Interactions of 6-gingerol and ellagic acid with the cardiac sarcoplasmic reticulum Ca2+-ATPase.

The inotropic/lusitropic effects of beta-adrenergic agonists on the heart are mediated largely by protein kinase A (PKA)-catalyzed phosphorylation of phospholamban, the natural protein regulator of the Ca2+ pump present in sarcoplasmic reticulum (SR) membranes. Gingerol, a plant derivative, is known to produce similar effects when tested in isolated cardiac muscle. The purpose of the present study was to compare the effects of gingerol and another plant derivative, ellagic acid, on the kinetics of the SR Ca2+ pump with those of PKA-catalyzed phospholamban phosphorylation to elucidate their mechanisms of Ca2+ pump regulation. As previously demonstrated for PKA, 50 microM gingerol or ellagic acid increased Vmax(Ca) of Ca2+ uptake and Ca2+-ATPase activity assayed at millimolar ATP concentrations in light cardiac SR vesicles. Unlike PKA, which decreases Km(Ca), neither compound had a significant effect on Km(Ca) in unphosphorylated vesicles. However, gingerol increased Km(Ca) in phosphorylated vesicles, in which Ca2+ uptake was significantly increased further at saturating Ca2+ and remained unchanged at subsaturating Ca2+. An inhibition of Ca2+ uptake by gingerol at micromolar MgATP concentrations was overcome with increasing MgATP concentrations. The stimulation of Ca2+ uptake attributable to gingerol in unphosphorylated microsomes at saturating Ca2+ was 30% to 40% when assayed at 0.05 to 2 mM MgATP and only about 12% in phosphorylated microsomes as well as in rabbit fast skeletal muscle light SR. The present results support the view that an ATP-dependent increase in Vmax(Ca) of the SR Ca2+ pump plays an important role in mediating cardiac contractile responses to gingerol and phospholamban-dependent beta-adrenergic stimulation.

Effect of chronic electrostimulation of rabbit skeletal muscle on calmodulin level and protein kinase activity.

(a) Chronic electrostimulation of fast-twitch skeletal muscles makes them resemble slow-twitch muscles. The involvement of second-messenger cascades in this muscle reprogramming is not well understood. The goal of this study was to examine protein kinase activities and calmodulin levels as a function of the duration of electrostimulation. (b) Fast-twitch rabbit muscle was subjected to continuous low-frequency electrostimulation for 2 weeks. The extensor digitorum longus was taken and examined for calmodulin concentration and cAMP-dependent (PKA). Ca(2+)-phospholipid-dependent (PKC) and Ca(2+)-calmodulin-dependent (CaM kinase or PKB) protein kinase activities. (c) Electrostimulation for 14 days led to a significant increase in total calmodulin level and PKB activity, both rising in the cytosolic fraction. Protein kinase C translocated to the membrane fraction, although total activity did not change. (d) These changes could be related with electrostimulation-induced changes in excitation-contraction coupling.

Kinetic differences in the phospholamban-regulated calcium pump when studied in crude and purified cardiac sarcoplasmic reticulum vesicles.

Phospholamban (PLN) phosphorylation contributes largely to the inotropic and lusitropic effects of beta-adrenergic agonists on the heart. The mechanical effects of PLN phosphorylation on the heart are generally attributed solely to an increase in the apparent affinity of the Ca pump in the sarcoplasmic reticulum (SR) membranes for Ca2+ with little or no effect on Vmax(Ca). In the present report, we compare the kinetic properties of the cardiac SR Ca pump in commonly studied crude microsomes with those of our recently developed preparation of light SR vesicles. We demonstrate that in crude microsomes, the increase in the apparent affinity of the pump for Ca2+ is larger, while the increase in Vmax(Ca) is smaller, than in purified vesicles. The greater phosphorylation-induced increase in apparent Ca2+ affinity in crude microsomes may be further enhanced by an ATP-sensitive inhibitory effect of ruthenium red on the activity of the pump at subsaturating, but not saturating, Ca2+ concentrations as a result of a greater inhibition in unphosphorylated microsomes. Upon increasing the ATP concentration from 1 to 5 mm, an inhibition by 10 micrometer ruthenium red is eliminated in phosphorylated microsomes and reduced in control microsomes. Addition of the phosphoprotein phosphatase inhibitor okadaic acid produces a considerable increase in the phosphorylation-induced effects in both crude and purified microsomes. We conclude that the use of purified cardiac SR vesicles is critical for the demonstration of a major increase in Vmax(Ca) in addition to an increase in the pump's apparent affinity for Ca2+ in response to phosphorylation of PLN by protein kinase A.

Comparison of the kinetic effects of phospholamban phosphorylation and anti-phospholamban monoclonal antibody on the calcium pump in purified cardiac sarcoplasmic reticulum membranes.

Protein kinase A- (PKA-) catalyzed phosphorylation of phospholamban (PLN), the protein regulator of the cardiac Ca pump, mediates abbreviation of systole in response to beta-adrenergic agonists. Investigators previously, however, have been unsuccessful in demonstrating an effect of PLN phosphorylation or anti-PLN monoclonal antibody (mAb), which is considered to mimic phosphorylation's well-known effect on Km(Ca), on microsomal Ca uptake at the (high) Ca2+ concentrations found intracellularly at peak systole. We therefore compared the effects of the catalytic subunit of PKA and anti-PLN mAb on the kinetics of Ca uptake in sucrose gradient-purified cardiac microsomes. Both treatments produced a 33-44% increase in Vmax(Ca) at 25 and 37 degrees C, and an 11-31% decrease in Km(Ca) with comparable changes in Ca2+-ATPase activity. An acceleration of E2P decomposition upon PLN phosphorylation may contribute to the increased Vmax(Ca) of Ca uptake at 25 degrees C but not at 37 degrees C, based on measurement of the kinetics of E2P decomposition and steady-state E2P formation from Pi at different temperatures. Our data document almost identical increases in Vmax(Ca) of microsomal Ca uptake with PLN phosphorylation or addition of anti-PLN mAb and hence provide insight into the kinetic mechanism of PLN's regulation of the cardiac sarcoplasmic reticulum Ca pump protein.

[On the evaluation of the city children's dentist clinic]. / Ob otsenke raboty gorodskoy detskoy stomatologicheskoy polikliniki.

The authors offer criteria to validate the choice of a pediatric dental clinic or another treatment-and-prophylactic institution, as exemplified by Moscow, as the base for the study of therapeutic and diagnostic activity. The mean values for a number of parameters were determined: hourly loading of dentists, frequency of x-raying and physiotherapeutic procedures, sanitization of the oral cavity and teeth, etc., and deviations of the similar indexes at other outpatient clinics from these values, expressed in the mean square values. Summation of the mean square deviations with consideration for their positive or negative signs, followed by ranking of the data, helped determine an institution occupying an intermediate position, which, in author's opinion, indicates its typical character and, hence, should be preferable.

Comparison of the effects of phospholamban and jasmone on the calcium pump of cardiac sarcoplasmic reticulum. Evidence for modulation by phospholamban of both Ca2+ affinity and Vmax (Ca) of calcium transport.

Regulation of the calcium pump of the cardiac sarcoplasmic reticulum by phosphorylation/dephosphorylation of phospholamban is central to the inotropic and lusitropic effects of beta-adrenergic agonists on the heart. In order to study the mechanism of this regulation, we first obtained purified ruthenium red-insensitive microsomes enriched in sarcoplasmic reticulum membranes. The kinetics of microsomal Ca2+ uptake after phospholamban phosphorylation or trypsin treatment, which cleaves the inhibitory cytoplasmic domain of phospholamban, were then compared with those in the presence of jasmone, whose effects on the kinetics of fast skeletal muscle Ca2+-ATPase are largely known. All three treatments increased Vmax (Ca) at 25 degrees C and millimolar ATP; phosphorylation and trypsin decreased the Km (Ca), while jasmone increased it. Trypsin and jasmone increased the rate of E2P decomposition 1.8- and 3. 0-fold, respectively. The effects of phospholamban phosphorylation and jasmone on the Ca2+-ATPase activity paralleled their effects on Ca2+ uptake. Our data demonstrate that phospholamban regulates E2P decomposition in addition to the known increase in the rate of a conformational change in the Ca2+-ATPase upon binding the first of two Ca2+. These steps in the catalytic cycle of the Ca2+-ATPase may contribute to or account for phospholamban's effects on both Vmax (Ca) and Km (Ca), whose relative magnitude may vary under different experimental and, presumably, physiological conditions.

Membrane phosphorylation protects the cardiac sarcoplasmic reticulum Ca(2+)-ATPase against chlorinated oxidants in vitro.

OBJECTIVE: The calcium (Ca) pump of cardiac sarcoplasmic reticulum (SR) membranes is vulnerable to oxidation and hence likely to be damaged by chlorinated compounds, specifically hypochlorite (NaOCl) and monochloramine (NH2Cl), the most potent oxidants produced upon neutrophil activation. This could occur during prolonged ischemia or myocardial infarction when tissue levels of catecholamines are high. Phospholamban (PLN), the phosphorylatable regulator of the Ca pump, plays a central role in the effects of beta-adrenergic agonists on the heart. The purpose of this study was to investigate a possible role of PLN in determining the pump's sensitivity to NaOCl and NH2Cl. METHODS: Ca-uptake and Ca(2+)-ATPase activities in purified phosphorylated and control canine cardiac microsomes, incubated at increasing concentrations of NaOCl or NH2Cl, were related to the extent of PLN phosphorylation by protein kinase A, which was quantitated by PhosphorImager analysis. RESULTS AND CONCLUSIONS: Our data indicate that microsomal phosphorylation protects the Ca pump fully against 10 microM NaOCl or NH2Cl, which inhibit Ca-uptake by 21-41% when assayed at 25 or 37 degrees C and saturating Ca2+ in unphosphorylated microsomes, and protects partially at higher oxidant concentrations. The protective effect of protein kinase A on Ca-uptake is proportional to the amount of phosphorylated PLN. No comparable protection against similar oxidative damage of the Ca pump is observed when light fast skeletal muscle microsomes, which lack PLN, are incubated under conditions favorable for phosphorylation nor when PLN's inhibition of the cardiac Ca pump is relieved by proteolytic cleavage of its cytoplasmic domain. Our findings contribute toward an understanding of possible endogenous protective mechanisms that may promote calcium homeostasis in myocardial cells in inflammatory states associated with neutrophil activation and may suggest an approach toward development of protective strategies against oxidative damage in the heart.

[Prevalence and cause of dental loss in children and adolescents]. / Rasprostranennost' i prichina utraty zybov u detei i podrostkov.

The author analyzes statistical data of examinations of the oral cavity and teeth and of sociological interviews of 739 schoolchildren of a Moscow school, carried out in March, 1997. Loss of teeth has been recorded; the data characterize the maxillodental status and dental health. Relationship between dental loss and such factors as sex, age, place of lost teeth (functional orientation in the maxilla and mandible), etc., has been established. The author emphasizes the need in regular check-ups of the oral cavity and teeth in various age groups of children and adolescents both in girls and boys and considers that both dentists and endocrinologists should take part in these check-ups.

[Assessment of the indicators of organizational activities of dental polyclinics in Moscow]. / Ob otsenke pokazatelei organizatsionnoi deiatel'nosti stomatologicheskikh poliklinik Moskvy.

Presents the method of comprehensive probability assessment of a number of indexes calculated on the basis of the data derived from annual reports of 36 dental clinics of Moscow for 1994. Correlations between the indexes and their signal deviations from the mean values were assessed and their integration and ranging carried out. This confirmed the possibility of a more effective utilization of reported data in adopting the managerial solutions and improvement of the medical and technological process.

[Use of methods of overall service theory in the organization of therapeutic and diagnostic process in the dental care system]. / Ob ispol'zovanii metodov teorii massovogo obsluzhivaniia pri organizatsii lechebno-diagnosticheskogo protsessa v sisteme stomatologicheskoi pomoshchi.

Consultation rate for emergency dental care in Moscow is analyzed. Consultation rate in limited time periods conformed to Poisson's law; this opens vistas for applying the theory of overall care in operative planning of emergency dental care.

[The mechanisms of the effect of furosemide on the water permeability of the frog bladder and on oxytocin-stimulated contractions of the rat uterus]. / Mekhanizmy vliianiia furosemida na vodnuiu pronitsaemost' mochevogo puzyria liagushki i stimulirovannye oksitotsinom sokrashcheniia matki krysy.

Furosemide increased the hydrosmotic water flow in the frog urinary bladder and promoted the ADH-like effect of inhibitors of phosphodiesterase cAMP, potentiated hydrosmotic effects of theophylline and serosal osmotic hypertonicity but failed to change the effect of pituitrin. Fur reversibly suppressed oxytocin-induced contractions in the rat myometrium, inhibited the activity of the frog urinary bladder PDE cAMP, whereas the activity of the enzyme from the rat medulla and myometrium was activated by saluretic. Incubation of the myometrium strips in Fur resulted in a decrease in the cAMP content of the tissue. The cAMP seems to play an important role both in the myometrium smooth muscle relaxation and in the oxytocin-activated contractions.

[The effect of OCl- and Ag+ on active and passive calcium transport in the sarcoplasmic reticulum]. / Deistvie OCl- i Ag+ na aktivnyi i passivnyi transport kal'tsiia v sarkoplazmaticheskom retikulume.

After hypochlorite (OCl-) treatment of the aortic smooth muscle sarcoplasmic reticulum (SR), the membrane microviscosity increases considerably in "bound" lipid regions in comparison with protein-free regions. OCl- induces the inhibition of active and the enhancement of passive calcium transport in SR. Treatment of SR vesicles with Ag+ and then with OCl- (but not in the reverse order) leads to the enhancement of the activating effect of OCl- on passive calcium release from the vesicles. It is concluded that the enhancement effect is due to the OCl(-)-induced increase in the passive permeability of the SR membrane for Ca2+ as a result of pore formation in the lipid bilayer.

[Second messengers in heart cells and smooth muscle vessels]. / Vtorichnye posredniki v kletkakh serdtsa i gladkikh myshts sosudov.

Advances in regulation by secondary messengers of Ca2+ level in cardiomyocyte and vascular smooth muscle cell cytosols with special reference to the major differences in regulatory effects in cells of the both types are reviewed. The effects of cAMP, cGMP, Ca2+, calmodulin, diacylglycerol and polyphosphoinositides on the Ca(2+)-channel, Ca(2+)-ATPase, plasmalemma, sarcoplasmic reticulum and outer membrane Na+/Ca2+ uniporter function are considered. Compartmentation of secondary messengers and protein kinase in cardiac and vascular smooth muscle cells should be taken into consideration during extrapolation of in vitro data to an in situ situation. The feasible role of impaired phosphorylation of membrane-bound proteins of cardiac and vascular smooth muscle cells in cardiac insufficiency and atherosclerosis is discussed.

[cAMP, calmodulin-dependent stimulation and stability to proteolysis of Ca 2+ transport in the heart sarcoplasmic reticulum]. / cAMP, kal'modulinzavisimaia stimuliatsiia i ustoichivost' k proteolizu transporta Ca 2+ v sarkoplazmaticheskom retikulume serdtsa.

The calmodulin content in cardiomyocyte cytosol of hypoxic myocardium is increased compared to normal level. This is unaccompanied by differences in the stimulating effect of calmodulin on Ca2+ transport in sarcoplasmic reticulum (SR) of ischemic heart. The decrease of the endogenous cAMP-dependent protein kinase activity in ischemia is associated with the lowered resistance to trypsinolysis of Ca2+ transport in SR (trypsin/microsomal protein ratio is 1:10) with simultaneous Ca-ATPase activation. In the presence of exogenous protein kinase and cAMP the protective effect of phosphorylation on Ca2+ transport in SR vesicles of hypoxic cardiomyocytes treated with trypsin for 10 min reaches the same level as in intact heart.

The increase in viscosity and peroxidation of sarcoplasmic reticulum membrane lipids in isadrine myocarditis.

The viscosity of membranes isolated from sarcoplasmic reticulum of rabbits with isadrine myocarditis was studied, using pyrene as a hydrophobic fluorescent probe. The increase in the viscosity of membranes from injured heart occurred at lower temperatures and was sharper than in the case of intact heart in both "free" and "bound" lipid domains. The increase in the lipid viscosity under myocarditis was associated with decreased Ca++, Mg++ -ATPase and cAMP-dependent protein kinase activities and with an elevated content of lipid peroxidation products.

[Ultrastructural and functional changes in the myocardium and coronary vessels after massive blood loss]. / Ul'trastrukturnye i funktsional'nye izmeneniia miokarda i ego sosudov posle massivnoi krovopoteri.

The ultrastructure of cardiomyocytes and circulatory bed has been compared to transmembrane cAMP-dependent Ca2+ transport in experiments on the hearts of 14 dogs immediately after massive blood loss. The results an hour after non-compensatory hemorrhage have shown extra- and intracellular myocardial edema, central destruction of sarcomers, steep increase in the volume of agranular sarcomplasmic reticulum and T-system, different degree of damage of other organoids, and also disturbances in the ultrastructure of venous capillary and postcapillary section. The biochemical techniques used have shown a decrease in Ca2+ transporting ability of sarcolemma due to its AMP-dependent regulation of cardiomyocytes. Excessive Ca2+ storage in cytosole promoted the appearance of "constriction bands" in myofibrils.