Pentagalloylglucose, an antisecretory component of Paeoniae radix, inhibits gastric H, K-ATPase

We purified a compound with strong inhibitory effect on H⁺, K⁺-ATPase from Paeoniae radix, which has been used in Japan for the treatment of gastritis and peptic ulcers. The compound was identified as 1,2,3,4,6,-penta-o-galloyl-β- -glucose by proton nuclear magnetic resonance, carbon-13 nuclear magnetic resonance, and fast atomic bombardment mass spectrometry. The IC₅₀ of the compound for H⁺, K⁺-ATPase was 166 nmol/l. Kinetic analyses indicated that the inhibition of the enzyme by pentagalloylglucose was noncompetitive with respect to K⁺. Pentagalloylglucose had relatively weak inhibitory effects for Mg⁺-ATPase (IC₅₀: >10 μmol/l) and Na⁺, K⁺-ATPase (IC₅₀: 2.7 μmol/l). Pentagalloylglucose also inhibited the accumulation of [¹⁴C]aminopyrine in parietal cells that had been isolated from guinea pig stomach and stimulated by 10 μmol/l histamine (IC₅₀: 7.8 μmol/l) and 1 mmol/l dbc-AMP (IC₅₀: 10 μmol/l). These results suggest that pentagalloylglucose is a potent inhibitor of H⁺, K⁺-ATPase and may be responsible for inhibition of acid secretion by Paeoniae radix.     

Mechanisms of gastric proton pump inhibition by calcium channel antagonists

The inhibitory effects of Ca channel antagonists on gastric acid secretion [[14C]-aminopyrine (AP) uptake ratio] have been analyzed in isolated rabbit parletal cells (PC). Secretagogue-stimulated AP uptake was inhibited by verapamil and diltiazem in a dose-dependent manner with IC50 values of 15 and 100 microM, respectively, both in the presence and absence of extracellular Ca. In contrast, nifedipine had no effect on AP accumulation. Verapamil decreased histamine-stimulated respiration with the same IC50 as observed for AP uptake. Imidazole, a weak base, by buffering the acid spaces in PC, reversed the inhibitory effect of verapamil on respiration. In the bullfrog gastric mucosa, forskolin-stimulated proton transport was inhibited by verapamil (10(-4) M) from the luminal but not the serosal side. This inhibitory effect was reversed by either elevating KCl concentration in, or removing the drug from, the secretory solution. Verapamil inhibited gastric microsomal H+,K(+)-adenosine triphosphatase (H+,K(+)-ATPase) and PC K(+)-stimulated p-nitrophenyl phosphatase activities with a higher potency than diltiazem. Inhibition of these enzymes by verapamil and diltiazem was pH dependent. The drugs competed with K+ in both H+,K(+)-ATPase and K(+)-stimulated p-nitrophenyl phosphatase reactions. Our data suggest that inhibition of the gastric proton pump by verapamil or diltiazem is not due to their Ca channel antagonism but to their interaction with the luminal high affinity K(+)-site of the H+,K(+)-ATPase under acidic conditions.     

Optimal conditions for measurement of Na+,K+-ATPase activity of human leucocytes

The Na+,K+-ATPase activity of human leucocytes was assayed by measuring the release of inorganic phosphate (Pi) from ATP. The maximum enzyme activity was achieved under the following conditions: concentration (mmol/l), Tris/HCl 50, Na 100, K 15, ATP 5, Mg 7, EDTA 1; pH 7.2 and temperature 37 degrees C, were optimal. Ouabain showed maximal inhibition at a concentration of 10-100 mumol/l. Ethanol, the solvent for ouabain, had a dose-related inhibitory effect. Heparin or citrate used as an anticoagulant gave similar results. Leucocyte samples could be stored at -20 degrees C for up to 6 days without loss of activity. Hypotonic lysis had advantages over sonication as the technique for cell disruption. The leucocyte Na+,K+-ATPase enzyme activity in healthy subjects was 186 mumol of Pi h-1g-1 of protein (median) with a range 136-243 mumol of Pi h-1g-1 of protein. The within-batch coefficient of variation was 6.4% and the between-batch precision was 9.6%.     

Sulfide and sulfoxide derivatives of substituted benzimidazoles inhibit acid formation in isolated gastric glands by different mechanisms

The sulfoxide and sulfide forms of three pairs of substituted benzimidazoles, including omeprazole, were investigated using isolated gastric glands and microsomal membranes containing H+,K+-adenosine Triphosphatase (ATPase). The sulfoxides inhibited stimulated aminopyrine (AP) uptake giving IC50 values between 0.47 and 1.1 microM, whereas the sulfides were less potent and showed a greater variation in IC50 values, 9.5 to 170 microM. The decrease in stimulated oxygen consumption induced by the sulfoxides was parallel to their inhibition of AP-uptake, with IC50 values of 0.40 to 3.7 microM, whereas the sulfides were virtually without effect, IC50 greater than 100 microM. The permeable buffers imidazole and 2,6-dimethylpyridine mimiced the effect of the sulfides on both AP accumulation and oxygen consumption. When tested on H+, K+-ATPase, an enzyme suggested to be the proton pump of the gastric mucosa, the sulfoxides inhibited the ATPase activity with IC50 values between 0.25 to 2.8 microM, in contrast to the sulfides, which were without inhibitory action. The sulfoxide-induced inhibition of AP-uptake in gastric glands and H+, K+-ATPase activity was prevented by the addition of beta-mercaptoethanol, whereas the mercaptane was without effect on sulfide-induced inhibition of AP-accumulation. When tested on vesicles containing H+, K+-ATPase, both the sulfide and sulfoxide derivatives dissipated the proton gradient generated by the enzyme, but only the sulfoxide-induced inhibition was prevented by the addition of beta-mercaptoethanol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of the mechanism for the inhibition of Na+,K(+)-adenosine triphosphatase by hyperglycemia involving activation of protein kinase C and cytosolic phospholipase A2.

Inhibition of Na+,K(+)-ATPase activity by hyperglycemia could be an important etiological factor of chronic complications in diabetic patients. The biochemical mechanism underlying hyperglycemia's inhibitory effects has been thought to involve the alteration of the protein kinase C (PKC) pathway since agonists of PKC can normalize hyperglycemia-induced inhibition of Na+,K(+)-ATPase activity. Paradoxically, elevated glucose levels and diabetes have been shown to increase PKC activities in vascular cells. The present study tested the hypothesis that the inhibition of Na+,K(+)-ATPase activity is mediated by the sequential activation of PKC and cytosolic phospholipase A2 (cPLA2). In cultured rat vascular smooth muscle cells (VSMC), increasing glucose levels in the medium from 5.5 to 22 mM elevated cPLA2 activity and increased [3H]arachidonic acid release and PGE2 production by 2.3-, 1.7- and 2-fold, respectively. Similar increases in cPLA2 activity were also induced by elevated glucose levels in human VSMC and rat capillary endothelial cells. The activation of cPLA2 was mediated by PKC since the increases in cPLA2 phosphorylation and enzymatic activity were inhibited by the PKC inhibitor GFX. In contrast, elevation of glucose levels decreased Na+,K(+)-ATPase activity as measured by ouabain-sensitive 86Rb uptake by twofold in rat VSMC. Surprisingly, both PMA, a PKC agonist, and GFX, a PKC inhibitor, were able to prevent glucose-induced decreases in 86Rb uptake. Further, the PLA2 inhibitor AACOCF3 abolished both glucose-induced activation of cPLA2 and the decrease in 86Rb uptake. These data indicated that hyperglycemia is inhibiting Na+,K(+)-ATPase activity by the sequential activation of PKC and cPLA2, resulting in the liberation of arachidonic acid and increased the production of PGE2, which are known inhibitors of Na+,K(+)-ATPase.     

Influence of angiotensin converting enzyme inhibitor (captopril) on kidney epithelial cells in vitro: studies on potassium (86Rb) influx and cellular proliferation

The effects of captopril on potassium influx and cellular proliferation in a dog kidney epithelial cell line (Madin-Darby canine kidney cells, MDCK) were studied. Na+K(+)-ATPase activity and the loop diuretic sensitive Na/K/2Cl- cotransport were measured using 86Rb as tracer substance. Cells were incubated with various concentrations of captopril (1-10 mmol/l). The furosemide sensitive Na/K/2Cl- cotransport was significantly decreased from 1 mmol/l onwards. Na+/K(+)-ATPase activity was lowered only when high amounts (10 mmol/l) of the drug were used. Cell proliferation was measured via [3H]thymidine incorporation. After incubation with 1 mmol/l captopril proliferation was strongly decreased (greater than 50%). Higher amounts (5-10 mmol/l) did not further suppress cell proliferation. The data suggest that natriuresis following ACE inhibition in vivo does not involve a direct effect of captopril on Na+K(+)-ATPase. However, the effect on cell proliferation may be of clinical relevance in respect to a possible mitogenic effect of angiotensin II.     

A rapid assay for the measurement of Na+,K(+)-ATPase inhibitors.

A rapid and automated assay for inhibitors of Na+, K(+)-ATPase was developed by determining the amount of inorganic phosphorus (Pi) released by Na+,K(+)-ATPase in a centrifugal analyzer. This method avoids long incubation, strong acids and centrifugation as in the conventional manual method. The coefficients of variation of intra- and inter-assay at ouabain concentration 0.5 mumol/L were 1.0 and 1.4%, respectively. The method is quick, reproducible and easy compared with current techniques.     

Impairment of sodium pump and Na/H exchanger in erythrocytes from non-insulin dependent diabetes mellitus patients: effect of tea catechins

BACKGROUND: Tea catechins (EGCG, EGC, ECG and EC) possess many important biological properties. We evaluated the effect of tea catechins on erythrocyte membrane Na(+)/K(+)-ATPase and sodium/hydrogen exchanger (NHE) activity in normal (control) and NIDDM subjects. METHODS: Erythrocyte membrane Na(+)/K(+)-ATPase and NHE activity were determined in normal and non-insulin dependent diabetes mellitus (NIDDM) patients. In vitro effect of tea catechins was studied by incubating membrane/intact erythrocytes in assay medium prior to Na(+)/K(+)-ATPase/NHE activity determination. RESULTS: A 24.2% decrease in the activity of Na(+)/K(+)-ATPase (p<0.001) and 39.37% increase in activity of NHE (p<0.02) were observed in NIDDM subjects compared to normal. Tea catechins inhibited the activity of Na(+)/K(+)-ATPase and NHE in both normal and NIDDM erythrocytes, the effect was concentration-dependent. The inhibitory effect of EGCG and ECG at micromolar concentrations was greater compared to EGC and EC on Na(+)/K(+)-ATPase. On NHE the inhibition of tea catechins was in the order: EC>EGC>ECG>EGCG at concentrations up to 10 micromol/l. CONCLUSIONS: This data may help to explain the anti-carcinogenic and cardioprotective effects of tea catechins. The effect of tea catechins on Na(+)/K(+)-ATPase and NHE may be explained due to a direct effect of these compounds on plasma membrane leading to a change in membrane fluidity.     

Characteristic features of proton pump inhibitors in the inhibition of gastric acid secretion: long-acting and complete inhibition]

Proton pump inhibitor is a compound recently applied for the treatment of peptic ulcers for its strong action to inhibit the gastric acid secretion. It works through inhibition of H+, K(+)-ATPase, so called proton pump, on the luminal surface of secretory canaliculi in the parietal cells, showing remarkable characteristics in the inhibition of gastric acid secretion; e.g., the long-acting and complete inhibition. At neutral pH, the unionized form of this compound as a weak base is lipophilic, and passes through the cell membrane to accumulate as the ionized form in an acidic environment in the secretory canaliculi of parietal cells, where it is transformed to an active molecule which binds covalently to the active site of H+, K(+)-ATPase, forming a highly stable complex. The long-acting and complete inhibition of gastric acid secretion by this compound is derived from this physico-chemical nature. The above characteristics of the proton pump inhibitor have been confirmed with the basal, stimulated and nocturnal gastric acid secretion and the 24-hour intragastric pH of healthy volunteers by several investigators prior to its nation-wide clinical trial in Japan. On the other hand, the increased endocrine and exocrine secretion, such as pepsin secretion and gastrin release, and the increased turnover of gastrointestinal endocrine cells by this compound have been reported in animal models, probably due to its accumulation in the acidic environment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sulphasalazine and derivatives, natural killer activity and ulcerative colitis

The effects of sulphasalazine, 5-aminosalicylic acid (5-ASA), sulphapyridine and azodisalicylic acid (ADS) in vitro on the natural killer (NK) activity of peripheral blood mononuclear cells (MNC) have been examined and compared with those of the lipoxygenase inhibitor, nordihydroguaiaretic acid (NDGA) and the cyclooxygenase inhibitor, indomethacin. Sulphasalazine, sulphapyridine and ADS inhibited NK activity with 50% inhibitory concentrations (IC50) of 0.7, 2.5 and 4.0 mmol/l respectively. The effect was rapidly reversible. In contrast, 5-ASA minimally inhibited NK activity at 50 mmol/l only. NDGA potently inhibited NK activity (IC50 27 mumol/l) but this was only partly reversible in short term incubations. Indomethacin had no effect at concentrations less than those inhibiting cyclo-oxygenase activity (1-10 mumol/l) but potently and reversibly inhibited NK activity at or above 25 mumol/l. The inhibitory effects observed were unlikely to be due to direct toxicity of effector cells as 5-ASA, sulphapyridine and ADS had no effect on the viability of peripheral blood MNC, whereas NDGA and indomethacin lysed MNC only at maximal concentrations tested. Though sulphasalazine produced MNC lysis at and above 1 mmol/l, the rapid reversibility of the inhibition of NK activity at 1 mmol/l suggested that lysis of NK cells contributed little to the suppressive effect at this concentration. The disparity of the therapeutic efficacy and effects on NK activity of sulphasalazine and its derivatives in vitro may suggest that NK activity is not a major pathogenic mechanism in ulcerative colitis.Any inhibitory effect on cellular immune function of indomethacin does not necessarily reflect an effect of cyclo-oxygenase inhibition.     

H(+)-ATPase and H(+), K(+)-ATPase]

Two different types of proton transporting ATPases, v-and p-type H(+)-ATPases engage in epithelial ion transport. Properties, function, molecular structure and distribution of these H(+)-ATPase (v-type) and H+, K(+)-ATPase (p-type) are summarized here. Intraorganellar spaces, such as lysosome, synapse, multivesicular body, are acidified by the vacuole-type H(+)-ATPases. Secretion of proton by some types of intercalated cells of kidney collecting tubules is due to H(+)-ATPase. Gastric proton secretion is due to H+, K(+)-ATPase. Proton secretion and absorption of potassium by distal colon is due to p-type ATPase.     

Studies on the intracellular pharmacodynamic properties of proton pump inhibitors and the inhibitory mechanism of acid secretion]

In recent studies, proton pump inhibitors, such as omeprazole, were found to be transformed into sulfenamide derivatives in the acid space of isolated parietal cells. It is considered that these sulfenamide derivatives mainly inhibit H+, K(+)-ATPase activity. To clarify the inhibitory mechanism of proton pump inhibitors, we studied the effect on acid secretion of the isolated parietal cells. Proton pump inhibitors inhibited histamine-, carbachol- and gastrin-stimulated 14C-aminopyrine accumulation. Db-cAMP stimulation was also inhibited by these inhibitors. Consequently, it is believed that the origin of H+, K(+)-ATPase was located in the final stage of the acid production.     

Decreased activity of the platelet Na+,K(+)-adenosine triphosphatase enzyme in allergic subjects

A pathogenic role and abnormal function have both been ascribed to the blood platelet in allergy, but the explanation for these observations is unknown. This study compared the cation-stimulated adenosine triphosphatase enzyme (ATPase) activities of platelets from allergic (n = 18), potentially allergic (asymptomatic, positive skin test, n = 5) and normal patients (n = 10), all of whom were without symptoms at the time of the study. Platelets were separated by centrifugation, were sonicated, and were assayed for cation-dependent ATPase activity by spectrophotometry. The mean Na+,K(+)-ATPase activity (in nanomoles per microgram protein per minute) of allergic subjects (0.94 +/- 1.28) was significantly lower than that of normal subjects (3.93 +/- 1.58). No Na+,K(+)-ATPase activity was detectable in platelets from eight of the allergic subjects. The Na+,K(+)-ATPase activity of potentially allergic subjects was intermediate between those of the allergic and normal subjects. A significant negative correlation (p less than 0.01) was observed between serum IgE levels and platelet Na+,K(+)-ATPase values, thus suggesting a relationship between the reduced platelet Na+,K(+)-ATPase and IgE immunoglobulin. No such differences were observed for the Ca+(+)- and Mg+(+)-stimulated ATPases. In vivo dysfunction of the plasma membrane Na+,K(+)-ATPase enzyme in allergic subjects could have profound effects on levels of intracellular cations and thus platelet activation and function.     

Sodium-potassium-activated adenosine triphosphatase of brain microsomes: modification of sodium inhibition by diphenylhydantoins

Effects of diphenylhydantoins on (Na(+) + K(+))-ATPase activity in rat and cat brain microsomes were studied. 5,5-diphenylhydantoin (DPH) in concentrations of 5-20 mug ml(-1) produces an apparent stimulation of the rat brain (Na(+) + K(+))-activated ATPase of 55-65% in media containing 50 mM Na(+), 0.15 mM K(+), 3 mM Mg(++), and 3 mM ATP. No effects are found on the Mg-ATPase. At constant K(+) levels of 0.05 mmole/liter and 0.15 mmole/liter, increasing the Na(+) concentration activates the enzyme similarly with and without DPH. However, Na(+) concentrations greater than 5 mmoles/liter and 10 mmoles/liter, respectively, which are inhibitory in these low K(+) media, produce less inhibition in the presence of DPH. In media containing 10 mM Na(+), the K(+) activation, on the other hand, is potentiated by DPH. In preparations from cat brain qualitatively similar results are obtained. No effect of DPH is seen on the inhibition produced by high K(+) in low Na(+) media. DPH produces an approximately constant apparent stimulation of 45% in the (Na(+) + K(+)) increments when these ions are varied simultaneously at a fixed ratio of 150 Na(+):1 K(+) with cat brain extracts. 5-(p-hydroxyphenyl)-5-phenylhydantoin (HPPH) has the same potency as DPH in reducing the Na(+) inhibition at high Na:K ratios. The hydantoins appear to act by decreasing the Na(+) inhibition that occurs at high Na:K ratios.     

Autocrine inhibition of Na+/K(+)-ATPase by nitric oxide in mouse proximal tubule epithelial cells.

An inducible nitric oxide synthase has recently been described in proximal tubule epithelium. To investigate the effects of proximal tubule NO on Na+/K(+)-ATPase, we induced NO production in mouse proximal tubule epithelial cells by treatment with lipopolysaccharide (LPS) and interferon-gamma (IFN gamma) followed by determinations of ouabain-sensitive ATPase activity. Na+/K(+)-ATPase activity decreased after 4 h of LPS/IFN gamma treatment, reaching maximal inhibition after 24 h (34% reduction in activity). The inhibition of Na+/K(+)-ATPase activity by LPS/IFN gamma was prevented by simultaneous incubation with N omega-nitro L-arginine and markedly blunted by removal of L-arginine from the medium. The NO donors sodium nitroprusside and SIN-1 also inhibited Na+/K(+)-ATPase activity to a similar extent than LPS/IFN gamma. However, treatment with 8-pCPT-cGMP only modestly reduced Na+/K(+)-ATPase activity. Interestingly, superoxide dismutase prevented the inhibitory effects of NO on Na+/K(+)-ATPase activity, suggesting a role for peroxynitrite in this inhibition. We conclude that NO generated by mouse proximal tubule epithelial cell iNOS inhibits Na/K ATPase activity in an autocrine fashion and that this inhibition is accompanied by a reduction in Na-dependent solute transport.     

Non-esterified fatty acids may regulate human leucocyte sodium pump activity

Human leucocyte sodium pump activity was studied in normal fasting subjects by measuring the ouabain-sensitive 22Na+ efflux rate constants. This 22Na+ efflux rate constant was inversely related to the fasting plasma non-esterified fatty acid level (rs = -0.73, P less than 0.0001). An oral glucose load (40 g/m2 surface area) led to an increase in the leucocyte ouabain-sensitive 22Na+ efflux rate constant after 2 h (1.97 +/- 0.25 to 2.44 +/- 0.19 h-1, P less than 0.0001, n = 11). There was a concomitant fall in the plasma non-esterified fatty acid level. Incubation of leucocytes in vitro with 100 mumol/l linoleic acid inhibited the leucocyte ouabain-sensitive 22Na+ efflux rate constant (1.52 +/- 0.27 vs 0.84 +/- 0.24 h-1, P less than 0.001, n = 8). The leucocyte Na+,K+-dependent adenosine triphosphatase (Na+,K+-ATPase) activity was inhibited in vitro by long chain non-esterified fatty acids, especially when unsaturated. Non-esterified fatty acids may account for some of the Na+,K+-ATPase inhibitory activity of plasma.     

Docosahexaenoic acid--induced vasorelaxation in hypertensive rats: mechanisms of action

The authors investigated the vasorelaxant properties of the omega-3 fatty acid, docosahexaenoic (DHA, 22:6n-3), and the possible involvement of endothelium-derived nitric oxide, prostanoids, opening of K+ channels, and/or modulation of calcium-mediated events. Isolated aorta from male spontaneously hypertensive rats (SHR) (age 16-17 weeks) were used to measure isometric tension. DHA-induced (1-100 mumol/l) relaxation was examined following contraction to norepinephrine (NE) (10(-6) mol/l) or high-K+ (80 mmol/l) solution in the presence and absence of various inhibitors and calcium-containing solution. DHA acid induced a significant vasorelaxant effect in both NE and high-K(+)-induced contracted SHR aortic rings, although DHA relaxations were greater in high-K(+)-induced contracted rings. In the absence of extracellular calcium, DHA (5-30 mumol/l) inhibited the initial phasic and sustained components of NE-induced contraction under different conditions. Inhibition of nitric oxide synthesis by N omega-nitro-L-arginine methyl ester hydrochloride (100 mumol/l) had no effect on DHA relaxations; however, indomethacin or nifedipine caused significant inhibition at > or = 30 mumol/l DHA. The K+ channel blocker, glibenclamide, but not tetraethyl-ammonium, also had an inhibitory effect on DHA-induced relaxation. These results indicate that DHA's vasorelaxant actions in SHR aorta are independent of endothelium-derived nitric oxide; however, at DHA concentrations > or = 30 mumol/l, vasodilatory prostanoids that activate ATP-sensitive K+ channels (KATP) may be involved. At lower concentrations, DHA-induced relaxation appears to be attributed to modulation of intracellular Ca2+ release and L-type Ca2+ channels in vascular smooth muscle cells. The vasorelaxant properties of DHA may contribute, in part, to the blood pressure-lowering effect of dietary fish oil in this hypertensive model.     

Physicochemical and functional modifications induced by obesity on human erythrocyte membranes

BACKGROUND: Na+,K(+)-ATPase activity was evaluated in relation to membrane composition and molecular organization in erythrocyte membranes from obese patients by the amphyphylic molecule 6-dodecanoyl-2-dimethylamino-naphthalene (Laurdan). Its possible relationship with fat distribution and hyperinsulinaemia was also investigated. DESIGN: Subjects were 10 obese men (OM), 12 women with subcutaneous obesity (FSO), 10 women with abdominal obesity (FAO) and 41 healthy lean subjects, 26 women (FC) and 15 men (MC). An oral glucose tolerance test was administered to all subjects to evaluate insulin secretion and glucose tolerance. RESULTS: Na+,K(+)-ATPase activity was increased in all obese patients. Values were higher in FSO and FAO than in FC (with FAO greater than FSO) and in OM than in MC. The erythrocyte membrane cholesterol-to-phospholipid ratio was increased in obese patients and was significantly different in FSO patients compared with FC. The erythrocyte membrane protein-to-phospholipid ratio was also increased in all obese subjects, reaching statistical significance only in FSO vs. FC. The liquid crystalline phase, as tested by Laurdan generalized polarization (GP), was decreased in obese patients, indicating the presence of greater molecular environmental order; all patients groups showed lower GP values than control subjects, but only FAO reached statistical significance compared with FC. There was no evident correlation between membrane Na+,K(+)-ATPase activity and insulin levels, nor did membrane composition and properties show any evident relationship with insulin levels. CONCLUSION: Both increased Na+,K(+)-ATPase activity and altered fluidity and lipid composition were observed in the erythrocyte membrane of all obese patients. These findings are in line with previous observations by our group and indicate that the changes in Na+,K(+)-ATPase activity observed in obese patients could be related to changes in plasma membrane organization and composition.     

Molecular characterization of the plasma membrane H(+)-ATPase, an antifungal target in Cryptococcus neoformans

The Cryptococcus neoformans PMA1 gene, encoding a plasma membrane H(+)-ATPase, was isolated from a genomic DNA library of serotype A strain ATCC 6352. An open reading frame of 3,380 nucleotides contains six introns and encodes a predicted protein consisting of 998 amino acids with a molecular mass of approximately 108 kDa. Plasma membranes were isolated, and the H(+)-ATPase was shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be slightly larger than the S. cerevisiae H(+)-ATPase, consistent with its predicted molecular mass. The plasma membrane-bound enzyme exhibited a pH 6.5 optimum for ATP hydrolysis, K(m) and V(max) values of 0.5 mM and 3.1 micromol mg(-1) min(-1), respectively, and an apparent K(i) for vanadate inhibition of 1.6 microM. ATP hydrolysis in plasma membranes and medium acidification by whole cells were inhibited by ebselen, a nonspecific H(+)-ATPase antagonist which was also fungicidal. The predicted C. neoformans protein is 35% identical to proton pumps of both pathogenic and nonpathogenic fungi but exhibits more than 50% identity to PMA1 genes from plants. Collectively, this study provides the basis for establishing the Cryptococcus H(+)-ATPase as a viable target for antifungal drug discovery.     

The effect of thyroid hormone on bile salt-independent bile flow and Na+, K+ -ATPase activity in liver plasma membranes enriched in bile canaliculi.

The relationship between bile salt-independent canalicular flow and ATPase activity in liver plasma membranes (LPM) enriched in bile canaliculi, was studied in control, hyperthyroid, and hypothyroid rats. Canalicular bile production was significantly increased in hyperthyroid rats (3.19 +/- 0.23 mul/min per g liver) compared to controls (2.27 +/- 0.24 mul/min per g liver), while it diminished in hypothyroid animals (1.58 +/- 0.17 mul/min per g liver). Although bile salt excretion was also increased in hyperthyroid animals (62.4 +/- 13.3 vs. 41.2 +/- 8.4 nmol/min per g liver), the stimulation in canalicular secretion was primarily related to enhancement of the bile salt-independent fraction of flow (2.47 mul/min per g liver in hyperthyroid rats vs. 1.67 mul/min per g liver in controls). LPM Na+, K+-ATPase activity doubled in hyperthyroid animals (21.5 +/- 5.8 vs. 10.7 +/- 3.1 mumol Pi/mg protein per h) while Mg++-ATPase activity remained unchanged and 5'-nucleotidase activity increased to a small but significant extent. In hypothyroid rats, bile salt excretion remained unchanged from control values so that the reduced secretion was entirely secondary to an inhibition of bile salt-independent secretion (1.19 mul/min per g liver). Na+, K+-ATPase activity in the LPMs from hypothyroid animals decreased by nearly 50% (5.4 +/- 1.6 mumol Pi/mg protein per h), although comparable reductions in the specific activity of Mg++-ATPase and 5'-nucleotidase were also observed. Administration of L-thyroxine to hypothyroid animals restored both bile salt-independent canalicular secretion and membrane enzymes to control values within 2 and 4 days, respectively. Sodium dodecyl sulfate gel electrophoresis demonstrated no significant changes in LPM protein fractions from any of the treatment groups. These studies indicate that thyroid hormone has a parallel effect on bile salt-independent canalicular secretion and LPM Na+, K+-ATPase activity, supporting the hypothesis that Na+ transport and Na+, K+-ATPase may be determinants of bile salt-independent canalicular flow.