Mechanisms of chronic pain, including neuropathic pain, are poorly understood. Upregulation of voltage-gated calcium channel (VGCC) alpha2delta1 subunit (Ca(v)alpha2delta1) in sensory neurons and dorsal spinal cord by peripheral nerve injury has been suggested to contribute to neuropathic pain. To investigate the mechanisms without the influence of other injury factors, we have created transgenic mice that constitutively overexpress Ca(v)alpha2delta1 in neuronal tissues. Ca(v)alpha2delta1 overexpression resulted in enhanced currents, altered kinetics and voltage-dependence of VGCC activation in sensory neurons; exaggerated and prolonged dorsal horn neuronal responses to mechanical and thermal stimulations at the periphery; and pain behaviors. However, the transgenic mice showed normal dorsal horn neuronal responses to windup stimulation, and behavioral responses to tissue-injury/inflammatory stimuli. The pain behaviors in the transgenic mice had a pharmacological profile suggesting a selective contribution of elevated Ca(v)alpha2delta1 to the abnormal sensations, at least at the spinal cord level. In addition, gabapentin blocked VGCC currents concentration-dependently in transgenic, but not wild-type, sensory neurons. Thus, elevated neuronal Ca(v)alpha2delta1 contributes to specific pain states through a mechanism mediated at least partially by enhanced VGCC activity in sensory neurons and hyperexcitability in dorsal horn neurons in response to peripheral stimulation. Modulation of enhanced VGCC activity by gabapentin may underlie at least partially its antihyperalgesic actions.

Ethylene responsive factors (ERFs) are important plant-specific transcription factors, some of which have been demonstrated to interact with the ethylene-responsive GCC box and the dehydration-responsive element (DRE); however, data on the roles of ERF proteins in connection with various signaling pathways are limited. In this research, we used the GCC box, an essential cis-acting element responsive to ethylene and methyl jasmonate (MeJA), as bait in a yeast one-hybrid system to isolate transcription factors from tomato (Lycopersicon esculentum Mill.). One of the cDNAs, which was designated Jasmonate and Ethylene Response Factor 1 (JERF1), encodes an ERF protein, containing a conserved ERF DNA-binding motif and functioning as a transcriptional activator in yeast through targeting to the nucleus in onion (Allium cepa L.) epidermal cells. Biochemical analysis revealed that JERF1 bound not only to the GCC box but also to the DRE sequence. Expression of the JERF1 gene in tomato was induced by ethylene, MeJA, abscisic acid (ABA) and salt treatment, indicating that JERF1 might act as a connector among different signal transduction pathways. Further research with transgenic JERF1 tobacco (Nicotiana tabacum L.) plants indicated that overexpressing JERF1 activated expression of GCC box-containing genes such as osmotin, GLA, Prb-1b and CHN50 under normal growth conditions, and subsequently resulted in enhanced tolerance to salt stress, suggesting that JERF1 modulates osmotic tolerance by activation of downstream gene expression through interaction with the GCC box or DRE.

Primary afferent neurons are functionally heterogeneous. To determine whether this functional heterogeneity reflects, in part, heterogeneity in the regulation of the concentration of intracellular Ca 2+ ([Ca 2+ ] i ), the magnitude and decay of evoked Ca 2+ transients were assessed in subpopulations of dorsal root ganglion (DRG) neurons with voltage clamp and fura-2 ratiometric imaging. To determine whether differences in evoked Ca 2+ transients among subpopulations of DRG neurons reflected differences in the contribution of Ca 2+ regulatory mechanisms, pharmacological techniques were employed to assess the contribution of influx, efflux, release and uptake pathways. Subpopulations of DRG neurons were defined by cell body size, binding of the plant lectin IB 4 and responsiveness to the algogenic compound capsaicin (CAP). Ca 2+ transients were evoked with 30 mM K + or voltage steps to 0 mV. There were marked differences between subpopulations of neurons with respect to both the magnitude and decay of the Ca 2+ transient, with the largest and most slowly decaying Ca 2+ transients in small-diameter, IB 4 -positive, CAP-responsive neurons. The smallest and most rapidly decaying transients were in large-diameter, IB 4 -negative and CAP-unresponsive DRG neurons. These differences were not due to a differential distribution of voltage-gated Ca 2+ currents. However, these differences did appear to reflect a differential contribution of other influx, efflux, release and uptake mechanisms between subpopulations of neurons. These results suggest that electrical activity in subpopulations of DRG neurons will have a differential influence on Ca 2+ -regulated phenomena such as spike adaptation, transmitter release and gene transcription. Significantly more activity should be required in large-diameter non-nociceptive afferents than in small-diameter nociceptive afferents to have a comparable influence on these processes.

BackgroundDiabetic complications may be associated with impaired time-dependent glycemic control. Therefore, long-term glycemic variability, assessed by variations in haemoglobin A1c (HbA1c), may be a potential risk factor for microvascular complications, such as diabetic peripheral neuropathy (DPN). We investigated the association of HbA1c variability with DPN in patients with type 2 diabetes.MethodsIn this cross-sectional study, 563 type 2 diabetic patients who had been screened for DPN and undergone quarterly HbA1c measurements during the year preceding enrolment were recruited. DPN was confirmed in patients displaying both clinical manifestations of neuropathy and abnormalities in a nerve conduction evaluation. HbA1c variability was assessed by the coefficient of variation of HbA1c (CV-HbA1c), and the mean of HbA1c (M-HbA1c) was calculated. In addition, medical history and clinical data were collected.ResultsAmong the recruited patients, 18.1% (n = 102) were found to have DPN, and these patients also presented with a higher CV-HbA1c than the patients without DPN (p < 0.001). The proportion of patients with DPN increased significantly from 6.9% in the first to 19.1% in the second and 28.5% in the third tertile of CV-HbA1c (p for trend < 0.001). After adjusting for initial HbA1c, M-HbA1c and other clinical factors via multiple logistic regression analysis, the odds ratios (ORs) for DPN in the second and third versus those in the first CV-HbA1c tertile were 3.61 (95% CI 1.62–8.04) and 6.48 (2.86–14.72), respectively. The area under the receiver operating characteristic (ROC) curve of CV-HbA1c was larger than that of M-HbA1c, at 0.711 (95% CI 0.659–0.763) and 0.662 (0.604–0.721), respectively. ROC analysis also revealed that the optimal cutoff value of CV-HbA1c to indicate DPN was 15.15%, and its corresponding sensitivity and specificity were 66.67% and 65.73%, respectively.ConclusionsIncreased HbA1c variability is closely associated with DPN in type 2 diabetic patients and could be considered as a potent indicator for DPN in these patients.

Available evidence indicates voltage-gated Na+ channels (VGSCs) in peripheral sensory neurons are essential for the pain and hypersensitivity associated with tissue injury. However, our understanding of the biophysical and pharmacological properties of the channels in sensory neurons is largely based on the study of heterologous systems or rodent tissue, despite evidence that both expression systems and species differences influence these properties. Therefore, we sought to determine the extent to which the biophysical and pharmacological properties of VGSCs were comparable in rat and human sensory neurons. Whole cell patch clamp techniques were used to study Na+ currents in acutely dissociated neurons from human and rat. Our results indicate that while the two major current types, generally referred to as tetrodotoxin (TTX)-sensitive and TTX-resistant were qualitatively similar in neurons from rats and humans, there were several differences that have important implications for drug development as well as our understanding of pain mechanisms.DOI: http://dx.doi.org/10.7554/eLife.23235.001

The biophysical properties and distribution of voltage-dependent, Ca 2+ -modulated K + (BK Ca ) currents among subpopulations of acutely dissociated DiI labeled cutaneous sensory neurons from the adult rat were characterized with whole cell patch clamp techniques. BK Ca currents were isolated from total K + current with iberiotoxin, charybdotoxin, or paxilline. There was considerable variability in biophysical properties of BK Ca currents. There was also variability in the distribution of BK Ca current among subpopulations of cutaneous DRG neurons. While present in each of the subpopulations defined by cell body size, IB4 binding or capsaicin sensitivity, BK Ca current was present in vast majority (>90%) of small diameter IB4+ neurons but was present in only a minority of neurons in subpopulations defined by other criteria (i.e., small diameter IB4−). Current clamp analysis indicated that in IB4+ neurons, BK Ca currents contribute to the repolarization of the action potential and adaptation in response to sustained membrane depolarization, while playing little role in the determination of action potential threshold. RT-PCR analysis of mRNA collected from whole DRG revealed the presence of multiple splice variants of the BK Ca channel α-subunit, rslo and all 4 of the accessory β subunits, suggesting that heterogeneity in the biophysical and pharmacological properties of BK Ca current in cutaneous neurons, reflects, at least in part, the differential distribution of splice variants and/or β subunits. Because even a small decrease in BK Ca current appears to have a dramatic influence on excitability, modulation of this current may contribute to sensitization of nociceptive afferents observed following tissue injury.

Previously, we reported on a tomato ERF transcription activator, TERF1, which was concluded to act as a linker between ethylene and osmotic signal pathways. We now report on the regulatory role of TERF1 in ABA sensitivity and drought response during seedling development. Northern blotting analysis indicated that the transcripts of TERF1 were significantly accumulated in response to drought, cold and ABA. TERF1 activated GCC box- or DRE-driven reporter gene expression in transient expression assay, subsequently increasing the tolerance to drought and the osmoticum, PEG6000, in tobacco expressing TERF1. Further tests showed that TERF1 did not affect the seed germination, but greatly enhanced the sensitivity during tobacco seedling development under ABA treatment. This ABA hypersensitivity in transgenic TERF1 tobacco is both indirect ethylene action and expressions of ABA responsive genes, demonstrating that TERF1 is a multifunctional ERF protein that can integrate different stress signal pathways.

In addition to conventional risks including diabetic duration, HOMA-IR and HbA1c, increased glycaemic variability assessed by MAGE is a significant independent contributor to DPN in type 2 diabetic patients.