Exposure of the yeast Saccharomyces cerevisiae to alkaline stress resulted in adaptive changes that involved remodeling the gene expression. Recent evidence suggested that the calcium-activated protein phosphatase calcineurin could play a role in alkaline stress signaling. By using an aequorin luminescence reporter, we showed that alkaline stress resulted in a sharp and transient rise in cytoplasmic calcium. This increase was largely abolished by addition of EGTA to the medium or in cells lacking Mid1 or Cch1, components of the high affinity cell membrane calcium channel. Under these circumstances, the alkaline response of different calcineurin-sensitive transcriptional promoters was also blocked. Therefore, exposure to alkali resulted in entry of calcium from the external medium, and this triggered a calcineurin-mediated response. The involvement of calcineurin and Crz1/Tcn1, the transcription factor activated by the phosphatase, in the transcriptional response triggered by alkalinization has been globally assessed by DNA microarray analysis in a time course experiment using calcineurin-deficient (cnb1) and crz1 mutants. We found that exposure to pH 8.0 increased at least 2-fold the mRNA levels of 266 genes. In many cases (60%) the response was rather early (peak after 10 min). The transcriptional response of 27 induced genes (10%) was reduced or fully abolished in cnb1 cells. In general, the response of crz1 mutants was similar to that of calcineurin-deficient cells. By analysis of a systematic deletion library, we found 48 genes whose mutation resulted in increased sensitivity to the calcineurin inhibitor FK506. Twenty of these mutations (42%) also provoked alkaline pH sensitivity. In conclusion, our results demonstrated that calcium signaling and calcineurin activation represented a significant component of the yeast response to environmental alkalinization.

BACKGROUND AND PURPOSEThe sigma‐1 (σ₁) receptor is a ligand‐regulated molecular chaperone that has been involved in pain, but there is limited understanding of the actions associated with its pharmacological modulation. Indeed, the selectivity and pharmacological properties of σ₁receptor ligands used as pharmacological tools are unclear and the demonstration that σ₁receptor antagonists have efficacy in reversing central sensitization‐related pain sensitivity is still missing.EXPERIMENTAL APPROACHThe pharmacological properties of a novel σ₁receptor antagonist (S1RA) were first characterized. S1RA was then used to investigate the effect of pharmacological antagonism of σ₁receptors onin vivonociception in sensitizing conditions and onin vitrospinal cord sensitization in mice. Drug levels and autoradiographic,ex vivobinding for σ₁receptor occupancy were measured to substantiate behavioural data.KEY RESULTSFormalin‐induced nociception (both phases), capsaicin‐induced mechanical hypersensitivity and sciatic nerve injury‐induced mechanical and thermal hypersensitivity were dose‐dependently inhibited by systemic administration of S1RA. Occupancy of σ₁receptors in the CNS was significantly correlated with the antinociceptive effects. No pharmacodynamic tolerance to the antiallodynic and antihyperalgesic effect developed following repeated administration of S1RA to nerve‐injured mice. As a mechanistic correlate, electrophysiological recordings demonstrated that pharmacological antagonism of σ₁receptors attenuated the wind‐up responses in spinal cords sensitized by repetitive nociceptive stimulation.CONCLUSIONS AND IMPLICATIONSThese findings contribute to evidence identifying the σ₁receptor as a modulator of activity‐induced spinal sensitization and pain hypersensitivity, and suggest σ₁receptor antagonists as potential novel treatments for neuropathic pain.

Chronic hypertension is associated with resistance artery remodelling and mechanical alterations. However, the contribution of elastin has not been thoroughly studied. Our objective was to evaluate the role of elastin in vascular remodelling of mesenteric resistance arteries (MRA) from spontaneously hypertensive rats (SHR). MRA segments from Wistar Kyoto rats (WKY) and SHR were pressurised under passive conditions at a range of physiological pressures with pressure myography. Confocal microscopy was used to determine differences in the quantity and organisation of elastin in intact pressure‐fixed arteries. To assess the contribution of elastin to MRA structure and mechanics, myograph‐mounted vessels were studied before and after elastase incubation. When compared with WKY, MRA from SHR showed: (1) a smaller lumen, (2) decreased distensibility at low pressures, (3) a leftward shift of the stress‐strain relationship, (4) redistribution of elastin within the internal elastic lamina (IEL) leading to smaller fenestrae but no change in fenestrae number or elastin amount. Elastase incubation (1) fragmented the structure of IEL in a concentration‐dependent fashion, (2) abolished all the structural and mechanical differences between strains, and (3) decreased distensibility at low pressures. The study shows the overriding role of elastin in determining vascular dimensions and mechanical properties in a resistance artery. In addition, it informs hypertensive remodelling. MRA remodelling and increased stiffness are accompanied by elastin restructuring within the IEL and elastin degradation reverses structural and mechanical alterations of SHR MRA. Differences in elastin organisation are, therefore, a central element in small artery remodelling in hypertension.

Metabotropic glutamate receptors (mGluRs) are mandatory dimers playing important roles in regulating CNS function. Although assumed to form exclusive homodimers, 16 possible heterodimeric mGluRs have been proposed but their existence in native cells remains elusive. Here, we set up two assays to specifically identify the pharmacological properties of rat mGlu heterodimers composed of mGlu2 and 4 subunits. We used either a heterodimer-specific conformational LRET-based biosensor or a system that guarantees the cell surface targeting of the heterodimer only. We identified mGlu2-4 specific pharmacological fingerprints that were also observed in a neuronal cell line and in lateral perforant path terminals naturally expressing mGlu2 and mGlu4. These results bring strong evidence for the existence of mGlu2-4 heterodimers in native cells. In addition to reporting a general approach to characterize heterodimeric mGluRs, our study opens new avenues to understanding the pathophysiological roles of mGlu heterodimers.