Elsa Phillips

Pharmacological differences between the human and rat vanilloid receptor 1 (VR1).

Vanilloid receptors (VR1) were cloned from human and rat dorsal root ganglion libraries and expressed in Xenopus oocytes or Chinese Hamster Ovary (CHO) cells. Both rat and human VR1 formed ligand gated channels that were activated by capsaicin with similar EC50 values. Capsaicin had a lower potency on both channels, when measured electrophysiologically in oocytes compared to CHO cells (oocytes: rat=1.90±0.20 μM; human=1.90±0.30 μM: CHO cells: rat=0.20±0.06 μM; human=0.19±0.08 μM). In CHO cell lines co‐expressing either rat or human VR1 and the calcium sensitive, luminescent protein, aequorin, the EC50 values for capsaicin‐induced responses were similar in both cell lines (rat=0.35±0.06 μM, human=0.53±0.03 μM). The threshold for activation by acidic solutions was lower for human VR1 channels than that for rat VR1 (EC50 pH 5.49±0.04 and pH 5.78±0.09, respectively). The threshold for heat activation was identical (42°C) for rat and human VR1. PPAHV was an agonist at rat VR1 (EC50 between 3 and 10 μM) but was virtually inactive at the human VR1 (EC50>10 μM). Capsazepine and ruthenium red were both more potent at blocking the capsaicin response of human VR1 than rat VR1. Capsazepine blocked the human but not the rat VR1 response to low pH. Capsazepine was also more effective at inhibiting the noxious heat response of human than of rat VR1.

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.

Spinal neurokinin NK1 receptor down-regulation and antinociception: effects of spinal NK1 receptor antisense oligonucleotides and NK1 receptor occupancy.

Abstract: To define the effects of antisense oligonucleotides on spinal neurokinin 1 (NK1) receptor function in nociceptive processing, several antisense oligonucleotides directed against the NK1 receptor mRNA were intrathecally injected into rats via an implanted catheter, and their effect on the behavioural response to formalin injected into the paw was assessed. We observed that there was no significant reduction of pain behaviour or immunostaining of spinal NK1 receptors after repeated daily intrathecal treatment with an antisense oligonucleotide. However, spinal application of substance P (SP) in the antisense oligonucleotide‐treated animals resulted in a profound and long‐lasting reduction in the behavioural response to formalin injection, and a parallel reduction in the NK1 receptor immunoreactivity normally observed in spinal dorsal horn. Intrathecal SP in the control groups, i.e., rats treated with an oligonucleotide containing four mismatched bases, the corresponding sense oligonucleotide, a mixture of the sense and the antisense oligonucleotides, in each case had no effect. The effects of SP were blocked by NK1 receptor antagonists and were not mimicked by NMDA. The mechanism underlying these effects is not clear. It may be due to partial degradation of the internalised receptors, which cannot be replaced by newly synthesised receptors because of the action of the NK1 antisense oligonucleotide.

B1 and B2 Bradykinin Receptors Encoded by Distinct mRNAs

Abstract: Bradykinin receptors have been subdivided into at least two major pharmacological subtypes, B1 and B2. The cDNAs encoding functional B2 receptors have recently been cloned, but no molecular information exists at present on the B1 receptor. In this article, we describe experiments examining the possible relationship between the mRNAs encoding the B1 and B2 types of receptor. We showed previously that the Human fibroblast cell line W138 expresses both B1 and B2 receptors. In this report, we describe oocyte expression experiments showing that the B1 receptor in W138 human fibroblast cells is encoded by a distinct mRNA ∼2 kb shorter than that encoding the B2 receptor. We have used an antisense approach in conjunction with the oocyte expression system to demonstrate that the two messages differ in sequence at several locations throughout the length of the B2 sequence. Taken together with the mixed pharmacology exhibited in some expression systems by the cloned mouse receptor, the data indicate that B1‐type pharmacology may arise from two independent molecular mechanisms.

Expression of Functional Bradykinin Receptors in Xenopus Oocytes

Abstract: mRNA prepared from various tissues and cultured cells was injected into Xenopus laevis oocytes. Three to five days after injection, the response of the oocytes to the peptide bradykinin was monitored. The oocytes were voltage clamped and the membrane currents generated on application of agonist were recorded. mRNA from NG108‐15, rat uterus, and human fibroblast cell line WI38 gave similar responses to bradykinin (1 μM), with an initial inward current (10–20 nA) followed by a prolonged period of membrane current oscillations. The same pattern of response was given by total RNA from rat dorsal root ganglia. No response to bradykinin (10 μM) was recorded from oocytes injected with rat brain mRNA, although these oocytes gave peak inward currents of about 75 nA in response to serotonin (10 μM). mRNA from both NG108–15 cells and rat uterus was fractionated on sucrose gradients. This resulted in an approximately fivefold increase in the size of the response compared to that given by unfractionated mRNA. The largest responses were given by mRNA fractions with a size of approximately 4.5 kb. Data were obtained consistent with the expression of both B1 and B2 receptors by WI38 human fibroblasts and with the expression of only the B2 type of receptor by NG108‐15 cells.

Characterization of bradykinin receptors solubilized from rat uterus and NG108-15 cells

In this paper we report the solubilization of bradykinin B2 binding sites from membranes prepared from NG 108-15 tumours and rat uterus with retention of binding activity. Digitonin was found to solubilize the receptors from both tissues, and the addition of CHAPS increased the yield of soluble receptor from rat uterus only. The affinity of a range of bradykinin analogues has been shown to have the same rank order for both the soluble and membrane receptors from both tissues, and in corresponding functional assays. In addition, the binding of bradykinin ligands to the soluble and membrane receptors is similarly modulated by the presence of sodium ions. We conclude that the soluble binding sites correspond to the physiological bradykinin B2 receptor present in these tissues.

Construction of a Physiologically Active Photoaffinity Probe Based on the Structure of Bradykinin: Labelling of Angiotensin Converting Enzyme but Not Candidate Bradykinin Receptors on NG108–15 Cells

Abstract: The peptides bradykinin and kallidin are released in response to noxious stimuli and mediate various physiological effects, including a direct stimulation of nociceptive afferent neurones. The nature of the receptor molecules through which these ligands act is presently unknown. We synthesised an iodinatable photoaffinity probe, N′‐4‐azido‐salicylylkallidin, and used it in an attempt to identify candidate bradykinin receptors on the NG108–15 neuroblastoma X glioma hybrid cell line. The ligand bound in subdued light to a particulate fraction of NG108–15 tumours and could be displaced by bradykinin with an IC50 of 0.33 nM. In a physiological assay, it behaved as an agonist equipotent with bradykinin. Gel analysis of the labelled products after photolysis of the iodinated ligand in the presence of NG108–15 cells or tumour membranes revealed bradykinin‐blockable labelling of a glycoprotein with an Mr of 166,000. The probe was also able to label purified commercial angiotensin converting enzyme. The band labelled in NG108–15 cells was immunoprecipitable with a polyclonal antiserum to angiotensin converting enzyme, an enzyme shown to be present in low amounts in these preparations by direct binding using the iodinatable specific ligand MK351A.

A radioimmunoassay for bradykinin based on monoclonal antibodies

We describe two monoclonal antibodies with recognise the peptides bradykinin and kallidin. These antibodies were used in a study to assess the feasibility of using monoclonal antibodies in place of conventional sera for radioimmunoassay. Antibody SBK1 recognises bradykinin with a Kd of 0.67 +/- 0.17 nM. It fails to recognise bradykinin from which any carboxyl terminal amino acid has been removed. Antibody SBK2 recognises bradykinin with a Kd of 58.11 +/- 7.55 nM; it recognises 1-8 and 1-7 bradykinin about 30% as effectively as the full-length sequence, and shorter carboxyl terminal truncated sequences with a progressively declining efficiency. In this model system, bradykinin concentrations of 0.2 nM could be reliably measured, and carboxyl truncated versions of the peptide could be distinguished from the parent molecule. The feasibility of using monoclonal antibodies to assay bradykinin and other small peptides is discussed in the light of these results.

An Iodinatable Photoaffinity Probe Based on the Structure of Kallidin

Binding sites for the peptides bradykinin (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg; Bk) and kallidin (N-lysyl-bradykinin) have been described on many tissues. These include various smooth muscle preparations such as uterus and ileum, blood vessels, and the processes of nociceptive afferent neurons (Erdos, 1979). In addition, binding sites have been described on cultured fibroblasts and on cell lines such as the neuroblastoma-glioma hybrid NG108-15, (Hamprecht, 1977) where they probably correspond to physiologically active Bk receptors.