Mg-dependent, Zn-ATPase: enzymatic characteristics, ion specificities and tissue distribution.

Mucosal crude microsomes, prepared from proximal rat small intestine, exhibited significant Mg-dependent, Zn-ATPase activity; Vmax = 23 micromoles Pi/mg protein/hr, Km = 160 nm, and Hill Coefficient, n = 1.5. Partial purification (approximately 10-fold) was achieved by detergent extraction, and centrifugation through 250 mm sucrose: Vmax = 268 units, Km = 1 nm, and n = 6. In partially purified preparations, the assay was linear with time to 60 min, and with protein concentration to 1 microg/300 microl. Activities at pH 8 and 8.5 were higher than at pH 7.2. The ATP Km was 0.7 mm, with an optimal ATP/Mg ratio of approximately 2. Ca elicited ATPase activity but did not augment the Zn-dependent activity. In partially purified preparations, the homologous salts of Co, Cd, Cu, and Mn exhibited no detectable activity. Vanadate inhibition studies yielded two component kinetics with a Ki of 12 microm for the first component, and 96 microm for the second component, in partially purified preparations. Tissue distribution analyses revealed gradients of activity. In the proximal half of the small intestine, Mg/Zn activity increased progressively from crypt to villus tip. In long axis studies, this activity decreased progressively from proximal to distal small bowel.

Na,K-ATPase in diabetic rat small intestine. Changes at protein and mRNA levels and role of glucagon.

Na,K-ATPase activity and isoform expression were measured in rat small intestinal mucosa taken from both normal and streptozocin-treated diabetic rats. Enzyme activity and abundance was 1.7-2.3-fold higher in rats diabetic for 2 wk than in controls. This was associated with 1.4-1.7-fold increases in small intestinal protein and DNA content. Ouabain inhibition curves of Na,K-ATPase were monophasic with Kis of 2.6 +/- 1.4 x 10(-4) and 2.0 +/- 1.2 x 10(-4) M for control and diabetic rats, respectively (NS). Northern blot analysis revealed a 2.5-fold increase in mRNA alpha 1 and a 3.4-fold increase in mRNA beta 1 in diabetic rats relative to controls. Two thirds of this increase occurred within 24h after injection of streptozocin. Immunoblots of intestinal enzyme preparations from diabetic and control rats indicated the presence of alpha 1 and beta 1 subunits but not of alpha 2 or alpha 3. Administration of glucagon (80 micrograms/kg) to normal rats daily for 14-16 d increased mRNA alpha 1 3.1-fold but did not increase mRNA beta 1 or enzyme activity. In experimental diabetes, alpha 1 and beta 1 isoforms of Na,K-ATPase are coordinately upregulated at both protein and mRNA levels, an effect which appears to be partially mediated by the associated hyperglucagonemia.

Diabetes mellitus and glucagon alter ouabain-sensitive Na(+)-K(+)-ATPase in rat small intestine.

Na(+)-K(+)-ATPase provides the driving force for cellular Na+ transport and exists in multiple isoforms that differ in ouabain sensitivities. We report that the Ki for ouabain inhibition of glucose-evoked short-circuit current, determined in intact rat ileal mucosa mounted in Ussing chambers, is higher in streptozocin-induced chronically diabetic rats than in age-matched controls. The changes in ouabain sensitivity seen in diabetes also occurred when intact ileum of age-matched controls was incubated in vitro with 2.8 x 10(-5) M glucagon for at least 80 min. The effect of glucagon was blocked by cycloheximide, indicating a role for protein synthesis. This suggests that changes in ouabain sensitivity seen in diabetes are produced by glucagon, the serum concentration of which increases in diabetes. Ouabain-dependent phosphorylation of Na(+)-K(+)-ATPase (backdoor phosphorylation) revealed a higher Km for phosphate in intestinal basolateral membranes obtained from diabetic rats compared with age-matched controls, again confirming a decrease in ouabain sensitivity. Furthermore, the mRNA encoding the alpha 1-isoform was upregulated 2.6-fold in chronically diabetic intestines. This suggests that the ouabain sensitivity seen during diabetes may be due to upregulation of the alpha 1-isoform, known to be less sensitive to ouabain than the other isoforms.

Anomalous mobilities of Na,K-ATPase alpha subunit isoforms in SDS-PAGE: identification by N-terminal sequencing.

Three isoforms of the alpha subunit of Na,K-ATPase, alpha 1, alpha 2, and alpha 3 have been characterized at the DNA, mRNA and protein levels. In admixtures, isoforms migrate as doublets (i.e. alpha 1 and another band originally designated alpha +, comprising alpha 2 + alpha 3) when analyzed by SDS-PAGE. As deduced from cDNA sequences their masses range from 111.7 to 112.6 kDa. With conventional protein standards, however, SDS-PAGE yields nominal masses of 85-105 kDa. In this system, the presence of a doublet that reacted with a polyclonal anti-Na,K-ATPase antibody in the kidney was interpreted as indicating two molecular or conformational species of the kidney alpha sub-unit (Siegel, G.J. and Desmond, T.J. (1989) J. Biol. Chem. 264, 4751-4754). We report that Na,K-ATPase purified from dog, guinea pig and rat kidney medulla or from rat brain, can yield two distinct bands when analyzed by SDS-PAGE or STS-PAGE, migrating between 85 and 105 kDa. An additional band migrating at 117 and 120 kDa appears often in enzyme purified from rat and guinea pig kidney medulla. The apparent molecular weights and relative intensities of these bands vary with temperature and duration of incubation during sample preparation. N-terminal sequencing and monospecific antibody probes revealed that the two distinct bands obtained from the kidney enzyme consist only of the alpha 1 isoform. The band appearing at 117-120 kDa also contains only the alpha 1 N-terminal sequence. In contrast, as reported earlier (Sweadner, K.J. (1979) J. Biol. Chem. 254, 6060-6067), the doublet seen in brain preparations consists of alpha 1 and alpha 2 or (alpha 2 + alpha 3). We conclude that monospecific antibody probes or N-terminal sequencing must be used to identify Na,K-ATPase isoforms by SDS- or STS-PAGE. In addition, gel conditions that may affect the mobilities of the isoforms are discussed.

Pharmacokinetic aspects of inorganic nitrate ingestion in man.

Inorganic nitrate and nitrite concentrations were monitored simultaneously in the plasma, erythrocytes, saliva and urine of five subjects following an oral dose of NaNO3 (470 mumols per kg body weight). There was an average 25-fold increase in plasma nitrate only 10 min. after ingestion. Its concentration rose to a peak level of 1.83 mM in 40 min., a value 49 times the preload level. Erythrocyte nitrate followed a similar pattern, but remained at about two thirds of the plasma values. Salivary nitrate showed a positive correlation with plasma nitrate and averaged 9 times the plasma level during 3 hr following ingestion. This is evidence of a concentration-dependent active secretion by the salivary glands. The mean nitrate clearance was 25.8 +/- 2.85 (S.E.M.) ml/min. corrected for a body area of 1.73 m2 (n = 17). The urinary nitrate/creatinine ratio increased 25 to 70 times after loading. These results indicate a predominantly tubular excretion of nitrate. Nitrite was not detectable in any of the body fluids studied except saliva, where it appeared to increase at the expense of nitrate.

Determination of inorganic nitrate in serum and urine by a kinetic cadmium-reduction method.

Nitrate in serum and urine was assayed by a modification of the cadmium-reduction method; the nitrite produced was determined by diazotization of sulfanilamide and coupling to naphthylethylene diamine. After samples were deproteinized with Somogyi reagent, the nitrate was reduced by Cu-coated Cd in glycine buffer at pH 9.7 (2.5 to 3 g of Cd granules for a 4-mL reaction mixture). The reduction followed pseudo-first-order reaction kinetics, a convenient time interval for assay being 75 to 90 min. Maximum reduction (85%) occurred at about 2 h. Detection limits in urine or serum were 2 to 250 mumol/L. This method does not require the reaction to go to completion, does not require expensive reagents or equipment, and can assay several samples simultaneously. Repeated assays of two serum pools gave CVs of 9.0% and 4.7% for nitrate concentrations of 31.4 and 80.2 mumol/L, respectively (n = 20 each). The mean concentration of nitrate was 1704.0 +/- 1294 (SD) mumol/L (n = 21) in untimed normal urine, 81.8 +/- 50.1 mumol/L in serum of 38 renal dialysis patients, and 51.2 +/- 26.4 mumol/L in serum of 38 controls.

Isoforms of Na,K-ATPase in Artemia salina: II. Tissue distribution and kinetic characterization.

To characterize the molecular properties conveyed by the isoforms of the alpha subunit of Na,K-ATPase, the two major transepithelial transporting organs in the brine shrimp (Artemia salina), the salt glands and intestines, were isolated in pure form. The alpha isoforms were quantified by ATP-sensitive fluorescein isothiocyanate (FITC) labeling. The salt gland enzyme exhibits only the alpha 1 isoform, whereas the intestinal enzyme exhibits both the alpha 1 and the alpha 2 isoforms. After 32 hours of development, Na,K-ATPase activity [in mumol Pi/mg protein/hr (1 mu)] in whole homogenates was 32 +/- 6 in the salt glands and 12 +/- 3 in the intestinal preparations (mean +/- SEM). The apparent half-maximal activation constants (K1/2) of the salt gland enzyme as compared to the intestinal enzyme were 3.7 +/- 0.6 mM vs. 23.5 +/- 4 mM (P less than 0.01) for Na+, 16.6 +/- 2.2 mM vs. 8.29 +/- 1.5 mM for K+ (P less than 0.01), and 0.87 +/- 0.8 mM vs. 0.79 +/- 1.1 mM for ATP (NS). The apparent Ki's for ouabain inhibition were 1.1 x 10(-4) M vs. 2 x 10(-5) M, respectively. Treatment of whole homogenates with deoxycholic acid (DOC) produced a maximal Na,K-ATPase activation of 46% in the salt gland as compared to 23% in the intestinal enzyme. Similar differences were found with sodium dodecyl sulfate (SDS). The two distinct forms of Na,K-ATPase isolated from the brine shrimp differed markedly in three kinetic parameters as well as in detergent sensitivity.(ABSTRACT TRUNCATED AT 250 WORDS)

Isoforms of Na,K-ATPase in Artemia saline: I. Detection by FITC binding and time course.

Partially purified Na,K-ATPase from whole nauplii at various stages of development, analyzed by SDS-PAGE, reveals a polydisperse beta and two alpha subunits (denoted alpha 1 and alpha 2). In the absence of Ca2+, ATP-inhibitable fluorescein isothiocyanate (FITC) labeling is restricted to the alpha subunit of this enzyme, even in crude naupliar homogenates. The intensity of the alpha-specific fluorescent signal (i.e., the sum of the yield from both alpha isoforms) is proportional to Na,K-ATPase activity during development. FITC-labeled subunits were detected at 8 hr of development prior to the detection of measurable Na,K-ATPase activity. The alpha 2/alpha 1 ratio changed from an initial value of 1.25 to a peak of 1.75 at 32 hr of development, then reverted to a ratio of 1.25 by 42 hr, and remained constant thereafter. Pulse chase studies with 35S-methionine indicated that the developmental increase in enzyme activity is coincident with amino acid incorporation into the alpha subunits, implying that enzyme synthesis is active during enzyme accumulation.

Hypophyseal-gonadal dysfunction in men with non-alcoholic liver cirrhosis.

Seven males with liver cirrhosis associated with hepatitis and one with schistosomal liver fibrosis were studied for hypophyseal gonadal dysfunction and compared to six age matched controls. Cirrhotics as a group had higher serum 17 beta estradiol levels (22.1 +/- 6.3 vs 7.8 +/- 0.8 pg/ml, p less than 0.05) which did not rise after four days of human chorionic gonadotropin (hCG) stimulation. Conversely, there was an adequate rise in serum testosterone level after hCG stimulation (332.8 +/- 99.7 ng/dl baseline to 887.6 +/- 67.1 ng/dl, p less than 0.01). Compared to the controls, cirrhotics had lower baseline serum follicle stimulating hormone (FSH) (3.6 +/- 1.7 vs. 10.2 +/- 1.5 mIu/ml, p less than 0.02) and higher serum prolactin (13.5 +/- 2.5 vs. 6.8 +/- 1.0 ng/ml, p less than 0.05). Pituitary dynamic function testing in cirrhotics revealed blunted response of luteinizing hormone (LH) and FSH, to luteinizing hormone releasing hormone (LHRH) in four out of eight subjects tested. We conclude that the mechanism of hypogonadism in non-alcoholic cirrhosis is mostly hypogonadotropic in origin rather than primary gonadal injury which is common in alcoholic cirrhosis.

Energetics of sodium transport in the urinary bladder of the toad. Effect of aldosterone and sodium cyanide.

Experiments were designed to determine whether the stimulatory effect of aldosterone on sodium transport involves an increase in tissue ATP. Urinary bladders that were removed from toads presoaked in 0.6% saline for 48-72 h, mounted as sacs, and maintained in open circuit except for brief observation of short circuit current every 30 min responded to 100 nM aldosterone added to the serosal bath with an increase in short circuit current to 170% of control hemibladders, which plateaus at 2-3 h. Tissue (ATP)/(ADP) X (Pi) measured in perchloric acid extracts increased to a maximum of 208% of controls (P less than 0.001) and ATP increased to 116% of controls (P less than 0.01) at 180 min. The short circuit current response to aldosterone paralleled the increase in ATP and (ATP)/(ADP) X (Pi) measured at 75, 120, 180, and 240 min. In bladders clamped at -150 mV, the short circuit current response to aldosterone was greater: 280% of controls (P less than 0.001) and tissue (ATP)/(ADP) X (Pi) increased to 191% of controls (P less than 0.001). In continuously short circuited bladders and bladders clamped at +75 mV, the short circuit current response to aldosterone and the change in ATP, ADP, or Pi were markedly diminished. 100 microM amiloride added to mucosal bath decreased the short circuit current to zero and inhibited the short circuit current response to aldosterone, whereas tissue ATP increased to 141% (P less than 0.05). 100, 250, and 500 microM NaCN dropped the short circuit current to 59, 35, and 24% of control values, respectively. Concurrently, tissue ATP measured at 60 min after the addition of NaCN dropped to 79, 66, and 56% of control values, respectively, and tissue ATP/ADP dropped to 68, 50, and 40%, respectively. The data revealed significant correlation between the change in the rate of sodium transport produced by aldosterone or NaCN as measured by the short circuit current and the concentration of ATP (r = 0.96, P less than 0.001), as well as ATP/ADP (r = 0.95, P less than 0.001). In conclusion, these results support the view that the stimulatory effects of aldosterone on sodium transport involve an increase in ATP or (ATP)/(ADP) X (Pi).

Aldosterone response in the turtle bladder is associated with an increase in ATP.

Urinary bladders from freshwater turtles, mounted as sacs, were stripped of their serosa and submucosa. This did not alter conductance. They were maintained in open circuit except for brief observation of short-circuit current (SCC) every 15 min. Potential difference (PD) averaged 68 +/- 14 mV and SCC 485 +/- 100 microA. Acetazolamide 10(-3) M increased SCC by 46 +/- 27 microA. Aldosterone 10(-7) M following acetazolamide resulted in a rise in SCC that began at about 75 min and reached a plateau between 3 and 5 h. SCC rose 127 +/- 15% compared with control bladder halves. ATP measured in perchloric acid extracts 5 h after addition of aldosterone increased by 33% (P less than 0.01) and (ATP)/(ADP) X (Pi) by 81% (P less than 0.01). These results support the view that the stimulatory effects of aldosterone on active sodium transport involve an increase in ATP and (ATP)/(ADP) X (Pi).

A functioning catecholamine-secreting vagal body tumor. A case report and review of the literature.

There are few cases of catecholamine-secreting paragangliomas of the neck reported in the literature, most of these being of the carotid body and glomus-jugulare type. This report cites the second case of a functioning vagal body tumor secreting norepinephrine predominantly and producing labile hypertension and symptoms of pheochromocytoma. A brief update on vagal body tumors and a review of the functioning paragangliomas of the head and neck are also presented.

Aldosterone action and sodium- and potassium-activated adenosine triphosphatase in toad bladder.

Urinary hemibladders obtained from toads soaked in water or saline were treated with aldosterone, 10(-6) M, either 1(1/2) or 16 h after mounting. After 2(1/2) h exposure to the hormone, short-circuit current was increased by 110-192% and open-circuit potential by 20-44% as compared with untreated paired hemibladders. Mucosal cells were then assayed for sodium-potassium-stimulated adenosine triphosphatase (ATPase). No increase occurred in activity per milligram protein or in the portion of total activity dependent on sodium. Activity at low sodium concentrations was also measured and analyzed by means of the Hill equation in terms of K, the apparent dissociation constant of the enzyme-sodium complex, and n, a number that expresses the degree of interaction between binding sites. Neither K nor n was significantly altered by aldosterone. A few experiments were also carried out at low ATP concentrations (0.3 mM); again no change in sodium-dependent activity was noted. The results indicate that aldosterone does not stimulate sodium transport by increasing the quantity of sodium-potassium adenosine triphosphatase in mucosal cells or the dependence of this activity on sodium or ATP concentrations.