Mechanism of protective effects of Ca++ channel blockers on energy deprivation contracture in cultured ventricular myocytes

To examine mechanisms of the protective effects of Ca++ channel blockers on energy deprivation contracture, we measured cystolic calcium ion concentration ([Ca++]i) (Indo-1 fluorescence), development of contracture (video motion detector) and ATP contents during exposure of cultured chick embryo ventricular cells to 1 mM cyanide (CN) and 20 mM 2-deoxyglucose (2-DG). The time periods required for [Ca++]i to reach 50% of [Ca++]i transient ([Ca++]i-50) and contracture were determined after exposure to 1) CN + 2-DG alone, 2) CN + 2-DG simultaneous with 1 microM verapamil (V-sim) and 3) verapamil followed by CN + 2-DG (V-pre). Time periods required to reach [Ca++]i-50 under these conditions were 4.2 +/- 0.4 min (CN + 2-DG alone), 3.8 +/- 0.4 min (NS vs. CN + 2-DG alone) (V-sim) and 6.4 +/- 1.1 min (P less than .05 vs. CN + 2-DG alone) (V-pre), respectively. Time periods required for contracture development were 4.4 +/- 0.3 min (CN + 2-DG alone), 4.4 +/- 0.6 min (NS vs. CN + 2-DG alone) (V-sim) and 9.3 +/- 1.2 min (P less than .05 vs. CN + 2-DG alone) (V-pre). Three minutes after metabolic inhibition, ATP contents declined from 32.3 +/- 0.7 nmol/mg of protein to 4.2 +/- 1.0 in CN + 2-DG alone, to 4.5 +/- 0.9 (NS vs. CN + 2-DG alone) with V-sim and to 8.3 +/- 2.2 (P less than .05 vs. CN + 2-DG alone) with V-pre.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ca2+ channel blockers enhance neurotensin (NT) binding and inhibit NT-induced inositol phosphate formation in prostate cancer PC3 cells

Neurotensin (NT) stimulates Ca2+ release and Ca2+ influx in many cells. Its contractile effects in smooth muscle are inhibited by removal of Ca2+ and by Ca2+ channel blockers (CCBs). To better understand NT signaling in prostate cancer PC3 cells, blockers of voltage-gated and store-operated Ca2+ channels (VGCC and SOCC) were tested for effects on NT-binding and signaling. Eight chemical types of agents, including VGCC-blocker nifedipine and SOCC-blocker SKF-96365 (1-[beta-[3-(4-methoxyphenyl)-propoxy]-4-methoxyphenyl]-1H-imidazole), enhanced cellular NT binding up to 3-fold, while inhibiting (by congruent with 70%) NT-induced inositol phosphate (IP) formation. The ability to enhance NT binding correlated with the ability to inhibit NT-induced IP formation, and both effects were relatively specific for NT. Although cellular binding for beta2-adrenergic, V1a-vasopressin, and epidermal growth factor receptors was not enhanced by these drugs, bombesin receptor binding was increased approximately equal to 19% and bombesin-induced IP formation was inhibited approximately equal to 15%. One difference was that the effect on NT binding was Ca2+-independent, whereas the effect on IP formation was Ca2+-dependent (in part). The Ca2+-dependent part of the IP response seemed to involve SOCC-mediated Ca2+ influx to activate phospholipase C (PLC)delta, while the Ca2+-independent part probably involved PLCbeta. Photoaffinity labeling of the NT receptor NTR1 was enhanced in CCB-treated cells. NTR1 affinity was increased but NTR1 number and internalization were unchanged. Since CCBs did not alter NT binding to isolated cell membranes, the effects in live cells were indirect. These results suggest that CCBs exert two effects: 1) they inhibit NT-induced IP formation, perhaps by preventing Ca2+ influx-dependent activation of PLCdelta; and 2) they enhance NTR1 affinity by an unexplained Ca2+-independent mechanism.     

Characterization of three abnormal factor IX variants (Bm Lake Elsinore, Long Beach, and Los Angeles) of hemophilia-B. Evidence for defects affecting the latent catalytic site.

Abnormal factor IX variant proteins were isolated from the plasmas of three unrelated severe hemophilia-B families that had been previously shown to contain functionally impaired molecules immunologically similar to normal factor IX. The families studied were: (1) a patient with markedly prolonged ox brain prothrombin time, designated factor IX Bm Lake Elsinore (IXBmLE); (b) three patients (brothers) with moderately prolonged ox brain prothrombin time, designated factor IX Long Beach (IXLB); and (c) a patient with normal ox brain prothrombin time designated factor IX Los Angeles (IXLA). Each variant molecule comigrates with normal factor IX (IXN) both in the sodium dodecyl sulfate and in the nondenaturing alkaline gel electrophoresis. All three variant proteins are indistinguishable from IXN in their amino acid compositions, isoelectric points, carbohydrate distributions and number of gamma-carboxyglutamic acid residues. Each variant protein undergoes a similar pattern of cleavage by factor XIa/Ca2+ and by factor VIIa/Ca2+/tissue factor, and is activated at a rate similar to that observed for IXN. All of the three variant proteins also react with an anti-IXN monoclonal antibody that interferes with the binding of activated IXN(IXaN) to thrombin-treated factor VIIIC. However, in contrast to IXaN, the cleaved IXBmLE has negligible activity (approximately 0.2%), and cleaved forms of IXLA and IXLB have significantly reduced activity (approximately 5-6%) in binding to antithrombin-III/heparin, and in activating factor VII (plus Ca2+ and phospholipid) or factor X (plus Ca2+ and phospholipid) +/- factor VIII. These data, taken together, strongly indicate that the defect in these three variant proteins resides near or within the latent catalytic site. This results in virtually a complete loss of catalytic activity of the cleaved IXBmLE molecule and approximately 95% loss of catalytic activity of the cleaved IXLA and IXLB molecules.     

Apparent binding of ionized calcium by various buffers

Using different substance concentrations in an aqueous 1.25 mmol/L solution of CaCl2 plus NaCl to a final solution ionic strength of 160 mmol/L, we tested six buffers for their effect on measurements of ionized calcium (Ca2+). Measured Ca2+ decreased with increasing ionic strength and pH. Increasing concentrations of Tris caused a positive Ca2+ electrode bias; the other five buffers caused a negative Ca2+ electrode bias with increasing concentration. 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid ("HEPES") at 10 mmol/L maintained pH 7.39 in the aqueous reference solutions containing 0.75 to 2.50 mmol of CaCl2 per liter, and showed a linear Ca2+ response among three different suppliers of Ca2+ instruments; it is thus a suitable buffer for a reference solution.     

A reliable potentiometric measurement of ionized calcium and pH on the ICA2 Radiometer in clinical practice

A second generation analyser for potentiometric measurement of ionized calcium and pH (ICA2 Radiometer, Copenhagen) was tested with respect to analytical performance in two different laboratories. Newly-developed aqueous buffered solutions were used at different Ca2+ concentrations as well as biological specimens prepared from human subjects. The overall precision of ICA2 was very good: CV = 0.0-0.4% for within-run and 0.4-1.7% for day-to-day precision with protein-free solutions and CV = 0.3-0.9% for within-run precision on biological specimens. The adjusted value of Ca2+ to pH 7.40 is an analytical advantage for which the calculation is correct but the assumption made for this algorithm may limit its clinical relevance. Measurements were comparable on ICA2 when performed on either whole blood, serum or plasma. Pre-analytical conditions should be observed such as the choice of anticoagulant and anaerobic conditions. Such a reliable and accurate instrument may promote the use of ionized calcium measurement in daily clinical practice.     

Neutral carrier electrode for continuous measurement of blood Ca2+ in the extracorporeal circulation

A neutral-carrier-based Ca2+-selective electrode exposed to whole blood exhibited an EMF-stability better than 0.13 mV (approximately 1% change of the initial activity) during 3.7 to 6.6 h. It has been used successfully to monitor ionic Ca2+ in the extracorporeal circulation during administration of calcitonin to dogs. Data simulation shows that the apparent concentration of ionic Ca2+ is affected by the concentration of Na+. A variation in [Na+] by +/- 5 mmol/L from a value of 143 mmol/L used for the calibration leads to uncertainties of +/- 0.8% in the Ca2+ concentration and +/- 0.2% in the Ca2+ activity if 3 mol/L KCl is used as the reference electrode solution. When an isotonic solution was used as reference electrolyte, errors of +/- 0.8% and +/- 1.7%, respectively, were observed.     

Blood pressure development of the spontaneously hypertensive rat after concurrent manipulations of dietary Ca2+ and Na+. Relation to intestinal Ca2+ fluxes.

The blood pressure of the spontaneously hypertensive rat (SHR) is influenced by the Ca2+ content of its diet. As the SHR's greater dependence on dietary calcium may reflect a defect in intestinal calcium absorption, we measured in vitro unidirectional Ca2+ flux (J) in the duodenum-jejunum (four segments each) of the SHR (n = 6) and the normotensive Wistar-Kyoto rat (WKY; n = 6) by a modified Ussing apparatus. Because of the known and postulated interactions between Ca2+ and Na+ in both intestinal and vascular tissue, we assessed in vivo the influence of a concurrent manipulation of Na+ intake (three levels: 0.25%, 0.45%, and 1.0%) on the blood pressure development of SHRs (n = 35) and WKYs (n = 35), between 6 and 20 wk of age, exposed to three levels of dietary calcium (0.1, 1.0, and 2%). Net calcium flux (Jnet) (mean +/- SEM) was significantly (P less than 0.01) lower in the SHR (-2.8 +/- 6.3 nmol/cm2 X h) than in the WKY (34.6 +/- 8.8 nmol/cm2 X h). The SHR's decreased Jnet resulted from a significantly (P less than 0.03) lower mucosa-to-serosa flux (Jm-s) in the SHR (41.0 +/- 5.6 nmol/cm2 X h) compared with the Jm-s of the WKY (70.1 +/- 9.1 nmol/cm2 X h). Serosa-to-mucosa flux for calcium did not differ between the SHR (43.8 +/- 6.6 nmol/cm2 X h) and the WKY (35.5 +/- 8.0 nmol/cm2 X h). The SHR's decreased (P less than 0.002) Jm-s was confirmed by additional measurements in SHRs and WKYs. Jm-s was 36.2 +/- 3.7 nmol/cm2 X h in the SHRs (n = 11) and 64.4 +/- 6.7 nmol/cm2 X h in the WKYs (n = 9). The provision of an increased dietary Ca2+ (2% by weight) and increased Na+ (1%) to the SHR prevented the emergence of hypertension (P less than 0.001) (mean +/- SEM systolic blood pressure at 20 wk of age; 135 +/- 5 mmHg for the 2% Ca2+, 1% Na+ SHR vs. 164 +/- 2 mmHg for the control diet SHR). Ca2+ (0.1%) and Na+ (0.25%) restriction accelerated the SHR's hypertension (192 +/- 2 mmHg) (P less than 0.001) and was associated with higher pressures in the WKY (146 +/- 4 mmHg in the restricted WKY vs. 134 +/- 4 mmHg in the control WKY). In a parallel group of 24 SHRs and 24 WKYs fed one of three diets (2% Ca2+/1% Na+; 1% Ca2+/0.45% Na+; or 0.1% Ca2+/0.25% Na+), the heart (P < 0.05) and kidney (P = 0.08) weight of the SHRs varied depending on the diet at 20 wk of age. Low Ca2+ and Na+ intake was associated with increased heart weight (1.6+/-0.9 g) compared with the normal diet for SHR (1.51+/-0.07 g). Increased Ca2+ and Na+ intake was associated with a significantly (P = 0.05) lower heart weight in the SHR (1.37+/-0.03 g) and in the WKY (1.35+/-0.06 g) compared with their normal diet controls. These findings show one mechanism for the SHR's depressor response to supplemental dietary Ca2+ and, in part, explain the sodium dependence of calcium's cardiovascular protective effect.     

Mechanism of calcium transport stimulated by chlorothiazide in mouse distal convoluted tubule cells.

Thiazide diuretics inhibit Na+ and stimulate Ca2+ absorption in renal distal convoluted tubules. Experiments were performed on immortalized mouse distal convoluted tubule (MDCT) cells to determine the mechanism underlying the dissociation of sodium from calcium transport and the stimulation of calcium absorption induced by thiazide diuretics. Control rates of 22Na+ uptake averaged 272 +/- 35 nmol min-1 mg protein-1 and were inhibited 40% by chlorothiazide (CTZ, 10(-4) M). Control rates of 36Cl- uptake averaged 340 +/- 50 nmol min-1 mg protein-1 and were inhibited 50% by CTZ. CTZ stimulated 45Ca2+ uptake by 45% from resting levels of 2.86 +/- 0.26 nmol min-1 mg protein-1. Bumetanide (10(-4) M) had no effect on 22Na+, 36Cl-, or 45Ca2+ uptake. Control levels of intracellular calcium activity ([Ca2+]i) averaged 91 +/- 12 nM. CTZ elicited concentration-dependent increases of [Ca2+]i to a maximum of 654 +/- 31 nM at 10(-4) M. CTZ reduced intracellular chloride activity ([Cl-]i), as determined with the chloride-sensitive fluorescent dye 6-methoxy-N-(3-sulfopropyl)quinolinium. The chloride channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, 10(-5) M) abolished the effect of CTZ on [Cl-]i. NPPB also blocked CTZ-induced increases of 45Ca2+. Resting membrane voltage, measured in cells loaded with the potential-sensitive dye 3,3'-dihexyloxacarbocyanine iodide [DiOC6(3)], averaged -72 +/- 2 mV. CTZ hyperpolarized cells in a concentration-dependent and reversible manner. At 10(-4) M, CTZ hyperpolarized MDCT cells by 20.4 +/- 7.2 mV. Reduction of extracellular Cl- or addition of NPPB abolished CTZ-induced hyperpolarization. Direct membrane hyperpolarization increased 45Ca2+ uptake whereas depolarization inhibited 45Ca2+ uptake. CTZ-stimulated 45Ca2+ uptake was inhibited by the Ca2+ channel blocker nifedipine (10(-5) M). We conclude that thiazide diuretics block cellular chloride entry mediated by apical membrane NaCl cotransport. Intracellular chloride, which under control conditions is above its equilibrium value, exits the cell through NPPB-sensitive chloride channels. This decrease of intracellular chloride hyperpolarizes MDCT cells and stimulates Ca2+ entry by apical membrane, dihydropyridine-sensitive Ca2+ channels.     

Effect of hematocrit and added heparin on ionized calcium in capillary blood samples from neonates

Sampling of capillary blood for determination of ionized calcium (Ca2+) in neonates requires that extra heparin be added to prevent clotting in the sampling tube and (or) in the Ca2+ analyzer. Because the additive dissolves in the plasma compartment, different hematocrit (erythrocyte volume fraction, EVF) values may cause different results for Ca2+. To study the effect of EVF and heparin additive, we repeatedly removed plasma, thereby increasing the EVF. These samples with different EVF's were aspirated into commercial capillary tubes containing heparin and, according to our routine procedure, an additional 10 microL (approximately 0.9 int. unit) of sodium heparin. We found a negative bias of 0.05-0.09 mmol/L in Ca2+, depending on the EVF. Adding saline instead of heparin gave the same effect, indicating that this bias was entirely due to dilution. We suggest compensating for this by adding 0.09 mmol/L to the actual value for ionized calcium when EVF exceeds 70%. The increase in Ca2+ in neonates on days 1 to 5 postpartum is physiological and not an effect of change in EVF.     

Inhibition of ryanodine receptors by 4-(2-aminopropyl)-3,5-dichloro-N,N-dimethylaniline (FLA 365) in canine pulmonary arterial smooth muscle cells

Ryanodine is a selective ryanodine receptor (RyR) blocker, with binding dependent on RyR opening. In whole-cell studies, ryanodine binding can lock the RyR in an open-conductance state, short-circuiting the sarcoplasmic reticulum, which restricts studies of inositol-1,4,5-trisphosphate receptor (InsP3R) activity. Other RyR blockers have nonselective effects that also limit their utility. 4-(2-aminopropyl)-3,5-dichloro-N,N-dimethylaniline (FLA 365) blocks RyR-elicited Ca2+ increases in skeletal and cardiac muscle; yet, its actions on smooth muscle are unknown. Canine pulmonary arterial smooth muscle cells (PASMCs) express both RyRs and InsP3Rs; thus, we tested the ability of FLA 365 to block RyR- and serotonin-mediated InsP3R-elicited Ca2+ release by imaging fura-2-loaded PASMCs. Acute exposure to 10 mM caffeine, a selective RyR activator, induced Ca2+ increases that were reversibly reduced by FLA 365, with an estimated IC50 of approximately 1 to 1.5 microM, and inhibited by 10 microM ryanodine or 10 microM cyclopiazonic acid. FLA 365 also blocked L-type Ca2+ channel activity, with 10 microM reducing Ba2+ current amplitude in patch voltage-clamp studies to 54 +/- 6% of control and 100 microM FLA 365 reducing membrane current to 21 +/- 6%. InsP3R-mediated Ca2+ responses elicited by 10 microM 5-hydroxytryptamine (serotonin) in canine PASMCs and 100 microM carbachol in human embryonic kidney (HEK)-293 cells were not reduced by 2 microM FLA 365, but they were reduced by 20 microM FLA 365 to 76 +/- 9% of control in canine PASMCs and 52 +/- 1% in HEK-293 cells. Thus, FLA 365 preferentially blocks RyRs with limited inhibition of L-type Ca2+ channels or InsP3R in canine PASMCs.     

Evaluation of a new automatic calcium ion analyzer.

We evaluated a new automatic analyzer for ionized calcium (Ca2+), the Orion Model SS-20, based on a flowthrough ion-exchange electrode. Ca2+ was measured in heparinized whole blood and serum. Within-day variation was 1.2%, day-to-day variation1.6%, and analytical recovery 92.4%. Over the phsiological range interference by K+ and Mg2+ was negligible; major changes in ionic strength, induced by changes in N+ concentration, made correction for a sodium error necessary. Within the physiological range, Ca2+ was inversely correlated to variations in pH. Therefore, to compare Ca2+ values, correction to an apparent pH of 7.40 should be made. The calcium binding effect of heparin was negligible when minimal (4.4 int. units/ml) concentrations of heparin were used. Storage of serum at 4 degrees C for one week resulted in a 4 degrees decrease in apparent serum Ca2+, primarily owing to an increase in pH during storage. In normal material, mean values for blood-and serum-Ca2+(1.10 and 1.07 mmol/liter, respectively) were close to results obtained by previous systems. Errors caused by disturbances in the fluid flow and non-function of half the electrodes we received were the major inconveniences of the analyzer. We conclude that this new analyzer gives decisive advantages in measurement of Ca2+, making this importnant analysis possible as a routine laboratory test for the first time.     

Cation-binding property of choroid plexus peptide IIF.

Preparations of the melanotropic-lipolytic peptide IIF from bovine choroid plexus contain 3% Ca, 1% Mg and less than 0.1% Na and K. Ca and Mg were removed by gel filtration on Sephadex G-10 in 1 N acetic acid. Capacity of the resulting cation-free peptide to bind Ca++, Mg++, Na+ and K+ was examined with the method of Hummel and Dreyer (Biochim. Biophys. Acta 63: 530-532, 1962). Peptide IIF bound a maximum of 3.7 to 4.4 mEq of Ca++, Mg++, Na+ or K+ per g of peptide. For each cation, linear Scatchard plot indicated one class of binding site. Association constants (liters per mole) were Ca++, 4.3 X 10(4); Mg++, 3.6 X 10(4); Na+, 4.8 X 10(2), K+, 1.9 X 10(2). Competitive binding experiments showed that all four cations occupied the same class of binding site.     

Selective loss of calcium permeability on maturation of reticulocytes.

Calcium and sodium permeability of human reticulocytes have been studied and compared to mature erythrocytes. Mature erythrocytes had extremely low Ca2+ permeability which was less than 0.1% of values published for squid axon or HeLa cells. Calcium entry was markedly increased in reticulocyte-rich suspensions and the uptake was linearly related to the percentage of reticulocytes present. The data suggest that reticulocytes are 43-fold more permeable to Ca2+ than mature cells although their Ca2+ concentration is not increased. Sodium influx into reticulocyte-rich suspensions was also increased in direct proportion to the percent of reticulocytes present. Reticulocytes are sixfold more permeable to Na+ than mature cells so the ratio of Ca2+:Na+ permeability falls by sevenfold as the reticulocyte changes to an erythrocyte. [3H]Ouabain binding was increased in reticulocyte-rich cell suspensions and the correlation suggested a value of about 4,000 sites per reticulocyte compared with 362+/-69 per mature cell. Maturation of the human reticulocyte produces disproportionate changes in cation permeability and in particular a selective loss of Ca2+ permeability.     

Superoxide generation and granule enzyme release induced by ionophore A23187. Studies on the early events of neutrophil activation

The responses of isolated human peripheral neutrophils to activation by the divalent cation ionophore A23187 (4 x 10(-8) M to 2 x 10(-5) M) were studied over the course of 15 min at 37 degrees C. Dose-dependent increases were noted in the following A23187-induced functions: (1) membrane depolarization, as assessed by a fluorescent cyanine dye, (2) altered membrane permeability to 22Na+ and 45Ca2+, (3) increased intracellular levels of cAMP, (4) O2-generation, and (5) granule enzyme release. Membrane depolarization and the cAMP response preceded the onset of O2- generation and exocytosis, which began after a latency period of approximately 30 sec. All five functions induced by 2 x 10(-5) M A23187 required approximately 1 mM Ca0 for optimal expression and declined substantially as Ca0 was reduced towards a nominal O mM. In contrast, the Na+ ionophore gramicidin D (0.1 to 10 microgram/ml) caused cell depolarization and increased 22Na+ ionophore gramicidin D (0.1 to 10 microgram/ml) caused cell depolarization and increased 22Na+ uptake but did not stimulate 45Ca2+ uptake, the cAMP response, O2- generation, or exocytosis of granule constituents. The observations dissociate membrane depolarization and altered Na+ permeability, on the one hand, from a Ca2+ influx, cAMP response, O2- release, and exocytosis on the other. These studies, then, are consistent with a hypothesis that a Ca2+ influx is required for the biologic expressions of cell activation.     

Taurine modulation of calcium binding to cardiac sarcolemma

A sarcolemma-enriched membrane fraction (SL) was prepared from the hearts of Sprague-Dawley rats and its ability to bind Ca++ was measured by equilibrium dialysis. We found that the effect of taurine on SL Ca++ binding varied with the buffer and with Na+ concentration. In Tris, in the presence of Na+ (140 mM), taurine (10 mM) increased the affinity but decreased the maximal binding of Ca++ (0.5-7 mM). In the absence of Na+, taurine decreased the affinity without altering the maximal binding. These effects on Ca++ binding were absent in bicarbonate or Krebs-Henseleit buffers. However, incubations with A23187, a Ca++ ionophore, and lanthanum, a Ca++ antagonist, indicated that SL membranes incubated in Tris, but not in buffers containing bicarbonate, were sealed vesicles with internal environments low in Ca++. High-affinity binding of Ca++ (10(-6)-10(-4) M) was measured in modified Krebs-Henseleit buffers. Taurine decreased Ca++ binding in a high-Na+ (145 mM), low-K+ (4.7 mM) buffer. Taurine increased Ca++ binding in both 4.7 mM Na+-145 mM K+ and 25 mM Na+-4.7 mM K+ buffers. Taurine also increased Ca++ binding in the presence of ATP. Thus, taurine increased high-affinity Ca++ binding in "intracellular" buffers, but it did not affect low-affinity Ca++ binding in "extracellular" buffers. These results suggest taurine may exert its cardiotonic actions through modulation of the high-affinity Ca++ binding sites on the internal aspect of the SL.     

Filling accuracy and imprecision of commercial evacuated sodium citrate coagulation tubes

Current recommendations advocate that blood tubes for coagulation testing should be filled not less than 90% of their nominal filling volume, since under- or over-filling >10% may generate unreliable results of some hemostasis assays. This study was hence aimed to explore filling accuracy and precision of commercial blood tubes. Between-lot variations of 3 different lots (20 tubes per lot) of 3.2% citrate blood tubes manufactured by Becton Dickinson, Greiner and Kima were studied. One additional lot from each manufacturer was assessed in triplicate (three series of 20 tubes), to assess within-lot variation. All tubes were first weighed empty and then filled with distilled water by a syringe, under ideal filling conditions. Filled tubes were weighed again, in duplicate. For each 20 tubes series, mean bias (deviation from the ideal tube filling volume) and imprecision (coefficient of variation; CV%) were calculated. All biases were within ±10%. Within-lot and between-lot variation in filling volume was acceptable, and comprised between 0.4 and 2.4%. Greiner tubes were the most accurate (bias, ?1.0 to 2.4%), followed by Kima (bias, ?7.8 to ?5.9%) and Becton Dickinson (bias, ?9.6 to 3.3%) tubes. The highest between-lot difference was noted for Becton Dickinson tubes (up to 12.9%), followed by Greiner and Kima tubes (up to 3.4 and 1.8%, respectively). Although coagulation tubes filling accuracy was within ±10% for all three tested manufacturers, the overall bias was found to be variable among manufacturers and lots. Major effort shall be made by blood tube manufacturers for improving standardization of their products.     

Role of calmodulin in platelet aggregation. Structure-activity relationship of calmodulin antagonists.

Two series of derivatives of N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), including a dechlorinated analog of W-7 (W-5) and various aminoalkyl chain analogs of W-7 (A-3, A-4, A-5, I-240, A-6) were synthesized and their structure-activity relationships with calmodulin antagonistic actions and their potencies in inhibiting human platelet aggregation in vitro were investigated. Their binding affinities to calmodulin in the presence of 100 microM Ca2+ were dependent both on the chlorination of the naphthalene ring and on the length of aminoalkyl chain. The ability of these derivatives to inhibit Ca2+-dependent phosphorylation of 20,000-dalton myosin light chain from platelets correlated well with the magnitude of their binding affinity to calmodulin. W-7(10-100 microM) inhibited in a dose-dependent manner platelet aggregation induced by collagen (2 micrograms/ml), ADP (5 microM), epinephrine (1 microgram/ml), sodium arachidonate (0.83 mM), thrombin (0.125 U/ml), and A-23187 (10 microM). The IC50 value (concentration producing 50% inhibition of aggregation) of W-7 was lower in arachidonate- and collagen-induced aggregation than in ADP- or epinephrine-induced aggregation. A good correlation between the potency in inhibition of collagen-induced aggregation by W-7 and its derivatives and their affinities to calmodulin was obtained (r = 0.94). Thus, the inhibitory mechanism of these compounds may be due to their effect on Ca2+-calmodulin-dependent processes, such as 20,000-dalton myosin light chain phosphorylation. These data also support the hypothesis that the calmodulin-mediated system has an important role in platelet function.     

Endothelin- and oxytocin-induced calcium signaling in cultured human myometrial cells.

The demonstration that endothelin (ET) induces rat uterine contraction, coupled with the observation that ET is present in human amniotic fluid, suggests that the myometrium may be an important target organ for this hormone. We show that in quiescent human myometrial cells ET produced a dose-dependent increase in cytosolic free Ca2+ (Cai2+), which was markedly attenuated when the cells were studied in Ca2(+)-free media. Preincubation with nicardipine, diltiazem, or verapamil reduced the ET-evoked Cai2+ transient by 30, 40, and 65%, respectively. The presence of voltage sensitive Ca2+ channels was demonstrated by Mn2+ quenching of fura-2. Activation of the Na+/H+ antiport could not be demonstrated with ET stimulation. In nonquiescent cells, the ET-evoked Cai2+ transient was significantly reduced, while the response to oxytocin was retained. This is at least partially explained by a reduction in Bmax (maximal binding capacity) for ET (mean +/- SEM) from 3,506 +/- 268 binding sites/cell in quiescent cells to 2,411 +/- 300 binding sites/cell, as well as 72% increase in Kd (equilibrium dissociation constant), in the nonquiescent cells. We conclude that, in human myometrial cells, ET and oxytocin modulate Cai2+ through independent receptors and propose that ET, like oxytocin, is an important endogenous modulator of uterine contractility.     

Activator-associated Ca2+-ATPase in erythrocyte membranes from cystic fibrosis patients

Erythrocyte membranes were prepared by a method which should ensure binding of an activator protein (calmodulin) to the calcium dependent membrane ATPase involved in calcium transport. The level of enzyme activity, assayed at optimum conditions, was 5-400 times higher than that found in previous investigations on cystic fibrosis patients. The Ca2+-ATPase activity of the cystic fibrosis patients was reduced by 15% compared to control subjects, whereas patients suffering from chronic pulmonary diseases did not deviate from controls. Even if a reduction of Ca2+ pumping activity occurs in other cells, a 15% decrease could hardly be the only cause of the changed calcium concentrations in secretions from cystic fibrosis patients.     

Tocolytic agents act on calcium channel current in single smooth muscle cells of pregnant rat uterus

Effects of Ca2+ channel blockers and clinically important tocolytic agents, Mg2+ and beta-agonists, were examined on the Ca2+ channel current recorded from freshly isolated single pregnant rat myometrial cells using the whole-cell voltage clamp method. Nifedipine inhibited the Ca2+ channel currents (Ba2+ current) dose-dependently. Inhibition by nifedipine was greater at higher (more positive) holding potentials and higher command potentials. According to the modulated receptor hypothesis, Kd values for resting state and inactivated state of channel were calculated to be 150-300 and 1.2-6.8 nM, respectively (at a command potential of -20 mV). Mg2+ applied in the bath dose-dependently inhibited the Ca2+ current recorded with 2 mM Ca2+ (Ki = 12 mM, at a command potential of -10 mV). Inhibition by Mg2+ was weaker at the higher command potentials. Voltage dependency observed for the Mg2+ block may be due to a shift of the activation curve (Mg2+ neutralizes the outer surface charge) and/or due to the location of the binding site for Mg2+ in the Ca2+ channel electric field. In contrast, high dose of isoproterenol (10 microM) did not produce significant change in the Ca2+ current. In summary, nifedipine and Mg2+ inhibited the Ca2+ channel, with opposite voltage dependencies, whereas isoproterenol had no effect on the Ca2+ current. Thus, beta-agonist may relax the uterine muscle by mechanisms other than inhibition of the Ca2+ channels.