Inflammatory mediators not only activate "pain-"sensing neurons, the nociceptors, to trigger acute pain sensations, more important, they increase nociceptor responsiveness to produce inflammatory hyperalgesia. For example, prostaglandins activate G(s)-protein-coupled receptors and initiate cAMP- and protein kinase A (PKA)-mediated processes. We demonstrate for the first time at the cellular level that heat-activated ionic currents were potentiated after exposure to the cAMP activator forskolin in rat nociceptive neurons. The potentiation was prevented in the presence of the selective PKA inhibitor PKI(14-22), suggesting PKA-mediated phosphorylation of the heat transducer protein. PKA regulatory subunits were found in close vicinity to the plasma membrane in these neurons, and PKA catalytic subunits only translocated to the cell periphery when activated. The translocation and the current potentiation were abolished in the presence of an A-kinase anchoring protein (AKAP) inhibitor. Similar current changes after PKA activation were obtained from human embryonic kidney 293t cells transfected with the wild-type heat transducer protein vanilloid receptor 1 (VR-1). The forskolin-induced current potentiation was greatly reduced in cells transfected with VR-1 mutants carrying point mutations at the predicted PKA phosphorylation sites. The heat transducer VR-1 is therefore suggested as the molecular target of PKA phosphorylation, and potentiation of current responses to heat depends on phosphorylation at predicted PKA consensus sites. Thus, the PKA/AKAP/VR-1 module presents as the molecular correlate of G(s)-mediated inflammatory hyperalgesia.

Interleukin 1 beta (IL-1 beta) is a proinflammatory cytokine that maintains thermal hyperalgesia and facilitates the release of calcitonin gene-related peptide from rat cutaneous nociceptors in vivo and in vitro. Brief applications of IL-1 beta to nociceptive neurons yielded a potentiation of heat-activated inward currents (Iheat) and a shift of activation threshold toward lower temperature without altering intracellular calcium levels. The IL-1 beta-induced heat sensitization was not dependent on G-protein-coupled receptors but was mediated by activation of protein kinases. The nonspecific protein kinase inhibitor staurosporine, the specific protein kinase C inhibitor bisindolylmaleimide BIM1, and the protein tyrosine kinase inhibitor genistein reduced the sensitizing effect of IL-1 beta whereas negative controls were ineffective. RT-PCR and in situ hybridization revealed IL-1RI but not RII expression in neurons rather than surrounding satellite cells in rat dorsal root ganglia. IL-1 beta acts on sensory neurons to increase their susceptibility for noxious heat via an IL-1RI/PTK/PKC-dependent mechanism.

The pro-inflammatory cytokine interleukin-6 (IL-6) together with its soluble receptor (sIL-6R) induces and maintains thermal hyperalgesia. It facilitates the heat-induced release of calcitonin gene-related peptide from rat cutaneous nociceptors in vivo and in vitro. Here we report that exposure of nociceptive neurons to the IL-6-sIL-6R complex or the gp130-stimulating designer IL-6-sIL-6R fusion protein Hyper-IL-6 (HIL-6) resulted in a potentiation of heat-activated inward currents (I(heat)) and a shift of activation thresholds towards lower temperatures without affecting intracellular calcium levels. The Janus tyrosine kinase inhibitor AG490, the selective protein kinase C (PKC) inhibitor, bisindolylmaleimide 1 (BIM1), as well as rottlerin, a selective blocker of the PKCdelta isoform, but not the cyclooxygenase inhibitor indomethacin, effectively reduced the effect. Reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization revealed expression of mRNA for the signal-transducing beta subunit of the receptor gp130 in neuronal somata, rather than satellite cells in rat dorsal root ganglia. Together, the results suggest that IL-6-sIL-6R acts directly on sensory neurons. It increases their susceptibility to noxious heat via the gp130/Jak/PKCdelta signalling pathway.

Calcium influx and the resulting increase in intracellular calcium concentration ([Ca(2+)](i)) can induce enhanced sensitivity to temperature increases in nociceptive neurons. This sensitization accounts for heat hyperalgesia that is regularly observed following the activation of excitatory inward currents by pain-producing mediators. Here we show that rat sensory neurons express calcium-dependent adenylyl cyclases (AC) using RT-PCR and nonradioactive in situ hybridization. Ionomycin-induced rises in [Ca(2+)](i)-activated calcium-dependent AC and caused translocation of catalytic protein kinase A subunit. Elevation of [Ca(2+)](i) finally resulted in a significant potentiation of heat-activated currents and a drop in heat threshold. This was not prevented in the presence of suramin that nonspecifically uncouples G protein-dependent receptors. The sensitization was, however, inhibited when the specific PKA antagonist PKI(14-22) was added to the pipette solution or when PKA coupling to A kinase anchoring protein (AKAP) was disrupted with InCELLect StHt-31 uncoupling peptide. The results show that heat sensitization in nociceptive neurons can be induced by increases in [Ca(2+)](i) and requires PKA that is functionally coupled to the heat transducer, mostly likely vanilloid receptor VR-1. This calcium-dependent pathway can account for the sensitizing properties of many excitatory mediators that activate cationic membrane currents.