Gillian M. Burgess

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.

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.

Activation of guanylate cyclase by bradykinin in rat sensory neurones is mediated by calcium influx: possible role of the increase in cyclic GMP

Abstract: Bradykinin, which activates polymodal nociceptors, increased cyclic GMP (cGMP) in a capsaiciiji‐sensitive population of cultured sensory neurones from rat dorsal root ganglia (DRG) by stimulating guanylate cyclase, but had no effect on cyclic AMP (cAMP). In nonneunbnal cells from DRG, bradykinin increased cAMP, but not cpMP. The bra‐dykinin‐induced increase in cGMP in thej neurones was completely blocked by removal of extracellular Ca2+, or by incubation of the cells with the calcium channel blockers nifedipine and verapamil. Pretreatment of the| neurones with either dibutyryl cGMP or sodium nitroprusside (which elevates cGMP) inhibited bradykinin‐induced formation of inositol phosphates. It is possible that cGMP could be involved in the regulation of polyphosphoinositide turnover in DRG neurones.

Capsaicin-Induced Ion Fluxes Increase Cyclic GMP but Not Cyclic AMP Levels in Rat Sensory Neurones in Culture

Abstract: Capsaicin, which induces fluxes of sodilim, calcium, and potassium ions in a subset of both neonatal and adult rat dorsal root ganglion neurones, increased cyclic GMP (cGMP) levels by a factor of 20 (EC50 0.07 μ) to 10‐20 pmol cGMP/mg protein in these cells. Cyclic AMP (CAMP) levels were unaffected. Nonneuronal cells derived from rat ganglia, and both neurones and nonneuronal cells from chick were unresponsive to capsaicin. Capsaicin‐induced cGMP elevation in rat dorsal root ganglion (DRG) neurones was unaffected by pertussis toxin, lowered by compounds that block voltage‐sensitive calcium channels, and was abolished by the removal of extracellular calcium. Calcium, guanidine, and rubidium fluxes were unaffected by treatment of DRG cells with sodium nitroprusside or dibutyryl cGMP. The cGMP response to capsaicin is thus a function of capsaicin‐evoked calcium uptake through voltage‐sensitive calcium channels. Elevated cGMP levels do not, however, contribute to capsaicin‐evoked ion fluxes or to their desensitisation.

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.

Regulation of bradykinin receptor gene expression in human lung fibroblasts.

In WI-38 human fibroblasts, interleukin-1 beta and tumour necrosis factor-alpha (TNF-alpha) increased bradykinin B(1) receptor mRNA, which peaked between 2 and 4 h, remaining elevated for 20 h. Binding of the bradykinin B(1) receptor selective ligand [3H]des-Arg(10)-kallidin, also increased, peaking at 4 h and remaining elevated for 20 h. The B(max) value for [3H]des-Arg(10)-kallidin rose from 280+/-102 fmol/mg (n=3) to 701+/-147 fmol/mg (n=3), but the K(D) value remained unaltered (control, 1.04+/-0.33 nM (n=3); interleukin-1 beta, 0.88+/-0.41 nM (n=3)). The interleukin-1 beta-induced [3H]des-Arg(10)-kallidin binding sites were functional receptors, as bradykinin B(1) receptor agonist-induced responses increased in treated cells. Bradykinin B(2) receptor mRNA and [3H]bradykinin binding were upregulated by interleukin-1 beta, but not TNF-alpha. The effect of interleukin-1 beta on bradykinin B(2) receptors was smaller than for bradykinin B(1) receptors. Cycloheximide prevented interleukin-1 beta-mediated increases in B(1) and B(2) binding, but not mRNA suggesting that de novo synthesis of a transcriptional activator was unnecessary.

Bradykinin evoked depolarization of a novel neuroblastoma × DRG neurone hybrid cell line (ND723)

Application of bradykinin (Bk) to neuroblastoma x dorsal root ganglion (DRG) neurone hybrid cells (ND7/23) evoked an inward (depolarizing) current associated with an increase in membrane conductance. This response was antagonized by D-Arg0,Hyp3,Thi5,8,D-Phe7-Bk, but was not mimicked by des-Arg9-Bk, indicating the involvement of B2-receptors. The response was unaltered by replacement of extracellular Na+ by N-methylglucamine. Replacement of extracellular Cl by gluconate shifted the estimate reversal potential to a more positive value, while the use of potassium acetate filled recording electrodes shifted the reversal potential to a more negative value, and reduced the response amplitude, indicating the importance of Cl- in the response. This response to Bk was mimicked by the calcium ionophore ionomycin. Bk stimulated the formation of inositol 1,4,5-trisphosphate (IP3), and increased the release of arachidonic acid. In addition, Bk produced an increase in [Ca2+]i, as determined by microspectrofluorimetry. This was due to the release of Ca2+ from intracellular stores, since the response was unaltered when the cells were bathed in Ca(2+)-free solution. In summary, Bk depolarizes ND7/23 cells, probably through the activation of a chloride conductance. It seems likely that this is secondary to the rise in cytosolic Ca2+ concentration, due to the release of Ca2+ from internal stores by IP3. This Ca(2+)-activated chloride response is present in some sensory neurones, although its role in the activation of sensory neurones by Bk is at present unclear.

Carbohydrate analysis of the B2 bradykinin receptor from rat uterus

Abstract: The B2 bradykinin receptor purified from rat uterus has an apparent molecular mass of 81 kDa. This is higher than the value of 42 kDa estimated from the sequence data of rat and human B2 receptors. Carbohydrate analysis of the rat B2 bradykinin receptor indicated that it was a sialoglycoprotein with three N‐linked complex oligosaccharide side chains. This was consistent with the sequence, which has three potential glycosylation sites. The receptor did not appear to possess O‐linked carbohydrate side chains. Removal of the N‐linked carbohydrates with endo‐β‐N‐acetylglucosaminidase yielded a core protein of 42–44 kDa. The presence of these N‐linked carbohydrates explains the discrepancy between the molecular size of the purified receptor protein and that estimated from the sequence. The sequence of the rat receptor suggests an isoelectric point of about pH 7.0, but the purified receptor had an isoelectric point of pH 4.5–4.7. Sialic acid residues on the N‐linked side chains are likely to be responsible for the acidic nature of the rat receptor. Carbohydrate does not appear to play a role in ligand‐receptor interactions, as deglycosylation did not alter the affinity of the B2 bradykinin receptor for bradykinin or the B2‐selective antagonist HOE‐140.

Selective labelling of bradykinin receptor subtypes in WI38 human lung fibroblasts.

1 Binding of the Bj bradykinin receptor radioligand, [3H]‐des‐Arg10‐kallidin (‐KD) and the B2 receptor radioligand [3H]‐bradykinin (‐BK) was investigated in membranes prepared from WI38 human foetal lung fibroblasts. 2 One‐site analysis of the saturation data for [3H]‐des‐Arg10‐KD gave an equilibrium dissociation constant (KD) value of 0.51 ± 0.12 nM and a maximum receptor density (Bmax) of 260 ± 49 fmol mg−1 of protein. [3H]‐des‐Arg10‐KD binding was displaced by ligands in the order: des‐Arg10‐KD > KD > > des‐Arg9[Leu8]‐BK > des‐Arg9‐BK > Hoe 140 > > BK, implying that it was binding selectively to B1 receptors. 3 One‐site analysis of the binding of [3H]‐BK to WI38 membranes indicated that it had a KD value of 0.25 ± 0.06 nM and a Bmax of 753 ± 98 fmol mg−1 of protein. The potencies for displacement of [3H]‐BK binding were: Hoe 140 > > BK = KD > > > des‐Arg10‐KD = des‐Arg9[Leu8]‐BK = des‐Arg9‐BK, which was consistent with binding to B2 receptors. 4 This is the first characterization of [3H]‐des‐Arg10‐KD binding to include both kinetic and equilibrium data, and demonstrates that [3H]‐des‐Arg10‐KD has a high affinity for human B1 bradykinin receptors and is sufficiently selective to be used as a radioligand for B1 receptors in human cells or tissues expressing an excess of B2 BK receptors.