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Bradykinin (BK) has long been recognized as an important mediator of pain and inflammation. In normal tissue bradykinin causes an acute sensation of pain by an action at B2 receptors, but in inflamed tissue the pharmacology of the response changes to that of B1 receptors. Attempts to demonstrate the presence of functional B1 receptors in sensory neurones have failed, however, and the actions of B1 agonists have therefore been presumed to be indirect. Here we show that specific B1 receptor activation causes translocation of the epsilon isoform of protein kinase C (PKC-epsilon) to the membrane of a small fraction of freshly isolated sensory neurones from rats and mice. The proportion of neurones in which PKC-epsilon translocation was observed increased to around 20% of neurones after 3 days in culture with the neurotrophins glial cell line derived neurotrophic factor (GDNF) and neurturin, but not with nerve growth factor (NGF). Using in situ hybridization we found that the proportion of neurones expressing B1 mRNA increased from close to zero to 20.4% after 8 h culture in GDNF. Neurones expressing functional B1 receptors were negative for the neuropeptides CGRP and substance P, but most expressed functional TRPV1 receptors for capsaicin (60%) and bound the lectin IB4 (68%), both markers characteristic of nociceptors. B1 activation enhanced the heat-activated membrane current approximately 3-fold, and the enhancement was much more prolonged than was the case with B2 activation, consistent with a role for B1 receptors in sustained pain. We conclude that GDNF and neurturin potently upregulate functional B1 receptor expression in small non-peptidergic nociceptive neurones.

1. The effects of activation of protein kinase C (PKC) on membrane currents gated by capsaicin, protons, heat and anandamide were investigated in primary sensory neurones from neonatal rat dorsal root ganglia (DRG) and in HEK293 cells (human embryonic kidney cell line) transiently or stably expressing the human vanilloid receptor hVR1. 2. Maximal activation of PKC by a brief application of phorbol 12-myristate 13-acetate (PMA) increased the mean membrane current activated by a low concentration of capsaicin by 1.65-fold in DRG neurones and 2.18-fold in stably transfected HEK293 cells. Bradykinin, which activates PKC, also enhanced the response to capsaicin in DRG neurones. The specific PKC inhibitor RO31-8220 prevented the enhancement caused by PMA. 3. Activation of PKC did not enhance the membrane current at high concentrations of capsaicin, showing that PKC activation increases the probability of channel opening rather than unmasking channels. 4. Application of PMA alone activated an inward current in HEK293 cells transiently transfected with VR1. The current was suppressed by the VR1 antagonist capsazepine. PMA did not, however, activate a current in the large majority of DRG neurones nor in HEK293 cells stably transfected with VR1. 5. Removing external Ca(2+) enhanced the response to a low concentration of capsaicin 2.40-fold in DRG neurones and 3.42-fold in HEK293 cells. Activation of PKC in zero Ca(2+) produced no further enhancement of the response to capsaicin in either DRG neurones or HEK293 cells stably transfected with VR1. 6. The effects of PKC activation on the membrane current gated by heat, anandamide and low pH were qualitatively similar to those on the capsaicin-gated current. 7. The absence of a current activated by PMA in most DRG neurones or in stably transfected HEK293 cells suggests that activation of PKC does not directly open VR1 channels, but instead increases the probability that they will be activated by capsaicin, heat, low pH or anandamide. Removal of calcium also potentiates activation, and PKC activation then has no further effect. The results are consistent with a model in which phosphorylation of VR1 by PKC increases the probability of channel gating by agonists, and in which dephosphorylation occurs by a calcium-dependent process.

Synaptic transmission between retinal photoreceptors and second-order neurones is controlled by an L-type Ca2+ conductance (gCa) in the photoreceptor inner segment. Modulation of this conductance therefore influences the flow of visual information to higher centres. Possible modulation of gCa by retinal factors was investigated using patch clamp and Ca2+ imaging. No significant modulation of gCa by retinal neurotransmitters nor by intracellular signalling pathways was found. gCa was inhibited by retinoids (all-trans retinal) and by polyunsaturated fatty acids (PUFAs) such as arachidonic acid and docosahexaenoic acid, which are known to be released in the retina by exposure to light. Some PUFAs tested are physiological substrates for the cyclo-oxygenase, lipoxygenase and epoxygenase pathways, but specific inhibitors of these pathways had no effect on the inhibition of gCa. Treatments designed to activate or inhibit G-protein-coupled pathways or protein kinases A and C similarly had no effect on the inhibition by PUFAs nor on gCa itself. Inhibitors of phosphatases 1 and 2A were also largely ineffective. The inhibition by PUFAs is, however, dependent on membrane potential, suggesting that it arises from a direct interaction of fatty acids with the Ca2+ channel. The effect was not use or frequency dependent, suggesting that the effect does not depend on channel gating state. Control by retinoids and by PUFAs may be an important mechanism by which the Ca2+ conductance, and consequently the transmission of the visual signal, is modulated at the first retinal synapse.