Low-threshold heat receptor in chick sensory neurons is upregulated independently of nerve growth factor after nerve injury.

In mammals, the cloned low-threshold heat receptor, vanilloid receptor subtype 1 (VR1), is involved in the genesis of thermal hyperalgesia after inflammation. However, there is evidence that VR1 is not involved in the thermal hyperalgesia that occurs after nerve injury. In search for other heat receptors which might be involved in this phenomenon, we previously demonstrated that chick dorsal root ganglion neurons, which are insensitive to capsaicin, respond to low-threshold heat. Here, we investigated whether expression of the low-threshold noxious heat receptor in chicks is regulated by nerve growth factor (NGF), as VR1 is in mammals. Heat (44 degrees C) responsiveness of isolated dorsal root ganglion neurons of chicks was investigated (i) under culture conditions for up to 4 days with and without NGF and (ii) after a tight ligation of the sciatic nerve for up to 6 days, using cobalt-uptake method. In every case, a significant upregulation in the proportion of heat-responsive neurons was observed. On the molecular level, there was an increase of chick VR1 mRNA level in dorsal root ganglion cells cultured for 3 days in medium lacking NGF. In rat dorsal root ganglion neurons cultured for 1-4 days without NGF, patch-clamp experiments revealed that after 1 day almost all neurons responding to heat also responded to capsaicin, whereas after 3-4 days, more than one-half of the heat-responsive neurons did not respond to capsaicin. These data suggest the existence of low-threshold heat receptors in chick dorsal root ganglion neurons, the expression of which is regulated independently of NGF.

No evidence for bradykinin B1 receptors in rat dorsal root ganglion neurons.

Bradykinin receptors are believed to contribute to hyperalgesia under conditions of neuropathic pain. Using calcium imaging we investigated responses to B1 and B2 agonists on isolated rat dorsal root ganglion neurons. No response to the B1 agonist was detected, whereas 12% of neurons responded to the B2 agonist. Northern blot analysis confirmed the lack of B1 receptor expression in dorsal root ganglia, as B1 mRNA was neither detected under normal conditions nor after nerve injury. In the calcium imaging experiments, agonists were applied with an elevated superfusion flow rate to avoid tachyphylaxis to the drug. Normal external solution applied at this flow rate constituted a mechanical stimulus causing a response in some neurons. Thus, in comparable set-ups mechanosensitivity has first to be tested to avoid masking effects.

Low-threshold heat response antagonized by capsazepine in chick sensory neurons, which are capsaicin-insensitive.

The heat-transducing receptor VR1 cloned from rat sensory neurons can be activated by both noxious heat and capsaicin. As the response of sensory neurons to capsaicin is species dependent, it is conceivable that the responses to noxious heat and to capsaicin are transduced by distinct receptors across different species. Therefore, we investigated responses to noxious heat from a capsaicin-insensitive (chick) and a capsaicin-sensitive (rat) species. In chick, whole-cell patch-clamp experiments in isolated dorsal root ganglion neurons revealed two populations of neurons with different thresholds to noxious heat, activated at approximately 43 degrees C and approximately 53 degrees C. In cobalt uptake experiments, the proportion of neurons showing a heat-induced response increased with increasing heat stimuli. Application of capsaicin (1-10 microM) did not result in inward currents or cobalt uptake. Rat neurons yielded comparable results in heat experiments, but were capsaicin-sensitive. Although chick neurons are insensitive to capsaicin, the competitive capsaicin antagonist capsazepine (1-10 microM) was effective in blocking heat-induced responses, verified by patch-clamp and cobalt uptake methods. The noncompetitive capsaicin antagonist ruthenium red (10 microM) reduced to almost nil the proportion of heat-responsive neurons identified with the cobalt uptake method. These findings suggest that chick DRG neurons express a low-threshold heat-transducing receptor with a pharmacological profile distinct from the low-threshold heat receptor VR1 cloned from rat DRG neurons. The data support the idea that there might be heat receptor subtypes with differences in the capsaicin binding site.

A non-pungent triprenyl phenol of fungal origin, scutigeral, stimulates rat dorsal root ganglion neurons via interaction at vanilloid receptors.

1. A [3H]-resiniferatoxin (RTX) binding assay utilizing rat spinal cord membranes was employed to identify novel vanilloids in a collection of natural products of fungal origin. Of the five active compounds found (scutigeral, acetyl-scutigeral, ovinal, neogrifolin, and methyl-neogrifolin), scutigeral (Ki=19 microM), isolated from the edible mushroom Albatrellus ovinus, was selected for further characterization. 2. Scutigeral induced a dose-dependent 45Ca uptake by rat dorsal root ganglion neurons with an EC50 of 1.6 microM, which was fully inhibited by the competitive vanilloid receptor antagonist capsazepine (IC50=5.2 microM). 3. [3H]-RTX binding isotherms were shifted by scutigeral (10-80 microM) in a competitive manner. The Schild plot of the data had a slope of 0.8 and gave an apparent Kd estimate for scutigeral of 32 microM. 4. Although in the above assays scutigeral mimicked capsaicin, it was not pungent on the human tongue up to a dose of 100 nmol per tongue, nor did it provoke protective wiping movements in the rat (up to 100 microM) upon intraocular instillation. 5. In accord with being non-pungent, scutigeral (5 microM) did not elicit a measurable inward current in isolated rat dorsal root ganglion neurons under voltage-clamp conditions. It did, however, reduce the proportion of neurons (from 61 to 15%) that responded to a subsequent capsaicin (1 microM) challenge. In these neurons, scutigeral both delayed (from 27 to 72 s) and diminished (from 5.0 to 1.9 nA) the maximal current evoked by capsaicin. 6. In conclusion, scutigeral and its congeners form a new chemical class of vanilloids, the triprenyl phenols. Scutigeral promises to be a novel chemical lead for the development of orally active, non-pungent vanilloids.

Dialdehyde sesquiterpenes and other terpenoids as vanilloids.

Selected naturally occurring unsaturated dialdehyde sesquiterpenes and related bioactive terpenoids were assayed for vanilloid-like activity. Out of the 25 compounds tested, eight inhibited completely the specific binding of [3H]resiniferatoxin by rat spinal cord membranes: binding affinities ranged from 0.6 microM for cinnamodial to 19.0 microM for hebelomic acid F. These values were comparable to the binding affinity of capsaicin (2.7 microM). With the exception of four ligands, compounds that inhibited resiniferatoxin binding to rat spinal cord membranes were also pungent on the human tongue where they showed cross-tachyphylaxis with capsaicin. As expected from their reactive nature, these compounds possess additional sites of action, as reflected in the complex behavior of the stimulation of calcium influx by cinnamodial and cinnamosmolide at high concentrations. This observation might explain the unexpectedly weak membrane depolarization by cinnamodial compared to capsaicin. We conclude that a range of sesquiterpene dialdehydes and related terpenoids, both pungent and non-pungent, may function as vanilloids. These compounds may represent a new chemical lead for the development of vanilloid drugs, structurally unrelated to either capsaicin or resiniferatoxin.

The proportion of isolated rat dorsal root ganglion neurones responding to bradykinin increases with time in culture.

The proportion of isolated rodent dorsal root ganglion neurones expressing bradykinin receptors increases transiently with time in culture. However, it has not yet been investigated whether these receptors are functioning. Therefore the responses of these neurones to bradykinin (1 microM) were investigated in patch-clamp experiments in the current clamp mode after 0.8 and 1.8 days under culture conditions. The proportion of neurones responding to bradykinin was 26% (5/19) at day 0.8 and increased to 73% (16/22) at day 1.8. The intensity of the response was assessed by counting the number of action potentials evoked by bradykinin within four fixed intervals of 500 ms duration during each experiment. It increased with time in culture from an average of 8 +/- 2 (SD) at day 0.8 to 16 +/- 6 at day 1.8, respectively. These results provide evidence for the induction of functioning bradykinin receptors in cultured dorsal root ganglion neurones with time in culture.

Plasticity in the expression of bradykinin binding sites in sensory neurons after mechanical nerve injury.

The pro-inflammatory mediator bradykinin plays an important role in hyperalgesia during inflammatory conditions. Here, we used unilateral ligation of the sciatic nerve to investigate whether the expression of bradykinin binding sites in isolated rat dorsal root ganglion neurons is changed following nerve injury. Under control conditions, the percentage of neurons expressing bradykinin binding sites increased from 52% at day 0.8 in culture to 93% at day 1.8 and decreased to 67% at day 3.8. Following nerve ligation either two or 10 days prior to the isolation of the somata, the percentage of neurons from ipsilateral ganglia that expressed bradykinin binding sites was already 87% and 86%, respectively, at day 0.8 in culture; this level was maintained at day 1.8 and decreased slightly at day 3.8. In control neurons, high densities of bradykinin binding sites on individual neurons were observed no sooner than at day 1.8, but already at day 0.8 following nerve ligation, due to a "de novo" expression of B1 receptors and augmentation of B2 receptors. Neurons from the contralateral side responded similarly to ipsilateral neurons after a two day nerve ligation, however, after either a 10 day ligation or a sham operation neurons responded similarly to control neurons. These data are the first evidence that expression of B1 receptors is induced and expression of B2 receptors is enhanced in sensory neurons following nerve ligation. Under pathophysiological conditions, increased expression of subtypes of bradykinin receptors in sensory neurons could contribute to chronic pain conditions.

Multiple capsaicin-evoked currents in isolated rat sensory neurons.

The response to capsaicin in functional assays suggests multiple sites of capsaicin action. This hypothesis is supported by the results of the present patch-clamp study of isolated dorsal root ganglion cells of the rat. The response to a prolonged application of capsaicin of different concentrations in an external solution with different ion compositions was investigated. Capsaicin evoked up to three distinct current components. The first and second current components could be activated independently. The third component occurred only in the presence of sodium and only in cells in which the second component was also elicited. In an extracellular solution with a physiological composition of ions and 300 nM capsaicin, the peaks of the three components, when evoked, occurred at 10.1 +/- 1.35 s (mean +/- S.E.M., n = 9), 44.0 +/- 2.64 s (n = 16) and 79.0 +/- 8.10 s (n = 5). The activation of the first and/or second current component depended on the concentration of capsaicin. A low concentration predominantly elicited the second component, while a high concentration activated the first and suppressed the second one. The third component seems to be a secondary response of the cell and was not investigated in detail. The activation and decay phases of the first two current components could be fitted by single exponential functions, whereas those of the third component could not. The first and second current components were carried by sodium and calcium. After tachyphylaxis, if the extracellular medium was then acidified to a pH of 6.3, the second component alone could then be elicited by capsaicin. The results demonstrate that capsaicin can elicit different current components that are distinguishable by their time-course, by the effects of acidification of the extracellular solution and by the concentration of capsaicin required to activate these currents. We postulate two distinct binding sites of capsaicin causing two distinct current components. This may account for the variety of physiological responses evoked by capsaicin and the variations in these responses between species.