State of transition to Ministry of Health governance of prison healthcare in the Council of Europe region.

OBJECTIVES: This study aimed to examine timebound prison healthcare governance amendments and current structures in Europe two decades after the World Health Organization (WHO) Declaration on Prison Health as part of Public Health adopted in Moscow on 24 October 2003 (Moscow Declaration), which recommended prison health care be closely linked with public health systems to ensure quality prison health care, connected health surveillance, and continuity of care. STUDY DESIGN: We present here a regional evolutionary mapping of the Council of Europe Member State transfer of prison healthcare governance to the auspices of the Ministry of Health. METHODS: The European Committee for the Prevention of Torture database and WHO Regional Office for Europe Health In Prison European Database were scrutinised for Council of Europe (CoE) Member State status regarding the Ministry responsible for prison healthcare governance and if this had changed since the adoption of the Moscow Declaration in 2003. RESULTS: As of October 2023, completed transfer of governance to the Ministry of Health nationally is documented in 13 CoE Member States and in one CoE Member State candidate (Kosovo). Partial transfer is documented in Spain (Catalonia and Basque Autonomous Community) and Switzerland (cantons of Geneva, Valais, Vaud, Neuchatel, and Basel-Stadt). Three CoE Member States operate joint governance of prison health care between Ministries (Malta, Portugal, Türkiye). Transfer is a lengthy process (up to 10 years). CONCLUSIONS: Successful transition requires political commitment, cooperation, needs assessment, resourcing, and evaluation. Monitoring of cost and prison healthcare standards, due process for complaints, and cooperation with independent/Committee against Torture inspections is critical.

Cognitive impairment in early stages of multiple sclerosis is associated with high cerebrospinal fluid levels of chitinase 3-like 1 and neurofilament light chain.

BACKGROUND AND PURPOSE: Chitinase 3-like 1 (CHI3L1) and neurofilament light chain (NF-L) are promising biomarkers of disability in multiple sclerosis (MS). However, their role in cognitive dysfunction remains elusive. Here, we aimed to correlate cerebrospinal fluid (CSF) levels of CHI3L1 and NF-L with cognitive status in MS. METHODS: Fifty one recently diagnosed patients were cognitively evaluated and CSF was collected. Levels of CHI3L1 and NF-L were determined by ELISA. Spearman's partial correlation coefficient was performed. RESULTS: After adjusting cognitive scores by age, anxiety and EDSS, association was detected between CHI3L1 levels and Trail Making Test A (rs = 0.348; p = 0.016) and between NF-L levels and Word List Generation (rs = -0.324; p = 0.025). CONCLUSION: High levels of CSF CHI3L1 and NF-L are associated with cognitive impairment in the early phases of MS.

Dominant isolated renal magnesium loss is caused by misrouting of the Na(+),K(+)-ATPase gamma-subunit.

Primary hypomagnesaemia is composed of a heterogeneous group of disorders characterized by renal or intestinal Mg(2+) wasting, often associated with disturbances in Ca(2+) excretion. We identified a putative dominant-negative mutation in the gene encoding the Na(+), K(+)-ATPase gamma-subunit (FXYD2), leading to defective routing of the protein in a family with dominant renal hypomagnesaemia.

[Intramammary use of antibiotics in dairy farms in the canton of Ticino before, during and after Staphylococcus aureus genotype B elimination]. / Intramammärer Antibiotikaeinsatz vor, während und nach einer Staphylococcus aureus Genotyp B Sanierung in Milchkuhbetrieben im Kanton Tessin.

INTRODUCTION: The aim of the study was to evaluate the use of intramammary antibiotics before, during and after the elimination of Staphylococcus aureus genotype B (SAGTB). Data on intramammary antibiotic use was collected in 65 dairy farms as part of a pilot project for SAGTB elimination in the canton of Ticino from 2017 to 2019. The investigated farms were divided into 46 affected farms (with at least one SAGTB-positive animal) and 19 control farms (SAGTB-free farms). Data on antibiotic use were requested from veterinarians and treatment incidence, as a measure of antibiotic use, was calculated based on medical records and veterinary prescriptions. In addition, the treatment incidence was calculated for 47 farms during alpine farming period. In 2018 (elimination year), the mean incidence of treatment during lactation in the SAGTB-positive farms was significantly higher than in the control farms (p=0,003). In 2019 no significantly lower antibiotic use during lactation or dry period was detected between 2017 (before elimination) and 2019 (after elimination). Alpine farming places where only S. aureus genotype B-negative animals had access to had a significantly lower antibiotic use during lactation (p=0,004). The new federal database (Antibiotics Information System in Veterinary Medicine, IS ABV) should allow continuous monitoring and to confirm the reduction of antibiotic use in the coming years.

INTRODUCTION: L'objectif de l'étude était d'évaluer la consommation d'antibiotiques intramammaires avant, pendant et après l'assainissement de Staphylococcus aureus génotype B (SAGTB). Dans le cadre d'un projet pilote d'assainissement SAGTB dans le canton du Tessin, l'étude a recueilli des données sur la consommation d'antibiotiques intramammaires de 2017 à 2019 dans 65 exploitations laitières réparties en 46 exploitations test (avec au moins un animal positif au SAGTB) et 19 exploitations témoins (exploitations exemptes de SAGTB). Les données relatives à la consommation d'antibiotiques ont été demandées aux vétérinaires et, grâce aux enregistrements et aux prescriptions des vétérinaires, il a été possible de calculer l'incidence du traitement en tant que mesure de la consommation d'antibiotiques. En outre, l'incidence des traitements pendant l'alpage a également pu être calculée pour 47 exploitations. En 2018 (année d'assainissement), l'incidence des traitements pendant la lactation était en moyenne significativement plus élevée dans les exploitations SAGTB-positives que pour les exploitations de contrôle (p=0,003). En comparant 2017 (avant l'assainissement) et 2019 (après l'assainissement), il n'y a pas eu de baisse significative de la consommation d'antibiotiques pendant la lactation et le tarissement en 2019. Dans les exploitations d'estivage, on a constaté l'année suivant l'assainissement (2019) une consommation d'antibiotiques significativement plus faible pendant la lactation pour les alpages qui n'accueillaient que des animaux négatifs à S. aureus génotype B (p=0,004). Il faut espérer qu'avec l'aide de la nouvelle base de données fédérale (Système d'information sur les antibiotiques en médecine vétérinaire, IS ABV), le monitoring pourra être poursuivi dans les années à venir et que la réduction de la consommation d'antibiotiques sera confirmée dans les années suivantes.

[Economic importance of antibiotic sale for private food animal practices in Switzerland - example veal production]. / Ökonomische Bedeutung des Umsatzes mit Antibiotika für private Nutztierpraxen in der Schweiz ­ Beispiel Kälbermast.

INTRODUCTION: The use of antibiotics in Swiss veal production is considered an established method for controlling bacterial infectious diseases. Although the veterinary profession aims to ensure animal welfare, the veterinary business income needs to be ensured at the same time. Against the background of increasing problems with resistant pathogens in human and veterinary medicine, the use of antibiotics should be significantly reduced and used more selectively. The associated economic consequences for food animal practitioners are unknown. The aim of this study was to determine the economic importance of antibiotic sale volume for private food animal practitioners in veal production. An anonymized questionnaire was sent to 120 mixed veterinary practices in Switzerland, which offered services to veal and beef cattle farmers. Questions involved the pharmaceutical sale volume, details on veterinary invoices from three farms with average, below and above average animal health throughout 2017. Twenty-nine complete questionnaires (response rate: 24.2%) and veterinary invoices of 84 farms were returned. The study is not representative, but it allows a rough assessment of the economic framework in Swiss livestock practice. The majority of the total turnover with livestock farms was generated by the sale of antibiotics (54%). Antibiotic sales per animal were higher as expected in farms with a below-average animal health than in farms with an average or above-average animal health. Consulting services turnover contributed only 0.5% to the total sale volume in veal farming. The results document, that antibiotic reduction measurements in veal and beef production will have economic consequences for veterinary livestock practices. In the medium term, the profitable existence of livestock veterinary practice requires a change to cost based consulting services.

INTRODUCTION: En Suisse, dans le cadre de la pratique de l'engraissement des veaux, l'utilisation d'antibiotiques est principalement considérée comme une méthode éprouvée pour lutter contre les maladies infectieuses bactériennes. L'activité vétérinaire vise à prévenir ou minimiser les souffrances chez les animaux. Mais pour autant, les vétérinaires restent des entrepreneurs qui doivent assurer un revenu adéquat de leur pratique. Dans un contexte de problèmes croissants rencontrés avec les agents pathogènes résistants en médecine humaine et vétérinaire, l'utilisation d'antibiotiques devrait être considérablement réduite et ciblée. Les conséquences économiques associées pour la pratique vétérinaire rurale sont peu connues. Le but de la présente étude était de déterminer l'importance économique des ventes d'antibiotiques pour les pratiques vétérinaires rurales privées en utilisant l'exemple de l'engraissement des veaux. À cet effet, un questionnaire anonymisé a été envoyé à 120 cabinets vétérinaires mixtes en Suisse, qui s'occupent entre autres d'exploitations d'engraissement de veaux, de broutards et de taurillons. Les participants ont été interrogés sur le niveau des ventes de médicaments. En outre, nous les avons invités à envoyer des factures vétérinaires concernant trois entreprises d'engraissement avec un niveau de santé animale inférieur, égal et supérieur à la moyenne tout au long de l'année 2017. Vingt-neuf questionnaires complets (taux de réponse: 24,2%) ainsi que des factures vétérinaires concernant au total 84 exploitations d'engraissement de veaux, de broutards ou de taurillons nous ont été retournés. L'étude n'est certes pas représentative, mais elle permet une évaluation approximative du cadre économique des pratiques rurales en Suisse. La majorité du chiffre d'affaires total sur les exploitations d'engraissement de veaux, de broutards ou taurillons a été générée par la vente d'antibiotiques (54%). Il apparait que les ventes d'antibiotiques étaient plus élevées dans les exploitations où la santé animale était inférieure à la moyenne que dans les établissements où elle était supérieure ou égale à la moyenne. Le chiffre d'affaires des prestations de conseil ne représentait que 0,5% du chiffre d'affaires total dans le domaine des veaux. Ces résultats montrent que les mesures visant à réduire les antibiotiques dans les conditions actuelles de production de veau et de boeuf auront probablement des conséquences économiques non négligeables sur la part des exploitations d'engraissement dans les revenus des pratiques vétérinaire rurales. A moyen terme, afin d'assurer la rentabilité d'une pratique rurale, une nouvelle orientation de l'activité vétérinaire sera nécessaire: la mise en place d'un service de conseils payants pour les exploitations d'engraissement, ayant pour but l'établissement de concepts de prévention.

A novel breast tissue density classification methodology.

It has been shown that the accuracy of mammographic abnormality detection methods is strongly dependent on the breast tissue characteristics, where a dense breast drastically reduces detection sensitivity. In addition, breast tissue density is widely accepted to be an important risk indicator for the development of breast cancer. Here, we describe the development of an automatic breast tissue classification methodology, which can be summarized in a number of distinct steps: 1) the segmentation of the breast area into fatty versus dense mammographic tissue; 2) the extraction of morphological and texture features from the segmented breast areas; and 3) the use of a Bayesian combination of a number of classifiers. The evaluation, based on a large kappa = 0.81 and 0.67 for the two data sets) between automatic and expert-based Breast Imaging Reporting and Data System mammographic density assessment.

[Importance of nocturia and its impact on quality of sleep and quality of life in patient with benign prostatic hyperplasia]. / Importancia de la nocturia y su impacto en la calidad del sueño y en la calidad de vida en el paciente con hiperplasia benigna de próstata.

INTRODUCTION: Nocturia is the main cause of insomnia or sleep interruption in adult men, which has a negative impact on daily activities, quality of life (QoL) and quality of sleep (QoS). The assessment of nocturia and its impact on QoL and QoS in patients suffering from benign prostatic hyperplasia (BPH) has been poor in terms of clinical research, moreover there is a lack of specific methods to assess this impact. OBJECTIVES: The objectives of BPH treatment should include the improvement of patient's QoL by controlling both diurnal and nocturnal symptoms. In order to assess how nocturia affects QoL and also QoS, some specific tools, such as N-QoL questionnaire or the number of Hours of Undisturbed Sleep (HUS), have been recently developed. Therefore, it would be interesting to assess how nocturia reduction due to LUTS/BPH treatment can impact on some objective parameters such as HUS and also how nocturia reduction improves QoS and QoL. This assessment should be developed during the application of pharmacological treatments in clinical practice by means of these specific tools. With the aim of tackling nocturia as an urologic problem in patients with LUTS/BPH, as well as knowing the physiology of sleep and the effect of nocturia on the sleep and QoL, a meeting of expert urologists, that gathered about fifty specialists of all around Spain, was held in Madrid. This article presents the main ideas and concepts exposed in this meeting. CONCLUSIONS: Nocturia is a symptom with a high prevalence in older patients with STUI/BPH. The PreNoc study has showed a Nocturia prevalence in Spain of 83% in patients > or =60 years old diagnosed of BPH. Nocturia is the most bothersome symptom in patients with STUI/BPH.

Hematologic disorders associated with primary mediastinal nonseminomatous germ cell tumors.

BACKGROUND: The association between primary germ cell tumors of the mediastinum (the space between the lung pleura that contains the heart and other chest viscera) and hematologic malignancies has been described by retrospective analysis of patients treated at individual clinical centers. To better characterize the risk of hematologic disorders in patients with extragonadal germ cell tumors and to describe the clinical and biologic features of the disorders, we studied an unselected population in a large, international, multicenter database. METHODS: Six hundred thirty-five patients treated at 11 centers in the United States and Europe from 1975 through 1996 were evaluated retrospectively. RESULTS: A hematologic disorder was observed in 17 patients with germ cell tumors. All cases developed among the 287 patients with primary mediastinal nonseminomatous germ cell tumors, giving an incidence rate in this group of 2.0% (95% confidence interval [CI] = 1.1%-3.1%) per year over a median follow-up time of 3 years. The risk of developing hematologic disorders was statistically significantly increased in patients with primary mediastinal nonseminomatous germ cell tumors in comparison with the age-matched general population (standardized incidence ratio = 250; 95% CI = 140-405). The median time to onset of hematologic neoplasia was 6 months (range, 0-47 months), and the median survival after diagnosis of the hematologic disorder was 5 months (range, 0-16 months) (two-sided P<.0001, comparing survival from the time of diagnosis of the germ cell tumor of patients with and without hematologic disorders). CONCLUSION: In our study, approximately one in 17 patients with primary mediastinal nonseminomatous germ cell tumors was affected by a hematologic disorder, whereas no cases were seen among 334 patients with other extragonadal germ cell tumors. The hematologic disorder had a statistically significant impact on prognosis, with none of the 17 reported patients surviving for more than 2 years.

Incidence of metachronous testicular cancer in patients with extragonadal germ cell tumors.

BACKGROUND: The frequency of subsequent testicular cancer (referred to as metachronous testicular cancer) in men who have had previous testicular cancer is relatively high. The rate of metachronous testicular cancer in men with extragonadal germ cell tumors (EGCTs), however, is largely unknown. We conducted a retrospective study of EGCT patients to determine the incidence, cumulative risk, and specific risk factors for metachronous testicular cancers. METHODS: A standardized questionnaire about patient characteristics, the extent of EGCT disease, any second malignancies, and treatments received was completed for 635 patients with EGCTs identified from the medical records of 11 cancer centers in Europe and the United States from 1975 through 1996. Comparisons with age group-specific data from the Saarland, Germany, population-based cancer registry were used to calculate the standardized incidence ratio (SIR). The Kaplan-Meier method was used to analyze survival data and cumulative risk. All statistical tests were two-sided. RESULTS: Sixteen EGCT patients (4.1%) developed metachronous testicular cancers, with a median time between diagnoses of 60 months (range, 14-102 months). The risk of developing metachronous testicular cancers was statistically significantly increased in patients with EGCTs (observed = 16; expected = 0.26; SIR = 62; 95% confidence interval [CI] = 36 to 99) and in subsets of EGCT patients with mediastinal location (SIR = 31; 95% CI = 8 to 59), retroperitoneal location (SIR = 100; 95% CI = 54 to 172), and nonseminomatous histology (SIR = 75; 95% CI = 43 to 123). The cumulative risk of developing a metachronous testicular cancer 10 years after a diagnosis of EGCT was 10.3% (95% CI = 4.9% to 15.6%) and was higher among patients with nonseminomatous EGCTs (14.3%; 95% CI = 6.7% to 21.9%) and retroperitoneal EGCTs (14.2%; 95% CI = 5.6% to 22.8%) than among patients with seminomatous EGCTs (1.4%; 95% CI = 0.0% to 4.2%) and mediastinal EGCTs (6.2%; 95% CI = 0.1% to 12.2%). CONCLUSIONS: Patients with EGCTs, particularly those with retroperitoneal or nonseminomatous tumors, but also those with primary mediastinal EGCTs, are at an increased risk of metachronous testicular cancer.

Reversible inactivation of (Na+ + K+)-ATPase by use of a cleavable bifunctional reagent.

1. Purified (Na+ + K+)-ATPase, prepared from rabbit kidney outer medulla, is incubated with the bifunctional NH2-directed reagent dimethyl 3,3'-dithiobis-propionimidate. This results in a cross-link between the subunits of the enzyme and a simultaneous reduction of the (Na+ + K+)-ATPase and K+-stimulated p-nitrophenylphosphatase activities. 2. The most abundant cross-link product is a dimer of the two different subunits of the enzyme. 3. Reduction of the disulfide cross-link by dithioerythritol results in partial recovery of the original subunit structure of the enzyme and of the (Na+ + K+)-ATPase and K+-stimulated p-nitrophenylphosphatase activities. 4. These results suggest that a free mobility of the subunits of the (Na+ + K+)-ATPase system relative to each other is essential for proper functioning of both enzyme activities.

Role of monovalent cations in fluid secretion from the exocrine rabbit pancreas.

The role of Na+ in fluid secretion by the isolated rabbit pancreas was investigated. The fluid secretion rate is reduced upon replacement of Na+ in the bathing medium by Li+, K+ or choline. The inhibition depends on the nature of the substituting cation, and is largest with choline. Upon replacement, the substituent cation appears in the secreted fluid, and the Na+ concentration in the secreted fluid is decreased in a mirror-like fashion. When Na+ is replaced by Li+ or choline, the secretory Na+ concentration is decreased, although less than in the bathing medium, and the K+ concentration is increased. When Na+ is replaced by K+, the Na+ and the K+ concentration in the secreted fluid are approximately equal to their bathing medium concentrations. In the Li+ and choline medium, stimulation of the pancreas by carbachol or CCK-8 increases the fluid secretion rate. In addition, it increases the Li+ or choline concentration, and decreases the Na+ and K+ concentrations in the secreted fluid. In normal and K+ medium, stimulation causes only a slight increase in fluid secretion rate, with no change in the secretory Na+ concentration. In normal medium, stimulation leads to a decrease in the secretory K+ concentration. The effects of replacing Na+ appear to be the result of a direct inhibition of the active HCO3- transport underlying secretion, and an indirect inhibition related to the permeability of the pancreas for the various cations. The stimulants are likely to act by increasing the permeability of the tight junctions.

A model for fluid secretion in the exocrine pancreas.

Fluid secretion by the isolated rabbit pancreas is strongly dependent on the presence of Na+ in the bathing medium. Substitution of Na+ by another cation such as Li+ or K+ causes an inhibition of fluid secretion rate and a change in the composition of the secreted fluid which is dependent on the nature of the substituent cation. Stimulation of the pancreas by CCK-8 or carbachol increases paracellular ion permeability and, in some cases, also fluid secretion rate. We present a simple, quantitative model for ion and water secretion which accounts for the effects observed upon Na+ substitution and stimulation. The main features are active, Na+-dependent transcellular HCO3- transport and passive, paracellular cation and anion permeation. The activity of the HCO3- pump is dependent on the energy status of the cell and on the Na+ concentration in the bathing medium, and is competitively inhibited by K+. The paracellular ion permeabilities can be modulated by stimulatory agonists. We examine the extent to which, according to the model, fluid secretion is controlled by the various system parameters such as ion permeabilities and ion pump activity, and by external parameters such as the ion concentrations in the bathing medium. In addition, calculation of the effects of changes in these parameters are carried out in order to gain more insight in the mechanisms of secretion.

Rat pancreatic adenylate cyclase. IV. Effect of hormones and other agents on cyclic AMP level and enzyme release.

1. The effects of secretin and pancreozymin-C-octapeptide and phosphodiesterase inhibitors on the concentration of adenosine 3',5'-cyclic monophosphate (cyclic AMP) and on the release of enzymes from rat pancreas have been studied. 2. In determininging cyclic AMP by means of the saturation assay of Brown et al. ((1971) Biochem. J. 121, 561-563) it is found essential to purify the pancreatic tissue extract by ion-exchange chromatography prior to the assay. 3. Injection of synthetic secretin or pancreozymin-C-octapeptide in anaesthetized rats in a secretory active dose (0.1 nmol) has no effect on the pancreatic cyclic AMP level. 4. Incubation for up to 10 min of pancreatic slices in Krebs-Ringer bicarbonate glucose medium containing 10(-2) M theophylline as phosphodiesterase inhibitor does not result in an increase of the cyclic AMP level. With 10(-2) M 1-methyl-3-isobutylxanthine as phosphodiesterase inhibitor the level is more than doubled after the first min of incubation and remains constant thereafter. 5. Addition of 3-10(-7) M secretin to slices incubated in the presence of 10(-2) M theophylline causes 84% increase of the cyclic AMP level above control, whereas the addition of 3-10(-7) M pancreozymin-C-octapeptide has no significant effect. In the presence of 10(-2) M 1-methyl-3-isobutylxanthine the latter hormone causes significant increases of up to 34% above control during 10 min of incubation. Secretin in this condition augments the cyclic AMP level by up to 296% above control during a 10 min incubation period. Addition of secretin and pancreozymin-C-octapeptide together has no greater effect than of secretin alone. 6. A broken cell fraction of rat pancreas contains adenylate cyclase activity which can be stimulated to 457 and 600% above the basal activity by 3-10(-7) M pancreozymin-C-octapeptide and secretin, respectively. Incubation of pancreatic slices with either hormone has no effect on the cyclic AMP phosphodiesterase activity in the homogenate of these slices. 7. Pancreozymin-C-octapeptide, dibutyryl cyclic AMP, 1-methyl-3-isobutylxanthine and carbamylcholine cause an elevated release of chymotrypsin from pancreatic slices incubated for 2 h in Krebs-Ringer bicarbonate medium, containing 10 mM glucose, while secretin, cyclic AMP and butyric acid have no significant effect. The release of the cytoplasmic enzyme lactate dehydrogenase is also elevated by dibutyryl cyclic AMP, 1-methyl-3-isobutylxanthine and carbamylcholine, but not significantly by pancreozymin-C-octapeptide. 8. The results support the role of cyclic AMP in the action of secretin, and do not exclude a mediating function of this nucleotide in the actions of pancreozymin in rat pancreas.

Rat pancreas adenylate cyclase V. Its presence in isolated rat pancreatic acinar cells.

(1) In order to determine the cellular localization of the secretin- and pancreozymin-sensitive adenylate cyclase in rat pancreas, the occurence of this enzyme system has been investigated in isolated pancreatic cells. (2) Digestion of rat pancreatic lobules with collagenase yields a preparation of isolated cells which upon differential morphological analysis appears to consist for 97% of acinar cells and to contain for fewer centro-acinar and ductal cells than undissociated lobules. (3) Expressed per mg protein, the isolated cells contain the same amount of DNA, chymotrypsin and lactic dehydrogenase as the undissociated tissue. The stimulated adenylate cyclase activity is nearly entirely recovered in the isolated acinar cells, as is also the case for the low Km adenosine 3',5-cyclic monophosphate phosphodiesterase activity and the adenosine 3',5'-cyclic monophosphate (cyclic AMP) content. Marked losses are noted for the basal adenylate cyclase and the high Km cyclic AMP phosphodiesterase activities. (4) Washing the isolated acinar cells in Krebs-Ringer bicarbonate medium containing 10 mM 1-methyl-3-isobutylxanthine causes a cyclic AMP level 2.6 times that in cells washed in Krebs-Ringer bicarbonate alone. The cyclic AMP level is further increased by subsequently incubating the cells for 10 min in the presence of 3-10(-7) M pancreozymin-C-octapeptide or secretin to values 1.7 or 4.7 times the control level in cells incubated for 10 min with 1-methyl-3-isobutylxanthine alone. (5) It is suggested that the adenylate cyclase of the acinar cells may be involved, with another factor, in the stimulation of enzyme secretion, whereas a ductular cyclase would function in the regulation of the bicarbonate-dependent fluid secretion.

Studies on (Na+ plus K+)-activated ATPase. XXXVII. Stabilization by cations of the enzyme-ouabain complex formed with Mg1+ and inorganic phosphate.

Dissociation of the (Na+ + K+)-ATPase ouabain complex, formed in the presence of Mg2+ and inorganic phosphate (Complex II), is inhibited by Mg2+ (21-45%) and the alkali cations Na+ (25-59%) and K+ (27-75%) when kidney cortex tissue (bovine, rabbit, guinea pig) is the enzyme source. Choline chloride at 200 mM, equivalent to the highest concentration of NaCl tested, does not inhibit. Dissociation of Complex II from brain cortex (bovine, rat, rabbit) or heart muscle (rabbit) is much less inhibited: 0-11% by Na+ and 11-19% by K+. The degree of inhibition is not directly related to the size of the dissociation rate constant (k-) of the various complexes, but rather to the extent of interaction between the cation and ouabain binding sites for these tissues. Inhibition curves for Na+ and K+ are sigmoidal. Half-maximal inhibition for rabbit brain and kidney cortex is at 30-40 mM Na+ and 6-10 mM K+, and the maximally inhibitory concentrations are 50-150 and 15-20 mM, respectively. Maximal inhibition by Na+ or K+ for these tissues is the same. For guinea pig kidney cortex Na+ and K+ are almost equally effective, but 150 mM K+ or 200 mM Na+ are still not saturating, and inhibition curves indicate high- and low-affinity binding sites for the alkali cations. The inhibition curve for Mg2+ is not sigmoidal. In the kidney preparations Mg2+ inhibits half-maximally at 0.4-0.5 mM, maximally at 1-3 mM. Maximal inhibition by Mg2+ is higher than by Na+ or K+ for rabbit kidney cortex and lower for guinea pig kidney cortex. There is no competition or additivity among the cations, indicating the existence of different binding sites for Mg2+ and the alkali cations. Complex II differs in stability in the extent of inhibition, in the dependence of inhibition on the cation concentration and in the absence of antagonism between Na+ and K+, from the ouabain complex formed via phosphorylation by ATP (Complex I). This indicates that the phosphorylation states for the complexes are clearly different.

Studies on (Na+ + K+)-activated ATPase. XLII. Evidence for two classes of essential sulfhydryl groups.

1. Preincubation of purified (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) preparations from rabbit kidney outer medulla with 5,5'-dithiobis-(2-nitrobenzoic acid) inhibits the (Na+ + 5+)-ATPase and K+-stimulated 4-nitro-phenylphosphatase activities. Phosphorylation of the enzyme by ATP and the Na+-stimulated ATPase activity are inhibited to the same extent as the (Na+ + K+)-ATPase activity, whereas the K+-stimulated 4-nitrophenylphosphatase activity is inhibited much less. 2. Titration with 5,5'-dithiobis-(2-nitrobenzoic acid) in sodium dodecyl sulphate shows the presence of 36 reactive sulfhydryl groups per molecule (Na+ + K+)-ATPase (Mr = 250 000). 3. Treatment with N-ethylmaleimide, resulting in complete inhibition of (Na+ + K+)-ATPase activity, leads to modification of 26 sulfhydryl groups, whereas treatment with 5,5'-dithiobis-(2-nitrobenzoic acid) results in modification of 12 sulfhydryl groups under the same conditions. 4. The reaction of N-ethylmaleimide with an essential SH-group is not prevented by previous blocking of sulfhydryl groups with 5,5'-dithiobis-(2-nitrobenzoic acid). 5. These findings indicate the existence of at least two classes of sulfhydryl groups on the enzyme, each containing at least one vital group. The difference between these classes consists in their different reactivity towards 5,5'-dithiobis-(2-nitrobenzoic acid) and N-ethylmaleimide.

Absence of (Na+,K+)-ATPase involvement in lactose production by lactating guinea pig mammary gland.

The role of the (Na+, K+)-ATPase system in lactose production by the lactating guinea pig mammary gland has been studied in vitro with slices of the gland. In this system there is an initial fast lactose release, mainly representing secretion of preformed lactose, followed by a continuous slow lactose release, representing mainly lactose synthesis. The latter process occurs at a rate of 1.6 to 2.4 g lactose/kg wet wr/h, which value is about half of the lactose production in vivo (3.9 g/kg set wt/h). Incubation of slices in the presence of 10-4 M ouabain does not influence the rate of overall lactose production. When determined separately, it does not change either the rate of secretion or that of synthesis. This pleads against a role of the (Na+, K+)-ATPase system in lactose secretion or synthesis, in particular it seems to rule out control of the rates of these processes by the intracellular potassium concentration. An explanation for the generally observed correlation between the lactose and potassium concentrations in milk, may be that both the maintenance of the intracellular potassium concentration and the lactose synthesis rate require the presence of ATP.

Inhibition by cyclic AMP of lactose production in lactating guinea pig mammary gland slices.

Addition of the cyclic AMP phosphodiesterase inhibitors theophylline (10- minus 2 M) or papaverine (10- minus 4 M) leads to a complete inhibition of lactose synthesis in incubated guinea pig mammary gland slices. Addition of 10- minus 5 M cyclic AMP or dibutyryl cyclic AMP results in 1 30-40% inhibition of the synthesis, which effect is not increased by applying higher concentrations of these compounds. A 30-40% inhibition can also be obtained with epinephrine (5 - 10- minus 5 M), or isoproterenol (10- minus 4 M), but the polypeptide hormones glucagon (10- minus 7 M), insulin (1 munit/ml) and relaxin (10 mug/ml) do not significantly affect lactose synthesis. Cytochalasin B (5 mug/ml) inhibits lactose production by 58and colchicine (10- minus 5 M) by 25%. These experiments suggest that an increase in the intracellular level of cyclic AMP either through its addition, through hormonal stimulation of its synthesis, or through inhibition of its intracellular breakdown, leads to an inhibition of lactose production in lactating mammary gland. This effect of cyclic AMP is similar to that of progesterone, which is known to inhibit lactation in vivo and the withdrawal of which at parturition has been postulated to initiate lactogenesis.

Rat pancreas adenylate cyclase. III. Its role in pancreatic secretion assessed by means of choleara toxin.

1. The role of adenylate cyclase in rat pancreas is further investigated by means of cholera toxin, which is known to activate the enzyme in several tissues. 2. Cholera toxin activates rat pancreatic adenylate cyclase in vitro upon preincubation of tissue slices with the toxin for more than 30 min, but not when it is merely present during the enzyme assay. The maximal effect is reached after 90 min pre-incubation. The half-maximally activating concentration is 3.5 mu-g/ml upon pre-incubation for 90 min. 3. After pre-treatment of pancreatic tissue slices with 2 mu-g/ml cholera toxin, further stimulation of adenylate cyclase activity can be obtained by adding pancreozymin-C-octapeptide, secretin, or fluoride to the assay medium, but the final activity with maximally effective concentrations of the hormones is not higher, and with fluoride even less, than that without the toxin pre-treatment. 4. The in vivo effects of the two hormones and of cholera toxin have been studied after cannulation of the pancreas. Pancreozymin-C-octapeptide (intravenously) markedly stimulates both flow rate and rate of protein secretion. Synthetic secretin (intravenously), in addition to its expected effect on flow rate, slightly stimulates protein secretion, which is not due to a wash-out effect. Cholera toxin, topically applied to the cannulated rat pancreas, causes a steady increase of the flow rate after a delay of 20--30 min. The rate of protein secretion is not affected or slightly decreased by the toxin. Pancreozymin-C-octapeptide, given intravenously 1 h after cholera toxin application, causes the same increase in flow rate and rate of protein secretion as would be expected without cholera toxin treatment. 5. The sodium and potassium levels in the pancreatic fluid after administration of secretin or cholera toxin do not change, while the chloride level decreases in both cases. 6. These observations indicate that the rat pancreas adenylate cyclase activity is a rate-limiting factor in the regulation of water and electrolyte secretion. A possible auxiliary role in the regulation of enzyme secretion cannot yet be excluded.

Pertussis toxin stimulates cholecystokinin-induced cyclic AMP formation but is without effect on secretagogue-induced calcium mobilization in exocrine pancreas.

The role of a pertussis toxin sensitive GTP-binding protein in mediating between cholecystokinin receptors and phosphatidylinositol 4,5-bisphosphate phosphodiesterase as well as in preventing cholecystokinin from increasing cellular cyclic AMP has been investigated using dispersed acini from rabbit pancreas. Pertussis toxin pretreatment (500 ng/ml, 2 h) did not affect cholecystokinin(octapeptide) (CCK-8)-induced increases in cytosolic free Ca2+ as judged from changes in fluorescence obtained from quin2-loaded acini. Although pretreatment with pertussis toxin was also without effect on resting acinar cell cyclic AMP levels, adenylate cyclase activity was increased, since inhibition of cyclic AMP phosphodiesterase activity by isobutylmethylxanthine (IBMX) resulted in an additional increase in cyclic AMP levels in toxin-treated acini, indicating that acinar cell adenylate cyclase activity is under some tonic inhibitory control by the pertussis toxin-sensitive inhibitory GTP-binding protein (Gi) of the adenylate cyclase system. CCK-8 gave an increase in cyclic AMP levels in both control (1.6-fold) and toxin-treated (2.3-fold) acini, leading to cyclic AMP levels in the toxin-treated acini 2-times as high as those in control acini. In the presence of IBMX, the cyclic AMP response to CCK-8 was again markedly enhanced in acini pretreated with the toxin (3.2- vs. 1.8-fold), resulting in cAMP levels in the toxin-treated acini 3.7-times those in the absence of IBMX, 2.5-times those in control acini in the presence of IBMX and 7.0-times those in control acini in the absence of IBMX. Neither the pretreatment with pertussis toxin, nor the presence of IBMX alone, nor the combination had an effect on basal amylase secretion. However, all three treatments potentiated the stimulatory effect of CCK-8 on amylase secretion and the amount of potentiation was proportional to the cyclic AMP levels reached. Our findings suggest that in the intact pancreatic acinar cell Gi inhibition of the catalytic subunit of the adenylate cyclase may largely be responsible for preventing cholecystokinin from increasing cellular cyclic AMP. They moreover show that cyclic AMP is a modulatory agent in rabbit pancreatic enzyme secretion, not able to stimulate secretion itself, but potentiating effects mediated by the phosphatidylinositol-calcium pathway.