br Materials and methods br
Materials and methods
Results The i.v. administration of urocortin 2 reduced the mean arterial blood pressure in a dose-dependent manner (Fig. 1). The ED50 for the urocortin 2 cumulative dose–response effect on mean arterial blood pressure was 0.014±0.004 mg kg−1. Urocortin 2 administration had no statistically significant effects on ret pathway rate in the same animals (Fig. 1). Antisauvagine-30, at doses up to 10 mg kg−1, had no cumulative dose–response effects on mean arterial blood pressure or heart rate (Fig. 1). The temporal profiles of the dose-dependent effects of urocortin 2 on mean arterial blood pressure and heart rate are shown in Fig. 2. Urocortin 2 produced a statistically significant reduction in mean arterial blood pressure as compared with vehicle administration [main treatment effect: F(3,12)=15.85, P<0.001; treatment×time interaction: F(18,72)=9.66, P<0.00001]. Urocortin 2 also induced a significant dose-dependent increase in heart rate in the same animals [treatment×time interaction: F(18,72)=2.97, P<0.001]. Administration of urocortin 2 at the two highest doses tested (0.03 and 0.1 mg kg −1) evoked the most marked and significant effects on mean arterial blood pressure and heart rate relative to vehicle-treated animals (P<0.05). The hypotensive and modest tachycardic responses were still present at 30 min postinjection although there was evidence of a gradual return toward baseline values. The temporal data for the effect of antisauvagine-30 pretreatment on urocortin 2 (0.1 mg kg −1) evoked changes in mean arterial blood pressure are shown in Fig. 3. The systemic administration of urocortin 2 (0.1 mg kg−1) at 30 min produced a significant reduction in mean arterial blood pressure when compared to vehicle treatment [main treatment effect: F(3,12)=5.96, P<0.01; treatment×time interaction: F(36,144)=10.99, P<0.00001]. The reduction in mean arterial blood pressure observed following urocortin 2 treatment was blocked by pretreatment with antisauvagine-30 at time 0. Administration of antisauvagine-30 had no significant effect on mean arterial blood pressure at any of the doses investigated. Antisauvagine-30 pretreatment failed to produce any statistically significant effects on heart rate in the same animals (data not shown).
Discussion The results of the current study demonstrate that the selective CRF2 receptor peptide agonist urocortin 2 produced a dose-dependent reduction in mean arterial blood pressure, an effect that was blocked by pretreatment with the selective CRF2 receptor peptide antagonist antisauvagine-30. Urocortin 2 also caused a concomitant dose-dependent increase in heart rate. Systemic administration of antisauvagine-30 alone had no effect on mean arterial blood pressure or heart rate at any of the doses examined. The anatomical localization of the CRF2 receptor may explain, at least in part, the effects of urocortin 2 on cardiovascular function observed in the present study. In the rodent, the CRF2 receptor subtype has two functional splice variants, CRF2(a) and CRF2(b), which are differentially distributed between peripheral tissues and the CNS. The mRNA for CRF2(a) is found almost exclusively in the brain (Lovenberg et al., 1995). In contrast, CRF2(b) mRNA is found predominantly in the periphery, where high levels of expression are found in the heart (aorta and myocardium, epicardium and arterioles of the atrium and ventricles) and skeletal muscle, with lower levels in the lungs and gastrointestinal tract Baigent and Lowry, 2000, Kishimoto et al., 1995, Lovenberg et al., 1995. CRF2(b) mRNA is also expressed in limited regions of the brain (Lovenberg et al., 1995). The observations that CRF2 mRNA is expressed in cardiac arterioles (Lovenberg et al., 1995) and also in a vascular smooth muscle cell line (Kageyama et al., 2000) suggests that the hypotensive effect of systemically administered urocortin 2 in the present study may be attributed to a marked fall in resistance in response to CRF2 activation by urocortin 2 in the peripheral vasculature, an effect that is blocked by pretreatment with the selective CRF2 receptor antagonist antisauvagine-30. The modest tachycardia observed may be a reflex response to the reduction in mean arterial blood pressure or a direct effect of urocortin 2 on CRF2 receptors in the heart. It is interesting to note that urocortin 2 did not significantly change heart rate in the cumulative dose–response study, but did significantly increase heart rate in the temporal profile study despite a comparable fall in mean arterial blood pressure. The reason for this difference is unclear, but one possible explanation may be that if the tachycardia is reflex in origin as suggested then it could be that it has subsided by the time the readings are taken in the cumulative dose–response study.