Na+, K+-ATPase of the canine mesenteric artery

Na+,K+-ATPase, the enzymatic moiety that operates as the electrogenic sodium-potassium pump of the cell plasma membrane, is inhibited by cardiac glycosides, and this specific interaction of a drug with an enzyme has been considered to be responsible for digitalis-induced vascular smooth muscle contraction. Although studies aimed at localization, isolation, and measurement of the Na+,K+-ATPase activity (or Na+, K- pump activity) indicate its presence in vascular smooth muscle sarcolemma, its characterization as the putative vasopressor receptor site for cardiac glycosides has depended on pharmacological studies of vascular response in vivo and on isolated artery contractile responses in vitro. More recently, radioligand-binding studies using [3H]ouabain have aided in the characterization of drug-enzyme interaction. Such studies indicate that in canine superior mesenteric artery (SMA), Na+,K+-ATPase is the only specific site of interaction of ouabain with resultant inhibition of the enzyme. The characteristics of [3H]ouabain binding to this site are similar to those of purified or partially purified Na+,K+-ATPase of other tissues, which suggests that if Na+,K+-ATPase inhibition is causally related to digitalis-mediated effects on vascular smooth muscle contraction, then therapeutic concentrations of cardiac glycosides could act to cause SMA vasoconstriction. The additional finding from radioligand-binding studies that Na+,K+-ATPase exists in much smaller quantities (density of sites per cell) in SMA than in either heart or kidney may have implications concerning its physiological, biochemical or pharmacological role in modulating vascular muscle tone.     

A nonelectrogenic H+ pump in plasma membranes of hog stomach.

Differential and density gradient centrifugation were used to prepare a vesicular membrane fraction from hog gastric mucosa enriched 17-fold with respect to cation-activated ATPase and 5'-AMPase. Fractionation of the gradient material by free flow electrophoresis resulted in a fraction 35-fold enriched in cation-activated ATPase and essentially free of 5'-AMPase and Mg2+ATPase. The addition of ATP to either fraction resulted in H+ uptake and Rb+ efflux. The ionophoric and osmotic sensitivity showed that these ion movements were due to transport rather than binding. The cation selectivity sequences, substrate specificities and action of inhibitors indicated that the transport was a function of K+ATPase activity. The characteristics of the ATP-dependent enhancement of SCN- uptake and 8-anilinonapthalene-1-sulfonate fluorescence in the presence of valinomycin and the action of ionophores and lipid-permeable ions suggested that the energy dependent K+:H+ exchange was effectively nonelectrogenic. Thus these vesicles contain a nonelectrogenic (H+ + K+)-ATPase, hence acid secretion by the stomach is probably due to an ATP-dependent H+ + K+ exchange.     

Half of the (Na+ + K+)-transporting-ATPase-associated K+-stimulated p-nitrophenyl phosphatase activity of gastric epithelial cells is exposed to the surface exterior.

Ouabain inhibited 86RbCl uptake by 80% in rabbit gastric superficial epithelial cells (SEC), revealing the presence of a functional Na+,K+-ATPase [(Na+ + K+)-transporting ATPase] pump. Intact SEC were used to study the ouabain-sensitive Na+,K+-ATPase and K+-pNPPase (K+-stimulated p-nitrophenyl phosphatase) activities before and after lysis. Intact SEC showed no Na+,K+-ATPase and insignificant Mg2+-ATPase activity. However, appreciable K+-pNPPase activity sensitive to ouabain inhibition was demonstrated by localizing its activity to the cell-surface exterior. The lysed SEC, on the other hand, demonstrated both ouabain-sensitive Na+,K+-ATPase and K+-pNPPase activities. Thus the ATP-hydrolytic site of Na+,K+-ATPase faces exclusively the cytosol, whereas the associated K+-pNPPase is distributed equally across the plasma membrane. The study suggests that the cell-exterior-located K+-pNPPase can be used as a convenient and reliable 'in situ' marker for the functional Na+,K+-ATPase system of various isolated cells under noninvasive conditions.     

Altered transverse tubule dihydropyridine receptor binding in malignant hyperthermia

Transverse tubule (TT) membrane vesicles have been isolated from the skeletal muscle of normal and malignant hyperthermia-susceptible (MHS) pigs. MHS and normal TT did not differ in the distribution of the major proteins, cholesterol, or phospholipid content, (Na+ + K+)-ATPase activity, [3H]ouabain binding, Ca2+-ATPase activity, Mg2+-ATPase activity, or [3H]saxitoxin binding. Furthermore, in the presence of micromolar Ca2+, MHS and normal TT did not differ significantly in the KD values for either [3H]nitrendipine binding (2.7 +/- 0.6 and 3.3 +/- 0.5 nM, respectively) or (-)-[3H]desmethoxyverapamil ([3H]D888) binding (7.2 +/- 0.9 and 6.4 +/- 0.6 nM, respectively). However, in contrast to normal TT, MHS TT exhibited a significantly decreased Bmax for both [3H]nitrendipine binding (26.4 +/- 5.4 for MHS versus 40.6 +/- 3.7 pmol/mg protein for normal TT) and [3H]D888 binding (17.8 +/- 7.0 for MHS versus 37.4 +/- 5.9 pmol/mg protein for normal TT). At calcium concentrations greater than 0.1 mM, there was a greater inhibition of [3H]nitrendipine binding to normal than to MHS TT such that binding was now similar for both preparations. As with purified TT, [3H]nitrendipine binding to MHS muscle homogenates was significantly less than to normal muscle homogenates (109 +/- 20 versus 211 +/- 19 fmol/mg protein, for MHS and normal TT, respectively); this difference was not apparent when 100 mM CaCl2 was included in the binding medium. We conclude that the altered MHS TT dihydropyridine receptor properties may reflect an adaptation of the TT voltage sensing mechanism to the abnormal sarcoplasmic reticulum calcium release channel regulation in MHS muscle.     

Evoked secretion of [3H]noradrenaline and ATP from nerve varicosities isolated from the myenteric plexus of the guinea pig ileum

Neuronal varicosities, isolated from the myenteric plexus of guinea pig ileum longitudinal muscle, were incubated with [3H]noradrenaline to label the contents of the noradrenergic secretory vesicles. Exposure of these varicosities to KCl, nicotine, or acetylcholine resulted in the Ca2+ -dependent release of [3H]noradrenaline. Veratridine also evoked a large efflux of [3H] from this preparation, but this release was only partially Ca2+ dependent. The alpha 2-adrenoceptor agonist, clonidine, inhibited the K+-, nicotine-, and acetylcholine-induced release of [3H]noradrenaline. Similarly, exogenously administered (-)noradrenaline was an effective inhibitor of the K+ -evoked release of [3H]noradrenaline. The alpha 2-adrenoceptor antagonist, yohimbine, antagonized the inhibitory actions of both clonidine and (-)noradrenaline on the K+ -evoked release of [3H]noradrenaline from myenteric varicosities. Nicotine, acetylcholine, KCl, and veratridine also released ATP from these guinea pig ileal myenteric varicosities. However, the evoked release of ATP was unaffected by clonidine. These results indicate that myenteric varicosities can take up and release [3H]noradrenaline and that they possess presynaptic alpha 2-adrenoceptors which, when activated, inhibit the release of [3H]noradrenaline. These receptors may play a role in modulating the release of noradrenaline in the myenteric plexus in vivo. In addition, the present results suggest that ATP and [3H]noradrenaline may not be released from the same population of secretory vesicles in neuronal varicosities isolated from guinea pig ileum longitudinal muscle.     

Reconstitution of the voltage-sensitive sodium channel of rat brain from solubilized components

The voltage-sensitive sodium channel of rat brain synaptosomes was solubilized with sodium cholate. The solubilized sodium channel migrated on a sucrose density gradient with an apparent S20,w of approximately 12 S, retained [3H]saxitoxin ([3H]STX) binding activity that was labile at 36 degrees C but no longer bound 125I-labeled scorpion toxin (125I-ScTX). Following reconstitution into phosphatidylcholine vesicles, the channel regained 125I-ScTX binding and thermal stability of [3H]STX binding. Approximately 50% of the [3H]STX binding activity and 58% of 125I-ScTX binding activity were recovered after reconstitution. The reconstituted sodium channel bound STX and ScTX with KD values of 5 and 10 nM, respectively. Under depolarized conditions, veratridine enhanced the binding of 125I-ScTX with a K0.5 of 20 microM. These KD and K0.5 values are similar to those of the native synaptosome sodium channel. 125I-ScTX binding to the reconstituted sodium channel, as with the native channel, was voltage dependent. The KD for 125I-ScTX increased with depolarization. This voltage dependence was used to demonstrate that the reconstituted channel transports Na+. Activation of sodium channels by veratridine under conditions expected to cause hyperpolarization of the reconstituted vesicles increased 125I-ScTX binding 3-fold. This increased binding was blocked by STX with K0.5 = 5 nM. These data indicate that reconstituted sodium channels can transport Na+ and hyperpolarize the reconstituted vesicles. Thus, incorporation of solubilized synaptosomal sodium channels into phosphatidylcholine vesicles results in recovery of toxin binding and action at each of the three neurotoxin receptor sites and restoration of Na+ transport by the reconstituted channels.     

Differential localization of 5-hydroxytryptamine3 and 5-hydroxytryptamine4 receptors in the human rectum

The functions of the 5-hydroxytryptamine3 (5-HT3) and 5-hydroxytryptamine4 (5-HT4) receptors in gastrointestinal tract are complex depending on the species and anatomical regions, and the localization of these receptors in the human rectum was unclear. We examined the localization of the 5-HT3 and 5-HT4 receptors in human rectum by in vitro receptor autoradiography using [125I](S)iodozacopride and [125I] SB207710 as a ligand, respectively. Specific [125I](S)iodozacopride binding sites were clearly evident in the myenteric plexus, whereas, low levels of [125I]SB207710 binding sites were distributed over the muscle but not to the myenteric plexus. The 5-HT3 receptor located on the myenteric plexus and the 5-HT4 receptor on the smooth muscle may participate in contractility and relaxation of human rectum, respectively.     

Impaired muscle Ca2+ and K+ regulation contribute to poor exercise performance post-lung transplantation.

Lung transplant recipients (LTx) exhibit marked peripheral limitations to exercise. We investigated whether skeletal muscle Ca2+ and K+ regulation might be abnormal in eight LTx and eight healthy controls. Peak oxygen consumption and arterialized venous plasma [K+] (where brackets denote concentration) were measured during incremental exercise. Vastus lateralis muscle was biopsied at rest and analyzed for sarcoplasmic reticulum Ca2+ release, Ca2+ uptake, and Ca2+-ATPase activity rates; fiber composition; Na+-K+-ATPase (K+-stimulated 3-O-methylfluorescein phosphatase) activity and content ([3H]ouabain binding sites); as well as for [H+] and H+-buffering capacity. Peak oxygen consumption was 47% less in LTx (P < 0.05). LTx had lower Ca2+ release (34%), Ca2+ uptake (31%), and Ca2+-ATPase activity (25%) than controls (P < 0.05), despite their higher type II fiber proportion (LTx, 75.0 +/- 5.8%; controls, 43.5 +/- 2.1%). Muscle [H+] was elevated in LTx (P < 0.01), but buffering capacity was similar to controls. Muscle 3-O-methylfluorescein phosphatase activity was 31% higher in LTx (P < 0.05), but [3H]ouabain binding content did not differ significantly. However, during exercise, the rise in plasma [K+]-to-work ratio was 2.6-fold greater in LTx (P < 0.05), indicating impaired K+ regulation. Thus grossly subnormal muscle calcium regulation, with impaired potassium regulation, may contribute to poor muscular performance in LTx.     

The Effect of Anti-Synaptosomal Membrane Antibodies on Neurotransmitter Uptake

Antibodies raised against synaptosomal plasma membranes of rat hippocampus (anti-HPC IgG) caused inhibition of [3H]noradrenaline, [3H]5-hydroxytryptamine, [3H]GABA and [3H]aspartate uptake into S1 fractions and slices of hippocampus and cerebral cortex, but not those of caudate nucleus and hypothalamus. Similar inhibition was not observed on using antibodies against synaptosomal membranes of rat caudate nucleus. Anti-HPC IgG raised against synaptosomal membranes of hippocampus failed to alter both spontaneous and K+-evoked release of [3H]noradrenaline. They did not interfere with the binding of [3H]desipramine (the potent noradrenaline-uptake inhibitor) and with the binding of [3H]dihydroalprenolol, thus excluding any interaction of the antibodies with drug receptors which are located on either the pre- or postsynaptic membrane. The anti-HPC IgG inhibit the enzymatic activity of [Na+-K+-]ATPase by 30% upon incubation of the antibodies with crude membrane preparations. A comparison of their inhibitory effects with those of the neurotoxin 6-hydroxydopamine suggests that the corresponding hippocampal specific antigens are located at a presynaptic site.     

Modulation of gastric H+,K+-transporting ATPase function by sodium

Gastric H+,K+-ATPase activity is not affected by Na+ at pH 7.0 but is significantly stimulated by Na+ at pH 8.5. For the stimulation at the latter pH, the presence of both Na+ and K+ were essential. Contrary the H+,K+-ATPase, the associated K+-pNPPase was inhibited by Na+ at both pH values. Sodium competes with K+ for the K+-pNPPase reaction. Also, unlike the H+, K+-ATPase activity the ATPase-mediated transport of H+ within the gastric microsomal vesicles was inhibited by Na+. For the latter event only the extravesicular and not the intravesicular Na+ was effective. The data suggest that the K+-pNPPase activity does not represent the phosphatase step of the H+,K+-ATPase reaction. In addition, the observed inhibition of vesicular H+ uptake by Na+ appears to be due to the displacement by Na+ of a cytosolic (extravesicular) H+ site responsible for the vectorial translocation of H+.     

Purification and functional reconstitution of the voltage-sensitive sodium channel from rabbit T-tubular membranes

The voltage-sensitive sodium channel has been purified from rabbit T-tubular membranes and reconstituted into defined phospholipid vesicles. Membranes enriched in T-tubular elements (specific [3H]nitrendipine binding = 41 +/- 9 pmol/mg of protein, n = 7) were isolated from fast skeletal muscle. After solubilization with Nonidet P-40, the sodium channel protein was purified to greater than 95% of theoretical homogeneity based on the specific activity of [3H]saxitoxin binding. Two subunits of Mr approximately 260,000 and 38,000 were found; these bands co-distributed with the peak of [3H]saxitoxin binding on sucrose gradients. The purified protein was reconstituted into egg phosphatidylcholine vesicles and retained the ability to gate specific 22Na+ influx in response to activation by batrachotoxin or veratridine. All activated fluxes were blocked by saxitoxin and tetrodotoxin. On sucrose gradients, the distribution of protein capable of functional channel activity paralleled the distribution of specific [3H]saxitoxin binding and of the Mr 260,000 and 38,000 components. The cation selectivity for the reconstituted, batrachotoxin-activated channel was Na+ greater than K+ greater than Rb+ greater than Cs+, with flux ratios of 1:0.13:0.02:0.008. Nine of 25 monoclonal antibodies raised against the rat sarcolemmal sodium channel cross-reacted with the rabbit T-tubular sodium channel in a solid-phase radioimmunoassay. Six of these antibodies showed specific binding to immunoblot transfers of T-tubular membrane proteins. Each labeled a single band at Mr approximately 260,000 corresponding in mobility to the large subunit of the sodium channel.     

Isolation and characterization of Sertoli cell plasma membranes and associated plasminogen activator activity

Plasma membranes were isolated from the cultured Sertoli cells of 20-day-old rat testes by differential centrifugation and sucrose density fractionation. The distribution and purity of subcellular components was determined by marker enzyme analysis of gradient fractions. The plasma membrane fraction showed an enrichment in two plasma membrane marker enzymes, 5'-nucleotidase and ouabain-sensitive Na+/K+-ATPase-specific activities, of 9- and 23-fold, respectively. Forty-two percent and 52% of the total cellular 5'-nucleotidase and ouabain-sensitive Na+/K+-ATPase activities, respectively, were found in the membrane fraction. The protein yield of plasma membrane was approximately 6% of the total cellular protein. Two-dimensional polyacrylamide gel electrophoresis was used to compare [35S] methionine- and [3H] glucosamine-labeled membrane proteins. The incorporation of [35S] methionine and [3H] glucosamine was increased in several proteins when the cultured Sertoli cells were treated with follicle-stimulating hormone, insulin, retinol, and testosterone. Isolated Sertoli cell membranes contained a membrane-associated form of plasminogen activator. Analysis of this plasminogen activator demonstrated that the membrane-associated enzyme existed primarily as a single 38,000-40,000-Mr form.     

Quantification of Rat Cerebral Cortex Na+,K+-ATPase: Effect of Age and Potassium Depletion

Na+,K(+)-ATPase concentration in rat cerebral cortex was studied by vanadate-facilitated [3H]ouabain binding to intact samples and by K(+)-dependent 3-O-methylfluorescein phosphatase activity determinations in crude homogenates. Methodological errors of both methods were evaluated. [3H]Ouabain binding to cerebral cortex obtained from 12-week-old rats measured incubating samples in buffer containing [3H]ouabain, and ouabain at a final concentration of 1 x 10(-6) mol/L gave a value of 11,351 +/- 177 (n = 5) pmol/g wet weight (mean +/- SEM) without any significant variation between the lobes. Evaluation of affinity for ouabain was in agreement with a heterogeneous population of [3H]ouabain binding sites. K(+)-dependent 3-O-methylfluorescein phosphatase activity in crude cerebral homogenates of age-matched rats was 7.24 +/- 0.14 (n = 5) mumol/min/g wet weight, corresponding to a Na+,K(+)-ATPase concentration of 12,209 +/- 236 pmol/g wet weight. It was concluded that the present methods were suitable for quantitative studies of cerebral cortex Na+,K(+)-ATPase. The concentration of rat cerebral cortex Na+,K(+)-ATPase showed approximately 10-fold increase within the first 4 weeks of life to reach a plateau of approximately 11,000-12,000 pmol/g wet weight, indicating a larger synthesis of Na+,K+ pumps than tissue mass in rat cerebral cortex during the first 4 weeks of development. K+ depletion induced by K(+)-deficient fodder for 2 weeks resulted in a slight tendency toward a reduction in K+ content (6%, p > 0.5) and Na+,K(+)-ATPase concentration (3%, p > 0.4) in cerebral cortex, whereas soleus muscle K+ content and Na+,K(+)-ATPase concentration were decreased by 30 (p < 0.02) and 32% (p < 0.001), respectively. Hence, during K+ depletion, cerebral cortex can maintain almost normal K+ homeostasis, whereas K+ as well as Na+,K+ pumps are lost from skeletal muscles.     

Effect of amiodarone on Na+-, K+-ATPase and Mg2+-ATPase activities in rat brain synaptosomes

Amiodarone hydrochloride is a diiodinated antiarrhythmic agent widely used in the treatment of cardiac disorders. With the increasing use of amiodarone, several untoward effects have been recognized and neuropathy following amiodarone therapy has recently been reported. The present studies were carried out to study the effect of amiodarone on rat brain synaptosomal ATPases in an effort to understand its mechanism of action. Na+, K+-ATPase and oligomycin sensitive Mg2+ ATPase activities were inhibited by amiodarone in a concentration dependent manner with IC50 values of 50 microM and 10 microM respectively. [3H]ouabain binding was also decreased in a concentration dependent manner with an IC50 value of 12 microM, and 50 microM amiodarone totally inhibited [3H]ouabain binding. Kinetics of [3H]ouabain binding studies revealed that amiodarone inhibition of [3H]ouabain binding is competitive. K+-activated p-nitrophenyl phosphatase activity showed a maximum inhibition of 32 per cent at 200 microM amiodarone. Synaptosomal ATPase activities did not show any change in rats treated with amiodarone (20 mg kg-1 day-1) for 6 weeks, when compared to controls. The treatment period may be short, since the reported neurological abnormalities in patients were observed during 3-5 years of treatment. The present results suggest that amiodarone induced neuropathy may be due to its interference with sodium dependent phosphorylation of Na+, K+-ATPase reaction, thereby affecting active ion transport phenomenon and oxidative phosphorylation resulting in low turnover of ATP in the nervous system.     

Saxitoxin and ouabain binding activity of isolated skeletal muscle membrane as indicators of surface origin and purity

A simple biochemical method for identifying and distinguishing transverse tubule and sarcolemma membranes in preparations of skeletal muscle microsomes is proposed and evaluated. This method is based on the previous observation that the ratio of ouabain to saxitoxin binding sites is five-fold higher in the sarcolemma than the transverse tubule. We measured [3H]saxitoxin and [3H]ouabain binding to microsomes of frog, rat and rabbit muscle in the presence of detergents to expose latent sites. A high density fraction (30--40% sucrose) of the membranes was identified as transverse tubule on the basis of a low ouabain/saxitoxin ratio and its association with sarcoplasmic reticulum. A low density fraction (20--30% sucrose) was identified as transverse tubule containing variable amounts of sarcolemma as judged by a higher ratio of ouabain/saxitoxin sites. Our results suggest that this ratio can be used to determine the surface origin of muscle membrane preparations. Several different methods for purifying transverse tubules were compared by this technique.     

Inactivation of Na+, K+ -ATPase from cattle brain by sodium fluoride

The influence of the physiological ligands and modifiers on the plasma membrane Na+, K+ -ATPase from calf brain inactivation by sodium fluoride (NaF) is studied. ATP-hydrolyzing activity of the enzyme was found to be more stable as to NaF inhibition than its K+ -pNPPase activity. The activatory ions of Na+, K+ -ATPase have different effects on the process of the enzyme inhibition by NaF. K+ intensifies inhibition, but Na+ does not affect it. An increase of [Mg2+free] in the incubation medium (from 0.5 to 3.0 mM) rises the sensitivity of Na+, K+ -ATPase to NaF inhibition. But an increase of [ATP] from 0.3 to 1.5 mM has no effect on this process. Ca and Mg ions modify Na+, K+ -ATPase inhibition by fluoride differently. Ca2+free levels this process, and Mg2+free on the contrary increases it. In the presence of Ca ions and in the neutral-alkaline medium (pH 7.0-8.5) the recovery of activity of the transport ATPase inhibited by-NaF takes place. Sodium citrate also protects both ATP-hydrolizing and K-pNPPase activity of the Na+, K+ -ATPase from NaF inhibition. Under the modifing membranous effects (the treatment of plasma membranes by Ds-Na and digitonin) the partial loss of Na+, K+ -ATPase sensitivity to NaF inhibition is observed. It is concluded that Na+, K+ -ATPase inactivation by NaF depends on the influence of the physiological ligands and modifiers as well as on the integrity of membrane structure.     

Specific inhibition of [3H] saxitoxin binding to skeletal muscle sodium channels by geographutoxin II, a polypeptide channel blocker

Geographutoxin II (GTX II), a peptide toxin isolated from Conus geographus, inhibited [3H]saxitoxin binding to receptor sites associated with voltage-sensitive Na channels in rat skeletal muscle homogenates and rabbit T-tubular membranes with K0.5 values of 60 nM for homogenates and 35 nM for T-tubular membranes in close agreement with concentrations that block muscle contraction. Scatchard analysis of [3H]saxitoxin binding to T-tubular membranes gave values of KD = 9.3 nM and Bmax = 300 fmol/mg of protein and revealed a primarily competitive mode of inhibition of saxitoxin binding by GTX II. The calculated KD values for GTX II were 24 nM for T-tubules and 35 nM for homogenates, respectively. In rat brain synaptosomes, GTX II caused a similar inhibitory effect on [3H]saxitoxin binding at substantially higher concentrations (K0.5 = 2 microM). In contrast, binding of [3H]batrachotoxin A 20-alpha-benzoate and 125I-labeled scorpion toxin to receptor sites associated with Na channels in synaptosomes was not affected by GTX II at concentrations up to 10 microM. Furthermore, [3H]saxitoxin binding to membranes of rat superior cervical ganglion was only blocked 10% by GTX II at 10 microM. These results indicate that GTX II interacts competitively with saxitoxin in binding at neurotoxin receptor site 1 on the sodium channel in a highly tissue-specific manner. GTX II is the first polypeptide ligand for this receptor site and the first to discriminate between this site on nerve and adult muscle sodium channels.     

Correlation between microsomal (Na+ + K+)-ATPase activity and [3H]ouabain binding to heart tissue homogenates.

ATP plus Mg2+ plus Na+ supported [3H]ouabain binding to canine left ventricular tissue homogenates and microsomal (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity from the same tissue were measured. A linear relationship was found between the initial velocity of [3H]ouabain binding to tissue homogenates and microsomal (Na+ + K+)-ATPase activity from the same tissue in the presence and absence of in vivo bound digoxin. In vivo bound digoxin reduced both measurements. With tissue from digoxin-free hearts, a linear relationship was also obtained between the initial velocity and the maximum level of [3H]ouabain binding to tissue homogenate. Binding of [3H]ouabain to whole tissue homogenate is a convenient method for estimating (Na+ + K+)-ATPase activity in small left ventricular biopsy samples.     

Subcellular distribution of K+-pNPPase (Na+,K+-ATPase) in the Golgi apparatus of developing rat cortical neurons

The distribution of K+-pNPPase (Na+,K+-ATPase) activity in the compartments of the Golgi apparatus in neurons of the cerebral cortex of young and adult Wistar rats was studied by ultrastructural cytochemistry. In adult rats, mainly the cis-most cisterna was associated with reaction deposits. In 10- and especially in 15-day-old rats, not only the cis-cisternae, but the cis- and trans-Golgi, as well as components of the Golgi stack, also revealed K+-pNPPase activity. The dynamic changes of K+ -pNPPase localization in the compartments of the neuronal Golgi complexes were discussed with respect to the biochemical evidence concerning the building, assembly and processing of Na+,K+-ATPase as plasma membrane glycoprotein. It was suggested that the high activity in the Golgi complexes seen in 15-day-old rats has to be associated with the advancing myelinization in this period and the necessity of Na+,K+-ATPase equipment of nodes of Ranvier.     

Cytochemical localization of ouabain-sensitive, K+ -dependent p-nitrophenylphosphatase and Ca++-stimulated adenosine triphosphatase activities in human parotid and submandibular glands

K+ -dependent p-nitrophenylphosphatase (pNPPase) and Ca++ -stimulated adenosine triphosphatase (ATPase) activities were studied in human parotid and submandibular glands using cytochemical methods at the ultrastructural level. In both glands, only the striated-duct epithelium showed K+ -pNPPase reaction product, thereby indicating the localization of Na+, K+ -ATPase. The precipitate was concentrated on the deep invaginations of the basolateral plasma membranes, in close association with their cytoplasmic surface. Ca++ -ATPase activity was also found on the basolateral plasma membranes, but two striking differences from the K+ -pNPPase distribution were observed: firstly, Ca++ -ATPase appeared in both acinar and ductal cells, and secondly, it was localized on the outer side of the plasma membranes.