Advances in Head and Neck Cancer Pain.

Head and neck cancer (HNC) affects over 890,000 people annually worldwide and has a mortality rate of 50%. Aside from poor survival, HNC pain impairs eating, drinking, and talking in patients, severely reducing quality of life. Different pain phenotype in patients (allodynia, hyperalgesia, and spontaneous pain) results from a combination of anatomical, histopathological, and molecular differences between cancers. Poor pathologic features (e.g., perineural invasion, lymph node metastasis) are associated with increased pain. The use of syngeneic/immunocompetent animal models, as well as a new mouse model of perineural invasion, provides novel insights into the pathobiology of HNC pain. Glial and immune modulation of the tumor microenvironment affect not only cancer progression but also pain signaling. For example, Schwann cells promote cancer cell proliferation, migration, and secretion of nociceptive mediators, whereas neutrophils are implicated in sex differences in pain in animal models of HNC. Emerging evidence supports the existence of a functional loop of cross-activation between the tumor microenvironment and peripheral nerves, mediated by a molecular exchange of bioactive contents (pronociceptive and protumorigenic) via paracrine and autocrine signaling. Brain-derived neurotrophic factor, tumor necrosis factor α, legumain, cathepsin S, and A disintegrin and metalloprotease 17 expressed in the HNC microenvironment have recently been shown to promote HNC pain, further highlighting the importance of proinflammatory cytokines, neurotrophic factors, and proteases in mediating HNC-associated pain. Pronociceptive mediators, together with nerve injury, cause nociceptor hypersensitivity. Oncogenic, pronociceptive mediators packaged in cancer cell-derived exosomes also induce nociception in mice. In addition to increased production of pronociceptive mediators, HNC is accompanied by a dampened endogenous antinociception system (e.g., downregulation of resolvins and µ-opioid receptor expression). Resolvin treatment or gene delivery of µ-opioid receptors provides pain relief in preclinical HNC models. Collectively, recent studies suggest that pain and HNC progression share converging mechanisms that can be targeted for cancer treatment and pain management.

Effects of activation of group III metabotropic glutamate receptors on spinal synaptic transmission in a rat model of neuropathic pain.

Chronic neuropathic pain remains an unmet clinical problem because it is often resistant to conventional analgesics. Metabotropic glutamate receptors (mGluRs) are involved in nociceptive processing at the spinal level, but their functions in neuropathic pain are not fully known. In this study, we investigated the role of group III mGluRs in the control of spinal excitatory and inhibitory synaptic transmission in a rat model of neuropathic pain induced by L5/L6 spinal nerve ligation. Whole-cell recording of lamina II neurons was performed in spinal cord slices from control and nerve-ligated rats. The baseline amplitude of glutamatergic EPSCs evoked from primary afferents was significantly larger in nerve-injured rats than in control rats. However, the baseline frequency of GABAergic and glycinergic inhibitory postsynaptic currents (IPSCs) was much lower in nerve-injured rats than in control rats. The group III mGluR agonist l(+)-2-amino-4-phosphonbutyric acid (l-AP4) produced a greater inhibition of the amplitude of monosynaptic and polysynaptic evoked EPSCs in nerve-injured rats than in control rats. l-AP4 inhibited the frequency of miniature EPSCs in 66.7% of neurons in control rats but its inhibitory effect was observed in all neurons tested in nerve-injured rats. Furthermore, l-AP4 similarly inhibited the frequency of GABAergic and glycinergic IPSCs in control and nerve-injured rats. Our study suggests that spinal nerve injury augments glutamatergic input from primary afferents but decreases GABAergic and glycinergic input to spinal dorsal horn neurons. Activation of group III mGluRs attenuates glutamatergic input from primary afferents in nerve-injured rats, which could explain the antinociceptive effect of group III mGluR agonists on neuropathic pain.

Stimulation of alpha(1)-adrenoceptors reduces glutamatergic synaptic input from primary afferents through GABA(A) receptors and T-type Ca(2+) channels.

Activation of the descending noradrenergic system inhibits nociceptive transmission in the spinal cord. Although both alpha(1)- and alpha(2)-adrenoceptors in the spinal cord are involved in the modulation of nociceptive transmission, it is not clear how alpha(1)-adrenoceptors regulate excitatory and inhibitory synaptic transmission at the spinal level. In this study, inhibitory and excitatory postsynaptic currents (IPSCs and EPSCs, respectively) were recorded from lamina II neurons in rat spinal cord slices. The specific alpha(1)-adrenoceptor agonist phenylephrine significantly increased the frequency of GABAergic spontaneous IPSCs in a concentration dependent manner, and this effect was abolished by the alpha(1)-adrenoceptor antagonist 2-(2,6-dimethoxyphenoxy)ethylaminomethyl-1,4-benzodioxane (WB4101). Phenylephrine also significantly reduced the amplitude of monosynaptic and polysynaptic EPSCs evoked from primary afferents. The inhibitory effect of phenylephrine on evoked monosynaptic glutamatergic EPSCs was largely blocked by the GABA(A) receptor antagonist picrotoxin and, to a lesser extent, by the GABA(B) receptor antagonist CGP55845. Furthermore, blocking T-type Ca(2+) channels with amiloride or mibefradil diminished the inhibitory effect produced by phenylephrine or the GABA(A) receptor agonist muscimol on monosynaptic EPSCs evoked from primary afferents. Collectively, these findings suggest that activation of alpha(1)-adrenoceptors in the spinal cord increases synaptic GABA release, which attenuates glutamatergic input from primary afferents mainly through GABA(A) receptors and T-type Ca(2+) channels. This mechanism of presynaptic inhibition in the spinal cord may be involved in the regulation of nociception by the descending noradrenergic system.

Signaling mechanisms mediating muscarinic enhancement of GABAergic synaptic transmission in the spinal cord.

Activation of muscarinic acetylcholine receptors (mAChRs) inhibits spinal nociceptive transmission by potentiation of GABAergic tone through M(2), M(3), and M(4) subtypes. To study the signaling mechanisms involved in this unique mAChR action, GABAergic spontaneous inhibitory postsynaptic currents (sIPSCs) of lamina II neurons were recorded using whole-cell patch clamp techniques in rat spinal cord slices. The mAChR agonist oxotremorine-M caused a profound increase in the frequency of GABAergic sIPSCs, which was abolished in the Ca(2+)-free solution. Inhibition of voltage-gated Ca(2+) channels with Cd(2+) and Ni(2+) largely reduced the effect of oxotremorine-M on sIPSCs. Blocking nonselective cation channels (NSCCs) with SKF96365 or 2-APB also largely attenuated the effect of oxotremorine-M. However, the KCNQ channel blocker XE991 and the adenylyl cyclase inhibitor MDL12330A had no significant effect on oxotremorine-M-induced increases in sIPSCs. Furthermore, the phosphoinositide-3-kinase (PI3K) inhibitor wortmannin or LY294002 significantly reduced the potentiating effect of oxotremorine-M on sIPSCs. In the spinal cord in which the M(3) subtype was specifically knocked down by intrathecal small interfering RNA (siRNA) treatment, SKF96365 and wortmannin still significantly attenuated the effect of oxotremorine-M. In contrast, SKF96365 and wortmannin both failed to alter the effect of oxotremorine-M on sIPSCs when the M(2)/M(4) mAChRs were blocked. Therefore, our study provides new evidence that activation of mAChRs increases synaptic GABA release through Ca(2+) influx and voltage-gated Ca(2+) channels. The PI3K-NSCC signaling cascade is primarily involved in the excitation of GABAergic interneurons by the M(2)/M(4) mAChRs in the spinal dorsal horn.

High-Density Nanosecond Charge Trapping in Thin Films of the Photoconductor ZnODEP.

An electrooptical memory effect is observed with solid thin films of the photoconductor zinc-octakis(beta-decoxyethyl) porphyrin (ZnODEP) sandwiched between two optically transparent electrodes. Upon irradiation with the simultaneous application of an electric field, electron-hole pairs are generated and separated within the photoconductive layer. These electron-hole pairs become "frozen" within the films when the irradiation is interrupted. These trapped charges can be released by irradiation of the cell, resulting in a transient short-circuit photocurrent. No cross talk between adjacent memory elements separated by approximately 0.2 micrometer (a density of 3 gigabits per square centimeter) was detected. The charge storage system is robust and nonvolatile. The response time for the write-read beam is in the subnanosecond range, and no refreshing is required for long-term retention of trapped charges.

Polyester Nanoparticle Encapsulation Mitigates Paclitaxel-Induced Peripheral Neuropathy.

Chemotherapy utilizing cytotoxic drugs, such as paclitaxel (PTX), is still a commonly used therapeutic approach to treat both localized and metastasized cancers. Unlike traditional regimens in which PTX is administered at the maximum tolerated dose, alternative regimens like metronomic dosing are beneficial by administering PTX more frequently and in much lower doses exploiting antiangiogenic and immunomodulatory effects. However, PTX-induced peripheral neuropathy and lack of patient compliant dosage forms of PTX are major roadblocks for the successful implementation of metronomic regimens. Because of the success of polyester nanoparticle drug delivery, we explored the potential of nanoparticle-encapsulated paclitaxel (nPTX) in alleviating peripheral neuropathy using a rat model. Rats were injected intraperitoneally with 2 mg/kg body weight of PTX or nPTX on four alternate days, and neuropathic pain and neuronal damage were characterized using behavioral assessments, histology, and immunohistochemistry. The reduction in tactile and nociceptive pressure thresholds was significantly less in nPTX-treated rats than in PTX-treated rats over a 16-day study period. Histological analysis showed that the degree of dorsal root ganglion (DRG) degeneration and reduction in motor neurons in the spinal cord was significantly lower in the nPTX group than the PTX group. Further, immunofluorescence data reveals that nPTX-treated rats had an increased density of a neuronal marker, ß-tubulin-III, reduced TUNEL positive cells, and increased high molecular weight neurofilament in the spinal cord, DRG, and sciatic nerves compared with PTX-treated rats. Therefore, this work has important implications in improving risk-benefit profile of PTX, paving the way for metronomic regimens.

Distinct inhibition of voltage-activated Ca2+ channels by delta-opioid agonists in dorsal root ganglion neurons devoid of functional T-type Ca2+ currents.

Both mu- and delta-opioid agonists selectively inhibit nociception but have little effect on other sensory modalities. Voltage-activated Ca(2+) channels in the primary sensory neurons are important for the regulation of nociceptive transmission. In this study, we determined the effect of delta-opioid agonists on voltage-activated Ca(2+) channel currents (I(Ca)) in small-diameter rat dorsal root ganglion (DRG) neurons that do and do not bind isolectin B(4) (IB(4)). The delta-opioid agonists [d-Pen(2),d-Pen(5)]-enkephalin (DPDPE) and deltorphin II produced a greater inhibition of high voltage-activated I(Ca) in IB(4)-negative than IB(4)-positive neurons. Furthermore, DPDPE produced a greater inhibition of N-, P/Q-, and L-type I(Ca) in IB(4)-negative than IB(4)-positive neurons. However, DPDPE had no significant effect on the R-type I(Ca) in either type of cells. We were surprised to find that DPDPE failed to inhibit either the T-type or high voltage-activated I(Ca) in all the DRG neurons with T-type I(Ca). Double immunofluorescence labeling showed that the majority of the delta-opioid receptor-immunoreactive DRG neurons had IB(4) labeling, while all DRG neurons immunoreactive to delta-opioid receptors exhibited Cav(3.2) immunoreactivity. Additionally, DPDPE significantly inhibited high voltage-activated I(Ca) in Tyrode's or N-methyl-d-glucamine solution but not in tetraethylammonium solution. This study provides new information that delta-opioid agonists have a distinct effect on voltage-activated Ca(2+) channels in different phenotypes of primary sensory neurons. High voltage-activated Ca(2+) channels are more sensitive to inhibition by delta-opioid agonists in IB(4)-negative than IB(4)-positive neurons, and this opioid effect is restricted to DRG neurons devoid of functional T-type Ca(2+) currents.

Kv1.1/1.2 channels are downstream effectors of nitric oxide on synaptic GABA release to preautonomic neurons in the paraventricular nucleus.

The paraventricular nucleus (PVN) of the hypothalamus is important for the neural regulation of cardiovascular function. Nitric oxide (NO) increases synaptic GABA release to presympathetic PVN neurons through the cyclic guanosine monophosphate (cGMP)/protein kinase G signaling pathway. However, the downstream signaling mechanisms underlying the effect of NO on synaptic GABA release remain unclear. In this study, whole-cell voltage-clamp recordings were performed on retrograde-labeled spinally projecting PVN neurons in rat brain slices. Bath application of the NO precursor l-arginine or the NO donor S-nitroso-N-acetylpenicillamine (SNAP) significantly increased the frequency of GABAergic miniature inhibitory postsynaptic currents (mIPSCs) in labeled PVN neurons. A specific antagonist of cyclic ADP ribose, 8-bromo-cyclic ADP ribose (8-Br-cADPR), had no significant effect on l-arginine-induced potentiation of mIPSCs. Surprisingly, blocking of voltage-gated potassium channels (Kv) with 4-aminopyridine or alpha-dendrotoxin eliminated the effect of l-arginine on mIPSCs in all labeled PVN neurons tested. The membrane permeable cGMP analog mimicked the effect of l-arginine on mIPSCs, and this effect was blocked by alpha-dendrotoxin. Furthermore, the specific Kv channel blocker for Kv1.1 (dendrotoxin-K) or Kv1.2 (tityustoxin-Kalpha) abolished the effect of l-arginine on mIPSCs in all neurons tested. SNAP failed to inhibit the firing activity of labeled PVN neurons in the presence of dendrotoxin-K, Kalpha. Additionally, the immunoreactivity of Kv1.1 and Kv1.2 subunits was colocalized extensively with synaptophysin in the PVN. These findings suggest that NO increases GABAergic input to PVN presympathetic neurons through a downstream mechanism involving the Kv1.1 and Kv1.2 channels at the nerve terminals.

Resistance to morphine analgesic tolerance in rats with deleted transient receptor potential vanilloid type 1-expressing sensory neurons.

Deletion of transient receptor potential vanilloid type 1 (TRPV1)-expressing afferent neurons reduces presynaptic mu opioid receptors but paradoxically potentiates the analgesic efficacy of mu opioid agonists. In this study, we determined if removal of TRPV1-expressing afferent neurons by resiniferatoxin (RTX), an ultrapotent capsaicin analog, influences the development of opioid analgesic tolerance. Morphine tolerance was induced by daily intrathecal injections of 10 microg of morphine for 14 consecutive days or by daily i.p. injections of 10 mg/kg of morphine for 10 days. In vehicle-treated rats, the effect of intrathecal or systemic morphine on the mechanical withdrawal threshold was gradually diminished within 7 days. However, the analgesic effect of intrathecal and systemic morphine was sustained in RTX-treated rats at the time the morphine effect was lost in the vehicle group. Furthermore, the mu opioid receptor-G protein coupling in the spinal cord was significantly decreased ( approximately 22%) in vehicle-treated morphine tolerant rats, but was not significantly altered in RTX-treated rats receiving the same treatment with morphine. Additionally, there was a large reduction in protein kinase Cgamma-immunoreactive afferent terminals in the spinal dorsal horn of RTX-treated rats. These findings suggest that loss of TRPV1-expressing sensory neurons attenuates the development of morphine analgesic tolerance possibly by reducing mu opioid receptor desensitization through protein kinase Cgamma in the spinal cord. These data also suggest that the function of presynaptic mu opioid receptors on TRPV1-expressing sensory neurons is particularly sensitive to down-regulation by mu opioid agonists during opioid tolerance development.

Regulation of synaptic input to hypothalamic presympathetic neurons by GABA(B) receptors.

The hypothalamic paraventricular (PVN) neurons projecting to the spinal cord and brainstem play an important role in the control of homeostasis and the sympathetic nervous system. Although GABA(B) receptors are present in the PVN, their function in the control of synaptic inputs to PVN presympathetic neurons is not clear. Using retrograde tracing and whole-cell patch-clamp recordings in rat brain slices, we determined the role of presynaptic GABA(B) receptors in regulation of glutamatergic and GABAergic inputs to spinally projecting PVN neurons. The GABA(B) receptor agonist baclofen (1-50 microM) dose-dependently decreased the frequency but not the amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) and inhibitory postsynaptic currents (sIPSCs). The effect of baclofen on sEPSCs and sIPSCs was completely blocked by 10 microM CGP52432, a selective GABA(B) receptor antagonist. Baclofen also significantly reduced the frequency of both miniature excitatory and miniature inhibitory postsynaptic currents (mEPSCs and mIPSCs). Furthermore, uncoupling pertussis toxin-sensitive G(i/o) proteins with N-ethylmaleimide abolished baclofen-induced inhibition of mEPSCs and mIPSCs. However, the inhibitory effect of baclofen on the frequency of mIPSCs and mEPSCs persisted in the presence of either Cd2+, a voltage-gated Ca2+ channel blocker, or 4-aminopyridine, a blocker of voltage-gated K+ channels. Our results suggest that activation of presynaptic GABA(B) receptors inhibits synaptic GABA and glutamate release to PVN presympathetic neurons. This presynaptic action of GABA(B) receptors is mediated by the N-ethylmaleimide-sensitive G(i/o) proteins, but independent of voltage-gated Ca2+ and K+ channels.

Signaling mechanisms of down-regulation of voltage-activated Ca2+ channels by transient receptor potential vanilloid type 1 stimulation with olvanil in primary sensory neurons.

Olvanil ((N-vanillyl)-9-oleamide), a non-pungent transient receptor potential vanilloid type 1 agonist, desensitizes nociceptors and alleviates pain. But its molecular targets and signaling mechanisms are little known. Calcium influx through voltage-activated Ca(2+) channels plays an important role in neurotransmitter release and synaptic transmission. Here we determined the effect of olvanil on voltage-activated Ca(2+) channel currents and the signaling pathways in primary sensory neurons. Whole-cell voltage-clamp recordings were performed in acutely isolated rat dorsal root ganglion neurons. Olvanil (1 microM) elicited a delayed but sustained inward current, and caused a profound inhibition (approximately 60%) of N-, P/Q-, L-, and R-type voltage-activated Ca(2+) channel current. Pretreatment with a specific transient receptor potential vanilloid type 1 antagonist or intracellular application of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid abolished the inhibitory effect of olvanil on voltage-activated Ca(2+) channel current. Calmodulin antagonists (ophiobolin-A and calmodulin inhibitory peptide) largely blocked the effect of olvanil and capsaicin on voltage-activated Ca(2+) channel current. Furthermore, calcineurin (protein phosphatase 2B) inhibitors (deltamethrin and FK-506) eliminated the effect of olvanil on voltage-activated Ca(2+) channel current. Notably, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, calmodulin antagonists, and calcineurin inhibitors each alone significantly increased the amplitude of voltage-activated Ca(2+) channel current. In addition, double immunofluorescence labeling revealed that olvanil induced a rapid internalization of Ca(V)2.2 immunoreactivity from the membrane surface of dorsal root ganglion neurons. Collectively, this study suggests that stimulation of non-pungent transient receptor potential vanilloid type 1 inhibits voltage-activated Ca(2+) channels through a biochemical pathway involving intracellular Ca(2+)-calmodulin and calcineurin in nociceptive neurons. This new information is important for our understanding of the signaling mechanisms of desensitization of nociceptors by transient receptor potential vanilloid type 1 analogues and the feedback regulation of intracellular Ca(2+) and voltage-activated Ca(2+) channels in nociceptive sensory neurons.

Local injection of endothelin-1 produces pain-like behavior and excitation of nociceptors in rats.

Neurobehavioral and neurophysiological actions of the peptide endothelin-1 (ET-1) were investigated after subcutaneous plantar hindpaw injections in adult male Sprague Dawley rats. Hindpaw flinching developed within minutes after ET-1 (8-16 nmol) injection, peaked at 30 min, lasted for 60 min, and was strongly inhibited by the endothelin-A (ET(A)) receptor antagonist, BQ-123 (3.2 m). In separate experiments, impulse activity of single, physiologically characterized sensory C-, Adelta-, and Abeta-fibers was recorded from the sciatic nerve in anesthetized rats after subcutaneous injections of endothelin-1 (1-20 nmol), alone or together with BQ-123 (3.2 m), into the plantar hindpaw receptive fields of these units. All nociceptive C-fibers (31 of 33 C-fibers studied) were excited by ET-1 (1-20 nmol) in a dose-dependent manner. For doses of 16-20 nmol, the mean latency for afferent activation after injection of ET-1 was 3.16 +/- 0.31 min, and the mean and maximum response frequency were 2.02 +/- 0.48 impulses (imp)/sec and 14.0 +/- 3.2 imp/sec, respectively. All 10 nociceptive Adelta-fibers (of 12 Adelta-fibers studied) also responded to 1-20 nmol of ET-1 in a dose-dependent manner with a mean latency of 3.5 +/- 0.12 min and mean response frequency of 3.3 +/- 2.3 imp/sec. In contrast, most Abeta-fibers (9 of 12) did not respond to ET-1. BQ-123, when coinjected with ET-1, blocked ET-1-induced activation in all C- and Adelta-fibers tested. These data demonstrate that subcutaneous administration of ET-1 to the rat plantar hindpaw produces pain-like behavior and selective excitation of nociceptive fibers through activation of ET(A) receptors.

Limitation of myocardial infarct size in pigs with a dual lipoxygenase-cyclooxygenase blocking agent by inhibition of neutrophil activity without reduction of neutrophil migration.

OBJECTIVES: The purpose of this study was to assess the effect of the dual cyclooxygenase-lipoxygenase blocking agent BW755C on the extent of myocardial infarction in the pig and to identify the mechanisms of any cardioprotective action of this drug. BACKGROUND: Activated neutrophils contribute to reperfusion injury after myocardial infarction and inhibition of neutrophil function can limit infarct size. METHODS: In 9 control and 10 study pigs pretreated with intravenous BW755C (10 mg/kg body weight) 30 min before coronary occlusion, ischemia was induced by a 50-min occlusion of the mid-left anterior descending coronary artery, followed by 3 h of reperfusion. Heart rate, arterial pressure, left ventricular end-diastolic pressure, the first derivative of left ventricular pressure (dP/dt) and regional myocardial blood flow were measured during control, occlusion and reperfusion periods. Infarct size was determined by histochemical staining; and myeloperoxidase activity, a marker for tissue neutrophil content, was assessed in normal and infarcted myocardium. The effect of BW755C on the function of isolated neutrophils stimulated with zymosan-activated serum was evaluated by measuring neutrophil degranulation, leukotriene B4 production, superoxide generation and chemotaxis. RESULTS: Hemodynamic function and regional myocardial blood flow were similar in control and BW755C-treated animals. BW755C significantly reduced myocardial infarct size compared with that in control animals, as measured by infarct/risk areas by histochemical staining (39 +/- 5% vs. 63 +/- 7%, p < 0.05). Myocardial myeloperoxidase activity was similar in normal, salvaged and infarcted areas in the control and treated groups, indicating that neutrophil accumulation in injured myocardium was unaltered by BW755C. However, this agent attenuated function of isolated, stimulated (zymosan-activated serum) neutrophils. At a concentration of 0.03 mg/ml, BW755C inhibited degranulation (-46%), leukotriene B4 production (-48%) and superoxide generation (-74%), but there was minimal inhibition of chemotaxis in vitro. CONCLUSIONS: These findings demonstrate that myocardial infarct size can be reduced by selective inhibition of neutrophil cytotoxic activity without affecting neutrophil migration into injured myocardium.

Activation of muscarinic receptors inhibits spinal dorsal horn projection neurons: role of GABAB receptors.

Spinally administered muscarinic receptor agonists or acetylcholinesterase inhibitors produce efficacious analgesia. However, the mechanisms of the antinociceptive actions of muscarinic agonists in the spinal cord are not fully known. Previous in vitro studies have shown that muscarinic agonists increase GABA release and reduce the glutamatergic synaptic input to lamina II interneurons through GABAB receptors in the spinal cord. In the present study, we studied the effect of muscarinic agents on dorsal horn projection neurons and the role of spinal GABAB receptors in their action. Single-unit activity of ascending dorsal horn neurons was recorded in the lumbar spinal cord of anesthetized rats. The responses of dorsal horn neurons to graded mechanical stimuli were determined before and after topical spinal application of muscarine and neostigmine. We found that topical application of 0.1-5 microM muscarine or 0.5-5 microM neostigmine significantly suppressed the evoked response of dorsal horn neurons in a concentration-dependent manner. The inhibitory effect of muscarine or neostigmine on dorsal horn neurons was completely abolished in the presence of 1 microM atropine and by intrathecal pretreatment with 1 microg pertussis toxin to inactivate inhibitory G proteins. Furthermore, the inhibitory effect of both muscarine and neostigmine on the evoked response of dorsal horn neurons was significantly attenuated in the presence of 1 microM CGP55845, a GABAB receptor antagonist. Collectively, these data suggest that muscarinic agents inhibit dorsal horn projection neurons through muscarinic receptors coupled to pertussis toxin-sensitive Gi/o proteins. The inhibitory action of muscarinic agonists on these dorsal horn neurons is mediated in part by spinal GABAB receptors.

Role of primary afferent nerves in allodynia caused by diabetic neuropathy in rats.

Both myelinated and unmyelinated afferents are implicated in transmitting diabetic neuropathic pain. Although unmyelinated afferents are generally considered to play a significant role in diabetic neuropathic pain, pathological changes in diabetic neuropathy occur mostly in myelinated A-fibers. In the present study, we first examined the role of capsaicin-sensitive C-fibers in the development of allodynia induced by diabetic neuropathy. We then studied the functional changes of afferent nerves pertinent to diabetic neuropathic pain. Diabetes was induced in rats by i.p. streptozotocin. To deplete capsaicin-sensitive C-fibers, rats were treated with i.p. resiniferatoxin (300 microg/kg). Mechanical and thermal sensitivities were measured using von Frey filaments and a radiant heat stimulus. Single-unit activity of afferents was recorded from the tibial nerve. Tactile allodynia, but not thermal hyperalgesia, developed in diabetic rats. Resiniferatoxin treatment did not alter significantly the degree and time course of allodynia. Post-treatment with resiniferatoxin also failed to attenuate allodynia in diabetic rats. The electrophysiological recordings revealed ectopic discharges and a higher spontaneous activity mainly in Adelta- and Abeta-fiber afferents in diabetic rats regardless of resiniferatoxin treatment. Furthermore, these afferent fibers had a lower threshold for activation and augmented responses to mechanical stimuli. Thus, our study suggests that capsaicin-sensitive C-fiber afferents are not required in the development of allodynia in this rat model of diabetes. Our electrophysiological data provide substantial new evidence that the abnormal sensory input from Adelta- and Abeta-fiber afferents may play an important role in diabetic neuropathic pain.

Intrathecal S-nitroso-N-acetylpenicillamine and L-cysteine attenuate nerve injury-induced allodynia through noradrenergic activation in rats.

Spinal norepinephrine release and activation of spinal alpha(2)-adrenergic receptors represent important components of descending control of nociception. Recent studies have shown that nitric oxide is capable of stimulating neuronal norepinephrine release in the presence of thiol-containing compounds such as L-cysteine. In the present study, we tested a hypothesis in a rodent model of neuropathic pain that intrathecal injection of the nitric oxide donor S-nitroso-N-acetylpenicillamine and L-cysteine produces an antiallodynic action mediated by the spinal alpha(2)-adrenergic receptors. Allodynia was induced in rats by ligation of the left lumbar L5/L6 spinal nerves. Mechanical allodynia was quantified by application of von Frey filaments to the left hindpaw. Intrathecal injection of 20-100microg of S-nitroso-N-acetylpenicillamine in the presence of 200microg of L-cysteine, but not D-cysteine, dose-dependently attenuated the allodynia. Intrathecal injection of a combination of 100microg of S-nitroso-N-acetylpenicillamine and 50-200microg of L-cysteine also inhibited the allodynia in a dose-dependent manner. Pretreatment with a nitric oxide scavenger, carboxy-PTIO, or depletion of norepinephrine with a specific neurotoxin, N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine, prevented the antiallodynic action of intrathecal S-nitroso-N-acetylpenicillamine and L-cysteine. Furthermore, the antiallodynic effect produced by intrathecal injection of a combination of S-nitroso-N-acetylpenicillamine and L-cysteine was abolished by pretreatment with intrathecal injection of a non-specific alpha-adrenergic receptor antagonist, phentolamine, or an alpha(2) receptor antagonist, idazoxan. This study provides the first functional evidence that spinal nitric oxide interacts with the thiol-containing compounds to produce an antiallodynic effect in neuropathic pain. We propose that such an action is mediated by endogenous norepinephrine and spinal alpha(2)-adrenergic receptors.

Spinal cyclooxygenase-2 is involved in development of allodynia after nerve injury in rats.

Increased spinal cyclooxygenase activity is associated with nociception induced by tissue inflammation. In the present study, we examined the changes of cyclooxygenase-1 and cyclooxygenase-2 protein expression in several regions of the CNS associated with pain perception, and the role of spinal cyclooxygenase activity in the development of allodynia following nerve injury. Allodynia was induced by ligation of the left L5 and L6 spinal nerves in rats. Using western blot analysis, we found that the cyclooxygenase-2 protein levels in the dorsal spinal cord and thalamus (but not in the ventral spinal cord, cingulate cortex and locus coeruleus) increased significantly one day after nerve ligation, compared with those in the sham animals. The cyclooxygenase-2 protein levels in the above tissues were similar in nerve-injured and sham animals three and 14 days after surgery. In contrast, cyclooxygenase-1 protein was not detectable in any of the neural tissues examined one, three, and 14 days after nerve injury. In the behavioral experiments, we observed that intrathecal injection of 100microg of indomethacin immediately or one day after nerve ligation attenuated the development of tactile allodynia. However, intrathecal injection of indomethacin had no effect on established allodynia two weeks after nerve injury.Collectively, our results suggest that cyclooxygenase-2 is preferentially up-regulated in the dorsal spinal cord and thalamus in response to nerve injury in rats. Spinal cyclooxygenase-2 probably plays an important role in the early development, but not in the maintenance, of tactile allodynia caused by the nerve injury in this rat model of neuropathic pain.

Formation of 6-nitro-norepinephrine from nitric oxide and norepinephrine in the spinal cord and its role in spinal analgesia.

Spinally released norepinephrine is thought to produce analgesia in part by stimulating alpha(2)-adrenergic receptors, which in turn leads to nitric oxide synthesis. Also, nitric oxide is known to react with norepinephrine in vivo in the brain to form 6-nitro-norepinephrine, which inhibits neuronal norepinephrine reuptake. In the present study, we tested the hypothesis that formation of 6-nitro-norepinephrine occurs in the spinal cord and that intrathecal administration of 6-nitro-norepinephrine produces analgesia by stimulating norepinephrine release. 6-Nitro-norepinephrine was present in rat spinal cord tissue and microdialysates of the dorsal horn and intrathecal space. Intrathecal norepinephrine injection increased 6-nitro-norepinephrine. 6-Nitro-norepinephrine also stimulated norepinephrine release in dorsal spinal cord in vitro. Intrathecal injection of 6-nitro-norepinephrine produced antinociception and interacted additively with norepinephrine for antinociception. Spinal noradrenergic nerve destruction increased antinociception from intrathecally injected norepinephrine, but decreased antinociception from 6-nitro-norepinephrine. These results suggest a functional interaction between spinal nitric oxide and norepinephrine in analgesia, mediated in part by formation of 6-nitro-norepinephrine. Stimulation of auto-inhibitory alpha(2)-adrenergic receptors at noradrenergic synapses decreases norepinephrine release. Paradoxically, alpha(2)-adrenergic agonist injection increases and alpha(2)-adrenergic antagonist injection decreases norepinephrine release in the spinal cord. 6-Nitro-norepinephrine may be an important regulator of spinal norepinephrine release and could explain the positive feedback on norepinephrine release after activation of spinal alpha(2)-adrenergic receptors.

New 9-stubstituted 3-N,O-diacetylhydroxylaminofluorenes: enhanced electrophilicity and mutagenicity. Derivatives of fluorene. 38.

The compound 2-acetamidofluorene is a potent hepatocarcinogen; its N-acetoxy derivative (2-NOAc-AAF), a model ultimate form of the carcinogen, is strongly mutagenic and is chemically reactive with nucleophiles. The isomeric 3-acetamidofluorene is a mammary carcinogen, but is not hepatocarinogenic; its N-acetoxy derivative (3-NOAc-AAF) is not reactive with nucleophiles. Derivatives of 3-NOAc-AAF containing electron-donating groups in positions conjugated with the 3-position have been synthesized. These show increased electrophilicity and mutagenicity. Thus, by electronic manipulation of the leaving-group capacity of the -OAc group of 3-NOAc-AAF, we have obtained compounds with increased biological activity, as well as increased chemical reactivity. Future experiments could show whether in vivo effects of these derivatives of 3-NOAc-AAF are more like the effects of 2-NOAc-AAF or of 3-NOAc-AAF.

Bradykinin contributes to the exercise pressor reflex: mechanism of action.

This study determined the receptors responsible for mediating bradykinin's effect on skeletal muscle afferents that cause the pressor reflex in anesthetized cats. In eight cats, 1 microgram of bradykinin was injected intra-arterially into the gracilis muscle before and after intravenous injection of a kinin B2-receptor antagonist (NPC 17731, 20 micrograms/kg). Initial injection of bradykinin reflexly increased mean arterial pressure by 23 +/- 7 mmHg, maximal change in pressure over time by 439 +/- 272 mmHg/s, and heart rate by 11 +/- 4 beats/min. The hemodynamic response to bradykinin was abolished by kinin B2-receptor blockade. Similar injection of the kinin B1-receptor agonist des-Arg9-bradykinin caused no cardiovascular responses (n = 6). In eight different animals, mean arterial pressure, maximal change in left ventricular pressure over time, and heart rate responses to 30 s of electrically stimulated hindlimb contraction were attenuated by 50 +/- 6, 55 +/- 7, and 41 +/- 8%, respectively, after kinin B2-receptor blockade. In eight other animals, mean arterial pressure, maximal change in left ventricular pressure over time, and heart rate responses were reduced by 58 +/- 8, 66 +/- 6, and 40 +/- 12%, respectively, after inhibition of prostaglandin synthesis with indomethacin (2.5-3 mg/kg iv) and were then abolished by subsequent B2-receptor blockade. These data suggest that bradykinin contributes to the exercise pressor reflex through its action on kinin B2 receptors located on the nerve endings of the muscle afferents.(ABSTRACT TRUNCATED AT 250 WORDS)