Michael R. Vasko

Pregabalin and gabapentin reduce release of substance P and CGRP from rat spinal tissues only after inflammation or activation of protein kinase C.

Gabapentin and pregabalin are amino acid derivatives of &ggr;‐amino butyric acid that have anticonvulsant, analgesic, and anxiolytic‐like properties in animal models. The mechanisms of these effects, however, are not well understood. To ascertain whether these drugs have effects on sensory neurons, we studied their actions on capsaicin‐evoked release of the sensory neuropeptides, substance P and calcitonin gene‐related peptide from rat spinal cord slices in vitro. Although release of immunoreactive peptides from non‐inflamed animals was not altered by either drug, prior in vivo treatment by intraplantar injection of complete Freunds adjuvant enhanced release from spinal tissues in vitro, which was attenuated by gabapentin and pregabalin. These drugs also reduced release of immunoreactive neuropeptides in spinal tissues pretreated in vitro with the protein kinase C activator, phorbol 12,13‐dibutyrate. Our results suggest that gabapentin and pregabalin modulate the release of sensory neuropeptides, but only under conditions corresponding to significant inflammation‐induced sensitization of the spinal cord.

Peripheral mechanisms of pain and analgesia

This review summarizes recent findings on peripheral mechanisms underlying the generation and inhibition of pain. The focus is on events occurring in peripheral injured tissues that lead to the sensitization and excitation of primary afferent neurons, and on the modulation of such mechanisms. Primary afferent neurons are of particular interest from a therapeutic perspective because they are the initial generator of noxious impulses traveling towards relay stations in the spinal cord and the brain. Thus, if one finds ways to inhibit the sensitization and/or excitation of peripheral sensory neurons, subsequent central events such as wind-up, sensitization and plasticity may be prevented. Most importantly, if agents are found that selectively modulate primary afferent function and do not cross the blood-brain-barrier, centrally mediated untoward side effects of conventional analgesics (e.g. opioids, anticonvulsants) may be avoided. This article begins with the peripheral actions of opioids, turns to a discussion of the effects of adrenergic co-adjuvants, and then moves on to a discussion of pro-inflammatory mechanisms focusing on TRP channels and nerve growth factor, their signaling pathways and arising therapeutic perspectives.

Ceramide, a putative second messenger for nerve growth factor, modulates the TTX‐resistant Na+ current and delayed rectifier K+ current in rat sensory neurons

Because nerve growth factor (NGF) is elevated during inflammation and is known to activate the sphingomyelin signalling pathway, we examined whether NGF and its putative second messenger, ceramide, could modulate the excitability of capsaicin‐sensitive adult and embryonic sensory neurons. Using the whole‐cell patch‐clamp recording technique, exposure of isolated sensory neurons to either 100 ng ml−1 NGF or 1 μM N‐acetyl sphingosine (C2‐ceramide) produced a 3‐ to 4‐fold increase in the number of action potentials (APs) evoked by a ramp of depolarizing current in a time‐dependent manner. Intracellular perfusion with bacterial sphingomyelinase (SMase) also increased the number of APs suggesting that the release of native ceramide enhanced neuronal excitability. Glutathione, an inhibitor of neutral SMase, completely blocked the NGF‐induced augmentation of AP firing, whereas dithiothreitol, an inhibitor of acidic SMase, was without effect. In the presence of glutathione and NGF, exogenous ceramide still enhanced the number of evoked APs, indicating that the sensitizing action of ceramide was downstream of NGF. To investigate the mechanisms of action for NGF and ceramide, isolated membrane currents were examined. Both NGF and ceramide facilitated the peak amplitude of the TTX‐resistant sodium current (TTX‐R INa) by approximately 1.5‐fold and shifted the activation to more hyperpolarized voltages. In addition, NGF and ceramide suppressed an outward potassium current (IK) by ≈35 %. Ceramide reduced IK in a concentration‐dependent manner. Isolation of the NGF‐ and ceramide‐sensitive currents indicates that they were delayed rectifier types of IK. The inflammatory prostaglandin, PGE2, produced an additional suppression of IK after exposure to ceramide (≈35 %), suggesting that these agents might act on different targets. Thus, our findings indicate that the pro‐inflammatory agent, NGF, can rapidly enhance the excitability of sensory neurons. This NGF‐induced sensitization is probably mediated by activation of the sphingomyelin signalling pathway to liberate ceramide(s), wherein ceramide appears to be the second messenger involved in modulating neuronal excitability.

Suppression of inflammatory and neuropathic pain by uncoupling CRMP-2 from the presynaptic Ca2+ channel complex

The use of N-type voltage-gated calcium channel (CaV2.2) blockers to treat pain is limited by many physiological side effects. Here we report that inflammatory and neuropathic hypersensitivity can be suppressed by inhibiting the binding of collapsin response mediator protein 2 (CRMP-2) to CaV2.2 and thereby reducing channel function. A peptide of CRMP-2 fused to the HIV transactivator of transcription (TAT) protein (TAT-CBD3) decreased neuropeptide release from sensory neurons and excitatory synaptic transmission in dorsal horn neurons, reduced meningeal blood flow, reduced nocifensive behavior induced by formalin injection or corneal capsaicin application and reversed neuropathic hypersensitivity produced by an antiretroviral drug. TAT-CBD3 was mildly anxiolytic without affecting memory retrieval, sensorimotor function or depression. At doses tenfold higher than that required to reduce hypersensitivity in vivo, TAT-CBD3 caused a transient episode of tail kinking and body contortion. By preventing CRMP-2–mediated enhancement of CaV2.2 function, TAT-CBD3 alleviated inflammatory and neuropathic hypersensitivity, an approach that may prove useful in managing chronic pain.

Prostacyclin enhances the evoked-release of substance P and calcitonin gene-related peptide from rat sensory neurons

Prostacyclin (PGI2) is a potent prostanoid producing various symptoms of inflammation, including an increased sensitivity to noxious stimulation. One component of these PGI2-mediated actions may involve activation or sensitization of sensory neurons to enhance release of neuroactive peptides. We, therefore, examined whether PGI2 and carba prostacyclin (CPGI2), a stable analog of PGI2, could alter the resting and evoked release of the neuropeptides, substance P (SP) and calcitonin gene-related peptide (CGRP) from embryonic rat sensory neurons grown in culture. Treating isolated sensory neurons with CPGI2 (10-1000 nM) for 30 min caused a 3-fold increase in the resting release of both peptides. One nM CPGI2, a concentration that did not alter the resting release, significantly enhanced neuropeptide release evoked by capsaicin, 100 nM bradykinin, or 40 mM KCl. Similarly, 10 nM PGI2 did not alter resting release, but augmented capsaicin-stimulated release of SP and CGRP 2-3 fold. In contrast, prostaglandin F2 alpha was ineffective in altering either resting or capsaicin-evoked peptide release. Our results demonstrate that low concentrations of PGI2 sensitize sensory neurons to other stimuli, whereas higher concentrations evoke release directly. This PGI2-induced augmentation of neuropeptide release may be one mechanism contributing to neurogenic inflammation.

Differential regulation of evoked peptide release by voltage-sensitive calcium channels in rat sensory neurons

To determine whether the sensitizing action of prostaglandins on sensory neurons are due to modulation of voltage-sensitive calcium channels (VSCC) we examined the effects of inhibiting these channels on PGE2-induced enhancement of evoked peptide release from isolated dorsal root ganglion neurons. The inhibitory effects of the VSCC blockers on stimulated release were dependent upon the type of chemical agent used to evoke the release. Bradykinin-stimulated release of immunoreactive substance P (iSP) and calcitonin gene-related peptide (iCGRP) was attenuated by the N-type VSCC blocker, omega-conotoxin GVIA (100 nM), but was unaffected by blockade of L-type (1 microM nifedipine) or P-type (200 nM omega-agatoxin IVA) VSCC. In contrast, potassium-stimulated release of peptides was inhibited by nifedipine, but not by omega-conotoxin GVIA or omega-agatoxin IVA. None of the VSCC blockers tested attenuated capsaicin-stimulated release of iSP and iCGRP. The combination of 1 microM nifedipine and 100 nM omega-conotoxin GVIA reduced the whole cell calcium current 89% +/- 1.7%. Administration of 100 nM PGE2 potentiated bradykinin- and capsaicin-evoked peptide release by 2-3-fold. Neither nifedipine nor omega-conotoxin GVIA attenuated the PGE2-mediated potentiation of bradykinin-evoked release, and neither omega-conotoxin GVIA nor omega-agatoxin IVA blocked the potentiation of capsaicin-evoked release induced by PGE2. These results indicate that the sensitizing actions of PGE2 as measured by enhanced peptide release, are not mediated by L-, N-, or P-type VSCC.

Activation of Protein Kinase C Augments Peptide Release from Rat Sensory Neurons

Abstract: To determine whether protein kinase C (PKC) mediates release of peptides from sensory neurons, we examined the effects of altering PKC activity on resting and evoked release of substance P (SP) and calcitonin gene‐related peptide (CGRP). Exposing rat sensory neurons in culture to 10 or 50 nM phorbol 12,13‐dibutyrate (PDBu) significantly increased SP and CGRP release at least 10‐fold above resting levels, whereas the inactive 4α‐PDBu analogue at 100 nM had no effect on release. Furthermore, 100 nM bradykinin increased peptide release approximately fivefold. Down‐regulation of PKC significantly attenuated the release of peptides evoked by either PDBu or bradykinin. PDBu at 1 nM or 1‐oleoyl‐2‐acetyl‐sn‐glycerol at 50 µM did not alter resting release of peptides, but augmented potassium‐ and capsaicin‐stimulated release of both SP and CGRP approximately twofold. This sensitizing action of PKC activators on peptide release was significantly reduced by PKC down‐regulation or by pretreating cultures with 10 nM staurosporine. These results establish that activation of PKC is important in the regulation of peptide release from sensory neurons. The PKC‐induced enhancement of peptide release may be a mechanism underlying the neuronal sensitization that produces hyperalgesia.

Implications of Apurinic/Apyrimidinic Endonuclease in Reactive Oxygen Signaling Response after Cisplatin Treatment of Dorsal Root Ganglion Neurons

Peripheral neuropathy is one of the major side effects of the anticancer drug cisplatin. Although previous work suggests that this neuropathy correlates with formation of DNA adducts in sensory neurons, growing evidence suggests that cisplatin also increases the generation of reactive oxygen species (ROS), which could cause DNA damage. Apurinic/apyrimidinic endonuclease/redox factor-1 (Ape1/Ref-1) is a multifunctional protein involved in DNA base excision repair of oxidative DNA damage and in redox regulation of a number of transcription factors. Therefore, we asked whether altering Ape1 functions would influence cisplatin-induced neurotoxicity. Sensory neurons in culture were exposed to cisplatin for 24 hours and several end points of toxicity were measured, including production of ROS, cell death, apoptosis, and release of the immunoreactive calcitonin gene-related peptide (iCGRP). Reducing expression of Ape1 in neuronal cultures using small interfering RNA (siRNA) enhances cisplatin-induced cell killing, apoptosis, ROS generation, and cisplatin-induced reduction in iCGRP release. Overexpressing wild-type Ape1 attenuates all the toxic effects of cisplatin in cells containing normal endogenous levels of Ape1 and in cells with reduced Ape1 levels after Ape1siRNA treatment. Overexpressing the redox deficient/repair competent C65-Ape1 provides partial rescue, whereas the repair-deficient Ape1 (N226A + R177A) does not protect neurons from cisplatin toxicity. We also observe an increase in phosphorylation of p53 after a decrease in Ape1 levels in sensory neuronal cultures. These results strongly support the notion that Ape1 is a potential translational target such that protecting Ape1 levels and particularly its DNA repair function could reduce peripheral neuropathy in patients undergoing cisplatin treatment.

A controlled trial of the cost benefit of computerized bayesian aminoglycoside administration

We studied the effect of a bayesian pharmacokinetic dosing program on the outcome of aminoglycoside therapy in patients with clinical infections. Patients were randomized to a control (dosing based on physician choice; n = 75) or experimental group (dosing based on the bayesian program; n = 72). Both groups used serum aminoglycoside concentration data when making dosing decisions. Improved response rates were seen in the experimental (60%; 42/68) compared with the control group (48%; 36/68). A higher, but not statistically significant, incidence of toxicity was found in the control (7/75; 9.7%) versus the experimental group (4/72; 5.1%). Mean length of total hospital stay was significantly longer for patients in the control group (20.3 days) compared with the experimental group (16.0 days) (p = 0.028). The variables from multivariate analysis with a significant impact on length of stay were patient group and length of aminoglycoside therapy. On the basis of a reduced length of stay, a potential cost savings of

Intrathecal NSAIDS attenuate inflammation-induced neuropeptide release from rat spinal cord slices

1311 per patient can be achieved.