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CARDIOVASCULAR

Mesenteric Vasoconstriction and Hindquarters Vasodilatation Accompany the Pressor Actions of Exendin-4 in Conscious Rats

Centre for Integrated Systems Biology and Medicine, School of Biomedical Sciences, University of Nottingham, Nottingham, United Kingdom

Received July 22, 2005; accepted October 11, 2005.

	Abstract

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The hemodynamic effects of the glucagon-like peptide-1 (GLP-1) receptor agonist, exendin-4, and putative underlying mechanisms were assessed in conscious male Sprague-Dawley rats. At a dose of 25 ng kg^-1 i.v., exendin-4 had little effect, but doses of 250 and 2500 ng kg^-1 had significant tachycardic effects (+66 ± 9 and +95 ± 16 beats min^-1 at 5 min, respectively) and pressor actions (+10 ± 2 and +12 ± 1 mm Hg), accompanied by substantial falls in mesenteric vascular conductance (-38 ± 3% and -47 ± 3%) and increases in hindquarters vascular conductance (+82 ± 14% and +126 ± 15%). The latter were likely due to adrenaline-mediated activation of ₂ adrenoceptors since they were abolished 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], or propranolol [(RS)-1-[(1-methylethyl)amino]-3-(1-naphthalenyloxy)-2-propanol], and absent in adrenal-demedullated rats. In the presence of -adrenoceptor antagonism, the tachycardic effects of exendin-4 were suppressed, but the pressor action was enhanced. Enhancement of the pressor action of exendin-4 was not seen in adrenal-demedullated rats or in animals given phentolamine in addition to propranolol, consistent with a component of the pressor action of exendin-4 being due to an adrenaline-mediated positive inotropic effect mediated by -adrenoceptors. The mesenteric vasoconstrictor effect of exendin-4 was unaffected by antagonism of -adrenoceptors, vasopressin receptors, angiotensin receptors, or GLP-1 receptors, although antagonism of the latter substantially inhibited the hindquarter vasodilator effects of exendin-4. These results are consistent with exendin-4 having cardiovascular effects through GLP-1 receptor-dependent and -independent mechanisms, some of which involve sympathoadrenal activation.

GLP-1 receptors are found not only in the pancreas but also in the stomach, lung, kidney, heart, and in several regions of the brain, many of which are intimately involved in cardiovascular regulation (for review, see Kieffer and Habener, 1999). Because of their beneficial effects on glucose metabolism, GLP-1 and other "incretin mimetics" are promising therapeutic agents for the treatment of type 2 diabetes (Drucker, 2001; Knudsen, 2004), but their potential cardiovascular actions and any underlying mechanisms have not been fully elucidated. Barragán et al. (1994, 1996, 1999) performed a series of experiments concerned with the cardiovascular actions of the full-length GLP-1-(1-37), truncated GLP-1-(7-36) amide, and the structurally related peptide, exendin-4, in anesthetized rats. They showed modest effects of GLP-1-(1-37), but clear-cut pressor and tachycardic effects of GLP-1-(7-36) amide and similar, but more prolonged, effects of exendin-4. Using a variety of pharmacological probes and differing routes of administration, they came to the conclusion that the cardiovascular effects of GLP-1-(7-36) amide were due to both central and peripheral loci of action, with the former involving vagal efferent activation causing release of regulatory peptides such as neuropeptide Y and the latter depending on stimulation of GLP-1 receptors in "peripheral cardiovascular sites" but not mediated through activation of adrenoceptors (Barragán et al., 1999). The latter statement was made on the strength of the fact that neither propranolol nor phentolamine attenuated the pressor response to GLP-1-(7-36) amide. In fact, both adrenoceptor antagonists enhanced the pressor response to GLP-1-(7-36) amide, although Barragán et al. (1994) offered no interpretation of this observation.

In contrast to the conclusions of Barragán et al. (1994, 1996, 1999), more recently, Yamamoto et al. (2002, 2003) reported that exendin-4, administered either peripherally or centrally, caused activation of autonomic pathways in sites consistent with the pressor and tachycardic effects being due to sympathoadrenal activation. As yet, there has been no reconciliation of these apparently conflicting findings. However, none of the published studies on GLP-1-(7-36) amide or exendin-4 have measured the underlying hemodynamic changes accompanying the pressor effects. In the light of the findings reported above, we hypothesized that the cardiovascular effects of exendin-4 are an amalgam of sympathoadrenal activation (with -adrenoceptor-mediated vasoconstriction offsetting -adrenoceptor-mediated vasodilatation), vagal withdrawal (Barragán et al., 1999), vasopressin release (Bojanowska and Stempniak, 2002), and renin release (Malendowicz et al., 2003). To test our hypothesis, experiments were performed in conscious, chronically instrumented rats in which we monitored the regional hemodynamic responses to exendin-4 in the absence and presence of selected pharmacological antagonists.

	Materials and Methods

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Animals and Surgical Procedures. All procedures were carried out with the approval of the University of Nottingham Local Ethical Review Committee, under Home Office Project and Personal Licence authority. Adult male Sprague-Dawley rats, weighing 300 to 350 g, were obtained from Charles River (Margate, Kent, UK) and housed in a temperature-controlled (21-23°C) environment with a 12-h light/dark cycle (lights on at 6:00 A) and free access to food (Teklad Global 18% Protein Rodent Diet; Harlan UK Limited, Bicester, Oxon, UK) and water. At least 7 days after arrival from the supplier, the animals underwent the first surgical procedure, which was for probe implantation. Under general anesthesia (fentanyl and medetomidine, 300 µg kg^-1 of each i.p., supplemented as required), miniature pulsed Doppler flow probes were sutured around the left renal artery, the superior mesenteric artery, and the distal abdominal aorta (to monitor hindquarters hemodynamics). Following surgery, reversal of anesthesia and provision of analgesia was achieved with atipamezole and nalbuphine, respectively (1 mg kg^-1 of each s.c.). At least 7 days after the first surgical procedure and following a satisfactory inspection from the Veterinary Surgeon, the animals underwent the second surgical procedure, which was for catheter implantation. Using the same anesthetic regime as above, catheters were implanted in the distal abdominal aorta, via the caudal (tail) artery, for arterial blood pressure monitoring and the derivation of heart rate, and in the right jugular vein for peptide and drug administration. Up to three separate intravenous catheters were placed in the jugular vein to enable concurrent administration of different compounds. Experiments were started 24 h after surgery for catheter implantation, with animals fully conscious and unrestrained in home cages, with free access to food and water.

Cardiovascular Recordings. Cardiovascular variables were recorded using a customized, computer-based system [hemodynamics data acquisition system (HDAS); University of Limburg, Maastricht, The Netherlands] connected to a transducer amplifier (model 13-4615-50; Gould Instrument Systems Inc., Cleveland, OH) and a Doppler flowmeter (Crystal Biotech, Holliston, MA) VF-1 mainframe (pulse repetition frequency, 125 kHz) fitted with high velocity (HVPD-20) modules. Raw data were sampled by HDAS every 2 ms, averaged every cardiac cycle, and stored to disc at 5-s intervals.

Experimental Protocols. In one group of rats (n = 8), on day 1, exendin-4 was administered as i.v. bolus doses of 25, 250, and 2500 ng kg^-1 in ascending order with 60 min between the first and second doses and 180 min between the second and third doses. In the same animals, exendin-4 (250 ng kg^-1) was given again on days 2, 3, and 4. On day 2, exendin-4 was given 90 min after the onset of infusion of the ₂ adrenoceptor antagonist, ICI 118551 (0.2 mg kg^-1 bolus, 0.1 mg kg^-1 h^-1 infusion). On day 3, it was given 90 min after the onset of infusion of the nonselective -adrenoceptor antagonist, propranolol (1 mg kg^-1 bolus, 0.5 mg kg^-1 h^-1 infusion), and on day 4, it was given 90 min after onset of infusion of propranolol (as above) together with the nonselective -adrenoceptor antagonist, phentolamine (1 mg kg^-1 bolus, 1 mg kg^-1 h^-1 infusion). In pilot experiments, we ascertained that repeated administration of exendin-4 (250 ng kg^-1) across 4 experimental days produced consistent results (data not shown). In some experiments (n = 3), blood glucose levels were tested (Glucotrend 2; Roche Diagnostics, Mannheim, Germany) before and 10, 60, 120, and 180 min following exendin-4 (250 ng kg^-1).

In separate groups of rats, exendin-4 (250 ng kg^-1) was given in the presence of vehicle (saline) on day 1 and again on day 3 in the presence of (n = 8) a combination of propranolol, phentolamine, and the vasopressin (V₁) receptor antagonist, d(CH₂)₅-O-Me-Tyr-AVP (10 µg kg^-1 bolus, 10 µg kg^-1 h^-1 infusion), or (n = 9) atropine (1 mg kg^-1 bolus, 1 mg kg^-1 h^-1 infusion), or (n = 7) the GLP-1 receptor antagonist, exendin-(9-39) (75 µgkg^-1). In some of the latter experiments (n = 4), on a 3rd experimental day, animals were given losartan (10 mg kg^-1) followed by exendin-(9-39) prior to administration of exendin-4.

In the experiments involving exendin-(9-39), which has a plasma half-life of 33 min after i.v. infusion in man (Edwards et al., 1999), it was administered 5 min before the agonist. In all other experiments, antagonist administration was started 90 min prior to administration of exendin-4. The effectiveness of doses of the adrenoceptor antagonists (Gardiner et al., 2002; Woolard et al., 2004), losartan (Batin et al., 1991), and d(CH₂)₅-O-Me-Tyr-AVP (Gardiner et al., 1989) have been shown by us previously. The dose of exendin-(9-39) was chosen on the basis of data from Barragán et al. (1996).

Although ICI 118551 is a highly selective ₂-adrenoceptor antagonist (K_i values for ₂-and ₁-adrenoceptors are 1.2 and 120 nM, respectively; Bilski et al., 1983), and its affinity at ₂-adrenoceptors is 550-fold greater than at ₁-adrenoceptors (Baker, 2005), the effects of ICI 118551 on the heart rate response to exendin-4 (see Results) caused us to reevaluate the selectivity of the dose used. A 10-fold lower dose of ICI 118551 (0.02 mg kg^-1, 0.01 mg kg^-1 h^-1) caused only 13% inhibition of the hindquarters hyperemic response to salbutamol (1 nmol kg^-1 min^-1 for 3 min) and 67% inhibition of the tachycardia, whereas a 2-fold lower dose (0.1 mg kg^-1, 0.05 mg kg^-1 h^-1) caused 78 and 76% inhibition of the salbutamol-induced hindquarters hyperemia and tachycardia, respectively. At the chosen dose (0.2 mg kg^-1, 0.1 mg kg^-1 h^-1), the cardiovascular effects of salbutamol were reduced by more than 95%. In addition, a 3-min infusion (1 nmol kg^-1 min^-1) of salmeterol (Ball et al., 1991), which shows more selectivity for ₂-adrenoceptors than salbutamol (Baker, 2005), caused similar degrees of tachycardia (+82 ± 14 beats min^-1) and hindquarters hyperemia (+98 ± 13%) to salbutamol (+81 ± 8 beats min^-1 and +117 ± 15%, respectively). Taken together, these findings indicate that a major component of the tachycardic response to ₂-adrenoceptor stimulation is a direct effect (Insulander et al., 2004) and that ICI 118551 is acting selectively at the chosen dose.

View larger version (33K): [in this window] [in a new window]   Fig. 1. Regional hemodynamic effects of i.v. bolus doses of exendin-4 (25 ng kg-1, closed circles; 250 ng kg-1, open circles; 2500 ng kg-1, closed squares) in conscious Sprague-Dawley rats (n = 8). Values are mean, and vertical bars represent S.E.M. N.B. for clarity the time-base has been changed between 30 and 60 min. *, P < 0.05 versus baseline (Friedman's test). Statistical comparisons of integrated responses are given in the text.

Data Analysis. Data were analyzed offline using software (Datview; University of Limburg, Maastricht, The Netherlands) that interfaced with HDAS. Average values were calculated at time points selected on the basis of the temporal profile of the response and extracted into a customized statistical package (Biomed, University of Nottingham, Nottingham, UK) for subsequent analysis. The baseline was taken as the average values over the 2- to 3-min period prior to administration of exendin-4. Responses to exendin-4 were then measured at 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 90, 120, 150, and 180 min postdosing. Since the data did not always fit a normal distribution, we used a nonparametric, two-way analysis of variance (Friedman's test; Theodorsson-Norheim, 1987) for within-group comparisons and Mann-Whitney (unpaired) or Wilcoxon's (paired) tests for between-group comparisons. The between-group comparisons of the effects of exendin-4 were performed on the integrated areas under or over the curves between 0 and 60 min. Vascular conductances were calculated from the mean arterial blood pressure (BP) and Doppler shift (flow) data. P 0.05 was taken as significant.

Materials. Exendin-4, exendin-(9-39), and d(CH₂)₅-O-Me-Tyr-AVP were purchased from Bachem (Bubendorf, Switzerland), and ICI 118551 and propranolol hydrochloride were obtained from Tocris Cookson Inc. (Bristol, UK). Phentolamine mesylate and atropine methyl nitrate were from Sigma Chemical (Poole, Dorset, UK). Losartan was from Merck (Whitehouse Station, NJ). Stock solutions of peptides were made up in sterile water for injection and diluted in sterile saline containing 1% bovine serum albumin. Injection volumes were 0.1 ml and infusion rates were 0.4 ml h^-1. Fentanyl citrate was from Janssen-Cilag (High-Wycombe, UK), medetomidine hydrochloride (Domitor) and atipamezole hydrochloride (Antisedan) were from Pfizer Central Research (Sandwich, Kent, UK), and nalbuphine hydrochloride (Nubain) was from Bristol-Myers Squibb Co. (Hounslow, UK).

	Results

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Dose-Dependent Hemodynamic Responses to Exendin-4. On day 1, resting cardiovascular variables prior to administration of the first dose of exendin-4 were: heart rate, 334 ± 7 beats min^-1; BP, 109 ± 2 mm Hg; renal Doppler shift, 7.5 ± 0.9 kHz; renal vascular conductance, 69 ± 9 (kHz mm Hg)10³; mesenteric Doppler shift, 8.1 ± 1.0 kHz; mesenteric vascular conductance, 74 ± 9 (kHz mm Hg)10³; hindquarters Doppler shift, 4.5 ± 0.5 kHz; and hindquarters vascular conductance, 42 ± 5 (kHz mm Hg)10³. At a dose of 25 ng kg^-1, exendin-4 had no consistent effects on BP or heart rate but caused a small, significant (P < 0.05), and selective reduction in mesenteric Doppler shift (-9 ± 2% at 4 min) due to a reduction in mesenteric vascular conductance (-13 ± 2% at 4 min).

At a dose of 250 ng kg^-1, exendin-4 caused a modest but long-lasting (significant up to 60 min) rise in BP and substantial tachycardia (Fig. 1). These effects were accompanied by a marked increase in hindquarters Doppler shift (+102 ± 14% at 5 min), due to a significant increase in hindquarters vascular conductance (Fig. 1). There was an initial, short-lived renal vasoconstriction (Fig. 1) but no consistent accompanying change in renal Doppler shift (data not shown). Thereafter, however, there was a gradual increase in renal Doppler shift (+23 ± 5% at 20 min; P < 0.05) due to renal vasodilatation (Fig. 1). In contrast, there was a rapid-onset fall in mesenteric Doppler shift (-31 ± 3% at 5 min; P < 0.05) caused by a fall in mesenteric vascular conductance which persisted for 60 min (Fig. 1). All integrated (0-60 min) cardiovascular effects of exendin-4 at 250 ng kg^-1 were significantly (P < 0.05) greater than at 25 ng kg^-1. There were no consistent changes in blood glucose levels following 250 ng kg^-1 exendin-4 (before, 4.7 ± 0.2 mM; 10 min, 4.9 ± 0.5 mM; 60 min, 5.2 ± 0.3 mM; 120 min, 5.1 ± 0.4 mM; 180 min, 5.6 ± 0.4 mM; n = 3).

After the highest dose of exendin-4 (2500 ng kg^-1), the maximal change in BP was not different from that seen after the middle dose, and although the duration of effect was longer (significantly different from baseline up to 120 min, Fig. 1), the integrated (0-60 min) BP response to 2500 ng kg^-1 (557 ± 118 mm Hg min) was not different from the response to 250 ng kg^-1 (458 ± 97 mm Hg min). However, the tachycardia and increases in hindquarters Doppler shift (+151 ± 16% at 5 min) and vascular conductance following the high dose of exendin-4 were greater in both magnitude and duration (Fig. 1) than following the middle dose; therefore, the integrated (0-60 min) effects on heart rate (6135 ± 810 and 3719 ± 760 beats, respectively) and hindquarters vascular conductance (7040 ± 867 and 3806 ± 620% min, respectively) were significantly (P < 0.05) different. The accompanying changes in renal Doppler shift (data not shown) and vascular conductance (Fig. 1) were similar to those seen with the middle dose of exendin-4, although the later-onset renal vasodilatation was more long-lasting. Similarly, there was a fall in mesenteric Doppler shift (-41 ± 5% at 5 min) and vascular conductance (Fig. 1) following the highest dose of exendin-4 (2500 ng kg^-1), and the changes were similar in magnitude, but more long-lasting, than those seen with the middle dose (250 ng kg^-1); hence, the integrated (0-60 min) changes in mesenteric vascular conductance at 2500 and 250 ng kg^-1 were significantly (P < 0.05) different (2302 ± 130 and 1095 ± 137% min, respectively).

View larger version (24K): [in this window] [in a new window]   Fig. 2. Regional hemodynamic effects of exendin-4 (250 ng kg-1 i.v.) in the absence (closed circles, data from Fig. 1) and presence (open circles) of (a) ICI 118551 (0.2 mg kg-1, 0.1 mg kg-1 h-1), (b) propranolol (1 mg kg-1, 0.5 mg kg-1 h-1) and (c) propranolol (1 mg kg-1, 0.5 mg kg-1 h-1) together with phentolamine (1 mg kg-1,1mgkg-1 h-1) in conscious Sprague-Dawley rats (n = 8). Values are mean, and vertical bars represent S.E.M. *, P < 0.05 versus baseline (Friedman's test). Statistical comparisons of integrated responses are given in the text.

In the presence of ICI 118551, there was no significant increase in hindquarters vascular conductance in response to exendin-4 (250 ng kg^-1), and the integrated (0-60 min) tachycardic response (1239 ± 395 beats) was markedly reduced compared with the control run (see above), but the integrated pressor effect (955 ± 122 mm Hg min) was augmented (Fig. 2a). The duration of the initial renal vasoconstriction in response to exendin-4 in the presence of ICI 118551 was enhanced (significant up to 10 min), although this effect was not accompanied by a consistent reduction in renal Doppler shift (data not shown). Mesenteric hemodynamic responses to exendin-4 were unaffected by ICI 118551 (Fig. 2a).

Effects of Exendin-4 in the Presence of Propranolol. On day 3, resting cardiovascular variables prior to administration of exendin-4 (250 ng kg^-1) in the presence of propranolol were: HR, 312 ± 4 beats min^-1; BP, 103 ± 2 mm Hg; renal Doppler shift, 7.8 ± 0.7 kHz; renal vascular conductance, 77 ± 7 (kHz mm Hg)10³; mesenteric Doppler shift, 7.3 ± 0.7 kHz; mesenteric vascular conductance, 71 ± 6 (kHz mm Hg)10³; hindquarters Doppler shift, 4.0 ± 0.5 kHz; and hindquarters vascular conductance, 38 ± 5 (kHz mm Hg)10³.

The tachycardic and hindquarters hyperemic vasodilator effects of exendin-4 (250 ng kg^-1) were abolished by propranolol, but the accompanying integrated (0-60 min) pressor effect was markedly enhanced (989 ± 92 mm Hg min), and there was an initial, short-lived hindquarters vasoconstriction (Fig. 2b). The duration of the initial renal vasoconstriction following exendin-4 was prolonged (significant up to 10 min) in the presence of propranolol (Fig. 2b), but there was no consistent reduction in renal flow (data not shown). Mesenteric hemodynamic responses to exendin-4 were not significantly affected by propranolol (Fig. 2b).

Effects of Exendin-4 in the Presence of Propranolol Plus Phentolamine. On day 4, resting cardiovascular variables prior to administration of the exendin-4 (250 ng kg^-1) in the presence of propranolol plus phentolamine were: heart rate, 320 ± 5 beats min^-1; BP, 99 ± 2 mm Hg; renal Doppler shift, 6.8 ± 0.6 kHz; renal vascular conductance, 70 ± 7 (kHz mm Hg)10³; mesenteric Doppler shift, 6.6 ± 0.7 kHz; mesenteric vascular conductance, 67 ± 6 (kHz mm Hg)10³; hindquarters Doppler shift, 4.6 ± 0.4 kHz; and hindquarters vascular conductance, 47 ± 5 (kHz mm Hg)10³.

The augmentation of the pressor effect of exendin-4 by propranolol (see above) was not observed in the additional presence of phentolamine, and the initial hindquarters vasoconstriction did not occur (Fig. 2c). Otherwise, the hemodynamic effects of exendin-4 in the presence of propranolol together with phentolamine were not different from those seen in the presence of propranolol alone (Fig. 2, compare b with c).

View larger version (25K): [in this window] [in a new window]   Fig. 3. Regional hemodynamic effects of exendin-4 (250 ng kg-1 i.v.) in the absence (closed circles) and presence (open circles) of propranolol (1 mg kg-1, 0.5 mg kg-1 h-1), phentolamine (1 mg kg-1,1mgkg-1 h-1), and d(CH2)5-O-Me-Tyr-AVP) (10 µg kg-1, 10 µg kg-1 h-1) in conscious Sprague-Dawley rats (n = 8). Values are mean, and vertical bars represent S.E.M. *, P < 0.05 versus baseline (Friedman's test).

The effects of exendin-4 (250 ng kg^-1) on day 1 in this group of naive rats were very similar to those seen in the initial series of experiments, and the effects of exendin-4 in the additional presence of d(CH₂)₅-O-Me-Tyr-AVP) were not different from those in the combined presence of propranolol and phentolamine (compare Fig. 2c with Fig. 3).

Effects of Exendin-4 in the Absence and Presence of Atropine. Resting cardiovascular variables in this group of rats prior to administration of exendin-4 (250 ng kg^-1)onday 1 (in the presence of saline) and on day 3 (in the presence of atropine) were: heart rate, 350 ± 14 and 438 ± 14 beats min^-1; BP, 110 ± 4 and 119 ± 2 mm Hg; renal Doppler shift, 8.5 ± 1.7 and 10.3 ± 1.5 kHz; renal vascular conductance, 76 ± 14 and 87 ± 13 (kHz mm Hg)10³; mesenteric Doppler shift, 6.0 ± 1.1 and 7.0 ± 0.9 kHz; mesenteric vascular conductance, 53 ± 9 and 58 ± 7 (kHz mm Hg)10³; hindquarters Doppler shift, 4.8 ± 0.5 and 4.9 ± 0.4 kHz; and hindquarters vascular conductance, 44 ± 4 and 41 ± 3 (kHz mm Hg)10³, respectively.

The effects of exendin-4 (250 ng kg^-1) on day 1 in this group of rats were very similar to those described above (Fig. 4). In the presence of atropine, the integrated (0-60 min) tachycardic (1376 ± 303 beats) and pressor (142 ± 31 mm Hg min) effects of exendin-4 were significantly (P < 0.05) smaller than in the absence of atropine (3994 ± 698 beats, 431 ± 125 mm Hg min, respectively), but the associated hemodynamic effects were not changed significantly (Fig. 4).

View larger version (25K): [in this window] [in a new window]   Fig. 4. Regional hemodynamic effects of exendin-4 (250 ng kg-1 i.v.) in the absence (closed circles) and presence (open circles) of atropine (1 mg kg-1, 1 mg kg-1 h-1) in conscious Sprague-Dawley rats (n = 9). Values are mean, and vertical bars represent S.E.M. *, P < 0.05 versus baseline (Friedman's test). Statistical comparisons of integrated responses are given in the text.

The effects of exendin-4 (250 ng kg^-1) on day 1 in this group of rats were very similar to those described above (compare Figs. 1, 2, 3, 4 with Fig. 5). In the presence of exendin-(9-39), the initial (at 5 min) increases in heart rate (+32 ± 7 beats min^-1) and hindquarters vascular conductance (+3 ± 6%) in response to exendin-4 were smaller (P < 0.05) than in the absence of exendin-(9-39) (+53 ± 12 beats min^-1, +72 ± 9% respectively). Thereafter, tachycardia developed such that the integrated (0-60 min) heart rate responses in the absence and presence of exendin-(9-39) were not different (3020 ± 629 and 2644 ± 335 beats, respectively), but the integrated (0-60 min) hindquarters vasodilator effects of exendin-4 were significantly (P < 0.05) attenuated [control, 2320 ± 433; with exendin-(9-39), 934 ± 411% min]. The effects of exendin-4 on BP and renal and mesenteric vascular conductances were not changed significantly by exendin-(9-39) (Fig. 5). The presence of losartan together with exendin-(9-39) had no additional effect on the responses to exendin-4 (data not shown).

View larger version (25K): [in this window] [in a new window]   Fig. 5. Regional hemodynamic effects of exendin-4 (250 ng kg-1 i.v.) given 5 min following administration of saline (0.1 ml) (closed circles) or exendin-(9-39) (75 µg kg-1) (open circles) in conscious Sprague-Dawley rats (n = 7). Values are mean, and vertical bars represent S.E.M. *, P < 0.05 versus baseline (Friedman's test). Statistical comparisons of integrated responses are given in the text.

In the sham-operated rats, the effects of exendin-4 (250 ng kg^-1) were as described above (Fig. 6). In adrenal-demedullated rats following administration of exendin-4, there was no significant tachycardia (0 ± 11 beats min^-1 at 5 min) and no significant increase in hindquarters vascular conductance (+3 ± 13% at 5 min), but the integrated (0-60 min) pressor response (551 ± 115 mm Hg min) and fall in mesenteric vascular conductance (912 ± 206%/min) were not different from the changes seen in the sham-operated rats (499 ± 143 mm Hg min and 780 ± 210% min, respectively; Fig. 6).

View larger version (24K): [in this window] [in a new window]   Fig. 6. Regional hemodynamic effects of exendin-4 (250 ng kg-1 i.v.) in sham-operated (closed circles, n = 5) and adrenal-demedullated (open circles, n = 6) conscious Sprague-Dawley rats. Values are mean, and vertical bars represent S.E.M. *, P < 0.05 versus baseline (Friedman's test). Statistical comparisons of integrated responses are given in the text.

	Discussion

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The primary aims of the present study were to evaluate the regional hemodynamic effects of the GLP-1 receptor agonist, exendin-4, in conscious rats, and to assess the possible involvement of adrenoceptors in the effects observed. As indicated in the Introduction, the conclusions of Barragán et al. (1994, 1996, 1999), from their studies on anesthetized rats, were that the pressor and tachycardic effects of GLP-1 receptor agonists did not involve adrenoceptors. In contrast, Yamamoto et al. (2002, 2003) produced evidence in favor of the cardiovascular actions of these peptides being due to sympathoadrenal activation in conscious, telemetered rats. Our findings provide a bridge between these apparently disparate views, in as much as we obtained results consistent with sympathoadrenal activation causing opposing hemodynamic effects through the mediation of - and -adrenoceptors. In addition, in the presence of adrenoceptor antagonists and in adrenal-demedullated rats, we still observed pressor and mesenteric vasoconstrictor effects of exendin-4.

It has been suggested that the clear tachycardic and pressor effects of these peptides seen in rats may be specific to rodents (Knudsen, 2004) because no such effects have been reported in man in chronic studies. However, there is evidence for acute pressor and tachycardic effects of GLP-1 in humans (Edwards et al., 1998), and, given the complex hemodynamic effects of exendin-4 found in the present study, it is feasible that a relative lack of change in BP could conceal substantial underlying hemodynamic responses to these peptides administered chronically in man.

Under control conditions, the response to exendin-4 consisted of dose-dependent tachycardia, accompanied by mesenteric vasoconstriction and hindquarters vasodilatation. It appeared the latter was largely mediated by ₂ adrenoceptors and opposed the former because, in the presence of a ₂ adrenoceptor antagonist, the hindquarters vasodilatation was abolished, and the pressor response was enhanced, whereas the mesenteric vasoconstriction was unaffected. In this circumstance, the tachycardia was also substantially reduced. It is unlikely that at the dose used, ICI 118551 was acting nonselectively (see Materials and Methods). Thus, inhibition of the tachycardic effect of exendin-4 by ICI 118551 was most likely due to antagonism of postjunctional ₂ adrenoceptor-mediated chronotropy, but it may also have involved inhibition of a prejunctional facilitatory mechanism and/or activation of vagally mediated baroreflex slowing of the heart driven by the increase in the pressor effect of exendin-4 in this condition.

A major role for ₂ adrenoceptors in the hindquarters vasodilator effect of exendin-4 is consistent with the ability of propranolol also to abolish that effect, thereby producing a picture very similar to that seen with ICI 118551. However, an indication of additional involvement of ₁ adrenoceptors came from the finding that propranolol also unmasked a significant, but modest, initial hindquarters vasoconstriction. This effect was likely adrenoceptor-mediated because it was not seen in the additional presence of phentolamine. Moreover, in the presence of propranolol and phentolamine, the marked augmentation of the pressor effect of exendin-4 seen in the presence of propranolol alone was not observed. However, we do not believe this additional effect of phentolamine on the pressor response was due only to suppression of the hindquarters vasoconstrictor action of exendin-4 because there was no hindquarters vasoconstrictor response to exendin-4 in the presence of ICI 118551, when the pressor effect of exendin-4 was augmented to a similar extent. Hence, the most likely explanation of the ability of phentolamine to suppress the increase in the pressor effect of exendin-4 caused by propranolol is that this was due to inhibition of another mechanism, e.g., a cardiac adrenoceptor-mediated positive inotropic effect, most likely attributable to adrenaline (Broadley et al., 1999). So, our results are consistent with the sympathoadrenal activation caused by exendin-4 resulting in substantial adrenaline release causing activation of cardiac and adrenoceptors and (largely) hindquarters ₂ adrenoceptors, with little evidence of activation of vascular adrenoceptors, either through this mechanism or through mediation of sympathetic efferent noradrenergic vasoconstrictor activity. The source of the adrenaline is likely to be from the adrenal medulla since the effects of exendin-4 in adrenal-demedullated animals closely resembled those seen in rats treated with propranolol and phentolamine. Since blood glucose was not reduced by exendin-4, it appears that sympathoadrenal activation was not due to hypoglycemia. We have no explanation for the lack of effect of propranolol on the tachycardic effects of GLP-1 in the experiments of Barragán et al. (1994), because the ability of exendin-4 to increase heart rate was clearly inhibited by ICI 118551 or propranolol in our experiments.

In the presence of propranolol and phentolamine and in adrenal-demedullated animals, the pressor effect of exendin-4 was accompanied by constriction only in the renal and mesenteric vascular beds, and, in the former, the fall in vascular conductance was not accompanied by consistent changes in flow, so the vasoconstriction could have been an autoregulatory response to the rise in pressure. However, the mesenteric vasoconstriction was clearly active (i.e., involved a substantial reduction in flow). Given the ability of GLP-1 receptor agonists to release vasopressin (Bojanowska and Stempniak, 2002) and given the potent effects of vasopressin to cause mesenteric vasoconstriction in conscious rats (Gardiner et al., 1988), we had anticipated that a V₁ receptor antagonist would suppress the mesenteric vasoconstrictor effects of exendin-4 in the presence of propranolol and phentolamine. However, this was not the case, despite some previous findings indicating an involvement of vasopressin in the pressor effects of GLP-1 receptor agonism (Isbil-Buyukcoskun and Gulec, 2004). We also observed that losartan did not influence the mesenteric vasoconstrictor effect of exendin-4, indicating that activation of the renin-angiotensin system was not responsible, despite adrenaline (see above) being a stimulus for such activation.

In light of these collective findings, we speculated that the most likely mechanism responsible for the residual mesenteric effect underlying the pressor action of exendin-4 was a direct vasoconstrictor action. However, preliminary studies on isolated mesenteric arterioles showed no vasoconstrictor action of exendin-4 (S. O'Sullivan, personal communication), and the only published evidence of a direct vascular effect of GLP-1 receptor agonism indicates it results in vasodilatation (Golpon et al., 2001; Nyström et al., 2005). Furthermore, although it is feasible that a direct cardiac action of exendin-4 could have a pressor effect by increasing cardiac output, present evidence indicates that activation of cardiac GLP-1 receptors has a negative inotropic effect (Vila Petroff et al., 2001).

We also considered the possibility that the residual mesenteric and pressor action of exendin-4 was due to the release of a vasoactive substance, such as neuropeptide Y, from the adrenal medulla, as has been observed in other experimental paradigms (Martin, 2005), but this is unlikely since the pressor and mesenteric vasoconstrictor effect of exendin-4 appeared quite normal in adrenal-demedullated rats. Furthermore, neuropeptide Y is a particularly potent renal vasoconstrictor (Gardiner et al., 1988), and yet marked renal vasoconstriction was not seen with exendin-4. However, it is feasible that neuropeptide Y released locally from sympathetic nerves supplying the mesenteric artery was responsible for the effects observed. A further possible explanation for the mesenteric and pressor action of exendin-4 is inhibition of the endogenous production of a vasodilator such as nitric oxide, although again, it is difficult to see why this should be restricted to the mesenteric vasculature unless it involved disruption of the function of the nitrergic innervation of the mesenteric vasculature.

The pressor and mesenteric vasoconstrictor actions of exendin-4 were also resistant to the GLP-1 receptor antagonist, exendin-(9-39), at a dose that clearly attenuated the hindquarters vasodilatation and, to a lesser extent, the tachycardia. In contrast, in the experiments of Barragán et al. (1996), exendin-(9-39) abolished the pressor and tachycardic effects of GLP-1 in anesthetized rats. Here, we used the same doses and timings of administration of exendin-4 and exendin-(9-39) as Barragán et al. (1996) and can only suggest that, perhaps, the underlying mechanisms are different in conscious and anesthetized animals. Other studies have reported effects of GLP-1 receptor agonism that are not inhibited by exendin-(9-39), raising the possibility of other receptor subtypes (e.g., Daniel et al., 2002). In addition, if some of the variation in results is due to different locations of GLP-1 receptors, our results indicate that the GLP receptors most accessible to peripherally administered exendin-(9-39) are those responsible for activation of the adrenal medulla following i.v. injection of exendin-4. Barragán et al. (1999) proposed that a component of the cardiovascular response to exendin-4 was due to centrally mediated vagal withdrawal, and our experiments with atropine are consistent with that suggestion since atropine reduced the tachycardia and the pressor response. Thus, the remaining tachycardic response to exendin-4, seen in the presence of exendin-(9-39), may have been due to centrally mediated vagal withdrawal. However, it is not clear why any such atropine-sensitive component was not seen in the experiments with the adrenoceptor antagonists or in adrenal-demedullated rats.

In conclusion, in conscious rats, exendin-4 causes substantial adrenoceptor-mediated hindquarters vasodilatation and tachycardia due to sympathoadrenal activation. However, the mesenteric vasoconstriction and accompanying pressor actions of exendin-4 do not appear to be due to activation of -adrenoceptors, V₁ receptors, or angiotensin receptors.

	Footnotes

 
Some of these results have been presented to the British Pharmacological Society Meeting, July 2005, Cambridge, UK.

doi:10.1124/jpet.105.093104.

ABBREVIATIONS: GLP, glucagon-like peptide; propranolol, (RS)-1-[(1-methylethyl)amino]-3-(1-naphthalenyloxy)-2-propanol; HDAS, hemodynamic data acquisition system; ICI 118551, (±)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol) hydrochloride; V₁, vasopressin; AVP, arginine vasopressin; losartan, 2-n-butyl-4-chloro-5-hydroxymethyl-1-[(2'-(1H-tetrazol-5-yl)-biphenyl-4-yl)methyl] imidazoline, potassium salt; BP, mean arterial blood pressure; phentolamine mesylate, (2-[N-(m-hydroxyphenyl)-p-toluidinomethyl]-imidazoline) methanesulfonate.

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

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