Expression and activity of the Ca(2+)-atpase enzyme in human neonatal erythrocytes.

The plasma membrane Ca(2+)-ATPase (PMCA) is one of the main regulators of Ca(2+) homeostasis. We studied the perinatal alteration of the abundance and the activity of PMCA molecules in human erythrocytes in pre-term and full-term neonates and children at the age of 1-4 years. The lower abundance of the 4b isoform was associated with lower enzyme activity in full-term neonates compared to children. Although the number of PMCA molecules was higher in pre-term neonates, their total PMCA activities were identical to those of full-term neonates. Our findings suggest that the abundance of PMCA molecules changes during the perinatal development. The same activity at higher enzyme molecule numbers might indicate a potential immaturity of the enzyme in the pre-term infant.

[Diabetes mellitus as a general membrane disease and its consequences]. / Diabetes mellitus mint általános membránbetegség és ennek következményei.

The metabolic disturbances and their consequences in diabetes mellitus are well known more or less in details too. However, our knowledge on the diabetic disorders in membrane functions are limited. These damages are connected mostly with the disregulation of the membrane protein syntheses due to deficiency of insulin. In this review the impairments of the Na(+)-pump and the Ca(2+)-transport mechanisms as well as the insulin-dependent glucose transporter GLUT4 will be discussed in diabetes. The capacity of these transporters could be decreased even more than 50 percent in diabetes. This is the reason why using the same dose of cardioactive steroids as if in not diabetic subjects--can cause toxic alterations on the heart in diabetic patients. Insulin regulates not only the expression of some membrane proteins but it can initiate the translocation of the Na(+)-pump and GLUT4 from the intracellular membrane compartments to the plasma membrane in muscle, heart and adipose tissue. Therefore the uptake of K+ and glucose into these tissues will increase significantly under the acute influence of insulin. Untreated diabetic patients generally show hyperkalemia. Forceful treatment with insulin of these subjects often causes severe hypokalemia as a consequence of sudden translocation of the Na(+)-pump. Different Ca(2+)-transport systems are also impaired in diabetes. These changes may result significantly higher free Ca2+ concentration in the cytoplasma of cardiomyocytes. This is one of the most important reason for the Ca2+ overloading and ultimately for heart death. According to authors opinion, beside the dangerous metabolic disorders, general membrane damage and extended disturbances in membrane functions are also very characteristic for diabetes. The acknowledgement of these alterations are very important for the exact planning of the up to date treatment of diabetes.

Streptozotocin-induced diabetes alters the oligomerization pattern of acetylcholinesterase in rat skeletal muscle.

AIMS/HYPOTHESIS: Diabetes mellitus has a serious effect on most of the properties of skeletal muscles. Changes in neuromuscular transmission are also involved in propagating the disease. METHODS: In our experiments, acetylcholinesterase was extracted from the fast extensor digitorum longus and slow soleus muscles of control, non-treated 6-week-diabetic and insulin-treated diabetic rats. The extracts were applied to velocity sedimentation and acetylcholinesterase activity was determined. RESULTS: We observed considerable differences in the distribution of individual acetylcholinesterase molecular forms in diabetic fast muscles. This included a 59% decline in G4 content together with a fivefold increase in A8 and a 53 % increase in A12 activity resulting in a shift of acetylcholinesterase profile characteristically towards slow muscles. These alterations were partly reversed by insulin treatment. CONCLUSION/INTERPRETATION: In slow muscles diabetes caused an increase in G4 activity without affecting the sedimentation profile. Decline in G4 content in fast muscles could contribute to enhanced desensitization of acetylcholine receptors in diabetes.

Determination of H+/K+-ATPase activity in human gastric biopsy specimens.

The aim of our work was to develop a method to determine the the H+/K+-ATPase activity of human gastric biopsy samples. Our method is based on the phosphatase activity and the K+-inducible property of the enzyme. K+-inducible pNPPase activity was determined from homogenated corpus and antrum biopsy samples. H+/K+-ase activity was calculated as the difference between the corpus and antrum K+-inducible pNPPase activities. Quality control measurements were done during 20 successive days from pooled homogenates. The total, between-day and between-run, within-day and within-run coefficients of variations were between 10 and 16%. The healthy mean and reference range of K+-inducible pNPPase activity in the corpus was 95.8 (95% CI: 83.4-108.2 mU/mg protein); in the antrum it was 28.3 (21.6-35.0) mU/mg protein. The calculated H+/K+-ATPase activity was 67.2 (56.9-77.5) mU/mg protein. The measured activities were independent of the age and gender. Summarizing our results we have concluded, that our novel method might be a potential tool to gather data about the functional acid producing capability of human gastric mucosa.

Developmental changes in erythrocyte Na(+),K(+)-ATPase subunit abundance and enzyme activity in neonates.

AIM: To study the relation between erythrocyte Na(+),K(+)-ATPase subunit isoform composition, Na(+),K(+)-ATPase activity, and cation pump function in preterm and term neonates. DESIGN: Erythrocyte Na(+),K(+)-ATPase subunit isoform abundance, Na(+),K(+)-ATPase activity, and cation pump function were studied in blood samples obtained from 56 preterm neonates of 28-32 weeks gestation (group 1), 58 preterm neonates of 33-36 weeks gestation (group 2), and 122 term neonates (group 3) during the first two postnatal days. RESULTS: alpha(1) isoform abundance was higher and beta(2) isoform abundance was lower in group 1 than in group 3 (p = 0.0002). alpha(2) and beta(1) isoform abundance did not change with maturation and there was no evidence for the presence of the alpha(3) isoform. Gestational age was inversely related to Na(+), K(+)-ATPase activity (p = 0.0001) and directly related to intracellular Na(+) concentration (p = 0.0025). CONCLUSIONS: Expression of the alpha(1) and beta(2) Na(+),K(+)-ATPase subunit isoforms is developmentally regulated. The increased abundance of alpha(1) isoforms of immature neonates translates to increased ATPase activity. The lower intracellular Na(+) concentration of immature neonates suggests that their erythrocyte Na(+),K(+)-ATPase cation pump function may also be increased.

Functional and structural properties of Na+/K(+)-ATPase enzyme in neonatal erythrocytes.

BACKGROUND: The Na+/K(+)-pump is the main regulator enzyme of intracellular monovalent cation concentration. There are only limited data available concerning its structure and function in healthy neonates, in comparison with data available regarding its structure and function in children. PATIENTS AND METHODS: Samples of 100 microL of anticoagulated blood were taken from 53 healthy neonates (age under 6th postnatal day, median age 3.5 days) and 61 healthy children (median age 12.4 months, range 6-36 months,). The Na+/K(+)-ATPase activity, its sensitivity to ouabain (a digoxin-analogue substance) and the expression of Na+/K(+) ATPase subunit isoforms were determined. RESULTS: The enzyme activity (429.2 +/- 17.2 versus 295.5 +/- 10.2 U, P < 0 x 0.001) and I50 value for ouabain inhibition (1.50 +/- 0.10 versus 0.96 +/- 0.10 mumol L-1, P < 0.05) was higher in neonates. More alpha 1 subunits (relative density: 1.16 +/- 0.10 versus 0.75 +/- 0.03, P < 0.001) and higher alpha 1/ alpha 2 ratio (4.14 +/- 0.21 versus 2.02 +/- 0.16, P < 0.01) were detected. CONCLUSION: This is the first study demonstrating changes of Na+/K(+) -ATPase molecules not only in enzyme activity, but also on protein level. Our results might contribute to the understanding of the resistance of neonatal cell membranes toward the pharmacodynamic actions of cardiac glycosides.

Changes in the expression of Na+/K+-ATPase isoenzymes in the left ventricle of diabetic rat hearts: effect of insulin treatment.

Na+/K+-ATPase related strophanthidin sensitive 3-O-methylfluorescein-phosphatase activity, [3H]ouabain binding and expression of Na+/K+-ATPase subunit isoforms were measured in the left ventricle of the heart of normal and streptozotocin-diabetic rats with and without insulin treatment. Compared to control animals, the enzyme activity was 0.75 +/- 0.09 and 0.62 +/- 0.06 times lower in rats diabetic for 2 and for 4 weeks, respectively. This was associated with a proportional decrease of the [3H]ouabain binding sites. Immunoblots indicated a 0.76 +/- 0.08 and 0.61 +/- 0.08-fold decrease of alpha1, a 0.68 +/- 0.09 and 0.41 +/- 0.04-fold decrease of alpha2 subunit in 2- and 4-week diabetic rats, respectively relative to controls. Beta1 subunit decreased proportionally 0.71 +/- 0.07 and 0.38 +/- 0.06-fold, and beta2 decreased 0.75 +/- 0.08 and 0.31 +/- 0.06-fold, respectively. Northern blot analysis revealed a significant reduction in mRNA level of Na+/K+-ATPase subunit isoforms after 2 and 4 weeks of diabetes (for alpha1 66.2 +/- 8.2 and 55.9 +/- 7.8% of controls for alpha2 91.7 +/- 12.1 and 41.1 +/- 7.1% of controls and for beta subunit 93.4 +/- 11.1 and 49.8 +/- 6.8% of controls, respectively). Although, mRNA levels of isoform reverted to even higher levels than the control values after insulin treatment, insulin caused only a partial recovery of enzyme activity, [3H]ouabain binding capacity and protein expression. We have obtained evidence that in cardiac left ventricle there are more than one type of Na+/K+-ATPase alpha and beta subunit isoforms which are affected in diabetes and by insulin treatment. The time course of diabetes induced changes and the degree of involvement suggest that the Na+/K+-ATPase isoforms are altered individually.

Alterations in the properties and isoform ratios of brain Na+/K(+)-ATPase in streptozotocin diabetic rats.

In this study we analysed the changes in the properties of rat cerebral cortex Na+K(+)-ATPase in streptozotocin induced diabetes (STZ-diabetes). Special attempt was made to determine whether insulin treatment of diabetic animals could restore the altered parameters of this enzyme. Na+/K(+)-ATPase activity was found to be decreased by 15% after 2 weeks, and by 37% after 4 weeks in diabetic rat brains with a parallel decrease in maximal capacity of low affinity ouabain binding sites. There was no significant change in the high affinity ouabain binding sites. The Kd values did not change significantly. Western blot analysis of brain Na+/K(+)-ATPase isoforms indicated a 61 +/- 5.8% and 20 +/- 2.8% decrease of the alpha 1 and alpha 3 isoforms, respectively in 4 weeks diabetic animals. Change in the amount of the alpha 2 isoform proved to be less characteristic. Both types of beta subunit isoform showed a significant decrease in four weeks diabetic rats. Our data indicate a good correlation in diabetic rats between changes in Na-/K(+)-ATPase activity, low affinity ouabain binding capacity and the level of alpha 1 isoform. While insulin treatment of diabetic animals restored the blood glucose level to normal, a complete reversal of diabetes induced changes in Na+/K(+)-ATPase activity, ouabain binding capacity and Na+/K(+)-ATPase isoform composition could not be achieved.

Transformation and recovery of rat skeletal muscles in pathologic hormonal states.

There are numerous controversial data published on muscle transformation in pathologic endocrine states. Information is consistent in respect of slow muscle sensitivity to diabetes, but data concerning characteristics of fast muscles are contradictory under the same conditions. Sensitivity of fast and slow muscles to diabetes was examined and compared in this study. Diabetes was induced by a single intravenous dose of Streptozotocin in Wistar rats. Contractile parameters of soleus and extensor digitorum longus muscle were determined by in vitro measurements. We collected data on reversibility of diabetic transformation using exogenous thyroxine and insulin treatment. Insulin was used both to prevent and revert diabetic transformations. We found that, according to the data previously published, soleus muscle is more sensitive to diabetes than the extensor digitorum longus (EDL). Changes in the EDL muscle proved not to be statistically significant, but the comparison of hyperthyroid and diabetic muscles to controls led us to the conclusion that there is a sublimital transformation in the EDL in diabetes. T4 and insulin were effective in preventing diabetic muscle transformation. Application of insulin not only prevented diabetic complications, but its use in animals suffering from diabetes for a longer time (8 weeks) resulted in a successful recovery of the muscle transformation.

Interference of the sulphonylurea antidiabeticum gliquidone with mitochondrial bioenergetics in the rat under in vitro conditions.

The hypoglycaemic sulphonylurea gliquidone and glibenclamide exerted a partial uncoupling effect on mitochondrial respiration of liver under in vitro conditions using various citrate cycle intermediates as substrates. Besides the uncoupling effect, gliquidone and glibenclamide caused a direct inhibition of ATP-as well as DNP-stimulated oxygen consumption. Both phenomena proved to be dose dependent. Respiratory control ratio decreased progressively with increasing concentrations of sulphonylureas mainly through the inhibition of ADP-stimulated respiration. Basal and DNP-stimulated ATP-ase activity of isolated mitochondria changed similarly to the respiratory parameters. Changes in membrane permeability of mitochondria and the inhibition of substrate uptake further support the assumption of structural and functional alteration of mitochondria by the hypoglycaemic compounds tested.

Interference of sulphonylurea antidiabetica with mitochondrial bioenergetics under in vivo conditions.

Sulphonylurea antidiabetica effectively inhibits the basal hepatic glucose production. Since it has been firmly established that lipophylic sulphonylurea drugs exerted an uncoupling effect on mitochondrial oxidative phosphorylation, a relationship between the reduction of hepatic gluconeogenesis and the insufficient energy supply due to sulphonylureas could be supposed. In this study we have investigated the effects of glibenclamide and gliquidone on mitochondrial bioenergetics in liver after peroral treatments of normal rats with different doses. The treatment of rats with 5 mg/kg glibenclamide or gliquidone daily for 14 days elicited only a marginal inhibition on mitochondrial oxidation capacity and remained without any effect on mitochondrial ATPase activity. Only the supermaximal dose 50 mg/kg for 14 day produced a significant damage in the mitochondrial functions. The basal respiration increased with 60-80 per cent, whereas the ADP- or DNP-stimulated oxygen consumption significantly decreased independently from the respiratory substrates investigated. Similar alterations were found in the mitochondrial ATPase activity after treatment with these drugs. No essential differences have been observed in the actions between glibenclamide and gliquidone. However, the lowest dose applied in this study is many times higher than the usual therapeutic dose. Consequently, glibenclamide and gliquidone do not interact with mitochondrial bioenergetic processes under therapeutic conditions. On the other hand, in different liver and kidney damages we have no sufficient knowledge whether these drugs can be accumulated in these organs and therefore their elevated concentration may interfere with the mitochondrial energy metabolism.

Effect of streptozotocin-induced diabetes on kidney Na+/K(+)-ATPase.

The maximal capacity of low affinity ouabain binding sites in kidney medulla was found to be increased by 20 +/- 3.8% after 2 weeks, and by 35 +/- 4.5% in 4 weeks diabetes. However, in kidney cortex no similar changes could be detected. Western blot analysis of Na+/K(+)-ATPase subunits in kidney medulla indicated a significant enhancement of both the alpha 1 and beta 1 subunit in two and four weeks diabetic rats (alpha 1: 35 +/- 3.1, 51 +/- 5.8% and beta 1: 31.3 +/- 5.2 and 43.2 +/- 6.8%, respectively). However, kidney cortex showed no significant change in any condition tested. In diabetes we could detect a significant change only in the medulla in case of the b subunit mRNA transcript, which showed 1.69 +/- 0.59 and 2.89 +/- 0.81 times increased in two and four weeks diabetic state, respectively. There was no change in the alpha 1 subunit mRNA abundance. Insulin treatment of diabetic animals did not result in a complete reversal of diabetes-induced changes in ouabain binding capacity or in the amount of Na+/K(+)-ATPase alpha 1 and beta 1 subunit protein and mRNA levels. Our data indicate a good correlation between changes in low affinity ouabain binding capacity and the level of alpha 1 isoform in diabetic rats, and suggest an important role of the b subunit in the regulation of enzyme quantity.

Age-dependent responses to chemosensory cues mediating kin recognition in dogs (Canis familiaris)

During individually administered 5-min tests conducted in a neutral cage, four age groups (n = 10 males and 10 females per group) of purebred beagles reacted to bedding from their home cage vs. bedding from another litter of the same age. The 20-24-day-old males and females preferred (p < 0.05) home cage bedding over strange cage bedding. Those aged 31-36 days or 66-72 days showed no reliable preference for either type of bedding. Among pups aged 52-56 days, the males preferred (p < 0.05) strange cage bedding, but the females showed no reliable preference. Chemosensory cues are sufficient as mediators of kin recognition in beagles, but their reactions to such cues vary with age-dependent factors, some stemming from changes in the strength of the mother-litter bond. The dogs providing the two types of bedding lived in the same room and on the same diet. Therefore, kin recognition could not have been mediated by different chemosensory cues produced by variations in these environmental factors.

Oxytocin regulates Ca2+ level in myometrium by influencing phosphoinositide metabolism.

The effect of oxytocin on phosphoinositide metabolism as well as on membrane protein phosphorylation in myometrial tissue was studied. Oxytocin enhanced the 32P incorporation into phospholipids in myometrial tissue. The effect of oxytocin on phosphoinositide metabolism was also detected in plasma membrane of 20 days pregnant rats. Phosphorylated membrane lipids have been analysed and phosphatidylinositol 4, 5-bisphosphate proved to be the main reaction product. Oxytocin enhanced the 32P incorporation into phospholipids measured in the first 30 sec then the labeling decreased more rapidly then in case of the control. The effect of oxytocin proved to be concentration dependent. The protein phosphorylation was also influenced by oxytocin. However the amount of alkylphosphate formed depended on the presence or absence of Ca2+, Ca2+-calmodulin and cyclic AMP, oxytocin influenced the protein phosphorylation in the presence of Ca2+-calmodulin only.

Comparison of Ca2+-extrusion systems in the myometrial plasma membrane of pregnant and non-pregnant rats.

Plasma membrane derived from the myometrial tissues of pregnant and non-pregnant rats accumulates Ca2+ both by Na+ and by ATP-dependent ways. Sodium-dependent Ca2+-uptake in plasma membranes obtained from 20 days pregnant rats reveals higher Ca2+ sensitivity than in plasma membrane of non-pregnant rats. Ca2+ concentration required for half maximal Ca2+-transport was 5.7 +/- 0.5 microM and 11.8 +/- 0.6 microM in plasma membrane of pregnant and non-pregnant rats, respectively. The initial rate of sodium-dependent Ca2+-uptake also differed in membrane preparations from pregnant and non-pregnant animals. ATP-dependent Ca2+-uptake in myometrial plasma membrane of pregnant rats was higher than in non-pregnant ones. The difference was abolished by calmodulin antagonist drugs. In calmodulin depleted preparations the administration of exogenous calmodulin restored Ca2+-uptake to the rate measured in calmodulin not-depleted ones. Membrane phosphorylation by endogenous protein kinases proved to be also different in the two preparations.

Effects on pseudopregnancy, pregnancy and interoestrous intervals of pharmacological suppression of prolactin secretion in female dogs and cats.

Cabergoline, a new ergoline derivative, is a potent prolactin inhibitor. In this review, results are combined from previously published and unpublished blind laboratory and open clinical studies with cabergoline in pseudopregnant, pregnant and lactating bitches, in bitches with normal and prolonged cycles, and in pregnant queens. Dose-response studies in nursing bitches, using puppy weight as an endpoint, revealed that a dose of 5 micrograms/kg/day orally (for 5 days) was the optimal dose with a minimum of side effects. This dose effectively lowered blood prolactin concentrations in pregnant bitches and was partly luteolytic during the 1st half of gestation, and fully luteolytic during the 2nd half of gestation. Consequently, pregnancies were terminated in the 2nd half of pregnancy in the bitch, and in the queen. Treatment successes with pseudopregnancy and true and false lactation, including cases of eclampsia, were greater than 90%. The same level of success was seen in bitches with prolonged cycles (anoestrus). A 7-10-day treatment period resulted almost uniformly in oestrus, and restored fertility in greater than 80% of all bitches mated. Cycles were occasionally shortened in bitches treated for false lactation. Attempts to shorten cycles routinely in beagle bitches, in a commercial breeding operation, with a dose of 5 micrograms/kg/day for 14 days during months 4, 5 or 6 of the cycle were unsuccessful.

Efficacy of oral epostane administration to terminate pregnancy in mated laboratory bitches.

Epostane was administered orally for 7 days at doses of 10-300 mg/dog/day, starting at early oestrus, mid-oestrus, or metoestrus in 162 mated beagle bitches. With the exception of the early oestrus group receiving 10 mg epostane dog/day x 7 days, the incidence of dogs whelping in all other treatments was statistically lower than that of the placebo-treated control group (P less than 0.01). The ED50 and ED90 values were 19.5 and 43.2 mg/dog, respectively, for administration at early oestrus, and 8.6 and 24.0 mg/dog, respectively, for administration at metoestrus. Progesterone concentrations were significantly decreased after administration at oestrus or metoestrus in all epostane-treated groups compared to those given placebo. There were no adverse reactions or clinically significant changes in laboratory parameters or health of the bitches related to the use of epostane. There were no changes in the reproductive performance of epostane-treated bitches at the next oestrus after treatment compared to placebo controls, and there were no reproductive problems noted in the progeny of epostane-treated bitches. These results suggest that the minimally effective dose of epostane for application during metoestrus is approximately 2.5 mg/kg and the effective dose range is about 2.5-5.0 mg/kg body weight. Oral epostane is potentially a novel agent for the safe interception of unwanted pregnancy in dogs.

The use of epostane, a 3(beta)-hydroxysteroid dehydrogenase delta 4-5 isomerase enzyme inhibitor, in oil suspension as a mismating agent in the dog.

Administration of epostane as a single subcutaneous injection in sesame oil prevented or terminated pregnancy in bred beagle bitches (P = 0.002). Doses of 15 and 20 mg/kg prevented or terminated pregnancy in all of 14 bred beagle bitches studied but demonstrated a high rate of serious abscess formation at the injection site. A dose of 10 mg/kg was partially effective in 3 of 12 subjects whelping live pups, and a dose of 2.5 mg/kg was completely ineffective in 12 of 12 subjects whelping live pups. Plasma progesterone levels were lowered in dose-related fashion with respect to both depth of depression (P < 0.01) and length of time (P < 0.05). Subjects in which pregnancy was maintained had significant differences (P < 0.01) in progesterone patterns compared with nonpregnant subjects, as measured by area under the curve. No clinically significant differences were seen between treated and control females with respect to return to estrus, subsequent fertility, or in a battery of serum electrolytes, blood parameters and serum enzymes.

Drug disposition and biotransformation in the developing beagle dog.

The ability of developing male and female beagle pups to biotransform and eliminate drugs was studied by administering single intravenous doses of acetaminophen (50 mg/kg body wt), phenobarbital (15 mg/kg body wt), or phenytoin (15 mg/kg body wt) to the same groups of dogs (n = 6-8/drug) at 4, 10, 20, 40, and 60 days of age. At suitable intervals after treatment, small (1.0 ml) blood samples were obtained via the jugular vein and centrifuged and the plasma was recovered and stored at -20 degrees C to await analysis. Acetaminophen proved to be the most interesting "probe" of function with the plasma elimination half-life (beta t/2) in 40- to 60-day-old pups being 4.5-fold shorter than at 4 days of age. The synthesis of sulfate-conjugated drug decreased with age. In older pups, the synthesis of the glucuronide-conjugated drug was predominant. The elimination of sulfated acetaminophen from plasma was slow at all ages whereas the rate of glucuronide disappearance increased with age. Phenobarbital was slowly eliminated from the plasma at all ages and there was no indication of p-hydroxylated metabolite formation. The plasma beta t/2 of phenytoin decreased dramatically with age, a 10-fold difference occurring between 4- and 60-day-old pups. para-Hydroxylated phenytoin (pHPPH) was detected only in the plasma of 4- and 10-day-old pups, the plasma beta t/2 decreasing with age. With the appropriate chemical and using the technique of collecting small, serial blood samples, this animal model can be potentially useful in perinatal toxicity studies.