K-dependent P-nitrophenylphosphatase Research Articles

Inhibition of Na,K-ATPase by Cadmium: Different Mechanisms in Different Species

The mechanism of action of Cd on Na,K-ATPase was investigated in two "classical" model systems, the shark rectal gland and rabbit kidney outer medulla. In lyophilized plasma membranes from dogfish rectal gland Cd inhibited Na,K-ATPase activity after 30 min of preincubation with an I 50 of 1.3 × 10 −5 M. K-Dependent p-nitrophenylphosphatase (pNPPase) activity was inhibited 50% by Cd at 9.4 × 10 −6 M. Neither Na nor K altered the interaction of the enzyme with Cd. Decreasing the ATP concentration, however, lowered the apparent sensitivity of Na,K-ATPase to Cd. The inhibitory effect was also significantly reduced when the Mg concentration present during the preincubation was increased from 0.5 to 6.0 mM. The apparent Cd sensitivity of the K-dependent pNPPase is lower at 10 mM Mg than at 1 mM Mg. In initial rate experiments 4 × 10 −5 M Cd increased the apparent K m of the enzyme for Mg significantly from 0.88 ± 0.29 mM to 1.73 ± 0.3 mM whereas the V max (167 ± 32 μmol/min × mg protein compared to 140 ± 16 μmol/min × mg protein) remained essentially unchanged. In lyophilized rabbit kidney outer medulla, Cd was found to inhibit Na,K-ATPase activity with an I 50 of 1.9 × 10 −5 M. K-Dependent pNPPase was inhibited 50% under identical conditions by Cd at a nominal concentration of 2.1 × 10 −4 M. Increasing K concentrations protected the enzyme from the inhibitory action of Cd as indicated by a 10-fold decrease in sensitivity of pNPPase when the K concentration was increased from 1 to 20 mM. K, 20 mM, delayed also the onset of inhibition by about 15 min at 37°C. These studies suggest that the mode of action of Cd on Na,K-ATPase varies in different species. In rectal gland Cd competes with a Mg site (or sites) critically involved in ATP and pNPP hydrolysis, whereas in rabbit renal medulla Cd interacts with a potassium-binding site. Differences in the protein sequence, protein conformation, and/or in the kind of protein membrane-lipid interaction might contribute to this diversity observed in the inhibitory mechanisms.

Detection of ouabain-insensitive H(+)-transporting, K(+)-stimulated p-nitrophenylphosphatase activity in rat gastric glands by cerium-based cytochemistry.

We employed a modification of our previously reported cerium-based cytochemical method for ouabain-sensitive, K-dependent p-nitrophenylphosphatase (Na-K ATPase) activity to detect ouabain-insensitive, K-stimulated p-nitrophenylphosphatase (K-pNPPase) activity in rat gastric glands. Biochemically, the enzyme activity of gastric glands incubated in a medium containing 50 mM Tricine buffer (pH 7.5), 50 mM KCl, 10 mM MgCl2, 2 mM CeCl3, 2 mM p-nitrophenylphosphate (pNPP), 2.5 mM levamisole, 10 mM ouabain, and 0.00015% Triton X-100, was optimal at pH 7.5-8.0 and decreased above pH 8.5. The amount of p-nitrophenol after incubation increased linearly in proportion to the amount of tissue in the medium. The enzyme activity was inhibited by omeprazole, sodium flouride (NaF), N-ethylmaleimide (NEM), and dicyclohexylcarbodiimide (DCCD). Heat-treated specimens had no enzyme activity. The enzyme activity increased with addition of K ions up to the concentration of 50 mM, and became constant above 50 mM. Cytochemically, the parietal cells of the gastric glands reacted positively for ouabain-insensitive K-pNPPase activity. Intense reaction was observed at the microvilli of the luminal surface and the intracellular canaliculi. The tubulovesicular system showed weak enzyme activity. The reaction products were found as fine, granular, electron-dense deposits in the cytoplasm just beneath the plasma membrane. The ouabain-insensitive K-pNPPase activity detected in this study appears, therefore, to be associated with that of H-transporting, K-stimulated adenosine triphosphatase (H-K ATPase).

Ultrastructural localization of ouabain-sensitive, K-dependent p-nitrophenyl phosphatase activity in monkey eccrine sweat gland.

We studied the electron microscopic localization of ouabain-sensitive, potassium-dependent p-nitrophenyl phosphatase (K-pNPPase) activity of the Na K-ATPase complex in Rhesus monkey eccrine sweat gland by use of the one-step lead citrate method of Mayahara et al. (Histochemistry 1980; 67:125). Reaction product was observed predominantly in the cytoplasmic side of the basolateral membranes of clear (secretory) cells, especially in the interdigitating membrane folds in the basal labryinth, and were completely abolished by 10 mM ouabain or by removal of K+. Little or no enzyme activity was noted in membrane processes in the intercellular canaliculi and in the secretory coil lumen. Basolateral membranes of the dark cells also showed moderate enzyme activity. The myoepithelial cell membrane was devoid of reaction product, except in a few membrane processes arising from the inner aspect of myoepithelial cells. In the coiled duct, K-pNPPase activity was present predominantly in the entire cell membrane of the peripheral ductal cells. The predominantly basolateral distribution of Na-K-ATPase in the eccrine sweat secretory cells is consistent with the concept that a Na-K-Cl co-transport model may be involved in the mechanism of eccrine sweat secretion.

Na-K-ATPase in the enamel organ: localization and possible roles in enamel formation.

Ouabain-sensitive, K-dependent p-nitrophenyl phosphatase (p-NPPase) activity was localized ultra-Ocytochemically in the lateral plasma membranes of secretory ameloblasts and the stratum intermedium and principally in the papillary layer cells of aldehyde-fixed rat incisor enamel organs by the one-step lead method. Daily intraperitoneal injection of ouabain (250 μg, 500 μg, and 1 mg/100 g body weight) for two weeks reduced p-NPPase activity in the enamel organ cells. However, the degree to which this activity was reduced appeared to vary among the experimental animals. Addition of ouabain to the cytochemical incubation medium completely inhibited p-NPPase activity in the tissues. Although long-term ouabain injection did not result in any morphological alterations of the enamel organ cells, it caused, in part, an appearance of electron-dense, homogeneous matrix-like substances (MS) in the extracellular spaces of the ameloblast layers at both the secretion and maturation stages. In addition, long-term ouabain injection appeared to have resulted in delayed maturation of enamel as measured by energy-dispersive x-ray analysis of Ca and P in surface enamel. These results suggest that Na-K-ATPase of enamel organ cells may participate in the net flow (removal) of organic matrix components and water from the enamel during the maturation stage of enamel formation. It is suggested that this flow is maintained by local osmotic gradients generated by Na-K-ATPase within the papillary layer.

Localization of K-NPPase and Li+ secretion in the exocrine pancreas of the pig.

To study the mode of transepithelial Na+ transport into pancreatic ducts during secretin-dependent NaHCO3 secretion, Na, K-ATPase was first localized within the exocrine pancreas of the pig using a cytochemical reaction for K-dependent p-nitrophenylphosphatase (K-NPPase). K-NPPase staining was confined to the lateral cell membrane bordering the intercellular spaces between ductal cells, negating the possibility of primary active, transcellular Na+ transport into pancreatic ducts. To assess how transepithelial Na+ transport may be coupled to HCO-3 secretion, net flux of Li+ into pancreatic juice was measured following intravenous systemic Li+ loading of 12 secretin infused, anaesthetized pigs. At plasma Li+ 32 (23-35) mmol l-1, Li+ displaced Na+ as accompanying cation to secreted HCO-3, and Li+/Na+ in pancreatic juice matched Li+/Na+ in arterial plasma. During superimposed inhibition of pancreatic water flux by hyperglycaemia, Li+ and Na+ were both transported against a transepithelial concentration gradient. Li+ reduced pancreatic HCO-3 secretion rate by 14 (-2 to -20)%, as well as Na,K-ATPase activity in a separate in vitro assay. The finding that Li+ substituted for Na+ in the secretion even during reduced osmotic water flow suggests that Na+ and Li+ are transported together with secreted HCO-3 into pancreatic juice by an electrogenic mechanism in addition to solvent drag and diffusion.

Localization of ATPases in retinal receptor cells.

Ouabain-sensitive, K-dependent p-nitrophenylphosphatase (K- NPPase ) activity of the Na-K-ATPase complex and the Ca-ATPase activity were studied ultracytochemically in the inner and outer segments of guinea pig photoreceptor cells by means of newly developed methods. The K- NPPase activity was demonstrated on the plasmalemma of the inner segments, whereas the Ca-ATPase activity was restricted to the matrix side of the inner membrane of the mitochondria which were accumulated in the inner segments. In contrast to the enzyme activities in the inner segments no such activity could be detected on the plasma and disk membranes of the outer segments. The functional meaning of the enzyme activities is discussed.

Cytochemical localization of ouabain-sensitive, K-dependent p-nitrophenylphosphatase in the rat hepatocyte.

To determine the localization of Na-K-ATPase activity in the rat hepatocyte, ouabain-sensitive, K-dependent p-nitrophenylphosphatase activity was studied cytochemically with a newly developed one-step lead citrate method devised by Mayahara et al. (1980). The liver was fixed with a mixture of 0.125% glutaraldehyde and 2% paraformaldehyde buffered with 30mM NaOH-PIPES instead of conventionally used 0.1M sodium cacodylate. Reaction products were localized predominantly on the sinusoidal and lateral membranes of the rat hepatocyte, which were remarkably abolished by addition of 10mM ouabain or by the deletion of K. They were localized on the cytoplasmic side of the membranes. Occasionally, reaction products were seen on the bile canalicular membrane, which were insensitive to ouabain and independent of K. These findings indicate that Na-K-ATPase is localized to the sinusoidal and lateral (basolateral) membranes of rat hepatocytes.

Ultracytochemical localization of ouabain-sensitive, potassium-dependent p-nitrophenylphosphatase activity in the lacrimal gland of the rat.

The electron-microscopic localization of ouabain-sensitive, K-dependent p-nitrophenylphosphatase (K-NPPase) activity of the Na - K-ATPase complex was studied in the exorbital lacrimal gland of the untreated rat with the use of a newly developed one-step lead-citrate method (Mayahara and Ogawa 1980; Mayahara et al. 1980). In the rat lacrimal gland fixed for 15 min in a mixture of 2% paraformaldehyde and 0.25% glutaraldehyde, an electron-dense reaction product was observed on the plasma membrane of the basal infoldings and the lateral interdigitations of the ductal cells. The most intense reaction product - and thus the major site of the Na - K-ATPase activity - was evident on the basolateral membranes of the cells of the large interlobular ducts; a weak reaction was seen on the basolateral, extensively folded plasma membranes of the small intercalated ducts; no reaction product was observed on the plasma membranes of the acinar cells. Addition of 1) 10 mM ouabain, 2) p-chloromercuri-phenyl-sulfonic acid (PCMB-S), 3) elimination of K-ions from the incubation medium, or 4) preheating abolished completely the K-NPPase reaction. The activity was also substrate-dependent. Mg-ATPase-activity was observed not only in the basolateral membranes of all ductal cells but also in the basal part of the acinar cells and on the walls of blood vessels. This reaction was neither inhibited by ouabain nor activated by K-ions. The precipitate of the Mg-ATPase-activity was localized at the extracellular side of the plasma membrane, whereas the K-NPPase-reaction product was restricted to the cytoplasmic side of the plasmalemma. In contrast, non-specific alkaline-phosphatase (ALPase) activity was missing in cells of the large interlobular ducts, but obvious on the apical plasmalemma of cells lining the small intercalated ducts. With respect to its localization and reactivity pattern the activity of the K-NPPase (member of the Na - K-ATase complex) differs markedly from the Mg-ATPase- and ALPase-activity.

Ultracytochemical study of the ouabain-sensitive, K-dependent p-nitrophenylphosphatase (Na-K-ATPase) activity in the developing rat kidney.

The ultracytochemical localization of ouabain-sensitive, K-dependent p-nitrophenylphosphatase (K-NPPase) activity of Na-K-ATPase was investigated in the fetal and neonatal rat kidneys with the one-step lead citrate method (17) in order to study the developmental changes in the site of the Na-pump.In fetal rat kidneys, the K-NPPase positive renal tubules first appeared in the inner cortex at 18 gestation days and increased with the maturation of the fetus. Electron microscopically, the reaction products were found on the basolateral plasma membranes of distal convoluted tubule cells at various levels of differentiation. In the kidney of 19 and more gestation days, K-NPPase positive tubules were found also in the ascending thick tubules of the loop of Henle in the medulla. In newborn rats, some of the ascending thick tubules with intense K-NPPase activity reached to the tip of the papilla. These tubules in the papilla gradually lost K-NPPase activity and redifferentiated into ascending thin tubules during the period of lactation. The clear separation of K-NPPase positive outer medulla and K-NPPase negative inner medulla was completed around 3 weeks after birth. The above results indicate that suckling rats have Na-pumps even in the inner medulla and thus have different urine concentration mechanisms from those in the adults.

Ultracytochemical localization of ouabain-sensitive, potassium-dependent p-nitrophenylphosphatase activity in guinea pig retina. II. Neurons and Mueller cells.

Ouabain-sensitive, K-dependent p-nitrophenylphosphatase (K-NPPase) activity, which reflects the second dephosphorylative step of Na-K-ATPase, was investigated histochemically under light and electron microscopes in neurons and Müller cells of the normal adult guinea pig retina with the newly eveloped one step lead citrate method (Mayahara et at., 1980). In guinea pig retina fixed for 5min in a mixture of 2% paraformaldehyde and 0.25% glutaraldehyde, electron dense reaction precipitates indicating K-NPPase activity were observed on plasma membranes of neurons and Müller cells. The most intensive K-NPPase reaction products were evident in the post-synaptic membranes of neurons in the outer plexiform layer, presynaptic membranes of neurons and post-synaptic membranes of ganglion cells and in the processes of Müller cells in the inner plexiform layer. This reaction was almost completely abolished by 10mM ouabain or elimination of K. The activity was also substrate-dependent, and completely inhibited by PCMB-S or preheating.It is noteworthy that another p-nitrophenylphosphatase activity which is ouabain-insensitive, Levamisole-resistant and K-independent was recognized on the endoplasmic reticulum, Golgi apparatus and nuclear envelope solely in Müller cells. This activity was also substrate-dependent, and completely inhibited by PCMB-S or preheating.Mg-ATPase activity was observed only on the synaptic membranes of neurons and ganglion cells in the inner plexiform layer. However, this reaction could be neither inhibited by ouabain nor activated by K. Non-specific alkaline phosphatase activity was not demonstrated on any part of the plasma membranes of neurons, ganglion cells or Müller cells.

A new one-step method for the cytochemical localization of ouabain-sensitive, potassium-dependent p-nitrophenylphosphatase activity

A new one-step method for the light and electron microscopic localization of the ouabain-sensitive, K-dependent p-nitrophenylphosphatase (K-NPPase) activity of the Na-K-ATPase complex is introduced. The incubation medium contains p-nitrophenylphosphate (NPP) as substrate, lead citrate as the capture reagent, and dimethylsulfoxide (DMSO) as an activator. It is usable at the optimal pH of the K-NPPase, which is about pH 9.0 in the presence of 25% of DMSO. The effects of fixation, lead concentration, and DMSO on the enzyme activity were studied using rat kidney as a test tissue. The fixation of tissues in a mixture of 2% paraformaldehyde and 0.5% glutaraldehyde for 60 min at 0 degrees--4 degrees C preserved 45% of the enzyme activity. In the absence of DMSO, lead citrate (4.0 mM) caused 82% inhibition of the enzyme activity in fixed tissue. However, the addition of DMSO (25%) caused about 3-fold activation of the remaining activity. Cytochemical demonstration of the ouabain-sensitive K-NPPase activity was successfully made by this method at both light and electron microscopic levels.

ULTRACYTOCHEMICAL LOCALIZATION OF OUABAIN-SENSITIVE, POTASSIUM-DEPENDENT <i>P</i>-NITROPHENYLPHOSPHATASE ACTIVITY IN THE GUINEA PIG RETINA

Ouabain-sensitive, K-dependent p-nitrophenylphosphatase activity was studied cytochemically in photoreceptor cells of the normal adult guinea pig retina with a newly developed one step lead citrate method (Mayahara et al., 1980). In guinea pig photoreceptor cells fixed for 5min in a mixture of 2% paraformaldehyde and 0.25% glutalaldehyde, electron dense reaction products were observed on plasma membranes of the inner segments and more proximal portion, but not on membranes of the outer segments. The most intensive reaction products were evident in the pre-synaptic membranes in the outer plexiform layer. It is also noteworthy that this activity was positive on the apical villi of the Muller cells, which were spread out distal to the outer limiting membrane into interstices between the inner segments. This reaction was almost completely abolished by 10mM ouabain or elimination of K. This activity was also substrate-dependent and completely inhibited by PCMBS or preheating. In contrast to K-dependent p-nitrophenylphosphatase activity, Mg-ATPase and non-specific alkaline phosphatase activity were not observed on every part of the plasma membranes of the photoreceptor cells.

Some properties of thyroidal membrane adenosinetriphosphatase and iodide uptake: effects of basic polyamino acids.

Poly L-lysine, poly L-ornithine, and histone significantly inhibited the iodide uptake by the thyroid slices, as previously reported. These basic polymers diminshed Na, K-ATPase and concomitantly markedly elevated Mg-ATPase activity in the NaI-treated microsomal preparation and the plasma membrane fraction obtained from thyroid. Poly L-glutamic acid, which was noneffetive to the iodide uptake in vitro, did not show such phenomenon. K-dependent p-nitrophenylphosphatase activity which is considered to reflect the terminal step of the reaction sequence of Na, K-ATPase was also inhibited by poly L-lysine. The effects mentioned above of poly L-lysine and other basic polyamino acids on membrane ATPase system were only found in the preparations from thyroid. The inhibitory effect of these reagents on thyroidal iodide uptake was discussed in terms of the change in membrane ATPase activities.