Role of nitric oxide in angiotensin IV-induced increases in cerebral blood flow

Regulatory Peptides 74 (1998) 185–192

Role of nitric oxide in angiotensin IV-induced increases in cerebral blood flow ´ Radhika Krishnan, Joseph W. Harding, John W. Wright* Eniko¨ A. Kramar, Program in Neuroscience, Departments of Psychology and Veterinary and Comparative Anatomy, Pharmacology and Physiology, Washington State University, Pullman, WA 99164 -4820, USA Received 16 December 1997; received in revised form 23 April 1998; accepted 30 April 1998

Abstract The present study investigated the effects of three newly synthesized AngIV analogs (Lysine 1 -AngIV, Norleucine 1 -AngIV, and Norleucinal) on cerebral blood flow (CBF) in anesthetized Sprague–Dawley rats utilizing laser-Doppler flowmetry. The results indicate that internal carotid infusions of AngIV, Norleucine 1 -AngIV, Norleucinal, and Lysine 1 -AngIV increased CBF above baseline by 25, 32, 33 and 44%, respectively, without changing systemic arterial blood pressure. In a second experiment separate groups of rats were pretreated with nitric oxide (NO) synthase inhibitor, Nw -nitro-L-arginine methyl ester ( L-NAME) or saline, followed by AngIV or Norleucinal for the purpose of evaluating the hypothesis that the mechanism of action of these compounds is linked to the release of NO. Pretreatment with saline followed by AngIV and Norleucinal increased CBF by 29 and 39%, respectively, while pretreatment with L-NAME blocked the vasodilatory effects of AngIV and Norleucinal, suggesting that the increment in blood flow induced by these compounds is dependent upon the synthesis and release of NO from vascular endothelial cells.  1998 Published by Elsevier Science B.V. Keywords: Laser-Doppler flowmetry; Cerebral microcirculation; Lysine 1 -AngIV; Norleucine 1 -AngIV; Norleucinal; Rats

1. Introduction An ischemic insult to the brain can lead to significant cell losses especially in the CA1 field of the hippocampus [1–4]. Within this field pyramidal neurons are lost in proportion to the severity of the ischemia, thus compromising this structure’s ability to mediate learning acquisition and memory processing [5–10]. The CA1 field is also targeted during the progression of Alzheimer’s disease (AD) with concomitant cognitive impairment [11,12]. These AD-associated cell losses have been attributed to insufficient microcirculation [13]. Thus, an understanding of the mechanisms regulating cerebral blood flow (CBF) may be relevant to these cognitive dysfunctions [3]. Earlier reports have suggested that the octapeptide, angiotensin II (AngII), may play a role in CBF regulation *Corresponding author. Tel.: 1 1 509 335 2329; fax: 1 1 509 335 5043; e-mail: [email protected]

[14–16]. Recent evidence supports the notion that an AngII degradative product, the hexapeptide angiotensin IV (AngIV), may also serve a critical function in the control ¨ of CBF [17,18]. Along these lines Naveri et al. [19] reported that intravenous infusions of AngIV in rats reversed the reduction in CBF resulting from experimentally-induced subarachnoid hemorrhage (SAH) by increasing CBF 45–80% above baseline over a 60-min period. Pretreatment with a non-selective AT 1 and AT 2 receptor antagonist, Sar 1 , Ile 8 AngII (Sarile), failed to block this AngIV-induced vasodilation response, suggesting that AngIV-induced elevations in blood flow are mediated by a receptor subtype other than AT 1 and AT 2 . Our laboratory has discovered what may be a novel angiotensin receptor subtype, classified AT 4 [20], that displays high affinity for AngIV [21,22], however, the molecular structure of this receptor has not been determined. The initial discovery was made in bovine adrenal tissue but has since been extended to include other tissues

0167-0115 / 98 / $19.00  1998 Published by Elsevier Science B.V. All rights reserved. PII: S0167-0115( 98 )00039-1

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such as cultured endothelial and smooth muscle cells [22–24]. Therefore, it is possible that this receptor subtype is responsible for mediating the AngIV-induced increases ¨ in CBF reported by Naveri et al. [19]. The development and availability of Losartan (Dup 753), a specific AT 1 receptor subtype antagonist [25], and a newly synthesized specific AT 4 receptor subtype antagonist, Divalinal-AngIV [26], now makes it possible to differentiate the roles of AngII and AngIV. We have utilized laser-Doppler flowmetry to demonstrate that intra-arterial infusions of AngII reduced CBF by 23%, whereas, infusions of AngIV increased CBF in a dose-dependent fashion [27]. Further, these AngII-induced reductions in CBF could be inhibited by pre-treatment with Losartan, while pre-treatment with Divalinal-AngIV completely abolished the AngIV-induced vasodilation response. There were no receptor antagonist cross-over effects suggesting that AngII acts at the AT 1 receptor to reduce CBF, while AngIV binds at the AT 4 subtype to increase CBF. As mentioned above, vascular endothelial cells possess AT 4 receptors [24]. It has also been shown that a vascular endothelial cell nitric oxide (NO)-mediated mechanism decreased infarction volume after middle cerebral artery occlusion by increasing regional CBF in rats [30]. Taken together, we speculated that AngIV-induced vasodilation may be caused by AngIV directly (or indirectly) activating the synthesis and release of NO in vascular endothelial cells. Results from a recent study by Patel and Block [31] support this notion in that AngIV enhanced the catalytic activity of endothelial cell NO synthase (ecNOS) and the production of cGMP in porcine pulmonary artery endothelial cells (PAEC). These investigators predicted that Divalinal-AngIV would block ecNOS activation, and consequently NO production in PAEC (J.M. Patel, personal communication). Given the recent findings on the effects of AngIV on cerebral circulation [19,22] it appears that AngIV participates in the regulation of CBF in the microvasculature. However, native AngIV possesses a short metabolic halflife (Harding, personal communication; [28]), consequently, there is a need to develop AngIV analogs that are resistant to systemic metabolic breakdown and display high-affinity binding at the AT 4 receptor. Our laboratory has investigated the structure-binding characteristics of AngIV with the AT 4 receptor [29], and we have recently synthesized several AngIV analogs. Thus, the purpose of the present study was to investigate the effects of AngIV and three AngIV analogs, Lysine 1 -AngIV (Lys 1 -AngIV), Norleucine 1 -AngIV (Nle 1 -AngIV) and Norleucinal on CBF regulation, and to evaluate the hypothesis that these compounds may stimulate the release of NO in cerebral circulation. We predicted that intra-arterial infusions of Lys 1 -AngIV, Nle 1 -AngIV and Norleucinal would increase CBF without influencing systemic blood pressure, and that an NO synthase inhibitor, Nw -nitro-L-arginine methyl ester ( L-NAME), would block these AngIV- and AngIV analoginduced responses in the cerebral microvasculature.

2. Materials and methods

2.1. Animals and maintenance Female Sprague–Dawley rats (Taconic Farms, Germantown, NY, derived), weighing 250–350 g, were bred and housed in group cages in an American Association for Accreditation of Laboratory Animal Care-approved vivarium under a 12:12 h light / dark cycle initiated at 07:00 h at 22618C with free access to Purina laboratory chow and water.

2.2. Surgery and instrumentation Each animal utilized in experiments 1 and 2 was initially pretreated with an intramuscular injection of diazepam (2.5 mg / kg, Parke Davis, St. Louis, MO) 10 min prior to anesthetization with ketamine hydrochloride (Ketaset, 100 mg / kg i.m., Phoenix Scientific, St. Joseph, MO). During surgery, anesthesia was maintained by supplemental injections of ketamine (50 mg / kg, i.m.) at 30-min intervals. Body temperature was monitored by a rectal probe and maintained at 3760.58C using a homeothermic blanket control unit (Harvard Apparatus, South Natick, MA). Surgery was performed as previously described [27]. Briefly, a ventral midline incision was made from the larynx to the manubrium and the left common carotid artery was catheterized (PE-60, Clay Adams) for the purpose of monitoring mean arterial pressure (MAP) via a blood pressure analyzer (Micro-Med, Louisville, KY). Next, the animal was turned onto its right-side in order to expose and catheterize the internal common carotid artery. A beveled tip infusion catheter (PE-10, Clay Adams) was inserted 0.8–1.2 cm into the internal carotid artery and directed toward the circle of Willis, thus permitting the infused compounds to influence the middle and anterior cerebral arteries. The catheter was anchored to the surrounding muscle and connected to a 1.0-ml syringe used to infuse the compounds via an infusion pump (Sage Instruments; model 355). During the last stage of surgery the animal was placed in a stereotaxic head holder and a left parietal craniectomy (3 3 3 mm) was performed such that a paper-thin layer of bone remained intact, thus avoiding epidural bleeding [30]. In order to measure cerebrocortical blood flow, the probe (0.9 mm o.d.) of a laser-Doppler flowmeter (Optokinetics Corp., Advanced Model ALF2100) was positioned over, but not in contact with, the thin layer of bone overlaying the posterior parietal cortex by the use of a micromanipulator (Stoeling, Model MM33). Care was taken to avoid positioning the probe over large surface vessels.

2.3. Compounds Sardinia et al. [29,32] have demonstrated that positions 1, 2 and 3 on the AngIV peptide chain contain essential information for high affinity binding to the AT 4 receptor,

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whereas, positions 4, 5 and 6 are more flexible. The AngIV analogs utilized in this study all had number 1 position substitutions and demonstrated high-affinity binding to the AT 4 receptor as illustrated in Table 1. AngIV analogs [Norleucine 1 ]-(CH 2 -NH)1 – 2 -Tyr-Ile-His-Pro-Phe (Norleucinal), [Norleucine 1 ]-Tyr-Ile-His-Pro-Phe (Nle 1 AngIV) and [Lysine 1 ]-Tyr-Ile-His-Pro-Phe (Lys 1 -AngIV) were synthesized in our laboratory employing a Vega (Coupler 250) amino acid synthesizer, followed by amino acid analyses. Their purities were estimated to be 73%, whereas the purity of AngIV (BaChem, Cat. [: H-8125) was determined by the supplier to be 71%. Corrections for purity were made when the compounds were prepared for use. The NOS inhibitor, Nw -nitro-L-arginine methyl ester ( L-NAME), was purchased from Sigma (St. Louis, MO; Cat. [: N 5751) and was indicated to be 100% pure.

2.4. Experiment 1: effects of intra-arterial infusions of AngIV analogs on CBF Forty rats were randomly assigned to five groups of eight animals each. Members of group 1 served as controls, while animals in groups 2–5 received doses of AngIV, Lys 1 -AngIV, Nle 1 -AngIV or Norleucinal as indicated below. Once the laser-Doppler probe was in position, baseline CBF and MAP were established during a 10-min infusion of 0.15 M NaCl (25 ml / min) via the internal carotid artery catheter. The testing period for all animals consisted of a 10-min treatment period followed by a 25-min recovery period. The control group received a 35-min infusion of 0.15 M NaCl (vehicle, 25 ml / min). Treatment groups 2–5 received a 10-min infusion of AngIV (100 pmol / 25 ml 0.15 M NaCl / min), Lys 1 -AngIV (100 pmol / 25 ml 0.15 M NaCl / min), Nle 1 -AngIV (100 pmol / 25 ml 0.15 M NaCl / min), or Norleucinal (100 pmol / 25 ml 0.15 M NaCl / min), followed by a second 25-min infusion of 0.15 M NaCl (25 ml / min).

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AngIV, while the potency and duration of Norleucinalinduced vasodilatory responses were comparable to Lys 1 AngIV. Forty-two animals were randomly assigned to six groups. Members of groups 1 (n 5 8) and 6 (n 5 6) served as controls, while groups 2, 3 (n 5 8 each) and 4, 5 (n 5 6 each) received an infusion of L-NAME and 0.15 M NaCl, respectively, followed by an infusion of either AngIV, or Norleucinal. The testing phase consisted of three successive 10-min infusion periods: pre-treatment, treatment and recovery. After baseline measures of CBF and MAP were established (10-min infusion of 0.15 M NaCl at 25 ml / min), control animals received a 10-min pretreatment infusion of L-NAME (10 nmol / 25 ml 0.15 M NaCl / min) or 0.15 M NaCl (25 ml / min) followed by a 10-min infusion of 0.15 M NaCl (25 ml / min). Treatment groups 2 and 3 received a 10-min pretreatment dose of L-NAME (10 nmol / 25 ml 0.15 M NaCl / min), while groups 4 and 5 received a pretreatment dose of 0.15 M NaCl (25 ml / min) followed by a 10-min infusion of either AngIV (100 pmol / 25 ml 0.15 M NaCl / min), or Norleucinal (100 pmol / 25 ml 0.15 M NaCl / min).

2.6. Statistical analyses The baseline blood pressure data set and the data set concerned with CBF for the groups subsequently treated with the AngIV analogs of experiment 1 were each analyzed by one-way analysis of variance (ANOVA). The baseline blood pressure data set of experiment 2 was analyzed by a one-way ANOVA. The maximum changes in blood pressure exhibited by members of groups 1–6 were analyzed by a 6 (groups) 3 2 (conditions) ANOVA with repeated measures on the second factor. The comparable data set concerned with maximum change in CBF for groups 1–6 was also analyzed by a 6 (groups) 3 2 (conditions) ANOVA with repeated measures on the second factor. Significant effects were analyzed by Neuman-Keuls post-hoc tests with a level of significance set at 0.05.

2.5. Experiment 2: pre-treatment with NOS inhibitor, LNAME 3. Results Given the results from experiment 1, only AngIV and Norleucinal were chosen to be further investigated in experiment 2 in order to limit the number of animals employed and because the magnitude and duration of AngIV-induced increases in CBF were similar to Nle 1 -

3.1. Experiment 1: AngIV analog-induced pressor and CBF effects The maximum changes in systemic blood pressure

Table 1 Molecular weights and inhibitory constants (k i ) for the binding of selected AT 4 receptor subtype ligands to heat-treated bovine adrenal membranes Peptide AngIV Norleucinal Norleucine 1 -AngIV Lys 1 -AngIV a Values are 6S.E.M. c, reduced peptide bond.

Structure Val-Tyr-Ile-His-Pro-Phe [Nle 1 ]c(CH 2 -NH)1 – 2 -Tyr-Ile-His-Pro-Phe [Nle 1 ]-Tyr-Ile-His-Pro-Phe [Lys]-Tyr-Ile-His-Pro-Phe

Molecular weight 774 774 789 804

k i (M)

Source 29a

2.6360.12 3 10 1.8060.2 3 10 210 3.5960.51 3 10 212 1.1469.8 3 10 210

BaChem BioSciences Kirshnan and Harding Kirshnan and Harding Kirshnan and Harding

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Table 2 Mean (6S.E.M.) baselevel, treatment, and maximum change in blood pressure measured in the groups utilized in experiments 1 and 2

Experiment 1 Group 1 Group 2 Group 3 Group 4 Group 5

Experiment 2 Group 1 Group 2 Group 3 Group 4 Group 5 Group 6

0.15 M saline AngIV Lys 1 -AngIV Nle 1 -AngIV Norleucinal

L-NAME / 0.15

M saline

L-NAME /AngIV L-NAME / Norleucinal Saline /AngIV Saline / Nle Saline / saline

during the infusion of AngIV and AngIV analogs in experiment 1 are listed for groups 1–5 in Table 2. The groups used to test for the effects of AngIV, and AngIV analogs, on systemic blood pressure and CBF did not differ regarding baseline blood pressure (overall mean6S.E.M. 5 110.061.3 mmHg; F 5 1.12, df 4 / 35, P . 0.10). The maximum change in MAP during the pretreatment and treatment period was also shown to have no groups effect (F 5 1.17, df 5 / 36, P . 0.10), treatment effect (F 5 0.30, df 1 / 36, P . 0.10) or interaction effect (F 5 0.82, df 5 / 36, P . 0.10). Fig. 1 illustrates the influence of intra-arterial infusions of AngIV and AngIV analogs upon CBF. A one-way ANOVA revealed that there were significant differences

Baselevel

Treatment

Maximum D

110.564.3 113.762.1 106.261.5 109.661.5 109.662.1

111.764.0 116.661.3 109.062.0 110.261.4 113.461.9

1.260.6 2.961.0 2.761.6 0.661.1 3.761.8

Baselevel

Pretreatment

Treatment

111.761.0 114.461.8 107.262.0 110.563.4 109.364.3 115.261.2

118.062.5 117.062.6 116.962.6 112.760.6 111.861.5 116.461.8

115.562.1 119.561.9 115.463.1 115.061.4 117.062.0 114.261.7

among these groups (F 5 24.04, df 4 / 35, P , 0.0001). Post-hoc analyses indicated that AngIV, Lys 1 -AngIV, Nle 1 AngIV and Norleucinal (100 pmol / min each) displayed significant maximum elevations in CBF (mean6S.E.M. 5 32.965.6, 43.964.4, 32.562.6, and 25.563.5%, respectively) during infusion as compared with the control group. Further, Lys 1 -AngIV differed from Norleucinal, but there was no difference between AngIV and Nle 1 -AngIV. A simple linear regression model was used to test for differences in slopes among AngIV, Nle 1 -AngIV, Lys 1 AngIV, and Norleucinal during the initial 3 min of the treatment period. It was determined that the linear regression best fit line for AngIV ( y 5 7.94x 1 0.5) and Nle 1 AngIV ( y 5 6.63x 1 1.33) were equivalent (P . 0.10), however, the best fit lines for Lys 1 -AngIV ( y 5 6.31x 1 18.13) and Norleucinal ( y 5 1.94x 1 2.46) were significantly different (P , 0.05). During the recovery period (saline infusion following drug treatment) mean (6S.E.M.) percent CBF changes for Lys 1 -AngIV and Norleucinal peaked at 47.5% (64.8) and 40.4% (65.1), respectively. An independent t-test indicated that there was no difference between these groups (t 5 1.01, df 14, P . 0.10). Further, mean percent CBF changes for Lys 1 -AngIV and Norleucinal (25 and 35%, respectively) were significantly higher than AngIV and Nle 1 -AngIV (12 and 18%, respectively) 25 min post-infusion as compared with the control group (F 5 3.16, df 4 / 35, P , 0.05).

3.2. Experiment 2: pre-treatment with NOS inhibitor, LNAME

Fig. 1. Mean (6S.E.M.) percent change in CBF from baseline during a 10-min intra-arterial infusion of AngIV, Lys 1 -AngIV, Nle 1 -AngIV, or Norleucinal (each 100 pmol / 25 ml 0.15 M NaCl / min) and 0.15 M NaCl (25 ml / min).

In order to eliminate L-NAME-induced hypertensive effects that may mask any AngIV-induced changes, a preliminary study generated a dose-response curve for systemic infusions of L-NAME. L-NAME concentrations of 0, 5, 10 and 20 nmol / 25 ml 0.15 M NaCl / min revealed mean systemic blood pressure changes of 0, 2 3, 8 and 20

´ et al. / Regulatory Peptides 74 (1998) 185 – 192 E. A. Kramar

mmHg, respectively. As a result we selected the 10 nmol dose for further investigation because it was the highest concentration we could infuse without accompanying significant CBF and MAP changes. During baseline blood pressure measurements there were no significant differences observed among groups 1–6 in experiment 2 (overall mean6S.E.M. 5 111.461.0 mmHg; F 5 1.67, df 5 / 36, P . 0.10) (see Table 2). The data set concerned with maximum blood pressure changes induced by the treatments utilized with groups 1–6 was analyzed by a 6 (groups) 3 2 (conditions) ANOVA. Results revealed that there were no group, treatment or interaction effects. Fig. 2 illustrates the mean percent CBF changes measured during pretreatment with 0.15 M NaCl (panel A) or L-NAME (panel B), followed by AngIV, Norleucinal, or 0.15 M NaCl. A 6 (group) 3 2 (conditions) ANOVA revealed that there was a significant groups effect (F 5 15.94, df 5 / 36, P , 0.0001), a treatment effect (F 5 43.81, df 1 / 36, P , 0.0001), and an interaction effect (F 5 19.36, df 5 / 36, P , 0.0001). Post-hoc analyses indicated that pretreatment with L-NAME (groups 2 and 3) blocked subsequent AngIV- and Norleucinal-induced changes in CBF.

Fig. 2. (A) Mean (6S.E.M.) percent change in CBF from baseline during a 10-min infusion of AngIV, Norleucinal (each 100 pmol / 25 ml 0.15 M NaCl / min), or saline (25 ml 0.15 M NaCl / min) following a 10-min pretreatment with saline (25 ml 0.15 M NaCl / min). (B) Mean (6S.E.M.) percent change in CBF from baseline during a 10-min intra-arterial infusion of AngIV, Norleucinal (each 100 pmol / 25 ml 0.15 M NaCl / min), or saline (25 ml 0.15 M NaCl / min) following a 10-min infusion with L-NAME (10 nmol / 25 ml 0.15 M NaCl / min).

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4. Discussion The recent discovery of an AT 4 receptor subtype that binds AngIV has generated several investigations concerning its roles within the brain-renin angiotensin system. Initial studies revealed that this AT 4 receptor is localized in species ranging from arthropods to mammals, and in a wide range of tissue beds including heart, lung, brain, bladder, spleen, thymus, adrenals, kidney, gut, spinal cord, pituitary and vascular endothelial and smooth muscle cells [23,24,33,34]. It appears that the AT 4 receptor is involved in facilitating memory acquisition and retrieval [35–37], regulation of blood flow [22,27,38–40], neurite outgrowth [41], angiogenesis (Miller-Wing et al., unpublished observation), kidney function ( [42]; Hamilton et al., unpublished observations), cardiac function [43] and stimulation of endothelial cell expression of plasminogen activator inhibitor (PAI-1) [44]. In order to better understand the physiologies and behaviors mediated by this AngIV/AT 4 system, our laboratory has developed pharmacological agents that bind to the AT 4 receptor with high-affinity while possessing metabolic stability. This objective led Sardinia et al. [29] in our laboratory to investigate the structure-binding characteristics of the AT 4 receptor. From this study, several prerequisite structural features for high-affinity binding to the AT 4 receptor were determined. Specifically, deletion of Val in position 1, or extension of the N-terminal with additional amino acids, significantly reduced affinity. Glycine mono-substitutions for each amino acid revealed that positions 1, 2 and 3, or the use of D-isomers at these positions, contained essential information that determined ligand affinity. In contrast, positions 4, 5 and 6 could accommodate various amino acid constituents without effecting the affinity of the molecule, but were necessary for ligand-receptor recognition. These findings initiated a detailed study of the first position of the AngIV molecule and revealed several important characteristics that would maximize AT 4 receptor binding [32]. First, a primary amine must be present in position 1 for high-affinity binding. Second, the presence of hydrophobic residues in position 1 (i.e. Norleucine 1 -AngIV) produced the greatest binding affinity to the AT 4 receptor. Further, a charged amino acid substitution (i.e. Lys 1 -AngIV) in position 1 yielded lower, albeit still reasonably high, affinity. Third, the N-terminal amino acid must be in the L-configuration. Lastly, an isostere substitution between positions 1 and 2 on the AngIV molecule (i.e. Norleucinal) increased metabolic stability with minimal effect on binding affinity. A recent laser-Doppler flowmetry study from our laboratory reported that infusions of the AngIV analogs, Lys 1 AngIV and Nle 1 -AngIV via the renal artery increased renal cortical blood flow without influencing systemic blood pressure [45]. Further, pretreatment with the NOS inhibitor, N G -monomethyl-L-arginine ( L-NMMA), blocked this elevation in renal blood flow suggesting that Lys 1 -AngIV-

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and Nle 1 -AngIV-induced vasodilation may activate an NO pathway. The present study extended this inquiry to the cerebral microvasculature. In experiment 1 three AngIV analogs (Lys 1 -AngIV, Nle 1 -AngIV, and Norleucinal) were tested to determine their effect on CBF. The results indicated that during the treatment period Norleucinal, Nle 1 -AngIV, AngIV, and Lys 1 -AngIV increased CBF by 25, 32, 33 and 44%, respectively, without influencing systemic blood pressure. Further, among these four compounds Lys 1 -AngIV induced a change in CBF at a faster rate than Norleucinal, suggesting that Lys 1 -AngIV was more effective at initially activating the AT 4 receptor than Norleucinal. However, during the recovery period Lys 1 -AngIV- and Norleucinalinduced vasodilatory responses reached comparable levels and remained elevated at least 25 min post-infusion. Taken together, these findings are consistent with results from previous investigations [19,27,45], and support the hypothesis that AngIV, and AngIV analogs, play a role in mediating vasodilation within the cerebral microvasculature. Previous studies concerned with identifying the mechanism of action responsible for AngIV-induced vasodilation remain in opposition. An earlier report by Haberl et al. [17] revealed that a vasodilation response occurred after topical application of AngIV in the presence of L-arginine in rabbit brain arterioles. This response was inhibited by pretreatment with methylene blue suggesting that AngIV may induce vasodilation through an endothelium-dependent process, specifically by stimulating the synthesis and release of endothelium-derived relaxing factor (EDRF), now also known as NO [46]. In support of this notion, the present study has shown that a NOS inhibitor, L-NAME, blocked subsequent AngIV- and Norleucinal-induced increases in CBF. Coleman et al. [45] and Yoshida et al. [40] have reported similar results concerning renal blood flow. ¨ In contrast with the present results, Naveri et al. [19] concluded that L-NAME reduced [ 3 H]citrulline by 58% in unperturbed basilar arteries, however, AngIV had no effect suggesting that NO was not a mediator of AngIV-induced vasodilation. The authors pointed out that while AngIVinduced vasodilation was measured in small cerebral arterioles using laser-Doppler flowmetry, measurement of NO’s activity was carried out using large cerebral arteries. Further, the hypertensive effect of L-NAME may have masked a possible antagonistic effect. This discrepancy concerning the role of NO in AngIV-induced vasodilation may be due to additional differences in methodology. Specifically, in the present investigation: (1) laser-Doppler flowmetry was employed to measure changes in CBF in cerebral microvasculature during infusions of L-NAME ¨ followed by AngIV and Norleucinal, while Naveri et al. [19] measured NOS activity in basal artery tissue following intravenous infusions of AngIV and L-NAME in rats with and without SAH. (2) L-NAME was used as a blocking agent for NO release to determine if AngIV-

induced vasodilation was dependent on NO release, while ¨ Naveri et al. [19] infused AngIV and L-NAME in order to measure [ 3 H]citrulline release. (3) We pretreated with a reasonably low dose of L-NAME (10 nmol / min) in order to avoid the possible confound associated with a hyperten¨ sive effect as seen by Naveri et al. [19]. A recent report by Patel and Block [31] provides additional support to the present results in that AngIV was found to increase the activation and production of ecNOS and cGMP, respectively, in porcine and human PAEC. Consistent with previous investigations [27,38,40,45] these researchers concluded that AngIV may play a functional role in the regulation of blood flow in pulmonary circulation, possibly through the release of NO. Although the present study provides additional information concerning the physiological activity of AngIV analogs within the cerebral vasculature, there were inconsistencies. The predicted efficacy of the AngIV analogs employed in this study was based on affinities established using bovine adrenal membranes as a source of AT 4 receptor. Recent data from our laboratory (Zhang, unpublished observation) indicate that the AT 4 receptor exists as multiple subtypes and that the bovine adrenal and guinea pig hippocampal receptors may be different. As such, the predictive value of adrenal data is suspect. However, until further neocortex and hippocampal information is available, we are forced to utilize present structure-binding data. In conclusion, the present study demonstrated that intraarterial infusions of AngIV, and three AngIV analogs, increased CBF without affecting systemic blood pressure in the rat, and this elevation appeared to be NO dependent. Thus, an endothelium-dependent process, such as the synthesis and release of NO, may be the mechanism by which these ligands produce vasodilation in the cerebral microvasculature. It should be acknowledged that the laser-Doppler flowmetry technique provides a measure of relative, not absolute blood flow, and therefore it will be necessary to use techniques that yield absolute blood flow changes in order to further test the present conclusions.

Acknowledgements We wish to extend our thanks to Bryan Slinker for statistical consultation. This investigation was supported by funds provided by Washington State University, Hedral Therapeutics, and The Laing Endowment for research on Alzheimer’s Disease.

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