Enhanced sensitivity to noxious stimuli is one of the characteristics of the inflammatory response and may result from an increase in the excitability of small-diameter sensory neurones that conduct nociceptive signals. Indeed, a number of pro-inflammatory agents that are synthesized and released in response to trauma can stimulate directly or augment the activation of sensory neurones (Treede et al. 1992). In this regard, prostaglandins represent an important group of agents that sensitize sensory neurones. Exposing various preparations of sensory neurones to prostaglandin E2 (PGE2) or prostaglandin I2 (PGI2) increases the number of action potentials elicited by noxious thermal, chemical and/or mechanical stimuli (Handwerker, 1976; Mense, 1981; Baccaglini & Hogan, 1983; Martin et al. 1987; Nicol & Cui, 1994; Nicol et al. 1997). These prostanoids also facilitate neurotransmitter release evoked by exposing sensory neurones to bradykinin, capsaicin or high extracellular potassium (Franco-Cereceda, 1989; Andreeva & Rang, 1993; Hingtgen & Vasko, 1994; Vasko et al. 1994).
Although the cellular mechanisms underlying the actions of these prostaglandins are not well understood, recent evidence suggests that these eicosanoids modulate ion channel activity that could enhance the excitability of sensory neurones. For example, PGE2 increases the amplitude of a tetrodotoxin (TTX)-resistant sodium current in adult and neonatal rat dorsal root ganglion (DRG) cells (Gold et al. 1996a; England et al. 1996). This prostanoid also suppresses a calcium-dependent slow after-hyperpolarization in adult rat nodose ganglia cells and DRG neurones (Fowler et al. 1985; Gold et al. 1996B). Furthermore, we demonstrated recently that PGI2 and PGE2 attenuate whole-cell potassium currents (IK), whereas the non-sensitizing prostanoid PGF2α is ineffective in sensory neurones (Nicol et al. 1997).
The question remains as to whether the prostaglandin-induced modulation of ion channels results in sensitization of sensory neurones. To initially address this issue, it is important to assess whether prostaglandin-induced sensitization and alterations in ion channels are mediated by the same transduction mechanisms. Because the sensitizing actions of PGE2 or PGI2 on sensory neurones are mediated by the cyclic AMP (cAMP) transduction cascade (Ferreira & Nakamura, 1979; Taiwo et al. 1989; Hingtgen et al. 1995; Cui & Nicol, 1995), modulatory effects of prostanoids on ion channels regulating membrane excitability should also be dependent on the cAMP pathway. We hypothesize that the sensitizing actions of PGE2 are also mediated by the suppression of IK. This notion is based, in part, on observations wherein cAMP modulates voltage-dependent IK in a variety of cell types. In the GH4C1 pituitary cell line, dibutyryl cAMP suppresses a delayed rectifier IK (Chung & Kaczmarek, 1995). Similarly, the current that arises from the expression of Kv3.2 (delayed rectifier potassium) channels in Chinese hamster ovary cells is inhibited by a membrane-permeant analogue of cAMP (Moreno et al. 1995). Activation of the cAMP pathway in mouse neurones isolated from the colliculus leads to a long-term (2-4 h) enhancement of excitability as exhibited by an increased duration of the action potential and a greater number of action potentials evoked by a depolarizing current pulse (Ansanay et al. 1995). These authors found that stimulation of protein kinase A (PKA) inhibits a delayed rectifier-like IK. Lastly, PGE2, through activation of the cAMP-PKA cascade, may play a role in the regulation of vascular smooth muscle tone. In smooth muscle cells isolated from the tail artery of the rat, PGE2 suppresses a non-inactivating IK (Ren et al. 1996). Therefore, taken together, the sensitivity of excitable cells and its modulation by different mediators will play an important role in the regulation of the physiological function of the cell.
To ascertain whether suppression of IK could also be a mechanism for prostaglandin-induced sensitization of sensory neurones, we examined the hypothesis that the cAMP pathway mediates the PGE2-induced decrease in IK in isolated rat embryonic sensory neurones. Our results demonstrate that the cAMP analogue, chlorophenylthio-adenosine cyclic 3′,5′-monophosphate (cpt-cAMP) inhibits a delayed rectifier-like IK in sensory neurones in a manner analogous to PGE2 and that the inhibitory effects of PGE2 on the whole-cell IK are blocked by the inhibition of PKA. Preliminary findings from this study have been reported in abstract form (Evans et al. 1996B).
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Our results demonstrate that cpt-cAMP and PGE2 suppress, in an analogous manner, potassium currents in embryonic rat sensory neurones. This suppression of IK is dependent on activation of the cAMP-PKA transduction cascade since pretreatment with the selective PKA inhibitor, PKI, completely abolished the suppressive effects of PGE2. Thus, these results support the growing evidence that the cAMP signalling pathway mediates the sensitizing actions of PGE2. This evidence includes findings wherein PGE2 elevates intracellular levels of cAMP in rat sensory neurones (Hingtgen et al. 1995). In addition, the PGE2-induced facilitation of both neuropeptide release and the number of action potentials evoked by bradykinin are attenuated by blocking the production of cAMP or inhibiting the activation of PKA (Hingtgen et al. 1995; Cui & Nicol, 1995). In behavioural assays measuring the effects of noxious stimulation in rats, the application of membrane-permeant analogues of cAMP produce a sensitization similar to the hyperalgesia evoked by PGE2 (Ferreira & Nakamura, 1979; Taiwo et al. 1989).
The question remains as to the specific mechanism of action whereby pro-inflammatory prostaglandins give rise to this sensitization in sensory neurones. These prostaglandins are known to modulate multiple types of membrane currents in various model systems. Examples of this mediation are as follows: a non-selective inward current activated by hyperpolarization termed Ih (Ingram & Williams, 1994), a calcium-dependent current that gives rise to a slow after-hyperpolarization (AHPslow) (Fowler et al. 1985; Gold et al. 1996B), calcium currents (Miwa et al. 1988; Alloatti et al. 1991; Mochizuki-Oda et al. 1991; Nicol et al. 1992), a TTX-resistant sodium current (Gold et al. 1996a; England et al. 1996), and outward potassium current (Ren et al. 1996; Nicol et al. 1997). However, several lines of evidence suggest that alterations of some of these currents are not the mechanism(s) by which PGE2 enhances the excitability of small-diameter sensory neurones. First, Ih is expressed mainly in medium to large cells, but not in small-diameter Aδ and C-type sensory neurones (Tokimasa et al. 1990). Second, the inhibitory effects of PGE2 on AHPslow were observed in only about half of the neurones sensitized by this prostanoid (Gold et al. 1996B). Also, PGE2 increased the excitability of neurones not expressing AHPslow, thus modulation of this current was not critical for sensitization. Finally, we reported recently that blockade of N-, L- or P-type voltage-dependent calcium channels did not attenuate the PGE2-mediated facilitation of peptide release from rat sensory neurones, indicating that these channels were not involved in PGE2-induced sensitization (Evans et al. 1996a).
Our findings are consistent with and expand on previous work that has shown that increasing levels of intracellular cAMP led to the suppression of voltage-dependent potassium currents in adult and neonatal DRG cells. Indeed, previous studies indicated a role for the cAMP signalling cascade in the enhancement of membrane excitability. In both chick (Dunlap, 1985) and mouse (Grega & Macdonald, 1987) sensory neurones grown in culture, activation of the cAMP pathway increased the duration of the action potential and this lengthening of the action potential resulted from the inhibition of IK. In sensory neurones isolated from adult rats and grown in culture, the application of membrane-permeant analogues of cAMP produced an inhibition (∼15 %) of the outward IK (Akins & McCleskey, 1993). The modulation of potassium currents as a means of controlling excitability in sensory neurones may be a general scheme. In Aplysia sensory neurones, the serotonin-induced sensitization is analogous to the effects of PGE2 on mammalian sensory neurones and results, in part, from the cAMP-mediated suppression of multiple potassium currents (Klein et al. 1982; Baxter & Byrne, 1989; Goldsmith & Abrams, 1992). Furthermore, in another sensory system, increased levels of cAMP led to the suppression of a TEA-sensitive IK in fungiform taste bud cells (Cummings et al. 1996).
The capacity of cAMP-PKA to decrease IK has also been observed in other neuronal preparations. For example, the activity of the type 2 BK (high conductance calcium-sensitive) potassium channel in recordings from planar bilayers is reduced greatly after exposure to PKA (Reinhart et al. 1991). In addition, PKA reduced the open probability of the human BK channel (hSlo) when it was co-expressed with the β subunit (hSloβ) and exhibited properties that were similar to the native type 2 channel (Dworetzky et al. 1996). In mouse anterior pituitary cells (AtT20 cells), cpt-cAMP reduced an IK believed to be carried by BK potassium channels by about 30 % (Shipston et al. 1996). In the presence of TEA, cpt-cAMP was ineffective at reducing IK. Thus, activation of the cAMP-PKA transduction cascade may be an important signalling mechanism in the regulation of membrane excitability in a variety of neurones.
The exact nature of the IK modulated by activation of PKA is difficult to ascertain. At least two different types of voltage-dependent IK have been described consistently in adult, neonatal and embryonic sensory neurones (Kostyuk et al. 1981; Kameyama, 1983; Akins & McCleskey, 1993; Nicol et al. 1997): an inactivating IA-type and a delayed rectifier-type current. In addition, recent work of Gold and co-workers suggests that additional types of IK exist (Gold et al. 1996c). Although a more careful characterization of the IK modulated by PGE2 and cAMP remains to be determined, our results suggest that the PGE2-/cAMP-modulated IK in rat sensory neurones may be a delayed rectifier-like current. This conclusion is based upon several lines of evidence. First, the current-voltage relations for both the PGE2- and cpt-cAMP-sensitive currents were quite similar, both began to exhibit activation at approximately -20 mV (Fig. 3). Second, the cpt-cAMP-sensitive current observed under various experimental conditions exhibited slow inactivation kinetics, which are characteristic of delayed rectifier currents. Third, the extent of inhibition by either PGE2 or cpt-cAMP did not depend on the level of the conditioning prepulse voltage and was approximately the same for either the peak or steady-state values. Lastly, cpt-cAMP effectively suppressed IK in the presence of 4-AP, whereas no further inhibition of IK was observed in the presence of TEA, an established inhibitor of delayed rectifier-like currents.
Our present work and that of Gold et al. (1996a) and England et al. (1996) demonstrate that PGE2 may enhance neuronal excitability by suppressing potassium currents and/or augmenting a TTX-resistant sodium current. Additionally, both the sensitizing actions of PGE2 and the effects of this prostanoid on potassium and TTX-resistant sodium currents are dependent on the activation of the cAMP transduction cascade (Cui & Nicol, 1995; Hingtgen et al. 1995; England et al. 1996). Therefore, taken together, activation of the cAMP signalling pathway gives rise to PKA-mediated modulation of the activity of multiple ion channels, which, in turn, results in the enhanced excitability of sensory neurones. This prostaglandin-induced modification of ion channel activity may then account for the heightened sensitivity to noxious stimulation exhibited in behavioural observations.