Spinal GABA(A) and GABA(B) receptor pharmacology in a rat model of neuropathic pain

BACKGROUND: This study tests the hypothesis that loss of spinal activity of gamma-aminobutyric acid (GABA) contributes to the allodynia and hyperalgesia observed after peripheral nerve injury. METHODS: Intrathecal catheters were implanted in male Sprague-Dawley rats. Antinociception was assessed by measuring withdrawal latency to immersion of the tail in a 52 degrees C water bath. Nerve injury was produced by ligation of the L5 and L6 spinal nerves. Testing was performed 4-14 days after spinal nerve ligation, when tactile allodynia and thermal hyperalgesia were established. Tactile allodynia was quantitated using the threshold to withdrawal of the hind paw on probing with von Frey filaments. Thermal hyperalgesia was quantitated using the latency to withdrawal of the hind paw from radiant heat. Motor function was tested using a rotarod apparatus. RESULTS: Spinal administration of the GABAA receptor antagonist bicuculline or the GABAB receptor antagonist phaclofen produced tactile allodynia and thermal hyperalgesia in normal rats. The GABAB receptor agonist baclofen, administered spinally, produced antinociception in the tail-flick test, whereas the GABAA receptor agonist isoguvacine did not. Isoguvacine and baclofen each reversed tactile allodynia and thermal hyperalgesia produced by spinal nerve ligation. Baclofen but not isoguvacine prolonged thermal withdrawal latency in nerve-injured rats beyond preoperative values. Baclofen but not isoguvacine impaired motor function. CONCLUSIONS: Pharmacologic inhibition of intrinsic GABA tone in normal rats resulted in tactile allodynia and thermal hyperalgesia, consistent with the hypothesis being tested. Exogenous administration of GABA agonists reversed spinal nerve ligation-induced allodynia and hyperalgesia, also consistent with this hypothesis. Isoguvacine produced specific antihyperalgesic and antiallodynic effects, whereas assessment of the effects of baclofen was complicated by motor dysfunction. Spinal GABAA agonists may provide a specific therapy for neuropathic pain.     

Blockade and activation of the human neuronal nicotinic acetylcholine receptors by atracurium and laudanosine

BACKGROUND: Curaremimetic nondepolarizing muscle relaxants are widely used in clinical practice to prevent muscle contraction either during surgery or during intensive care. Although primarily acting at the neuromuscular junction, these compounds can cause adverse effects, including modification of cardiac rhythm, arterial blood pressure, and in the worst cases, triggering of seizures. In this study, we assessed the interaction of atracurium and its metabolite, laudanosine, with neuronal nicotinic receptors. METHODS: The human neuronal nicotinic receptors alpha4beta2, alpha3beta4, alpha3alpha5beta4, and alpha7 are heterologously expressed in Xenopus laevis oocytes, and the effect of atracurium and its degradation product, laudanosine, were studied on these receptors. RESULTS: Atracurium and laudanosine inhibited in the micromolar range the major brain alpha4beta2 receptor and the ganglionic alpha3beta4 or alpha3beta4alpha5 and the homomeric alpha7 receptors. For all four receptors, inhibition was rapid and readily reversible within less than 1 min. Atracurium blockade was competitive at alpha4beta2 and alpha7 receptors but displayed a noncompetitive blockade at the alpha3beta4 receptors. Inhibition at this receptor subtype was not modified by alpha5. Laudanosine was found to have a dual mode of action; first, it competes with acetylcholine and, second, it blocks the ionic pore by steric hindrance. At low concentrations, these two drugs are able to activate both the alpha4beta2 and the alpha3beta4 receptors. CONCLUSION: Adverse effects observed during atracurium administration may be attributed, at least partly, to an interaction with neuronal nicotinic receptors.     

Dose-dependent enhancement of in vivo GABA(A)-benzodiazepine receptor binding by isoflurane

BACKGROUND: Abundant in vitro and animal model data suggest the postsynaptic gamma-aminobutyric acid receptor type A (GABA(A)-R) is an important target for volatile general anesthetics, but the relevance of these models is untested in humans. Because benzodiazepines have also been shown to act via a specific GABA(A)-R site, they provide sensitive probes for the GABA(A)-R. Availability of the 11C-labeled benzodiazepine ligand, flumazenil, allowed us to quantitatively test in humans whether the volatile anesthetic isoflurane affects GABA(A)-Rs in vivo in a dose-dependent manner. METHODS: 11C-flumazenil positron emission tomography scans were obtained in 12 healthy subjects while awake (control condition) and anesthetized with either 1.0 or 1.5 minimum alveolar concentration isoflurane (n = 7 and 5, respectively; isoflurane conditions). Regions of interest included areas of high, intermediate, and low GABA(A)-benzodiazepine site density. For each subject and experimental condition, the binding of 11C-flumazenil, expressed as distribution volume (which linearly correlates to maximal binding site density and apparent ligand affinity), was obtained by curve fitting using a two-compartment model. RESULTS: The ratio of distribution volume increased significantly in each examined region during the isoflurane conditions compared with control conditions (P < 0.01, one-tailed t test). Furthermore, the increases in ratio of distribution volume during the 1.5-minimum alveolar concentration isoflurane condition were significantly greater than those measured during 1.0 minimum alveolar concentration isoflurane inhalation (P < 0.002, one-tailed t test). CONCLUSIONS: Isoflurane exposure appeared to enhance receptor-specific 11C-flumazenil binding in a dose-dependent manner. The results suggest the possibility that a conformational change of the GABA(A)-R is involved in the mechanism of action of isoflurane in the living human brain.     

Modulation of GABA(A) receptor function by nonhalogenated alkane anesthetics: the effects on agonist enhancement,direct activation,and inhibition

At clinically relevant concentrations, ethers, alcohols, and halogenated alkanes enhance agonist action on the gamma-aminobutyric acid(A) (GABA(A)) receptor, whereas nonhalogenated alkanes do not. Many anesthetics also directly activate and/or inhibit GABA(A) receptors, actions that may produce important behavioral effects; although, the effects of nonhalogenated alkane anesthetics on GABA(A) receptor direct activation and inhibition have not been studied. In this study, we assessed the abilities of two representative nonhalogenated alkanes, cyclopropane and butane, to enhance agonist action, directly activate, and inhibit currents mediated by expressed alpha(1)beta(2)gamma(2L) GABA(A) receptors using electrophysiological techniques. Our studies reveal that cyclopro- pane and butane enhance agonist action on the GABA(A) receptor at concentrations that exceed those required to produce anesthesia. Neither nonhalogenated alkane directly activated nor inhibited GABA(A) receptors, even at concentrations that approach their aqueous saturated solubilities. These results strongly suggest that the behavioral actions of nonhalogenated alkane anesthetics do not result from their abilities to enhance agonist actions, directly activate, or inhibit alpha(1)beta(2)gamma(2L) GABA(A) receptors and are consistent with the hypothesis that electrostatic interactions between anesthetics and their protein binding sites modulate GABA(A) receptor potency. IMPLICATIONS: When normalized to either their in vivo anesthetic potencies or hydrophobicities, cyclopropane and butane are 1-1.5 orders of magnitude less potent enhancers of agonist action on alpha(1beta2gamma2L) GABA(A) receptors than isoflurane. Additionally, cyclopropane and butane fail to directly activate or inhibit receptors, even at near aqueous saturating concentrations. Thus, it is unlikely that either enhancement or inhibition of the most common GABA(A) receptor subtype in the brain accounts for the behavioral activities of cyclopropane and butane.     

Histamine H(1) receptor activation inhibits the proliferation of human prostatic adenocarcinoma DU-145 cells

BACKGROUND: Histamine stimulates cell proliferation in some tumor cell lines through the activation of H(1) receptors coupled to phosphoinositide hydrolysis. We therefore set out to study the presence of H(1) receptors in the prostate cancer cell line DU-145 and the effect of their stimulation on cell growth. METHODS: The presence of histamine receptors was studied by radioligand binding. Phosphoinositide hydrolysis was assessed by measuring [(1)H]-inositol phosphate ([(1)H]-IPs) accumulation and changes in the intracellular concentration of free Ca(2+) ([Ca(2+)](i)). Proliferation was assessed by cell counting and by [(1)H]-thymidine incorporation. RESULTS: DU-145 cells express H(1) receptors (110+/-14 fmol/mg of protein) whose stimulation results in [(1)H]-IPs accumulation (602+/-23% of basal, EC(50) 2.2+/-0.4 microM) and calcium mobilization (resting level 96+/-5 nM, Delta[Ca(2+)](i) 517+/-32 nM, EC(50) 6.2+/-0.1 microM). Incubation with histamine (100 microM, 24 hr) resulted in a decrease in both cell number and [(1)H]-thymidine incorporation, blocked by the H(1) antagonist mepyramine (1 microM). CONCLUSIONS: Histamine inhibits the proliferation of DU-145 cells through the activation of H(1) receptors coupled to phosphoinositide hydrolysis.     

Biphasic effect of GABA on rat sperm acrosome reaction: involvement of GABA(A) and GABA(B) receptors

The functional relationship between GABA(A) and GABA(B) receptors in regulating acrosome reaction (AR) of rat spermatozoa was demonstrated by studying the differential effects of a GABA(B) agonist and an antagonist on the process. AR rates were determined using the chlortetracycline staining assay. The induction of AR in rat sperm by GABA was found to be a biphasic phenomenon; i.e., AR rates increased with increasing GABA concentrations up to <5 micro M and at higher concentrations of the neurotransmitter (>5 micro M), there was a reductionin the AR rates. This biphasic phenomenon is apparently due to the differential interaction of the neurotransmitter with GABA receptor subtypes in a dose-dependent manner; i.e., GABA(A) receptors (stimulatory) are primarily activated at low concentration of GABA, while GABA(B) receptors (inhibitory) become activated at higher concentrations. This hypothesis is supported by the present findings that treatment with saclofen, a GABA(B) receptor antagonist, did not influence the AR rates effected by GABA at low concentrations; while the AR rates were maintained at the maximum level at higher concentrations of GABA, resulting in the elimination of the biphasic phenomenon. Baclofen, a GABA(B) receptor agonist, blocks the AR activating action of GABA at both low and high concentrations. It would appear that the induction of AR in rat sperm by GABA is regulated by the proportionality of activated GABA(A) and GABA(B) receptors acting as a yin-yang control.     

Nonhalogenated anesthetic alkanes and perhalogenated nonimmobilizing alkanes inhibit alpha(4)beta(2) neuronal nicotinic acetylcholine receptors

The nonhalogenated anesthetic alkanes, cyclopropane and butane, do not enhance gamma-aminobutyric acid-elicited GABAergic currents, suggesting that these agents produce anesthesia via interactions with other molecular targets. Perhalogenated nonimmobilizing alkanes, such as 1,2-dichlorohexafluorocyclobutane and 2,3-dichlorooctafluorobutane, also fail to enhance GABAergic currents, but display specific behavioral effects that are distinct from those of structurally similar anesthetics. At concentrations predicted to be anesthetic, 1,2-dichlorohexafluorocyclobutane and 2,3-dichlorooctafluorobutane produce amnesia but fail to produce immobility. Neuronal nicotinic acetylcholine (nACh) receptors are sensitive to many anesthetics and are thought to have an important role in learning and memory. We postulated that neuronal nACh receptors might mediate the common amnestic action of nonhalogenated and perhalogenated alkanes. To test the hypothesis that neuronal nACh receptors have a role in mediating the behavioral effects of general anesthetics and nonimmobilizers, we quantified the inhibitory potencies of nonhalogenated anesthetic alkanes and perhalogenated nonimmobilizing alkanes on currents mediated by alpha(4)beta(2) neuronal nACh receptors. Our studies reveal that anesthetics and nonimmobilizers significantly inhibit alpha(4)beta(2) neuronal nACh receptors at concentrations that suppress learning and with potencies that correlate with their hydrophobicities. These results support the hypothesis that alpha(4)beta(2) neuronal nACh receptors mediate the amnestic actions of alkanes but not their immobilizing actions. IMPLICATIONS: The results of this study suggest that the immobilizing actions of general anesthetics do not result from the inhibition of alpha(4beta2) neuronal nicotinic acetylcholine receptors. However, the inhibition of neuronal nicotinic acetylcholine receptors may account for the amnestic activities of general anesthetics and nonimmobilizers.     

Halothane suppression of spinal sensory neuronal responses to noxious peripheral stimuli is mediated,in part,by both GABA(A) and glycine receptor systems

BACKGROUND: A major effect of general anesthesia is lack of response in the presence of a noxious stimulus. Anesthetic depression of spinal sensory neuronal responses to noxious stimuli is likely to contribute to that essential general anesthetic action. The authors tested the hypothesis that gamma-aminobutyric acid receptor type A (GABA(A)) and strychnine-sensitive glycine receptor systems mediate halothane depression of spinal sensory neuronal responses to noxious stimuli. METHODS: Extracellular activity of single spinal dorsal horn wide dynamic range (WDR) neurons was recorded in decerebrate, spinal cord transected rats. Neuronal responses to noxious (thermal and mechanical) and nonnoxious stimuli were examined in the drug-free state. Subsequently, cumulative doses (0.1-2.0 mg/kg) of bicuculline (GABA(A) antagonist) or strychnine (glycine antagonist) were administered intravenously in the absence or presence of 1 minimum alveolar concentration (MAC) of halothane. RESULTS: Halothane, 1.1%, depressed the response of WDR neurons to both forms of noxious stimuli. Antagonists, by themselves, had no effect on noxiously evoked activity. However, bicuculline and strychnine (maximum cumulative dose, 2.0 mg/kg) partially but significantly reversed the halothane depression of noxiously evoked activity. Similar results were seen with most, but not all, forms of nonnoxiously evoked activity. In the absence of halothane, strychnine significantly increased neuronal responses to low threshold receptive field brushing. CONCLUSION: Halothane depression of spinal WDR neuronal responses to noxious and most nonnoxious stimuli is mediated, in part, by GABA(A) and strychnine-sensitive glycine systems. A spinal source of glycine tonically inhibits some forms of low threshold input to WDR neurons.     

Glucose-dependent regulation of gamma-aminobutyric acid (GABA A) receptor expression in mouse pancreatic islet alpha-cells

The mechanism(s) by which glucose regulates glucagon secretion both acutely and in the longer term remain unclear. Added to isolated mouse islets in the presence of 0.5 mmol/l glucose, gamma-aminobutyric acid (GABA) inhibited glucagon release to a similar extent (46%) as 10 mmol/l glucose (55%), and the selective GABA(A) receptor (GABA(A)R) antagonist SR95531 substantially reversed the inhibition of glucagon release by high glucose. GABA(A)R alpha4, beta3, and gamma2 subunit mRNAs were detected in mouse islets and clonal alphaTC1-9 cells, and immunocytochemistry confirmed the presence of GABA(A)Rs at the plasma membrane of primary alpha-cells. Glucose dose-dependently increased GABA(A)R expression in both islets and alphaTC1-9 cells such that mRNA levels at 16 mmol/l glucose were approximately 3.0-fold (alpha4), 2.0-fold (beta3), or 1.5-fold (gamma2) higher than at basal glucose concentrations (2.5 or 1.0 mmol/l, respectively). These effects were mimicked by depolarizing concentrations of K(+) and reversed by the L-type Ca(2+) channel blocker nimodipine. We conclude that 1) release of GABA from neighboring beta-cells contributes substantially to the acute inhibition of glucagon secretion from mouse islets by glucose and 2) that changes in GABA(A)R expression, mediated by changes in intracellular free Ca(2+) concentration, may modulate this response in the long term.     

Local GABA(A) receptor blockade reverses isoflurane's suppressive effects on thalamic neurons in vivo

Many in vitro effects of volatile anesthetics are known, but the mechanisms of action are still under debate. Because suppression of sensory perception is one of the major goals of general anesthesia, we studied the effects of isoflurane on the processing of somatosensory information in anesthetized rats. Local iontophoretic administration of the gamma-aminobutyric acid-A (GABA(A)) receptor antagonist bicuculline in the thalamic ventral posteromedial nucleus reversed suppressive effects of isoflurane on thalamocortical relay neurons (TCNs). The action potential discharges of TCNs (n = 23) in response to defined mechanical stimulation of receptive fields seen with small concentrations of isoflurane (0.79% +/- 0.01%, mean +/- SEM) were suppressed under large concentrations (1.44% +/- 0.04%). In addition, the tonic response pattern was lost, which initially encoded the information about the stimulus features. In 70% of TCNs, bicuculline administration reestablished the initially present tonic response pattern under large isoflurane concentrations. These results indicate that isoflurane suppresses somatosensory information transfer at the thalamic level in vivo, apparently by enhancing thalamic GABA(A) receptor-mediated inhibition.     

Both cerebral GABA(A) receptors and spinal GABA(A) receptors modulate the capacity of isoflurane to produce immobility

We previously demonstrated that intrathecal administration of the noncompetitive gamma-aminobutyric acid type A (GABA(A)) receptor antagonist picrotoxin increased isoflurane MAC (the minimum alveolar concentration of anesthetic producing immobility in 50% of animals) by a maximum (ceiling effect) of approximately 40%. We also found that IV administration of picrotoxin increased MAC by more than 60%, without evidence of a ceiling effect. The larger increase with IV administration suggested a role of cerebral GABA(A) receptors. Accordingly, in this study we examined the effect of intracerebroventricular administration of picrotoxin in rats, finding that picrotoxin infusion into the third ventricle increased isoflurane MAC by a maximum of approximately 40%, without finding a ceiling effect. In addition, we concurrently infused picrotoxin into the intrathecal and intracerebroventricular spaces, producing an increase in MAC in excess of 70%, also with no evidence of a ceiling effect. The dose-response relationship for the intrathecal-intraventricular infusion paralleled that of the IV infusion but was shifted to the left by an order of magnitude. We conclude that both cerebral and spinal GABA(A) receptors modulate the capacity of inhaled anesthetics to produce immobility. Because other studies have shown that the spinal cord, and not the brain, mediates the capacity of inhaled anesthetics to produce immobility, these results call into question the relevance of GABA(A) receptors to the immobilizing action of isoflurane.     

Melanoma inhibits macrophage activation by suppressing toll-like receptor 4 signaling

BACKGROUND: Activated macrophages defend against tumors by secreting cytokines to recruit secondary immune cells, presenting antigen to T cells, and by direct tumor cytotoxicity. Peritoneal macrophages harvested from melanoma-bearing mice are less cytotoxic to melanoma cells, and produce less superoxide, nitric oxide, and tumor necrosis factor-alpha (TNF-alpha) than those from nontumor-bearing mice. Similar impairment of macrophage activation occurs in vitro using media harvested from cultured melanoma cells. Stimulation of Toll-like receptor 4 (TLR-4) activates macrophages and results in the release of TNF-alpha. We hypothesized that melanoma inhibits macrophage activation by suppressing TLR-4 signaling. STUDY DESIGN: Melanoma conditioned media (MCM) was generated from B16 melanoma cells. Peritoneal macrophages from TLR-4 competent or TLR-4 incompetent mice were exposed to control or MCM for 24 hours; then stimulated with lipopolysaccharide. TNF-alpha secretion, TNF-alpha mRNA production, nuclear factor-kappaB (NF-kappaB) activation, and TLR-4 surface expression were measured. RESULTS: Peritoneal macrophages exposed to MCM produced considerably less TNF-alpha in response to stimulus than controls (691 pg/mL versus 2,066 pg/mL, p < 0.001). TNF-alpha production by TLR-4 incompetent macrophages was not affected by MCM (454 pg/mL versus 480 pg/mL). Stimulated TNF-alpha mRNA and activated NF-kappaB were decreased in MCM treated C57BL/6 macrophages (by 38% and 33%, respectively). TLR-4 surface expression, however, was not decreased by exposure to MCM. CONCLUSIONS: Melanoma inhibits macrophage activation by suppressing TLR-4 signaling downstream of the TLR-4 receptor.     

Evidence for GABA(A) receptor agonistic properties of ketamine: convulsive and anesthetic behavioral models in mice

We examined the potentiation by ketamine of the gamma-aminobutyric acid(A) (GABA(A)) receptor function using convulsive and anesthetic behavioral models in adult male ddY mice. General anesthetic potencies were evaluated by a rating scale, which provided the data for anesthetic scores, loss of righting reflex, duration, and recovery time. All drugs were administered intraperitoneally. Small subanesthetic doses of ketamine did inhibit tonic seizures induced by a large dose of the GABA(A) receptor antagonist bicuculline (8 mg/kg). The 50% effective dose value was 15 (95% confidence limits 10-22) mg/kg. Even large anesthetic doses (100-150 mg/kg) did not suppress clonic seizures in 50% of the animals. The GABA(A) receptor agonist, muscimol (0.32-1.12 mg/kg), potentiated ketamine-induced anesthesia in a dose-dependent fashion (P < 0.05). Similarly, the benzodiazepine receptor agonist, diazepam (1-3 mg/kg), augmented ketamine anesthesia in a dose-dependent manner (P < 0.05). Bicuculline (2-5 mg/kg) dose-dependently antagonized ketamine-induced anesthesia (P < 0.05). Neither the benzodiazepine receptor antagonist, flumazenil (2-20 mg/kg), nor the GABA synthesis inhibitor, L-allylglycine (200 mg/kg), affected the anesthetic action of ketamine. These results suggest that ketamine has GABA(A) receptor agonistic properties and that ketamine-induced anesthesia is mediated, at least in part, by GABA(A) receptors. Implications: We examined the potentiation by ketamine of the gamma-aminobutyric acid(A) receptor function using convulsive and anesthetic behavioral models in mice. Subanesthetic doses of ketamine-inhibited tonic convulsions induced by the gamma-aminobutyric acid(A) receptor antagonist bicuculline. The gamma-aminobutyric acid(A) receptor agonist, muscimol, potentiated ketamine-induced anesthesia. Bicuculline antagonized ketamine anesthesia, but the benzodiazepine receptor antagonist, flumazenil, and the gamma-aminobutyric acid synthesis inhibitor, L-allyglycine, did not. The effects of ketamine on the gamma-aminobutyric acid(A) receptors appear to correlate with its anesthetic actions.     

Systemic capsaicin inhibits neuronal activation in the brainstem during postoperative ileus in the mouse

Introduction Neuronal inhibitory reflex mechanisms contribute to postoperative ileus after abdominal surgery. During this condition, sensory neurons in the brainstem are activated. We aimed to determine the contribution of capsaicin-sensitive afferents to central vagal sensitivity in mice during postoperative ileus. Materials and methods Under enflurane anesthesia, C57BL/6 mice were laparotomized and the small bowel was manipulated to induce ileus or was left untouched as a sham-treatment group. A subgroup of ileus animals was pre-treated with Capsaicin (1 μm/kg, i.p.) 48 h before small bowel manipulation. The animals were killed 24 h later and the brainstem was removed for Fos immunohistochemistry, which was quantified in the nucleus of the solitary tract (nTS). Spontaneous jejunal motility was recorded in vitro. Leukocyte infiltration in the intestinal muscularis was studied by myeloperoxidase staining as an index of postoperative inflammation. Results There were 30±9 Fos-positive neurons counted in the nTS after ileus and 6±2 in sham controls (Bregma −7.70 mm, P=0.01). A reduction to 8±3 was observed after Capsaicin pre-treatment in ileus animals (P<0.05). Peak amplitudes of spontaneous jejunal motility were 2±0.3 cmH₂O during postoperative ileus, 3±0.6 cmH₂O after ileus with capsaicin pre-treatment, and 10±2 cmH₂O in control animals (N=6, both P<0.05). The number of leukocytes infiltrating the muscularis was 39±9/mm² during ileus and 1.8±1/mm² in controls (mean±SEM, P<0.01, N=6). After capsaicin, this number increased to 72±28/mm² in ileus animals (P<0.05 vs control animals, N=7). Conclusion The inhibition of capsaicin-sensitive vagal afferent pathways appears to boost rather than to attenuate the inflammatory response during postoperative ileus, while intestinal motility remained unchanged. This suggests a protective role of the capsaicin-sensitive afferent innervation for the inflammatory phase of postoperative ileus. Best of Forum Papers presented at the Annual Meeting of the German Society of Surgery, 2–5 May 2006, Berlin, Germany     

Nicotine inhibits apoptosis and stimulates proliferation in aortic smooth muscle cells through a functional nicotinic acetylcholine receptor

Atherosclerosis and neointimal hyperplasia formation are induced by alterations in the homeostatic balance between cell growth and cell death. Apoptosis is a physiological cell death process that, when deregulated, may be involved in many pathological conditions. Cigarette smoking is a primary risk factor for vascular disease and nicotine seems to exert its atherogenic effects in part through the increase of smooth muscle cell (SMC) proliferation. The aim of this study was to investigate the effect of nicotine on SMC apoptosis. Nicotine added for 24 and 72 h to serum deprived cell cultures resulted in a decrease of apoptotic SMCs. The inhibition was direct and not mediated by platelet-derived growth factor, basic fibroblast growth factor, and transforming growth factor beta(1), autocrinally released by nicotine-treated SMCs, because it was not influenced by addition of specific neutralizing antibodies. Apoptosis inhibition as well as the proliferation increase, and basic fibroblast growth factor expression on nicotine-treated SMCs were blocked by nicotinic acetylcholine receptor antagonists, including alpha-bungarotoxin, a competitive antagonist of alpha subunits of nicotinic receptor. In conclusion, we propose that nicotine could lead to the increase of neointimal SMCs in vascular lesions by inducing the inhibition of physiological SMC apoptosis and the increase of SMC proliferation. We also showed that nicotine signaling occurs as a result of activation of the classical nicotine receptor pathways.     

Perineural alpha(2A)-adrenoceptor activation inhibits spinal cord neuroplasticity and tactile allodynia after nerve injury

BACKGROUND: Nerve injury in animals increases alpha(2)-adrenoceptor expression in dorsal root ganglion cells and results in novel excitatory responses to their activation, perhaps leading to the phenomenon of sympathetically maintained pain. In contrast to this notion, peripheral alpha(2)-adrenoceptor stimulation fails to induce pain in patients with chronic pain. We hypothesized that alpha(2) adrenoceptors at the site of nerve injury play an inhibitory, not excitatory role. METHODS: Partial sciatic nerve ligation was performed on rats, resulting in a reduction in withdrawal threshold to tactile stimulation. Animals received perineural injection at the injury site of clonidine, saline, or clonidine plus an alpha(2)-adrenergic antagonist, and withdrawal threshold was monitored. Immunohistochemistry was performed on the sciatic nerve ipsi- and contralateral to injury and on the spinal cord. RESULTS: Clonidine reduced this hypersensitivity in a dose-dependent manner, and this was blocked by an alpha(2A)-preferring antagonist. Perineural clonidine injection had a slow onset (days) and prolonged duration (weeks). Systemic or intrathecal clonidine, or transient neural blockade with ropivacaine, had short lasting or no effect on hypersensitivity. alpha(2A)-adrenoceptor immunostaining was increased near the site of peripheral nerve injury, both in neurons and in immune cells (macrophages and T lymphocytes). Phosphorylated cAMP response element binding protein (pCREB) in lumbar spinal cord was increased ipsilateral to nerve injury, and this was reduced 1 week after perineural clonidine injection. CONCLUSIONS: These data suggest that peripheral alpha(2) adrenoceptors are concentrated at the site of peripheral nerve injury, and their activation receptors produce long-lasting reductions in abnormal spinal cord gene activation and mechanical hypersensitivity.     

Nitrous oxide and xenon increase the efficacy of GABA at recombinant mammalian GABA(A) receptors

We investigated the interactions between recombinant gamma-aminobutyric acid receptor complex (GABA(A)R) and nitrous oxide (N(2)O) or xenon (Xe). Human embryonic kidney cells (HEK 293) were transfected with rat cDNA for alpha(1)beta(2)gamma(2L) or for alpha(1)beta(2) recombinant GABA(A)R subunits. Patch clamp techniques were used in the whole-cell mode to evaluate the effect of N(2)O and Xe on GABA-induced currents. A piezo-driven "liquid filament switch" was used for fast application. Both N(2)O (100%, 29.2 mM) and Xe (100%, 3.9 mM) reversibly increased GABA-induced currents through the alpha(1)ss(2)gamma(2L) and the alpha(1)beta(2) GABA(A)R channels. The potentiating effect of N(2)O or Xe on peak currents was prominent at small GABA concentrations (10(-7) to 10(-5) M). The addition of N(2)O or Xe increased the efficacy of GABA (10(-7) to 10(-3) M). Both N(2)O and Xe significantly decreased the risetime((10%-90%)) of the currents elicited by small GABA concentrations. At the concentrations used, neither N(2)O nor Xe had an intrinsic effect. We conclude that, similar to other anesthetics, both N(2)O and Xe increase the efficacy of GABA at the GABA(A)R and enhance inhibitory GABAergic synaptic transmission.     

Excitatory amino acid, GABA(A), and GABA(B) binding sites in human striate cortex.

Receptor autoradiography was used to study the laminar distribution of excitatory amino acid, GABA(A), and GABA(B) binding sites in human striate cortex. Binding sites for all these receptor subtypes were found within striate cortex, but there were marked differences in the laminar distribution of binding sites. NMDA binding sites were most dense in layers 1-4C, with highest density in layer 4C and lower levels in layers 5 and 6. Among non-NMDA binding sites, alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid binding sites had highest levels in layers 1-3, intermediate levels in layers 5 and 6, and lowest levels in layers 4B and 4C. Kainate and metabotropic binding sites were more uniformly distributed across cortical laminae, with a trend toward highest kainate binding in layers 5 and 6. GABA(A)/benzodiazepine binding sites had highest levels in layers 2, 3, and 4C, with intermediate levels in 4B and lowest levels in layers 1, 5, and 6. GABA(B) binding sites were uniformly distributed across laminae. There was no evidence of a "columnar" or "blob" pattern of any binding site within any of the laminae. With the exception of kainate, metabotropic excitatory amino acid, and GABA(B) binding sites, the laminar distribution of binding sites within striate cortex was different than that seen in adjacent visual cortex.