Demonstration of an inwardly rectifying K+ current component modulated by thyrotropin-releasing hormone and caffeine in GH3 rat anterior pituitary cells

 Reduction of an inwardly rectifying K⁺ current by thyrotropin-releasing hormone (TRH) and caffeine has been considered to be an important determinant of electrical activity increases in GH₃ rat anterior pituitary cells. However, the existence of an inwardly rectifying K⁺ current component was recently regarded as a misidentification of an M-like outward current, proposed to be the TRH target in pituitary cells, including GH₃ cells. In this report, an inwardly rectifying component of K⁺ current is indeed demonstrated in perforated-patch voltage-clamped GH₃ cells. The degree of rectification varied from cell to cell, but both TRH and caffeine specifically blocked a fraction of current with strong rectification in the hyperpolarizing direction. Use of ramp pulses to continuously modify the membrane potential demonstrated a prominent blockade even in cells with no current reduction at voltages at which M-currents are active. Depolarization steps to positive voltages at the maximum of the inward current induced a caffeine-sensitive instantaneous outward current followed by a single exponential decay. The magnitude of this current was modified in a biphasic way according to the duration of the previous hyperpolarization step. The kinetic characteristics of the current are compatible with the possibility that removal from inactivation of a fast-inactivating delayed rectifier causes the hyperpolarization-induced current. Furthermore, the inwardly rectifying current was blocked by astemizole, a potent and selective inhibitor of human ether-á-go-go -related gene (HERG) K⁺ channels. Along with other pharmacological and kinetic evidence, this indicates that the secretagogue-regulated current is probably mediated by a HERG-like K⁺ channel. Addition of astemizole to current-clamped cells induced clear increases in the frequency of action potential production. Thus, an inwardly-rectifying K⁺ current and not an M-like outward current seems to be involved in TRH and caffeine modulation of electrical activity in GH₃ cells. Received: 15 May 1997 / Received after revision and accepted: 24 July 1997     

Noradrenergic and opioidergic alterations in neuropathy in different rat strains

The Fischer 344 (F344) rat strain differs from the Lewis strain in the response to neuropathic pain. Recently, we found that F344 rats totally recover from mechanical allodynia induced by chronic constriction injury (CCI) of the sciatic nerve 28 days after surgery whereas Lewis rats are initiating their recovery at this time point. Thus, the use of this neuropathic pain model in these different rat strains constitutes a good strategy to identify possible target genes involved in the development of neuropathic pain. Since differences between Lewis and F344 rats in their response to pain stimuli in acute pain models have been related to differences in the endogenous opioid and noradrenergic systems, we aimed to determine the levels of expression of key genes of both systems in the spinal cord and dorsal root ganglia (DRG) of both strains 28 days after CCI surgery. Real time RT-PCR revealed minimal changes in gene expression in the spinal cord after CCI despite the strain considered, but marked changes in DRG were observed. A significant upregulation of prodynorphin gene expression occurred only in injured DRG of F344 rats, the most resistant strain to neuropathic pain. In addition, we found a significant downregulation of tyrosine hydroxylase and proenkephalin gene expression levels in both strains whereas δ-opioid receptor was found to be significantly downregulated only in injured DRG of Lewis rats although the same trend was observed in F344 rats. The data strongly suggest that dynorphins could be involved in strain differences concerning CCI resistance.     

A functional survey of the enhancer activity of conserved non-coding sequences from vertebrate Iroquois cluster gene deserts

Recent studies of the genome architecture of vertebrates have uncovered two unforeseen aspects of its organization. First, large regions of the genome, called gene deserts, are devoid of protein-coding sequences and have no obvious biological role. Second, comparative genomics has highlighted the existence of an array of highly conserved non-coding regions (HCNRs) in all vertebrates. Most surprisingly, these structural features are strongly associated with genes that have essential functions during development. Among these, the vertebrate Iroquois (Irx) genes stand out on both fronts. Mammalian Irx genes are organized in two clusters (IrxA and IrxB) that span >1 Mb each with no other genes interspersed. Additionally, a large number of HCNRs exist within Irx clusters. We have systematically examined the enhancer activity of HCNRs from the IrxB cluster using transgenic Xenopus and zebrafish embryos. Most of these HCNRs are active in subdomains of endogenous Irx expression, and some are candidates to contain shared enhancers of neighboring genes, which could explain the evolutionary conservation of Irx clusters. Furthermore, HCNRs present in tetrapod IrxB but not in fish may be responsible for novel Irx expression domains that appeared after their divergence. Finally, we have performed a more detailed analysis on two IrxB ultraconserved non-coding regions (UCRs) duplicated in IrxA clusters in similar relative positions. These four regions share a core region highly conserved among all of them and drive expression in similar domains. However, inter-species conserved sequences surrounding the core, specific for each of these UCRs, are able to modulate their expression.     

Encoding of electrical, thermal, and mechanical noxious stimuli by subnucleus reticularis dorsalis neurons in the rat medulla

1. In anesthetized rats, recordings were made within the medullary subnucleus reticularis dorsalis (SRD) from neurons that exhibited convergence of nociceptive inputs from the entire body. Neurons with total nociceptive convergence (TNC) responded to suprathreshold percutaneous electrical stimuli (2-ms duration) with an early and a late peak due to activation of A delta- and C-fibers, respectively, no matter which part of the body was stimulated. Neurons with partial nociceptive convergence (PNC) responded to the same stimuli with an A delta-peak regardless of which part of the body was stimulated and with a C-peak of activation from some, mainly contralateral, parts of the body. The characteristics of the responses of these neurons to the application of graded intensities of electrical, thermal, and mechanical stimuli were analyzed. 2. All TNC neurons and 85% of PNC neurons responded to A delta- and C-fiber activation following percutaneous electrical stimulation of the contralateral hindpaw. With regard to A delta-fiber-evoked responses, a linear relationship between the logarithm of the applied current and the magnitude of the responses was found within the 0.25- to 6.0-mA and 0.5- to 24-mA ranges for TNC and PNC neurons, respectively; however, these curves were essentially similar. With regard to C-fiber-evoked responses, such a linear relationship was found within the 1.5- to 6.0-mA range for both types of SRD neurons, although the TNC neurons presented larger C-fiber-evoked responses than did the PNC neurons. 3. TNC and PNC neurons linearly increased their discharges during the application of noxious thermal stimuli to the contralateral hindpaw within the range 44-52 degrees C; the mean responses evoked by noxious heat from TNC neurons were of higher magnitude than those from PNC neurons. The majority of SRD neurons presented long-lasting afterdischarges, especially with the highest temperature employed (52 degrees C). 4. TNC neurons monotonically increased their discharges during graded mechanical or thermal stimulation of the tail. When mechanical stimuli were applied, a linear relationship was found between the logarithm of the strength of the mechanical stimulus and the neuronal discharges, in the 5.3- to 7.4-N/cm2 range. With thermal stimulation, TNC neurons linearly increased their discharges in the 44-52 degrees C range. When increasing amounts of the tail were immersed in a 50 degrees C waterbath, TNC neurons increased their discharges within a restricted range of tail surface areas (0.9-5.7 cm2); further increases in the stimulated surface size were not followed by increases in firing rate.(ABSTRACT TRUNCATED AT 400 WORDS)     

Metabolic polymorphisms and the micronucleus frequency in buccal epithelium of adolescents living in an urban environment

Micronuclei and other biomarkers were evaluated in oral cells from 11- to 16-year-old girls living in a foster home in the central area of México City. Variables analyzed for possible association with these biomarkers include smoking habits, body mass index, metabolic polymorphisms for NAT1 and GSTM1 and whether the cells were obtained from the cheek or pharynx. The results indicated that individuals having the NAT1*10 homozygous genotype showed a significant increase in chromatin buds and binucleated cells. When the damage in the cheek was compared with damage in the pharynx, a significant increase in micronuclei and binucleated cells was found for the latter tissue in all the individuals analyzed.     

Vaccination of the Leishmania major susceptible BALB/c mouse. I. The precise selection of peptide determinant influences CD4+ T cell subset expression

BALB/c mice are susceptible to cutaneous lelshmanlasls uponinfection with Leishmania major while C57BL/6 are not. Thereis a major promastigote surface protease (PSP or gp63) whichis available in both native and recomblnant forms, and for whichthe primary amlno acid sequence is known. Immunization withPSP has been shown to offer some protection against challengewith the live organism. Therefore, we attempted to develop apeptide vaccine with PSP peptldes. In the first experiments,recall prollferatlve responses to PSP were measured using aset of 15mer peptldes spanning the entire PSP molecule whichallowed designation of major determinant regions in BALB/c,C57BL/6, and CBA mice. Several of these determinants were promiscuousand shared almost the identical core amlno acid residues inthe different strains. Immunization with major determinant peptldeswas recalled vigorously with L. major soluble antigen as wellas with PSP. The response to peptide was almost entirely T_h1as measured by a localized ELISA assay for single-cell productionof IFN-. A similar assay for IL-5, which overcomes problemsof sensitivity and inhibition by lymphoklnes produced by T_h1cells, Indicates very little production of T_h1 cells even byBALB/c. It was found that if a major responsive peak was examinedby recall with overlapping peptldes, the highest, central peptidegave a mainly Th1 response while the boundary, less efficientpeptldes gave more of a T_h2 response. Possible reasons for thiswere discussed. These results point to the importance of selectingthe exactly appropriate peptide in considering a vacclnogenthat might protect susceptible individuals. Even the choiceof a somewhat immunogenlc peptide within the determinant envelopemight actually exacerbate infection by steering the responsein a T_h2 direction.