ATPase activity and uptake of calcium by a fraction of plasma membrane of rabbit myometrium at functional rest and in pregnancy

Study of the dependence of 45CA2+ binding on its concentration demonstrated that both at functional rest (FR) and in pregnancy the Ca-binding ability of rabbit myometrium subcellular fractions is decreased in the following order: sarcolemma greater than microsomes greater than mitochondria approximately nuclei. The values of Kd for the sarcolemmal Ca2+-binding site complex are identical in both cases (80-87 micro M); however, the maximal calcium-binding capacity of pregnancy is 1.5 times less than in FR. In the plasma membrane fraction of pregnant rabbits the adenosine triphosphatase activity is decreased. The rate of ATP-dependent 45Ca2+ uptake in sarcolemmal fraction is significantly higher than in other subcellular fractions in both conditions. It is concluded that at FR and in pregnancy sarcolemma plays a key role in regulation of ionized calcium concentration in myometrium cells.     

Mechanical Properties of the Frog Sarcolemma

The elastic properties of cylindrical segments of sarcolemma were studied in single striated fibers of the frog semitendinosus muscle. All measurements were made on membranes of retraction zones, cell segments from which the sarcoplasm had retracted. Quantitative morphological studies indicated that three deforming forces interact with the intrinsic elastic properties of the sarcolemma to determine membrane configuration in retraction zone segments. The three deforming forces, namely intrazone pressure, axial fiber loads, and radial stresses introduced by retracted cell contents, could all be experimentally removed, permitting determination of the “undeformed” configuration of the sarcolemma. Analysis of these results indicated that membrane of intact fibers at rest length is about four times as wide and two-thirds as long as undeformed membrane. Membrane geometry was also studied as a function of internal hydrostatic pressure and axial loading to permit calculation of the circumferential and longitudinal tension-strain (T-S) diagrams. The sarcolemma exhibited nonlinear T-S properties concave to the tension axis in both directions. Circumferential T-S slopes (measures of membrane stiffness) ranged from 1500 to greater than 50,000 dynes/cm over the range of deformations investigated, while longitudinal T-S slopes varied from 23,000 to 225,000 dynes/cm. Thus, the membrane is anisotropic, being much stiffer in the longitudinal direction. Certain ramifications of the present results are discussed in relation to previous biomechanical studies of the sarcolemma and of other tissues.     

An electron paramagnetic resonance study of skeletal muscle membrane fluidity in malignant hyperthermia

Skeletal muscle sarcolemma (SL), transverse tubule (TT) and heavy sarcoplasmic reticulum (HSR) membranes were isolated from malignant hyperthermia susceptible (MHS) and normal pigs, and the rotational dynamics of lipid hydrocarbon chain motion was examined by electron paramagnetic resonance (EPR) spectroscopy. The stearic acid spin probe 16-SASL was incorporated into MHS and normal membranes and both the order parameter (S) and effective correlation time (tau r) of probe motion were calculated from spectra recorded over the temperature range of 2 to 40 degrees C. At any given temperature, TT membranes exhibited significantly greater values for both the S and tau r of probe motion than did SL, which exhibited significantly greater values than did HSR membranes. The order of decreasing S and tau r values for 16-SASL mobility correlated with the decreasing cholesterol content of these membranes (TT greater than SL greater than HSR), however there was no difference in the S or tau r values for a given membrane fraction isolated from both MHS and normal muscle. Arrhenius plots of 16-SASL mobility in SL, TT and HSR were linear from 2 to 40 degrees C, indicating no abrupt thermotropic change in the lipid hydrocarbon phase of any of the membrane types studied. Apparent activation energies (Ea), calculated from the Arrhenius plots, were similar for MHS and normal membranes derived from a given cellular location. However, the Ea of probe motion for TT membranes (2.3 +/- 0.1 and 2.4 +/- 0.1 kcal/mol/degree for MHS and normal, respectively) was significantly less than for SL (3.4 +/- 0.4 and 2.9 +/- 0.1 kcal/mol/degree for MHS and normal, respectively) which, in turn, was significantly less than the Ea for HSR (3.7 +/- 0.1 and 3.7 +/- 0.1 kcal/mol/degree for MHS and normal, respectively). Since 16-SASL motion was similar in MHS and normal membranes, we conclude that there is no evidence for a generalized membrane defect affecting lipid mobility in these MHS muscle membranes.     

Gastrin metabolism in neonatal pigs and grower-pigs.

1. Half-life (1.7 +/- 0.1 min), distribution volume (146 +/- 12 ml/kg) and metabolic clearance rate (28 +/- 1 ml/kg/min) of little gastrin (G17) in neonatal pigs (N = 6; 3-12 days old) were significantly different from those in grower-pigs (N = 4; 161-170 days old) (2.4 +/- 0.1 min; 58 +/- 2 ml/kg; 7.9 +/- 0.3 ml/kg/min, respectively). 2. Half-life (33 +/- 4 min) and distribution volume (265 +/- 33 ml/kg) of big gastrin (G34) in neonatal pigs were greater but not significantly different from those in grower-pigs (24 +/- 2 min; 217 +/- 20 ml/kg, respectively). 3. Half-life of G17 in liver extracts from pigs 2-90 days old (40.4 +/- 4.2 min) was significantly longer than in kidney extracts (22.0 +/- 1.7 min). Half-lives of G34 in liver and kidney extracts from pigs 10-90 days old (78 +/- 6; 74 +/- 4 min, respectively) were significantly shorter than the corresponding values for 2-day-old pigs (134 +/- 3; 149 +/- 9 min, respectively). 4. Since G34 is the major circulating form of gastrin in neonatal pigs the relative longer half-life of G34 to G17 in these animals may contribute to the higher circulating gastrin concentration compared with that in older animals.     

Further characterization of light and heavy sarcoplasmic reticulum vesicles. Identification of the ‘sarcoplasmic reticulum feet’ associated with heavy sarcoplasmic reticulum vesicles

Light and heavy sarcoplasmic reticulum vesicles were isolated from rabbit leg muscle using a combination of differential centrifugation and isophycnic zonal ultracentrifugation. Light sarcoplasmic reticulum vesicles obtained from the 30–32.5% and heavy sarcoplasmic reticulum vesicles obtained from the 38.5–42% sucrose regions of the linear sucrose gradient were determined to be free of surface and mitochondrial membrane contamination by marker enzyme analysis and electron microscopy. Thin sections of the light vesicles revealed empty vesicles of various sizes and shapes. Freeze-fracture replicas of the light vesicles showed an asymmetric distribution of intramembranous particles with the same orientation and distribution as the longitudinal sarcoplasmic reticulum in vivo. Heavy vesicles appeared as rounded vesicles of uniform size filled with electron dense material, similar to that seen in the terminal cisternae of the sarcoplasmic reticulum. The cytoplasmic surface of the membrane was decorated by membrane projections, closely resembling the ‘feet’ which join the sarcoplasmic reticulum to the transverse tubules in the intact muscle fiber. Freeze-fracture replicas of the heavy vesicles revealed an asymmetric distribution of particles which in some areas of the vesicle's surface are larger and less densely aggregated than those of the light vesicles. In the best quality replicas, some regions of the luminal leaflet were not smooth but showed evidence of pits. These structural details are characteristic of the area of sarcoplasmic reticulum membrane which is covered by the ‘feet’ in the intact muscle.Heavy vesicles contained greater than six times the calcium content of light vesicles, 54 vs. 9 nmol Ca²⁺/μl of water space. After KCl washing both contained less than 4 nmol Ca²⁺/μl of water space. Although they transported at the same rate and the same total amount of calcium, the rate of passive Ca²⁺ efflux from the heavy vesicles was double that of light vesicles. The higher rate of calcium efflux from the heavy vesicles was inhibited by dantrolene, an inhibitor of Ca²⁺ release. High resolution sodium dodecyl sulfate gel electrophoresis showed that the light vesicles contained predominantly Ca²⁺-ATPase along with several approx. 55 000-dalton proteins and a 5000-dalton proteolipid, while the heavy vesicles contained Ca²⁺-ATPase and calsequestrin along with several approx. 55 000-dalton proteins, extrinsic 34 000- and 38 000-dalton proteins, intrinsic 30 000- and 33 000-dalton proteins and two proteolipids of 5000 and 9000 daltons. KCl washing of the heavy vesicles removed both the approx. 34 000- and 38 000-dalton proteins, and the ‘sarcoplasmic reticulum feet’ were no longer seen on the heavy vesicles. The KCl supernatant was enriched in the 34 000- and 38 000-dalton proteins, indicating that these proteins are possible components of the sarcoplasmic reticulum feet. The biochemical and morphological data strongly support the view that the light vesicles are derived from the longitudinal sarcoplasmic reticulum and that the heavy vesicles are derived from the terminal cisternae containing junctional sarcoplasmic reticulum membrane with the intact ‘sarcoplasmic reticulum feet’.     

Plasma membranes from cardiac cells in culture. Enzymatic radio-iodination, evaluation of preparation and properties of the sarcolema.

Plasma membranes from heart (sarcolemma) were prepared by the method of Kidwai, A.M. (1975) Methods in Enzymology (Fleischer, S. and Packer, L., eds.), Vol XXXIA, pp. 134--144, Academic Press, New York). On many occasions the sarcolemmal fraction identified by the enzyme markers such as (Na+ + K+)-ATPase banded at heavier densities (d greater than 1.25 g/ml) than expected for plasma membrane (d less than 1.15 g/ml). Radio-iodination of the membrane was added as an independent marker and conditions for the reproducible preparation of the sarcolemma were studied. Cultured heart cells were enzymatically iodinated under conditions which did not affect viability and labeled primarily the sarcolemma. The distribution of radioactivity in homogenates of cultured cells on the density gradient corresponded to that of the enzymes' activity. The best sarcolemma preparation was obtained with 0.3 M KCl extraction of heart homogenates in the presence of 0.05 M pyrophosphate, especially if the salt was also present during the fractionation by density gradient centrifugation. Alterations in the density were also observed with erythrocytes and cultured liver cells' plasma membrane. The data suggests a meta-stable state of the plasma membranes due to handling or storage which could cause alterations of some of their physical properties (e.g. density).     

Characterization of membrane-bound adenosinetriphosphatase activity of sarcolemma-enriched fraction from vascular smooth muscle

Membrane-bound adenosinetriphosphatase (ATPase) activities of the sarcolemma-enriched fraction from bovine aorta were characterized. The membranes, isolated by a sucrose density gradient method, were enriched about 31-fold in sodium- and potassium-stimulated, magnesium-dependent ATPase (Na,K-ATPase) activity, and about 8-fold in 5'-nucleotidase activity compared to the homogenate, suggesting that the isolated membranes were substantially enriched with the sarcolemma. The membranes exhibited about 31, 33 and 42 mumol Pi/mg protein/h of Na,K-ATPase, magnesium-dependent ATPase and calcium-dependent ATPase activities, respectively, in the presence of 4 mmol/l ATP. The sarcolemma-enriched membranes required considerably high concentrations of well-known inhibitors for Na,K-ATPase such as vanadate (more than 1 mumol/l), lanthanum (more than 1 mmol/l) and calcium (10 mmol/l), to induce a significant inhibition in the Na,K-ATPase activity. Treatments of the membrane with physical disruptions and sodium dodecyl sulfate or deoxycholate reduced the total Na,K-ATPase activity, and did not expose fully the ouabain sensitivity of the Na,K-ATPase. These results indicate that there are marked differences in the properties of the ATPase between vascular smooth muscle sarcolemma and cardiac sarcolemma.     

Crucial stages of embryogenesis of <Emphasis Type="Italic">R. arvalis</Emphasis>: Part 1. Linear measurements of embryonic structures

Investigations of individual variability have allowed us to reveal the crucial (=nodal) stages in embryogenesis of the moor frog (Rana arvalis Nills.). These crucial stages are: the late gastrula stage (stages 18–20), the hatching stages (stages 32–33) and, apparently, early metamorphosis (stage 39). Moreover, we have found that each embryonic structure passes through its specific crucial stages. For example, stage 34 is crucial for the trait “tail width” but is internodal for all other embryonic traits. At this stage, larva passes from an attached to a free-swimming life style. We also found considerable differences between the different frog populations in the the level of developmental variability. These differences were associated with internodal developmental stages.     

Differential alterations in ATP-supported calcium transport activities of sarcoplasmic reticulum and sarcolemma of aging myocardium

Two membrane fractions, one enriched in sarcoplasmic reticulum and the other enriched in sarcolemma, were isolated from the myocardium of young (3–4-months-old) and aged (24–25-months old) rats. ATP-supported Ca²⁺ binding and accumulating activities as well as (Mg²⁺ + Ca²⁺)-ATPase activities of these membrane fractions were studied in an effort to determine the influence of age on the Ca²⁺ pump function of the two myocardial membrane systems. Sarcoplasmic reticulum from aged hearts showed significantly reduced (approx. 50%) rates of ATP-supported (oxalate-facilitated) Ca²⁺ accumulation compared to sarcoplasmic reticulum from young hearts; the amount of Ca²⁺ accumulated by this membrane of aged heart at steady state was also lower. On the other hand, sarcolemma from aged hearts displayed 2-fold higher rates of ATP-supported Ca²⁺ accumulation compared to sarcolemma from young hearts; at steady state, sarcolemma from aged hearts accumulated significantly higher amounts of Ca²⁺ than did sarcolemma from young hearts. Similar age-related differences were also observed in the ATP-dependent Ca²⁺ binding activities of the two membranes, determined in the absence of oxalate. The divergent age-associated changes in Ca²⁺ binding and accumulating activities of sarcoplasmic reticulum and sarcolemma were seen at varying Ca²⁺ concentrations (0.24–39.1 μM).With either membrane, kinetic analysis showed 2-fold age-related differences in the V values for ATP-supported Ca²⁺ accumulation (V (nmol Ca²⁺/mg protein per min): sarcoplasmic reticulum — young, 119 ± 8; aged, 59 ± 5; sarcolemma — young, 11 ± 2; aged, 21 ± 3); the concentrations of Ca²⁺ required for half-maximal velocities did not differ significantly with age (K_0.5 for Ca²⁺ (μM): sarcoplasmic reticulum — young, 2.5 ± 0.20; aged, 2.9 ± 0.25; sarcolemma — yount, 2.7 ± 0.25; aged, 3.2 ± 0.30). Kinetic parameters of ATP-dependent Ca²⁺ binding also indicated that the velocity of Ca²⁺ binding but not the concentration of Ca²⁺ required for half-maximal binding was altered due to aging. At identical Ca²⁺ concentrations, the combined Ca²⁺ accumulating activity of sarcoplasmic reticulum and sarcolemma from aged hearts was significantly lower (38–47%) than the combined Ca²⁺ accumulating activity of the two membranes from young hearts. No significant age-related differences were observed in the ATP-independent (passive) Ca²⁺ binding (or accumulation) by sarcoplasmic reticulum and sarcolemma, the (Mg²⁺ + Ca²⁺)-ATPase activities of these membranes, their polypeptide composition or relative purity. These results indicate that differential alterations occur in the ATP-supported Ca²⁺ pump activities of sarcoplasmic reticulum and sarcolemma in aging myocardium and such alterations may be due to age-associated changes in the efficacy of coupling ATP hydrolysis to Ca²⁺ transport. Further, the age-related increment in the Ca²⁺ pump activity of sarcolemma is inadequate to fully compensate for the diminished Ca²⁺ pump activity of sarcoplasmic reticulum. It is, therefore, suggested that deterioration of the Ca²⁺ pump function of sarcoplasmic reticulum may contribute to the increased relaxation time observed in aging heart.     

Gonadotropin-releasing hormone 1 directly affects corpora lutea lifespan in Mediterranean buffalo (Bubalus bubalis) during diestrus: presence and in vitro effects on enzymatic and hormonal activities

The expression of gonadotropin-releasing hormone (GNRH) receptor (GNRHR) and the direct role of GNRH1 on corpora lutea function were studied in Mediterranean buffalo during diestrus. Immunohistochemistry evidenced at early, mid, and late luteal stages the presence of GNRHR only in large luteal cells and GNRH1 in both small and large luteal cells. Real-time PCR revealed GNRHR and GNRH1 mRNA at the three luteal stages, with lowest values in late corpora lutea. In vitro corpora lutea progesterone production was greater in mid stages and lesser in late luteal phases, whereas prostaglandin F2 alpha (PGF2alpha) increased from early to late stages, and PGE2 was greater in the earlier-luteal phase. Cyclooxygenase 1 (prostaglandin-endoperoxide synthase 1; PTGS1) activity did not change during diestrus, whereas PTGS2 increased from early to late stages, and PGE2-9-ketoreductase (PGE2-9-K) was greater in late corpora lutea. PTGS1 activity was greater than PTGS2 in early corpora lutea and lesser in late luteal phase. In corpora lutea cultured in vitro, the GNRH1 analog (buserelin) reduced progesterone secretion and increased PGF2alpha secretion as well as PTGS2 and PGE2-9-K activities at mid and late stages. PGE2 release and PTGS1 activity were increased by buserelin only in late corpora lutea. These results suggest that GNRH is expressed in all luteal cells of buffalo, whereas GNRHR is only expressed in large luteal phase. Additionally, GNRH directly down-regulates corpora lutea progesterone release, with the concomitant increases of PGF2alpha production and PTGS2 and PGE2-9-K enzymatic activities.     

Identification of the Na+/CA2+ exchanger of calf heart sarcolemma with the help of specific antibodies

The Na+/Ca2+ exchanger of calf heart sarcolemma has been identified in solubilized membrane preparations with the help of specific antibodies as a molecule of approximate Mr of 30 KDa. The conclusion supports the previous proposal by Soldati et al. (J. Biol. Chem. 260, 13321-13327, 1985) that the exchanger is a molecule of Mr about 33 KDa. Antibodies (IgG) were raised in rabbits by injecting proteins electroeluted from different regions of preparative SDS gels of solubilized heart sarcolemma. After purification the IgG against the proteins of the 30 KDa region recognized the 33 KDa component but also proteins of Mr about 70 and 140 KDa. Conversely, antibodies against the 140 KDa protein(s) also recognized the 70 and the 33 KDa proteins. However, if the solubilized sarcolemma extract was treated with DTT prior to the transfer to nitrocellulose the 140 KDa protein was not seen. Both the antibodies against the 30 KDa and those against the 140 KDa proteins inhibited the Na+/Ca2+ exchange activity of sarcolemma vesicles. It is proposed that the basic unit of the Na+/Ca2+ exchanger of heart sarcolemma is a monomer of Mr about 33 KDa, the functionally active exchanger being a tetramer in which the four 33 KDa subunits are held together by disulfide bonds. In the monomer-tetramer transition an intermediate dimeric state of Mr 70 KDa is also formed.     

Granulocytes in the endometrium of post-partum women

Endometrial samples of women at various stages of gonadal activity after parturition were examined for the presence and numbers of endometrial granulocytes. Although samples at all the stages contained significant numbers of the granulocytes (i.e. greater than 7/high-power field), the 100% values for late-proliferative and adaptation hyperplasia were significantly higher than the values for the resting (81.8%), early (82.4%) and mid- (87.9%) proliferative and secretory (83.3%) phases. We suggest that this correlates with the suggestion that the granulocytes constitute a receptor system for oestrogens.     

Quantitative retention of membrane lipids in the freeze-fracture replica

SDS-digested freeze-fracture replicas have been used as a substrate for immunoelectron microscopy to determine localization of membrane proteins and lipids. We, as well as others, have noticed that replicas prepared by first evaporating carbon are labeled more efficiently than conventional preparations in which platinum/carbon is evaporated first followed by carbon. In the present study, we examined whether the superior labeling in the carbon-first replica is caused by better retention of membrane molecules during SDS digestion. We used phosphatidylcholine liposomes as a model sample and measured the amount of inorganic phosphorus retained in the SDS-digested replica. The result showed that there was equivalent retention of inorganic phosphate among replicas prepared in different ways, indicating that labeling intensity on the replica did not correlate with the retention ratio. Interestingly, despite a similar retention ratio, replicas made of carbon alone gave far less labeling for ganglioside GM1 and phosphatidylcholine than replicas prepared by carbon followed by platinum/carbon. These results suggest that probes can bind to lipids captured by carbon more efficiently than those captured by platinum. Nonetheless, evaporation of platinum after carbon is indispensable for proper labeling.     

Heart sarcolemmal Ca2+ transport in endotoxin shock: II. Mechanism of impairment in ATP-dependent Ca2+ transport

The role of the phosphorylation and dephosphorylation of sarcolemma and that of the alteration of membrane lipids in the endotoxin-induced impairment of the ATP-dependent Ca²⁺ transport in canine cardiac sarcolemma were investigated. The results indicate that the ATP-dependent Ca²⁺ transport in canine cardiac sarcolemma was decreased by 30–35% 4h after endotoxin administration. Phosphorylation of sarcolemma by the catalytic subunit of the cAMP-dependent protein kinase or calmodulin stimulated ATP-dependent Ca²⁺ transport in both groups, however, the phosphorylation-stimulated activities remained significantly lower in endotoxic animals. Dephosphorylation of sarcolemma decreased ATP-dependent Ca²⁺ transport in both groups, yet, the time required to reach maximal dephosphorylation was reduced from 120 to 90 min 4 h post-endotoxin. Analysis of sarcolemmal membranes reveals that phosphatidylcholine and phosphatidylethanolamine contents were decreased while their respective lysophosphatide levels were increased significantly after endotoxin injection. Digestion of control heart sarcolemma with phospholipase A₂ inhibited Ca²⁺ transport and the inhibition was reversible by phosphatidylcholine. The inhibition caused by the in vivo administration of endotoxin was completely reversible by the addition of phosphatidylcholine. Based on these data, it is concluded that endotoxin administration impairs ATP-dependent Ca²⁺ transport in canine cardiac sarcolemma and that the impairment may be due to i) a defective phosphorylation of sarcolemma; ii) a reduced number of Ca²⁺ pumps; iii) an accelerated dephosphorylation of sarcolemma; and iv) an alteration in membrane phospholipid profile in response to phospholipase A activation.     

An outer envelope membrane component of the plastid protein import apparatus plays an essential role in Arabidopsis

T ranslocon at the o uter envelope membrane of c hloroplasts, 34  kDa (Toc34) is a GTP-binding component of the protein import apparatus within the outer envelope membrane of plastids. The Arabidopsis genome encodes two homologues of Toc34, designated atToc33 and atToc34. In this report, we describe the identification and characterization of two atToc34 knockout mutants, plastid protein import 3-1 ( ppi3-1 ) and ppi3-2 . Aerial tissues of the ppi3 mutants appeared similar to the wild type throughout development, and contained structurally normal chloroplasts that were able to efficiently import the Rubisco small subunit precursor (prSS) in vitro . The absence of an obvious ppi3 phenotype in green tissues presumably reflects the ability of atToc33 to substitute for atToc34 in the mutant, and the relatively high level of expression of the atTOC33 gene in these tissues. In the roots, where atTOC33 is expressed at a much lower level, significant growth defects were observed in both mutants: ppi3 roots were approximately 20–30% shorter than wild-type roots. Attempts to identify a double homozygote lacking atToc34 and atToc33 (by crossing the ppi3 mutants with ppi1 , an atToc33 knockout mutant) were unsuccessful, indicating that the function provided by atToc33/atToc34 is essential during early development. Plants that were homozygous for ppi1 and heterozygous for ppi3 displayed a chlorotic phenotype much more severe than that of the ppi1 single mutant. Furthermore, the siliques of these plants contained approximately 25% aborted seeds, indicating that the double homozygous mutation is embryo lethal. The data demonstrate that atToc33/atToc34 performs a central and essential role during plastid protein import, and indicate that the atToc34 isoform is relatively more important for plastid biogenesis in roots.     

Modifications of the envelope of Chlamydia psittaci during its developmental cycle: freeze-fracture study of complementary replicas.

Examination of complementary replicas obtained by freeze-fracture of Chlamydia psittaci revealed, at the level of the plasma membrane, a progressive differentiation of "crate-like formations," which likely correspond to transmembranal pores. Recognition of "early" and "late" stages observed in the intermediate bodies permitted detailed study of the developmental cycle of this chlamydia.     

Crucial stages of embryogenesis of R. arvalis: Part 1. Linear measurements of embryonic structures

Investigations of individual variability have allowed us to reveal the crucial (= nodal) stages in embryogenesis of the moor frog (Rana arvalis Nills.). These crucial stages are: the late gastrula stage (stages 18-20), the hatching stages (stages 32-33) and, apparently, early metamorphosis (stage 39). Moreover, we have found that each embryonic structure passes through its specific crucial stages. For example, stage 34 is crucial for the trait "tail width" but is internodal for all other embryonic traits. At this stage, larva passes from an attached to a free-swimming life style. We also found considerable differences between the different frog populations in the the level of developmental variability. These differences were associated with internodal developmental stages.     

Separation of cardiac plasmalemma into cell surface and T-tubular components. Distribution of saxitoxin- and nitrendipine-binding sites

To compare surface sarcolemmal with T-tubular distributions of [3H]saxitoxin (STX)- and [3H]nitrendipine (NTD)-binding sites, we centrifuged membrane vesicles from sheep and bovine ventricles on a 10-40% linear sucrose gradient from which fractions were assayed for STX and NTD binding; for markers of surface sarcolemma (ouabain-sensitive Na,K-ATPase activity, [3H]quinuclidinyl benzilate binding); and for markers of junctional sarcoplasmic reticulum known to be preferentially associated with T-tubules (ryanodine-sensitive Ca2+ uptake, calsequestrin, an Mr 300,000 putative phosphorylatable "foot" protein, and electron microscopically visible junctional sarcoplasmic reticulum-plasmalemma complexes). We identified three distinct peaks in the sucrose gradient, each characterized by significant high and low affinity STX- and high affinity NTD-binding: Peak I (approximately 19% sucrose), highly enriched in surface sarcolemma; Peak III (approximately 36% sucrose), enriched in junctional sarcoplasmic reticulum markers and hence in junctional sarcoplasmic reticulum complexes with T-tubule; and Peak II (approximately 27% sucrose), showing greatest specific STX binding and only moderate NTD binding, enriched in T-tubular membrane, unassociated with junctional sarcoplasmic reticulum. For ventricular myocytes, the ratio NTD sites/STX sites was 2.5 for surface sarcolemma, but only approximately 1.0 for T-tubules. Unlike data published for mammalian skeletal muscle, sheep and beef cardiac NTD receptors were not significantly more concentrated in T-tubular than in surface plasmalemma.     

Quantity of functionally changed cells as an identificator of the moment of the organizmus transfer to the next period of development

As "the threshold gears are peculiar to all processes which are flowing past in the world", the problem of looking up of quantitative criterion determining transition of a biological system from one condition to another, is one of the main problems of natural sciences. For analysis of the given problem we investigated some stages of forwardness of marine aster Asterias rubens (mean and late blastula, early and late gastrula, early larva), dis- tinguishing among themselves by morphological characters. Change of structure of cell populations at each stage of development A. rubens analyzed with the help of method of quantitative assessment of functional condition of cells genome. The method is based on cytophotometryc analysis of cell populations coloured by Feulgen (quantifying of DNA in cell) and Naftol yellow S (quantifying of histones in a cell). As the criterion of estimation of functional condition of the cell was used the parameter CFAGEN, coefficient of functional activity of cell genome deduced from histone/DNA ratio after estimation of the absorbency of the nucleus coloured by two chromophores. The data obtained showed that at the moment of organism transition from one stage of development (late blastula) to another (early gastrula) the difference in CFAGEN values between these stages was statistically authentic. Thus comparison of cell distribution histograms has show that cell population conforming to the early gastrula contains 33 % cells with the level of genome functional activity that differs from this one in the cell population conforming to the late blastula. Just in the same moment, the transition of an organism from the stage of late gastrula to the stage of larva is accompanied by statistically authentic distinction in CFAGEN between these stages and availability of more than 1/3 cells (34 %) of the population at the larva stage showing genome functional activity different from that at the late gastrula stage. Thus, it is established, that the biological system passes to qualitatively new condition, if quantity of members of this system changes (increaser or decreases comparatively to a standard point of reference: the norm, previous system status and etc.) not less than on 1/3.     

The role of phosphatidylinositol-3-kinases P85/P110 and hVPS34 in endocytosis of EGF-receptor complexes

The previous data (Zheleznova et al., 2001) did not enable the authors to conclude which particular wortmannin sensitive PI-3-kinase--p85/p110 (I class PI-3-K) or hVPS34 (III class PI-3-K)--may be involved in the regulation of EGF-receptor endocytosis. In the present work, we have shown that upon stimulation of EGF-receptor endocytosis additional structures stained with antibody against p85 appear in A431 cells, but the p85-positive compartment never co-localized with EGF-receptor-containing compartments either in control or in wortmannin-treated cells. At the same time, wortmannin treatment prevented association of hVPS34 with endosomal membranes. We have also found that early endosomal markers--Rab5 and EEA1 (membrane association of the latter depends on Rab5 and hVPS34)--co-localized with EGF-receptor in the juxtranuclear region during late stages of endocytosis, both in control and upon wortmannin treatment. These observations favor our suggestions that the transition of EGF-receptors from early to late endosomes may occur directly in this juxtranuclear region and be tightly associated with the formation of so called multivesicular bodies (MVB), which are late endosomes per se. We suggest that wortmannin may have no effect on early EEA1-dependent stage of the receptor endocytosis but blocks a transition of EGF-receptor complexes into the late endosomes by inhibiting activity of hVPS34 and removing it from membranes. The hVPS34 product PI-3-K, according to the known data, is involved in the formation of internal vesicles of MVB. Accumulation of EGF-receptors in these vesicles is believed to be necessary for the receptor degradation.