Helicobacter pylori augments the acid inhibitory effect of omeprazole on parietal cells and gastric H(+)/K(+)-ATPase

BACKGROUND: In duodenal ulcer patients, intragastric acidity during omeprazole treatment is significantly lower before Helicobacter pylori eradication than after cure. AIMS: To determine if H pylori enhances the acid inhibitory potency of omeprazole in isolated parietal cells and on H(+)/K(+)-ATPase. METHODS: Rat parietal cells and pig gastric membrane vesicles enriched in H(+)/K(+)-ATPase activity were incubated with H pylori and the H pylori fatty acid cis 9,10-methyleneoctadecanoic acid (MOA), and the inhibitory effects of omeprazole on parietal cell acid production, H(+)/K(+)-ATPase enzyme activity, and ATPase mediated proton transport were assessed. RESULTS: In isolated parietal cells, H pylori and MOA increased the acid inhibitory potency of omeprazole 1.8 fold. H pylori did not affect the inhibitory potency of omeprazole on H(+)/K(+)-ATPase enzyme activity. In proton transport studies, H pylori (intact bacteria and sonicate) and MOA accelerated the onset of the inhibitory effect of omeprazole and enhanced the proton dissipation rate in response to omeprazole. H. pylori itself increased proton permeability at the vesicle membrane. CONCLUSION: Our results show that H pylori augments the acid inhibitory potency of omeprazole in parietal cells and enhances omeprazole induced proton efflux rate from gastric membrane vesicles. We suggest that omeprazole unmasks the permanent effect of H pylori on proton permeability at the apical parietal cell membrane, which is counteracted in the absence of a proton pump inhibitor by a reserve H(+)/K(+)-ATPase capacity.     

Gastrin receptor genes are expressed in gastric parietal and enterochromaffin-like cells ofMastomys natalensis

Although gastric enterochromaffin-like (ECL) carcinoid tumors are known to develop in patients with long-standing hypergastrinemia, the expression of the gastrin receptor gene in ECL cells has not yet been demonstrated. Therefore, this study was designed to examine gastrin receptor gene expression in ECL cells.Mastomys gastric mucosal cells isolated by enzyme dispersion were separated into 10 fractions (F1–10) by centrifugal elutriation. Each fraction was examined histologically to determine whether they contained ECL and/or parietal cells and Northern blot analysis was used to confirm the presence of histidine decarboxylase and H⁺, K⁺-ATPase gene expression. ECL cells were found only in fractions 1 and 2, whereas parietal cells were detected in fractions 6–10. Gastrin receptor gene expression was demonstrated in both parietal cell-rich and ECL cell-rich fractions. In addition, the gastrin receptor cDNA sequences obtained from the two of the fractions (F1 and 8) were identical. These results suggest that gastrin receptor genes are expressed in ECL cells as well as in parietal cells and that these receptors are identical.This study was supported by a grant-in-aid for scientific research from the Ministry of Education, Science, and Culture, and a grant from the Ministry of Health and Welfare, Japan.     

Evidence for apical Na+/H+ exchanger in bovine main pancreatic duct

The finding of a high Pco ₂ in basally secreted pancreatic juice of man and dog raises the hypothesis of proton secretion from ductal epithelial cells presumably through a Na⁺/H⁺ exchanger. To test this possibility, H⁺ luminal secretion and Na⁺ movements were measuredin vitro on samples of bovine pancreatic ducts mounted in Ussing-type chambers. The rate of luminal acidification measured by the pH stat method, using bicarbonate-free media gassed with 100% O₂, reached 2.75 Eq/cm²/hr. Proton secretion was blocked in the presence of 1 mM amiloride or in the absence of Na⁺ (replaced by choline) in the mucosal solution. Study of transepithelial²²Na fluxes in short-circuited tissue, bathed on both sides by control Ringer solution, gassed by 95% O₂-5% CO₂ demonstrated a net sodium transport from the mucosal to the interstitial side of the duct (net²²Na flux=3.23±0.8 Eq/cm²/hr). This net sodium transport was electroneutral and blocked by mucosal amiloride (0.5–1 mM/liter) or by interstitial ouabain (1 mM/liter). These results are consistent with the existence of a Na⁺/H⁺ exchanger on the luminal side of the bovine main pancreatic duct.     

Famotidine,a histamine-2-receptor antagonist,inhibits the increase in rat gastric H+/K+-ATPase mRNA induced by intravenous infusion of gastrin 17 and histamine

We examined the effects of gastrin and histamine on rat gastric H⁺/K⁺-ATPase, the enzyme responsible for H⁺ secretion, gene expressionin vivo. Gastrin 17 (G 17) or histamine dihydrochloride (histamine) was continuously infused through the femoral vein of anesthetized rats. Gastric H⁺/K⁺-ATPase mRNA levels were measured using northern blot analysis. Infusion of G 17 and histamine increased the H⁺/K⁺-ATPase mRNA level significantly compared with basal control level or vehicle control level (P<0.01). However, pretreatment with famotidine, a potent histamine-2 (H₂)-receptor antagonist, inhibited the increase of rat gastric H⁺/K⁺-ATPase mRNA following G 17 and histamine infusion. These findings indicate that both histamine and G 17 increase expression of H⁺/K⁺-ATPase mRNA by activating H₂ receptor on the parietal cell.     

Potassium Channelopathies and Gastrointestinal Ulceration

Potassium channels and transporters maintain potassium homeostasis and play significant roles in several different biological actions via potassium ion regulation. In previous decades, the key revelations that potassium channels and transporters are involved in the production of gastric acid and the regulation of secretion in the stomach have been recognized. Drugs used to treat peptic ulceration are often potassium transporter inhibitors. It has also been reported that potassium channels are involved in ulcerative colitis. Direct toxicity to the intestines from nonsteroidal anti-inflammatory drugs has been associated with altered potassium channel activities. Several reports have indicated that the long-term use of the antianginal drug Nicorandil, an adenosine triphosphate-sensitive potassium channel opener, increases the chances of ulceration and perforation from the oral to anal regions throughout the gastrointestinal (GI) tract. Several of these drug features provide further insights into the role of potassium channels in the occurrence of ulceration in the GI tract. The purpose of this review is to investigate whether potassium channelopathies are involved in the mechanisms responsible for ulceration that occurs throughout the GI tract.     

Prospective study of efficacy and safety of lansoprazole in Zollinger-Ellison syndrome

Lansoprazole, a new substituted benzimidazole H⁺, K⁺-ATPase inhibitor, profoundly inhibits gastric acid secretion and has potential use in the management of diseases such as Zollinger-Ellison syndrome (ZES). In the present study we evaluated the efficacy and safety of lansoprazole in controlling acid hypersecretion in 20 patients with ZES. The starting dose was 60 mg once daily. Control of acid hypersecretion was defined as the dose required to reduce acid secretion to <10 meq/hr in the last hour before the next dose. Doses were adjusted upwards until effective control was achieved. Patients not controlled with 120 mg once daily were placed on twice daily lansoprazole. Most patients (90%) required lansoprazole once daily. During long-term follow-up (mean 18.5 months), 25% of patients required upward dose adjustments and 25% of patients required twice daily lansoprazole. Following cessation of therapy, the mean time for gastric acid output to reach half basal acid output was 39.1 hr. Lansoprazole was well-tolerated without side effects. Clinical chemistry and hematological studies were unchanged, and no gastric carcinoids developed. These results demonstrate that lansoprazole is a safe and effective inhibitor of gastric acid hypersecretion in patients with Zollinger-Ellison syndrome. Because it has a long duration of action, lansoprazole can be used to control gastric acid hypersecretion in most patients with Zollinger-Ellison syndrome using a once daily dosing schedule.     

Increased IL-4- and IL-17-producing CD8+ cells are related to decreased CD39+CD4+Foxp3+ cells in allergic asthma

Objective: In allergic asthma, regulatory T cell (Treg) number and function are decreased. Antigen-primed CD8⁺ T cells play an indispensable role in the full development of airway inflammation and airway hyper-responsiveness (AHR) occurring in asthma. In this study, we investigated the relationship between subpopulations of CD8⁺ T cells and CD39⁺ Tregs. Methods: Female C57BL/6 mice were used to develop the model of allergic asthma. Experimental mice were immunized with ovalbumin (OVA) by intra-peritoneal (i.p) injection and then challenged with OVA by intra-tracheal administration. Control mice were immunized with vehicle by i.p injection and challenged with OVA. Airway inflammation was determined by histology and AHR was measured by an invasive method. Levels of interferon (IFN)-γ, IL-4, and IL-17 in bronchoalveolar lavage fluid (BALF) were determined by enzyme-linked immunosorbent assay. The frequencies of CD8⁺IFN-γ⁺ cells (Tc1), CD8⁺IL-4⁺ cells (Tc2), CD8⁺IL-17⁺cells (Tc17), and CD39⁺Tregs were measured by flow cytometry. The correlation between CD39⁺Tregs and Tc subsets was analyzed by Pearson’s test. Results: Experimental mice displayed phenotypes of allergic asthma, including inflammatory cell infiltration into the lungs, goblet cell hyperplasia, increased airway resistance, and increased IL-4 and IL-17 in BALF. Compared to control mice, experimental mice displayed lower CD39⁺Tregs and Tc1 but higher Tc2 and Tc17. There was a negative correlation between CD39⁺Tregs and Tc2 or Tc17. Conclusion: In allergic asthma, increased Tc2 and Tc17 are possibly related to insufficient CD39⁺Tregs.     

Ouabain-Like and Non-Ouabain-Like Factors in Plasma of Patients with Essential Hypertension

Circulating inhibitor of Na⁺, K⁺-ATPase and ouabain-like immuno-reactivity were studied in patients with essential hypertension. In the plasma of patients, two types of Na⁺, K⁺-ATPase inhibitors (ouabain-like and non-ouabain-like inhibitors) and ouabain-like immunoreactivity were detected. Ouabain-like inhibitor was clearly detected at a low KC1 concentration (0.1 mM) in the assay buffer, and non-ouabain-like inhibitor was detected at a high KC1 concentration (10 mM). The plasma level of ouabain-like inhibitor correlated significantly with that of ouabain-like immunoreactivity (p<0.001) and with a mean blood pressure (p<0.01). The plasma level of non-ouabain-like inhibitor was not correlated with the levels of either ouabain-like immunoreactivity or mean blood pressure. The level of plasma ouabain-like inhibitor did not correlate with that of plasma non-ouabain like inhibitor. Both ouabain-like inhibitor and ouabain-like immunoreactivity in the plasma of patients with essential hypertension were significantly higher than those in normotensive subjects, but the plasma level of non-ouabain-like inhibitor in patients with essential hypertension was not higher than that in normotensive subjects. These results suggest that the plasma from patients with essential hypertension contains ouabain-like factor(s) which is important to mantain the high blood pressure.     

Cardiovascular remodelling and red blood cell Li+/Na+ countertransport in patients with type 1 diabetes and essential hypertension

Patients with type 1 (insulin-dependent) diabetes may develop a specific cardiac disease characterized by functional and structural abnormalities. The pathogenesis of the cardiac disease is poorly understood but cardiac and renal complications may coexist. Patients with overt diabetic nephropathy have increased red cell Li⁺/Na⁺ countertransport (CT), which reflects abnormal kinetic properties of the red cell membrane Na⁺/H⁺ exchange. Since the activation of Na⁺/H⁺ exchange has a key role in cell proliferation and cell growth, as well as in the regulation of cell sodium and cell pH and in the renal reabsorption of Na⁺ and bicarbonate, we have looked for relationships between red cell Li⁺/Na⁺ CT, Na⁺/H⁺ exchange and cardiovascular remodeling in patients with type 1 diabetes, essential hypertension and idiopathic familiar cardiomyopathy. In type 1 diabetes the maximal velocity of Li⁺/Na⁺ CT is positively correlated with interventricular septum thickness and the left ventricular wall to lumen ratio. Similar results were obtained in patients with essential hypertension. In these patients an increased Li⁺/Na⁺ CT is also associated with severe and drug-resistant hypertension and with significant vascular remodelling, estimated by the minimal post-ischaemic vascular resistance in the calf. Finally, Li⁺/Na⁺ CT is negatively correlated with diastolic relaxation of the left ventricle in familiar hypertrophic cardiomyopathy. From these data it appears that widespread abnormal kinetic properties of Na⁺/H⁺ exchange, estimated by increased red cell Li⁺/Na⁺ CT, may have epistatic effects on the pathogenesis of cardiac complications of type 1 diabetes and essential hypertension.     

Alterations of Na/K-ATPase function in caveolin-1 knockout cardiac fibroblasts

Recent studies have demonstrated that the Na⁺/K⁺-ATPase is not only an ion pump, but also a membrane receptor that confers the ligand-like effects of cardiotonic steroids (CTS) such as ouabain on protein kinases and cell growth. Because CTS have been implicated in cardiac fibrosis, this study examined the role of caveolae in the regulation of Na⁺/K⁺-ATPase function and CTS signaling in cardiac fibroblasts. In cardiac fibroblasts prepared from wild-type and caveolin-1 knockout [Cav-1(−/−)] mice, we found that the absence of caveolin-1 did not affect total cellular amount or surface expression of Na⁺/K⁺-ATPase α1 subunit. However, it did increase ouabain-sensitive ⁸⁶Rb⁺ uptake. While knockout of caveolin-1 increased basal activities of Src and ERK1/2, it abolished the activation of these kinases induced by ouabain but not angiotensin II. Finally, ouabain stimulated collagen synthesis and cell proliferation in wild type but not Cav-1(−/−) cardiac fibroblasts. Thus, we conclude that caveolae are important for regulating both pumping and signal transducing functions of Na⁺/K⁺-ATPase. While depletion of caveolae increases the pumping function of Na⁺/K⁺-ATPase, it suppresses CTS-induced signal transduction, growth, and collagen production in cardiac fibroblasts.     

Some Properties of Erythrocyte Na+ -K+ -ATPase in Essenial Hypertension

The activity and some allosteric properties of Na⁺ -K⁺ -ATPase in erythrocytes and their membrane preparations (ghosts) from 57 patients with essential hypertension and 36 normotensive controls were studied. To reveal enzyme activity in whole erythrocytes the cells were pretreated with detergent Tween-20. It was found that in the patient erythrocytes the Na⁺ -K⁺ -ATPase activity was 33% less as compared to the control group. Moreover, in the patient erythmcytes the sensitivity of the enzyme to high concentrations of MgCI₂ was decreased. In contrast, no analogous changes of the enzyme were revealed in the patient ghosts. It is suggested that the erythrocytes of patients with essential hypertension contain an inhibitor of Na⁺ -K⁺ -ATPase.     

MdERDL6-mediated glucose efflux to the cytosol promotes sugar accumulation in the vacuole through up-regulating TSTs in apple and tomato

Sugar transport across tonoplasts is essential for maintaining cellular sugar homeostasis and metabolic balance in plant cells. It remains unclear, however, how this process is regulated among different classes of sugar transporters. Here, we identified a tonoplast H⁺/glucose symporter, MdERDL6-1, from apples, which was highly expressed in fruits and exhibited expression patterns similar to those of the tonoplast H⁺/sugar antiporters MdTST1 and MdTST2. Overexpression of MdERDL6-1 unexpectedly increased not only glucose (Glc) concentration but also that of fructose (Fru) and sucrose (Suc) in transgenic apple and tomato leaves and fruits. RNA sequencing (RNA-seq) and expression analyses showed an up-regulation of TST1 and TST2 in the transgenic apple and tomato lines overexpressing MdERDL6-1. Further studies established that the increased sugar concentration in the transgenic lines correlated with up-regulation of TST1 and TST2 expression. Suppression or knockout of SlTST1 and SlTST2 in the MdERDL6-1–overexpressed tomato background reduced or abolished the positive effect of MdERDL6-1 on sugar accumulation, respectively. The findings demonstrate a regulation of TST1 and TST2 by MdERDL6-1, in which Glc exported by MdERDL6-1 from vacuole up-regulates TST1 and TST2 to import sugars from cytosol to vacuole for accumulation to high concentrations. The results provide insight into the regulatory mechanism of sugar accumulation in vacuoles mediated by the coordinated action of two classes of tonoplast sugar transporters.

Soluble sugars play important roles not only as nutrients but also as signal molecules in plant growth and development (1). In fleshy fruits, sucrose (Suc) and monosaccharides (glucose [Glc] and fructose [Fru]) are also central to fruit quality as they are the main nutrients and sweetening compounds. The distribution of sugars in plant cells is dependent on several transport steps across cell membranes in addition to long-distance translocation through the phloem (2).In most plants, Suc is transported from photosynthetically active leaves (source), through the phloem, to nonphotosynthetic sink organs, such as fruits, roots, and stems. Suc is then either directly transported into sink cells or taken up in the form of hexoses (Glc and Fru) after cleavage by an extracellular invertase (3, 4). Within the sink cells, soluble sugars are first metabolized to meet the requirements for energy and carbon skeleton production. Excessive sugars are generally converted into starch for storage in the plastid or imported into vacuoles for transient or long-term storage mediated by vacuolar sugar transporters (2, 5). The vacuole occupies up to 90% of the cell volume, where most sugars are compartmentalized, typically in the parenchyma cells of many sugar-accumulating organs such as sugar cane stems, sugar beet roots, and fresh fruits of tomato, apple, and orange (5–7). While considerable effort has been made to understand sugar transport and accumulation in plants, our knowledge of the mechanism by which cells accumulate high concentrations of sugars in the vacuole remains rather limited.Sugar input into vacuoles is largely determined by different classes of sugar transporters located on the tonoplast (6). The tonoplast monosaccharide transporter (TMT), now renamed as tonoplast sugar transporter (TST), and the vacuolar glucose transporter (vGT) families encode proteins to mediate sugar influx into vacuoles as H⁺/sugar antiporters (8–10). In Arabidopsis, AtvGT1 is a tonoplast transporter specific for Glc with a role in seed germination and flowering (10), while AtTMT1/2 exhibits a broad substrate specificity and could transport Glc, Fru, and Suc into the vacuole (8, 11). More recently, SWEETs (sugars will eventually be exported transporters) have been identified as energy independent uniporters from Arabidopsis, rice, and other species, mediating concentration-dependent sugar influx or efflux across membranes (12). In the SWEET family of Arabidopsis, AtSWEET16 and AtSWEET17 were localized on the tonoplasts of root and leaf cells, where the former transported Suc, Glc, and Fru (13), while the latter appeared specific to Fru (14).Among the transporters that had been identified, only one member from the TST subfamily, namely, the TST2 subfamily, has been shown to play important roles in sugar accumulation (9, 15, 16). TSTs from different plants displayed different substrate specificities. For example, the watermelon ClTST2 protein exhibited similar transport activity for Suc, Fru, and Glc (16), while the BvTST2.1 protein from sugar beets has a specific affinity for Suc and is responsible for Suc accumulation in the taproots (9).Apart from the H⁺/sugar antiporter, TSTs, two types of H⁺/sugar symporters, the Suc exporter SUC4 (17) and the Glc exporter early response to dehydration like 6 (ERDL6), have also been identified to function on the tonoplast (18, 19). The operation of H⁺-coupled importers (e.g., TSTs, as an antiporter) and exporters (e.g., SUC4 and ERDL6, as symporters) on the tonoplast regulates sugar homeostasis between vacuole and cytoplasm (6). In Arabidopsis, AtSUC4 is weakly expressed in Suc-accumulating leaf mesophyll cells, and high SUC4 activity is unfavorable for sugar accumulation (17). It was reported that AtERDL6 acts as a Glc exporter in Arabidopsis to transfer Glc from the vacuole to the cytosol, as indicated by the finding that the corresponding erdl6 mutant accumulated more Glc in the vacuole (18, 19).It is noteworthy that orthologous genes of AtERDL6 exhibited higher expression levels in a wide range of sugar-accumulating fleshy fruits, such as those from pineapples (20), tomatoes (21), oranges (22), and apples (23, 24). The expression level of these ERDL6 orthologs genes generally matches with the fruit sugar content. In apples, the mRNA level and protein abundance of multiple MdERDL6 family members were strongly and positively correlated with Suc and Fru concentrations in the fruits, in parallel to high expression of MdTST1/2 (23, 24). The high expression of MdERDL6 genes in the sugary apple fruits is intriguing given that ERDL6 is an exporter of Glc from vacuoles and its overexpression in Arabidopsis impaired cold tolerance and seed germination (19). It remains unknown if ERDL6s contribute to sugar accumulation in fruits and, in particular, how ERDL6s and TSTs may coregulate sugar levels in the vacuoles.To explore the potential roles and cross talks of tonoplast H⁺/sugar symporters and antiporters in sugar accumulation, we first characterized MdERDL6-1 expression patterns and the transport activities of the encoded protein. This was followed by overexpression of MdERDL6-1 in apple and tomato (Solanum lycopersicum cv. Micro-Tom). The data obtained indicated that MdERDL6-1 functions as a H⁺/Glc symporter on the tonoplast to export Glc from the vacuole into the cytosol. Overexpression of MdERDL6-1 in apples and tomatoes substantially increased Glc, Fru, and Suc concentrations in leaves and fruits. This promoting effect was owing to the increased expression of TST1 and TST2 in the transgenic lines because the positive effect of MdERDL6-1 overexpression on sugar level was abolished or reduced once TST1 and TST2 were knocked out or suppressed, respectively. Overall, these results, together with other findings documented in this paper, identified a tonoplast H⁺/Glc symporter from apple that exports Glc from vacuoles and established that the increased Glc efflux mediated by MdERDL6-1 into the cytosol up-regulated TST1 and TST2 expression to promote sugar import into the vacuoles. The findings provide insights into the regulatory mechanism on sugar transport between cytosol and vacuole across tonoplast and offer potential opportunities to improve fruit sugar levels, hence fruit quality and yield.     

Effect of phenylalanine and p-chlorophenylalanine on Na+, K+-ATPase activity in the synaptic plasma membrane from the cerebral cortex of rats

Na⁺, K⁺-ATPase activity was measured in synaptic plasma membrane from cerebral cortex of Wistar rats subjected to experimental phenylketonuria, i.e., chemical hyperphenylalaninemia induced by subcutaneous administration of 5.2 μmol phenylalanine /g body weight (twice a day) plus 0.9 μmol p-chlorophenylalanine /g body weight (once a day). The treatment was performed from the 6^th to the 14^th postpartum day and rats were killed 12 h after the last injection. Synaptic plasma membrane from cerebral cortex was prepared by a discontinuous density sucrose gradient for Na⁺, K⁺-ATPase activity determination. The results showed that the enzyme activity was decreased by 30% in animals subjected to experimental phenylketonuria when compared to control. Thein vitro effects of the drugs on Na⁺, K⁺-ATPase activity were also investigated. Phenylalanine and p-chlorophenylalanine inhibited the enzyme activity and this inhibition was reversed by alanine. In addition, competition between phenylalanine and p-chlorophenylalanine for binding to the enzyme was observed, suggesting a common binding site for these substances. Our results suggest that reduction of Na⁺, K⁺-ATPase activity may be one of the mechanisms related to the brain dysfunction observed in human PKU.     

Endothelin-1 induced hypertrophic effect in neonatal rat cardiomyocytes: involvement of Na+/H+ and Na+/Ca2+ exchangers

Endothelin-1 (ET-1) is a potent agonist of cell growth that also stimulates Na(+)/H(+) exchanger isoform 1 (NHE-1) activity. It was hypothesized that the increase in intracellular Na(+) ([Na(+)](i)) mediated by NHE-1 activity may induce the reverse mode of Na(+)/Ca(2+) exchanger (NCX(rev)) increasing intracellular Ca(2+) ([Ca(2+)](i)) which in turn will induce hypertrophy. The objective of this work was to test whether the inhibition of NHE-1 or NCX(rev) prevents ET-1 induced hypertrophy in neonatal rat cardiomyocytes (NRVMs). NRVMs were cultured (24 h) in the absence (control) and presence of 5 nmol/L ET-1 alone, or combined with 1 mumol/L HOE 642 or 5 mumol/L KB-R7943. Cell surface area, (3)H-phenylalanine incorporation and atrial natriuretic factor (ANF) mRNA expression were increased to 131 +/- 3, 220 +/- 12 and 190 +/- 25% of control, respectively (P < 0.05) by ET-1. [Na(+)](i) and total [Ca(2+)](i) were higher (8.1 +/- 1.2 mmol/L and 636 +/- 117 nmol/L, respectively) in ET-1-treated than in control NRVMs (4.2 +/- 1.3 and 346 +/- 85, respectively, P < 0.05), effects that were cancelled by NHE-1 inhibition with HOE 642. The rise in [Ca(2+)](i) induced by extracellular Na(+) removal (NCX(rev)) was higher in ET-1-treated than in control NRVMs and the effect was prevented by co-treatment with HOE 642 or KB-R7943 (NCX(rev) inhibitor). The ET-1-induced increase in cell area, ANF mRNA expression and (3)H-phenylalanine incorporation in ET-1-treated NRVM were decreased by NHE-1 or NCX(rev) inhibition. Our results provide the first evidence that NCX(rev) is, secondarily to NHE-1 activation, involved in ET-1-induced hypertrophy in NRVMs.     

Relative abundance of alpha2 Na(+) pump isoform influences Na(+)-Ca(2+) exchanger currents and Ca(2+) transients in mouse ventricular myocytes

In the mouse, genetic reduction in the Na(+), K(+)-ATPase alpha1 or alpha2 isoforms results in different functional phenotypes: heterozygous alpha2 isolated hearts are hypercontractile, whereas heterozygous alpha1 hearts are hypocontractile. We examined Na(+)/Ca(2+) exchange (NCX) currents in voltage clamped myocytes (pipette [Na(+)]=15 mM) induced by abrupt removal of extracellular Na(+). In wild-type (WT) myocytes, peak exchanger currents were 0.59+/-0.04 pA/pF (mean+/-S.E.M., n=10). In alpha1(+/-) myocytes (alpha2 isoform increased by 54%), NCX current was reduced to 0.33+/-0.05 (n=9, P<0.001) indicating a lower subsarcolemmal [Na(+)]. In alpha2(+/-) myocytes (alpha2 isoform reduced by 54%), the NCX current was increased to 0.89+/-0.11 (n=8, P=0.03). The peak sarcolemmal Na(+) pump currents activated by abrupt increase in [K(+)](o) to 4 mM in voltage clamped myocytes in which the Na(+) pump had been completely inhibited for 5 min by exposure to 0 [K(+)](o) were similar in alpha1(+/-) (0.86+/-0.12, n=10) and alpha2(+/-) myocytes (0.94+/-0.08 pA/pF, n=16), and were slightly but insignificantly reduced relative to WT (1.03+/-0.05, n=24). The fluo-3 [Ca(2+)](i) transient (F/F(o)) in WT myocytes paced at 0.5 Hz was 2.18+/-0.09, n=34, was increased in alpha2(+/-) myocytes (F/F(o)=2.56+/-0.14, n=24, P=0.02), and was decreased in alpha1(+/-) myocytes (F/F(o)=1.93+/-0.08, n=28, P<0.05). Thus the alpha2 isoform rather than the alpha1 appears to influence Na(+)/Ca(2+) exchanger currents [Ca(2+)](i) transients, and contractility. This finding is consistent with the proposal that alpha2 isoform of the Na pump preferentially alters [Na(+)] in a subsarcolemmal micro-domain adjacent to Na(+)/Ca(2+) exchanger molecules and SR Ca(2+) release sites.     

In vitro insulin action on different ATPases of erythrocyte membranes in normal and diabetic rats

Summary Thein vitro effect of porcine insulin on Na⁺+K⁺-, Ca²⁺- and Mg²⁺-ATPases of the rat erythrocyte membrane of normal and alloxan-induced diabetic rats was investigated. Na⁺+K⁺- and Ca²⁺-stimulated enzyme activities were significantly decreased in diabetic rats in comparison to normal animals. The specific activities of both these ATPases in the latter group were markedly reduced on pre-incubating the ghosts with insulin. Similar treatment of the erythrocyte membranes of diabetic animals, however, resulted in a significant increase of these activities. These qualitatively different effects of the hormone in the two groups increased progressively with hormone concentration and duration of pre-incubation. Mg²⁺-stimulated ATPase activity was not significantly affected in diabetes or by insulin.