[Evaluating the risk factors of inferior alveolar nerve injury following removal of the mandibular third molars].

Objective: To evaluate the risk factors of inferior alveolar nerve injury (IANI) after surgical removal of the mandibular third molars (M3) and present a new risk scoring system to predict the probability of IANI. Methods: Patients who underwent extraction of M3 in the Stomatology Hospital, Zhejiang University School of Medicine from April 2017 to December 2019 were involved. The investigators enrolled a sample composed of 949 mandibular third molars. Prediction model was used for univariate and multivariate analysis of gender, age, M3, inferior alveolar canal (IAC), and the contact between M3 and IAC, to assess the risk factors of IANI. Combined with the risk factors determined by the outcomes of prediction model, the risk scoring system was constructed. The diagnostic performance of each cut-off score was examined to conduct a risk stratification of IANI risk scores. The predictive ability and reliability of the model were evaluated. Results: In prediction model, twenty nine cases (4.4%, 29/664) experienced postoperative IANI. Number of root (P<0.01), depth of impaction (P<0.05), contact between M3 and IAC (P<0.01) and their contact position (P<0.05) were statistically significant as contributing risk factors of IANI. Specifically, the incidence of temporary IANI was higher in those who aged under 25 years (P<0.001), while female suffer more permanent injury (P<0.05). Based on the IANI risk scoring system, patients were stratified into low-risk, middle-risk and high-risk groups at cutoff scores of 3 and 4. The area under the receiver operator characteristic curve of the risk scoring system were 0.81 [95%CI (0.70-0.90), P=0.002] and 0.80 [95%CI (0.68-0.92), P=0.007] towards good discrimination. Conclusions: Age, gender, number of root, depth of impaction, and contact between M3 and IAC were risk factors of IANI. IANI risk scoring system might help in preoperative assessment, recognition of high-risk cases and decision-making to reduce IANI.

[Establishment of index system for population based SARS-CoV-2 nucleic acid screening].

Objective: To establish an index system of population based SARS-CoV-2 nucleic acid screening, and provide reference to determine the screening coverage appropriately. Methods: The literature review and brain storming sessions were used to develop the basic frame and index system of population based SARS-CoV-2 nucleic acid screening. Based on Delphi method and Analytic Hierarchy Process, 21 domestic experts were selected for two rounds of consultation to determine the index system of population based SARS-CoV-2 nucleic acid screening and its weight. Results: The positive indexes of experts in two rounds of consultations were both 100%. The experts' authority coefficients (Cr) were 0.88±0.08 and 0.89±0.07, respectively. And the range of coefficient of variation (CV) were (0.08, 0.24), (0.09, 0.25). The Kendall's W coordination coefficients were 0.34 and 0.22 respectively, which were statistically significant. The index system of population based SARS-CoV-2 nucleic acid screening was established, which had 4 first-level indexes, 11 second-level indexes and 58 third-level indexes. Besides, the weight of each index was determined. Conclusion: The index system of population based SARS-CoV-2 nucleic acid screening has been established, which can provide scientific reference for the health administration to determine the coverage of population based SARS-CoV-2 nucleic acid screening when local COVID-19 epidemic occurs.

[Efficacy and safety of ticagrelor versus clopidogrel in Chinese patients with acute coronary syndrome treated with glycoprotein â ¡b/â ¢a receptor antagonist].

Objective: To explore the efficacy and safety of ticagrelor versus clopidogrel in acute coronary syndrome (ACS) Chinese patients using glycoprotein Ⅱb/Ⅲa inhibitor (GPI). Methods: The data from CCC-ACS (Improving Care for Cardiovascular Disease in China-ACS) project were systematically reviewed in ACS patients with GPI. The patients were divided into ticagrelor and clopidogrel groups. A logistic analysis and propensity score matching (PSM) were performed to compare occurrences of major cardiovascular events (MACE) and bleeding events between the two groups during hospitalization. Results: A total of 63 641 ACS patients were collected from 150 hospitals. Logistic regression analyses showed that there was no statistically significant difference in the reduction of MACE between ticagrelor and clopidogrel when using GPI (OR=0.881, 95%CI 0.599-1.296; P=0.521). However, major bleeding rate was higher in the ticagrelor group than that in the clopidogrel group (OR=1.401, 95%CI 1.075-1.852; P=0.013). Similar results were observed after PSM. No statistic difference in MACE between the ticagrelor and clopidogrel group (OR=0.919, 95%CI 0.613-1.376; P=0.681). Major bleeding rate was higher in the ticagrelor group (OR=1.559, 95%CI 1.130-2.150; P=0.007). Conclusion: In ACS patients with GPI, ticagrelor did not reduce MACE, but increased the major bleeding risk compared with clopidogrel.

Immunohistochemical and in-situ hybridization study of hypoxia inducible factor-1alpha and angiopoietin-1 in a rabbit model of mandibular distraction osteogenesis.

The endogenous release of angiogenic factors in a rabbit mandibular distraction osteogenesis model was investigated. The spatial and temporal expression of hypoxia inducible factor-1alpha (HIF-1alpha) and angiopoietin-1 (Ang-1) was compared at different phases. The lengthened calluses were harvested on post-osteotomy days 13, 20, 34 and 48, and then stained with haematoxylin & eosin. Immunohistochemical (IHC) and in-situ hybridization (ISH) examination of HIF-1alpha and Ang-1 staining was performed. The ossification in the distracted gap was predominantly intramembranous and slightly endochondral. Expression of HIF-1alpha and Ang-1 was mainly detected in the cytoplasm of fibroblast-like cells, osteoblasts and immature osteocytes on day 13 and 20, but declined with bone maturation. HIF-1alpha was also detected in the nuclei of some osteoblasts. These results suggest that the production of HIF-1alpha and Ang-1 in the distracted gap may contribute to new bone formation during gradual distraction of the mandible.

Cardiac performance in inbred rat genetic models of low and high running capacity.

1. Previous work demonstrating that DA inbred rats are superior to COP inbred rats in aerobic treadmill running capacity has indicated their utility as genetic models to explore this trait. We tested the general hypothesis that intermediate phenotypes of cardiac function and calcium metabolism are responsible for the difference in capacity between these strains. 2. Logical cardiac trait differences were estimated at a tissue (isolated papillary muscle), cellular (isolated left ventricular cells), and biochemical level of organization. 3. DA hearts were found to give significantly higher values than COP hearts for: (1) maximal developed tension (38.3 % greater), and rates of tension change in contraction (61 %) or relaxation (59 %) of isolated papillary muscle, (2) fractional shortening (50 %), amplitude of the Ca(2+) transient (78.6 %), and caffeine-induced release of Ca(2+) from the sarcoplasmic reticulum (SR; 260 %) in isolated ventricular myocytes, and (3) Na(+),K(+)-ATPase activity of isolated myocytes (17.3 %). 4. Our results suggest that these trait differences may prove useful for further studies into the genes responsible for natural variations in both ventricular function and aerobic endurance capacity. Understanding the genetic basis of aerobic capacity will help define the continuum between health and disease.

P-glycoprotein expression in squamous cell carcinoma of the oral and maxillofacial region.

OBJECTIVE: To investigate the mechanism of drug resistance in oral and maxillofacial carcinoma to chemotherapy. MATERIALS AND METHODS: Forty cases of squamous carcinoma in the oral and maxillofacial region were examined for the multidrug resistance gene product P-glycoprotein using a monoclonal antibody, MRK16. RESULT: P-glycoprotein was detected in 62.5% of all samples. P-glycoprotein expression was related to the chemotherapy and the differentiation of carcinoma. P-glycoprotein expression was higher in the post-chemotherapy group than in the non-chemotherapy group (P < 0.05). Well-differentiated tumors expressed P-glycoprotein more frequently (P < 0.05). P-glycoprotein expression was compared with clinical response to chemotherapy. The accuracy rate of prediction was 75%. CONCLUSION: P-glycoprotein plays an important role in mechanism of drug resistance of squamous cell carcinoma in the oral and maxillofacial region.

Inhibition of the growth of WI-38 fibroblasts by benzyloxycarbonyl-Leu-Leu-Tyr diazomethyl ketone: evidence that cleavage of p53 by a calpain-like protease is necessary for G1 to S-phase transition.

The effect of a calpain-selective cell permeant inhibitor, benzyloxycarbonyl Leu-Leu-Tyr diazomethylketone (ZLLY-CHN2), on the serum-stimulated growth of WI-38 human fibroblasts has been investigated. Only cell permeant protease inhibitors with activity against calpains prevented progression into S-phase. Protein blotting experiments indicated that p53 immunoreactivity increased in late G1 cells treated with ZLLY-CHN2. The content of p21Waf1/Cip1 CDK inhibitor also increased, providing a mechanism for the observed failure to enter S-phase. Further studies indicated that p53 could be degraded by a ZLLY-CHN2-sensitive protease immediately prior to S-phase, but that proteolysis did not occur after this critical time point. Chelation of extracellular Ca2+ by addition of EGTA inhibited the p53 degradation. Consistent with proteolysis of p53 in late G1 phase, mu-calpain immunoreactivity transiently accumulated in cell nuclei at this time. ZLLY-CHN2 did not appear to increase p53 mRNA in WI-38 cells. Purified mu-calpain required only 1 to 3 microM Ca2+ to proteolyze p53 in WI-38 cell lysates. These results indicate that ZLLY-CHN2 inhibits progression of WI-38 cells into S-phase by inactivating a calpain-like protease that is responsible for proteolysis of constitutively expressed p53 in late G1.

[Phenomenon of intrinsic rhythm of luteinizing hormone release from isolated anterior pituitary gland of female rat].

The intrinsic nature of rthymic release of luteinizing hormone (LH) of isolated human and rat anterior pituitary gland reported independently by Macro Gambacciani and Xie in 1987 can be more directly demonstrated by a computer programme of Time Series-HSY Hidden Periodic Analytic Approach for continuous monitoring the LH output of the perfusate from a perfusion system with in vitro anterior pituitary of SD female rat. The results are as follows: (1) Under various reproductive conditions the average frequency (min/cycle) and amplitude (ng/ml) of the intrinsic rhythm of LH release were quite different: In proestrous group the frequency and amplitude were the highest, being intermediate in the ovariectomized group and lowest in the lactation group. (2) The intrinsic rhythm of LH release could be changed by either peptide or steroid hormones. In proestrous group with 30 min of gonadotropin-releasing hormone (GnRH), stimulation would reduce both frequency and amplitude. In case of lactation, the frequency was unchanged, but amplitude lowered, while in the ovariectomized rat pituitary, the 30 min GnRH stimulation decreased the frequency of release only. The intrinsic rhythm of the LH release could also be influenced by steriod hormones (Ru486 and Anordrin). With 120 min before removal of the anterior pituitary gland the rats receiving i.m. injection of Ru486 (2 mg/kg bw) or Anordrin (2 mg/kg), the results showed that Ru486 decreased frequency, while Anordrin decreased only the frequency to a less extent, both without amplitude affected. (3) Verapamil and EGTA added to the perfusion system did not abolish but only decreased the rhythmic phenomenon by using proestrous pitutary. This suggests that participation of Ca2+ may take place in the intrinsic release of LH. The above results indicated that the intrinsic rhythm of LH release of isolated anterior pituitary gland is different from various reproductive hormonal conditions and capable of being modified by exogenous hormones. The physiological function of the intrinsic rhythm of LH release of anterior pituitary gland remains to be elucidated.

Heterogeneity of the aggregation response of human platelets to arginine vasopressin.

Previous reports have alluded to variability in the aggregation response of normal human platelets to the neuropeptide arginine vasopressin (AVP). Since it has not been well documented, the current studies were undertaken to characterize this response. AVP (1-100 nM) produced a concentration-dependent aggregation response. Although the aggregation response to 100 nM AVP did not correlate with age or sex, there was a bimodal response distribution based on the presence or absence of a second wave of aggregation. In kinetic studies, the apparent km of AVP was 18.3 +/- 5.4 nM. There was a significant inverse relationship between the maximal aggregation response to 100 nM AVP and the km (r = -0.82). One hundred nanomolar AVP increased the intracellular calcium concentration of platelets by 406 +/- 120 nM in calcium free buffer and by 658 +/- 233 nM in the presence of 1.0 mM CaCl2. The aggregation response to 100 nM AVP correlated most strongly with the transmembrane influx of calcium (r = 0.84). In individuals whom 100 nM AVP was able to generate a second wave of aggregation, the selective protein kinase C inhibitor bis-indolylmaleimide significantly decreased the platelet aggregation response. Thus, there is significant heterogeneity in the aggregation response of normal human platelets to AVP. Based on our kinetic studies and the effects of PKC inhibition on the aggregation response to AVP, we would hypothesize that the variability of the aggregation response of normal human platelets to AVP is related to a polymorphism of the platelet AVP V1 receptor.

Oxidant-induced activations of nuclear factor-kappa B and activator protein-1 in cardiac myocytes.

Activator protein-1 (AP-1) and nuclear factor-kappa B (NF-kappa B), two transcription factors that respond to a wide range of signals, have been shown to be activated by H2O2 in several cell lines. Since H2O2 and related oxidants are implicated in reperfusion injury to the heart, we wished to know if NF-kappa B is present in the myocardium and if cardiac AP-1 and NF-kappa B also respond to oxidants. Rat neonatal cardiac myocytes were exposed to H2O2, and changes in c-fos and c-jun mRNAs, immunoreactive c-Fos and c-Jun proteins (components of AP-1), and immunoreactive p50 subunit of NF-kappa B were determined. Changes in nuclear activities of AP-1 and NF-kappa B were also measured by electrophoretic mobility shift assays. When myocytes were exposed to nonlethal concentrations of H2O2, c-fos and c-jun mRNAs were rapidly induced, reaching peak values at 30-60 min. The levels of c-Fos and c-Jun proteins increased in nuclei as revealed by immunostaining, and DNA binding activity of nuclear AP-1 increased. The presence of p50 subunit of NF-kappa B and its H2O2-induced shift from cytoplasm to nucleus were shown by immunostaining. H2O2-induced myocyte nuclear proteins capable of binding to a DNA probe containing the NF-kappa B element were also demonstrated. The findings suggest that altered expressions of cardiac genes regulated by AP-1 and NF-kappa B may be components of oxidant-induced injury to the heart or a part of the heart's adaptive response to oxidative stress.

The alpha-subunit of the Na,K-ATPase has catalytic activity independent of the beta-subunit.

All catalytic activities of the Na,K-ATPase have been ascribed to the alpha-subunit; however, normal activity requires the presence of the beta-subunit. Using recombinant baculoviruses to infect insect cells, we demonstrate that the alpha-subunit, without the beta-subunit, has catalytic activity. During the normal catalytic cycle of the Na,K-ATPase, the alpha-subunit is transiently phosphorylated by ATP at an aspartate residue. This phosphorylation requires Na+, in the presence of K+ the enzyme undergoes rapid dephosphorylation. In contrast, phosphorylation of the independent alpha-subunit by ATP occurs in the presence of Mg2+, does not require Na+ or K+, and is not affected by ouabain. The phosphorylation is, however, inhibited by EGTA and increasing ionic strength. Chemical properties of the alpha-subunit phosphointermediate are consistent with phosphorylation at the normal aspartyl residue. Membranes from cells infected with the recombinant alpha baculovirus exhibit an EGTA-sensitive Mg(2+)-ATPase activity that is not present in the uninfected cells. The Mg(2+)-ATPase of the alpha-infected cells is reduced under conditions of high ionic strength and completely inhibited by EGTA. Thus the phosphorylation of the unassociated alpha-subunit is representative of the ATPase activity of the enzyme. These results suggest that the alpha-subunit of the Na,K-ATPase can catalyze an activity not normally associated with the enzyme and demonstrate that the bea-subunit plays an important role in conferring normal activity to the enzyme complex.

Glucocorticoid receptor conversion to high affinity nuclear binding and transcription enhancement activity in Chinese hamster ovary cells subjected to heat and chemical stress.

The untransformed glucocorticoid receptor (GR) is a heteromeric complex containing two molecules of the 90-kDa heat shock protein (hsp90) and one molecule of the 56-kDa heat shock protein (hsp56). In the absence of hormone, this complex is found in the cytosolic fraction of cells, and upon hormone-binding the complex dissociates and the GR is recovered in the nuclear pellet fraction. Given the association of heat shock proteins with the cytosolic form of the GR, we have examined the effects of heat shock on GR subcellular localization and transcription enhancement activity in a series of Chinese hamster ovary (CHO) cells which express either low levels of endogenous GR (CHOd cells), high levels of the mouse GR (WCL2 cells), or high levels of mutated mouse GR unable to bind DNA (NB cells). It was found that heat shock treatment of WCL2 cells results in wild-type mouse GR that is recovered almost entirely within the nuclear pellet fraction, a response similar to that seen in hormone-treated cells. In contrast, heat shock treatment of NB cells results in complete loss of GR from the cytosolic fraction, but almost no shift of GR to the nuclear nuclear pellet. These results indicate that heat shock-mediated conversion to high-affinity nuclear binding by the wild-type GR requires a functional DNA-binding domain, and that heat shock will result in loss of GR to proteolysis in the absence of nuclear sequestration. Analysis of MMTV-CAT reporter gene expression in these cells revealed that heat or chemical shock, in comparison to hormone-treatment, results in a small induction of MMTV-CAT expression in the WCL2 cells, but not in the CHOd or NB cells. These results indicate that cellular stress can cause at least a partial induction of hormone-independent GR-mediated gene expression.

Submicrostructure and typing of female genital condylomata.

Thirty biopsies from female genital condylomata were examined by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) to study structural characteristics and typing of condylomata. It was found that cytoplasmic clearing was marked in acuminate condylomata, diffuse interstitial and epithelial proliferation in nodular condylomata (flat condylomata), and invagination of the lesions into the interstitial tissue or glandular ducts in endophytic condylomata. In nodular condylomata, SEM also showed some structural features similar to those of intra-epithelial neoplasia. Microridges on the surface of squamous cells had villiform of granular changes. On the surface of a percentage of squamous or columnar cells, there were holes with a diameter of about 3 to 5 microns. A number of giant cells were seen among other cells. The cervical squamatization zone contained groups of special cells covered with dense microvilli. TEM of nodular condylomata revealed some pictures resembling active proliferation of tumor cells, such as enlarged or irregular nuclei (large N/C ratio), evaginated or invaginated nuclear membranes, condensed chromatin attached to the inner part of the nuclear membrane, transparent nucleoplasm, and frequent nucleosomes and karyokinesis. Virus particles with the morphological characteristics of HPV (naked hexagon-like particles with an average diameter of 45-50 nm) were seen in some nuclei with markedly condensed chromatin. It is suggested that HPV-induced genital condylomata, especially nodular one (flat condylomata), entail a potential progression to malignancy.

Functional expression of the alpha 2 and alpha 3 isoforms of the Na,K-ATPase in baculovirus-infected insect cells.

Multiple isoforms of both the alpha and beta subunits of Na,K-ATPase have been identified. Elucidating their roles has been complicated by the fact that most tissues express multiple isoforms and purification techniques specific for each isoform have not been achieved. The baculovirus expression system, which uses the baculovirus Autographica californica to infect insect cells, is an ideal system for studying the Na,K-ATPase isoforms since high amounts of foreign proteins can be produced and some insect cell lines have low levels of endogenous Na,K-ATPase. Recombinant baculoviruses containing the cDNAs for the alpha 2, alpha 3, and beta 1 isoforms of the rat Na,K-ATPase were prepared and used to infect Sf-9 cells, an insect cell line derived from the ovary of the fall armyworm Spodoptera frugiperda. By using this system, Na,K-ATPase alpha 2 and alpha 3 subunits that were antigenically and electrophoretically indistinguishable from the native subunits were produced. When each subunit is expressed independently in the Sf-9 cells, it is primarily delivered to the plasma membrane. Although the isolated expression of each Na,K-ATPase subunit did not render active Na,K-ATPase molecules, the coexpression of alpha 2 or alpha 3 with beta 1 resulted in catalytically active molecules. This activity could be measured as a ouabain-sensitive ATPase activity or directly demonstrated using either [gamma-32P]ATP or 32Pi to identify the phosphorylated intermediates of the alpha 2 and alpha 3 isoforms. [3H]Ouabain binding studies showed that both isoforms are capable of binding the cardiotonic steroid with high affinity, alpha 3 being more sensitive to ouabain. These results demonstrate that the baculovirus system is suitable for the expression of the Na,K-ATPase isoforms and should provide a useful method for the characterization of the enzymatic properties of each isoform.

Expression, targeting, and assembly of functional Na,K-ATPase polypeptides in baculovirus-infected insect cells.

Deciphering the roles of the individual subunits of the heteromeric Na,K-ATPase in the structure, function, and assembly of this enzyme has been complicated because most expression systems have endogenous levels of Na,K-ATPase activity. This problem has become even more significant in light of the recent identification of multiple isoforms for both the alpha and beta subunits. The baculovirus expression system, which uses the baculovirus Autographica californica to infect insect cells, affords two distinct advantages for the expression of the Na,K-ATPase; some insect cells have little or no levels of Na,K-ATPase, and baculovirus-infected cells produce extremely high levels of foreign protein. We have made two separate recombinant baculoviruses containing the rodent alpha 1 or beta 1 cDNAs and used them to infect the insect cell line Sf-9. The infected Sf-9 cells produce Na,K-ATPase subunit protein on the order of 5-10 micrograms of protein/ml of cultured cells. The rodent alpha 1 polypeptide produced in the Sf-9 cells is indistinguishable electrophoretically and antigenically from the native subunit. The expressed beta 1 subunit is also antigenically identical but has a higher electrophoretic mobility due to differential glycosylation by the infected Sf-9 cell. In contrast to other systems, when expressed alone, each individual Na,K-ATPase subunit is targeted to the infected Sf-9 plasma membrane. In contrast, when infected with a virus that induces the heavy chain of murine IgG, the infected Sf-9 cell retains the polypeptide in the endoplasmic reticulum. However, when both IgG light and heavy chains are expressed, the polypeptides are properly processed and secreted. When the Na,K-ATPase alpha 1 and beta 1 polypeptides are simultaneously expressed, they form detergent-resistant complexes that are functional. Ouabain-sensitive ATPase activity on the order of 5 mumol Pi/mg/h in infected Sf-9 membranes was dependent on the expression of both the alpha 1 and beta 1 subunits. Sodium-dependent phosphorylated intermediates were detected that were potassium- and ouabain-sensitive. No increase in ouabain-sensitive activity or phosphorylated intermediates was detected when either subunit was expressed alone. The alpha 1 beta 1-coinfected cells were also able to transport ions, as detected in 86Rb uptake experiments. Thus, the recombinant Na,K-ATPase expressed in insect cells is biologically active and is suitable for structural and functional analysis.

A second messenger role for monoacylglycerols is suggested by their activating effects on the sodium pump.

Receptor-mediated activation of the sodium pump has been noted in several intact tissues. To test the hypothesis that this may be due to the direct effects of the second messenger diacylglycerols on the pump, we studied the effects of various long-chain acylglycerols on the purified Na+/K(+)-ATPase. With optimal ATP, acylglycerols had no effect on enzyme activity. When ATP was suboptimal, tri- and diacylglycerols had no effects, but monoacylglycerols caused up to 3-fold increase in ATPase activity. Using sealed vesicles of red cell membranes and cardiac sarcolemma, stimulation of the ion transport function of the enzyme by monoacylglycerols in the presence of suboptimal ATP was also shown. Since the sodium pump may not be saturated with ATP in the intact cell, the possibility arises that monoacylglycerols are the second messengers for the receptor-mediated regulation of the pump.

Studies on the specificity of the effects of oxygen metabolites on cardiac sodium pump.

Isolated myocytes of rat heart, and sealed sarcolemmal vesicles of bovine heart, were used to examine the selectivity of the effects of partially reduced oxygen species (generated by a mixture of xanthine and xanthine oxidase) on cardiac sodium pump and several other ion transporters of the plasma membrane. When myocytes were exposed to xanthine plus xanthine oxidase, there were time-dependent inhibitions of ouabain-sensitive 86Rb+ uptake and (Na+ + K+)-ATPase activity that could be prevented by allopurinol, or by catalase and superoxide dismutase; suggesting the involvements of H2O2 or oxygen free radicals in the inhibition of the pump. This inhibition preceded any significant decrease in cellular ATP or in the number of viable cells. While ouabain increased 45Ca2+ uptake by myocytes as expected, exposure to xanthine plus xanthine oxidase decreased 45Ca2+ uptake; suggesting that the Na+, Ca2(+)-exchanger of the intact myocytes is also inhibited by oxygen metabolites. Simultaneous inhibitions of the pump, the Na+, Ca2(+)-exchange, the Na+, H(+)-exchange, and the Na+, Pi-cotransport activities also occurred in sarcolemmal vesicles that were treated with xanthine plus xanthine oxidase. These findings indicate that inactivations of the sodium pump and other sarcolemmal ion carriers are early events in the oxidant-induced damage to the cardiomyocyte. In the rat heart myocytes, a fraction of (Na+ + K+)-ATPase that seems to be more sensitive to ouabain, was inactivated more rapidly upon exposure of myocytes to xanthine plus xanthine oxidase; raising the possibility of the existence of different pump populations with different sensitivities to extracellularly generated oxygen metabolites.

Determination of total (Na+ + K+)-ATPase activity of isolated or cultured cells.

The aim of this work was to determine if the total (Na+ + K+)-ATPase of the plasma membrane of a cell population could be assayed without cell homogenization and partial purification of the enzyme. Several types of intact cells that were placed in an assay medium containing MgATP, Na+, and K+ hydrolyzed little or none of the added ATP. When the cells were pretreated with the ionophore alamethicin and then placed in the assay medium, they exhibited an ouabain-sensitive (Na+ + K+)-ATPase activity that increased and reached a limiting value with increasing alamethicin concentration. Since alamethicin did not increase the activity of the purified membrane-bound (Na+ + K+)-ATPase, its effects on the intact cells are probably due to the formation of large channels within the plasma membrane that allow the free access of the components of the assay medium to the intracellular domains of (Na+ + K+)-ATPase. Utilizing whole cells treated with alamethicin, total (Na+ + K+)-ATPase activity was determined in clonal pheochromocytoma cells (PC12), neuroblastoma x glioma hybrid cells (NG108-15), and myocytes isolated from adult and neonatal rat hearts. With the use of this whole-cell assay, the ouabain sensitivities of the enzymes in adult and neonatal rat heart myocytes were determined and found to be the same as those that have been determined with the use of partially purified enzymes.