Hypoalgesia and altered inflammatory responses in mice lacking kinin B1 receptors

Abstract

Kinins are important mediators in cardiovascular homeostasis, inflammation, and nociception. Two kinin receptors have been described, B1 and B2. The B2 receptor is constitutively expressed, and its targeted disruption leads to salt-sensitive hypertension and altered nociception. The B1 receptor is a heptahelical receptor distinct from the B2 receptor in that it is highly inducible by inflammatory mediators such as bacterial lipopolysaccharide and interleukins. To clarify its physiological function, we have generated mice with a targeted deletion of the gene for the B1 receptor. B1 receptor-deficient animals are healthy, fertile, and normotensive. In these mice, bacterial lipopolysaccharide-induced hypotension is blunted, and there is a reduced accumulation of polymorphonuclear leukocytes in inflamed tissue. Moreover, under normal noninflamed conditions, they are analgesic in behavioral tests of chemical and thermal nociception. Using whole-cell patch-clamp recordings, we show that the B1 receptor was not necessary for regulating the noxious heat sensitivity of isolated nociceptors. However, by using an in vitro preparation, we could show that functional B1 receptors are present in the spinal cord, and their activation can facilitate a nociceptive reflex. Furthermore, in B1 receptor-deficient mice, we observed a reduction in the activity-dependent facilitation (wind-up) of a nociceptive spinal reflex. Thus, the kinin B1 receptor plays an essential physiological role in the initiation of inflammatory responses and the modulation of spinal cord plasticity that underlies the central component of pain. The B1 receptor therefore represents a useful pharmacological target especially for the treatment of inflammatory disorders and pain.

Figure 1

Targeting of the kinin B1-receptor gene. (A) Schematic representation of the targeting strategy with the wild-type B1 locus, the recombination vector, and the predicted structure of the targeted gene. The probe used for Southern blot and the length of the EcoRI restriction fragments are indicated. (B) Southern blot analysis of tail DNA from offspring of a cross between two heterozygous B1-deficient mice digested with EcoRI and hybridized with the aforementioned probe. (C) RNase protection assay detecting B1, IL-1β, and β-actin mRNA in untreated animals or 5 h after injection of 10 μg/kg LPS. (D) Isometric responses of isolated ileum from wild-type (WT) and B1-deficient mice (KO) to BK and des-Arg⁹-BK (1 μM each). Only the B2 receptor-dependent contractile response to BK is preserved in B1-deficient mice.

Figure 2

Blood pressure. (A) Mean arterial pressure (MAP) measured by carotid artery cannulation in conscious animals is not different between wild-type (+/+, n = 8) and B1-deficient mice (−/−, n = 8). (B) Blood pressure decline after i.p. injection of 250 μg/kg LPS in wild-type (+/+, n = 11) and B1-deficient mice (−/−, n = 9) is significantly different until 35 min after LPS administration but not thereafter. Data are means ± SEM and were analyzed by analysis of variance followed by Dunnetts' test. *, P < 0.05 compared with +/+ animals.

Figure 3

Acute inflammatory response induced by intrapleural injection of des-Arg⁹-BK (A-–C) or carrageenan (D–F) obtained in wild-type (+/+) or B1-deficient mice (−/−). Plasma extravasation (A and D) and the invasion of mononuclear (B and E) and PMN (C and F) leukocytes was determined. Data are means ± SEM and were analyzed by analysis of variance followed by Dunnetts' test (n = 5). **, P < 0.01; *, P < 0.05 compared with +/+ animals.

Figure 4

Responses to thermal (A and B) and chemical (C and D) noxious stimuli. Thermal nociception was tested in the tail-flick (A) and the hot-plate test at three different temperatures (B) and chemical nociception by intradermal injection of formalin (2.5%) (C) and capsaicin (1.6 mg) (D) in wild-type (+/+) and B1-deficient mice (−/−). Data are means ± SEM and were analyzed by analysis of variance followed by Dunnetts' test (n = 4–6). ***, P < 0.001; **, P < 0.01; *, P < 0.05 compared with +/+ animals.

Figure 5

Sensory neuron and spinal cord electrophysiology. Examples are shown of inward currents to a heat ramp (top bar from 24 to 49°C) in an isolated nociceptor from a wild-type (A) and B1 receptor-deficient neuron (B). The proportion of both IB₄-positive and -negative nociceptors responding to the heat stimulus was unchanged in the mutants (C) and the mean magnitude of the inward current was also not altered (D). Application of des-Arg⁹-BK to isolated neonatal spinal cord from wild-type mice increased the C-fiber-evoked component of the VRP (F). The magnitude of the VRP returned to control values 20 min after washout of des-Arg⁹-BK. In B1-deficient mice, wind-up was significantly reduced compared with control (G). Examples of VRP wind-up from wild-type (+/+) and B1 −/− mice are shown in E. Data are means ± SEM and were analyzed by using the t test. **, P < 0.005; *, P < 0.05 compared with control.