both P ⬎ 0.5). L-NAME alone did, however, increase the phase peak (P ⫽ 0.007), and BQ-788 had no further effect (P ⬎ 0.8). Neither L-NAME (P ⫽ 0.082) nor subsequent BQ-788 (P ⬎ 0.3) significantly altered fractional compensation achieved by the myogenic mechanism. Although BQ-788 had little effect on RBF dynamics when administered during L-NAME infusion, it appeared to augment myogenic autoregulation when infused alone. As shown in Fig. 2H, the phase peak was increased (P ⫽ 0.03), the slope of gain reduction was numerically, although not significantly, in-

creased (P ⫽ 0.067); fractional compensation also increased numerically, although not significantly (P ⫽ 0.065). Subsequent L-NAME infusion reduced fractional compensation (P ⫽ 0.017) and the slope of gain reduction (P ⫽ 0.017) but did not significantly affect the phase peak (P ⫽ 0.3). DISCUSSION

The present study examined two renal vascular responses in which there is evidence for significant interaction between

Fig. 2. RBF dynamics acquired in experiment 3 during control and intrarenal infusion of L-NAME and BQ-788. The control record is in black, the second record is in red (L-NAME on left, BQ-788 on right), and the third record (L-NAME plus BQ-788) is in blue. A–D: data acquired when L-NAME was infused first. E–H: data acquired when BQ-788 was infused first. A and E: power spectra of renal perfusion pressure were similar in both sequences. B and F: coherence approached unity at frequencies between 0.1 and 0.5 Hz under all conditions. At lower frequencies, coherence was reduced by L-NAME, but not by BQ-788. The slope of gain reduction by the myogenic mechanism (C) was high during control and was not altered by L-NAME or by BQ-788. L-NAME modulated the myogenic autoregulation, shown by the greater reduction of gain (C) and by the increased phase peak (D). When BQ-788 was infused during NOS inhibition, it had no visible effect on RBF dynamics. However, when infused alone, BQ-788 tended to increase the slope of gain reduction (G, P ⫽ 0.067) and increased the associated phase peak (H). Subsequent L-NAME reduced the slope of gain reduction but did not reduce the phase peak.


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nitric oxide and endothelin. There is a considerable literature showing that prior administration of endothelin receptor antagonists significantly inhibits, but does not block, the vasoconstrictor response to NOS inhibition (e.g., see Refs, 1, 25, and 31). This finding has been variably interpreted to indicate that endothelin mediates the renal vascular response to NOS inhibition (1, 31) or that there is a postreceptor interaction between the two signal pathways (25). We are aware of only one previous study examining potential interactions between endothelin and nitric oxide as modulators of renal autoregulation (25), although both are well-known modulators of the myogenic component of autoregulation (11, 18, 20, 22, 40, 41). The current results lead to two major conclusions. First, the effects on vascular tone of L-NAME and endothelin receptor blockade are consistent only with a postreceptor interaction. Second, the modulation of autoregulation that involves an interaction between nitric oxide and ET-B receptors, first reported by Kramp et al. (25), is shown to occur in the myogenic mechanism. Previous studies showed that prior blockade of ET-A and ET-B receptors blunts the constriction induced by inhibition of NOS (1, 25, 31). A similar result is seen in the present study (Table 1). However, when PD-145,065 was infused after L-NAME, there was no reversal of the constriction induced by L-NAME. The dose of PD-145,065 employed was clearly adequate to prevent activation of ET receptors by exogenous endothelin-1 and was infused for ⬃90 min, presumably long enough to assure receptor occupancy by the antagonist. Failure to reverse the constriction induced by L-NAME indicates that endothelin cannot be a mediator of this response to NOS inhibition. Instead, this pattern is consistent with the presence of an interaction between nitric oxide and endothelin-1 at the postreceptor level (25). Results from experiments 2 and 3 in which the endothelin receptor subtype selective antagonists BQ-123 (ET-A) and BQ-788 (ET-B) were used with L-NAME are also consistent with interactive effects. When infused alone, BQ-123 displayed, at most, a modest contribution to vascular tone. A similarly small and variable response was also seen when BQ-123 was infused during NOS inhibition, again consistent with a relatively minor ET-A-mediated contribution to renal vascular tone. In this experiment, the combined effects of L-NAME and BQ-123 were additive and provide no evidence for an interaction between nitric oxide and ET-A-mediated events. Schmidt et al. (34) have reported in humans that BQ-123 can partially reverse renal vasoconstriction due to nonselective NOS inhibition. The time course of their experiments is such that, had this effect occurred in the present study, it would have been visible. We do not explain the apparent discrepancy, but we note it. In contrast, BQ-788 caused strong vasoconstriction whether infused alone or in the presence of L-NAME, indicating a tonic vasodilator effect of ET-B activation that is substantially independent of nitric oxide. Because nonselective endothelin receptor blockade blunted the constrictor response to L-NAME, the constrictor response to BQ-788 alone is not explicable in simply additive terms. One possible explanation for this finding arises from the fact that ET-B is a clearance receptor (10) so that BQ-788 can result in elevated circulating endothelin levels and increased occupancy of ET-A receptors. Because drugs were infused directly into the renal artery, or into ⬃10% of cardiac output, we feel it is unlikely that the concentration of AJP-Regul Integr Comp Physiol • VOL

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BQ-788 reached blocking levels in the lungs, an important site of endothelin clearance (10). We suggest instead that BQ-788induced vasoconstriction reflects an ET-A-mediated event that normally is damped by ET-B activation, consistent with the conclusion drawn by Just et al. (19). This event is unlikely to be nitric oxide-dependent because there is no interaction apparent between L-NAME and BQ-123, and because the constrictor response to NOS inhibition is not affected by BQ-788. The nature of this pathway is not clear, although it is unlikely to involve prostaglandins or an epoxygenase product (20). Although agonist-induced vasoconstriction and pressuredependent autoregulation are related phenomena, the relationship is not necessarily close. Vasopressin, ANG II, and phenylephrine can all cause substantial renal vasoconstriction; however, none of them affects kinetics or dynamics of the myogenic mechanism in vivo (17, 33, 42). In addition, both endothelin and nitric oxide modulate the myogenic mechanism under conditions where vasoconstriction is unlikely to be significant (22, 33). Consequently, a study concerning RBF does not necessarily contain implications about autoregulation and nor does an autoregulation study necessarily contain implications about the regulation of RBF. Less effective myogenic autoregulation was observed in experiment 2 (ET-A blockade) than occurred in experiment 3 (ET-B blockade). The reasons underlying the difference in the efficiency of myogenic autoregulation in the two experiments are unclear, although we have shown elsewhere that the intrarenal arterial cannula itself can impair RBF dynamics, and this may contribute to the difference (33). Parenthetically, susceptibility to manipulation is a general phenomenon, as discussed in Ref. 26. However, the expected response to L-NAME (increased gain slope, increased phase peak, increased fractional compensation) was present in both experiments when L-NAME was given alone, although somewhat obscured in experiment 3 by the unusually strong rate-sensitive component of myogenic autoregulation that was apparent during the control period. Blockade of ET-A receptors by BQ-123 caused, at most, a minor reduction in the effectiveness of myogenic autoregulation and did not interfere in any way with the response to L-NAME. Thus additive responses most simply explain the combined effects of NOS inhibition and ET-A receptor blockade on the myogenic mechanism. Previous studies have shown that ET-A inhibition, per se, did not affect autoregulation assessed by the pressure ramp experiment (4, 25). Consistent with this finding, in this present study, ET-A inhibition did not affect RBF dynamics. The steady-state experiment used previously showed that the left shift of the lower limit of autoregulation that is induced by L-NAME was preserved, although perhaps reduced in amplitude, in the presence of ET-A inhibition (25). The time series analysis used here detected that NOS inhibition augmented the myogenic mechanism both in the absence and in the presence of BQ-123. Thus both experimental designs show pronounced modulation of autoregulation by NOS inhibition but little effect on autoregulation of ET-A inhibition or interaction between the two. A more complex picture emerges from combined NOS inhibition plus ET-B receptor inhibition by BQ-788. Both NOS inhibition and ET-B inhibition by BQ-788 modulated the myogenic mechanism. In the case of NOS inhibition, this is the expected response (17, 33, 40), while in the case of ET-B inhibition, it can be explained as an inhibition of endothelial 292 • JANUARY 2007 •

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NOS coupled to ET-B. Blockade of ETB receptors in the presence of L-NAME had no further effect on the myogenic mechanism, most likely because no further modulation was possible. Importantly, however, there was no impairment of the myogenic mechanism in this experimental sequence. In contrast, NOS inhibition after ET-B blockade caused negative modulation of the myogenic mechanism, consistent with the impaired autoregulation seen by Kramp et al. (25). The prima facie explanation of this result would be that removal of ET-B influence exposes an action of ET-A signaling that impairs the myogenic mechanism (and autoregulation generally). However, such an interpretation is not consistent with the results of the experiment in which ET-A receptors were blocked, or with previously published studies (25). We do not have an entirely satisfactory explanation for this rather complex interaction, although several conclusions can be drawn. In our experiments, as in those of other laboratories (19, 25), it is clear that individual effects of ET-A and ET-B selective antagonists, when added together, are not the same as those of nonselective endothelin antagonists. This is true for effects on vascular tone (19, 25, and present results), for autoregulation (25), and for the myogenic mechanism (present results). In each case, it is also difficult to parse the interaction between endothelin blockade and NOS inhibition. Both for vasoconstriction and for modulation of the myogenic mechanism the interaction between nitric oxide and endothelin is clearly nonadditive (i.e., nonlinear) and because it is asymmetrical, cannot be explained simply by convergence on a common signal pathway. Instead, one must postulate that there is some degree of hierarchical organization and that nitric oxide acts downstream to ET-B activation. As discussed by Just et al. (19) in considerable detail, all of these results strongly suggest postreceptor interactions not only between ET-A- and ET-Bmediated events, but also between these and nitric oxidemediated events. In summary, the current results are largely consistent with, and provide an explanation for, the finding of Kramp et al. (25) that combined NOS inhibition and ET-B receptor blockade impairs autoregulation. They localize the effect to the myogenic mechanism and show that, like the vasoconstrictor interaction, it is asymmetrical; while prior ET-B receptor blockade alters the autoregulatory response to NOS inhibition, subsequent ET-B receptor blockade does not affect augmentation of myogenic autoregulation by NOS inhibition. ACKNOWLEDGMENTS Present address of Y. Shi: Pulmonary Medicine and Critical Care, Brigham and Women’s Hospital at Harvard Medical School, Boston, MA 02115. GRANTS This study was funded by Grants in Aid (to W. A. Cupples) from the Canadian Institutes for Health Research and from the Kidney Foundation of Canada. REFERENCES 1. Banting JD, Friberg P, Adams MA. Acute hypertension after nitric oxide synthase inhibition is mediated primarily by increased endothelin vasoconstriction. J Hypertens 14: 975–981, 1996. 2. Baumann JE, Persson PB, Ehmke H, Nafz B, Kirchheim HR. Role of endothelium-derived relaxing factor in renal autoregulation in conscious dogs. Am J Physiol Renal Fluid Electrolyte Physiol 263: F208 –F213, 1992. AJP-Regul Integr Comp Physiol • VOL

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