Urokinase-type Plasminogen Activator Is a Therapeutic Target for Overcoming Sorafenib Resistance in Hepatoma Cells.

BACKGROUND/AIM: Sorafenib is a multikinase inhibitor approved as a first-line therapy for hepatocellular carcinoma. This study examined the sorafenib resistance mechanism. MATERIALS AND METHODS: Hepatoma HepG2 cells were exposed to sorafenib, and the biological activity of the conditioned media was analyzed using cell proliferation/apoptosis assays, multiplex immunoassays, ELISA, and western blot analyses. The effect of urokinase-type plasminogen activator (uPA) inhibitors or siRNA-mediated gene silencing was examined in culture experiments and a mouse xenograft tumor model. RESULTS: Sorafenib increased uPA secretion, which was abrogated by an Akt inhibitor. The growth-inhibitory effect of sorafenib was significantly enhanced by the uPA inhibitors UK122 and amiloride. Sorafenib-induced apoptosis was increased 2.4-fold in uPA siRNA-transduced cells (p<0.05). Combined therapy with sorafenib and amiloride significantly decreased tumor volumes [mean volume: 759 mm3 (sorafenib) vs. 283 mm3 (sorafenib plus amiloride), p<0.05]. CONCLUSION: uPA may play a critical role in sorafenib resistance.

Antinociceptive activity of 3ß-6ß-16ß-trihydroxylup-20 (29)-ene triterpene isolated from Combretum leprosum leaves in adult zebrafish (Danio rerio).

Drugs used to treat pain are associated with adverse effects, increasing the search for new drugs as an alternative treatment for pain. Therefore, we evaluated the antinociceptive behavior and possible neuromodulation mechanisms of triterpene 3ß, 6ß, 16ß-trihydroxylup-20(29)-ene (CLF-1) isolated from Combretum leprosum leaves in zebrafish. Zebrafish (n = 6/group) were pretreated with CLF-1 (0.1 or 0.3 or 1.0 mg/mL; i.p.) and underwent nociception behavior tests. The antinociceptive effect of CFL-1 was tested for modulation by opioid (naloxone), nitrergic (L-NAME), nitric oxide and guanylate cyclase synthesis inhibitor (methylene blue), NMDA (Ketamine), TRPV1 (ruthenium red), TRPA1 (camphor), or ASIC (amiloride) antagonists. The corneal antinociceptive effect of CFL-1 was tested for modulation by TRPV1 (capsazepine). The effect of CFL-1 on zebrafish locomotor behavior was evaluated with the open field test. The acute toxicity study was conducted. CLF-1 reduced nociceptive behavior and corneal in zebrafish without mortalities and without altering the animals' locomotion. Thus, CFL-1 presenting pharmacological potential for the treatment of acute pain and corneal pain, and this effect is modulated by the opioids, nitrergic system, NMDA receptors and TRP and ASIC channels.

Amiloride Alleviates Neurological Deficits Following Transient Global Ischemia and Engagement of Central IL-6 and TNF-α Signal.

BACKGROUND: Central pro-inflammatory cytokine (PIC) signal is involved in neurological deficits after transient global ischemia induced by cardiac arrest (CA). The present study was to examine if blocking acid sensing ion channels (ASICs) using amiloride in the Central Nervous System can alleviate neurological deficits after the induction of CA and further examine the participation of PIC signal in the hippocampus for the effects of amiloride. METHODS: CA was induced by asphyxia and then cardiopulmonary resuscitation was performed in rats. Western blot analysis and ELISA were used to determine the protein expression of ASIC subunit ASIC1 in the hippocampus, and the levels of PICs. As noted, it is unlikely that this procedure is clinically used although amiloride and other pharmacological agents were given into the brain in this study. RESULTS: CA increased ASIC1 in the hippocampus of rats in comparison with control animals. This was associated with the increase in IL-1ß, IL-6 and TNF-α together with Caspase-3 and Caspase-9. The administration of amiloride into the lateral ventricle attenuated the upregulation of Caspase-3/Caspase-9 and this further alleviated neurological severity score and brain edema. Inhibition of central IL-6 and TNF-α also decreased ASIC1 in the hippocampus of CA rats. CONCLUSION: Transient global ischemia induced by CA amplifies ASIC1a in the hippocampus likely via PIC signal. Amiloride administered into the Central Nervous System plays a neuroprotective role in the process of global ischemia. Thus, targeting ASICs (i.e., ASIC1a) is suggested for the treatment and improvement of CA-evoked global cerebral ischemia.

Gallic acid, a phenolic compound isolated from Mimosa bimucronata (DC.) Kuntze leaves, induces diuresis and saluresis in rats.

Although present in the leaves of Mimosa bimucronata (DC.) and many other medicinal plants commonly used to augment urinary volume excretion, the effects of gallic acid as a diuretic agent remain to be studied. Wistar rats were orally treated with vehicle, hydrochlorothiazide, or gallic acid. The effects of gallic acid in the presence of hydrochlorothiazide, furosemide, amiloride, L-NAME, atropine, and indomethacin were also investigated. Diuretic index, pH, conductivity, and electrolyte excretion were evaluated at the end of the experiment (after 8 or 24 h). Gallic acid induced diuretic and saluretic (Na+ and Cl-) effects, without interfering with K+ excretion, when orally given to female and male rats at a dose of 3 mg/kg. These effects were associated with increased creatinine and conductivity values while pH was unaffected by any of the treatments. Plasma Na+, K+, and Cl- levels were not affected by any of the acute treatments. The combination with hydrochlorothiazide or furosemide was unable to intensify the effects of gallic acid when compared with the response obtained with each drug alone. On the other hand, the treatment with amiloride plus gallic acid amplified both diuresis and saluresis, besides to a marked potassium-sparing effect. Its diuretic action was significantly prevented in the presence of indomethacin, a cyclooxygenase inhibitor, but not with the pretreatments with L-NAME or atropine. Although several biological activities have already been described for gallic acid, this is the first study demonstrating its potential as a diuretic agent.

Essential role of Kir5.1 channels in renal salt handling and blood pressure control.

Supplementing diets with high potassium helps reduce hypertension in humans. Inwardly rectifying K+ channels Kir4.1 (Kcnj10) and Kir5.1 (Kcnj16) are highly expressed in the basolateral membrane of distal renal tubules and contribute to Na+ reabsorption and K+ secretion through the direct control of transepithelial voltage. To define the importance of Kir5.1 in blood pressure control under conditions of salt-induced hypertension, we generated a Kcnj16 knockout in Dahl salt-sensitive (SS) rats (SSKcnj16-/-). SSKcnj16-/- rats exhibited hypokalemia and reduced blood pressure, and when fed a high-salt diet (4% NaCl), experienced 100% mortality within a few days triggered by salt wasting and severe hypokalemia. Electrophysiological recordings of basolateral K+ channels in the collecting ducts isolated from SSKcnj16-/- rats revealed activity of only homomeric Kir4.1 channels. Kir4.1 expression was upregulated in SSKcnj16-/- rats, but the protein was predominantly localized in the cytosol in SSKcnj16-/- rats. Benzamil, but not hydrochlorothiazide or furosemide, rescued this phenotype from mortality on a high-salt diet. Supplementation of high-salt diet with increased potassium (2% KCl) prevented mortality in SSKcnj16-/- rats and prevented or mitigated hypertension in SSKcnj16-/- or control SS rats, respectively. Our results demonstrate that Kir5.1 channels are key regulators of renal salt handling in SS hypertension.

Na+/H+ Exchanger Regulates Amino Acid-Mediated Autophagy in Intestinal Epithelial Cells.

BACKGROUND/AIMS: Dysfunctional autophagy has been reported to be associated with aberrant intestinal metabolism. Amino acids can regulate autophagic activity in intestinal epithelial cells (IECs). Na+/H+-exchanger 3 (NHE3) has been found to participate in the absorption of amino acids in the intestine, but whether NHE3 is involved in the regulation of autophagy in IECs is unclear. METHODS: In the present study, an amino acid starvation-induced autophagic model was established. Then, the effects of alanine and proline with or without the NHE inhibitor 5-(N-ethyl-N-isopropyl) amiloride (EIPA) were evaluated. Autophagy was examined based on the microtubule-associated light chain 3 (LC3) levels, transmission electron microscopy (TEM), tandem GFP-mCherry-LC3 construct, sequestosome-1 (SQSTM1, P62) mRNA and protein levels, and autophagy-related gene (ATG) 5, 7, and 12 expression levels. The autophagic flux was evaluated as the ratio of yellow (autophagosomes) to red (autolysosomes) LC3 puncta. RESULTS: Following amino acid starvation, we found the LC3-II and ATG expression levels were enhanced in the IEC-18 cells. An increase in the number of autophagic vacuoles was concomitantly observed by TEM and confocal microscopy. Based on the results, supplementation with either alanine or proline depressed autophagy in the IEC-18 cells. Consistent with the elevated LC3-II levels, ATG expression increased upon NHE3 inhibition. Moreover, the mCherry-GFP-LC3 autophagic puncta representing both autophagosomes and autolysosomes per cell increased after EIPA treatment. CONCLUSIONS: These results demonstrate that NHE (most likely NHE3) may participate in the amino acid regulation of autophagy in IECs, which would aid in the design of better treatments for intestinal inflammation.

Amiloride Is Effective in the Management of Abiraterone-Induced Mineralocorticoid Excess Syndrome without Interfering with Its Antineoplastic Activity.

BACKGROUND: The administration of abiraterone acetate (abiraterone) leads to an adrenocorticotropic hormone (ACTH)-driven increase in mineralocorticoid hormones, requiring glucocorticoid supplementation that may stimulate the growth of prostate cancer (PCa). Amiloride is a drug that selectively reduces the aldosterone-sensitive Na+/K+ exchange and could be effective in the management of mineralocorticoid excess syndrome (MCES). METHODS: The efficacy of amiloride + hydrochlorothiazide (HCT) in the clinical management of abiraterone-induced MCES was assessed in 5 consecutive patients with castration-resistant PCa (CRPC). Then, using the in vitro experimental model of PCa cell lines, the possible effects of drugs usually used in the clinical management of CRPC patients on PCa cell viability were investigated. RESULTS: Amiloride/HCT led to a complete disappearance of all clinical and biochemical signs of abiraterone-induced MCES in the 5 treated patients. The in vitro study showed that abiraterone treatment significantly decreased cell viability of both androgen receptor (AR)-expressing VCaP (vertebral-cancer of the prostate) and LNCaP (lymph node carcinoma of the prostate) cells, with no effect on AR-negative PC-3 cells. Prednisolone, spironolactone, and eplerenone increased LNCaP cell viability, while amiloride reduced it. The non-steroid aldosterone antagonist PF-03882845 did not modify PCa cell viability. CONCLUSIONS: The combination of amiloride/HCT was effective in the management of abiraterone-induced MCES. Amiloride did not negatively interfere with the abiraterone inhibition of PCa cell viability in vitro.

Feeding through your gills and turning a toxicant into a resource: how the dogfish shark scavenges ammonia from its environment.

Nitrogen (N) appears to be a limiting dietary resource for elasmobranchs, required not only for protein growth but also for urea-based osmoregulation. Building on recent evidence that the toxicant ammonia can be taken up actively at the gills of the shark and made into the valuable osmolyte urea, we demonstrate that the uptake exhibits classic Michaelis-Menten saturation kinetics with an affinity constant (Km) of 379 µmol l-1, resulting in net N retention at environmentally realistic ammonia concentrations (100-400 µmol l-1) and net N loss through stimulated urea-N excretion at higher levels. Ammonia-N uptake rate increased or decreased with alterations in seawater pH, but the changes were much less than predicted by the associated changes in seawater PNH3 , and more closely paralleled changes in seawater NH4+ concentration. Ammonia-N uptake rate was insensitive to amiloride (0.1 mmol l-1) or to a 10-fold elevation in seawater K+ concentration (to 100 mmol l-1), suggesting that the mechanism does not directly involve Na+ or K+ transporters, but was inhibited by blockade of glutamine synthetase, the enzyme that traps ammonia-N to fuel the ornithine-urea cycle. High seawater ammonia inhibited uptake of the ammonia analogue [14C]methylamine. The results suggest that branchial ammonia-N uptake may significantly supplement dietary N intake, amounting to about 31% of the nitrogen acquired from the diet. They further indicate the involvement of Rh glycoproteins (ammonia channels), which are expressed in dogfish gills, in normal ammonia-N uptake and retention.

Combination therapy with cystic fibrosis transmembrane conductance regulator modulators augment the airway functional microanatomy.

Recently approved therapies that modulate CFTR function have shown significant clinical benefit, but recent investigations regarding their molecular mechanism when used in combination have not been consistent with clinical results. We employed micro-optical coherence tomography as a novel means to assess the mechanism of action of CFTR modulators, focusing on the effects on mucociliary clearance. Primary human airway monolayers from patients with a G551D mutation responded to ivacaftor treatment with increased ion transport, airway surface liquid depth, ciliary beat frequency, and mucociliary transport rate, in addition to decreased effective viscosity of the mucus layer, a unique mechanism established by our findings. These endpoints are consistent with the benefit observed in G551D patients treated with ivacaftor, and identify a novel mechanism involving mucus viscosity. In monolayers derived from F508del patients, the situation is more complicated, compounded by disparate effects on CFTR expression and function. However, by combining ion transport measurements with functional imaging, we establish a crucial link between in vitro data and clinical benefit, a finding not explained by ion transport studies alone. We establish that F508del cells exhibit increased mucociliary transport and decreased mucus effective viscosity, but only when ivacaftor is added to the regimen. We further show that improvement in the functional microanatomy in vitro corresponds with lung function benefit observed in the clinical trials, whereas ion transport in vitro corresponds to changes in sweat chloride. Functional imaging reveals insights into clinical efficacy and CFTR biology that significantly impact our understanding of novel therapies.

Regulating effect of TongXie-YaoFang on colonic epithelial secretion via Cl- and HCO3- channel.

AIM: To investigate the pharmacological effect of TongXie-YaoFang (TXYF) formula, a Chinese herbal formula, on Diarrhea-predominant irritable bowel syndrome (D-IBS) rats. METHODS: In a neonatal maternal separation plus restraint stress (NMS + RS) model of D-IBS, male Sprague Dawley rats were randomly divided into two groups (NMS + RS group and TXYF-formula group) with no handlings were used as controls (NH group). Starting from postnatal day 60, rats in TXYF-formula group were administered TXYF-formula (4.92 g/100 g bodyweight) orally twice a day for 14 consecutive days while NH group and NMS + RS group were given distilled water. Using short-circuit current technology, we observed 5-HT-induced changes of current across ion channels, such as cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel, epithelial Na+ channel (ENaC), Ca2+-dependent Cl- channel (CACC), Na+-K+-2Cl- co-transporter (NKCC), and Na+-HCO3- co-transporter (NBC), in the colonic epithelium of three groups after exposure to drugs and specific blockers with a Power Lab System (AD Instruments International). RESULTS: Under basal conditions, the changes of short-circuit current (∆Isc, µA/cm2) induced by 5-HT were similar in NH group and TXYF-formula group, and both higher than NMS + RS group (70.86 µA/cm2 ± 12.32 µA/cm2, 67.67 µA/cm2 ± 11.68 µA/cm2vs 38.8 µA/cm2 ± 7.25 µA/cm2, P < 0.01, respectively). When CACC was blocked by 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid, 5-HT-induced ∆Isc was smaller in NMS + RS group than in NH group and TXYF-formula group, respectively (48.41 µA/cm2 ± 13.15 µA/cm2vs 74.62 µA/cm2 ± 10.73 µA/cm2, 69.22 µA/cm2 ± 11.7 µA/cm2, P < 0.05, respectively). The similar result could be obtained when ENaC was blocked by Amiloride (44.69 µA/cm2 ± 12.58 µA/cm2vs 62.05 µA/cm2 ± 11.26 µA/cm2, 62.11 µA/cm2 ± 12.01 µA/cm2, P < 0.05, respectively). However, when CFTR Cl- channel was blocked by 1,1-dimethyl piperidinium chloride (DPC), 5-HT-induced ∆Isc did not significantly differ in three groups (42.28 µA/cm2 ± 10.61 µA/cm2vs 51.48 µA/cm2 ± 6.56 µA/cm2vs 47.75 µA/cm2 ± 7.99 µA/cm2, P > 0.05, respectively). The similar results could also be obtained in three groups when NBC and NKCC were respectively blocked by their blockers. CONCLUSION: TXYF-formula can regulate the Cl- and HCO3- secretion of colonic mucosa via CFTR Cl- channel, Cl-/HCO3- exchanger, NBC and NKCC co-transporters.

Indomethacin, amiloride, or eplerenone for treating hypokalemia in Gitelman syndrome.

Patients with Gitelman syndrome (GS), an inherited salt-losing tubulopathy, are usually treated with potassium-sparing diuretics or nonsteroidal anti-inflammatory drugs and oral potassium and magnesium supplementations. However, evidence supporting these treatment options is limited to case series studies. We designed an open-label, randomized, crossover study with blind end point evaluation to compare the efficacy and safety of 6-week treatments with one time daily 75 mg slow-release indomethacin, 150 mg eplerenone, or 20 mg amiloride added to constant potassium and magnesium supplementation in 30 patients with GS (individual participation: 48 weeks). Baseline plasma potassium concentration was 2.8±0.4 mmol/L and increased by 0.38 mmol/L (95% confidence interval [95% CI], 0.23 to 0.53; P<0.001) with indomethacin, 0.15 mmol/L (95% CI, 0.02 to 0.29; P=0.03) with eplerenone, and 0.19 mmol/L (95% CI, 0.05 to 0.33; P<0.01) with amiloride. Fifteen patients became normokalemic: six with indomethacin, three with eplerenone, and six with amiloride. Indomethacin significantly reduced eGFR and plasma renin concentration. Eplerenone and amiloride each increased plasma aldosterone by 3-fold and renin concentration slightly but did not significantly change eGFR. BP did not significantly change. Eight patients discontinued treatment early because of gastrointestinal intolerance to indomethacin (six patients) and hypotension with eplerenone (two patients). In conclusion, each drug increases plasma potassium concentration in patients with GS. Indomethacin was the most effective but can cause gastrointestinal intolerance and decreased eGFR. Amiloride and eplerenone have similar but lower efficacies and increase sodium depletion. The benefit/risk ratio of each drug should be carefully evaluated for each patient.

Cardiac sympathetic afferent stimulation induces salt-sensitive sympathoexcitation through hypothalamic epithelial Na+ channel activation.

The cardiac sympathetic afferent (CSA), which plays an important role in heart-brain communication for sympathoexcitation, is stimulated in heart failure. Additionally, high salt intake leads to further sympathoexcitation due to activation of hypothalamic epithelial Na(+) channels (ENaCs) in heart failure. In the present study, we stimulated the CSA in adult male mice by epicardial application of capsaicin and using ethanol as a control to determine whether CSA stimulation led to activation of hypothalamic ENaCs, resulting in salt-induced sympathoexcitation. Three days after capsaicin treatment, an upregulation of hypothalamic α-ENaCs, without activation of mineralocorticoid receptors, was observed. We also examined expression levels of the known ENaC activator TNF-α. Hypothalamic TNF-α increased in capsaicin-treated mice, whereas intracerebroventricular infusion of the TNF-α blocker etanercept prevented capsaicin-induced upregulation of α-ENaCs. To examine brain arterial pressure (AP) sensitivity toward Na(+), we performed an intracerebroventricular infusion of high Na(+)-containing (0.2 M) artificial cerebrospinal fluid. AP and heart rate were significantly increased in capsaicin-treated mice compared with control mice. CSA stimulation also caused excitatory responses with high salt intake. Compared with a regular salt diet, the high-salt diet augmented AP, heart rate, and 24-h urinary norepinephrine excretion, which is an indirect marker of sympathetic activity with mineralocorticoid receptor activation, in capsaicin-treated mice but not in ethanol-treated mice. Treatment with etanercept or the ENaC blocker benzamil prevented these salt-induced excitatory responses. In summary, we show that CSA stimulation leads to an upregulation of hypothalamic α-ENaCs mediated via an increase in TNF-α and results in increased salt sensitivity.

A minireview on NHE1 inhibitors. A rediscovered hope in oncohematology.

BACKGROUND: Na(+)/H(+) exchanger-1 (NHE-1) is involved in pH regulation and is up-regulated in different malignancies. Activation of NHE-1 is one way for allowing cells to avoid intracellular acidification and protect them against apoptosis. Inhibitors of NHE-1 are able to decrease intracellular pH and induce apoptosis. Some statins can also act by partial inhibition of NHE-1. This review presents progress in understanding the mechanisms of action of these inhibitors, connections with certain genetic mutations and acquired treatment resistance, as well as new patents on them. METHODS: A MEDLINE search for original and review articles using key terms, Na(+)/H(+) exchanger, leukemia, cariporide, and amiloride. Recent patents with NHE-1 inhibitors published by United States Patent and Trademark Office are also presented. RESULTS AND CONCLUSIONS: Sorafenib is used for the treatment of acute myeloid leukemia patients carrying internal tandem duplication of fms-like tyrosine kinase 3 (FLT3-ITD) mutation. 5-(N, N-hexamethylene)-amiloride can increase the suppression of FLT3 signaling by sorafenib. NHE-1 inhibitors are able to increase the sensitivity of chronic myeloid leukemia cells to tyrosine kinase inhibitors, including through the inhibition of P-glycoprotein. NHE-1 inhibitors are promising adjuvant drugs for overcoming acquired resistance to treatment in various malignant hemopathies.

Sodium-hydrogen exchanger inhibitory potential of Malus domestica, Musa × paradisiaca, Daucus carota, and Symphytum officinale.

BACKGROUND: The involvement of sodium-hydrogen exchangers (NHE) has been described in the pathophysiology of diseases including ischemic heart and brain diseases, cardiomyopathy, congestive heart failure, epilepsy, dementia, and neuropathic pain. Synthetic NHE inhibitors have not achieved much clinical success; therefore, plant-derived phytoconstituents may be explored as NHE inhibitors. METHODS: In the present study, the NHE inhibitory potential of hydroalcoholic and alkaloidal fractions of Malus domestica, Musa × paradisiaca, Daucus carota, and Symphytum officinale was evaluated. The different concentrations of hydroalcoholic and alkaloidal extracts of the selected plants were evaluated for their NHE inhibitory activity in the platelets using the optical swelling assay. RESULTS: Among the hydroalcoholic extracts, the highest NHE inhibitory activity was shown by M. domestica (IC50=2.350 ± 0.132 µg/mL) followed by Musa × paradisiaca (IC50=7.967 ± 0.451 µg/mL), D. carota (IC50=37.667 ± 2.517 µg/mL), and S. officinale (IC50=249.330 ± 1.155 µg/mL). Among the alkaloidal fractions, the highest NHE inhibitory activity was shown by the alkaloidal fraction of Musa × paradisiacal (IC50=0.010 ± 0.001 µg/mL) followed by D. carota (IC50=0.024 ± 0.002 µg/mL), M. domestica (IC50=0.031 ± 0.005 µg/mL), and S. officinale (IC50=4.233 ± 0.379 µg/mL). The IC50 of alkaloidal fractions was comparable to the IC50 of synthetic NHE inhibitor, EIPA [5-(N-ethyl-N-isopropyl)amiloride] (IC50=0.033 ± 0.004 µg/mL). CONCLUSIONS: It may be concluded that the alkaloidal fractions of these plants possess potent NHE inhibitory activity and may be exploited for their therapeutic potential in NHE activation-related pathological complications.

Acid sensing ion channel 1 in lateral hypothalamus contributes to breathing control.

Acid-sensing ion channels (ASICs) are present in neurons and may contribute to chemoreception. Among six subunits of ASICs, ASIC1 is mainly expressed in the central nervous system. Recently, multiple sites in the brain including the lateral hypothalamus (LH) have been found to be sensitive to extracellular acidification. Since LH contains orexin neurons and innervates the medulla respiratory center, we hypothesize that ASIC1 is expressed on the orexin neuron and contributes to acid-induced increase in respiratory drive. To test this hypothesis, we used double immunofluorescence to determine whether ASIC1 is expressed on orexin neurons in the LH, and assessed integrated phrenic nerve discharge (iPND) in intact rats in response to acidification of the LH. We found that ASIC1 was co-localized with orexinA in the LH. Microinjection of acidified artificial cerebrospinal fluid increased the amplitude of iPND by 70% (pH 7.4 v.s. pH 6.5:1.05±0.12 v.s. 1.70±0.10, n = 6, P<0.001) and increased the respiratory drive (peak amplitude of iPND/inspiratory time, PA/Ti) by 40% (1.10±0.23 v.s. 1.50±0.38, P<0.05). This stimulatory effect was abolished by blocking ASIC1 with a nonselective inhibitor (amiloride 10 mM), a selective inhibitor (PcTX1, 10 nM) or by damaging orexin neurons in the LH. Current results support our hypothesis that the orexin neuron in the LH can exert an excitation on respiration via ASIC1 during local acidosis. Since central acidification is involved in breathing dysfunction in a variety of pulmonary diseases, understanding its underlying mechanism may improve patient management.

Pharmacological characterization of the diuretic effect of Hibiscus sabdariffa Linn (Malvaceae) extract.

ETHNOPHARMACOLOGICAL RELEVANCE: Hibiscus sabdariffa L. (Malvaceae) populary known in Mexico as "Jamaica", "flor de Jamaica", has widely used in Mexican Traditional Medicine as antihypertensive and diuretic, although the latter activity has been reported the present work show evidence about the diuretic, natriuretic and potassium-sparing effects. AIM OF THE STUDY: To evaluate the diuretic activity of Hibiscus sabdariffa aqueous extract on in vivo and in situ models. MATERIALS AND METHODS: The Hibiscus sabdariffa aqueous extract was administrated in increasing doses and evaluated the diuresis produced and disposal of electrolytes. Moreover, in isolated kidney was determined the renal filtration rate with plant extract, furosemide and amiloride. RESULTS: The yield of Hibiscus sabdariffa aqueous extraction was 28.3% and the chemical standardization from 1 g of extract was: 56.5 mg delphinidin-3-O-sambubioside, 20.8 mg/g cyanidin-3-O-sambubioside, 3.2 mg/g quercetin, 2.1 mg/g rutin and 2.7 mg/g chlorogenic acid. The diuretic and natriuretic effect of Hibiscus sabdariffa aqueous extract showed a dose-dependent behavior. The pharmacological constants of natriuretic effect was ED50=86 mg/kg and Emax=0.9 mEq/100 g/5 h. In the model of kidney in situ was observed that renal filtration increased 48% with the aqueous extract of Hibiscus sabdariffa and an additive effect when was perfuse with furosemide. CONCLUSION: The compound presents in Hibiscus sabdariffa as quercetin had effect on the vascular endothelium causing oxide nitric release, increasing renal vasorelaxation by increasing kidney filtration. Therefore, the diuretic effect of Hibiscus sabdariffa may be mediated by nitric oxide release.

Hypericum perforatum differentially affects corticosteroid receptor-mRNA expression in human monocytic U-937 cells.

A dysregulation of the hypothalamic-pituitary-adrenocortical (HPA) axis represents a prominent finding in major depression, possibly related to a dysfunction of the corticosteroid receptor system. Antidepressants are involved in the restoration of the altered feed-back mechanism of the HPA-axis, probably via normalization of corticosteroid receptor functions. Since Hypericum perforatum has antidepressive properties, we here examined its putative actions on glucocorticosteroid receptor mRNA levels in human blood cells as a peripheral model for neuroendocrine effects in human brain cells. Our data show that Hypericum (LI 160) affects the cellular mRNA levels of both, the glucocorticoid receptor (GR)-α and its inhibitory counterpart, the GR-ß, at clinically-relevant concentrations. Under these conditions, a bimodal effect was observed. Dose-response studies suggest a rather small effective concentration range and time-effect data show a primary and transient up-regulation of GR-α mRNA levels and a down-regulation of GR-ß mRNA levels after 16 h of treatment. The sodium channel blocker benzamil neutralized the effects of Hypericum, pointing to an at least partial mechanism of action via this pathway. In conclusion, Hypericum treatment differentially affects GR-mRNA levels in the human system. Our data suggest a bimodal effect on GR, resulting in a time-and dose-related modification of GR-mediated cellular effects. Such a mechanism has been alleged as an important way of action for a number of antidepressants. It is the first time that a specific effect on both receptors, especially on the subtype of GR-ß, is shown under antidepressive treatment in a human system under in vitro conditions.

Antinociception produced by Thalassia testudinum extract BM-21 is mediated by the inhibition of acid sensing ionic channels by the phenolic compound thalassiolin B.

BACKGROUND: Acid-sensing ion channels (ASICs) have a significant role in the sensation of pain and constitute an important target for the search of new antinociceptive drugs. In this work we studied the antinociceptive properties of the BM-21 extract, obtained from the sea grass Thalassia testudinum, in chemical and thermal models of nociception in mice. The action of the BM-21 extract and the major phenolic component isolated from this extract, a sulphated flavone glycoside named thalassiolin B, was studied in the chemical nociception test and in the ASIC currents of the dorsal root ganglion (DRG) neurons obtained from Wistar rats. RESULTS: Behavioral antinociceptive experiments were made on male OF-1 mice. Single oral administration of BM-21 produced a significant inhibition of chemical nociception caused by acetic acid and formalin (specifically during its second phase), and increased the reaction time in the hot plate test. Thalassiolin B reduced the licking behavior during both the phasic and tonic phases in the formalin test. It was also found that BM-21 and thalassiolin B selectively inhibited the fast desensitizing (τ < 400 ms) ASIC currents in DRG neurons obtained from Wistar rats, with a nonsignificant action on ASIC currents with a slow desensitizing time-course. The action of thalassiolin B shows no pH or voltage dependence nor is it modified by steady-state ASIC desensitization or voltage. The high concentration of thalassiolin B in the extract may account for the antinociceptive action of BM-21. CONCLUSIONS: To our knowledge, this is the first report of an ASIC-current inhibitor derived of a marine-plant extract, and in a phenolic compound. The antinociceptive effects of BM-21 and thalassiolin B may be partially because of this action on the ASICs. That the active components of the extract are able to cross the blood-brain barrier gives them an additional advantage for future uses as tools to study pain mechanisms with a potential therapeutic application.

Effects of sodium selenite and amiloride on calvarial calcification in closing small cranial defects.

OBJECT: Fresh autogenous bone graft is the most preferred osteoplastic material, whether the purpose is cosmetic, psychological, or for the protection of the brain. These grafts are not rejected and do not react immunologically. The aim of this study was to evaluate the efficacy of autogenous fat rolled with bone dust derived from the bur hole in closing small cranial defects. Additionally, the authors examined the morphological and biochemical effects of Na selenite and amiloride on calvarial calcification. METHODS: The study group consisted of 20 domestic pigs. These animals were randomly divided into 4 groups. A bur hole with a diameter of 10 mm was made at the right parietal region in all animals, and then the periosteum around the bur hole was cauterized following exposure of the dura mater. The dura was coagulated with bipolar cautery. Group 1 (controls): only a bur hole was opened, and it was then closed with a mixture of the bone dust that had been created during the opening of the bur hole and fat tissue that was taken from the animal's neck. Group 2 (amiloride): 1 nmol/g body weight of amiloride was applied subcutaneously within 15 minutes after closure of the bur hole with bone dust and fat, and then amiloride was applied once a day for 4 weeks. Group 3 (Na selenite): 30 nmol/g body weight of Na selenite was applied subcutaneously within 30 minutes after closure of the bur hole with bone dust and fat, and then Na selenite was applied once a day for 4 weeks. Group 4 (amiloride and Na selenite): 1 nmol/g body weight of amiloride was applied subcutaneously at 15 minutes, and 30 nmol/g body weight of Na selenite was applied subcutaneously at 30 minutes after closure of the bur hole with bone dust and fat, and these 2 injections were repeated once a day for 4 weeks. At the end of 4 weeks, the animals were anesthetized to evaluate the closure of the bur hole. Tissue samples were obtained for ultrastructural and biochemical examination. RESULTS: The defect was covered with diffuse connective tissue in the control group. Although multiple capillary vessels were present, the authors did not observe osteogenic differentiation. Histological examination of the second group revealed osteogenic changes. Although new matrix was formed, calcification was not detected. The authors observed fibroblast, collagen fibers, and dense connective tissue filled with capillary in the third group of pigs, which had undergone Na selenite application. Calcification was not detected in this group. Both connective and osteogenic tissue were observed in specimens obtained in the fourth group, which had undergone amiloride and Na selenite application. CONCLUSIONS: The authors experimentally evaluated the supplementary osteogenic effects of Na selenite and amiloride by using them separately and together. The findings seem promising as a lead-in to new studies in restoring cranial defects.

The effects of amiloride, a Na+-H+ exchange inhibitor, on iliac artery stenosis after balloon injury in rabbits.

This study was designed to explore the effects of amiloride, a Na+-H+ exchange (NHE) inhibitor, on vessel stenosis by observing the expression of NHE-1 protein in vascular smooth muscle (VSM) after balloon injury and the effects of amiloride on VSM cell proliferation, migration, and excretion of extracellular matrices (ECMs). A total of 32 adult male New Zealand white rabbits were randomly divided into a balloon injury group (BG), an amiloride-treated group (AG), and a sham-operated group (SG). The left iliac artery was injured by inflating a 2.5 mm x 20 mm Foley catheter in BG and AG rabbits; in SG rabbits, the Foley catheter was inserted but not inflated. Amiloride (5 mg x kg(-1) x d(-1)) was injected intraperitoneally in AG and the same volume of distilled water was used in BG 3 days before balloon injury and for 28 days after the injury. The left iliac artery was stained by hematoxylin-eosin, alpha-actin, and Masson's trichrome to observe the vessel cava, neointima, media layer, and ECMs. NHE-1 proteins of the VSM were detected by Western blotting. A narrowing of the arterial cava, neointima formation, and thickened VSM layer were observed 28 days after balloon injury in BG and AG. However, in AG, the vessel cava was not as narrowed as that of BG and the intimal areas were to a lesser extent than in BG. In AG, the alpha-actin-positive areas and the ECM areas in the neointima were increased compared with SG, but to a lesser extent than in BG. The expression of NHE-1 protein in VSM was increased in BG and AG after balloon injury; however, the levels in AG were significantly less than in BG. In conclusion, VSM cell proliferation, migration, and excretion of ECMs contributed to vessel stenosis in the BG and AG rabbits. The expression of NHE-1 protein in VSM increased after balloon injury. Amiloride, an inhibitor of NHE-1, can limit the development of vessel stenosis through inhibition of VSM cell proliferation, migration, and excretion of ECMs.