Chronic intrathecal infusion of mibefradil, ethosuximide and nickel attenuates nerve ligation-induced pain in rats.

BACKGROUND: T-type Ca(2+) channels (TCC) are important for pain transmission, especially the Ca(V)3.2 subtype. In this study, we examined the effects of intrathecal TCC blockers in the L5/6 spinal nerve ligation pain rat model. METHODS: Under isoflurane anaesthesia, rats received right L5/6 spinal nerve ligation and intrathecal catheters (attached to an infusion pump) were sited. After surgery, saline, mibefradil, ethosuximide or NiCl2 were given intrathecally for seven days. The right hindpaw withdrawal thresholds to von Frey hair stimuli and withdrawal latencies to radiant heat were measured before and once daily for seven days after surgery. Double immunofluorescence and western blotting were used to examine the expression of Ca(V)3.2 in dorsal root ganglion (DRG) and spinal cord. RESULTS: On post-ligation day seven, rats receiving mibefradil, ethosuximide or NiCl2 had significant higher median withdrawal thresholds (15.0, 10.2, and 10.9 g) and latencies (8.0, 7.6 and 7.6 s) than saline-treated rats (1.6 g and 4.3 s, respectively). Ca(V)3.2 was expressed in parvalbumin(+), IB4(+), CGRP(+) and VR1(+) neurones in DRG and most neurones in spinal dorsal horn. Ca(V)3.2 was up-regulated in the right L5/6 DRG and spinal cord seven days after nerve ligation. CONCLUSIONS: In this study, we demonstrated that intrathecal TCC blockers attenuate the development of nerve injury-induced mechanical allodynia and thermal hyperalgesia. Our data suggest that continuous intrathecal infusion of TCC or Ca(V)3.2 blockers may be a promising alternative for the management of nerve injury-induced pain.

Chronic intrathecal infusion of gabapentin prevents nerve ligation-induced pain in rats.

BACKGROUND: Gabapentin is an anticonvulsant and adjuvant analgesic. It is effective in several pain studies. Neuropathic pain is the most difficult type of pain to treat. In this study, we examined if intrathecal gabapentin could prevent nerve injury-induced pain. METHODS: Under isoflurane anaesthesia, male Sprague-Dawley rats (200-250 g) underwent right L5/6 spinal nerve ligation and placement of an intrathecal catheter connected to an infusion pump. After surgery, intrathecal saline or gabapentin (20 µg h(-1)) was given for 7 days (n=8 per group). The right hind paw withdrawal threshold to von Frey filament stimuli and withdrawal latency to radiant heat were determined before (baseline) and once daily for 7 days after surgery. Haematoxylin and eosin and toluidine blue staining were used to evaluate the neurotoxicity of gabapentin (40 µg h(-1)). RESULTS: Seven days after nerve ligation, the affected paw withdrawal threshold and latency of saline-treated rats decreased from the baseline 11.7 (11.7-22.2) [median (inter-quartile range)] to 1.6 (0.9-3.2) g and 10.8 (10.5-11.2) to 4.3 (4.2-7) s, respectively. Rats receiving gabapentin (20 µg h(-1)) had higher withdrawal threshold [9.9 (9.9-19.3) g] and latency [11.5 (9.7-11.9) s] on day 7 after ligation. No obvious histopathological change or growth retardation was detected after intrathecal gabapentin (40 µg h(-1)) infusion. CONCLUSIONS: We showed a preventative effect of intrathecal gabapentin on the development of nerve injury-induced mechanical allodynia and thermal hyperalgesia. Our data suggest that continuous intrathecal gabapentin may be considered as an alternative for the prevention of nerve injury-induced pain.

Localization of A-type K+ channel subunit Kv4.2 in rat brain.

Kv4.2, a voltage-gated K+ (Kv) channel subunit, has been suggested to be the key component of the subthreshold A-type K+ currents (I(SA)s) recorded from the specific subcellular compartments of certain CNS neurons. To correlate Kv4.2 localization with the I(SA)s detected, immunohistochemistry will be useful. Although the Kv4.2 immunostaining pattern in the hippocampus and cerebellum has been reported, the Kv4.2 antibody used was not specific. Furthermore, Kv4.2 localization in other brain regions remains unclear. In this report, we first demonstrated the specificity of a new Kv4.2 antibody, and then used it to examine Kv4.2 localization throughout adult rat brain by immunohistochemistry. At the cellular level, Kv4.2 was found in neurons but not glias. At the subcellular level, Kv4.2 was localized in the somatodendritic compartment of most neurons examined. Nevertheless, our preliminary data indicated that Kv4.2 might be also present in the axon/terminal compartment. At the functional level, our data indicates that Kv4.2 localization and I(SA) correlate quite well in some CNS neurons, supporting that Kv4.2 is the key component of some I(SA)s recorded in vivo.

gas7: A gene expressed preferentially in growth-arrested fibroblasts and terminally differentiated Purkinje neurons affects neurite formation.

Growth arrest-specific (gas) genes are expressed preferentially in cells that enter a quiescent state. gas7, which we identified in serum-starved murine fibroblasts, is reported here to be expressed in vivo selectively in neuronal cells of the mature cerebral cortex, hippocampus, and cerebellum. gas7 transcripts encode a 48-kDa protein containing a structural domain that resembles sequences of OCT2, a POU transcription factor implicated in neuronal development, and synapsins, which have a role in modulating neurotransmitter release. Using in situ hybridization and immunocytochemical analysis, we show that GAS7 expression occurs prominently in cerebellar Purkinje cells and that inhibition of production in terminally differentiating cultures of embryonic murine cerebellum impedes neurite outgrowth from maturing Purkinje cells. Conversely, GAS7 overexpression in undifferentiated neuroblastoma cell cultures dramatically promotes neurite-like outgrowth. Collectively, our results provide evidence for an association between expression of this gas gene and neuronal development.

Expression of B-type endothelin receptor gene during neural development.

Mutations of the B-type endothelin receptor (ETRB) gene have been found to cause defects in the development of enteric neurons, which resulted in aganglionic megacolon in rodents and humans. To determine the distribution of ETRB mRNA during neural development, mainly in the CNS, in situ hybridization was applied at various developmental stages of rat. ETRB gene was abundantly expressed prenatally in the ventricular and subventricular zones, as well as postnatally in the ependymal and subependymal cells. ETRB mRNA was also strongly detected prenatally in the dorsal root ganglia, as well as postnatally in the cerebellar Bergmann glial cells and epithelial cells of choroid plexus. Our data suggest that ETRB acts as a regulator in the differentiation, proliferation, or migration of neural cells during development.

Cloning, expression and CNS distribution of Kv4.3, an A-type K+ channel alpha subunit.

A full-length K+ channel cDNA of Kv4.3, with an open reading frame of 611 amino acids, was isolated from rat hippocampus. Functional expression of Kv4.3 cDNA in Xenopus oocytes revealed an A-type K+ channel. In the central nervous system, Kv4.3 is most prominently expressed in the retrosplenial cortex, medial habenula, anterior thalamus, hippocampus, cerebellum, as well as lateral geniculate and superior colliculus, which are important for vision. The abundant expression of Kv4.3 in many CNS neurons supports its important role as a major component of subthreshold A currents in the control of action potentials and thus neuronal excitability.

Isolation of a cDNA clone encoding a KATP channel-like protein expressed in insulin-secreting cells, localization of the human gene to chromosome band 21q22.1, and linkage studies with NIDDM.

The metabolism of glucose in insulin-secreting cells leads to closure of ATP-sensitive K+ channels (KATP), an event that initiates the insulin secretory process. Defects in insulin secretion are a common feature of non-insulin-dependent diabetes mellitus (NIDDM), and the beta-cell KATP that couples metabolism and membrane potential is a candidate for contributing to the development of this clinically and genetically heterogeneous disorder. We screened a hamster insulinoma cDNA library by low-stringency hybridization with a probe coding for the G-protein-coupled inwardly rectifying K+ channel GIRK1/KGA and isolated clones encoding a protein, KATP-2, whose sequence is 90% similar to that of the recently described KATP-1, an ATP-sensitive K+ channel expressed in heart and other tissues. RNA blotting showed that KATP mRNA was present in insulin-secreting cells and brain but not in heart. To assess the contribution of KATP-2 to the development of NIDDM, the human KATP-2 gene (symbol KCNJ7) was isolated and mapped to chromosome band 21q22.1 by fluorescence in situ hybridization. A simple tandem repeat DNA polymorphism, D21S1255, was identified in the region of the KATP-2 gene, and linkage studies between this marker and NIDDM were carried out in a group of Mexican-American sib pairs with NIDDM. There was no evidence for linkage between D21S1255 and NIDDM, indicating that KATP-2 is not a major susceptibility gene in this population.

Contrasting subcellular localization of the Kv1.2 K+ channel subunit in different neurons of rat brain.

In the nervous system, a wide diversity of K+ channels are formed by the oligomeric assembly of subunits encoded by a large number of K+ channel genes. The physiological functions of a specific K+ channel subunit in vivo will be dictated in part by its subcellular location within neurons. We have used a combined in situ hybridization and immunocytochemical approach to determine the subcellular distribution of Kv1.2, a member of the Shaker subfamily of K+ channel genes. In contrast to other characterized K+ channel subunits, Kv1.2 protein shows a complex differential subcellular distribution in neurons of rat brain. In some of these neurons (e.g., hippocampal and cortical pyramidal cells, and Purkinje cells), Kv1.2 is concentrated in dendrites, while in others (e.g., cerebellar basket cells), Kv 1.2 is predominantly, if not exclusively, localized to nerve terminals. Furthermore, Kv1.2 immunoreactivity was also detected in certain axon tracts. We hypothesize that the differential sorting of Kv1.2 could result from association of Kv1.2 with varying heterologous K+ channel subunits in different cell types, with the implication that Kv1.2 may participate in distinct heteromultimeric K+ channels in different subcellular domains. The findings suggest that Kv1.2-containing K+ channels may play diverse functional roles in several neuronal compartments, regulating presynaptic or postsynaptic membrane excitability, depending on the neuronal cell type.

Subcellular segregation of two A-type K+ channel proteins in rat central neurons.

In the mammalian nervous system, K+ channels regulate diverse aspects of neuronal function and are encoded by a large set of K+ channel genes. The roles of different K+ channel proteins could be dictated by their localization to specific subcellular domains. We report that two K+ channel polypeptides, Kv1.4 and Kv4.2, which form transient (A-type) K+ channels when expressed in Xenopus oocytes, are segregated in rat central neurons. Kv1.4 protein is targeted to axons and possibly terminals, while Kv4.2 is concentrated in dendrites and somata. This differential distribution implies distinct roles for these channel proteins in vivo. Their localizations suggest that Kv1.4 and Kv4.2 may regulate synaptic transmission via presynaptic, or postsynaptic mechanisms, respectively.

Differential expression of K+ channel mRNAs in the rat brain and down-regulation in the hippocampus following seizures.

K+ channels are major determinants of membrane excitability. Differences in neuronal excitability within the nervous system may arise from differential expression of K+ channel genes, regulated spatially in a cell type-specific manner, or temporally in response to neuronal activity. We have compared the distribution of mRNAs of three K+ channel genes, Kv1.1, Kv1.2, and Kv4.2 in rat brain, and examined activity-dependent changes following treatment with the convulsant drug pentylenetetrazole. Both regional and cell type-specific differences of K+ channel gene expression were found. In addition, seizure activity caused a reduction of Kv1.2 and Kv4.2 mRNAs in the dentate granule cells of the hippocampus, raising the possibility that K+ channel gene regulation may play a role in long-term neuronal plasticity.

Characterization of a mammalian cDNA for an inactivating voltage-sensitive K+ channel.

A cDNA clone encoding a K+ channel polypeptide with 72% amino acid sequence identity to Drosophila Shal was isolated from rat hippocampus. Functional expression of the cDNA in Xenopus oocytes generated 4-amino-pyridine-sensitive K+ channels displaying rapid inactivation kinetics. The fastest component of inactivation was slowed by the deletion of 3 basic residues in the amino-terminal region. Northern blots revealed that the mRNA encoding this K+ channel polypeptide was expressed at a similar level in the brain and in the heart. In situ hybridization revealed that the mRNA encoding this K+ channel appeared concentrated in the hippocampus, dentate gyrus, and habenular nucleus in the brain. Thus, this K+ channel polypeptide is likely to form some of the A-type K+ channels expressed in the mammalian nervous system and heart.

Localization of the control region for expression of exotoxin A in Pseudomonas aeruginosa.

The 2,760-base-pair (bp) PstI-EcoRI segment of the chromosome of Pseudomonas aeruginosa PA103 which carries the exotoxin A structural gene was expressed from an internal promoter when cloned in a pUC9 derivative and transformed into a nontoxigenic mutant of P. aeruginosa PAO1. The unique terminal EcoRI site was deleted, and a new EcoRI site was substituted for a PvuI site located 107 bp 5' to the transcription initiation site. Following EcoRI cleavage, Bal31 deletions were generated from this site, and an EcoRI linker sequence, GGAATTCC, was inserted in place of the deleted DNA. Mutants with deletions located 73 bp or more upstream of the transcription initiation site retained normal expression, whereas in mutants with deletions extending into the region 69 bp or less upstream of this site, exotoxin synthesis was greatly reduced. From a KpnI site located 473 bp 3' to the transcription initiation site, a similar series of Bal31 deletion mutants were generated in which the inserted EcoRI linker sequence was located within the same 72-bp region. Pairs of mutants from the two deletion series were identified in which the EcoRI linker was located at the same sequence, and these mutant pairs were ligated to derive a series of constructs in which the EcoRI linker sequence GGAATTCC was substituted for an 8-bp sequence within the 72-bp region. Some of these linker-substituted mutants showed greatly reduced exotoxin A synthesis. The results are consistent with a binding site for a positive activator contiguous with the binding site for an RNA polymerase.

Pharmacological study on angusticeps-type toxins from mamba snake venoms.

Five angusticeps-type toxins, F7, F8 and C10S2C2 from Dendroaspis angusticeps and C and FS2 from D. polylepis polylepis, were tested for action on the chick biventer cervicis nerve-muscle, the frog rectus abdominis muscle and the mouse phrenic nerve-diaphragm preparations. In the chick muscle, none of these toxins exhibited any stimulatory effect up to 100 micrograms/ml. In the frog muscle, the response to acetylcholine, but not to carbachol, was enhanced dose dependently by F7 and C. No appreciable effect was observed with the other three toxins. In the mouse diaphragm, also only F7 and C augmented responses to indirect stimulation and produced spontaneous fasciculations. On tetanic stimulation, a marked Wedensky inhibition was observed. Their stimulatory effect was abolished by d-tubocurarine. In the presence of d-tubocurarine as well as in the denervated mouse diaphragm, neither toxin increased responses to direct stimulation. In low-calcium (0.6 mM) or high magnesium (4.2 mM) medium, the stimulatory effect of both toxins was markedly attenuated. The resting membrane potential of the mouse diaphragm was not changed. The amplitude and frequency of MEPPs and the quantal content and the half-decay time of EPPs was increased. Both toxins also produced a stimulatory effect on the isolated guinea-pig ileum, which was abolished by atropine. In the rat atrial preparation, both toxins caused negative inotropic and chronotropic effects, which were reversed by atropine. If pretreated with atropine, these effects were completely prevented. Both F7 and C markedly inhibited the cholinesterase activity of the homogenized mouse diaphragm and frog rectus abdominis muscle but not that of the chick biventer cervicis muscle.(ABSTRACT TRUNCATED AT 250 WORDS)