Canine Renal Medulla Research Articles

Inhibition of the Na,K-ATPase of canine renal medulla by several local anesthetics

The ATPase activity of Na,K-ATPase-enriched membranes from canine renal medulla was determined in the absence of local anesthetic and in the presence of procaine, chloroprocaine, bupivacaine, mepivacaine, lidocaine, and two quaternary derivatives of lidocaine (QX-222 and QX-314) at 37∘C. Chloroprocaine (IC50= 13 mM) had slightly greater potency than procaine (IC50= 17.7 mM). Bupivacaine (IC50= 6.7 mM) was more potent than its congener mepivacaine (IC50> 10 mM, the solubility limit). QX-222 (IC50> 600 mM) and QX-314 (IC50= 132 mM) had less potency than lidocaine (IC50= 30.4 mM). This study supports the interpretation that the uncharged forms of local anesthetics are much more potent inhibitors of Na,K-ATPase activity than the cationic forms.

EFFECTS OF LOCAL ANAESTHETICS ON THE ACTIVITY OF THE Na,K-ATPASE OF CANINE RENAL MEDULLA

The purpose of this study is to characterize the effects of local anaesthetics on Na,K-ATPase activity. The ATPase activity of Na,K-ATPase-enriched membranes from canine renal medulla was determined in the absence and in the presence of lidocaine, procaine, tetracaine, benzocaine, bupivacaine, prilocaine, and procainamide at 37 and 25°C. All of these local anaesthetics, except benzocaine, inhibit the activity of the Na,K-ATPase of canine renal medulla at both 25 and 37°C. Benzocaine inhibits Na,K-ATPase activity at 37°C, but stimulates activity at 25°C. The influence of lidocaine on stimulation of Na,K-ATPase activity by Na+and K+was investigated. Lidocaine increases the apparentK0.5 of the Na,K-ATPase for both Na+and K+and decreases the Vmaxvalues for both ions. IC50values for lidocaine increase with increasing concentrations of both Na+and K+. The data indicate that lidocaine diminishes the affinity of the Na,K-ATPase for Na+and K+and that binding of Na+or K+decreases the potency of lidocaine as an inhibitor of the Na,K-ATPase. Lidocaine markedly decreases the affinity of the Na,K-ATPase for ouabain, but only slightly diminishes the maximum amount of ouabain bound. Unprotonated lidocaine is apparently a more potent inhibitor than is the protonated form. 2000 Academic Press@p$hr

EFFECTS OF GENERAL ANAESTHETICS ON THE ACTIVITY OF THE Na,K-ATPASE OF CANINE RENAL MEDULLA

Several previous studies have reported inhibition of Na,K-ATPase activity by chlorpromazine, phenobarbital and pentobarbital, thiopental, and monoketones. The purpose of this study is to investigate the influences of other general anaesthetics on Na,K-ATPase activity. The ATPase activity of Na,K-ATPase-enriched membranes from canine renal medulla was determined at 37°C in the absence and in the presence of hexanol, diethylether, halothane, and propofol. The influence of hexanol on stimulation of Na,K-ATPase activity by Na+and K+was investigated. Hexanol, diethylether, halothane, and propofol inhibited the activity at 37°C of the Na,K-ATPase of canine renal medulla. The IC50values at 37°C were: hexanol, 12.3 m m; diethylether, 170 m m; halothane, 7.35 m m; propofol, 0.127 mm . Hexanol increased the K0.5of the Na,K-ATPase for K+at 37°C, but did not affect theK0.5 for Na+. At lower [K+] hexanol was a more potent inhibitor than at higher [K+].pc 1999 Academic Press@p$hr

Characterization of partially purified prostaglandin E 2 receptor from the canine renal medulla: evidence for physical association of the receptor protein with the inhibitory guanine nucleotide-binding protein

Prostaglandin (PG) E 2 binding protein, a putative PGE 2 receptor, was purified 26-fold with 0.4% recovery from canine renal outer medullary membranes solubilized with 12% digitonin with the sequential use of a Superose 12, Wheat Germ Agglutinin (WGA) Affigel 10, DEAE-5PW and Ampholine column chromatographies. The final preparation retained the binding activity specific for PGE 2, but lost most of the sensitivity to guanosine-5′-(γ-thio)triphosphte (GTPγS). An antibody against α subunit of the inhibitory guanine nucleotide-binding protein (αGi) 1 and αGi 2 or that against common sequences of α subunit of guanine nucleotide-binding proteins (αG common) reacted a 41 kDa protein in the sample at each step of purification, but failed to do so in the final preparation. An antibody against αGi 3 or αGo had no effect. In fact, peaks of the binding activity and immunoreactivity for αGi 1,2 were chromatographically separated by isoelectric focusing. Moreover, antibodies against αG common or αGi 1,2, but not that against αGi 3 and αGo, precipitated PGE 2 binding activity in the active fractions of WGA-Affigel 10 column chromatography. These results suggest that the PGE 2 receptor is an acidic glycoprotein and that Gi 1 or Gi 2 is physically associated with the PGE 2 receptor and dissociates from the receptor protein during purification procedures.

Determination of subunit molecular weights of canine renal (Na+,K+)-ATPase by low-angle laser light scattering coupled with high performance gel chromatography in the presence of sodium dodecyl sulfate.

Subunit molecular weights of the (Na+,K+)-ATPase of canine renal outer medulla were estimated in the presence of sodium dodecyl sulfate (SDS) by the measuring system consisted of components connected in the following sequence: a TSK-gel G3000 SW column, a UV spectrophotometer, a low-angle laser light scattering photometer, and a differential refractometer. Polypeptide molecular weights of the alpha- and beta-subunits were determined to be 117,900 and 39,400, respectively. The measurement required the extinction coefficient at 280 nm of the sample polypeptide in addition to the outputs from the three detectors. The extinction coefficients at 280 nm of the alpha- and beta-subunits were determined to be 0.931 and 1.41 ml X mg-1 X cm-1, respectively by the quantitative amino acid analysis. The above procedure seems to be most appropriate to determine uniquely the composition of subunits molecular weights of an oligomeric membrane protein.

Association of a solubilized prostaglandin E2 receptor from renal medulla with a pertussis toxin-reactive guanine nucleotide regulatory protein.

Prostaglandin E2 (PGE2) was found to bind specifically, reversibly, and in a protein-dependent manner to a single class of high affinity (KD approximately equal to 20 nM) binding sites in membranes prepared from canine renal outer medulla. PGE2 binding activity was solubilized from these membranes in a stable form (t1/2 greater than 14 days) in the absence of ligand in 75% yields using digitonin. The characteristics of PGE2 binding to membranes and solubilized protein were similar with respect to pH dependence, KD for PGE2, and order of potency of prostaglandins (PGE2 approximately PGE1 greater than PGF2 alpha greater than PGD2) in inhibiting the binding of [3H]PGE2. Importantly, the extents of binding of PGE2 to membranes and to a solubilized preparation partially purified by chromatography on wheat germ agglutinin-Affi-Gel 10 were both increased about 2-fold by GDP and GTP and its analogs. Treatment of the digitonin-solubilized PGE2 binding activity with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS) rendered the binding activity insensitive to stimulation by GTP and decreased the apparent molecular weight of the peak of PGE2 binding activity from about 175,000 to about 65,000. These results suggest that the PGE2 binding activity resides in a protein which is tightly associated with, but distinct from, a guanine nucleotide regulatory (N) protein. PGE2 (greater than or equal to 10 nM) was found to stimulate GTPase activity of renal outer medullary membranes, and this stimulation was eliminated by pretreatment of membranes with pertussis toxin and NAD, but not cholera toxin and NAD. Treatment of both particulate and solubilized preparations of PGE2 binding activity with pertussis toxin plus NAD also eliminated the ability of GTP to stimulate PGE2 binding. This evidence indicates that it is the inhibitory guanine nucleotide regulatory protein, Ni, with which the PGE2 binding activity is associated. Thus, this PGE2 binding activity is an inhibitory PGE2 receptor, quite possibly one that mediates inhibition of vasopressin-induced cAMP formation in the medullary thick ascending limb and/or collecting tubule of the kidney.

Modified cation activation of the (Na+K)-ATPase following treatment with thimerosal

Treatment of the Na,K-ATPase enzyme, isolated from canine renal outer medulla, with thimerosal (ethylmercurithiosalicylate) resulted in significant inhibition of the overall Na,K-ATPase with only slight, if any, inhibition of the Na-ATPase and ATP:ADP exchange activities. The K-stimulated PNPPase activity was stimulated [see G. R. Henderson and A. Askari (1977) Arch. Biochem. Biophys., 182, 221–226] . Examination of the Na dependence of the ATPase and ATP:ADP exchange activities revealed an Na-independent, ouabain-sensitive activity that was inhibited by Na in the range 0–10 m m. At ⩾ 10 m m concentration of Na the treated and modified enzymes showed similar activities. The apparent affinity of the modified enzyme for ATP in the presence of 100 m m Na was the same as that of the untreated enzyme (0.2–0.3 μ m). In the absence of Na, the modified enzyme hydrolyzed ATP with a relatively low affinity (about 120 μ m). The enhancement of p-nitrophenylphosphatase (PNPPase) activity measured in the presence of K ions was due to the appearance of K-independent, ouabain-sensitive PNPP activity. The modification was without major affect on the apparent affinity of the enzyme for K ions in the PNPPase activity. Treatment of the thimerosal-modified enzyme with dithiothreitol removed (or greatly reduced) the cation-independent, ouabain-sensitive activities and the Na,K-ATPase activity returned. Modification of a set of enzyme -SH groups in the Na,K-ATPase enzyme made it able to hydrolyze ATP in the absence of Na ions and PNPP in the absence of K ions. The -SH groups modified by thimerosal are evidently critical to the major dephosphoenzyme conformational changes but are not involved in the major transport conformational change between phosphoenzymes.

Enzyme Cytochemistry of Ventricular Choroid Plexus

The peroxidase-antiperoxidase (PAP) method was employed to determine the ultrastructural localization of Na+, K+-adenosinetriphosphatase (ATPase) in normal adult canine choroidal epithelium. The α- and β-subunits of Na+, K+-ATPase were prepared from Na+, K+-ATPase holoenzyme, which was purified from canine renal outer medulla. Rabbit antisera to both subunits were then produced. The intense immunoreactive electron-dense products were confined along the apical plasmalemma, including that of microvilli of choroidal epithelial cells. This finding is compatible with that of the authors' previous report by light microscopic immunohistochemistry. It can be inferred that the apical border of the epithelium is a very important site for cerebrospinal fluid secretion.

Enzyme histochemistry on ventricular choroid plexus (Part VII). Immunohistochemical localization of Na+, K+-ATPase

To determine the immunohistochemical localization of Na+, K+-ATPase in the normal adult canine and rat choroidal epithelium, the indirect immunoperoxidase technique was employed. Rabbit antiserum to purified canine renal outer medulla Na+, K+-ATPase was prepared. The specificity of the antiserum was confirmed by Ouchterlony's method, biochemical inhibitory effect, and au toradio graphic immunoblotting method. When the rabbit immunoglobulin that was purified from the Na+, K+-ATPase antiserum through DEAE (diethylaminoethyl) -cellulose ion exchange chromatography was used for immunoperoxidase staining of the choroid plexus, intense immunoreactive staining was present on the epithelial cells of the choroid plexus in both animals, but was not found in the tissue around the vessels. Staining was especially confined to the apical surfaces of the epithelial cells. Both the canine and rat choroid plexus are rich in Na+, K+-ATPase, and it can be estimated that the enzyme plays an important role in cerebrospinal fluid secretion.

Molecular weights of αβ-protomeric and oligomeric units of soluble (Na +, K +)-ATPase determined by low-angle laser light scattering after high-performance gel chromatography

The (Na +, K +)-ATPase of canine renal outer medulla was solubized with a nonionic surfactant, octaethylene glycol n-dodecyl ether (C 12E 8), in the presence of 0.2 M sodium ion. The solubilized ATPase retained 74% of the enzymatic activity expressed before solubilization. Molecular species of the solubilized ATPase were analyzed by high-performance chromatography through a TSK-GEL G3000SW column in the presence of 1 mg/ml C 12E 8 at 23°C. The eluate was monitored by one or two monitors Chosen from the following: an ultraviolet absorption monitor, a precision differential refractometer and a low-angle laser light scattering photometer. The three kinds of elution pattern thus obtained can best be interpreted by assuming the presence of at least four kinds of protein component with molecular weights 1 740 000 ± 230 000, 836 000 ± 82 000, 286 000 ± 30 000 and 123 000 ± 8 000, respectively. Among them, those with the last two molecular weight were the major components. The amounts of the first three components were found to increase with time during the incubation before application to the column at the expense of that of the last one. The amounts of the last two were 18 and 73%, respectively, when measure immediately after the solubilization. A stoichiometric composition of 1:1 molar ratio for the α and β polypeptide chains was obtained for the two major components as well as for the intact ATPase by high-performance gel chromatography in the presence of sodium dodecyl sulfate using the same column as above. The (Na +, K +)-ATPase was, thus, indicated to be solubilized with C 12E 8 to give the αβ-protomer and its dimer as the main components.

Enzyme histochemistry on ventricular choroid plexus (Part III). The localization of glucose-6-phosphatase and thiamine pyrophosphatase (author's transl)

To determine the immunohistochemical localization of Na+, K+-ATPase in the normal adult canine and rat choroidal epithelium, the indirect immunoperoxidase technique was employed. Rabbit antiserum to purified canine renal outer medulla Na+, K+-ATPase was prepared. The specificity of the antiserum was confirmed by Ouchterlony's method, biochemical inhibitory effect, and au toradio graphic immunoblotting method. When the rabbit immunoglobulin that was purified from the Na+, K+-ATPase antiserum through DEAE (diethylaminoethyl) -cellulose ion exchange chromatography was used for immunoperoxidase staining of the choroid plexus, intense immunoreactive staining was present on the epithelial cells of the choroid plexus in both animals, but was not found in the tissue around the vessels. Staining was especially confined to the apical surfaces of the epithelial cells. Both the canine and rat choroid plexus are rich in Na+, K+-ATPase, and it can be estimated that the enzyme plays an important role in cerebrospinal fluid secretion.

Tryptic inactivation of the ouabain binding site of canine kidney Na +, K +-ATPase and its effect on catalytic function

Trypsin treatment of the purified Na +, K +-ATPase from canine renal outer medulla causes loss of ADP-ATP exchange activity when digestion takes place in 0.1 M KCl. Activity surviving this treatment remains inhibitable by ouabain. Addition of ATP to such digestion mixtures stabilizes the Na +, K +-ATPase in a different conformation (Na +-form). Under these conditions ADP-ATP exchange activity is protected, and becomes ouabain-insensitive. Quantitative analysis of the cleavage products and rates of loss of ouabain binding and exchange activity suggest that catalytically inactive trypsinolysis products can bind ouabain, and that the 85,000 dalton fragment associated with ouabain-insensitive ADP-ATP exchange activity cannot bind ouabain. Cleavage to produce the 85,000 dalton fragment therefore destroys the ouabain binding site.

Purification of distinct plasma membranes from canine renal medulla

Two types of plasma membrane were purified from canine distal renal medulla by the techniques of differential and zonal density-gradient centrifugation followed by free-flow electrophoresis. One group of plasma membranes was identified as basal-laterally derived based on a 30-fold enrichment of Na-K-ATPase, a 20-fold enrichment of vasopressin-stimulated adenylate cyclase, and a 33-fold enrichment of [3H]vasopressin binding sites. The second type of plasma membrane was free of these markers, but had a cholesterol and phospholipid composition similar to them. Alkaline phosphatase also had a similar distribution in the two fractions. This lighter membrane fraction contained a membrane-bound cyclic AMP-dependent protein kinase as well as substrate for this kinase. In addition there was a 26-fold enrichment of specific activity of an anion (SO32-)-activated ATPase which was insensitive to mitochondrial ATPase inhibitor protein, in contrast to the mitochondrial fraction of the tissue. Based on the relative preponderance of collecting duct tissue in the distal medulla and the yield of membrane protein, these membranes are tentatively identified as containing apical membranes of the collecting duct.

Two soluble forms of guanosine 5'-(beta,gamma-imino)triphosphate and fluoride-activated adenylate cyclase.

Soluble and membrane-bound adenylate cyclase from the canine renal medulla are very slowly activated by guanosine 5'-(beta,gamma-imino)triphosphate (Gpp(NH)p) or fluoride. At 25 degrees, 8 to 10 h of incubation of the soluble enzyme, and 6 to 8 h of incubation of the membrane-bound enzyme are required to reach the maximal activity. The dependence of activation on concentration of Gpp(NH)p changes with time. Half-maximal activation of soluble adenylate cyclase occurs at 3 +/- 1 X 10(-6) M Gpp(NH)p if the activity is measured without preincubation with Gpp(NH)pp and at 4 +/- 2 X 10(-8) M if it is measured after 6 to 22 h of incubation with Gpp(NH)p at 25 degrees. The activation occurs over a rather broad range of nucleotide concentration and cannot, therefore, be simply interpreted to reflect a function of nucleotide binding. Two forms of soluble adenylate cyclase are resolved by Sepharose gel filtration. One form, with a Stokes radius of 71 A, is rapidly activated by Gpp(NH)p; the other, with a Stokes radius of 56 A, requires long incubation with Gpp(NH)p to be activated. The sedimentation coefficient of the two forms is 7.3 S. The apparent molecular weight of the rapidly activated form is 200,000 while that of the slowly activated form is 160,000. The interrelationship of these two species of adenylate cyclase is not clear and is under investigation.

Involvement of microtubules and microfilalments in the action of vasopressin in canine renal medulla

AbstractVasopressin‐stimulated cyclic AMP content and the uptake of 3H2O and 22Na into canine renal medullary slices were measured. Cyclic AMP was increased threefold by 9 × 10−9M vasopressin in isotonic (290 mOsm/kg H2O) Krebs‐Ringers bicarbonate. A significant increase in vasopressin‐stimulated 3H2O uptake began at 2.75 min after hormone addition and lasted until 5.00 min. Colchicine (1 × 10−5 M) inhibited the vasopressin‐ stimulated 3H2O uptake. This effect required a minimum preincubation period of 30–40 min in colchicine‐containing medium. Colchicine had no effect on basal or vasopressin‐stimulated cyclic AMP (10 mM). Lumicolchicine (10−5M) had no effect on either vasopression‐ or dibutyryl cyclic AMP‐stimulated 3H2O uptake. 14 C‐colchicine bound predominantly to the cytosol fraction enriched in microtubules, while virtually no binding was observed on plasma membranes. Loght‐microscopic examinations of cross sections of tissue slices showed that a majority of vasopressin‐treated collecting tybulels and some control tubules had occluded lumens. Colchicine‐treated cells, in the presence of vasopressin, had open lumens indicating a blockage of the vasopressin‐induced water transport. Cells treated with cytochalasin B (1 μgm/ml) also had open lumens in the presence of vasopressin. Cytochalasin B also blocked vasopressin and dibutyryl cyclic AMP‐stimulated 3H2O uptake into collecting duct cells but had no effect on vasopressin stimulated cyclic AMP levels. It was concluded that microtubules and possibley microfilaments are involved in the subcellular mechanism by which vasopresssin increases the permeability of the collecting duct to water.

Effects of (Na+ + K+)-ATPase-specific antibodies of enzymatic activity and monovalent cation transport.

Antibodies have been obtained that specifically interact with the transport enzyme (Na+ + K+)-activated ATPase. The antigen used was purified (Na+ + Ka+)-ATPase from canine renal medulla. Purified gamma globulin from immunized animals, but not from control animals or preimmune serum, inhibited (Na+ + Ka+)-ATPase from canine renal medullar with reduction of activity to 33 +/- 4 (SD)% in concentration-dependent manner. Maximum inhibition occurred in less than 5 minutes at 37 degrees C. The Mg++ -dependent, nonouabain inhibited component of activity (Mg++ -ATPase) was unaffected. Fab fragments obtained by papain cleavage of the gamma globulin fraction had similar inhibitory activity and specificity. These antibodies also produced varying degrees of concentration-related inhibition of canine myocardial, calf brain, and human red cell ghost (Na++ + Ka+)-ATPase, but not Mg++-ATPase activity.

Interaction of ethacrynic acid and cysteine with renal medullary adenylate cyclase

Results of the present study indicate that (1) ethacrynic acid, dihydroethacrynic acid, and the cysteine adduct of ethacrynic acid inhibit plasma membrane-bound adenylate cyclase from canine renal medulla; (2) reaction with a sulfhydryl group is not essential for inhibition by ethacrynic acid and its derivatives, but may contribute quantitatively to the inhibition; and (3) cysteine enhances the activity of renal medullary adenylate cyclase in the presence of a vasopressin analog or sodium fluoride. Observations support the view that ethacrynic acid and its cysteine adduct interfere with the action of vasopressin on the distal nephron at the site of renal medullary adenylate cyclase.

Reconstitution of Active Transport Catalyzed by the Purified Sodium and Potassium Ion-stimulated Adenosine Triphosphatase from Canine Renal Medulla

Abstract Microsomal sodium and potassium ion-stimulated adenosine triphosphatase ((Na+ + K+)-ATPase) was prepared from canine renal medulla by the method of Kyte. The two polypeptides of the enzyme were co-purified to homogeneity by solubilization with sodium cholate in the presence of egg lecithin and by removal of contaminating protein by sedimentation. This purified (Na+ + K+)-ATPase was reconstituted into lipid vesicles by slow removal of the cholate. A fraction of this enzyme was oriented in the vesicle membranes in such a way as to catalyze active uptake of 22Na+, dependent on externally added ATP and inhibitable by internally trapped cardiac glycosides, to a level 3-fold higher (60 mm) than the initial concentration of Na+ within the vesicles (20 mm). Double label experiments with 42K+ and 22Na+ indicated that the ratio of K+ efflux to Na+ uptake is far below the 2:3 ratio of K+ influx to Na+ efflux observed in nerve axons and erythrocyte ghosts. Parallel experiments employing 36Cl- and 22Na+ demonstrated that 36Cl- is co-transported along with 22Na+ in the amount necessary to maintain bulk electrical neutrality of charge transported across the membrane. The mechanism of selective transport of Cl- along with actively pumped Na+, rather than exchange of K+ for Na+, cannot be explained by the observation that the permeability of the vesicles to Cl- is roughly 2-fold higher than that for K+. It appears that this reconstituted (Na+ + K+)-ATPase either is capable of pumping Cl- as well as Na+ or is equipped with some specific mechanism for translocating Cl- along with actively pumped Na+.

Effects of an antibody to a highly purified Na+, K+-ATPase from canine renal medulla: separation of the "holoenzyme antibody" into catalytic and cardiac glycoside receptor-specific components.

An antiserum was prepared against a highly purified Na(+), K(+)-adenosine triphosphatase (ATP phosphohydrolase, EC 3.6.1.3). Purification and fractionation yielded two globulin components, one of which appears specific for a digitalis glycoside receptor site or binding conformation and the other for a catalytic site or conformation.

Properties of the Two Polypeptides of Sodium- and Potassium-dependent Adenosine Triphosphatase

Abstract The (Na+ + K+)-dependent adenosine triphosphatase from canine renal medulla is composed of lipids and of two polypeptide chains. The larger one has a molecular weight of 135,000, NH2-terminal glycine, and has a high content of non-polar amino acids. The smaller polypeptide chain has NH2-terminal alanine and it is a sialoglycoprotein. The two polypeptides are present in a mass ratio of 1.7:1 for the ratio of large to small chain. This mass ratio probably corresponds to a molar ratio of one large chain to two small chains. The two chains are close to one another in the purified enzyme because they can be covalently crosslinked by dimethyl suberimidate. Finally, the large polypeptide chain, which has all of the properties of a membrane-bound enzyme, is soluble in simple aqueous solvents in the absence of detergents and lipids, although it no longer has enzymatic activity.