Harvey, Justine Sarah; (1994) Signal transduction pathways activated by the noxious agents, bradykinin and resiniferatoxin in sensory neurones. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Bradykinin-induced formation of [3H]inositol-l,4,5 trisphosphate ([3H]IP3) in cultured neonatal rat dorsal root ganglion neurones desensitised after brief exposure to bradykinin. This was characterised by an increase in the EC50 value and a reduction in the maximum response for bradykinin-induced [3H]IP3 formation. There was no change in the affinity of the bradykinin receptor on the neurones for [3H]bradykinin but desensitisation was accompanied by a reduction in the number of binding sites. Mimicking bradykinin-induced elevation of cyclic GMP with dibutyryl cGMP and activation of protein kinase C with phorbol 12,13-dibutyrate reduced bradykinin-induced [3H]IP3 formation, but had no effect on [3H]bradykinin binding. Preventing bradykinin-induced formation of cyclic GMP with the nitric oxide synthase inhibitor N-nitro-L-arginine reduced bradykinin-induced desensitisation. Similarly, preventing activation of protein kinase C with GF109203X also reduced bradykinin-induced desensitisation. Simultaneous pretreatment with maximal concentrations of dibutyryl cGMP and phorbol 12, 13-dibutyrate had a greater effect on the maximum response to bradykinin than each agent alone, mimicking the effect of pretreatment with bradykinin, implying that their mechanisms of action did not entirely overlap. Pretreatment with a combination of dibutyryl cGMP and phorbol 12,13-dibutyrate had no effect on [3H]bradykinin binding. Pretreatment with neuropeptide Y, which also activates phospholipase C, reduced bradykinin-induced [3H]IP3 formation, and vice versa, implying that both heterologous and homologous desensitisation were occurring. These studies show that the sensitivity of dorsal root ganglion neurones to Bk is regulated by cyclic GMP and protein kinase C through different sites, providing strong evidence for a role for cyclic GMP in desensitisation and weaker evidence for a role for PKC. An additional mechanism might be responsible for the loss of [3H]bradykinin binding sites seen following bradykinin-induced desensitisation. Resiniferatoxin activates sensory neurones via the capsaicin receptor. [3H]resiniferatoxin binding was detected in membranes (KD value of 1.8 ± 0.2 nM), but not in the cytosolic fraction, of rat dorsal root ganglia . Resiniferatoxin caused translocation of protein kinase C in cultures of neonatal rat dorsal root ganglion neurones (EC50 value of 18 ± 3 nM). This response was greatly reduced, but not abolished, in the absence of external calcium. Raising [Ca2+]i with ionomycin and KCl also resulted in translocation of protein kinase C. These results suggested that the effect of resiniferatoxin on translocation was indirect. Resiniferatoxin did, however, cause direct activation of a Ca2+ and lipid-dependent kinase, or kinases, isolated from the cytosol of adult rat dorsal root ganglia at concentrations higher (100 nM-10 mM) than those required for [3H]resiniferatoxin binding and translocation of protein kinase C in cultured neurones.

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