Roxanne Y. Walder

Diaspirins that cross-link beta chains of hemoglobin: bis(3,5-dibromosalicyl) succinate and bis(3,5-dibromosalicyl) fumarate.

Two double-headed aspirins, bis(3,5-dibromosalicyl) succinate and bis(3,5-dibromosalicyl) fumarate, have been found to be potent acylating agents of intracellular hemoglobin (A or S) in vitro. Furthermore, each of these reagents cross-links beta chains of hemoglobin, probably at the beta cleft. The modified hemoglobins show increased oxygen affinities and reduced gelation or sickling tendencies.

Role of ASIC3 in the primary and secondary hyperalgesia produced by joint inflammation in mice.

&NA; The acid sensing ion channel 3 (ASIC3) is critical for the development of secondary hyperalgesia as measured by mechanical stimulation of the paw following muscle insult. We designed experiments to test whether ASIC3 was necessary for the development of both primary and secondary mechanical hyperalgesia that develops after joint inflammation. We used ASIC3 −/− mice and examined the primary (response to tweezers) and secondary hyperalgesia (von‐Frey filaments) that develops after joint inflammation comparing to ASIC3 +/+ mice. We also examined the localization of ASIC3 to the knee joint afferents innervating the synovium using immunohistochemical techniques before and after joint inflammation. We show that secondary mechanical hyperalgesia does not develop in ASIC3 −/− mice. However, the primary mechanical hyperalgesia of the inflamed knee joint still develops in ASIC3 −/− mice and is similar to ASIC3 +/+ mice. In knee joint synovium from ASIC3 +/+ mice without joint inflammation, ASIC3 was not localized to joint afferents that were stained with an antibody to protein gene product (PGP) 9.5 or calcitonin gene‐related peptide (CGRP). ASIC3 was found, however, in synoviocytes of the knee joint of uninflamed mice. In ASIC3 +/+ mice with joint inflammation, ASIC3 co‐localized with PGP 9.5 or CGRP in joint afferents innervating the synovium. We conclude that the decreased pH that occurs after inflammation would activate ASIC3 on primary afferent fibers innervating the knee joint, increasing the input to the spinal cord resulting in central sensitization manifested behaviorally as secondary hyperalgesia of the paw.

ASIC1 and ASIC3 Play Different Roles in the Development of Hyperalgesia After Inflammatory Muscle Injury

UNLABELLED Acid-sensing ion channels (ASICs) respond to acidosis that normally occurs after inflammation. We examined the expression of ASIC1, ASIC2, and ASIC3 mRNAs in lumbar dorsal root ganglion neurons before and 24 hours after carrageenan-induced muscle inflammation. Muscle inflammation causes bilateral increases of ASIC2 and ASIC3 but not ASIC1 (neither ASIC1a nor ASIC1b) mRNA, suggesting differential regulation of ASIC1 versus ASIC2 and ASIC3 mRNA. Similar mRNA increases were observed after inflammation in knockout mice: ASIC2 mRNA increases in ASIC3-/- mice; ASIC2 and ASIC3 mRNAs increase in ASIC1-/- mice. Prior behavioral studies in ASIC3-/- mice showed deficits in secondary hyperalgesia (increased response to noxious stimuli outside the site of injury) but not primary hyperalgesia (increased response to noxious stimuli at the site of injury). In this study, we show that ASIC1-/- mice do not develop primary muscle hyperalgesia but develop secondary paw hyperalgesia. In contrast, and as expected, ASIC3-/- mice develop primary muscle hyperalgesia but do not develop secondary paw hyperalgesia. The pharmacological utility of the nonselective ASIC inhibitor A-317567, given locally, was tested. A-317567 reverses both the primary and the secondary hyperalgesia induced by carrageenan muscle inflammation. Thus, peripherally located ASIC1 and ASIC3 play different roles in the development of hyperalgesia after muscle inflammation. PERSPECTIVE This study shows changes in ASIC mRNA expression and behavioral hyperalgesia of C57Bl/6 (wild type), ASIC1-/-, and ASIC3-/- mice before and after the induction of muscle inflammation. A-317567 was effective in reversing hyperalgesia in these animals, suggesting the potential of ASICs as therapeutic targets for muscle inflammatory pain.

Development of antisickling compounds that chemically modify hemoglobin S specifically within the 2,3-diphosphoglycerate binding site

Abstract In this paper we describe a class of affinity reagents that react specifically with hemoglobin S at the 2,3-diphosphoglycerate binding site and effectively disrupt the sickling process. The prototype of these compounds is the bifunctional acylating agent bis(3,5-dibromosalicyl) fumarate. Previously it was shown that this compound reacts selectively with oxyhemoglobin to cross-link the β chains of the tetramer ( Walder et al. , 1979 a ). The site of cross-linking has now been established by X-ray crystallographic studies to be from Lys82 β 1 to Lys82 β 2 , spanning the DPG ‡ binding site. The oxygen binding properties of the cross-linked hemoglobin are very similar to those of native hemoglobin A in the absence of DPG. However, in the presence of DPG, the oxygen affinity of the cross-linked hemoglobin is increased due to the blockade of the DPG site by the interchain bridge. This effect may indirectly interfere with the sickling process in vivo . More importantly the cross-link modification directly inhibits the polymerization of deoxyhemoglobin S. The solubility of the cross-linked derivative is increased by approximately 35% relative to that of native deoxyhemoglobin S. The X-ray crystallographic studies reveal that the cross-link strongly perturbs the acceptor site for Val6β in the lateral contact between molecules of deoxyhemoglobin S within the double strand of the sickle cell fiber. Our current hypothesis is that this structural perturbation weakens the lateral contact and is responsible for the observed increase in solubility. Due to the hydrophobic properties of the halogen substituents bis(3,5-dibromosalicyl) fumarate is able to traverse the red-cell membrane and, therefore, may be active in vivo . These studies provide the basis for further design and development of affinity reagents directed to the DPG binding site that may prove useful in the clinical management of sickle cell disease.

Mice defective in Trpm6 show embryonic mortality and neural tube defects

The syndrome of hypomagnesemia with secondary hypocalcemia is caused by defective TRPM6. This protein is an ion channel that also contains a kinase in its C-terminus. It is usually diagnosed in childhood and, without treatment with supplemental Mg, affected children suffer from mental retardation, seizures and retarded development. We developed a mouse lacking Trpm6 in order to understand in greater detail the function of this protein. In contrast to our expectations, Trpm6(-/-) mice almost never survived to weaning. Many mice died by embryonic day 12.5. Most that survived to term had neural tube defects consisting of both exencephaly and spina bifida occulta, an unusual combination. Feeding dams a high Mg diet marginally improved offspring survival to weaning. The few Trpm6(-/-) mice that survived were fertile but matings between Trpm6(-/-) mice produced no viable pregnancies. Trpm6(+/-) mice had normal electrolytes except for modestly low plasma [Mg]. In addition, some Trpm6(+/-) mice died prematurely. Absence of Trpm6 produces an apparently different phenotype in mice than in humans. The presence of neural tube defects identifies a previously unsuspected role of Trpm6 in effecting neural tube closure. This genetic defect produces one of very few mouse models of spina bifida occulta. These results point to a critical role of Trpm6 in development and suggest an important role in neural tube closure.

Linkage of infantile Bartter syndrome with sensorineural deafness to chromosome 1p.

Bartter syndrome (BS) is a family of disorders manifested by hypokalemic hypochloremic metabolic alkalosis with normotensive hyperreninemic hyperaldosteronism. We evaluated a unique, inbred Bedouin kindred in which sensorineural deafness (SND) cosegregates with an infantile variant of the BS phenotype. Using a DNA-pooling strategy, we screened the human genome and successfully demonstrated linkage of this unique syndrome to chromosome 1p31. The genes for two kidney-specific chloride channels and a sodium/hydrogen antiporter, located near this region, were excluded as candidate genes. Although the search for the disease-causing gene in this family continues, this linkage further demonstrates the genetic heterogeneity of BS. In addition, the cosegregation of these phenotypes allows us to postulate that a single genetic alteration may be responsible for the SND and the BS phenotype. The identification and characterization of this gene would lead to a better understanding of the normal physiology of the kidney and the inner ear.

Selective targeting of ASIC3 using artificial miRNAs inhibits primary and secondary hyperalgesia after muscle inflammation.

Summary Artificial miRNAs targeting mouse acid‐sensing ion channel 3 (ASIC3) reduce pH sensitivity of heteromeric ASIC1/3 channels, ASIC3 protein, and mRNA expression, and prevent the development of hyperalgesia after muscle inflammation. Abstract Acid‐sensing ion channels (ASICs) are activated by acidic pH and may play a significant role in the development of hyperalgesia. Earlier studies show ASIC3 is important for induction of hyperalgesia after muscle insult using ASIC3−/− mice. ASIC3−/− mice lack ASIC3 throughout the body, and the distribution and composition of ASICs could be different from wild‐type mice. We therefore tested whether knockdown of ASIC3 in primary afferents innervating muscle of adult wild‐type mice prevented development of hyperalgesia to muscle inflammation. We cloned and characterized artificial miRNAs (miR‐ASIC3) directed against mouse ASIC3 (mASIC3) to downregulate ASIC3 expression in vitro and in vivo. In CHO‐K1 cells transfected with mASIC3 cDNA in culture, the miR‐ASIC3 constructs inhibited protein expression of mASIC3 and acidic pH‐evoked currents and had no effect on protein expression or acidic pH‐evoked currents of ASIC1a. When miR‐ASIC3 was used in vivo, delivered into the muscle of mice using a herpes simplex viral vector, both muscle and paw mechanical hyperalgesia were reduced after carrageenan‐induced muscle inflammation. ASIC3 mRNA in DRG and protein levels in muscle were decreased in vivo by miR‐ASIC3. In CHO‐K1 cells co‐transfected with ASIC1a and ASIC3, miR‐ASIC3 reduced the amplitude of acidic pH‐evoked currents, suggesting an overall inhibition in the surface expression of heteromeric ASIC3‐containing channels. Our results show, for the first time, that reducing ASIC3 in vivo in primary afferent fibers innervating muscle prevents the development of inflammatory hyperalgesia in wild‐type mice, and thus, may have applications in the treatment of musculoskeletal pain in humans.

Blood pressure and survival of a Chromosome 7 congenic strain bred from Dahl rats

Abstract11 β-hydroxylase (Cyp11b1) mutations were previously linked to altered steroid biosynthesis and blood pressure in Dahl salt-resistant (R) and Dahl salt-sensitive (S) rats. In the present work, interval mapping identified a putative blood pressure quantitative trait locus (QTL) near Cyp11b1 in an F1(SxR)xS population (LOD = 2.0). Congenic rats (designated S.R-Cyp11b) were constructed by introgressing the R-rat Cyp11b1 allele into the S strain. S.R-Cyp11b rats had significantly lower blood pressure and heart weight compared with S rats, proving the existence of a blood pressure QTL on Chromosome (Chr) 7 despite the fact that QTL linkage analysis of blood pressure never achieved stringent statistical criteria for significance. To test the effects of the introgressed region on blood pressure and survival, S.R.-Cyp11b and S rats were maintained on a 4% NaC1 diet until they died or became moribund. Analysis of variance (ANOVA) indicated significant strain differences in blood pressure and days survived (P < 0.0001 for both) as well as gender differences in days survived (P = 0.0003). Kaplan-Meier survival analysis also found significant strain (P < 0.0001) and gender (P = 0.007) differences in days survived. However, when the effects of blood pressure were removed, significant strain differences in survival essentially disappeared. This suggests that the increased survival of S.R-Cyp11b rats was largely due to their decreased blood pressure and thus strongly corroborates the existence of a blood pressure QTL on Chr 7 near or at Cyp11b1.

Acid-sensing ion channel 3 expressed in type B synoviocytes and chondrocytes modulates hyaluronan expression and release

Background Rheumatoid arthritis is an inflammatory disease marked by intra-articular decreases in pH, aberrant hyaluronan regulation and destruction of bone and cartilage. Acid-sensing ion channels (ASICs) are the primary acid sensors in the nervous system, particularly in sensory neurons and are important in nociception. ASIC3 was recently discovered in synoviocytes, non-neuronal joint cells critical to the inflammatory process. Objectives To investigate the role of ASIC3 in joint tissue, specifically the relationship between ASIC3 and hyaluronan and the response to decreased pH. Methods Histochemical methods were used to compare morphology, hyaluronan expression and ASIC3 expression in ASIC3+/+ and ASIC3−/− mouse knee joints. Isolated fibroblast-like synoviocytes (FLS) were used to examine hyaluronan release and intracellular calcium in response to decreases in pH. Results In tissue sections from ASIC3+/+ mice, ASIC3 localised to articular cartilage, growth plate, meniscus and type B synoviocytes. In cultured FLS, ASIC3 mRNA and protein was also expressed. In FLS cultures, pH 5.5 increased hyaluronan release in ASIC3+/+ FLS, but not ASIC3−/− FLS. In FLS from ASIC3+/+ mice, approximately 50% of cells (25/53) increased intracellular calcium while only 24% (14/59) showed an increase in ASIC3−/− FLS. Of the cells that responded to pH 5.5, there was significantly less intracellular calcium increases in ASIC3−/− FLS compared to ASIC3+/+ FLS. Conclusion ASIC3 may serve as a pH sensor in synoviocytes and be important for modulation of expression of hyaluronan within joint tissue.

Changes in expression of NMDA-NR1 receptor subunits in the rostral ventromedial medulla modulate pain behaviors.

&NA; NMDA receptors have an important role in pain facilitation in rostral ventromedial medulla (RVM) and the NR1 subunit is essential for its function. Studies suggest that the NMDA receptors in RVM are critical to modulate both cutaneous and muscle hypersensitivity induced by repeated intramuscular acid injections. We propose that increased expression of the NR1 subunit in the RVM is critical for the full development of hypersensitivity. To test this we used recombinant lentiviruses to over‐express the NR1 subunit in the RVM and measured nociceptive sensitivity to cutaneous and muscle stimuli. We also downregulated the expression of NR1 in the RVM and measured the hyperalgesia produced by repeated‐acid injections. Increasing the expression of NR1 in the RVM reduces cutaneous and muscle withdrawal threshold, and decreasing the expression of NR1 in the RVM increases the muscle withdrawal threshold and prevents the development of hyperalgesia in an animal model of muscle pain. These results suggest that the NR1 subunits in the RVM are critical for modulating NMDA receptor function, which in turn sets the ‘tone’ of the nervous systems response to noxious stimuli and tissue injury.