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The in vitro methylation of three liver cytosolic proteins was found to be selectively stimulated by calmodulin. This effect was also seen, although to a much smaller degree, in kidney and lung, but not in testes, brain, or spleen. The three methylated proteins affected by calmodulin have apparent Mr = 29,000, 32,000, and 45,000. The stimulation of methylation by calmodulin was greatest for the Mr 29,000 protein; there was an equal degree of methylation of the other two proteins. Dialysis of liver cytosolic fractions also stimulated the methylation of these proteins; the methylation of the Mr 32,000 and 45,000 proteins was stimulated to a greater extent by dialysis than by calmodulin. The degree of stimulation of methylation of the Mr 29,000 protein by calmodulin and dialysis was equivalent, but the addition of calmodulin to dialyzed liver cytosolic fractions gave additive effects on the stimulation of methylation of the Mr 29,000 protein, but not of either the Mr 32,000 or 45,000 proteins. Troponin C stimulated the methylation of the Mr 29,000 protein, but not the Mr 32,000 or 45,000 proteins, whereas parvalbumin stimulated methylation of the Mr 32,000 protein, but not the Mr 29,000 or 45,000 proteins. The effects of calmodulin and dialysis on protein methylation are cation-dependent and substrate-specific; methylation of the Mr 29,000 was supported by Mn2+, Ca2+, and Co2+, and to a lesser degree by Mg2+, Ni2+, and Zn2+. Methylation of the Mr 32,000 protein was supported only by Mn2+ and Mg2+ and methylation of the Mr 45,000 protein by Mn2+, Mg2+, Ca2+, Ni2+, and Zn2+, and to a much smaller extent by Fe2+. In extracts of fetal liver, stimulation of protein methylation by calmodulin or dialysis was restricted to the Mr 45,000 protein. In regenerating liver, stimulation of the methylation of all three proteins was observed, but the stimulation provided by dialysis plus calmodulin was much less than that observed in preparations from intact adult liver, suggesting a possible negative correlation between the rate of cell division and calmodulin-dependent methylation of these hepatic proteins. These results are consistent with the presence in liver of a minimum of three distinct N-methyltransferases and a dialyzable inhibitor which antagonizes calmodulin-dependent protein methylation.

We have investigated the ability of various divalent cations to carry current through membrane channels in ciliates activated by mechanical stimulation and by membrane depolarization. Both types of channels are identified as Ca2+ channels. Whereas Ba2+ ions and Sr2+ ions can replace Ca2+ ions as charge carriers during either kind of current, Mg2+ ions can carry the current evoked by mechanical stimulation but not the current elicited by membrane depolarization. Mn2+ ions did not carry either of these currents, and they reduced the currents carried by Ca2+ ions. Our experiments suggest that the mechano-sensitive and the voltage-sensitive channels exhibit different selectivities for divalent cations.

Incubation of C57BL/6 thymus-derived macrophages (TDM phi) with syngeneic thymocytes resulted in binding of thymocytes to macrophages and rosette formation. Up to 60% of the TDM phi formed rosettes with thymocytes after 6 hr of interaction at 4 degrees C. Rosette formation of the immature PNA+ thymocyte fraction was up to fivefold higher than that of PNA- and cortisone-resistant thymocytes. Pretreatment of PNA- thymocytes with neuraminidase enhanced thymocyte binding to macrophages up to sevenfold, whereas a marked reduction of rosette formation was seen following (1) incubation of thymocytes with tunicamycin; (2) incubation of macrophages with 20 mM D-galactose, GLCNaC, or GalNaC; (3) treatment of macrophages or thymocytes with trypsin; (4) treatment of macrophages with anti-1-Ab mAb and its F(ab')2 fragment; (5) treatment of thymocytes with anti-Lyt-2.2 mAb; and (6) addition of EDTA and EGTA to the interacted two cell populations.

The electrophoretic mobilities of proteins F and H1 from the outer membrane of Pseudomonas aeruginosa (mucoid and non-mucoid) in polyacrylamide gel electrophoresis were affected by the quality of sodium dodecyl sulphate (SDS) used. In particular, the sodium tetradecyl sulphate impurity present in crude SDS influenced the mobilities of F and H1. These observations explain conflicting reports on changes in outer membrane proteins with strain and growth conditions. Synthesis of H1 was induced by growth in magnesium depleted medium but repressed when calcium or manganese were added to magnesium depleted medium.

It has been shown that when long-acting forces are applied, the intercellular contacts in the small intestine epithelium are destroyed at the values of these forces lower than the adhesion ones obtained at the application of short-term loads. Starting with some threshold value of the load viscose-elastic properties of intercellular contacts are shown up. Thus the threshold varies essentially for different cells of the population: 4 variations are observed. A removal of bivalent cations from the tissue significantly decreases the threshold value of the forces and decreases the viscosity while the excessive concentrations of bivalent cations increase the threshold value of forces, the membrane viscosity and contact components.

Serum amyloid P-component (SAP) is a major acute phase protein of mice which we have previously shown increases the bactericidal activity of elicited, inflammatory macrophages (M phi). The presence of specific receptors for mouse SAP on M phi was demonstrated and the receptor-ligand (SAP) interaction characterized. Purified 125I-labeled mouse SAP binds to elicited M phi with the characteristics of a receptor-mediated event, i.e., the binding was saturable, specific, and reversible. A single type of receptor population was detected with an affinity of 5 x 10(-8) M (KD) and the calculated number of receptor sites per cell was approximately 10(5). Binding of SAP to M phi required Ca2+ or Mg2+ and was inhibited at a pH less than or equal to 5.6. Activated M phi from mice given BCG bind less SAP than nonactivated M phi. Activation of M phi with mouse interferon-gamma (IFN-gamma) or lipopolysaccharide (LPS) also decreased their SAP binding capacity. SAP is a glycosylated protein with a high mannose content; therefore mannose and other sugars were tested for inhibition of binding. Specific binding of SAP was inhibited by less than 1 mM concentrations of mannose 6-P, mannose 1-P, and mannose; however, other monosaccharides did not inhibit the binding. Removal of the oligosaccharide from SAP with an endoglycosidase specific for N-linked carbohydrate reduced the binding of SAP to M phi. The pattern of inhibition by sugars, the divalent cation requirement, and the sensitivity to low pH indicate that the receptor binding SAP is the cation-dependent mannose 6-P receptor, or a closely related receptor. The results suggest that SAP may alter or trigger M phi functions associated with inflammation by binding to glycoprotein receptors.

Sperm nucleus decondensing activity (NDA) of eggs has been investigated using the gametes of the sea urchin, Lytechinus variegatus. To assay for NDA, isolated sperm heads were exposed to homogenate from mature unfertilized ova and then examined with phase-contrast optics. NDA is retained by dialysis and requires Ca++ at the time of homogenization as well as at the time of assay. Other divalent cations (Mg++, Sr++, Ba++, Mn++) can substitute for Ca++ but are much less effective. NDA is stable to cold (-20 degrees C, -76 degrees C) but not to heat (56 degrees C). The pH activity optimum is in the range 7-9. NDA is present in the supernatant after centrifugation at 150,000 g for 105 minutes. It elutes shortly following the void volume on a Sephacryl S-200 column, with an approximate M.W. of 100,000 daltons.

Reintroduction of Ca ions to cat adrenal glands perfused at room temperature with Krebs solution lacking Ca and Mg, evoked a catecholamine secretory response that was directly proportional to the concentration of Ca reintroduced. This secretory response inactivated quickly, was abolished by nM concentrations of nifedipine and was potentiated dramatically by nM concentrations of BAY-K-8644. Excess Ca antagonized the inhibitory effects of nifedipine and this drug inhibited competitively the potentiating effects of BAY-K-8644. These data suggest 1st) that extracellular divalent cations deprivation activates specific Ca channels; 2nd) that the dihydropyridine BAY-K-8644 increases the release of catecholamines by Ca reintroduction by activating and/or delaying the inactivation of Ca channels; and 3rd) that the access of the dihydropyridine-type Ca agonist and antagonists to their "intra-channel" site of action requires the pre-activation of Ca channels.

Voltage clamp experiments were carried out on the sinus venosus of the frog by means of the double mannitol gap technique. The ionic mechanism underlying the slowly hyperpolarization-activated inward current was investigated by changing the concentration and species of alkali cations and divalent cations in the bathing solution. Adding Rb or Cs in concentration of 10-20 mM to the control solution led to a dose-dependent increase in the inward current, as does elevating the external concentration of K from 2.5 to 25 mM. After the inward current had been nearly suppressed by completely substituting Tris for Na in the external medium, it was partially restored after a subsequent addition of K, Rb or Cs to the Na-free medium. Various alkaline earths or transition metals added to the bathing solution markedly depressed the magnitude of the inward current. This inhibitory effect varied with concentration and nature of divalent cations added. It also depended on the concentration and species of alkali cations present in the external solution. From these observations it was proposed that the conductance responsible for the inward rectification in frog sinus venosus does not discriminate among monovalent cations. The results support the existence of a weak-field-strength site located in the permeation pathway. Divalent cation may exert their inhibitory effect by competing with permeant ions for this site.

Previous studies from this laboratory described myocardial depression in an arterially perfused rabbit interventricular septum following perfusion with acute septic plasma. Calcium is critical for maintenance of cardiac contractility on a beat-to-beat basis. We have investigated calcium flux in the septal tissue to determine whether altered calcium flux explains the impaired cardiac function during sepsis. Twenty-two rabbit septa were perfused with control and septic perfusate (cryo-precipitated plasma + RBCs) and calcium flux determined in seven experiments. Perfusate cations (Ca++, Na+, K+, and H+) were measured, tissue function and arterial pressure were monitored. Developed tension decreased 46%, acceleration of tension change fell 42%, and arterial pressure decreased 26%, all highly significant. All septa recovered after return to control perfusate. The septic perfusate Ca++ was significantly lower than control perfusate, while K+ and H+ were significantly elevated. Ion flux studies, however, could not demonstrate altered calcium flux associated with the depressed contractility.

The modulation of solubilized type 1 and type 2 benzodiazepine receptors from cow brain by gamma-aminobutyric acid (GABA), divalent cations, and anions has been evaluated. GABA stimulates [3H] flunitrazepam binding of both receptor subtypes, whereas divalent cations and anions selectively stimulate solubilized type 2 receptors. Of numerous anions examined, only chloride, bromide, and iodide enhance [3H] flunitrazepam binding. Chloride and bromide increase mainly receptor affinity for [3H]flunitrazepam, whereas iodide largely influences Bmax values. Divalent cations also stimulate soluble type 2 receptors. Calcium, zinc, manganese, barium, and magnesium have similar potencies in enhancing [3H]flunitrazepam binding, whereas copper and nickel are about 4 to 5 times more potent. The 2- to 3-fold increase in type 2 receptor binding by divalent cations involves change in numbers of binding sites. Effects of combinations of GABA, calcium, and chloride suggest that they may exert their modulating effects on type 2 receptors through different mechanisms. GABA, calcium, and chloride also protect [3H]flunitrazepam binding from heat inactivation, indicating a close link in the native state between the GABA, ions, and the benzodiazepine recognition sites. Since physiologic concentrations of calcium and chloride influence type 2 receptors, these ions may be involved in those pharmacologic effects of benzodiazepines mediated by type 2 sites.

Soluble immune response suppressor (SIRS) is an immunosuppressive protein which requires activation to SIRSox by peroxide. This observation suggested that SIRS could be a metalloprotein. To investigate this possibility, purified hybridoma-derived SIRS was treated with chelating agents and dialyzed prior to activation with H2O2. EDTA, EGTA, or desferrioxamine prevented suppression of murine plaque-forming cell responses by SIRSox, whereas sodium citrate and penicillamine did not inhibit suppression. Suppressive activity was reconstituted by subsequent treatment of SIRS with FeSO4 (0.5 microM), NiSO4 (500 microM), or MgSO4 (500 microM), but not by FeCl3, MnSO4, CuSO4, ZnSO4, CaCl2, or CrCl3. Reconstitution of activity occurred only if FeSO4 was added at least 3 hr prior to treatment with H2O2. These data indicate that SIRS requires a divalent metal ion, probably ferrous iron, for activity, and suggest that SIRS is a metalloprotein.

The effects of H+ and divalent cations on the O2 equilibrium of hexameric hemocyanin from a spiny lobster, Panulirus japonicus, were examined. The hemocyanin showed the normal Bohr effect. When divalent cations were removed by EDTA treatment, the protein showed a fivefold increase in the O2 affinity and a considerable decrease in the cooperativity. Several cooperativity models were tested for the conformity with the observed O2-binding isotherms by the least-square curve fitting. Among the models examined, the three-state concerted model was found to be most consistent with the results. It was postulated that in the absence of divalent cations deoxyhemocyanin is mainly in the intermediate-affinity state. The arthropod hemocyanins were found to be classifiable into two groups according to their functional responses to the divalent cations. It was suggested that the cations act differently on the allosteric transitions of the two groups of hemocyanins.

Dolichol kinase activity is effectively solubilized by extracting calf brain microsomes with 2% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), a zwitterionic detergent. The solubilized kinase catalyzes the enzymatic phosphorylation of dolichols with either CTP or dCTP serving as phosphoryl donor in the presence of Ca2+. Similar Km values were calculated for CTP (7.7 microM) and dCTP (9.1 microM). Dolichol phosphorylation was inhibited by CDP and dCDP, but not CMP, ADP, GDP, or UDP. A kinetic analysis of the inhibitory effect of CDP revealed a pattern characteristic of competitive inhibition. Dolichol kinase activity was markedly stimulated by the addition of R-dolichol (C95) or S-dolichol(C95). The apparent Km value for R-dolichol(C95) and S-dolichol(C95) was 9 microM, but the Vmax for the phosphorylation reaction was 40% higher with S-dolichol(C95). Incubation of the CHAPS extract with [gamma-32P]CTP and exogenous undecaprenol(C55) resulted in the enzymatic synthesis of a radiolabeled product that was mild acid-labile and chromatographically identical to undecaprenyl monophosphate. An enzymatic comparison with a variety of polyprenol substrates indicates that the solubilized kinase prefers long-chain (C90-95) polyprenols with saturated alpha-isoprene units. The effect of exogenous phosphoglycerides on the kinase activity in the dialyzed CHAPS extracts has also been evaluated. These studies describe the properties and polyprenol specificity of stable, solubilized preparations of dolichol kinase that should be useful for further purification of the enzyme.

Apamin is a bee venom neurotoxin of 18 amino-acids containing two disulfide bridges. Current clamp and voltage clamp experiments have shown that externally applied apamin blocks specifically at low concentration (0.1 microM) the Ca2+-dependent slow K+ conductance which mediates the long-lasting after-hyperpolarization in neuroblastoma cells and rat muscle cells in culture. The apamin-sensitive Ca2+-dependent slow K+ conductance is voltage-dependent and tetraethylammonium (TEA) insensitive. It is distinct from the high conductance Ca2+-dependent K+ channel revealed by patch clamp experiments. Biochemical characterization of the apamin receptor in rat striated muscle, neuroblastoma cells, rat synaptosomes, smooth muscles and hepatocytes was carried out with the use of a radiolabelled monoiodo-apamin derivative (125I-apamin) of high specific radioactivity (2 000 Ci/mmol). The dissociation constant of the apamin-receptor complex is between 15 and 60 pM for all tissue preparations. The density of binding sites is very low; it varied between 1 and 40 fmol/mg of protein. Radiation inactivation analysis indicates a molecular weight for the apamin receptor of 250 000 daltons whereas affinity labelling with 125I-apamin results in covalent labelling of a single polypeptide chain with a molecular weight of about 30 000 daltons. We conclude that the apamin-sensitive Ca2+-dependent K+ channel is probably a large oligomeric structure containing one subunit of 30 000 daltons.

The outer layer of the bacteriophage phi 6 nucleocapsid (NC) was removed by EDTA and reassociated with the core in the presence of Ca2+ or Mg2+. The core was relatively inaccessible to trypsin digestion, was composed of protein P1, and was in the dodecahedral framework reported previously. (H.T. Steely, Jr., and D. Lang, J. Virol. 51:479-483, 1984; Y. Yang and D. Lang, J. Virol. 51:484-488, 1984). The double-stranded RNA genome became RNase sensitive after EDTA treatment of the nucleocapsid.

Drug-monoclonal antibody conjugates have been evaluated for their specificity and toxicity towards tumour cells in vitro and in vivo; however, few studies have investigated their mode of entry into cells and mechanism of action. In this study the uptake and toxic effect of three different Methotrexate-monoclonal antibody (MTX-MoAb) conjugates (MTX-anti-transferrin receptor (TFR), MTX-anti-Ly-2.1 and MTX-anti-L3T4) were examined and compared with free MTX. It was concluded that MTX and these MTX-MoAb conjugates gain entry into tumour cells and are processed by different mechanisms, considering the following results: alterations in temperature had a greater effect on the toxicity of MTX-MoAb than on MTX; in addition, MTX and MTX-MoAb had different rates of action on cells; the specific MTX transport inhibitor, p-chloromercuribenzene sulphonate (pCMS), reduced MTX toxicity but had no effect on specific MTX-MoAb conjugates; the concentration of various ions (Ca2+, Mg2+ and Mn2+) effected the entry of MTX-MoAb but had no effect on free MTX. MTX enters by its own carrier mechanism, while MTX-MoAb conjugates enter by endocytosis with release of MTX at the lysosomal membrane, demonstrated by the ability of chloroquine and NH4Cl (which inhibit lysosomal function) to inhibit the action of MTX-MoAb but not MTX. Therefore, these MTX-MoAb conjugates are not degraded at the surface but bind to their receptor and then enter the cell by endocytosis as one entity; the MTX-MoAb conjugates are then degraded within the lysosomes, resulting in the release of free MTX into the cytoplasm where it acts on dihydrofolate reductase (DHFR) to inhibit cell metabolism.

Basal and stimulated adenylate cyclase specific activity was characterized in gill plasma membrane of freshwater-adapted trout by measuring the conversion of [alpha-32P]ATP into [alpha-32P]cyclic AMP. Both basal and isoproterenol- or sodium fluoride-stimulated enzyme activities were linear with time and protein concentration. The optimum activities were obtained using a pH buffer of 7.5 and a temperature of 20 degrees. The Km for ATP was 0.5 mM in the presence or absence of the stimulators. The presence of 10(-5) M guanosine-5'-triphosphate and 4 X 10(-3) M MgCl2 (2.41 X 10(-3) M free Mg2+) was required to optimize not only the basal activity but also the stimulation ratio (test/control) produced by these agents. On the contrary, Ca2+ was inhibitory. IC50 for CaCl2 was 5 X 10(-4) M (10(-7) M free Ca2+) in the presence or absence of the stimulators. Under these conditions, the basal adenylate cyclase specific activity was 400-450 pmol/mg protein/10 min. A maximal stimulation was produced by isoproterenol or PGE1 10(-5) M (50% increase over basal activity) or by glucagon 5.7 X 10(-10) M (30%). In addition, this enzyme displayed high sensitivity to sodium fluoride which induced a particularly large maximal effect (370%) at a concentration of 10(-2) M.

The role of Na+ and Ca2+ as well as the influence of other metal ions on the lipase action were studied with the aid of immobilisation techniques. By this method it is possible to distinguish between the action of these ions on the lipase molecule itself and their action on the substrate or product. It could be shown that both alkali and alkali earth metal ions, especially Na+, Ca2+ and Mg2+ stabilize active states of the enzyme which were detected by immobilization to controlled pore glass beads. Though Na+ as well as Ca2+ and Mg2+ stabilize the enzyme significantly, they differ in their efficacy. Reasons for this are discussed and the data compared to the findings of other authors who performed their studies with the native enzyme.

The electrophysiological properties of the cell membrane of rat spermatogenic cells were studied using the whole-cell variation of the patch-electrode voltage-clamp technique. In late primary spermatocytes and early spermatids isolated from adult testis, a transient inward current followed by a slowly developing outward current was produced when the membrane potential was made more positive than -60 mV. Early spermatogenic cells which consist of spermatogonia and early spermatocytes were isolated either from new-born rats (12-14 days old) of from adult cryptorchid rats 15-21 days after the operation. In early spermatogenic cells, some showed a slowly developing outward current with negligible initial inward current, while others showed a recognizable inward current followed by the slowly developing outward current. The inward currents are identified as Ca2+-carried current, since replacement of external Ca2+ with Mn2+ reversibly diminished the current whereas Ba2+ or Sr2+ substituted for Ca2+. The reversal potential of the outward current changed from -65 to -12 mV when [K+]o was raised from 5 to 100 mM. The outward current was independent of [Ca2+]o and was blocked by tetraethylammonium chloride. Thus the current was identified as membrane-potential-dependent K+ current. During spermatogenesis from spermatogonia to early spermatids, the density of Ca2+ current increased while the K+ current density decreased significantly.

The interaction of 1,4-dihydropyridine derivatives with their receptors on voltage-dependent calcium channels in cardiac membranes was studied to determine if there are basic differences in the binding properties of ligands that cause inhibition or activation of calcium channels. The binding characteristics of 6 pure stereoisomers, (-) and (+)202-791, (-) and (+)Bay k 8644, (-) and (+)PN 200-110, as well as racemic Bay k 8644 and nitrendipine, were compared. Competition studies using the cold ligands and 3 different radiolabelled dihydropyridines, (+)[3H]PN 200-110, (+/-)[3H]nitrendipine, and (+/-)[3H]Bay k 8644, showed that, for each combination tested, the labelled dihydropyridine could be displaced by the cold dihydropyridine. The binding reactions were markedly affected by temperature. The Kd values for most compounds were significantly higher (5-19 times) at 0 degrees than at 37 degrees C. In contrast, the affinity of (+)PN 200-110 was similar at 0 degrees and 37 degrees C, but slightly higher at 25 degrees C. A thermodynamic analysis indicated that the binding of the two pure isomers that are Ca2+-channel activators ("agonists"), (-)Bay k 8644 and (+)202-791, was driven entirely by enthalpy and was associated with an unfavorable decrease in entropy. This was in marked contrast to the binding of the inhibitors ("antagonists"). The binding of (+)PN 200-110 and nitrendipine at low temperatures was driven largely or entirely by entropy. Other antagonist-binding reactions were driven mainly by enthalpy but were associated with favorable increases in entropy. The affinity of the three radiolabelled ligands for the dihydropyridine receptor differed 100 times and appeared to be due to large differences in dissociation rate constants for each of the ligands. The rates of dissociation of (+)[3H]PN 200-110 and (+/-)[3H]nitrendipine, but not of (+/-)[3H]Bay k 8644, were significantly slowed by diltiazem, a calcium-channel inhibitor that binds to another receptor on the calcium channel. The results show that there were marked differences in the binding of the various dihydropyridines and suggest that the energetics of binding of Ca2+-channel activators and inhibitors may be fundamentally different.

Growth of nontransformed 3T3MIT fibroblasts in media containing 200 mM urea leads to the rapid acquisition of the transformed adhesive phenotype as evidenced by an increased rate of divalent cation-independent cell aggregation. The increased rate of divalent cation-independent cell aggregation of urea treated 3T3MIT cells shares many properties with the high rate of aggregation of transformed cells including a sensitivity to treatment with trypsin or hyaluronidase and a reduction in the presence of exogenously added hyaluronic acid. Reversal of the urea-induced increase in aggregation occurs within 24 hours in the absence of urea and can be blocked by 0.2 micrograms/ml cycloheximide. In the presence of cycloheximide, low rates of aggregation can be restored by the addition of urea-conditioned supernatents. The results of these experiments suggest that the loss of an aggregation-inhibitory activity during growth in media containing 200 mM urea is responsible for the increased rate of divalent cation-independent cell aggregation. After removal of this aggregation-inhibitory activity, the normally lowly adhesive 3T3MIT cells become phenotypically transformed with regards to the rate of divalent cation-independent cell aggregation.

Membrane potentials of mouse parathyroid cells were measured by means of the intracellular microelectrode method. The membrane potential in external Krebs solution containing 2.5 mM of Ca++ was -23.6 +/- 0.4 mV (mean +/- standard error of mean). The low concentration of Ca++ (1.0 mM) caused hyperpolarization of the membrane potential to -61.7 +/- 0.8 mV. The membrane potential was proportional to the logarithm of the concentration of K ion in the solution of low Ca ion. The concentration of external Na+, C1- and HPO4-- had no effect on the membrane potential. The sigmoidal transition of membrane potentials was induced by the change of Ca ion concentration in the range from 2.5 to 1.0 mM. The change of the membrane potentials in low Ca ion is originated from increase in potassium permeability of the cell membrane. The similar sigmoidal changes of the membrane potentials were observed in the solution containing 4 to 3 mM of Sr ion. The Mg and Ba ion showed smaller effect on the membrane potential. The Goldman equation was extended to divalent ions. Appling the extended membrane potential equation, ratios of the permeability coefficients were obtained as follows: PK/PCa = 0.067 for 2.5 mM Ca++, 0.33 for 1.0 mM Ca++; PK/PSr = 0.08 for 4 mM Sr++ and 0.4 for 3 mM Sr++; PK/PMg = 0.5; PK/PBa = 0.67 for all range of concentration. The Hill constants of Sr ion and Ca ion were 20; the relationship between Sr ion and Ca ion was competitive. The Hill constants of Mg and Ba ion were 1 each. The Hill constant of Ca ion was depend of the temperature; nmax = 20 at 36 degrees C, n = 9 at 27 degrees C, n = 2 at 22 degrees C. The enthalpy of Ca-binding reaction was obtained from the Van't Hoff plot as 0.58 kcal. The activation energies of the K+ permeability increase were obtained from the Arrhenius plots as 3.3 kcal and 4 kcal. The difference, 0.7 kcal, corresponds to the enthalpy change of this reaction, of which value is close to that of the Ca-binding reaction.

Increased adherence of sickle erythrocytes to vascular endothelium has been suggested by Hebbel and his colleagues to play a role in vasocclusive events of sickle cell disease. To define the role of cell membrane changes and plasma factors in cell adherence, a micropipette technique previously developed by us to obtain a direct, quantitative measure of cell adherence was used to evaluate the adhesivity of different morphologic classes of sickle cells to endothelial cells in various suspending media. Irregularly shaped, deformable sickle cells were four- to fivefold more adherent than discoid sickle cells, whereas rigid irreversibly sickled cells were least adherent. Sickle erythrocytes adhered to endothelial cells when suspended in autologous citrated or heparinized plasma but were totally nonadherent when suspended in autologous EDTA plasma. Removal of the divalent cation chelator and addition of calcium to EDTA plasma restored its ability to promote adhesion, implying an absolute requirement for divalent cations in sickle cell adherence. Sickle cells also did not adhere to endothelial cells in protein-free media containing divalent cations, suggesting an additional requirement for plasma proteins. Removal of collagen-binding proteins from citrated sickle plasma resulted in a three- to fivefold reduction in its ability to promote cell adhesion, suggesting an important role for these plasma proteins in sickle cell adherence. The results of this study imply that sickle cell adherence to vascular endothelium is a complex process in which temporal changes in the numbers of cells identified to be most adhesive and the plasma concentration of protein(s) involved in the adhesive process determine the extent of in vivo sickle cell adherence.

The effects of external Zn+2 and other divalent cations on K channels in squid giant axons were studied. At low concentration (2 mM) Zn+2 slows opening kinetics without affecting closing kinetics. Higher concentrations (5-40 mM) progressively slow opening and speed channel closing to a lesser degree. In terms of "shifts," opening kinetics are strongly shifted to the right on the voltage axis, and off kinetics much less so. The shift of the conductance-voltage relation along the axis is intermediate. Zinc's kinetic effects show little sign of saturation at the highest concentration attainable. Zn does not alter the shape of the instantaneous current-voltage relation of open channels. Some other divalent cations have effects similar to Zn+2, Hg2+ being the most potent and Ca+2 the least. After treatment with Hg+2, which is irreversible, Zn+2 still slows opening kinetics, which suggests that each channel has at least two sites for divalent cation action. The results are not compatible with a simple theory of fixed, uniform surface charges. They suggest that external cations interact directly with a negatively charged element of the gating apparatus that moves inward from the membrane's outer surface during activation. Examination of normal kinetics shows that there is a slow step somewhere in the chain leading to channel opening. But the slowest step must not be the last one.

The activation of placental AC by either Mg2+ or Mn2+, in the presence and absence of NaF, followed sigmoidal saturation kinetics. Mn2+ enhanced maximally the NaF-stimulated Mg2+-dependent AC activity. The apparent Km of Mg2+-dependent AC for ATP was 0.4 mM, with and without NaF addition. GTP and GMP-P(NH)P stimulated the Mg2+-dependent AC in a dose-dependent manner with half-maximal stimulation taking place at concentrations of approximately 2 microM. In the presence of GMP-P(NH)P (10 microM) the kinetics of the AC dependence on Mg2+ ion concentration changed from sigmoidal to hyperbolic. Most of the AC activity (greater than 83 per cent) was associated with the particulate fractions of placental homogenate. For better reproducibility, the AC assay was performed using sonicated particulate fraction preparations; sonication did not alter the response of AC to stimuli to a variety of agents used in these experiments; freezing and thawing, however, obliterated the stimulation by beta-adrenergic agonists. Placental AC activity was inhibited by p-hydroxymercuriphenyl sodium sulphonate in a dose-dependent fashion, and the inhibition was reversed by dithiothreitol. Mg2+-dependent AC was inhibited by 0.5 mM phenylhydrazine (95 per cent). Mg2+-dependent AC activity was responsive to stimulation by epinephrine, without and with GTP addition, with half-maximal stimulation taking place at a concentration of 2 microM. The stimulatory effect of epinephrine was blocked by propranolol in a dose-dependent manner but was not blocked by phentolamine. Oestrone, oestradiol-17 beta, 2-hydroxyoestreone, 2-hydroxyoestradiol-17 beta, dehydroepiandrosterone sulphate, and progesterone, as well as oxytocin, did not alter either the basal or GMP-P(NH)P-stimulated Mg2+-dependent AC activities. Preincubation of 20 000 g particulate fraction with either NaF or GMP-P(NH)P, followed by washing, resulted in preparations that remained stimulated without the requirement of any further additions.

Using the Technicon Autocounter, the mechanisms involved in collagen-induced platelet aggregation in vivo have been studied without the interference of an anticoagulant. Extracellular divalent cation was essential for in vivo platelet aggregation. Non-steroidal anti-inflammatory drugs completely inhibited the aggregation induced by collagen in platelet-rich plasma in in vitro or ex vivo studies. In vivo only a maximum of 50% inhibition was achieved when release of thromboxane A2 (TXA2) was completely inhibited. Therefore in vivo, collagen causes aggregation through more than one pathway which operate independently of each other and which are all dependent on extracellular divalent cation. In vivo, when different doses of collagen were compared, aggregation produced by low doses of collagen was more dependent upon prostaglandin endoperoxide/TXA2 formation.