Locus coeruleus modulates thalamic nociceptive responses via adrenoceptors

This study investigated the parafascicular (PF) neuronal nociceptive responses and their modulation following electrical stimulation of the locus coeruleus (LC) and intrathecal (i.t.) or intracerebroventricular (i.c.v.) administration of two alpha-adrenoceptor antagonists, the alpha2-antagonist, yohimbine, and the alpha1-antagonist, prazosin. The main results were as follows: (1) the nociceptive evoked discharges in PF neurons were suppressed by preceding stimulation of LC; (2) the suppressive effect of LC stimulation on PF neurons was replaced by a facilitatory effect following pretreatment of i.t. yohimbine in 14 units tested, while i.t. prazosin failed to alter the LC-induced suppression, even when the prazosin dose was doubled; (3) i.c.v. pretreatment with prazosin strengthened the suppressive effect of LC stimulation on PF neurons; (4) i.c.v. norepinephrine (NE) administration induced, in PF neurons, a biphasic response to noxious stimulation; an early, brief (about 10 min) inhibitory effect followed by a late, long-lasting facilitatory effect; and (5) i.c.v. pretreatment of yohimbine or prazosin prevented the inhibitory or facilitatory responses released by NE, respectively. These results provide evidence that: (1) the LC-descending projections exhibit a suppressive effect on nociceptive transmission at the spinal level through alpha2-receptors; and (2) the LC-ascending projections exhibit dual effects, facilitatory and inhibitory, at the medial thalamus (PF) level through alpha1- and alpha2-receptors, respectively.     

Neutralization of conservative charged transmembrane residues in the Na+/glucose cotransporter SGLT1

Our goal was to identify pairs of charged residues in the membrane domains of the Na+/glucose cotransporter (SGLT1) that form salt bridges, to obtain information about packing of the transmembrane helices. The strategy was to neutralize Glu225, Asp273, Asp294, and Lys321 in helices 6-8, express the mutants in oocytes, measure [14C]-alphaMDG uptake, and then attempt to find second-site mutations of opposite charge that restored function. alphaMDG uptake by E225A was identical to that by SGLT1, whereas transport was reduced by over 90% for D273A, D294A, and K321A and was not restored in the double mutants D273A/K321A or D294A/K321A. This suggested that K321 did not form salt bridges with D273 or D294 and that E225 was not involved in salt-bridging. Neutralization of K321 dramatically changed the Na+ uniport and Na+/glucose cotransport kinetics. The maximum rate of uniport in K321A increased 3-5-fold with a decrease in the apparent affinity for Na+ (70 vs 3 mM) and no change in apparent H+ affinity (0.5 microM). The change in Na+ affinity caused a +50 mV shift in the charge/voltage (Q/V) and relaxation time constant (tau)/voltage curves in the presteady-state kinetics. The presteady-state kinetics in H+ remained unchanged. The lower Na+ affinity resulted also in a 200-fold increase in the apparent K0.5 for alphaMDG and phlorizin. Replacements of K321 with alanine, valine, glutamine, arginine, or glutamic acid residues changed the steady-state kinetics in a similar way. Therefore, we suggest that K321 determines, directly or indirectly, (i) the rate and selectivity of SGLT1 uniport activity and (ii) the apparent affinities of SGLT1 for Na+, and indirectly sugar in the cotransport mode.     

Nociceptive c-fos expression in supraspinal areas in avoidance of descending suppression at the spinal relay station

The number and distribution of Fos-like-immunoreactive neurons in different supraspinal brain areas induced by formalin injection into one hindpaw was estimated in rats with transected dorsal half of the spinal cord at the thoracic level in an attempt to avoid most of the descending modulatory actions. The results showed that: (i) after spinal lesion, the peripheral noxious inputs, going up mainly through the ventral spinal cord, elicited a more widespread and densely located Fos-like-immunoreactive neurons in subcortical areas, many of them showed no Fos expression when noxious stimulation was given in rats with intact spinal cord; (ii) at the same time, a small number of subcortical areas, such as the lateral ventroposterior thalamic nucleus and dorsal raphe nucleus, exhibited no significant increase of nociceptive Fos-like immunoreactive neurons after spinal lesion as compared to that with intact spinal cord; and (iii) there appeared a prominent expansion of cortical areas with densely located Fos-like-immunoreactive neurons in spinal-lesioned rats as compared with the limited labelled areas in the control group with intact spinal cord. These results indicate that: (i) in avoiding the spinally descending modulatory mechanisms, more widespread supraspinal and cortical neurons will be recruited and activated in response to the noxious stimulation; and (ii) the descending systems exert differential actions on the spinal targets which project nociceptive signals to different supraspinal regions. The implication of these facts is discussed.     

Imaging and tissue biodistribution of 99mTc-labeled adenovirus knob (serotype 5)

Hepatic sequestration of systemically administered adenoviral vectors reduces the number of viral particles available for delivery to other tissues. The biological basis of this phenomenon was investigated using a new in vivo technique which permitted imaging in real time. Recombinant adenovirus serotype 5 knob (Ad5K) was radiolabeled with the gamma-emitter 99mTc (half-life = 6 h). Scatchard analysis of the 99mTc-Ad5K showed specific, high-affinity binding to U293 cells (Kd = 1.4 +/- 0.5 nM), demonstrating that the radiolabeling process had no effect on receptor binding. In vivo dynamic imaging with an Anger gamma camera revealed that the liver binding followed an exponential rise to maximum, with a measured 100% extraction efficiency. Initially, the liver binding capacity was 3.1 +/- 0.4 micrograms Ad5K, equivalent to approximately 17,000 Ad5K molecules per liver cell. Liver binding was blocked by preincubation of Ad5K with neutralizing anti-Ad5K antibody; a 50% reduction in liver uptake was demonstrated by imaging. Unlabeled Ad5K was more effective in blocking liver uptake of 99mTc-Ad5K, whereas irrelevant unlabeled Ad3K had no effect. Imaging data for the liver uptake studies were in agreement with biodistribution determined by removing and measuring tissues. These data demonstrated that in vivo imaging is a sensitive tool for measuring changes to liver tropism. Similar imaging techniques can be applied to adenovirus vectors to measure specific targeting for gene therapy.     

The role of dispositional and situational factors in cognitive interference

Two studies investigated the cross-temporal stability and cross-situational consistency of cognitive interference. In Study 1, 70 college students reported on the intrusive thoughts they experienced during 2 course examinations and a self-reflective task. In Study 2, 55 college athletes reported on intrusive thoughts following 2 course examinations and 2 regular season football games. Major findings were (a) cognitive interference showed both cross-temporal stability and cross-situational consistency, (b) it was predicted by a measure of dispositional intrusive thinking, (c) the link between cognitive interference and dispositional intrusive thinking was not accounted for by indices of depressive symptomatology and trait anxiety, and (d) the degree of cross-situational consistency of cognitive interference and the content of these intrusive thoughts were influenced by situational factors.     

Glycosphingolipids as novel targets for T-cell suppression by the B subunit of recombinant heat-labile enterotoxin

Heat-labile enterotoxin subunit B (LTB) is a noncatalytic protein derived from Escherichia coli that binds to ganglioside GM1, a glycosphingolipid on the surface of mammalian cells. In this study, the effects of recombinant LTB (rLTB) on murine lymphocytes were examined in vitro. T and B cells readily bound fluorescein isothiocyanate-labeled rLTB. CD8+ T cells bound twice as much as CD4+ T cells and B cells. Exposure of T-cell subsets and B cells to rLTB abrogated mitogen-driven proliferation. CD8+ T cells were more susceptible to rLTB than either CD4+ T cells or B cells. There were differences in the sensitivity of lymphocytes from various strains of mice to rLTB. This was attributed to qualitative and quantitative differences in the CD4+ T cells. rLTB induced apoptosis in both T-cell subsets, but the level was significantly higher in CD8+ T cells. Apoptosis peaked at around 8 h after exposure to rLTB and incubation at 37 degrees C. Binding to ganglioside GM1 was essential for suppression, since rLTB/G33D, a mutant which does not bind GM1, failed to inhibit proliferation or induce apoptosis. Naive T cells, which were acutely sensitive to rLTB, became more resistant after activation. Conversely, activated T cells regained their sensitivity to rLTB when they reverted back to a resting state. A 1-h pulse with rLTB was sufficient to inhibit T-cell proliferation and cytotoxic-T-lymphocyte generation in primary mixed lymphocyte reaction cultures. CD8+ T cells were preferentially depleted in these cultures. rLTB also induced functional modifications in T cells as indicated by inhibition of gamma interferon secretion after polyclonal activation. Thus, rLTB may have immunomodulatory properties independent of its ability to induce apoptosis.     

Interaction of the adenovirus 14.7-kDa protein with FLICE inhibits Fas ligand-induced apoptosis

Adenovirus type 5 encodes a 14.7-kDa protein that protects infected cells from tumor necrosis factor-induced cytolysis by an unknown mechanism. In this report, we demonstrate that infection of cells with an adenovirus vector expressing Fas ligand induced rapid apoptosis that was blocked by coinfection with a virus expressing 14. 7K. Moreover, AdFasL/G infection resulted in the rapid activation of DEVD-specific caspases, and caspase activation was blocked by coinfection with Ad14.7/G. Cell death induced by the overexpression of Fas ligand, Fas-associated death domain-containing protein (FADD)/MORT1, or FADD-like interleukin-1beta-converting enzyme (FLICE)/caspase-8 in a virus-free system was efficiently blocked by 14.7K expression. Moreover, we demonstrate that 14.7K interacts with FLICE. These results support the idea that FLICE is a cellular target for the 14.7-kDa protein.     

Differential downstream functions of protein kinase Ceta and -theta in EL4 mouse thymoma cells

Sensitive EL4 mouse thymoma cells (s-EL4) respond to phorbol esters with growth inhibition, adherence to substrate, and production of cytokines including interleukin 2. Since these cells express several of the phorbol ester-sensitive protein kinase C (PKC) isozymes, the function of each isozyme remains unclear. Previous studies demonstrated that s-EL4 cells expressed substantially more PKCeta and PKCtheta than did EL4 cells resistant to phorbol esters (r-EL4). To examine potential roles for PKCeta and PKCtheta in EL4 cells, wild type and constitutively active versions of the isozymes were transiently expressed using a Sindbis virus system. Expression of constitutively active PKCeta, but not PKCtheta, in s- and r-EL4 cells altered cell morphology and cytoskeletal structure in a manner similar to that of phorbol ester treatment, suggesting a role for PKCeta in cytoskeletal organization. Prolonged treatment of s-EL4 cells with phorbol esters results in inhibition of cell cycling along with a decreased expression of most of the PKC isozymes, including PKCtheta. Introduction of virally expressed PKCtheta, but not PKCeta, overcame the inhibitory effects of the prolonged phorbol ester treatment on cell cycle progression, suggesting a possible involvement of PKCtheta in cell cycle regulation. These results support differential functions for PKCeta and PKCtheta in T cell activation.     

ABT-594 [(R)-5-(2-azetidinylmethoxy)-2-chloropyridine]: a novel, orally effective antinociceptive agent acting via neuronal nicotinic acetylcholine receptors: II. In vivo characterization

The antinociceptive effects of ABT-594, a novel nicotinic acetylcholine receptor (nAChR) ligand, were examined in rats in models of acute thermal (hot box) and persistent chemical (formalin test) pain. Also, the effects of ABT-594 treatment on motor function and electroencephalogram (EEG) were determined. In the hot box and formalin test (i.e., phase 1 and 2), acute treatment with ABT-594 (0.03, 0.1 and 0.3 mumol/kg i.p.) produced significant dose-dependent antinociceptive effects. In the hot box, the efficacy of ABT-594 was maintained after a repeated dosing paradigm (5 days b.i.d.i.p.). ABT-594 was fully efficacious in the formalin test when administered before formalin, and also retained significant efficacy (0.3 mumol/kg i.p.) when administered after formalin injection. The antinociceptive effects of ABT-594 in the hot box and formalin tests were attenuated by pretreatment with the nAChR antagonist, mecamylamine, and in animals treated with the nAChR antagonist chlorisondamine, given centrally (10 micrograms/rat i.c.v. 5 days before), but not in animals pretreated with the opioid receptor antagonist, naltrexone. Acute treatment with ABT-594 produced an initial decrease in open-field locomotor activity, which was absent in animals dosed repeatedly (5 days b.i.d.) with ABT-594. Also, acute treatment with ABT-594 decreased body temperature and decreased the amount of time the animals could maintain balance in an edge-balance test. These effects were no longer present in animals dosed repeatedly with ABT-594. At antinociceptive doses, ABT-594 produced activation of free running EEG in contrast to the sedative-like effects of morphine. Full antinociceptive efficacy was maintained in both the hot box and formalin tests after oral administration, whereas the effects on motoric performance were attenuated. In conclusion, these data demonstrate that ABT-594 is a potent antinociceptive agent with full efficacy in models of acute and persistent pain and that these effects are mediated predominately by an action at central neuronal nAChRs. In addition, antinociceptive effects were maintained after repeated dosing, whereas effects of ABT-594 on motor and temperature measures were attenuated in animals treated repeatedly with ABT-594. Thus, compounds acting at nAChRs may represent a novel approach for the treatment of a variety of pain states.