Humphrey P. Rang

Capsazepine: a competitive antagonist of the sensory neurone excitant capsaicin.

1 Capsazepine is a synthetic analogue of the sensory neurone excitotoxin, capsaicin. The present study shows the capsazepine acts as a competitive antagonist of capsaicin. 2 Capsazepine (10 μm) reversibly reduced or abolished the current response to capsaicin (500 nm) of voltage‐clamped dorsal root ganglion (DRG) neurones from rats. In contrast, the responses to 50 μm γ‐aminobutyric acid (GABA) and 5 μm adenosine 5′‐triphosphate (ATP) were unaffected. 3 The effects of capsazepine were examined quantitatively with radioactive ion flux experiments. Capsazepine inhibited the capsaicin (500 nm)‐induced 45Ca2+ uptake in cultures of rat DRG neurones with an IC50 of 420 ± 46 nm (mean ± s.e.mean, n = 6). The 45Ca2+ uptake evoked by resiniferatoxin (RTX), a potent capsaicin‐like agonist was also inhibited. (Log concentration)‐effect curves for RTX (0.3 nm‐1 μm) were shifted in a competitive manner by capsazepine. The Schild plot of the data had a slope of 1.08 ± 0.15 (s.e.) and gave an apparent Kd estimate for capsazepine of 220 nm (95% confidence limits, 57–400 nm). 4 Capsazepine also inhibited the capsaicin‐ and RTX‐evoked efflux of 86Rb+ from cultured DRG neurones. The inhibition appeared to be competitive and Schild plots yielded apparent Kd estimates of 148 nm (95% confidence limits, 30–332 nm) with capsaicin as the agonist and 107 nm (95% confidence limits, 49–162 nm) with RTX as agonist. 5 A similar competitive inhibition by capsazepine was seen for capsaicin‐induced [14C]‐guanidinium efflux from segments of adult rat vagus nerves (apparent Kd = 690 nm; 95% confidence limits, 63 nm‐1.45 μm). No significant difference was noted in the apparent Kd estimates for capsazepine in assays on cultured DRG neurones and vagus nerve as shown by the overlap in the 95% confidence limits. 6 Capsazepine, at concentrations up to 10 μm, had no significant effects on the efflux of 86Rb+ from cultured DRG neurones evoked either by depolarization with high (50 mm) K+ solutions or by acidification of the external medium to pH 5.0–5.6. Similarly capsazepine had no significant effect on the depolarization (50 mm KCl)‐induced efflux of [14C]‐guanidinium from vagus nerve preparations. 7 Ruthenium Red was also tested for antagonism against capsaicin evoked [14C]‐guanidinium release from vague nerves and capsaicin induced 45Ca2+ uptake in cultures of DRG neurones. In contrast to capsazepine the inhibition by Ruthenium Red (10–500 nm in DRG and 0.5–10 μm in vagus nerve experiments) was not consistent with a competitive antagonism, but rather suggested a more complex, non‐competitive inhibition.

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.

A comparison of capsazepine and ruthenium red as capsaicin antagonists in the rat isolated urinary bladder and vas deferens

1 The ability of capsazepine, a recently developed capsaicin receptor antagonist, to prevent the effects of capsaicin on the rat isolated urinary bladder (contraction) and vas deferens (inhibition of electrically‐evoked twitches) was compared to that of ruthenium red, a dye which behaves as a functional antagonist of capsaicin. 2 In the rat bladder, capsazepine (3–30 μm) produced a concentration‐dependent rightward shift of the curve to capsaicin without any significant depression of the maximal response to the agonist. By contrast, ruthenium red (10–30 μm) produced a non‐competitive type of antagonism, characterized by marked depression of the maximal response attainable. Similar findings were obtained in the rat isolated vas deferens in which capsazepine (10 μm) produced a rightward shift of the curve to capsaicin while ruthenium red (3 μm) depressed the maximal response to the agonist. 3 At the concentrations used to block the effect of capsaicin, neither capsazepine nor ruthenium red affected the contractile response of the rat urinary bladder produced by either neurokinin A or electrical field stimulation or the twitch inhibition produced by rat α‐calcitonin gene‐related peptide (αCGRP) in the vas deferens. 4 These findings provide additional evidence that both capsazepine and ruthenium red are valuable tools for exploration of the function of capsaicin‐sensitive primary afferent neurones. The antagonism of the action of capsaicin by capsazepine is entirely consistent with the proposed interaction of this substance with a vanilloid receptor located on primary afferents, while the action of ruthenium red apparently involves a more complex, non‐competitive antagonism.

The putative role of vanilloid receptor-like protein-1 in mediating high threshold noxious heat-sensitivity in rat cultured primary sensory neurons

High threshold noxious heat‐activated currents and vanilloid receptor‐like protein‐1 expression were studied in rat cultured primary sensory neurons to find out the molecule(s) responsible for high threshold noxious heat‐sensitivity. The average temperature threshold and amplitude of high threshold noxious heat‐activated currents were 51.6 ± 0.13 °C and −2.0 ± 0.1nA (at a holding potential of −60 mV), respectively. The current–voltage relationship of high threshold noxious heat‐activated currents was linear at positive membrane potentials, while it showed a weak inward rectification at negative membrane potentials. The average reversal potential measured in control intracellular and extracellular solutions was 4.5 ± 0.9 mV (n = 6). Ionic substitutions revealed that the high threshold noxious heat‐activated current is a nonselective cationic current with calculated ionic permeabilities of Cs+ : Na+ : Ca2+ (1 : 1.3 : 4.5). Consecutive stimuli reduced the heat threshold from 52.2 ± 1 to 48.4 ± 1.4 °C and then to 44 ± 0.7 °C (n = 3). High threshold noxious heat‐activated currents could dose‐dependently and reversibly be reduced by ruthenium red (100 nm−10 µm) but not by capsazepine (10 µm). The average longest diameter of high threshold noxious heat‐sensitive neurons was 31.48 ± 0.5 µm (A = ≈778 µm2; n = 77). Twenty‐three percent of the total neuronal population expressed vanilloid receptor‐like protein‐1. The average area of the vanilloid receptor‐like protein‐1‐immunopositive cells was 1696 ± 65.3 µm2 (d = ∼46 µm). Vanilloid receptor‐like protein‐1‐expressing neurons did not express the vanilloid receptor 1. Comparison of our data with results obtained in vanilloid receptor‐like protein‐1‐expressing non‐neuronal cells and previous immunohistochemical findings suggests that high threshold noxious heat‐activated currents are produced by vanilloid receptor‐like protein‐1 and that high threshold heat‐sensitive dorsal root ganglion neurons are the perikarya of type I noxious heat‐sensitive fibers.

Comparison of intracellular calcium signals evoked by heat and capsaicin in cultured rat dorsal root ganglion neurons and in a cell line expressing the rat vanilloid receptor, VR1.

The cloning of the receptor for capsaicin, vanilloid receptor 1, has shown it to be non-selective cation channel with a high calcium permeability which can be opened by noxious heat as well as capsaicin. Here we compare the calcium signals produced by native and recombinant capsaicin receptors when activated by either heat or capsaicin by imaging intracellular calcium levels ([Ca2+](i)) in rat dorsal root ganglion neurons and Chinese hamster ovary cells transfected with the rat vanilloid receptor, vanilloid receptor 1. Vanilloid receptor 1 transfected cells and a subset of dorsal root ganglion neurons responded to both capsaicin and to heating to 50 degrees C with rapid, substantial and reversible rises in [Ca2+](i). All except one of the dorsal root ganglion neurons responsive to capsaicin also showed sensitivity to heat, and most, but not all, heat-sensitive neurons also responded to capsaicin. Both capsaicin and heat responses were dependent on the presence of extracellular Ca2+. Non-transfected Chinese hamster ovary cells and non-responsive dorsal root ganglion neurons showed only small rises in [Ca2+](i) in response to heat which did not depend on the presence of external Ca2+. Responsive dorsal root ganglion neurons and vanilloid receptor 1 transfected cells showed a clear temperature threshold, above which [Ca2+](i) increased rapidly. This was estimated to be 42.6+/-0.3 degrees C for vanilloid receptor 1 transfected cells and 42.0+/-0.6 degrees C for dorsal root ganglion neurons. The competitive capsaicin antagonist capsazepine (10microM) abolished [Ca2+](i) increases stimulated by capsaicin in both dorsal root ganglion neurons and vanilloid receptor 1 transfected cells. However, responses to heat of a similar magnitude in the same cells were inhibited by only 37% by capsazepine (10microM). In vanilloid receptor 1 transfected cells, Ruthenium Red (10microM) blocked responses to both capsaicin and heat. These results demonstrate that imaging of [Ca2+](i) can identify dorsal root ganglion neurons which are responsive to both heat and capsaicin. They show that heat and capsaicin responses mediated by native and recombinant capsaicin receptors are similar with respect to the characteristics and pharmacology examined, suggesting that expression of recombinant vanilloid receptor 1 in cell lines accurately reproduces the properties of the native receptor.

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.

Comparison of currents activated by noxious heat in rat and chicken primary sensory neurons.

The vanilloid receptor 1 (VR1) gene is responsible for both capsaicin-, and low threshold (LT) noxious heat-sensitivity in mammalian primary sensory neurons. Although, birds lack capsaicin-sensitivity they express LT noxious heat-sensitivity. Here, we compared LT noxious heat-activated whole-cell currents produced by rat and chicken cultured dorsal root ganglion neurons in order to find out the similarities and differences in the LT noxious heat transduction mechanisms between the two species. No significant differences between rat and chicken neurons were found in the mean cell diameter of the LT noxious heat-sensitive cells (20.4+/-0.8 microm, n=19 and 20.6+/-0.6 microm, n=11, respectively) and the average threshold (45.7+/-0.5 degrees C, n=19 and 46.1+/-0.7 degrees C, n=11, respectively) and peak amplitude (-2.9+/-0.6 nA, n=19 and -2.1+/-0.6 nA, n=11, respectively) of the heat-evoked responses. The current-voltage curves of the responses both in rat and chicken cells reversed at the same range (-19.5+/-3.8 mV, n=4 and -15.5+/-1. 2 mV, n=3, respectively) and showed strong outward rectification at negative membrane potentials. While all LT noxious heat-sensitive rat cells responded to capsaicin, none of the chicken neurons produced detectable response to it. These findings suggest that a VR1 homologue which lacks to sequence for capsaicin-sensitivity is possibly the LT noxious heat transducer in chicken.