Levo-tetrahydropalmatine retards the growth of ectopic endometrial implants and alleviates generalized hyperalgesia in experimentally induced endometriosis in rats.

One primary goal of medical treatment of endometriosis is to alleviate pain and there is a pressing need for new therapeutics for endometriosis with better efficacy and side-effect profiles. Levo-tetrahydropalmatine (l-THP) has been used as a sedative or analgesic for chronic pains in China since 1970s. In this study, we sought to evaluate the efficacy of l-THP, with or without valproic acid (VPA), in a rat model of endometriosis. We surgically induced endometriosis in 55 adult female rats. Two weeks after, all rats were further divided into 5 groups randomly: untreated, low- and high-dose of l-THP, VPA, and l-THP + VPA. Response latency in hotplate test was measured before the surgery, before and after 3-week treatment of respective drugs. All rats were then sacrificed for analysis. The average lesion size and the immunoreactivity to N-methyl-D-asparate receptor 1 (NMDAR1), acid-sensing ion channel 3 (ASIC3), calcitonin gene-related peptide (CGRP), c-Fos, tyrosine kinase receptor A (TrkA), and histone deacetylase 2 (HDAC2) in dorsal root ganglia (DRG), to phorphorylated p65, HDAC2, TrkA, and CGRP in ectopic endometrium and to phorphorylated p65 and CGRP in eutopic endometrium were evaluated. We found that rats receiving l-THP, with or without VPA, had significantly reduced lesion size and exhibited significantly improved response to noxious thermal stimulus. The treatment also significantly lowered immunoreactivity to all mediators involved in central sensitization and to HDAC2 in DRG, to TrkA and CGRP in ectopic endometrium, and to CGRP in eutopic endometrium. In summary, l-THP reduces lesion growth and generalized hyperalgesia. Thus, l-THP may be a promising therapeutics for endometriosis.

Effects of water restriction on gene expression in mouse renal medulla: identification of 3betaHSD4 as a collecting duct protein.

To identify novel gene targets of vasopressin regulation in the renal medulla, we performed a cDNA microarray study on the inner medullary tissue of mice following a 48-h water restriction protocol. In this study, 4,625 genes of the possible approximately 12,000 genes on the array were included in the analysis, and of these 157 transcripts were increased and 63 transcripts were decreased by 1.5-fold or more. Quantitative, real-time PCR measurements confirmed the increases seen for 12 selected transcripts, and the decreases were confirmed for 7 transcripts. In addition, we measured transcript abundance for many renal collecting duct proteins that were not represented on the array; aquaporin-2 (AQP2), AQP3, Pax-8, and alpha- and beta-Na-K-ATPase subunits were all significantly increased in abundance; the beta- and gamma-subunits of ENaC and the vasopressin type 1A receptor were significantly decreased. To correlate changes in mRNA expression with changes in protein expression, we carried out quantitative immunoblotting. For most of the genes examined, changes in mRNA abundances were not associated with concomitant protein abundance changes; however, AQP2 transcript abundance and protein abundance did correlate. Surprisingly, aldolase B transcript abundance was increased but protein abundance was decreased following 48 h of water restriction. Several transcripts identified by microarray were novel with respect to their expression in mouse renal medullary tissues. The steroid hormone enzyme 3beta-hydroxysteroid dehydrogenase 4 (3betaHSD4) was identified as a novel target of vasopressin regulation, and via dual labeling immunofluorescence we colocalized the expression of this protein to AQP2-expressing collecting ducts of the kidney. These studies have identified several transcripts whose abundances are regulated in mouse inner medulla in response to an increase in endogenous vasopressin levels and could play roles in the regulation of salt and water excretion.

Increased expression but not targeting of ENaC in adrenalectomized rats with PAN-induced nephrotic syndrome.

Sodium retention is a hallmark of nephrotic syndrome (NS). Puromycin aminonucleoside (PAN)-induced NS is associated with high aldosterone levels and increased ENaC expression and apical targeting. However, the mechanisms associated with increased apical targeting of ENaC in NS remain undefined, and it is unclear whether this is secondary to high aldosterone levels and whether aldosterone and/or apical ENaC targeting are important for the development of sodium retention. This study aimed at uncovering 1) whether aldosterone is essential for sodium retention in PAN-induced NS, 2) whether ENaC expression or apical targeting is secondary to high aldosterone levels, and 3) the role of aldosterone in the dysregulation of sodium transporters in NS. Puromycin treatment of adrenalectomized (ADX) rats supplemented with dexamethasone induced sodium retention despite the absence of aldosterone. Immunocytochemical analyses revealed an absence of enhanced apical targeting of ENaC subunits in PAN-treated ADX (ADX-PAN) rats, with distribution of labeling similar to adrenalectomized dexamethasone-treated control rats (ADX). Moreover, ENaC subunit abundance was increased in ADX-PAN rats. The abundance of aquaporin-2 was unchanged, whereas apical targeting was enhanced. Key sodium transporters were downregulated as previously observed in nonadrenalectomized puromycin-treated rats (Kim SW, Wang W, Nielsen J, Praetorius J, Kwon TH, Knepper MA, Frøkiaer J, and Nielsen S. Am J Physiol Renal Physiol 286: F922-F935, 2004), whereas the global expression of the alpha1-subunit of the Na-K-ATPase was unchanged. In conclusion, PAN treatment in the absence of aldosterone induced sodium retention, increased ENaC expression, but did not change the subcellular distribution of ENaC. This indicates that the previously observed enhanced apical targeting of ENaC in PAN-induced NS (Kim SW, Wang W, Nielsen J, Praetorius J, Kwon TH, Knepper MA, Frøkiaer J, and Nielsen S. Am J Physiol Renal Physiol 286: F922-F935, 2004) is caused by aldosterone and that development of sodium retention can occur in the absence of aldosterone in NS.

Changes in electrophysiological properties and sodium channel Nav1.3 expression in thalamic neurons after spinal cord injury.

Spinal cord contusion injury (SCI) is known to induce pain-related behaviour, as well as hyperresponsiveness in lumbar dorsal horn nociceptive neurons associated with the aberrant expression of Na(v)1.3, a rapidly repriming voltage-gated sodium channel. Many of these second-order dorsal horn neurons project to third-order neurons in the ventrobasal complex of the thalamus. In this study we hypothesized that, following SCI, neurons in the thalamus undergo electrophysiological changes linked to aberrant expression of Na(v)1.3. Adult male Sprague-Dawley rats underwent contusion SCI at the T9 thoracic level. Four weeks post-SCI, Na(v)1.3 protein was upregulated within thalamic neurons in ventroposterior lateral (VPL) and ventroposterior medial nuclei, where extracellular unit recordings revealed increased spontaneous discharge, afterdischarge, hyperresponsiveness to innocuous and noxious peripheral stimuli, and expansion of peripheral receptive fields. Altered electrophysiological properties of VPL neurons persisted after interruption of ascending spinal barrage by spinal cord transection above the level of the injury. Lumbar intrathecal administration of specific antisense oligodeoxynucleotides generated against Na(v)1.3 caused a significant reduction in Na(v)1.3 expression in thalamic neurons and reversed electrophysiological alterations. These results show, for the first time, a change in sodium channel expression within neurons in the thalamus after injury to the spinal cord, and suggest that these changes contribute to altered processing of somatosensory information after SCI.

Sodium transporter abundance profiling in kidney: effect of spironolactone.

Renal tubule profiling studies were carried out to investigate the long-term effects of administration of spironolactone, a mineralocorticoid receptor antagonist, on abundances of the major Na transporter and Na channel proteins along the rat renal tubule. Oral administration of spironolactone for 7 days to NaCl-restricted rats did not significantly alter abundances of Na transporters expressed proximal to the macula densa, while substantially decreasing the abundances of the thiazide-sensitive Na-Cl cotransporter (NCC), the alpha-subunit of the amiloride-sensitive epithelial Na channel (ENaC), and the 70-kDa form of the gamma-subunit of ENaC. A dependency of NCC expression on aldosterone was confirmed by showing increased NCC expression in response to aldosterone infusion in adrenalectomized rats. Immunoperoxidase labeling of ENaC in renal cortex confirmed that dietary NaCl restriction causes a redistribution of ENaC to the apical domain of connecting tubule cells and showed that high-dose spironolactone administration does not block this apical redistribution. In contrast, spironolactone completely blocked the increase in alpha-ENaC abundance in response to dietary NaCl restriction. We conclude that the protein abundances of NCC, alpha-ENaC, and the 70-kDa form of gamma-ENaC are regulated via the classical mineralocorticoid receptor, but the subcellular redistribution of ENaC in response to dietary NaCl restriction is not prevented by blockade of the mineralocorticoid receptor.

Molecular mechanisms underlying ionic remodeling in a dog model of atrial fibrillation.

The rapid atrial rate during atrial fibrillation (AF) decreases the ionic current density of transient outward K+ current, L-type Ca2+ current, and Na+ current, thereby altering cardiac electrophysiology and promoting arrhythmia maintenance. To assess possible underlying changes in cardiac gene expression, we applied competitive reverse transcriptase-polymerase chain reaction to quantify mRNA concentrations in dogs subjected to 7 (group P7 dogs) or 42 (group P42 dogs) days of atrial pacing at 400 bpm and in sham controls. Rapid pacing reduced mRNA concentrations of Kv4.3 (putative gene encoding transient outward K+ current; by 60% in P7 and 74% in P42 dogs; P<0.01 and P<0.001, respectively, versus shams), the alpha1c subunit of L-type Ca2+ channels (by 57% in P7 and 72% in P42 dogs; P<0.01 versus shams for each) and the alpha subunit of cardiac Na+ channels (by 18% in P7 and 42% in P42; P=NS and P<0.01, respectively, versus shams) genes. The observed changes in ion channel mRNA concentrations paralleled previously measured changes in corresponding atrial ionic current densities. Atrial tachycardia did not affect mRNA concentrations of genes encoding delayed or Kir2.1 inward rectifier K+ currents (of which the densities are unchanged by atrial tachycardia) or of the Na+,Ca2+ exchanger. Western blot techniques were used to quantify protein expression for Kv4.3 and Na+ channel alpha subunits, which were decreased by 72% and 47%, respectively, in P42 dogs (P<0.001 versus control for each), in a manner quantitatively similar to measured changes in mRNA and currents, whereas Na+,Ca2+ exchanger protein concentration was unchanged. We conclude that chronic atrial tachycardia alters atrial ion channel gene expression, thereby altering ionic currents in a fashion that promotes the occurrence of AF. These observations provide a potential molecular basis for the self-perpetuating nature of AF.