Two novel peripheral membrane proteins, pasin 1 and pasin 2, associated with Na+,K(+)-ATPase in various cells and tissues.

Purification of pig kidney Na+,K(+)-ATPase at low concentrations of SDS (0.5%) allowed copurification of several peripheral membrane proteins. Some of these associated proteins were identified as components of the membrane cytoskeleton. Here we describe two novel globular proteins of of Mr 77,000 (pasin 1) and Mr 73,000 (pasin 2) which copurify and coimmunoprecipitate with Na+,K(+)-ATPase and can be stripped off Na+,K(+)-ATPase microsomes by 1 M KCl. Pasin 1 and pasin 2 were detected by immunoblot analysis in various cells and tissues including erythrocytes and platelets. Immunostaining revealed colocalization of pasin 1 and Na+,K(+)-ATPase along the basolateral cell surface of epithelial cells of kidney tubules and parotid striated ducts (titers of pasin 2 antibodies were too weak for immunocytochemistry). In erythrocytes, pasin 1 and pasin 2 are minor components bound to the cytoplasmic surface of the plasma membrane. Pasin 1 showed the same electrophoretic mobility as protein 4.1b. However, both proteins have different isoelectric points (pasin 1, pI 6; protein 4.1, pI 7), different chymotryptic fragments, and are immunologically unrelated. Short pieces of sequence obtained from pasin 1 and pasin 2 were not found in any other known protein sequence. The occurrence of pasin 1 and pasin 2 in diverse cells and tissues and their association with Na+,K(+)-ATPase suggests a general role of these proteins in Na+,K(+)-ATPase function.

Mechanisms of inhibition of DNA synthesis by 2-chlorodeoxyadenosine in human lymphoblastic cells.

2-Chloro-2'-deoxyadenosine 5'-triphosphate (CldATP) is a strong inhibitor of the reduction of ADP, CDP, UDP and GDP by ribonucleotide reductase in extracts of CCRF-CEM with 50% inhibition at concentrations of 0.1 to 0.3 microM. In cells exposed to 0.3 microM 2-chloro-2'-deoxyadenosine (CldAdo), the intracellular concentration of CldATP reaches 2 microM within 15 min, and DNA synthesis by the cells is inhibited 90% within 30 min. At concentrations of extracellular CldAdo that inhibit DNA synthesis, there is also marked inhibition of intracellular conversion of cytidine to deoxycytidine nucleotides indicating significant intracellular inhibition of ribonucleotide reductase. Exposure of cells to 0.3 microM CldAdo decreases dCTP by 63% in 30 min, dATP and dTTP by 20% and dGTP by a smaller amount. Similar decreases in these pools occur when other inhibitors of ribonucleotide reductase are present at concentrations causing similar inhibition of DNA synthesis. Deoxycytidine treatment of cells inhibited by CldAdo restores dCTP and other pools, but restoration of DNA synthesis is incomplete, indicating that there is another mechanism for inhibition of DNA synthesis in addition to depletion of deoxyribonucleotide pools. This alternate mechanism is probably related to the incorporation of CldAdo into DNA that occurs despite a 25-times lower intracellular level of CldATP than dATP.

Colocalization and coprecipitation of ankyrin and Na+,K+-ATPase in kidney epithelial cells.

Interactions between integral proteins of the plasma membrane and the cytoskeleton may be important for localizing certain membrane proteins in a nonrandom fashion at specialized domains of the cell surface. Here, we show that ankyrin, the key protein for the linkage of the erythrocyte anion exchanger (band 3) to the spectrin-based membrane cytoskeleton, is also present in kidney distal tubular cells where ankyrin is precisely colocalized with Na+,K+-ATPase. Both proteins are confined to the basolateral plasma membrane and are absent from the apical membrane, the junctional complex and the membrane surface that contacts the basal lamina. Purified Na+,K+-ATPase of sheep and pig kidney contains a binding site for erythrocyte ankyrin as demonstrated by immunoprecipitation experiments. A band 3-like binding site for ankyrin is likely, since binding of ankyrin to Na+,K+-ATPase could be inhibited in a competitive fashion by the isolated cytoplasmic domain of erythrocyte band 3.

Demonstration of immunoreactive forms of erythrocyte protein 4.2 in nonerythroid cells and tissues.

Protein 4.2 is a major component of the erythrocyte membrane cytoskeleton. Here we show that immunoreactive forms of human (Mr 72,000) and pig (Mr 75,000) protein 4.2 are also associated with the plasma membrane of various nonerythroid cells and tissues, such as platelets, brain, and kidney. Protein 4.2 can be extracted from platelet membranes under the same conditions (pH 11, 1 M KI, 1 M urea) which are required to extract protein 4.2 from the erythrocyte plasma membrane. The demonstration of protein 4.2 in nucleated cells that contain also several other proteins of the erythrocyte membrane cytoskeleton indicates some general principles underlying the molecular construction of the plasma membrane in erythrocytes and nonerythroid cells.

Targeting of phosphomannosyl-deficient arylsulfatase A to lysosomes of I-cell fibroblasts.

Fibroblasts from I-cell disease, a genetically-determined lysosomal storage disease, are shown to contain large amounts of phase-dense lysosomes. These lysosomes accumulated acridine orange and were specifically labeled with antibodies to arylsulfatase A. In normal skin fibroblasts the number of arylsulfatase-containing lysosomes was considerably lower. By immunocytochemistry, metabolic labeling and enzyme assay, the arylsulfatase A in I-cell fibroblasts was shown to be synthesized, stored and secreted at a level that was several-fold higher than that present in heterozygous I-cell or normal fibroblasts. Arylsulfatase A in I-cell fibroblasts differed from arylsulfatase in normal fibroblasts by the absence of endoglycosidase H-sensitive phosphorylated oligosaccharides. These findings indicate that arylsulfatase A in I-cells is targeted to lysosomes by a mechanism that does not appear to involve the phosphorylated mannose marker.

Association of kidney and parotid Na+, K(+)-ATPase microsomes with actin and analogs of spectrin and ankyrin.

Kidney Na+,K(+)-ATPase has been recently shown to bind erythroid ankyrin and to colocalize with ankyrin at the basolateral cell surface of kidney epithelial cells. These observations suggest that Na+,K(+)-ATPase is linked via ankyrin to the spectrin/actin-based membrane cytoskeleton. In the present study we show that Na+,K(+)-ATPase and analogs of spectrin, ankyrin and actin copurify from detergent extracts of pig kidney and parotid gland membranes. Actin, spectrin and ankyrin were extracted from purified Na+,K(+)-ATPase microsomes at virtually identical conditions as their counterparts from the erythrocyte membrane, i.e., 1 mM EDTA (spectrin, actin) and 1 M KCl (ankyrin). Visualization of the stripped proteins by rotary shadowing revealed numerous elongated spectrin-like dimers (100 nm) and tetramers (215 nm), a fraction of which (17%) was associated with globular (10 nm) ankyrin-like particles. Like erythrocyte ankyrin, kidney ankyrin was cleaved into a soluble 72 kDa fragment and a membrane-bound 90 kDa fragment. Consistent with our previous immunocytochemical findings on the pig kidney, Na+,K(+)-ATPase and ankyrin were found to be colocalized at the basolateral plasma membrane of striated ducts and acini of the pig parotid gland. The present findings confirm and extend the recently proposed concept that in polarized epithelial cells Na+,K(+)-ATPase may serve as major attachment site for the spectrin-based membrane cytoskeleton to the basolateral cell domain. Connections of integral membrane proteins to the cytoskeleton may help to place these proteins at specialized domains of the cell surface and to prevent them from endocytosis.

The effects of cimetidine on theophylline pharmacokinetics at steady state.

The effects of cimetidine on the pharmacokinetics of theophylline were studied in seven healthy subjects at steady state after oral dosing. Aminophylline (200 mg) was administered every six hours for three days (A), then aminophylline (200 mg) and cimetidine (300 mg) were administered every six hours for seven days (A-C). Multiple serum theophylline determinations were made after the last dose on each regimen using an enzyme immunoassay. Cimetidine decreased the apparent total body clearance of theophylline an average of 29 percent (range: 18-47 percent) from 39.5 +/- 11.7 to 28.1 +/- 3.4 ml/hr/kg (p less than 0.02). There was no significant change in the volume of distribution; however, the terminal half-life increased from 7.3 +/- 1.5 to 10.1 +/- 2.1 hr (p less than 0.01) during A and A-C, respectively. The increase in the average steady state serum theophylline concentration of 34 percent (range: 23-87 percent) observed in our subjects may have important clinical implications.

The effect of thyrotropin and cAMP on DNA synthesis and cell growth of human thyrocytes in monolayer culture.

Monolayer cultures of human thyrocytes from normal tissue (n = 10), and adenomas (n = 7), differentiated (n = 4), poorly differentiated (n = 2), and undifferentiated (n = 3) thyroid cancers were established to assess the significance of thyrotropin (TSH) and cAMP (adenosine 3',5'-cyclic monophosphate) on cell growth and DNA (deoxyribonucleic acid) synthesis. Cell growth of thyrocytes from normal and adenomatous tissues increased more rapidly (p less than 0.01) after TSH (0.1 IU/ml) was added but was unaffected by cAMP (10(-4) mol/L). In these cells, TSH also enhanced DNA synthesis twofold to twelvefold (p less than 0.01). The adenylate cyclase (AC) inhibitor, 2',3' dideoxyadenosine (ddA), increased DNA synthesis 1.3 to 6 times at a concentration of 2 X 10(-4) mol, whereas the membrane/passable cAMP analogue, dibutyryl-cAMP, and the AC stimulator, forskolin, failed to show any effect on DNA synthesis up to a concentration of 10(-5) mol/L (p less than NS). When administered simultaneously, TSH (1/2 maximum) and ddA (20 mumol) had no cumulative effect on DNA synthesis (p = NS). TSH stimulation in cancerous thyroid tissue (n = 11) demonstrated a lack of TSH response in seven of 11 monolayer cultures with no apparent correlation to cancer differentiation, patient age, or sex. Thus TSH was demonstrated to stimulate DNA synthesis and cell growth of human thyrocytes in monolayer cultures independent of the AC system. However, the TSH effect on cell growth and DNA synthesis was unpredictable in thyrocytes from cancerous tissues.

Acute effects of furosemide on blood electrolytes and hemodynamics in dogs.

The effects of furosemide on the hemodynamics, blood electrolytes, and urinary output in 5 anesthetized dogs were studied. There were no significant changes in blood Na+ or Ca++ levels, but K+ decreased significantly after 15 minutes of furosemide treatment. There were no significant changes in the blood pressure, heart rate, left ventricular systolic pressure, index of left ventricular contractility [(dp/dt)/IIP], or systemic vascular resistance. Left ventricular dp/dt decreased for 30 to 60 minutes. Later the dp/dt and (dp/dt)/IIP of left ventricular pressure exceeded control values, although increases were not significant. Left ventricular work index and stroke volume decreased significantly between 30 and 90 minutes. The cardiac output and cardiac index also decreased. Left ventricular end-diastolic pressure decreased significantly only at 30 minutes. Cardiac function remained unchanged and consistent with the electrolytes changes. Although there was a marked diuresis, which normally must have significantly decreased the effective blood volume and hence the myocardial contractility, the cardiac function remained unchanged. These results suggests that furosemide might have a direct effect on the myocardium. Clinical improvement in patients might be the result of a direct effect on the myocardium aside from its effect due to diuresis.

Cardiovascular function in dogs with acute hypokalemia.

The effects of acute hypokalemia on plasma electrolytes, cardiovascular function, osmolality, and hematocrit were investigated in anesthetized dogs for 2 hours. There was progressive increase in the total systemic vascular resistance, but no change in the osmolality and blood hematocrit. All of the hemodynamic parameters decreased except the preferred index of myocardial contractility, [dp/dt]/IIP, which increased during hypokalemia. The changes in this index of myocardial contractility were associated with changes in the plasma potassium but not the plasma sodium. The results suggest that hypokalemia-induced increases in myocardial contractility might be associated with an increased influx of Ca++ as a result of hypokalemia-induced inhibition of sarcolemmal Mg++ -dependent, Na+-K+-ATPase.

[Expert assessment of medical malpractice]. / Begutachtung ärztlicher Behandlungsfehler.

Conciliation boards are advisory commissions of the medical societies. They deal with clarification of extrajudicial patient claims based on factual or putative treatment mistakes. In perhaps 90% of the cases, an extrajudicial solution is possible, whereby the federal average for recognition is 30%. Within the framework of this study, first the legal basis for the liability of doctors is explained, especially the importance of patient information and consent as well as the responsibility for documentation. The material of the conciliation board of the medical society of Saxony consists of 1375 medical expert opinions rendered between 1 January 1992 and 23 September 2000, of which 165 cases were due to orthopedic treatment. The most common health injuries resulting in compensation claims are described.

Incorporation of 2-halogeno-2'-deoxyadenosine 5-triphosphates into DNA during replication by human polymerases alpha and beta.

Extension of synthetic primers by purified human polymerase alpha (Hpol alpha) with the (+)-strand of M13mp18 DNA as template encounters numerous specific pause sites on the M13 template. Some of these are regions of template secondary structure, at others the template codes for incorporation of the same base in multiple consecutive positions, but at some the responsible feature in the sequence is not obvious. 2-Chloro-dATP (CldATP) substitutes efficiently for dATP in such chain extension, with 2-chloroadenine (ClA) incorporation into many positions coding for A. However, there are more sites where extension is interrupted than with all four normal nucleotide substrates, particularly (but not exclusively) at template secondary structure and sites of multiple consecutive ClA insertion. DNA synthesis from normal substrates by Hpol beta in this system shows less frequent and less marked pauses, but with CldATP substituted for dATP chain extension is limited because of marked slowing of extension at sites of multiple consecutive ClA insertion. With either polymerase, the rate of extension is decreased even more at such regions when bromo-dATP is used as substrate. Some misincorporation of ClA instead of G or T can occur at certain sites in absence of the corresponding normal substrate, but misincorporation as C is rare. CldATP is a very weak inhibitor of chain extension by Hpol alpha, but a somewhat better inhibitor of Hpol beta. These results may account in part for the inhibition of DNA synthesis in cells exposed to 2-chlorodeoxyadenosine or 2-bromodeoxyadenosine.

Identification of a binding motif for ankyrin on the alpha-subunit of Na+,K(+)-ATPase.

Cytoskeleton membrane associations are important for a variety of cellular functions. The anion exchanger of erythrocytes (AE1) and Na+,K(+)-ATPase of polarized epithelial cells provide well studied examples of how integral membrane proteins are anchored via the linker molecule ankyrin to the spectrin-based membrane cytoskeleton. In the present study we have generated several recombinant fragments of the large (third) cytoplasmic domain (CD3) of Na+,K(+)-ATPase to define binding sites of ankyrin on CD3 at a molecular level. We provide evidence that a cluster of four amino acids, ALLK, is essential for binding of ankyrin to both recombinant CD3 and to native Na+,K(+)-ATPase. Once bound, conformational changes might uncover further binding sites for ankyrin on Na+,K(+)-ATPase. A motif related to the ALLK cluster is also present in the cytoplasmic domain of AE1 where this sequence (ALLLK) turned out to be also important for ankyrin binding. These motifs are highly conserved during evolution of both Na+,K(+)-ATPase and AE1, further underlining their potential role in cytoskeleton to membrane linkage.

Bacterial exotoxins and endothelial permeability for water and albumin in vitro.

Effects of Staphylococcus aureus alpha-toxin and Pseudomonas aeruginosa cytotoxin on the permeability of an endothelial monolayer were studied. Porcine pulmonary artery endothelial cells were grown on a polycarbonate membrane, mounted in a chamber, and exposed to a continuous hydrostatic pressure of 10 cmH2O. On application of this trans-endothelial pressure, endothelial monolayer became "sealed," i.e., the filtration rate for water decreased and the reflection coefficient for albumin increased, reaching a plateau after 1-2 h. Sealed monolayer had a hydraulic conductivity of 2.1 X 10(-6) cm.s-1.cmH2O and an albumin reflection coefficient of 0.73. Permeability of the monolayer was increased on addition of an excess of EDTA and reversed on readdition of calcium. Within 60-90 min after addition of 1 microgram/ml alpha-toxin, the filtration rate increased 75-fold, and the albumin reflection coefficient dropped to 0.20. These changes in permeability were accompanied by cell retraction and formation of large intercellular gaps between endothelial cells. Effects of alpha-toxin were abolished by preincubation with neutralizing antibodies and by inhibitors of calmodulin function. Pseudomonas aeruginosa cytotoxin (25 and 50 micrograms/ml) also increased the permeability of the endothelial monolayer, but it was only about one-third as effective as alpha-toxin.

Cytoskeleton and epithelial polarity.

The membrane surface of polarized epithelial cells can be divided in apical and basolateral domains that differ in molecular composition and function. Components of the cytoskeleton are involved in critical steps of both generation and maintenance of cell polarity. Generation of polarity is controlled by microtubules that serve as uniformly aligned and polarized cytoplasmic guiding structures for the vectorial and selective transport of Golgi-derived carrier vesicles to the apical cell surface. Targeting of membrane proteins to the basolateral cell surface does not depend on microtubules but follows the constitutive bulk flow of membranes. Once inserted into the lipid bilayer several membrane proteins such as the kidney anion exchanger 1 (AE1) and the sodium pump become immobilized at specialized microdomains of the lateral cell surface. Evidence is provided that both membrane proteins are linked via ankyrin to the spectrin-based membrane cytoskeleton that underlies the basolateral membrane domain. Linkage of these and other integral membrane proteins to the cytoskeleton may not only place them to specialized sites of the plasma membrane but may also prevent these transporters from clustering and endocytosis, thus helping them to stay at the cell surface. In search of sequence motifs involved in binding of integral membrane proteins to components of the cytoskeleton we found that the binding interface of AE1 to protein 4.1 (an actin and spectrin cross-linking protein) consists of a cluster of five amino acid residues, namely IRRRY in AE1 and LEEDY on protein 4.1. This motif may play a more general role in cytoskeleton membrane linkages.

Immunochemical characterization of a band 3-like anion exchanger in collecting duct of human kidney.

Poly- and monoclonal antibodies have been prepared against the cytoplasmic domain (43 kDa) and the 17-, 20-, and 35-kDa fragments of the membrane-spanning domain of the human erythrocyte anion exchanger, band 3. The antibodies were used to localize and further characterize analogues of band 3 in the human kidney. We report here that the basolateral membrane of intercalated cells of the connecting tubules and collecting ducts contains an analogue of band 3 that appears to be highly homologous to the erythrocyte anion exchanger. This band 3-like protein is probably important for reabsorption of bicarbonate in the collecting duct system and thus for acidification of the forming urine. The band 3-like protein of the intercalated cells contain immunoreactive sites of both the cytoplasmic domain and the three major fragments of the membrane-spanning domain of erythrocyte band 3. Although no immunological differences were detected between the membrane-spanning domains of band 3 in erythrocytes and intercalated cells, there are at least three sites along the cytoplasmic domain of kidney band 3 that differ from erythrocyte band 3 in either amino acid composition or posttranslational modifications. The main kidney analogue of band 3 that contains epitopes of the cytoplasmic domain as well as the 17- and 35-kDa membrane-spanning domain of erythroid band 3 is a polypeptide with an apparent molecular mass of 100-110 kDa. Further immunoreactive polypeptides at approximately 180, approximately 140, approximately 38, approximately 25-30 kDa that were detected at lower stringency and higher sensitivity of the immunoblotting procedure may be members of a multigene family that encodes a series of related proteins.

Thyrotropin (TSH) stimulates cell growth and DNA synthesis in monolayer cultures of human thyrocytes independent of the adenylate-cyclase system.

Monolayer cultures of human thyrocytes from normal thyroids (n = 13), thyroid adenomas (n = 8), differentiated (n = 7), poorly and undifferentiated (n = 5) thyroid cancers as well as thyroid cancer metastases (n = 2) were established to assess the significance of TSH and cAMP on cell growth and DNA synthesis. Cell growth was stimulated by 0.1 IU bTSH/ml and inhibited by 1.0 IU bTSH/ml (P less than 0.01), while dibutyryl-cAMP (dbcAMP) failed to show any effect on cell growth at the concentrations (10-5; 10-3 mol/l), tested. Neither did the adenylate-cyclase inhibitor dideoxy-adenosine (ddA) (2 X 10-5 mol/l) stimulate thyrocyte growth. DNA synthesis, however, measured indirectly by [3H]thymidine incorporation, was stimulated not only by TSH 2-12-fold, but also by ddA 1.3-7-fold (P less than 0.01), and was not affected by dbcAMP. TSH had no effect on [3H]thymidine incorporation in fibroblasts and c-cells from c-cell carcinomas. The stimulatory effect of TSH on thyrocyte growth and DNA synthesis was unpredictable in thyrocytes from cancerous tissues (n = 14) with no obvious correlation to tumour differentiation or stage. Thus, we showed that TSH is a promotor for cell growth and DNA synthesis in monolayer cultures of human thyrocytes from normal and adenomatous human thyroid tissues with no obvious correlation to the AC system. This TSH effect is unpredictable, however, in thyrocytes from human thyroid cancer.

Adenylate cyclase stimulation and [3H]thymidine incorporation in human thyroid tissues and thyrocyte cultures: the effect of IgG preparation from patients with different thyroid disorders.

Primary cell cultures of normal and adenomatous human thyroid tissues were incubated with TSH or ammonium sulphate precipited IGG fractions (1 mg/ml) of sera from patients with different thyroid diseases (Graves' disease: active n = 7 in remission n = 12; thyroid autonomy n = 39; simple euthyroid goitre n = 15) and were compared to controls (n = 26). [3H]thymidine incorporation in primary thyrocyte cultures demonstrated a typical bell shape curve after incubation with EGF and TSH with a maximal effect at 10-100 microIU/ml. This effect, however, was inconsistent and positive only in 2 of 7 primary cultures. Only TSH positive cultures were used for IgG studies. 16-28% of IGG fractions from sera of thyroid patients caused high (more than X + 5 SD of controls) stimulation of [3H]thymidine incorporation. Dose response curves of IgG fractions of 19 additional patients (Graves' disease in remission n = 15; thyroid autonomy n = 4) showed an increase in [3H]thymidine incorporation at 0.1 mg protein/ml for 10 patients and at low concentrations of 10-5 mg/ml for 5 patients. There was a good correlation (r = 0.72) (P less than 0.0001) between positive findings in TSH-binding inhibition (TBII) and AC-stimulation (TSI) IGG fractions but none between stimulation of [3H]thymidine incorporation and any other thyroid specific immunoglobulin nor thyroid function nor any other available data. Immunoglobulins stimulating [3H]thymidine incorporation differ therefore from TBII and TSI. The growth effect of these immunoglobulins, however, has yet to be determined.

Binding of the stress protein alpha B-crystallin to cardiac myofibrils correlates with the degree of myocardial damage during ischemia/reperfusion in vivo.

Stress proteins are assumed to protect cells against various kinds of stresses including ischemia. In this study, we focused on the behaviour of the most abundant myocardial stress protein, alpha B-crystallin, during ischemia and reperfusion of the pig heart in vivo, alpha B-crystallin constitutes 1-2% of the soluble protein pool and underwent, during severe but reversibly damaging ischemia (25 min), complete translocation to the Z-line area of myofibrils. Irreversibly damaging ischemia (60 min) was accompanied by extreme stretching of the majority of myofibrils, and by concomitant extension of alpha B-crystallin localization from the Z-line area to I-bands. This I-band shift correlated with displacement of the T12 epitope of titin from the vicinity of Z-lines into I-bands, indicating that the primary binding sites for alpha B-crystallin might also be located in juxtaposition to Z-lines and move into the I-bands during extreme sarcomeric stretching. During reperfusion after 25 min of ischemia, alpha B-crystallin disappeared rapidly from myofibrils: whereas reperfusion after irreversibly damaging ischemia (60 min) resulted in dissociation of alpha B-crystallin only from those myofibrils and myocardiocytes that were still able to contract, and alpha B-crystallin remained bound to the overstretched, damaged myofibrils no longer capable of contraction. The time course of translocation of alpha B-crystallin to myofibrils during ischemia correlated with phosphorylation of approximately 20% of the entire alpha B-crystallin pool. However, disappearance of alpha B-crystallin from myofibrils during reperfusion was not accompanied by dephosphorylation, indicating that phosphorylation alone does not explain myofibrillar binding of alpha B-crystallin. Ischemia-induced myofibrillar targeting of alpha B-crystallin probably requires additional structural and posttranslational modifications of myofibrillar components in juxtaposition to I-bands.