Redundant Signaling Mechanisms Contribute to the Vasodilatory ...

Articles in PresS. Am J Physiol Renal Physiol (August 24, 2004). doi:10.1152/ajprenal.00194.2004

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Redundant Signaling Mechanisms Contribute to the Vasodilatory Response of the Afferent Arteriole to Proteinase Activated Receptor-2 (PAR2)

Xuemei Wang , Morley D. Hollenberg and Rodger Loutzenhiser Smooth Muscle Research Group Department of Pharmacology and Therapeutics University of Calgary

Running Tile: PAR2 response of the afferent arteriole

Address correspondence to: Dr. Rodger Loutzenhiser Department of Pharmacology and Therapeutics University of Calgary, Faculty of Medicine 3330 Hospital Drive N.W Calgary, Alberta, T2N 4N1 Canada Telephone: (403) 220 8862 Fax: (403) 270 2211 E-mail: [email protected]

Copyright © 2004 by the American Physiological Society.

2 Abstract We previously demonstrated that stimulation of proteinase-activated receptor 2 (PAR2) by SLIGRL-NH2 elicits afferent arteriolar vasodilation, in part, by elaborating NO suggesting an endothelium-dependent mechanism. In the present study we characterized the NO-independent component of this response, using the in vitro perfused hydronephrotic rat kidney. SLIGRL-NH2 (10 Fmol/L) dilated afferent arterioles pre-constricted with angiotensin II and the initial transient component of this response was resistant to NO synthase (NOS) and cyclooxygenase inhibition. This NO-independent response was not prevented by treatment with 10 nmol/L charybdotoxin and 1 :mol/L apamin, a manipulation that prevents the EDHF-like response of the afferent arteriole to acetylcholine, nor was it blocked by the addition of 1 mmol/L tetraethylammonium (TEA) or 50 :mol/L 17-octadecynoic acid, treatments that block the EDHF-like response to bradykinin. To determine if the PAR2 response additionally involves the electrogenic Na+/K+ ATPase, responses were evaluated in the presence of 3 mmol/L ouabain. In this setting SLIGRLNH2 induced a biphasic dilation in control and a transient response following NOS inhibition. The latter was not prevented by charybdotoxin plus apamin or by TEA alone, but was abolished by combined treatment with charybdotoxin, apamin and TEA. This treatment did not prevent the NO-dependent dilation evoked in the absence of NOS inhibition. Our findings indicate a remarkable redundancy in the signaling cascade mediating PAR2 -induced afferent arteriolar vasodilation, suggesting an importance in settings such as inflamation or ischemia, in which vascular mechanisms might be impaired and the PAR system is thought to be activated.

Key words: nitric oxide; SLIGRL-NH2; K channels; ouabain-sensitive Na+/K+ATPase; tetrathylammonium; apamin; charybdotoxin; 17-octadecynoic acid, protease

3 Introduction Proteinase activated receptors (PARs) are a novel class of G-protein-linked receptors that are activated by proteolytic unmasking of a tethered ligand amino acid sequence within the amino-terminal domain (6,18). Specific tethered ligand sequences have been identified for each of the four family members (PAR1-4) (18, 27, 36). PAR2 is unique in that it is activated by trypsin, but not by thrombin (18, 27) and is thought to play a prominent role in physiologic or pathophysiologic processes such as inflamation (reviewed in 5, 36,42). The synthetic peptide SLIGRL-NH2, based on the rodent PAR2 tethered ligand sequence, has been found to be a highly selective PAR2 agonist that can activate PAR2 with the absence of proteolysis and without affecting other receptor systems (18). The role of PAR2 in the kidney is largely unknown, but this area is of considerable interest as the expression levels of PAR2 in the kidney are particularly high (3, 35). PAR2 is expressed in renal epithelia, and has been shown to activate a chloride conductance in cortical collecting duct cells (2). However, PAR2 is also highly expressed in renal vascular endothelial cells and vascular smooth muscle cells (2). Previous studies from our laboratory using the isolated perfused rat kidney model (15) and the in vitro perfused hydronephrotic rat kidney preparation (41) have demonstrated a potential role for PAR2 in the regulation of renal hemodynamics, as PAR2 activation in these preparations exerts a potent renal vasodilatory response. PAR2 is suggested to play a prominent role in the cardiovascular system, impacting on tissue perfusion and angiogenesis (30,31). Both trypsin and the selective PAR2-activating peptide SLIGRL-NH2 induce endothelium-dependent vasodilation in isolated rat aorta, mouse mesenteric, and renal arteries (21, 29, 39). In some, but not all vessels, a component of the endothelium-dependent response is resistant to treatment with cyclooxygenase (COX) and nitric

4 oxide synthase (NOS) inhibition, prompting speculation that PAR2 activation causes the release of an endothelium-dependent hyperpolarizing factor (EDHF) (15, 21, 29, 30, 41). In the isolated perfused rat kidney both SLIGRL-NH2 and trypsin elicit renal vasodilation that is only partially attenuated by COX and NOS inhibition (15). Using the isolated perfused hydronephrotic rat kidney preparation we have previously shown that the NO-independent afferent arteriolar dilation evoked by SLIGRL-NH2 is prevented by elevated extracellular K+ consistent with the proposed role of an EDHF (41), but also consistent with a direct smooth muscle action. Recent findings from our laboratory indicate that multiple pathways contribute to the afferent arteriolar responses attributed to EDHF, in that we observed distinct EDHF-like responses of this vessel to acetylcholine and bradykinin (43, 44). The EDHF-like response to acetylcholine is fully abolished by the combined administration of charybdotoxin plus apamin (43), a treatment which eliminates EDHF responses in a wide variety of vessel types (reviewed in 11, 28). However, we found that this treatment, though necessary, was not sufficient to abolish the EDHF-like response of the afferent arteriole to bradykinin (44). To eliminate the bradykinin response it was necessary to use a combination of charybdotoxin and apamin plus either 1 mmol/L tetraethylammonium (TEA) or 50 :mol/L 17-octadecynoic acid (17-ODYA) (44). Either treatment alone, was ineffective. These findings are a clear indication of the complex nature of the responses attributed to EDHF in the afferent arteriole. The present study was undertaken to characterize the NO-independent vasodilator actions of PAR2 activation on the afferent arteriole. Since, in other vascular beds, this NO-independent component is endothelium-dependent and has been ascribed to and EDHF, we anticipated that the characteristics of this response might be similar to that previously reported for acetylcholine or

5 bradykinin. Our findings were surprising in that the characteristics of the NO-independent response to SLIGRL-NH2 were clearly distinct from the EDHF pathways associated with either acetylcholine or bradykinin. Moreover, our study revealed that a remarkably redundant and complex mechanism mediates the afferent arteriolar vasodilation evoked by PAR2 activation. The reason for this redundancy is not readily apparent, but this finding may implicate an underlying importance in settings where normal vascular responses might be impaired.

Methods The effects of the PAR2-activating peptide agonist SLIGRL-NH2 on the renal afferent arterioles was investigated in vitro perfused hydronephrotic kidney. The left ureters of 6-7 week old (80-100 gram) male Sprague Dawley rats were ligated under halothane-induced anesthesia to induced hydronephrosis in order to facilitate direct observations of the afferent arterioles. After 68 weeks the hydronephrotic kidney was harvested. The left renal artery was cannulated in situ and the kidney was excised and transferred to a heated chamber on the stage of an inverted microscope, without disrupting perfusion. The renal perfusate consisted of Dulbecco’s modified Eagle’s Medium (Sigma, DME) Base containing 30 mmol/L bicarbonate, 5 mmol/L glucose, and 5 mmol/L HEPES. The perfusate was equilibrated with 95% air / 5% CO2. Temperature and pH were maintained at 37o C and 7.40 respectively. Medium was pumped on demand through a heat exchanger to a pressurized reservoir connected to the renal arterial cannula. Perfusion pressure was monitored within the renal artery (26) and maintained at 80 mmHg in all experiments. Kidneys were allowed to equilibrate for at least one hour before study. A fibre optic probe was used to stabilize and transilluminate a portion of the membranous renal cortex for observations of renal microvascular responses. Afferent

6 arteriolar diameters were measured by on-line image processing (25). Afferent arteriolar diameter measurements were obtained at each pixel and were averaged over the entire segment length (~ 20 microns). These data were collected at a scanning rate of ~3 Hz. Mean diameters, thus obtained, over the plateau of the response were then averaged for each experimental point. The synthetic PAR2-activating peptide SLIGRL-NH2 , >95% pure by HPLC and mass spectral criteria, was prepared by the peptide synthesis facility at the University of Calgary ([email protected]). Stock solutions of SLIGRL-NH2 were prepared in 25 mmol HEPES, pH 7.40, and concentrations were verified by quantitative amino acid analysis. Stock solutions of 17octadecynoic acid (17-ODYA, obtained from Sigma, St Louis, MO) were prepared in ethanol. Freshly prepared stock solution of angiotensin II , L-NAME, TEA, charybdotoxin, and apamin (all obtained from Sigma) were prepared with sterile water. Inhibition of the rat Na+/K+ ATPase isoform requires high concentrations of ouabain. For these experiments, 3 mmol/L ouabain (Sigma) was prepared by direct addition to the perfusate. All reagents were added to the perfusate which emptied through the renal vein into the tissue bath. Accordingly, all agents reach both adluminal and abluminal vessel surfaces. Experiments employing apamin, charybdotoxin, and SLIGRL-NH2 required the use of a recirculating perfusion system to conserve reagents. Antibiotics (penicillin/ streptomycin, Invitrogen, Carlsbad, CA) were added to the recirculating perfusate in these experiments. Kidneys were perfused using a single-pass system and switched to the recirculating system when these agents were employed. Finally, in addition to causing smooth muscle membrane depolarization by inhibition of the electrogenic Na/K ATPase, ouabain can evoke the release of neurotransmitters by a similar action on nerves. Therefore, in all experiments using ouabain, 10 :mol/L phentolamine and 10 :mol/L propranolol (Sigma) were added to the perfusate.

7 All values are presented as the mean ± SEM. Differences between treatment groups were evaluated by one way ANOVA and Bonferroni’s t-test, when multiple comparisons were evaluated. Differences exhibiting P values less than 0.05 were considered to be statistically significant.

Results Effects of PAR2 agonists SLIGRL-NH2 and trypsin on afferent arteriole during angiotensin II induced vasoconstriction To assess the characteristics of vasorelaxation elicited by PAR2 activation SLIGRL-NH2 (10 :mol/L) was administered to control kidneys and to kidneys that had been pre-treated with 10 :mol/L ibuprofen and 100 :mol/L L-NAME. Afferent arteriolar tone was established by pre-

constricting the afferent arteriole with 0.1 nmol/L angiotensin II (Ang II). In the controls, SLIGRL-NH2 elicited a biphasic response, characterized by an initial transient component and followed by a reduced but sustained vasodilation. As shown in the tracing depicted in figure 1A, Ang II reduced afferent arteriolar diameter from 20 to 8 :m and SLIGRL-NH2 fully restored diameter at the initial peak response (20 :m). The diameter, spontaneously returned to 16 :m over the next several minutes. As shown in figure 1B, SLIGRL-NH2 elicited only a transient response following pre-treated with 100 :mol/L L-NAME plus 10 :mol/L ibuprofen,. Thus in this tracing Ang II reduced afferent arteriolar diameter from 16 to 4 :m and SLIGRL-NH2 caused a transient increase in diameter to 13 :m which returned to 4 :m within 3-5 minutes. Figures 2A&B illustrate tracings from similar experiments assessing responses to trypsin (2 nmol/L). In figure 2A, Ang II reduced afferent arteriolar diameter from 17 to 6.5 microns. Trypsin caused a rapid vasodilation to 16 microns which persisted for approximately 5 minutes. Proteolytic

8 activation of the PAR2 receptor, as would occur with trypsin, but not SLIGRL-NH2, is associated with rapid receptor internalization (see 36), perhaps contributing to the transient nature of the response to this agonist. In the presence of COX/NOS inhibition (figure 2B), the response to trypsin was much more transient. In this tracing, Ang II reduced diameter from 20 to 5 :m and trypsin caused a transient increase to 14 :m which rapidly returned to 5 :m. The persistence of a transient vasodilatory response to PAR2 activation in the presence of NOS and COX blockade is consistent with previous reports. We had previously shown that this NO-independent component is blocked by KCl-induced depolarization and others have attributed such responses to an EDHF (15, 29, 30, 41). We have observed NO-independent afferent arteriolar responses to both acetylcholine and bradykinin that exhibit similar temporal characteristics (43,44). In the case of acetylcholine, this EDHF-like response was fully abolished by combined treatment with charybdotoxin plus apamin (43), whereas with bradykinin, charybdotoxin plus apamin and the addition of either TEA or 17-ODYA was required to fully abolish the response (44). However, as shown in figures 3A&B, these treatments did not eliminate the NO-independent response to SLIGRL-NH2. These studies were conducted in the presence of COX and NOS blockade (100 :mol/L L-NAME plus 10 :mol/L ibuprofen). Kidneys were treated with 10 nmol/L charybdotoxin, 1 :mol/L apamin and either 1 mmol/L TEA (figure 3A) or TEA plus 50 :mol/L 17-ODYA (figure 3B). Afferent arteriolar tone was increased by the administration of 0.1 nmol/L Ang II. As shown, neither of these treatments was capable of eliminating the response to SLIGRL-NH2, though each treatment attenuated the response. In concert with our previous observations (43,44), these findings suggest that the NO-independent response to SLIGRL-NH2 involves a separate or an additional mechanism which is not seen with either acetylcholine or bradykinin.

9 To determine if these unusual characteristics of the NO-independent response to SLIGRLNH2 were dependent on the condition that basal afferent arteriolar tone was established by angiotensin II, we performed additional experiments using either pressure or barium to establish basal tone. In each case, SLIGRL-NH2 evoked similar responses. For these studies kidneys, were pre-treated with a "cocktail" containing 100 :mol/L L-NAME, 10 :mol/L ibuprofen, 10 nmol/L charybdotoxin, 1 :mol/L apamin and 1 mmol/L TEA. Figure 4 illustrates the response obtained when elevated renal arterial pressure (RAP) was used to establish tone. The administration of the cocktail described above reduced diameters from a control of 18.7 ± 1.5 :m to 16.4 ± 1.4 :m. In this setting, elevating RAP from 80 to 160 mmHg reduced diameters to 7.8 ± 1.2 :m. The administration of 10 :mol/L SLIGRL-NH2 evoked a transient increase in diameter to 13.6 ± 1.2 :m (P=0.001, n=4), which spontaneously abated as diameters returned to 7.4 ± 1.2 :m. Similar

results were obtained when barium was used to establish basal tone. In this series diameters were 17.5 ± 1.4 :m in the control state and 16.5 ± 1.2 :m following the exposure to the cocktail of blockers. Barium (100 :mol/L) constricted the arterioles to 9.0 ± 2.1 :m and the administration of 10 :mol/L SLIGRL-NH2 elicited a transient increase in diameter to 12.9 ± 2.2 :m (P=0.015, n=4). Diameters spontaneously returned to 8.3 ± 1.8 :m in the continued presence of the PAR2 agonist. Thus under conditions in which basal afferent arteriolar tone was established by either angiotensin II, elevated pressure, or barium-induced depolarization (see ref. 4), SLIGRL-NH2 elicited a residual vasodilation in the combined presence of L-NAME, ibuprofen, charybdotoxin, apamin and TEA. Role of ouabain-sensitive pump in NO-independent response to SLIGRL-NH2 The literature suggests that in addition to mechanisms sensitive to the administration of K channel blockers or inhibition of cytochrome P450, some EDHF-like responses are blocked by

10 ouabain (reviewed in 11,28), suggesting an involvement of the electrogenic Na+/K+ ATPase. We therefore examined whether ouabain would affect the NO-independent responses induced by PAR2 activation. We had previously shown that ouabain elicits afferent arteriolar vasoconstriction (4), so in these experiments ouabain (3 mmol/L) was administered to elicit afferent arteriolar tone and the effects of SLIGRL-NH2 were evaluated. In this setting, SLIGRL-NH2 evoked a biphasic vasodilation in controls and a transient response in the presence of COX/NOS blockade (figures 5 A and 5B, left panels). The magnitude of the initial response was not altered by 100 :mol/L LNAME plus10 :mol/L ibuprofen. As shown in figure 5A (right panel), ouabain reduced afferent arteriolar diameters from 18.8 ± 0.8 :m to 4.9 ± 0.7 :m in controls and SLIGRL-NH2 returned diameters to 18.0 ± 1.1 (n=6). In a separate series of kidneys pre-treated with L-NAME and ibuprofen (figure 5B, right panel), ouabain reduced afferent arteriolar diameter from 18.1 ± 0.8 to 5.5 ± 0.7 :m and SLIGRL-NH2 increased diameters to 16.1 ± 0.3 (n=5). The corresponding vasodilation expressed as percent were 94 ± 4% in controls and 84 ± 5% following L-NAME (P=0.14). We next examined the effects of K channel blocking agents on the NO-independent response elicited by SLIGRL-NH2 in the presence of ouabain. As depicted in figure 6A, the combination of charybdotoxin plus apamin, did not prevent the response to SLIGRL-NH2. In these studies ouabain reduced afferent arteriolar diameters from 17.7 ± 0.8 to 6.6 ± 1.3 µm (P=0.0002) and SLIGRL-NH2 returned diameters to 12.3 ± 1.6 Fm (P=0.025 versus control, n=5). Similarly, the administration of TEA alone was not sufficient to block this response. Thus, as shown in figure 6B, in TEA-treated kidneys ouabain reduced afferent arteriolar diameters from 20.0 ± 1.0 to 6.9 ±1.0 :m (P=0.0004) and SLIGRL-NH2 returned the diameters to 14.2 ± 1.3 :m (P=0.001, n=6). Thus neither charybdotoxin plus apamin nor TEA was capable of preventing the

11 NO-independent response to SLIGRL-NH2 when added alone, in this setting. However, when added together in the presence of ouabain the combination of TEA, charybdotoxin, and apamin completely abolished this response. As depicted in figure 7A, in the presence of these blockers, ouabain reduced afferent arteriolar diameters from 18.7 ± 0.8 to 5.2 ± 0.7 :m and the subsequent administration of SLIGRL-NH2 had no effect (6.0 ± 0.7 Fm, P=0.40, n=7). We had previously shown that the EDHF-like response of the afferent arteriole to bradykinin could be prevented by the combination of charybdotoxin and apamin plus either TEA or the cytochrome P450 inhibitor, 17-ODYA (50 :mol/L) (44). However, as shown in figure 7, 17-ODYA did not mimic the inhibition by of TEA of the response to SLIGRL-NH2. In the presence of ibuprofen and L-NAME, charybdotoxin, apamin and 17-ODYA, ouabain reduced afferent arteriolar diameters from 18.1 ± 0.4 to 4.7 ± 0.3 Fm. In this setting, SLIGRL-NH2 increased the diameter to 11.1 ± 1.3 Fm (P=0.0001 vs. ouabain, figure 6B) suggesting that cytochrome P450 products, such as an ecosatetraenoic acid (EET), do not appear to mediate the TEA-sensitive component of the NO-independent response to SLIGRL-NH2. The above observations indicated that a combination of ouabain, charybdotoxin, apamin and TEA fully blocked the response of the afferent arteriole to SLIGRL-NH2 in the presence of NOS blockade. We next examined the effects of this treatment regime on the response when NOS was not inhibited. The results of these experiments are presented in figure 8. In kidneys that had been pre-treated with the combination of TEA, apamin plus charybdotoxin, ouabain reduced afferent arteriolar diameters from 19.8 ± 0.3 to 8.7 ± 1.0 Fm. In this setting, SLIGRLNH2 returned the diameters to 14.0 ±1.2 Fm (P=0.036 vs. ouabain alone, n=4). Thus, this treatment did not prevent the NO-dependent response to the PAR2 agonist. Figure 9 summarizes the studies described above examining the effects of various

12 treatments on the SLIGRL-NH2-induced vasodilation in afferent arterioles constricted in the presence of ouabain. To facilitate comparisons, the data are expressed as the percent dilation of ouabain-induced vasoconstriction. As illustrated, the only combination of treatments that fully abolished the response to SLIGRL-NH2 was L-NAME, ouabain, charybdotoxin, apamin, and TEA (7 ± 3%, P=0.40 vs. ouabain alone). All other combinations attenuated the vasodilation (TEA, charybdotoxin, apamin, without L-NAME 52 ± 9%; L-NAME plus: TEA 57 ± 11%, charybdotoxin and apamin 53 ± 6%, charybdotoxin, apamin and 17-ODYA 51 ± 11%), but did not fully abolish the response. Discussion The present study demonstrates the complex nature of the mechanisms mediating PAR2 induced afferent arteriolar vasodilation. A component of this response appeared to be endothelium-dependent in that it was prevented by L-NAME, suggesting an involvement of endothelium-derived NO. A second component was prevented by combined treatment with apamin plus charybdotoxin, a manipulation that has been shown to block endothelium-dependent hyperpolarization in other vessel types (see 28 for review). The remaining components were sensitive to TEA and ouabain, and could reflect either additional endothelium-dependent mechanisms or direct actions of PAR2 activation on the underlying smooth muscle. Within the circulatory system, PAR2 is expressed on the endothelium and on vascular smooth muscle myocytes (7). In a number of vascular preparations, PAR2 induced vasodilation is eliminated by removal of the endothelium (21, 29, 30, 33, 39). Unfortunately we cannot remove the endothelium in our preparation without affecting vascular reactivity. L-NAME prevented the sustained phase of the afferent arteriolar response to PAR2 stimulation, and the NO-independent component that remained was transient in character, was not blocked by

13 cyclooxygenase, but was abolished by depolarizing concentrations of extracellular K+. These characteristics are similar to responses attributed to EDHF in other preparations (21, 29, 33, 41). There is limited information on the nature of the EDHF involved in the responses of other vessel types to PAR2 agonists. Nakayama et al. (33) found that in the porcine coronary artery, the EDHF-like response to trypsin was fully abolished by the combined treatment with charybdotoxin plus apamin. Similarly, Kawabata et al. (21) found the combination of charybdotoxin plus apamin to abolish the NO-independent dilator effects of SLIGRL-NH2 on gastric mucosal blood flow in the rat.

McGuire et al (29) also found this treatment to abolish the EDHF-like response

to SLIGRL-NH2 in the mouse mesenteric arteriole. However, these authors also found the combination of ouabain and barium (30 :mol/L) to attenuate this response. In the latter study, TEA, iberiotoxin and cytochrome P450 inhibition all had no effect. In contrast, McLean et al (30) found that the NO-independent component of the response of the isolated perfused rat heart to SLIGRL or trypsin could be attenuated by TEA, charybdotoxin plus apamin, eicosatetraynoic acid, nordihydroguaiaretic acid, baicalein, capsaicin and capsazepine, suggesting a complex and redundant signaling pathway involving K+ channels, lipoxygenase-derived ecosanoids and vanilloid receptors. Thus, while some common aspects of the PAR2-induced EDHF-like responses are apparent, for example the charybdotoxin/apamin-sensitive component, it appears that responses observed in different vascular beds display distinct characteristics. We have previously characterized the nature of the EDHF-like responses of the afferent arteriole to acetylcholine (43) and bradykinin (44) and these findings along with the present results are summarized in figure 10. Each study employed the in vitro perfused hydronephrotic rat kidney model under identical experimental conditions, in which basal tone was established with angiotensin II. The open bars depict the peak dilations elicited under control conditions

14 while the closed bars depict peak responses following inhibition of NOS and COX. Each vasodilator elicited a transient response under the latter conditions and, in each case, the peak dilations were similar to the peak responses seen in controls. Moreover these transient dilations were fully blocked by elevated extracellular potassium (16, 41, 44), consistent with the possible involvement of an EDHF. A common feature of all three agents was that a component of the dilation was blocked by treatment with apamin plus charybdotoxin (figures 9 & 10). We and others have suggested that this apamin/charybdotoxin-sensitive mechanism involves the activation of small (SKCa) and intermediate (IKCa) Ca-activated K channels that are located on the endothelium and that evoke smooth muscle hyperpolarization via myoendothelial gap junctions (e.g., 9, also discussed in 11, 28). However, as further shown in figure 10, there are marked differences in other aspects of the NO-independent responses to these three agents. The response to acetylcholine was fully abolished by treatment with apamin plus charybdotoxin, whereas this treatment alone did not prevent the EDHF-like response to bradykinin. Rather, the bradykinin response involves one component that is blocked by charybdotoxin plus apamin and a second which is prevented by either TEA (1 mmol/L) or with 17-ODYA (further discussed in 44). The NO-independent response to SLIGRL-NH2 differed from that of either acetylcholine or bradykinin, in that it persisted in the combined presence of charybdodoxin, apamin, TEA and 17ODYA, indicating an additional mechanism that is not evoked by either of these agents. As shown in figure 9, this additional mechanism was prevented by ouabain. Ouabain, which blocks the electrogenic Na+/K+ ATPase, has been shown to inhibit responses attributed to EDHF (reviewed in 11, 28) including those evoked by PAR2 (e.g. 29). In the present study we found that while ouabain fully prevented the NO-independent response to PAR2 stimulation in the combined presence of TEA, apamin and charybdotoxin (figure 7, figure 9), this treatment did not

15 prevent the NO-dependent response seen in the absence of L-NAME (figure 8), suggesting a specificity of action, rather than a general suppression of PAR2 activation. We cannot ascertain from our studies if the ouabain-sensitive component of the PAR2 induced afferent arteriolar dilation is endothelial-dependent. However, mechanisms linking activation of the overlying endothelium to a stimulation of smooth muscle Na+/K+ ATPase have been suggested. Edwards et al. (10) suggested that K+ efflux from the overlying endothelium in response to the activation of SKCa and IKCa causes an elevation of extracellular K+ near the sarcolemma of the underlying smooth muscle myocytes and that this elevation in K+ elicits hyperpolarization and vasodilation by stimulating the electrogenic Na+/K+ ATPase. While this hypothesis might seem an attractive means of explaining the involvement of the ouabain-sensitive component of actions of PAR2 activation there are a number of discrepancies that suggest this is not the case. First, while elevated extracellular K+ does indeed elicit afferent arteriolar vasodilation, we have previously shown that this response is not prevented by ouabain, but rather is fully abolished by barium (4), suggesting that the dilation is mediated primarily by alterations in the inward rectifying K channel (KIR). Thus the properties of the response elicited by SLIGRL-NH2 are quite distinct from that induced by elevations in extracellular K+. Secondly, we observed that although the responses elicited by acetylcholine, bradykinin and SLIGRL-NH2 were all sensitive to charybdotoxin and apamin, indicating an involvement of SKCa and IKCa, only the response to SLIGRL-NH2 was sensitive to ouabain. If K+ efflux via SKCa and IKCa stimulates the electrogenic Na+/K+ ATPase why would not all three of these agents exhibit a component that is ouabain-sensitive? Finally, it should be noted that the proposed role of K+ as an EDHF remains controversial and many observations underlying this premise are not confirmed by others (reviewed in 28), even by laboratories using the same vascular preparation (e.g., 1). A study by

16 Pratt et al. (37) suggests an alternate mechanism whereby a vasodilator agent acting on the endothelium might cause a stimulation of the electrogenic Na+/K+ ATPase. These investigators found that ouabain inhibited both bradykinin- and 14,15 EET-induced dilation of the bovine coronary artery and suggested that endothelium-derived EETs act via a ouabain-sensitive mechanism in this vessel. However as shown in figure 3B, we found that 17-ODYA, which would inhibit EET formation, did not prevent the ouabain-sensitive component of the afferent arteriole to SLIGRL-NH2. It is equally possible that the ouabain-sensitive component of the PAR2-induced response is not endothelium-dependent, but rather involves a direct action mediated by PAR2 receptors located on the afferent arteriolar myocyte. For example, studies assessing the effects of adenosine on endothelium-denuded aorta indicate a direct vasorelaxant effect that is ouabainsensitive (14). Similarly, the relaxant effects of isoproterenol on isolated detrusor smooth muscle cells (32) and the effects of bradykinin on cultured tracheal smooth muscle myocytes (8) have both been suggested to involve a stimulation of smooth muscle Na+/K+ ATPase via mechanisms that clearly could not involve the endothelium. Future studies assessing the expression of PAR2 receptors on afferent arteriolar myocytes and studies examining the direct effects of PAR2 activation on these cells would be of interest in this regard. We found TEA was required to completely abolish the response to PAR2 (figure 9). This TEA-sensitive component could also involve either a direct action or an endothelium-dependent mechanism. We found TEA to similarly inhibit a component of the afferent arteriolar response to bradykinin and this action was mimicked by 17-ODYA (figure 10). Several studies have implicated EETs in the EDHF-like response to bradykinin (12, 13, 19) and the vasodilator actions of 11,12 EET on the afferent arteriole are blocked by 1 mmol/L TEA (20, 45). Thus, a model in

17 which bradykinin stimulates the release of an EET, whose formation can be prevented by 17ODYA and whose smooth muscle vasodilator actions can be blocked by TEA could explain the bradykinin response (see 44 for further discussion). In the present study, we found that TEA also blocks a component of the NO-independent response to SLIGRL-NH2. However, unlike our observations with bradykinin, 17-ODYA had no effect on the PAR2 response (figure 7). Accordingly, TEA must be affecting another signaling pathway. In this regard it should be noted that, at the concentration used in our studies (1 mmol/L), TEA selectively blocks large conductance Ca activated K channels (BKCa) (23) and that a variety of vasodilatory agents, other than EETs, have been demonstrated to act by altering the activity of BKCa (reviewed in 40). Thus the TEA-sensitive component may involve a mechanism whereby activation of PAR2 receptors, located either on the myocyte or on the endothelium, is linked to a stimulation of smooth muscle BKCa channels. The role of NO in the response of the afferent arteriole to PAR2 activation merits further discussion. As seen with other vessels, it appears that NO-independent mechanisms, contribute predominantly to the initial phasic component of the response. The sustained component is fully blocked by L-NAME, reflecting an obligate role of sustained NO formation (figure 1). However, as shown in figures 9 & 10, when exposed to the combination of ouabain, TEA and charybdotoxin plus apamin, SLIGRL-NH2 elicited a transient NO-dependent afferent arteriolar vasodilation. We observed similar transient NO-dependent responses to both acetylcholine (43) and bradykinin (44) when the EDHF component of each agent was blocked (see figure 10). Why does one not see a sustained NO-dependent response in this setting? This likely reflects the obligate role of K+ channel activation in Ca2+ signaling of the endothelial cell. SKCa and IKCa are thought to play essential roles in the sustained entry of Ca2+ in the activated endothelial cell (see

18 34 for review). Accordingly, blockade of these K+ channels would be anticipated to affect the sustained, Ca2+-dependent activation of eNOS but would have less effect on the initial response, which depends on the release of intracellular Ca2+ stores (34). Finally, what could be the physiologic or pathophysiologic significance of the PAR2 induced vascular responses of the afferent arteriole? Currently, little is known regarding the role of PARs in the kidney, though PAR2 is abundantly expressed in this organ (3,2). PAR1 and PAR2 exert apposing effects on renal blood flow and GFR in the isolated perfused rat kidney (15) and thus have the capability of bidirectional control of renal hemodynamics. In other organs, evidence implicates an important role of PARs in inflammatory responses and in responses to tissue injury (reviewed in 5, 36,42). The endogeneous activators of PAR2 are proteinases, such as trypsin and mast cell tryptase. Increased numbers of renal mast cells are associated with glomerulonephritis, diabetic nephropathy and renal graft rejection (17,22,38) and urinary proteinase activity is reported to be elevated in patients with acute and chronic renal failure (24). Does the present study provide any insights into the potential role of PARs in such settings? One very curious aspect of our findings relates to the remarkable redundancy of the mechanisms mediating the actions of PAR2 on the afferent arteriole. Why would such multiple and potentially overlapping vasodilatory mechanisms have evolved? A similar complexity of PAR2 induced dilatory responses has been reported for the coronary circulation (30). An important observation in this regard is the finding by McLean et al. (30) that vasodilator responses of the perfused heart to acetylcholine are impaired following ischemia, whereas PAR2 responses are preserved, sugesting a potential importance in the conditions associated with ischemia-reperfusion. Did the redundancy in vasodilatory mechanisms seen in the afferent arteriole evolve to preserve vascular responsiveness of this vessel in pathophysiologic settings? These are all interesting questions for

19 future investigations. In conclusion, the present study demonstrates that redundant mechanisms contribute to the afferent arteriolar vasodilatory response to PAR2 activation. Nitric oxide plays a prominent role in the sustained response, whereas an NO-independent component, mediated by mechanisms involving K+ channels that are sensitive to blockade by charybdotoxin plus apamin (e.g., IKCa and SKCa) and by TEA (e.g., BKCa), and by a mechanism that is dependent on the electrogenic Na+/K+ ATPase. This remarkable redundancy may indicate an important role in settings associated with altered vascular reactivity, similar to that suggested to exist in the coronary circulation (30).

Acknowledgments: These studies were supported by grants from the Canadian Institutes for Health Research (RL, MH), the Heart and Stroke Foundation of Alberta & Nunavut (RL, MH), the Kidney Foundation of Canada (MH) and by a University-Industry grant in conjunction with Servier International (MH). RL is a Alberta Heritage Foundation for Medical Research Scientist.

20 References 1. Andersson DA, Zygmunt PM, Movadeh P, Andersson TLG, and Högestätt ED. Effects of inhibitors of small- and intermediate-conductance calcium-activated potassium channels, inwardly-rectifying potassium channels and Na+/K+ ATPase on EDHF relaxations in the rat hepatic artery. Br J Pharmacol 129:1490-1496, 2000. 2. Bertog M, Letz B, Kong W, Steinhoff M, Higgins MA, Bielfeld-Ackermann A, Fromter E, Bunnett NW and Korbmacher C. Basolateral proteinase-activated receptor (PAR-2) induces chloride secretion in M-1 mouse renal cortical collecting duct cells. J Physiol 521:3-17, 1999. 3. Bohm SK, Kong W, Bromme D, Smeekens SP, Anderson DC, Connolly A, Kahn M, Nelken NA, Coughlin SR, Payani DG and Bunnett NW. Molecular cloning, expression and potential functions of the human proteinase-activated receptor-2. Biochem J 314:1009-1016, 1996. 4. Chilton L, Loutzenhiser R. Functional evidence of an inward rectifier potassium current in the renal afferent arteriole. Circ Res 88:152-158, 2001 5. Cocks TM, Moffatt JD. Protease-activated receptors: sentries for inflammation? Trends Pharmacol Sci 21:103–108, 2000. 6. Coughlin SR. Thrombin signalling and protease-activated receptors. Nature 407: 258–264, 2000. 7. Damiano BP, D'Andrea MR, de Garavilla L, Cheung WM and Andrade-Gordon P. Increased expression of protease activated receptor-2 (PAR-2) in balloon-injured rat carotid artery. Thromb Haemost 81:808-814, 1999. 8. Dodson AM and Rhoden KJ. Bradykinin increases Na+-K+ pump activity in cultured

21 guinea-pig tracheal smooth muscle cells. Br J Pharmacol 133:1339-1345, 2001. 9. Doughty J M, Plane F, and Langton PD. Charybdotoxin and apamin block EDHF in rat mesenteric artery if selectively applied to the endothelium. Am J Physiol 276:H1107H1112, 1999. 10. Edwards G, Dora KA, Gardener MJ, Garland CJ and Weston AH. K+ is an endothelium-derived hyperpolarizing factor in rat arteries. Nature 396:269-272, 1998. 11. Feletou M and Vanhoutte PM. Endothelium-dependent hyperpolarization of vascular smooth muscle cells. Acta Pharmacol Sin 21:1-18, 2000. 12. Fulton D, Mahboubi K, McGiff JC, Quilley J. Cytochrome p450-dependent effects of bradykinin in the rat heart. Br J Pharmacol 114: 99-102, 1995. 13. Gauthier KM, Deeter C, Krishna UM, Reddy YK, Bondlela M, Falck JR and Campbell WB. 14,15-Epoxyeicosa-5(Z)-enoic acid: a selective epoxyeicosatrienoic acid antagonist that inhibits endothelium-dependent hyperpolarization and relaxation in coronary arteries. Circ Res 90:1028-1036, 2002. 14. Grbovic L and Radenkovic M. Analysis of adenosine vascular effect in isolated rat aorta: possible role of Na+/K+-ATPase. Pharmacol Toxicol 92:265-271, 2003. 15. Gui, Y, Loutzenhiser R, Hollenberg M. Bidirectional regulation of renal hemodynamics by proteinase-activated receptors 1 and 2. Am J Physiol 285: F95-F104, 2003. 16. Hayashi K, Loutzenhiser R, Epstein M, Suzuki H and Saruta T: Multiple factors contribute to acetylcholine-induced vasodilation during myogenic, norepinephrine- and KCl-induced afferent arteriolar vasoconstriction: studies in the isolated perfused hydronephrotic kidney. Circ Res 75:821-828, 1994. 17. Hiromura K, Kurosawa M, Yano S and Naruse T. Tubulointerstitial mast cell infiltration in

22 glomerulonephritis. Am J Kidney Dis 32:593-599, 1998. 18. Hollenberg MD and Compton SJ. International union of pharmacology. XXVIII. Proteinase-activated receptors. Pharmacol Rev 54: 203-217, 2002. 19. Imig JD Falck JR, Wei S, Capdevila JH. Epoxygenase metabolites contribute to nitric oxide-independent afferent arteriolar vasodilation in response to bradykinin. J Vasc Res 38: 247-255, 2001. 20. Imig JD, Navar LG, Roman RJ, Reddy KK and Falck JR. Actions of epoxygenase metabolites on the preglomerular vasculature. J Am Soc Nephrol 7:2364-2370, 1996. 21. Kawabata A, Kubo S, Nakaya Y, Ishiki T, Kuroda R, Sekiguchi F, Kawao N and Nishikawa H. Distinct roles for protease-activated receptors 1 and 2 in vasomotor modulation in rat superior mesenteric artery. Cardiovasc Res 61:683-692, 2004. 22. Lajoie, G, Nadasdy T, Laszik Z, Blick KE and Silva FG. Mast cells in acute cellular rejection of human renal allografts. Mod Pathol 9:1118-1125, 1996. 23. Langton PD, Nelson MT, Huang Y, Standen NB. Block of calcium-activated potassium channels in mammalian arterial myocytes by tetraethylammonium ions. Am J Physiol 260:H927-H934, 1991. 24. Lee DY, Park SK, Yorgin PD, Cohen P, Oh Y and Rosenfeld RG. Alterations in insulin-like growth factor-binding proteins (IGFBPs) and IGBBP-3 protease activity in serum and urine from acute and chronic renal failure. J Clin Endocrinol Metab 79:1376-1382, 1994. 25. Loutzenhiser R. In situ studies of renal arteriolar function using the in vitro perfused hydronephrotic rat kidney, Internat Rev Exp Pathol 36:145-160, 1996. 26. Loutzenhiser R, Horton C, and Epstein M. Flow-induced errors in estimating perfusion pressure of the isolated rat kidney. Kid Internat 22:693-696, 1982.

23 27. Macfarlane SR, Seatter MJ, Kanke T, Hunter GD, and Plevin R. Proteinase-activated receptors. Pharmacol Rev 53:245-282, 2001. 28. McGuire JJ, Ding H and Triggle CR. Endothelium-derived relaxing factors: a focus on endothelium-derived hyperpolarizing factor(s). Can J Physiol Pharmacol 79:443-470, 2001. 29. McGuire JJ, Hollenberg MD, Andrade-Gordon P and Triggle CR. Multiple mechanisms of vascular smooth muscle relaxation by the activation of proteinase-activated receptor 2 in mouse mesenteric arterioles. Br J Pharmacol 135:155-169, 2002. 30. McLean PG, Aston D, Sarkar D and Ahluwalia A. Protease-activated receptor-2 activation causes EDHF-like coronary vasodilation: selective preservation in ischemia/reperfusion injury: involvement of lipoxygenase products, VR1 receptors, and C-fibers. Circ Res 90:465-472, 2002. 31. Milia AF, Salis MB, Stacca T, Pinna A, Madeddu P, Trevisani M, Geppetti P and Emanueli C. Protease-activated receptor-2 stimulates angiogenesis and accelerates hemodynamic recovery in a mouse model of hindlimb ischemia. Circ Res 91:346-352, 2002. 32. Nakahira Y, Hashitani H, Fukuta H, Sasaki S, Kohri K and Suzuki H. Effects of isoproterenol on spontaneous excitations in detrusor smooth muscle cells of the guinea pig. J Urol 166:335-340, 2001 33. Nakayama T, Hirano K, Nishimura J, Takahashi S and Kanaide H. Mechanism of trypsin-induced endothelium-dependent vasorelaxation in the porcine coronary artery. Br J Pharmacol 134:815-826, 2001. 34. Nilius B and Droogmans G. Ion channels and their functional role in vascular endothelium. Physiol Rev 81:1415-1459, 2001.

24 35. Nystedt S, Emilsson K, Wahlestedt C, and Sundelin J. molecular cloning of a potential proteinase-activated receptor. Proc Natl Acad Sci USA 91: 9208-9212, 1994. 36. Ossovskaya VS, Bunnett NW. Protease-activated receptors: contribution to physiology and disease. Physiol Rev 84:579-621, 2004. 37. Pratt PF, Li P, Hillard CJ, Kurian J and Campbell WB. Endothelium-independent, ouabain-sensitive relaxation of bovine coronary arteries by EETs. Am J Physiol 280:H1113-H1121, 2001. 38. Roberts IS, Brenchley PE. Mast cells: the forgotten cells of renal fibrosis. J Clin Pathol 53:858-862, 2000. 39. Roy SS, Saifeddine M, Loutzenhiser R, Triggle CR, Hollenberg MD: Dual endothelium-dependent vascular activities of proteinase-activated receptor-2-activating peptides: evidence for receptor heterogeneity. Br J Pharmacol 123:1434-1440, 1998. 40. Schubert R and Nelson MT. Protein kinases: tuners of the BKCa channel in smooth muscle. Trends Pharmacol Sci 22:505-512, 2001. 41. Trottier G, Hollenberg M, Wang X, Gui Y, Loutzenhiser K, and Loutzenhiser R. PAR-2 elicits afferent arteriolar vasodilation by NO-dependent and NO-independent actions. Am J Physiol 282: F891-F897, 2002. 42. Vergnolle N, Wallace JL, Bunnett NW, Hollenberg MD. Protease-activated receptors in inflammation, neuronal signaling and pain. Trends Pharmacol Sci. 22:146-152, 2001. 43. Wang X and Loutzenhiser R. Determinants of the renal microvascular response to ACh, afferent and efferent arteriolar action of EDHF. Am J Physiol 282: F124-F132, 2002. 44. Wang, X, Trottier G and Loutzenhiser R. Determinants of the renal afferent arteriolar actions of bradykinin: Evidence for multiple pathways mediating the vasodilator responses

25 attributed to EDHF. Am J Physiol 285:F540-549, 2003. 45. Zou AP, Fleming JT, Faalck JR, Jacobs ER, Gebremedhin D, Harder DR, Roman RJ. Stereospecific effects of epoxyeicosatrienoic acids on renal vascular tone and K+-channel activity. Am J Physiol 270: F822-F832, 1996.

26

L-NAME + Ibu

A

Ang II (0.1 nM)

Ang II (0.1 nM) 22

SLIGRL-NH2 (10 µM)

B

18

18

14

Afferent 14 Arteriolar Diameter 10 (µm)

Afferent Arteriolar 10 Diameter 6 (µm)

2

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6

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5

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20

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SLIGRL-NH2 (10 µM)

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10

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Figure1. Original tracings depicting the response of the afferent arteriole to the PAR2-selective agonist SLIGRL-NH2. Note in panel A (control), that in the presence of angiotensin II (Ang II) induced vasoconstriction, SLIGRL-NH2 elicits a biphasic response, characterized by an initial peak response that was followed by a sustained, but reduced, level of vasodilation. In B, NO synthase and cyclooxygenase were inhibited by 100 :mol/L LNAME and 10 :mol/L ibuprofen (Ibu). In this setting, SLIGRL-NH2 induced only transitory (peak) vasodilation.

27

L-NAME + Ibu Ang II (0.1 nM)

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Trypsin (1 Unit)

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Trypsin (2 nmol/L)

22 18

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Figure 2. Original tracings illustrating the effects of PAR2 activation by trypsin. Note in panel A (control) that trypsin (2 nmol/L) produced a vasodilation that spontaneously abated after 5-10 minutes (see text for discussion). In a separate preparation, pre-treated with LNAME (100 :mol/L) and ibuprofen (10 :mol/L, Ibu), trypsin evoked only a transient response (B). Afferent arteriolar tone was induced by 0.1 nmol/L angiotensin II (Ang II).

28

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L-NAME + Ibu TEA ChTX + Ap 17-ODYA

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6 2

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Figure 3. Original tracings (left) and mean data (right) illustrating the NO-independent response to SLIGRL-NH2 (10 :mol/L) seen in tissues pre-treated with 100 :mol/L L-NAME and 10 :mol/L ibuprofen (Ibu). As shown in A, this response persisted in the combined presence of 1 mmol/L tetraethylammonium chloride (TEA), 100 nmol/L charybdotoxin (ChTX) and 1 :mol/L apamin (Ap) (n=6). Panels B illustrate that the addition of 50 :mol/L 17-octadecynoic acid (ODYA) to this mixture also fails to prevent this response

(n=7). Afferent arteriolar tone induced by 0.1 nmol/L angiotensin II (Ang II).

29

L-NAME + Ibu + TEA + ChTX + Ap RAP Increased to 160 mmHg

Afferent Arteriolar Diameter (µm)

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22 20 18 16 Afferent 14 Arteriolar 12 Diameter 10 (µm) 8 6 4 2 0 20

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Figure 4. Residual dilator response to SLIGRL-NH2 in the presence of L-NAME, ibuprofen, TEA, charybdotoxin and apamin, but in the absence of angiotensin II. In these experiments basal tone was established by elevating renal arterial pressure (RAP) from 80 mmHg to 160 mmHg. Tracing illustrating transient nature of the response is shown on left. Mean data (n=4) are shown on right.

30

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Ouabain 3 mM SLIGRL-NH2

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Afferent 14 Arteriolar Diameter 12 10 (µm) 8

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Figure 5. Original tracings illustrating ouabain-induced afferent arteriolar vasoconstriction and SLIGRL-NH2 (10 :mol/L) induced vasodilation in controls (A) and in the presence (B) of 100 Fmol/L L-NAME and 10 :mol/L ibuprofen (Ibu). Note the biphasic response in A versus the transient response in B. Right panels depict mean values of ouabain-induced vasoconstriction and initial peak responses to SLIGRL-NH2 in each setting (n=6; A and n=5, B).

31

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L-NAME + Ibu + ChTX + Ap

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Figure 6. Panel A demonstrates that the combination of 10 nmol/L charybdotoxin (ChTX) and 1 :mol/L apamin (Ap) did not prevent the NO-independent response to SLIGRL-NH2 in the presence of ouabain (3 mmol/L). Similarly, as demonstrated in panel B, TEA (1 mmol/L) did not prevent this response when added alone. All preparations pre-treated with 100 :mol/L L-NAME and 10 :mol/L ibuprofen (Ibu). Mean data (right) obtained from 5

preparations (A) and 6 preparations (B).

32

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L-NAME + Ibu + TEA + ChTX + Ap Ouabain 20 20

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Figure 7. Panel A illustrates that when added in combination TEA (1 mmol/L), charybdotoxin (ChTX, 10 nmol/L), and apamin (Ap, 1 Fmol/L) abolished the NO-independent response to SLIGRL-NH2 (10 :mol/L) in the presence of ouabain (3 mmol/L). As shown in panel B, the action of TEA was not mimicked by the cytochrome P450 inhibitor 17octadecynoic acid (ODYA, 50 :mol/L). All preparations were pre-treated with 100 :mol/L L-NAME and 10 :mol/L ibuprofen (Ibu). Mean data obtained from 7

preparations (A) and 5 preparations (B) depicted on right.

33

Ibu + TEA + ChTX + Ap Ouabain 22 SLIGRL-NH2

Afferent 18 Arteriolar Diameter 14 (µm) 10 6 0

22 18

5

10 15 Time, min

20

Ibu + TEA ChTX + Ap Ouabain SLIGRL-NH2

Afferent Arteriolar 14 Diameter (µm)

10 6

Figure 8. The vasodilator response to SLIGRL-NH2 (10 :mol/L) in the combined presence of ouabain (3 mmol/L), TEA (1 mmol/L), charybdotoxin (ChTX, 10 nmol/L), and apamin (Ap, 1Fmol/L), but in the absence of L-NAME. Note that SLIGRL-NH2 evokes a transient NO-dependent vasodilation in this setting (compare to figure 7A). The preparations were pre-treated with 10 :mol/L ibuprofen (n=4).

34

L-NAME + Ibuprofen

100 80 % Dilation of 60 Ouabain-Induced Vasoconstriction by SLIGRL-NH2 40 20 0 TEA + Apamin + ChTX

TEA

Apamin TEA 17-ODYA + ChTX + Apamin + Apamin + ChTX + ChTX

Figure 9. Summary of findings illustrating the effects of various treatments on the response to SLIGRL-NH2 in afferent arterioles that were pre-treated with 3 mmol/L ouabain. Data are expressed as the percent dilation of the ouabain-induced afferent arteriolar vasoconstriction. Note that in the presence of ouabain, the combination of L-NAME, TEA and ChTX+Apamin fully abolished the response, whereas TEA and ChTX +Apamin only attenuated the response when added separately. The effects of 17-ODYA and ChTX+Apamin were similar to those of ChTX+Apamin alone.

35 Acetylcholine

L-NAME + Ibuprofen

100 80 % Dilation of Ang II-Induced 60 Vasoconstriction 40 20 0

Bradykinin

Apamin

ChTX

Apamin + ChTX

L-NAME + Ibuprofen 100

80 % Dilation of Ang II-Induced 60 Vasoconstriction 40 20 0 Apamin + ChTX

TEA

17-ODYA

TEA 17-ODYA + Apamin + Apamin + ChTX + ChTX

SLIGRL-NH2 100

L-NAME + Ibuprofen

80 % Dilation of Ang II-Induced 60 Vasoconstriction 40 20 0

TEA + Apamin + ChTX

TEA + Apamin + ChTX + 17-ODYA

Figure 10. Summary of our findings comparing the EDHF-like responses of the afferent arteriole to acetylcholine (data from 43) and bradykinin (data from 44) to the NO-independent response to SLIGRL-NH2. Open bars depict peak responses under control conditions and solid bars depict responses following inhibition of NOS and COX. All data were obtained under identical conditions using the in vitro perfused hydronephrotic rat kidney. Afferent arteriolar tone was established in each case by the administration of angiotensin II. Note that the combination of apamin and charybdotoxin (ChTX) fully abolished the EDHF-like response to acetylcholine (top panel), but not to bradykinin (center panel). The EDHFlike response to bradykinin was abolished by the addition of either TEA or 17-ODYA to the mixture of ChTX plus AP (center). However, combined treatment with these agents did not abolish the NO-independent response to SLIGRL-NH2 (bottom).