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CARDIOVASCULAR

Regional Hemodynamic Effects of the N-(2-Benzoylphenyl)-L-tyrosine Peroxisome Proliferator-Activated Receptor- Ligand, GI 262570 [(S)-2-(2-Benzoylphenylamino)-3-[4-[2-(5-methyl-2-phenyl-2-oxazol-4-yl)ethoxy]phenyl]propionic Acid], in Conscious Rats

Centre for Integrated Systems Biology and Medicine, School of Biomedical Sciences, University of Nottingham Medical School, Queen's Medical Centre, Nottingham, United Kingdom (S.M.G., T.B.); and GlaxoSmithKline, Research Triangle Park, North Carolina (D.J.R.N., P.G.B., K.K.B.)

Received March 23, 2004; accepted May 3, 2004.

	Abstract

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This study provides novel data on the regional hemodynamic effects of the peroxisome proliferator-activated receptor- activator, GI 262570 [(S)-2-(2-benzoylphenylamino)-3-[4-[2-(5-methyl-2-phenyl-2-oxazol-4-yl)ethoxy]phenyl]propionic acid], in conscious, male Sprague-Dawley rats. Administration of GI 262570 twice daily for 4 days caused a slowly developing, modest fall in mean arterial blood pressure, associated with a progressive, hyperemic hindquarters vasodilatation, but with no consistent changes in renal or mesenteric hemodynamics. The hindquarters vasodilator effect of GI 262570 was not inhibited by the ₂-adrenoceptor antagonist, ICI 118551 ((±)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl) amino]-2-butanol hydrochloride), and was still apparent in the presence of the -adrenoceptor antagonist, phentolamine. Neither the latter, nor antagonism of angiotensin (AT₁) and endothelin (ET_A and ET_B) receptors unmasked vasodilator responses to GI 262570 in the renal or mesenteric vascular beds. In the presence of GI 262570, vasodilator responses to acetylcholine and vasoconstrictor responses to methoxamine were normal. Furthermore, the cardiovascular responses to nonselective nitric-oxide synthase inhibition were not influenced by GI 262570. Collectively, these results indicate that the vasodilator action of GI 262570 is specific to the hindquarters vascular bed (of those studied), does not involve - or ₂-adrenoceptors, and is not associated with a change in basal or stimulated nitric oxide release.

A novel series of N-(2-benzoylphenyl)-L-tyrosine compounds has been identified as potent PPAR- agonists (Cobb et al., 1998; Collins et al., 1998; Henke et al., 1998) and shown to be antidiabetic in vivo (Brown et al., 1999). Of this series, GI 262570, a nonthiazolidinedione, tyrosine-based insulin sensitizer, is the most potent in vitro (Willson et al., 2000), but there are few published data on the in vivo cardiovascular effects of this compound (Grillot et al., 2001; Callahan et al., 2002). Such data would be of value because of the potential link between muscle hemodynamics and glucose disposal resulting from PPAR--mediated increases in insulin sensitivity (Baron, 1994). Given the dearth of information about the hemodynamic consequences of PPAR- activation in the nondiabetic state generally, and the relative lack of detail about the cardiovascular actions of GI 262570 in particular, the first objective of the present study was to delineate the regional hemodynamic responses to acute and chronic administration of GI 262570 in conscious rats. Since those initial experiments revealed a striking, selective, hindquarters hyperemic vasodilator effect of GI 262570, further experiments were designed to explore mechanisms.

First, we investigated the possibility that the hindquarters vasodilator effect of GI 262570 was mediated by ₂-adrenoceptors, since GI 262570-induced changes in insulin-mediated glucoregulation could influence adrenal medullary adrenaline release, and adrenaline-induced ₂-adrenoceptor activation is particularly prominent in the skeletal muscle vascular bed (Gardiner and Bennett, 1988; Gardiner et al., 1991b).

Second, we determined whether or not activation of compensatory vasoconstrictor systems (renin-angiotensin, endothelin, sympathetic), in the presence of GI 262570, might oppose any vasodilator influence in renal and mesenteric vascular beds. In addition, using this approach, we were able to determine whether GI 262570-induced inhibition of such vasoconstrictor mechanisms in the hindquarters was responsible for the vasodilatation observed specifically in that region.

Third, we explored the possibility that up-regulation of basal and/or stimulated nitric oxide release was involved in the cardiovascular actions of GI 262570.

	Materials and Methods

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Male, Sprague-Dawley rats (initial weights 300-325 g) were obtained from Charles River (Margate, Kent, UK) at least 2 weeks before undergoing any procedure. During that period they were group-housed (4-5/cage) in a temperature-controlled environment (20-22°C), with a 12-h light/dark cycle (lights on at 6:00 AM) and free access to tap water and food (BeeKay Foods, Hull, UK; Standard Rat Diet BK 001E). The procedures were approved by the University of Nottingham Ethical Review Committee and were performed under Home Office Project License authority.

Surgical Preparation. Following the settling-in period, rats (now weighing 350-400 g) were anesthetized (fentanyl and medetomidine, 300 µg kg^-1 i.p. of each, supplemented as required), and had pulsed Doppler probes implanted around the left renal and superior mesenteric arteries, and the distal abdominal aorta, through a mid-line laparotomy (Gardiner and Bennett, 1988; Gardiner et al., 1991b). Postsurgery, anesthesia was reversed and analgesia provided with atipamezole and nalbuphine, respectively (1 mg kg^-1 s.c. of each). Thereafter, animals were housed individually for 7 to 14 days before implantation of intra-arterial (abdominal aorta via ventral caudal artery) and intravenous (right jugular vein) catheters. Procedures for anesthesia, reversal, and analgesia for catheterization were as above. Following catheterization, animals were housed in individual home cages for the whole of the experimental protocol with access to food and water ad libitum.

Cardiovascular Recordings. Experiments began the day after catheter implantation. From previous studies using the approaches described here, we have seen no evidence of hangover effects of the anesthetic regimen (Wakefield et al., 2003; Woolard et al., 2004). Furthermore, in the present study, hemodynamic responses to vasoconstrictor and vasodilator challenges were consistent across the four experimental days in vehicle-infused rats (see Results). Animals were connected (via a tether system) to a transducer amplifier (Gould Instrument Systems Inc., Cleveland, OH) and Doppler flow-meter (Crystal Biotech, Northborough, MA) which were, in turn, connected to a custom-designed microprocessor system (Instrumentation Laboratories, University of Maastricht, Maastricht, The Netherlands). The system was set to sample the signals (arterial blood pressure and renal, mesenteric, and hindquarters Doppler shifts) every 2 ms, average them every cardiac cycle, and store them to disc at 5-s intervals. Recordings were made as described under Data Analysis (below).

Experiment 1: Regional Hemodynamic Responses to GI 262570. GI 262570 is not soluble in saline; furthermore, polyethylene glycol, as used for oral dosing (e.g., Yang et al., 2003), cannot be used i.v. Therefore, GI 262570 (2 mg ml^-1) was solubilized in a vehicle containing glycine (225.2 mg), NaOH (0.1 M, 12.1 ml), NaCl (805.9 mg), and sterile water (to make up to 100 ml). GI 262570 or vehicle was infused (0.4 ml h^-1 i.v.) continuously for 2 h, twice daily (between 7:45 AM and 9:45 AM, and between 1:30 PM and 3:30 PM) for 4 days. The continuous infusions of vehicle or GI 262570 were given to different rats (n = 9 in each group) through 0.22-µm filters. This infusion rate of GI 262570 produced a daily drug exposure in the rats which was equivalent to that achieved by oral dosing (8 mg kg^-1 b.i.d.) of nondiabetic rats and shown to have hemodynamic effects (Callahan et al., 2002). This dose has also been shown to be maximally efficacious for glucose lowering in diabetic rodents (Brown et al., 2000), although above what would be expected for efficacy in patients with type 2 diabetes mellitus (Fiedorek et al., 2000).

Experiment 2: Effects of ICI 118551 on Regional Hemodynamic Responses to GI 262570. Rats were given GI 262570 together with the ₂-adrenoceptor antagonist, ICI 118551 ((±)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol hydrochloride) (0.2 mg kg^-1; 0.1 mg kg^-1 h^-1; n = 8), or its vehicle (saline, 0.4 ml h^-1; n = 8), over the same time course as the first experiment; i.e., ICI 118551 or its vehicle was administered for the 2-h periods during which GI 262570 was given. The dose of ICI 118551 used has been shown to have little or no effect on baseline hemodynamics but to cause marked inhibition of the hindquarters vasodilator responses to exogenous and endogenous ₂-adrenoceptor agonists (Gardiner and Bennett, 1988; Gardiner et al., 1992, 2002b).

Experiment 3: Effects of ZD 7155 (5,7-Diethyl-3,4-dihydro-1-[[2'-(1H-tetrazol-5-yl)[1,1'-biphenyl]-4-yl]methyl]-1,6-naphthyridin-2(1H)-one Hydrochloride) and SB 209670 [(+)-(1S,2R,3S)-3-(Carboxymethoxy-4-methoxyphenyl)-1-13,4-methylenedioxyphenyl)-5-(prop-1-yloxy)indane-2-carboxylic Acid] or Phentolamine on Regional Hemodynamic Responses to GI 262570. There were six experimental groups in this part of the study. Groups 1 and 2 were given GI 262570 (2 mg ml^-1 at 0.4 ml h^-1, n = 8) or vehicle (as above, n = 8) twice daily, at the same times as in experiment 1, together with sterile saline (0.4 ml h^-1), which was the vehicle for the antagonists used (see below).

Groups 3 and 4 were given the AT₁-receptor antagonist, ZD 7155 (Junggren et al., 1996) as a bolus at a dose of 2 mg kg^-1 (i.e., 0.8 mg in 0.1 ml). In pilot studies (n = 2), we confirmed that this dose of ZD 7155 blocked responses to exogenous angiotensin II (50 pmol). Simultaneously, the ET_A/ET_B-receptor antagonist, SB 209670, was administered as a primed infusion (600 µgkg^--1 bolus, 600 µg kg^-1 h^-1 infusion; bolus in 0.1 ml, infusion at 0.4 ml h^-1) (Gardiner et al., 1996), and the administration of GI 262570 (n = 8) or vehicle (n = 8) was begun (as above), such that the infused drugs reached the animal together, 15 min later, and the 2-h measurement period began from that time point. We adopted this approach to optimize the chances of seeing any influence of the endogenous vasoconstrictor systems. Since no such effect was observed (see Results), we considered it unnecessary to examine the effects of inhibiting either the renin-angiotensin system or endothelin alone.

Groups 5 and 6 were given phentolamine as a primed infusion (1 mg kg^-1 bolus, 1 mg kg^-1 h^-1 infusion; bolus in 0.1 ml, infusion at 0.4 ml h^-1) (Janssen et al., 1991), and at the same time the infusion of GI 262570 (n = 8) or vehicle (n = 8) was begun (as above).

Experiment 4: Responses to Acetylcholine, Methoxamine, and N^G-Nitro-L-arginine Methyl Ester (L-NAME) in the Presence of GI 262570. After the last recording period following the afternoon infusion session on all four experimental days, all animals in experiment 3 were challenged with randomized 3-min infusions (0.15 ml min^-1) of acetylcholine (27 µg ml^-1) and methoxamine (200 µg ml^-1). Between each infusion, sufficient time (>15 min) was left for all variables to return to their preinfusion values. On the fourth experimental day only, and at least 15 min after the last infusion of either acetylcholine or methoxamine, a bolus injection of L-NAME was given (10 mg kg^-1) (Gardiner et al., 1990) and recordings continued for an additional 30 min.

Data Analysis. On each experimental day, data were recorded continuously for about 10 h. For measurement purposes, the experimental day was divided into five blocks: 1) the initial 30- to 45-min period, 2) the first 2-h infusion period, 3) the interval of about 3 to 4 h between the infusions, 4) the second infusion period, and 5) the terminal recording period of about 1 h. The data collected in the first measurement period of the first day were taken as baseline, and all subsequent values were expressed as changes relative to these baseline values. Responses to acetylcholine or methoxamine were measured during the last minute of the 3-min infusion, when all variables were steady. Responses to L-NAME were measured 10 min after injection, when steady state had been achieved.

The measured variables were extracted into a custom-designed statistical package that performed all arithmetical calculations (means, standard errors, etc.) and allowed appropriate statistical analysis. As described elsewhere (Gardiner and Bennett, 1988), under the conditions of our experiments, percentage changes in Doppler shift are directly related to percentage changes in blood flow and are used to calculate percentage changes in vascular conductance, where vascular conductance is Doppler shift/mean blood pressure.

Within a group, changes relative to baseline were assessed using Friedman's test (Theodorsson-Norheim, 1987); between-group analysis was by the Mann-Whitney U test, applied to integrated responses (areas under or over curves). The significance level was set at P 0.05.

Drugs. GI 262570 was provided by GlaxoSmithKline (Research Triangle Park, NC) as a sterile solution (2 mg ml^-1) in a glycine-based vehicle. SB 209670 was a gift from Dr. E. Ohlstein (GlaxoSmithKline, Research Triangle, NC). ICI 118551 and ZD 7155 were purchased from Tocris Cookson Inc. (Bristol, UK). Phentolamine [(2-[N-(m-hydroxyphenyl)-p-toluidinomethyl]-imidazoline) methanesulfonate], acetylcholine chloride, methoxamine hydrochloride, and L-NAME were purchased from Sigma Chemical (Poole, Dorset, UK).

Anesthetic, analgesic, and reversing agents were as follows: fentanyl citrate (Martindale, Essex, UK), medetomidine hydrochloride (Domitor; Pfizer, Kent, UK), nalbuphine hydrochloride (Nubain; Bristol-Myers Squibb, Hounslow, UK), and atipamezole hydrochloride (Antisedan; Pfizer).

	Results

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Experiment 1: Regional Hemodynamic Responses to GI 262570
Resting cardiovascular variables for these groups of animals before treatment on day 1 are shown in Table 1, and Fig. 1 illustrates all the data as changes relative to these baseline values, throughout the experiment.

View larger version (35K): [in this window] [in a new window]   Fig. 1. Cardiovascular responses (mean ± S.E.) to vehicle (open circles, n = 9) or GI 262570 (closed circles, n = 9). Infusions of GI 262570 (2 mg ml-1) or glycine-based vehicle were given twice daily for 2 h on four experimental days. On each experimental day, data were recorded continuously for about 10 h. For measurement purposes, the experimental day was divided into five blocks: 1) the initial 30- to 45-min period, 2) the first 2-h infusion period, 3) the interval of about 3 to 4 h between the infusions, 4) the second infusion period, and 5) the terminal recording period of about 1 h. The first measurement period on day 1 was taken as the original baseline. , P < 0.05 versus original baseline (Friedman's test); #, P < 0.05 for integrated responses (areas under or over curves) (Mann-Whitney U test).

Across the 4 days of the experiment, the group of animals receiving vehicle showed no significant changes in their arterial blood pressure, heart rate, renal flow, or vascular conductance (Fig. 1). However, hindquarters flow and vascular conductance tended to drift downward, and some of the reductions on day 3 were significant (Fig. 1). Mesenteric flow and vascular conductance showed clear circadian variation in the vehicle-infused rats, values being highest at the beginning of the daily recording period (about 7:00 AM) (Fig. 1). We have noted this phenomenon elsewhere (Gardiner et al., 1995) and have attributed it to a waning of postprandial hyperemia during the day, with the rats sleeping during the light phase, having eaten during the dark phase (6:00 PM to 6:00 AM).

In the animals given GI 262570, there was an initial rise and then a delayed, but progressive fall in mean arterial blood pressure and variable increases in heart rate over the four experimental days. For both variables, the integrated changes (areas under or over curves) were significantly different from those in animals given vehicle (Fig. 1). Although animals given GI 262570 showed a fall in renal flow, the integrated change was not different from that in the vehicle-treated group, and there was no difference in renal vascular conductance between the two groups (Fig. 1). The pattern of change in mesenteric flow was not different between GI 262570- and vehicle-treated groups, but the former showed relatively less mesenteric vasoconstriction, specifically since they showed a more marked "postprandial" mesenteric vasodilatation. Hence, the integrated changes in mesenteric vascular conductance in the two groups were significantly different (Fig. 1). The most striking effect of GI 262570 was in the hindquarters, where it caused substantial increases in flow and vascular conductance that were significantly greater than the changes seen in vehicle-treated animals (Fig. 1).

Experiment 2: Effects of ICI 118551 on Regional Hemodynamic Responses to GI 262570
Resting cardiovascular variables for these groups of animals before treatment on day 1 are shown in Table 1, and Fig. 2 illustrates all the data as changes relative to these baseline values, throughout the experiment.

View larger version (34K): [in this window] [in a new window]   Fig. 2. Cardiovascular effects (mean ± S.E.) of GI 262570 in the absence (closed circles, n = 8) and presence (closed triangles, n = 8) of 2-adrenoceptor antagonism with ICI 118551. See Fig. 1 legend for explanation of measurement periods. , P < 0.05 versus original baseline (Friedman's test).

Animals receiving the vehicle for ICI 118551 together with GI 262570 showed hemodynamic changes qualitatively similar to those receiving GI 262570 in experiment 1 (Fig. 2). Moreover, in those animals given ICI 118551 together with GI 262570, there was no significant effect on any of the hemodynamic actions of the latter compound, and certainly not on its hyperemic, vasodilator action in the hindquarters (Fig. 2).

Experiment 3: Effects of ZD 7155 and SB 209670, or Phentolamine, on Regional Hemodynamic Responses to GI 262570
Resting cardiovascular variables for these groups of animals before treatment on day 1 are shown in Table 1 and Figs. 3, 4, 5 illustrate all the data as changes relative to these baseline values, throughout the experiments.

View larger version (35K): [in this window] [in a new window]   Fig. 3. Cardiovascular responses (mean ± S.E.) to vehicle (open circles, n = 8) or GI 262570 (closed circles, n = 8) given in the presence of saline. See Fig. 1 legend for explanation of measurement periods. , P < 0.05 versus original baseline (Friedman's test); #, P < 0.05 for integrated responses (areas under or over curves) (Mann-Whitney U test).

View larger version (39K): [in this window] [in a new window]   Fig. 4. Cardiovascular responses (mean ± S.E.) to vehicle (open circles, n = 8) or GI 262570 (closed circles, n = 8) given in the presence of angiotensin (AT1) receptor antagonism (with ZD 7155) and endothelin (ETA and ETB) receptor antagonism (with SB 209670). See Fig. 1 legend for explanation of measurement periods. , P < 0.05 versus original baseline (Friedman's test); #, P < 0.05 for integrated responses (areas under or over curves) (Mann-Whitney U test).

View larger version (35K): [in this window] [in a new window]   Fig. 5. Cardiovascular responses (mean ± S.E.) to vehicle (open circles, n = 8) or GI 262570 (closed circles, n = 8) given in the presence of -adrenoceptor antagonism (with phentolamine). See Fig. 1 legend for explanation of measurement periods. , P < 0.05 versus original baseline (Friedman's test); #, P < 0.05 for integrated responses (areas under or over curves) (Mann-Whitney U test).

Effects of GI 262570 or Vehicle
As in the previous study, GI 262570 caused clear hyperemic vasodilatation in the hindquarters together with increases in heart rate, and both these effects were significantly greater than that seen with vehicle (Fig. 3). However, compared with the previous study, the hypotensive effect of GI 262570 was only slight, and not different from that of vehicle, and there was no postprandial mesenteric hyperemia (Fig. 3).

Effects of ZD 7155 + SB 209670 on Responses to GI 262570
Coadministration of ZD 7155 and SB 209670 caused significant hypotension and tachycardia, accompanied by increases in renal, mesenteric and hindquarters flows, although the times of onset of these effects differed (Fig. 4). However, there was dilatation in all three vascular beds during the first infusion period on the first experimental day (Fig. 4). A waning of the effects of ZD 7155 and SB 209670 between experimental days (i.e., when they were not being administered) was apparent (Fig. 4). The increases in renal flow and vascular conductance seen in the presence of ZD 7155 and SB 209670 were significantly less when GI 262570 was coadministered, but the latter had an additional dilator effect in the hindquarters vascular bed (Fig. 4).

Effects of Phentolamine on Responses to GI 262570
On initial administration, on the first experimental day, phentolamine caused a transient hypotension and a substantial tachycardia (Fig. 5). These effects were accompanied by slight, transient reductions in renal and mesenteric flows and vascular conductances, but very marked increases in hindquarters flow and vascular conductance (Fig. 5). The effects of phentolamine waned with repeated administration over the four experimental days (Fig. 5). In the presence of phentolamine, GI 262570 caused enhanced hypotension and tachycardia, and the integrated vasodilator changes in the hindquarters were significantly greater in the presence of phentolamine and GI 262570 than in the presence of phentolamine alone (Fig. 5).

Experiment 4: Responses to Acetylcholine, Methoxamine, and L-NAME in the Presence of GI 262570
Responses to Acetylcholine. In animals given saline and the vehicle for GI 262570, there was a marked increase in renal vascular conductance at the end of a 3-min infusion of acetylcholine (+37 ± 5, +35 ± 4, +39 ± 6, and +48 ± 5% on days 1, 2, 3, and 4, respectively); the corresponding changes in animals treated with GI 262570 were not significantly different (+44 ± 13, +44 ± 9, +43 ± 8, and +51 ± 8%, respectively).

Responses to Methoxamine. On each experimental day, infusion of methoxamine had significant pressor and brady-cardic effects, accompanied by renal, mesenteric, and hindquarters vasoconstriction, and these effects were not influenced by GI 262570 (data not shown).

Responses to L-NAME. In animals given saline and vehicle, 10 min after injection of L-NAME, there was marked hypertension (+55 ± 5 mm Hg), bradycardia (-58 ± 8 beats min^-1), and falls in renal, mesenteric, and hindquarters vascular conductances (-56 ± 4, -72 ± 7, and -60 ± 5%, respectively); the corresponding changes in animals given saline and GI 262570 were not significantly different (hypertension, +51 ± 2 mm Hg; bradycardia, -87 ± 9 beats min^-1; falls in renal, mesenteric, and hindquarters vascular conductances, -52 ± 5, -67 ± 1, and -52 ± 3%, respectively).

	Discussion

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The degree to which the cardiovascular actions of thiazolidinediones involve changes in insulin sensitivity is unresolved, as is the possibility that some of the effects of these drugs are independent of PPAR- (see the Introduction). In the light of hepatic effects seen with troglitazone, a series of chiral, nonracemate, N-(2-benzoylphenyl)-L-tyrosine PPAR- agonists have been synthesized (Henke et al., 1998). They are more potent than troglitazone and hence may show greater therapeutic effect in type 2 diabetes mellitus (Henke et al., 1998). However, nothing is known of the putative regional hemodynamic effects of these compounds in the nondiabetic state. Therefore, the first objective of the present work was to assess the regional hemodynamic changes occurring in conscious, chronically instrumented rats treated over 4 days with the N-(2-benzoylphenyl)-L-tyrosine PPAR- ligand, GI 262570 (Henke et al., 1998), or its vehicle.

Our first experiment demonstrated clearly that GI 262570 selectively increased hindquarters flow (increased skeletal muscle blood flow) and vascular conductance progressively over a 4-day, intermittent infusion protocol. The magnitude of this effect, and its time course, fit with the proposition that the previously observed effects of GI 262570 on cardiac output and total peripheral conductance (Callahan et al., 2002) were attributable to the hindquarters hyperemic action of the drug.

The selectivity of the vasodilator action of GI 262570 for the hindquarters vascular bed raised the possibility that ₂-adrenoceptor activation contributed to this effect, since we have shown marked vasodilator responses to ₂-adrenoceptor agonists in the hindquarters vascular bed (Gardiner and Bennett, 1988; Gardiner et al., 1991b), and adrenomedullary adrenaline release would be expected to activate this process particularly effectively (Gardiner et al., 1992).

For this reason, in the second experiment, we assessed the effects of the ₂-adrenoceptor antagonist, ICI 118551, on the hindquarters vasodilator effects of GI 262570. Those experiments showed very clearly that ICI 118551 was entirely without inhibitory effect on the hindquarters vasodilator action of GI 262570, consistent with no role of ₂-adrenoceptors in this phenomenon. Moreover, these observations indicated that a putative vasodilator effect of insulin, mediated through -adrenoceptors (Creager et al., 1985), possibly as a result of insulin-mediated sensitization of adenylyl cyclase activation (e.g., Feldman, 1993), did not contribute to the hindquarters vasodilator action of GI 262570. Nonetheless, the apparent selectivity of the vasodilator effect of GI 262570 in the hindquarters echoes the insulin-mediated glucose uptake which occurs principally in skeletal muscle (see Baron, 1994, for review). However, as Yki-Järvinen and Utriainen (1998) have pointed out, physiological levels of insulin that substantially stimulate muscle glucose uptake have little effect on skeletal muscle blood flow, consistent with the former event being independent of the latter, although changes in microvascular hemodynamics might be important (Vincent et al., 2002).

The findings above raised the possibility that the apparent selectivity of the vasodilator action of GI 262570 for the hindquarters was due to the drug-induced changes in hemodynamics evoking compensatory activation of vasoconstrictor mechanisms (renin-angiotensin, sympathetic) which offset any direct GI 262570-induced vasodilatation in the renal and mesenteric vascular beds. In addition, it is feasible that endothelin could have contributed to such an effect, since GI 262570, which increases insulin sensitivity, could thereby stimulate endothelin release (Misurski et al., 2001; Verma et al., 2001), although there is also evidence that PPAR- activators inhibit endothelin release from endothelial cells (Delerive et al., 1999; Satoh et al., 1999; Fukunaga et al., 2001).

For these reasons, the third experiment was designed to assess effects of GI 262570 in the absence and presence of antagonists of AT₁-, and ET_A- and ET_B-receptors (with ZD 7155 and SB 209670, respectively), or of antagonism of ₁-and ₂-adrenoceptors (with phentolamine), to detect any signs of unmasking of renal and/or mesenteric vasodilator actions of the drug and, at the same time, to determine whether GI 262570 exerted greater or lesser hindquarters vasodilator effects in the presence of the antagonists.

In the presence of ZD 7155 and SB 209670, there was no unmasking of a renal or mesenteric vasodilator effect of GI 262570. Indeed, the latter appeared to suppress the increases in renal flow and vascular conductance caused by coadministration of ZD 7155 and SB 209670. It is feasible that this was due to GI 262570 causing renal "steal" through further increase in hindquarters flow and vascular conductance (see below), although this phenomenon was not apparent on the first experimental day, when the suppression of the ZD 7155- and SB 209670-induced increases in renal flow and vascular conductance were present (Fig. 4). Since coadministration of ZD 7155 and SB 209670 did not augment the vasodilator effect of GI 262570, we did not consider it necessary to examine the effects of either antagonist alone. However, we acknowledge that, with the use of selective antagonists of ET_A- and ET_B-receptors, one could gain more information regarding a possible vascular effect of endothelin.

Although there is some evidence that glitazones can inhibit Ca²⁺ channels and thereby suppress the action of vasoconstrictors (e.g., Buchanan et al., 1995), the observation that additional hypotensive and hindquarters vasodilator effects of GI 262570 were seen in the presence of ZD 7155 and SB 209670 indicates that these effects normally do not depend upon inhibition of the constrictor actions of angiotensin II and/or endothelin.

We noted less marked recovery in blood pressure following coadministration of ZD 7155 and SB 209670 in the presence of GI 262570, compared with that in its absence (see Figs. 3 and 4 at the start of each experimental day). Considering the long duration of action of the AT₁-receptor antagonist used (Junggren et al., 1996), it is likely that the recovery seen in the vehicle-treated animals was due to a waning of the action of SB 209670. Therefore, these data could indicate a modulation by GI 262570 of either endothelin's action, or the pharmacokinetics of SB 209670.

The dramatic hemodynamic effect of phentolamine administration alone is consistent with previous studies from our laboratory, in which we have shown a substantial component of the hindquarters vasodilatation to be dependent on activation of ₂-adrenoceptors (Gardiner and Bennett, 1988; Woolard et al., 2004). It was interesting to see, here, that with repeated phentolamine administration, the degree of hindquarters vasodilatation, but not the tachycardia, waned.

In the presence of phentolamine, GI 262570 still caused additional hindquarters vasodilatation; moreover, GI 262570 did not modify vasoconstrictor responses to methoxamine. These findings are consistent with the hindquarters vasodilator effects of GI 262570 being due to something other than inhibition of -adrenoceptor-mediated tone. Furthermore, as in the case of the experiments performed in the presence of ZD 7155 and SB 209670, there was no renal or mesenteric vasodilator action of GI 262570 unmasked in the presence of phentolamine. Thus, collectively, the results indicate that the vasodilator effect of GI 262570 is specific for the hindquarters (of the vascular beds monitored) and is largely independent of angiotensin (AT₁) and endothelin (ET_A and ET_B) receptors, and ₁- and ₂-adrenoceptors.

We know from previous studies that the renal vasodilator effect of acetylcholine infused over 3 min (as in the present study) is inhibited by the nitric-oxide synthase inhibitor, L-NAME, and is therefore likely to be due to stimulated release of NO (Gardiner et al., 1991a). The lack of effect of GI 262570 on either acetylcholine-induced renal vasodilatation or L-NAME-induced hemodynamic changes thus suggests that the compound did not influence either stimulated or basal release of NO. This is somewhat surprising, considering that insulin increases vascular NO production, although such an effect would not be expected to have been confined to the hindquarters (Hilzenrat et al., 2001). But, although an NO-dependent component of the vasodilator action of insulin is widely acknowledged (see Hsueh and Law, 1999, for review; Bursztyn et al., 2000; Molinari et al., 2001), there is evidence that the vasodilator action of thiazolidenediones on rat coronary vessels (Uchida et al., 2000) and the human forearm (Fujishima et al., 1998) is NO-independent. In fact, there is evidence that PPAR- ligands may suppress inducible nitric-oxide synthase expression while increasing heme oxygenase-1 expression in rat glial cells (Kitamura et al., 1999). Thus, one could hypothesize that carbon monoxide may contribute to the vasodilator action of compounds such as GI 262570. A very recent study (Yang et al., 2003) reported that 10 days' treatment with GI 262570 produced significant elevation of plasma and skeletal muscle nitrate and nitrite levels, but these data are not inconsistent with our findings since no significant changes in the measured indices of NO activity were seen after 4 days of treatment (Yang et al., 2003).

In conclusion, although PPAR- has been localized to cardiovascular tissues, our data showing marked differences in regional hemodynamic effects of GI 262570, and the lack of effect thereupon of pharmacological interventions, perhaps make it unlikely that the compound exerts a primary vascular action. We acknowledge the fact that our study is limited to the normal state, and that effects in pathological conditions, such as diabetes and hypertension, may differ from those described here.

	Acknowledgements

 
We thank Professor Paul Greenhaff for comments on the manuscript.

	Footnotes

 
This study was undertaken with financial support from GlaxoSmithKline. Some of the results reported herein have been presented to the British Pharmacological Society (Gardiner et al., 2002a).

doi:10.1124/jpet.104.068817.

ABBREVIATIONS: PPAR-, peroxisome proliferator-activated receptor-; GI 262570, (S)-2-(2-benzoylphenylamino)-3-[4-[2-(5-methyl-2-phenyl-2-oxazol-4-yl)ethoxy]phenyl]propionic acid; ICI 118551, (±)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol hydrochloride; ZD 7155, 5,7-diethyl-3,4-dihydro-1-[[2'-(1H-tetrazol-5-yl)[1,1'-biphenyl]-4-yl]methyl]-1,6-naphthyridin-2(1H)-one hydrochloride; SB 209670, (+)(1S,2R,3S)-3-(carboxymethoxy-4-methoxyphenyl)-1-13,4-methylenedioxyphenyl)-5-(prop-1-yloxy)indane-2-carboxylic acid; L-NAME, N^G-nitro-L-arginine methyl ester; AT₁, angiotensin type 1 receptor; ET_A or ET_B, endothelin type A or B receptor; NO, nitric oxide.

Address correspondence to: Professor Sheila M. Gardiner, School of Biomedical Sciences, University of Nottingham Medical School, Queen's Medical Centre, Nottingham NG7 2UH, UK. E-mail: sheila.gardiner{at}nottingham.ac.uk

	References

Top Abstract Materials and Methods Results Discussion References

 

Baron AD (1994) Hemodynamic actions of insulin. Am J Physiol 267: E187-E202.

Bishop-Bailey D (2000) Peroxisome proliferator-activated receptors in the cardiovascular system. Br J Pharmacol 129: 823-834.[CrossRef][Medline]

Brown KK, Faison WL, Hashim M, Harrington WW, Binz J, and Oliver W Jr (2000) Antidiabetic efficacy of GI 262570 in rodent models of type 2 diabetes. Diabetes 49 (Suppl 1): A278.

Brown KK, Henke BR, Blanchard SG, Cobb JE, Mook R, Kaldor I, Kliewer SA, Lehmann JM, Lenhard JM, Harrington WW, et al. (1999) A novel N-Aryl tyrosine activator of the peroxisome proliferator-activated receptor-gamma reverses the diabetic phenotype of the Zucker diabetic fatty rat. Diabetes 48: 1415-1424.[Abstract]

Buchanan TA, Meehan WP, Jeng YY, Yang D, Chan TM, Nadler JL, Scott S, Rude RK, and Hsueh WA (1995) Blood pressure lowering by pioglitazone. J Clin Investig 96: 354-360.

Bursztyn M, Mekler J, Peleg E, and Bernheim J (2000) Subpressor dose of L-NAME unmasks hypertensive effects of chronic hyperinsulinemia. Hypertension 36: 872-877.[Abstract/Free Full Text]

Callahan MF, Brown KK, Baer PG, and Smith TL (2002) Effects of PPAR agonist, GI 262570, on cardiac output (CO), arterial blood pressure (MAP) and systemic vascular resistance (SVR) in conscious rats (Abstract). Circulation 106: 560.

Cobb JE, Blanchard SG, Boswell GE, Brown KK, Charifson PS, Cooper JP, Collins JL, Dezube M, Henke BR, Hull-Ryde EA, et al. (1998) N-(2-Benzoylphenyl)-L-tyrosine PPAR agonists. 3. Structure-activity relationship and optimization of the N-Aryl substituent. J Med Chem 41: 5055-5069.[CrossRef][Medline]

Collins JL, Blanchard SG, Boswell GE, Charifson PS, Cobb JE, Henke BR, Hull-Ryde EA, Kazmierski WM, Lake DH, Leesnitzer LM, et al. (1998) N-(2-Benzoylphenyl)-L-tyrosine PPAR agonists. 2. Structure-activity relationship and optimization of the phenyl alkyl ether moiety. J Med Chem 41: 5037-5054.[CrossRef][Medline]

Creager MA, Liang C-S, and Coffman JD (1985) -Adrenergic-mediated vasodilator response to insulin in the human forearm. J Pharmacol Exp Ther 235: 709-714.[Abstract/Free Full Text]

Delerive P, Martin-Nizard F, Chinetti G, Trottein F, Fruchart JC, Najib J, Duriez P, and Staels B (1999) Peroxisome proliferator-activated receptor activators inhibit thrombin-induced endothelin-l production in human vascular endothelial cells by inhibiting the activator protein-1 signalling pathway. Circ Res 85: 394-402.[Abstract/Free Full Text]

Feldman RD (1993) Insulin-mediated sensitization of adenylyl cyclase activation. Br J Pharmacol 110: 1640-1644.[Medline]

Fiedorek FT, Wilson CG, Frith L, Patel J, and Abou-Donia M (2000) Monotherapy with GI 262570, a tyrosine-based non-thiazolidinedione PPAR gamma agonist, improves metabolic control in Type 2 diabetes mellitus patients. Diabetes 49 (Suppl 1): A38.

Fujishima S, Ohya Y, Nakamura Y, Onaka U, Abe I, and Fujishima M (1998) Troglitazone, an insulin sensitizer, increases forearm blood flow in humans. Am J Hypertens 11: 1134-1137.[CrossRef][Medline]

Fukunaga Y, Itoh H, Doi K, Tanaka T, Yamashita J, Chun T-H, Inoue M, Masatsugu K, Sawada N, Saito T, et al. (2001) Thiazolidinediones, peroxisome proliferator-activated receptor agonists, regulate endothelial cell growth and secretion of vasoactive peptides. Atherosclerosis 158: 113-119.[CrossRef][Medline]

Gardiner SM and Bennett T (1988) Regional hemodynamic responses to adrenoceptor antagonism in conscious rats. Am J Physiol 255: H813-H824.

Gardiner SM, Compton AM, Kemp PA, and Bennett T (1990) Regional and cardiac haemodynamic effects of N^G-nitro-L-arginine methyl ester in conscious, Long Evans rats. Br J Pharmacol 101: 625-631.[Medline]

Gardiner SM, Kemp PA, and Bennett T (1991a) Effects of N^G-nitro-L-arginine methyl ester on vasodilator responses to acetylcholine, 5'-N-ethylcarboxamidoadenosine or salbutamol in conscious rats. Br J Pharmacol 103: 1725-1732.[Medline]

Gardiner SM, Kemp PA, and Bennett T (1991b) Effects of N^G-nitro-L-arginine methyl ester on vasodilator responses to adrenaline or BRL 38227 in conscious rats. Br J Pharmacol 104: 731-737.[Medline]

Gardiner SM, Kemp PA, Bennett T, Bose C, Foulkes R, and Hughes B (1992) Involvement of ₂-adrenoceptors in the regional haemodynamic responses to bradykinin in conscious rats. Br J Pharmacol 105: 839-848.[Medline]

Gardiner SM, Kemp PA, March JE, Baer PG, Brown KK, Nunez D, and Bennett T (2002a) Regional haemodynamic effects of the PPAR activator, GI 262570, in conscious rats (Abstract). Br J Pharmacol 137: 29P.[CrossRef]

Gardiner SM, Kemp PA, March JE, and Bennett T (1995) Cardiac and regional haemodynamics, inducible nitric oxide synthase (NOS) activity and the effects of NOS inhibitors in conscious, endotoxaemic rats. Br J Pharmacol 116: 2005-2016.[Medline]

Gardiner SM, Kemp PA, March JE, and Bennett T (1996) Temporal differences between the involvement of angiotensin II and endothelin in the cardiovascular responses to endotoxaemia in conscious rats. Br J Pharmacol 119: 1619-1627.[Medline]

Gardiner SM, March JE, Kemp PA, and Bennett T (2002b) Influence of the CB₁ receptor antagonist, AM 251, on the regional haemodynamic effects of WIN-55212-2 or HU 210 in conscious rats. Br J Pharmacol 136: 581-587.[CrossRef][Medline]

Grillot DAM, Grondin P, Saintillan Y, and Mayoux E (2001) Antihypertensive effect of PPAR agonist GW 1929 in spontaneously hypertensive rats: a putative mechanism. Diabetes 50 (Suppl 2): 459P.

Henke BR, Blanchard SG, Brackeen MF, Brown KK, Cobb JE, Collins JL, Harrington WW, Hashim MA, Hull-Ryde EA, Kaldor I, et al. (1998) N-(2-Benzoylphenyl)-L-tyrosine PPAR agonists. 1. Discovery of a novel series of potent antihyperglycemic and antihyperlipidemic agents. J Med Chem 41: 5020-5036.[CrossRef][Medline]

Hilzenrat N, Yaari A, Maislos M, and Sikuler E (2001) Splanchnic and systemic haemodynamic effects of sustained euglycemic hyperinsulinemia in rats. Scand J Clin Lab Investig 61: 383-388.[Medline]

Hsueh WA and Law RE (1999) Insulin signalling in the arterial wall. Am J Cardiol 84: 21J-24J.[CrossRef][Medline]

Janssen PJ, Gardiner SM, Compton AM, and Bennett T (1991) Mechanisms contributing to the differential haemodynamic effects of bombesin and cholecystokinin in conscious, Long Evans rats. Br J Pharmacol 102: 123-134.[Medline]

Junggren IL, Zhao X, Sun X, and Hedner T (1996) Comparative cardiovascular effects of the angiotensin II type 1 receptor antagonists ZD 7155 and losartan in the rat. J Pharm Pharmacol 48: 829-833.[Medline]

Kitamura Y, Kakimura J-I, Matsuoka Y, Nomura Y, Gebicke-Haerter PJ, and Tamiguchi T (1999) Activators of peroxisome proliferator-activated receptor- (PPAR) inhibit inducible nitric oxide synthase expression but increase heme oxygenase-1 expression in rat glial cells. Neurosci Lett 262: 129-132.[CrossRef][Medline]

Klappacher GW and Glass CK (2002) Roles of peroxisome proliferator-activated receptor in lipid homeostasis and inflammatory responses of macrophages. Curr Opin Lipidol 13: 305-312.[CrossRef][Medline]

Martens FMAC, Visseren FLJ, Lemay J, de Koning EJP, and Rabelink TJ (2002) Metabolic and additional vascular effects of thiazolidinediones. Drugs 62: 1463-1480.[CrossRef][Medline]

Misurski DA, Wu SQ, McNeill JR, Wilson TW, and Gopalakrishnan V (2001) Insulin-induced biphasic responses in rat mesenteric vascular bed: role of endothelin. Hypertension 37: 1298-1302.[Abstract/Free Full Text]

Molinari C, Battaglia A, Bona G, Grossini E, Mary DASG, Ruggeri P, Stoker JB, and Vacca G (2001) Mechanisms of the renal vasodilation caused by insulin in anesthetized pigs. Life Sci 69: 1699-1708.[CrossRef][Medline]

Mudaliar S and Henry RR (2001) New oral therapies for type 2 diabetes mellitus: the glitazones or insulin sensitizers. Annu Rev Med 52: 239-257.[CrossRef][Medline]

Rival Y, Benéteau N, Taillandier T, Pezet M, Dupont-Passelaigue E, Patoiseau J-F, Junquéro D, Colpaert FC, and Delhon A (2002) PPAR and PPAR activators inhibit cytokine-induced nuclear translocation of NF-B and expression of VCAM-1 in EAhy926 endothelial cells. Eur J Pharmacol 435: 143-151.[CrossRef][Medline]

Rosen ED and Spiegelman BM (2001) PPAR: a nuclear regulator of metabolism, differentiation and cell growth. J Biol Chem 276: 37731-37734.[Free Full Text]

Satoh H, Tsukamoto K, Hashimoto Y, Hashimoto N, Togo M, Hara M, Maekawa H, Isoo N, Kimura S, and Watanabe T (1999) Thiazoldinediones suppress endothelin-l secretion from bovine endothelial cells: a new possible role for PPAR on vascular endothelial function. Biochem Biophys Res Commun 254: 757-763.[CrossRef][Medline]

Shiojiri T, Wada K, Nakajima A, Katayama K, Shibuya A, Kudo C, Kadowaki T, Mayumi T, Yura Y, and Kamisaki Y (2002) PPAR ligands inhibit nitrotyrosine formation and inflammatory mediator expressions in adjuvant-induced rheumatoid arthritis mice. Eur J Pharmacol 448: 231-238.[CrossRef][Medline]

Spiegelman BM (1998) PPAR-: adipogenic regulator and thiazolidinedione receptor. Diabetes 47: 507-514.[Abstract]

Theodorsson-Norheim E (1987) Friedman and Quade tests: BASIC computer program to perform non-parametric two-way analysis of variance and multiple comparisons on ranks of several related samples. Comput Biol Med 17: 85-99.[CrossRef][Medline]

Uchida K, Ogino K, Shimoyama M, Hisatome I, and Shigemasa C (2000) Acute hemodynamic effects of insulin-sensitizing agents in isolated perfused rat hearts. Eur J Pharmacol 400: 113-119.[CrossRef][Medline]

Verma S, Yao L, Stewart DJ, Dumont AS, Anderson TJ, and McNeill JH (2001) Endothelin antagonism uncovers insulin-mediated vasorelaxation in vitro and in vivo. Hypertension 37: 328-333.[Abstract/Free Full Text]

Vincent MA, Dawson D, Clark ADH, Lindner JR, Rattigan S, Clark MG, and Barrett EJ (2002) Skeletal muscle microvascular recruitment by physiological hyperinsulinemia precedes increases in total blood flow. Diabetes 51: 42-48.[Abstract/Free Full Text]

Wakefield ID, March JE, Kemp PA, Valentin J-P, Bennett T, and Gardiner SM (2003) Comparative regional haemodynamic effects of the nitric oxide synthase inhibitors, S-methyl-L-thiocitrulline and L-NAME, in conscious rats. Br J Pharmacol 139: 1235-1243.[CrossRef][Medline]

Willson TM, Brown PJ, Sternbach DD, and Henke BR (2000) The PPARs: from orphan receptors to drug discovery. J Med Chem 43: 527-550.[CrossRef][Medline]

Willson TM, Lambert MH, and Kliewer SA (2001) Peroxisome proliferator-activated receptor and metabolic disease. Annu Rev Biochem 70: 341-367.[CrossRef][Medline]

Woolard J, Bennett T, Dunn WR, Heal DJ, Aspley S, and Gardiner SM (2004) Acute cardiovascular effects of sibutramine in conscious rats. J Pharmacol Exp Ther 308: 1102-1110.[Abstract/Free Full Text]

Yang B, Clifton LG, McNulty JA, Chen L, Brown KK, and Baer PG (2003) Effects of a PPAR agonist, GI262570, on renal filtration fraction and nitric oxide level in conscious rats. J Cardiovasc Pharmacol 42: 436-441.[CrossRef][Medline]

Yki-Järvinen H and Utriainen T (1998) Insulin-induced vasodilatation: physiology or pharmacology? Diabetologia 41: 369-379.[CrossRef][Medline]

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