Clare Davis

Vanilloid receptor TRPV1, sensory C-fibers, and vascular autoregulation: a novel mechanism involved in myogenic constriction.

Myogenic constriction describes the innate ability of resistance arteries to constrict in response to elevations in intraluminal pressure and is a fundamental determinant of peripheral resistance and, hence, organ perfusion and systemic blood pressure. However, the receptor/cell-type that senses changes in pressure on the blood vessel wall and the pathway that couples this to constriction of vascular smooth muscle remain unclear. In this study, we show that elevation of intraluminal transmural pressure of mesenteric small arteries in vitro results in a myogenic response that is profoundly suppressed following ablation of sensory C-fiber activity (using in vitro capsaicin desensitization resulted in 72.8±10.3% inhibition, n=8; P<0.05). Activation of C-fiber nerve endings by pressure was attributable to stimulation of neuronal vanilloid receptor, TRPV1, because blockers of this channel, capsazepine (71.9±11.1% inhibition, n=9; P<0.001) and ruthenium red (46.1±11.7% inhibition, n=4; P<0.05), suppressed the myogenic constriction. In addition, this C-fiber dependency is likely related to neuropeptide substance P release and activity because blockade of tachykinin NK1 receptors (66.3±13.7% inhibition, n=6; P<0.001), and not NK2 receptors (n=4, NS), almost abolished the myogenic response. Previous studies support a role for 20-hydroxyeicosatetraenoic acid (20-HETE) in myogenic constriction responses; herein, we show that 20-HETE–induced constriction of mesenteric resistance arteries is blocked by capsazepine. Together, these results suggest that elevation of intraluminal pressure is associated with generation of 20-HETE that, in turn, activates TRPV1 on C-fiber nerve endings resulting in depolarization of nerves and consequent vasoactive neuropeptide release. These findings identify a novel mechanism contributing to Bayliss’ myogenic constriction and highlights an alternative pathway that may be targeted in the therapeutics of vascular disease, such as hypertension, where enhanced myogenic constriction plays a role in the pathogenesis.

Prostaglandin E2-induced sensitization of bradykinin-evoked responses in rat dorsal root ganglion neurons is mediated by cAMP-dependent protein kinase A.

Primary cultures of neonatal rat dorsal root ganglion (DRG) neurons were used to examine the mechanisms underlying both the direct activation and the sensitization of sensory neurons by prostanoids. Prostaglandin E2 (PGE2) elevated cytosolic calcium concentration ([Ca2+]i) in a subpopulation of small (< 19 μm) diameter, capsaicin‐sensitive DRG neurons. PGE2 also stimulated substance P (SP) release from DRG cultures. In contrast to bradykinin, PGE2 did not stimulate phosphoinositidase C (PIC) and the PGE2‐evoked increase in [Ca2+]i was dependent on extracellular calcium. Pre‐treatment with PGE2 potentiated bradykinin‐evoked increases in [Ca2+]i in small diameter neurons and increased the number of cells that responded to low concentrations of bradykinin. A similar effect was seen with prostaglandin I2 (PGI2) but not prostaglandin F2α (PGF2α). PGE2 pretreatment also potentiated bradykinin‐evoked release of SP, inducing a leftward shift in the bradykinin concentration–response curve and an increase in the maximum response. PGE2 stimulated adenylyl cyclase activity in DRG cultures, at concentrations and times consistent with those required to observe both the direct and sensitizing effects of the prostanoid on [Ca2+]i responses. Furthermore, the direct and sensitizing effects of PGE2, on both [Ca2+]i responses and SP release, were mimicked by the membrane permeant cAMP analogue dibutyryl cAMP and inhibited by H89, an inhibitor of cAMP‐dependent protein kinase A (PKA). These observations are consistent with the hypothesis that both direct activation and sensitization of sensory neurons by prostanoids, such as PGE2, are mediated by PKA‐dependent phosphorylation mechanisms.

Cloning and functional characterization of the guinea pig vanilloid receptor 1

We have cloned a guinea pig Vanilloid receptor 1 (VR1) from a dorsal root ganglion cDNA library and expressed it in CHO cells. The receptor has been functionally characterized by measuring changes in intracellular calcium produced by capsaicin, low pH and noxious heat. Capsaicin produced a concentration-dependent increase in intracellular calcium in guinea pig VR1-CHO cells with an estimated EC(50) of 0.17 +/- 0.0065 micro M, similar to that previously reported for rat and human VR1. Olvanil and resiniferatoxin were also effective agonists (EC(50) values of 0.0087 +/- 0.0035 micro M and 0.067 +/- 0.014 micro M, respectively), but 12-phenylacetate 13-acetate 20-homovanillate (PPAHV) and anandamide showed little agonist activity up to 10 micro M. As with human and rat VR1, guinea pig VR1 was also activated by pH below 6.0 and by noxious heat (>42 degrees C). Capsazepine acted as an antagonist of capsaicin responses in guinea pig VR1-CHO cells (IC(50) of 0.324 +/- 0.041 micro M ), as seen at rat VR1. However, in contrast to its lack of activity against pH and heat responses at rat VR1, capsazepine was an effective antagonist of these responses at guinea pig VR1. Capsazepine displayed an IC(50) of 0.355 +/- 25 micro M against pH 5.5, and provided complete blockade of heat responses at 1 micro M. Thus, capsazepine can significantly inhibit calcium influx due to heat and pH 5.5 at guinea pig VR1 and human VR1 but is inactive against these activators at rat VR1.

Bradyzide, a potent non-peptide B2 bradykinin receptor antagonist with long-lasting oral activity in animal models of inflammatory hyperalgesia

Bradyzide is from a novel class of rodent‐selective non‐peptide B2 bradykinin antagonists (1‐(2‐Nitrophenyl)thiosemicarbazides). Bradyzide has high affinity for the rodent B2 receptor, displacing [3H]‐bradykinin binding in NG108‐15 cells and in Cos‐7 cells expressing the rat receptor with KI values of 0.51±0.18 nM (n=3) and 0.89±0.27 nM (n=3), respectively. Bradyzide is a competitive antagonist, inhibiting B2 receptor‐induced 45Ca efflux from NG108‐15 cells with a pKB of 8.0±0.16 (n=5) and a Schild slope of 1.05. In the rat spinal cord and tail preparation, bradyzide inhibits bradykinin‐induced ventral root depolarizations (IC50 value; 1.6±0.05 nM (n=3)). Bradyzide is much less potent at the human than at the rodent B2 receptor, displacing [3H]‐bradykinin binding in human fibroblasts and in Cos‐7 cells expressing the human B2 receptor with KI values of 393±90 nM (n=3) and 772±144 nM (n=3), respectively. Bradyzide inhibits bradykinin‐induced [3H]‐inositol trisphosphate (IP3) formation with IC50 values of 11.6±1.4 nM (n=3) at the rat and 2.4±0.3 μM (n=3) at the human receptor. Bradyzide does not interact with a range of other receptors, including human and rat B1 bradykinin receptors. Bradyzide is orally available and blocks bradykinin‐induced hypotension and plasma extravasation. Bradyzide shows long‐lasting oral activity in rodent models of inflammatory hyperalgesia, reversing Freunds complete adjuvant (FCA)‐induced mechanical hyperalgesia in the rat knee joint (ED50, 0.84 μmol kg−1; duration of action >4 h). It is equipotent with morphine and diclofenac, and 1000 times more potent than paracetamol, its maximal effect exceeding that of the non‐steroidal anti‐inflammatory drugs (NSAIDs). Bradyzide does not exhibit tolerance when administered over 6 days. In summary, bradyzide is a potent, orally active, antagonist of the B2 bradykinin receptor, with selectivity for the rodent over the human receptor.

B1 bradykinin receptors and sensory neurones

1 The location of the B1 bradykinin receptors involved in inflammatory hyperalgesia was investigated. 2 No specific binding of the B1 bradykinin receptor ligand [3H]‐des‐Arg10‐kallidin was detected in primary cultures of rat dorsal root ganglion neurones, even after treatment with interleukin‐1β (100 iu ml−1). 3 In dorsal root ganglion neurones, activation of B2 bradykinin receptors stimulated polyphosphoinositidase C. In contrast, B1 bradykinin receptor agonists (des‐Arg9‐bradykinin up to 10 μm and des‐Arg10‐kallidin up to 1 μm) failed to activate polyphosphoinositidase C, even in neurones that had been treated with interleukin‐1β (100 iu ml−1), prostaglandin E2 (1 μm) or prostaglandin I2 (1 μm). 4 Dorsal root ganglion neurones removed from rats (both neonatal and 14 days old) that had been pretreated with inflammatory mediators (Freunds complete adjuvant, or carrageenan) failed to respond to B1 bradykinin receptor selective agonists (des‐Arg9‐bradykinin up to 10 μm and des‐Arg10‐kallidin up to 1 μm). 5 Bradykinin (25 nM to 300 nM) evoked ventral root responses when applied to peripheral receptive fields or central terminals of primary afferents in the neonatal rat spinal cord and tail preparation. In contrast, des‐Arg9‐bradykinin (50 nM to 500 nM) failed to evoke ventral root depolarizations in either control rats or in animals that developed inflammation following ultraviolet irradiation of the tail skin. 6 The results of the present study imply that the B1 bradykinin receptors that contribute to hypersensitivity in models of persistent inflammatory hyperalgesia are located on cells other than sensory neurones where they may be responsible for releasing mediators that sensitize or activate the nociceptors.

Antihyperalgesic activity of a novel nonpeptide bradykinin B1 receptor antagonist in transgenic mice expressing the human B1 receptor

1 We describe the properties of a novel nonpeptide kinin B1 receptor antagonist, NVP‐SAA164, and demonstrate its in vivo activity in models of inflammatory pain in transgenic mice expressing the human B1 receptor. 2 NVP‐SAA164 showed high affinity for the human B1 receptor expressed in HEK293 cells (Ki 8 nM), and inhibited increases in intracellular calcium induced by desArg10kallidin (desArg10KD) (IC50 33 nM). While a similar high affinity was observed in monkey fibroblasts (Ki 7.7 nM), NVP‐SAA164 showed no affinity for the rat B1 receptor expressed in Cos‐7 cells. 3 In transgenic mice in which the native B1 receptor was deleted and the gene encoding the human B1 receptor was inserted (hB1 knockin, hB1‐KI), hB1 receptor mRNA was induced in tissues following LPS treatment. No mRNA encoding the mouse or human B1 receptor was detected in mouse B1 receptor knockout (mB1‐KO) mice following LPS treatment. 4 Freunds complete adjuvant‐induced mechanical hyperalgesia was similar in wild‐type and hB1‐KI mice, but was significantly reduced in mB1‐KO animals. Mechanical hyperalgesia induced by injection of the B1 agonist desArg10KD into the contralateral paw 24 h following FCA injection was similar in wild‐type and hB1‐KI mice, but was absent in mB1‐KO animals. 5 Oral administration of NVP‐SAA164 produced a dose‐related reversal of FCA‐induced mechanical hyperalgesia and desArg10KD‐induced hyperalgesia in hB1‐KI mice, but was inactive against inflammatory pain in wild‐type mice. 6 These data demonstrate the use of transgenic technology to investigate the in vivo efficacy of species selective agents and show that NVP‐SAA164 is a novel orally active B1 receptor antagonist, providing further support for the utility of B1 receptor antagonists in inflammatory pain conditions in man.

The pharmacology of T-kinin and des-Arg11-T-kinin in primary cultures of rat bladder smooth muscle cells

T-kinin and its putative carboxypeptidase product des-Arg(11)-T-kinin are members of the kinin family that are unique to the rat. Primary cultures of rat bladder smooth muscle cells were used to investigate the pharmacology of these peptides. Calcium imaging experiments showed that rat bladder smooth muscle cells responded to both bradykinin and des-Arg(9)-bradykinin with an increase in [Ca(2+)](i) and responses to both agonists could be observed in the same cell. A more detailed pharmacological characterisation with a range of bradykinin receptor agonists and antagonists using 45Ca(2+) efflux confirmed the presence of both B(1) and B(2) bradykinin receptors. Using this cellular model, we confirm that T-kinin is a bradykinin B(2) receptor agonist and show for the first time that des-Arg(11)-T-kinin is a potent and selective bradykinin B(1) receptor agonist. In addition, using cells expressing the cloned rat and human bradykinin B(2) receptors plus the Ca(2+)-sensitive protein aequorin, T-kinin was shown to be selective for the rat over the human bradykinin B(2) receptor.