Platelet microparticles and calcium homeostasis in acute coronary ischemias.

UNLABELLED: Elevation of free cytoplasmic calcium is the common pathway of platelet activation, leading to shape change, shedding of platelet microparticles (PMP), aggregation, and secretion of internal granules, including expression of CD62p on the surface. Platelet activation is well documented in unstable angina (UA) and acute myocardial infarction (MI). We investigated the following markers of platelet activation in 55 patients undergoing coronary angiography for suspected CAD: free cytoplasmic calcium, [Ca2+]cyt, PMP, CD62p expression, and platelet/leukocyte (P/L) interaction. [Ca2+]cyt was measured by Fluo-3 and the other measurements were by flow cytometry. Patients were classified into three groups: unstable angina (UA, n = 11), recent myocardial infarction (MI, n = 11), and patient controls (CTL, n = 33). Blood was drawn before infusion of heparin through femoral lines at the time of catheterizaton for assays. ( RESULTS: (1) PMP values were significantly higher in both UA and MI than in CTL, P < 0.05. There was no difference between UA and MI. (2) P/L interaction was significantly elevated only in UA, P < 0.05. (3) CD62p expression on free platelets did not differ significantly between any of the three groups. (4) The resting [Ca2+]cyt, thrombin-induced Ca2+ influx, and release of Ca2+ from internal stores were all significantly higher in platelets from the combined patient group (UA + MI) than in the patient control group, P < 0.001 CONCLUSIONS: Results on calcium hemostasis and PMP were significantly different in patients with acute coronary syndromes than those with stable angina or no coronary ischemia; this may reflect underlying pathophysiology of acute coronary ischemia. P/L interaction was higher only in the UA group, suggesting a role of leukocytes in UA.

Variability of the thrombin- and ADP-induced Ca2+ response among human platelets measured using fluo-3 and fluorescent videomicroscopy.

The intracellular free Ca2+ concentration ([Ca2+]cyt) of individual human platelets localized between siliconized glass cover slips was determined at rest and after stimulation with thrombin and ADP using the Ca2+ indicator fluo-3 (0.97 +/- 0.30 mmol/l cell volume) with fluorescence video microscopy. Resting [Ca2+]cyt in the presence of 2 mM external Ca2+ showed only small inter-platelet variability ([Ca2+]cyt = 86 +/- 30 (S.D.) nM). Resting [Ca2+]cyt of individual fluo-3-loaded platelets measured as a function of time had a S.D. of 10 nM or 12% (S.D./mean). Individual platelets showed no affinity for the siliconized support and their [Ca2+]cyt showed no tendency to oscillate in either the resting or in the activated state. When 0.2 U/ml thrombin or 20 microM ADP were added, all platelets showed a characteristic Ca2+ transient whereby [Ca2+]cyt increased to peak values within 8-12 sec and then declined. The Ca2+ transients measured with fluo-3 were in approximate synchrony but peak [Ca2+]cyt values showed large inter-platelet variability. The ensemble average peak [Ca2+]cyt for thrombin and ADP were 672 +/- 619 (S.D.) nM and 640 +/- 642 (S.D.) nM, respectively. Thus inter-platelet variations (S.D./mean) were 92% or 100% as large as the average measured values. Mathematically-constructed averages of the single platelet experiments agreed reasonably well with platelet-averaged values obtained in parallel experiments with stirred platelet suspensions in a plastic cuvette, measured with a conventional spectrofluorometer. Peak [Ca2+]cyt values reflecting dense tubular Ca2+ release alone (external Ca2+ removed) also showed large interplatelet variation (171 +/- 105 (S.D.) nM with thrombin and 183 +/- 134 (S.D.) nM with ADP). Dense tubular Ca2+ release induced by cyclopiazonic acid (a dense tubular Ca2+-ATPase inhibitor) gave peak [Ca2+]cyt of 289 +/- 170 nM. Thus the size of the dense tubular Ca2+ pool has an inter-platelet variation of 59% (S.D./mean). Variability of the dense tubular pool size accounts for some, but not all, of the large interplatelet variation in peak (Ca2+]cyt seen with thrombin and ADP activation.

Intravenous lecithin-coated microcrystals of dantrolene are effective in the treatment of malignant hyperthermia: an investigation in rats, dogs, and swine.

Dantrolene effectively treats malignant hyperthermia (MH) hut the current form, Dantrium, must be dissolved to a 0.33 mg/mL, pH 9.5 solution. This study describes lecithin-coated microcrystal formulations of sodium dantrolene (MC-NaD) and neutral dantrolene (MC-D) which reconstitute to 200 mg/mL within 1 min. In rats, the pharmacokinetics and pharmacodynamics of MC-NaD and Dantrium were similar: half-lives of 3.1 h, volume distributions of 0.54 and 0.59 L/kg, and 95% effective dose (ED95) values for depression of skeletal muscle twitch height (ED95T) of 2.6 +/- 0.7 and 2.8 +/- 0.5 mg/kg. In swine, the ED95T values for MC-NaD and Dantrium were also similar (2.8 +/- 0.4 vs 2.7 +/- 0.6 mg/kg), but MC-D and Dantrium were only similar at doses more than 2.5 mg/kg (ED95T: 3.5 +/- 0.4 vs 2.7 +/- 0.5 mg/kg). In susceptible swine, MC-NaD successfully treated five of six MH episodes and prevented MH in three of four swine. However, MC-NaD caused marked pulmonary hypertension in swine, while MC-D caused only a mild response that was eliminated by filtration. Likewise, MC-D caused no pulmonary response in dogs. These observations suggest that MC-D has potential to improve the treatment of MH.

Na(+)-independent Active H(+) Extrusion and HCO(3)(-)/C1(-) Exchange Contribute to Cytoplasmic pH Regulation in the Human Platelet.

Homeostatic regulation of cytoplasmic pH (pH(eyt)) against acid and alkaline challenges was studied in the human platelet using the intracellular indicator 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Activation of a Na(+)/H(+) exchanger in the plasma membrane is a known mechanism by which the platelet resists cytoplasmic acidification. The present study demonstrates an additional Na(+)-independent H extrusion mechanism which persists at low external Na(+) concentration in the presence of 100 µM ouabain. This mechanism is inhibited by rotenone, oligomycin and an inhibitor of glycolysis, and is tentatively identified as a plasmalemmal H(+)-ATPase. The Na(+)-independent H extrusion mechanism partially restores cytoplasmic pH (pH(eyt)) after the cytoplasm is acidified by addition of 1 µM nigericin. The restoration process is energy-dependent and has a t(1/2) of 7-21 min. The Na(+)-independent H(+) extrusion mechanism is also shown capable of maintaining pH(eyt) ≊ 6.0 against an acidic pH, of 5.3 in an energy-dependent manner. The present study also revealed a Na(+)-independent, DIDS-inhibitable Cl(-)/HCO(3)(-) exchange activity. It removes alkaline equivalents from the cytoplasm with a half-time of 2.0 ± 0.4 min after alkaline loading with 25 mM NH(4)Cl. Its activity was also revealed in chloride to gluconate and gluconate to chloride perturbations of the external medium which raised or lowered pH(eyt) by 0.17 ± 0.05 or 0.14 ± 0.04 units, respectively. The activity of the anion exchanger allows the platelet to maintain pH(ext) = 7.00 ± 0.11 at the alkaline pH(ext) of 8.25. The combined activities of the Na(+)-independent H(+) extrusion and Cl(-)/HCO(3)(-) exchange mechanisms make the platelet cytoplasm very resistant to changes in external pH. For variation of pH(ext) between 5.0 and 8.5, pH(eyt) varies between 6.0 and 7.0, or roughly one-third as much.

Ultra-long-duration local anesthesia produced by injection of lecithin-coated tetracaine microcrystals.

This study was designed to determine if microencapsulated tetracaine would provide a longer duration of local anesthesia than nonmicroencapsulated (neat) tetracaine. Local anesthesia was determined by monitoring the response of the rat to tail clamping after the installation of a subcutaneous ring block. Ten percent microencapsulated tetracaine was found to provide local anesthesia of the tail for a 43-hour duration. Ten percent tetracaine solution was toxic. One percent tetracaine solution provided a tail block lasting 8 hours. Lecithin membranes without drug provided no block. This study demonstrates that lecithin-coated tetracaine microcrystals produce a local anesthetic effect that is ultra-long in duration, reversible, and not systemically toxic.

The Ca(2+)-extruding ATPase of the human platelet creates and responds to cytoplasmic pH changes, consistent with a 2 Ca2+/nH+ exchange mechanism.

The Ca(2+)-extruding ATPase pump of the human platelet was studied in situ by measuring Ca2+ extrusion from quin2-overloaded platelets (Johansson, J.S., Haynes, D.H. 1988. J. Membrane Biol. 104:147-163). Cytoplasmic pH (pHcyt) was measured by BCECF fluorescence in parallel experiments. The pump was studied by raising the cytoplasmic free Ca2+ to 2.5 microM and monitoring active Ca2+ extrusion into a Ca(2+)-free medium. The pump was shown to perturb pHcyt, to not respond to changes in membrane potential and to respond to imposed changes in pHcyt in a manner consistent with the Ca2+ pump acting as a 2 Ca2+/nH+ exchanger. (i) Raising the external pH (pHext) from 7.40 to 7.60 lowers the Vmax of the pump in basal condition (Vmax,1) from 110 +/- 18 to 73 +/- 12 microM/min (= mumol/liter cell volume/min). (ii) Lowering pHext to 7.13 raised Vmax,1 to 150 +/- 15 microM/min. (iii) In an N-methyl-D-glucamine (NMDG+) medium, the pump operation against high [Ca2+]cyt acidifies the cytoplasm by -0.36 +/- 0.10 pH units, and the pump becomes self-inhibited. (iv) Use of nigericin to drive pHcyt down to 6.23 reduces the Vmax,1 to 18 +/- 11 microM/min. (v) Alkalinization of the cytoplasm by monensin in the presence of Na+ raises the Vmax,1 (basal state with Km,1 = 80 nM) to 136 +/- 24 microM/min, but also activates the pump fourfold (Vmax,2 = 280 +/- 28 microM/min; Km,2 = 502 +/- 36 nM). (vi) Transient elevation of pHcyt by NH4Cl at high [Ca2+]cyt activates the pump eightfold (Vmax,2 > or = 671 +/- 350 microM/min). The large activation by alkaline pHcyt at high [Ca2+]cyt can be explained by Ca(2+)-calmodulin activation of the pump (Valant, P.A., Adjei, P.N., Haynes, D.H. 1992. J. Membrane Biol. 130:63-82) and by increased Ca2+ affinity of calmodulin at high pH.

Actions of thapsigargin on the Ca(2+)-handling systems of the human platelet. Incomplete inhibition of the dense tubular Ca2+ uptake, partial inhibition of the Ca2+ extrusion pump, increase in plasma membrane Ca2+ permeability, and consequent elevation of resting cytoplasmic Ca2+.

Thapsigargin (Tg) effects on Ca2+ handling in the intact human platelet were studied using Quin2 and chlorotetracycline to measure free cytoplasmic and dense tubular (DT) Ca2+ concentrations ([Ca2+]cyt and [Ca2+]dt, respectively). Tg inhibits Ca2+ uptake by the DT Ca(2+)-ATPase pumps, but incompletely, lowering the Vm to 32% of control (IC50,Tg = 0.18 +/- 0.10 microM). The kinetics of loss of DT Ca2+, transient increases in [Ca2+]cyt, and lowered steady-state [Ca2+]dt after Tg addition are all explained by pump inhibition, with no effect on the rate constant of Ca2+ leakage across the DT membrane (kleak,DT = 1.14 min-1). Tg lowers by 30% the Vm of the Ca2+ extrusion pump located in the plasma membrane (PM), as shown by a Quin2-based method measuring active Ca2+ extrusion (Johansson, J. S., and Haynes, D. H. (1988) J. Membr. Biol. 104, 147-163). This effect (IC50,Tg = 0.45 +/- 0.06 microM), together with a 24 +/- 16% increase in kleak,PM,Ca (to 3 x 10(-4) min-1), accounts for a Tg-dependent sustained elevation [Ca2+]cyt (to 708 +/- 78 nM) which is independent of DT Ca2+ status or history. Thrombin and Tg release 30 and 70% (respectively) of the DT Ca2+ available at any instant, independent of order of challenge, consistent with a single class of DT with respect to these agents.

Rapid Ca2+ extrusion via the Na+/Ca2+ exchanger of the human platelet.

This communication reports the kinetics of the Na+/Ca2+ exchanger and of the plasma membrane (PM) Ca2+ pump of the intact human platelet. The kinetic properties of these two systems were deduced by studying the rate of Ca2+ extrusion and its Na+ dependence for concentrations of cytoplasmic free Ca2+ ([Ca2+]cyt) in the 1-10-microM range. The PM Ca(2+)-ATPase was previously characterized (Johansson, J.S. Haynes, D.H. 1988. J. Membrane Biol. 104:147-163) for [Ca2+]cyt < or = 1.5 microM with the fluorescent Ca2+ indicator quin2 (Kd = 115 nM). That study determined that the PM Ca2+ pump in the basal state has a Vmax = 0.098 mM/min, a Km = 80 nM and a Hill coefficient = 1.7. The present study extends the measurable range of [Ca2+]cyt with the intracellular Ca2+ probe, rhod2 (Kd = 500 nM), which has almost a fivefold lower affinity for Ca2+. An Appendix also describes the Mg2+ and pH dependence of the Kd and fluorescence characteristics of the commercially available dye, which is a mixture of two molecules. Rates of active Ca2+ extrusion were determined by two independent methods which gave good agreement: (i) by measuring Ca2+ extrusion into a Ca(2+)-free medium (above citation) or (ii) by the newly developed "ionomycin short-circuit" method, which determines the ionomycin concentration necessary to short circuit the PM Ca2+ extrusion systems. Absolute rates of extrusion were determined by knowledge of how many Ca2+ ions are moved by ionomycin per minute. The major findings are as follows: (i) The exchanger is saturable with respect to Ca2+ with a Km = 0.97 +/- 0.31 microM and Vmax = 1.0 +/- 0.6 mM/min. (ii) At high [Ca2+]cyt, the exchanger works at a rate 10 times as large as the basal Vmax of the PM Ca2+ extrusion pump. (iii) The exchanger can work in reverse after Na+ loading of the cytoplasm by monensin. (iv) The PM Ca2+ extrusion pump is activated by exposure to [Ca2+]cyt > or = 1.5 microM for 20-50 sec. Activation raises the pump Vmax to 1.6 +/- 0.6 mM/min and the Km to 0.55 +/- 0.24 microM. (v) The Ca2+ buffering capacity of the cytoplasm is 3.6 mM in the 0.1 to 3 microM range of [Ca2+]cyt. In summary, the results show that the human platelet can extrude Ca2+ very rapidly at high [Ca2+]cyt. Both the Na+/Ca2+ exchanger and Ca2+ pump activation may prevent inappropriate platelet activation by marginal stimuli.

Protein kinase C stimulates dense tubular Ca2+ uptake in the intact human platelet by increasing the Vm of the Ca(2+)-ATPase pump: stimulation by phorbol ester, inhibition by calphostin C.

The effects of protein kinase C (PKC) on Ca2+ transport were investigated in human intact platelets. The indicator quin2 was used to measure the free cytoplasmic Ca2+ concentration ([Ca2+]cyt) and to search for possible PKC effects on the Ca(2+)-ATPase extrusion pump located in the plasma membrane. The Ca2+ indicator chlorotetracycline (CTC) was used to study PKC effects on the dense tubular Ca(2+)-ATPase uptake pump. The activity of PKC was stimulated by phorbol 12-myristate 13-acetate (PMA) and was inhibited with calphostin C. Neither PKC activation nor inhibition had any effect on [Ca2+]cyt or the Ca2+ extrusion pump. Substantial activation of the dense tubular pump was observed with PMA. In resting platelets bathed in 2 mM external Ca2+ giving [Ca2+]cyt = 102-106 nM, activation of PKC by PMA (100 nM) increases the rate and extent of dense tubular Ca2+ uptake to 1.62 +/- 0.35 and 1.25 +/- 0.3 times control value (respectively). The Vm of the dense tubular pump was measured by using ionomycin to manipulate [Ca2+]cyt. It is shown that PMA increases the Vm by a factor of 1.7 +/- 0.4 but has no effect on the Km value (= 180 nM). An unexpected finding was that PKC activity supports a portion of the basal activity of the dense tubular Ca2+ pump in resting platelets. Preincubation with the inhibitor calphostin C (100 nM) decreases the rate and extent of dense tubular Ca2+ uptake in resting platelets by 38 +/- 5% and 29 +/- 21% (respectively). This is due to a 28 +/- 9% decrease in the Vm of the dense tubular pump. This suggests that there is a low level of stimulation of dense tubular Ca2+ pump mediated by PKC in resting platelets.

Cyclic AMP stimulates Ca(2+)-ATPase-mediated Ca2+ extrusion from human platelets.

The effect of cAMP on active Ca2+ extrusion across the plasma membrane of intact human platelets was studied using quin2, a fluorimetric indicator of free Ca2+ in the cytoplasmic compartment ([Ca2+]cyt). Elevations of cAMP were achieved by incubation with dibutyryl-cAMP or by forskolin, which was found to selectively elevate cAMP without affecting cGMP levels. Progress curves of Ca2+ extrusion from quin2-overloaded platelets were measured. The rate vs. [Ca2+]cyt characteristic was calculated as previously described (Johansson, J.S. and Haynes, D.H. (1988) J. Membr. Biol. 104, 147-163). Forskolin, at a maximally effective concentration of 10 microM, was shown to stimulate Ca2+ extrusion by increasing by a factor of 1.6 +/- 0.5 the Vm of a saturable component, previously identified with a Ca(2+)-Mg(2+)-ATPase located in the plasma membrane. Neither the Km (80 nM) or Hill coefficient (1.7 +/- 0.3) of the Ca(2+)-ATPase was affected. Forskolin had no effect on the linear, non-saturable component of extrusion (previously identified with a Na+/Ca2+ exchanger) over the [Ca2+]cyt range examined (50-1500 nM). Dibutyryl-cAMP (Bt2-cAMP, 1 mM) stimulated the Ca(2+)-Mg(2+)-ATPase component of Ca2+ extrusion by a factor of 2.0 +/- 0.6. Separate experiments showed that 10 microM forskolin reduces the resting [Ca2+]cyt from 112 nM to 96 nM. Mathematical analysis showed that this can be accounted for by the above-mentioned increase in Vm of the pump, countered by a 37-74% increase in the rate constant for passive Ca2+ leakage across the plasma membrane. The results suggest two mechanisms by which prostacyclin-induced elevation of cAMP inhibits platelet aggregation: (a) lowering of resting [Ca2+]cyt and (b) increasing the rate of Ca2+ extrusion after the initial influx or triggered release event.

Stimulation of dense tubular Ca2+ uptake in human platelets by cAMP.

Elevation of intracellular cAMP is shown to increase the rate (V) and maximal extent of Ca2+ uptake by the dense tubules in intact human platelets. Elevation of [cAMP] was accomplished by preincubation with the adenylate cyclase activator forskolin or with dibutyryl-cAMP (Bt2-cAMP). The free concentration of Ca2+ in the dense tubular lumen ([Ca2+]dt) was monitored using the fluorescence of chlorotetracycline (CTC) according to protocols developed in this laboratory. The free cytoplasmic Ca2+ concentration ([Ca2+]cyt) was monitored in parallel experiments with quin2. Both [Ca2+]cyt and [Ca2+]dt were analyzed in terms of competition between pump and leak mechanisms in the plasma membrane (PM) and dense tubular membrane (DT). When platelets are incubated in media with approx. 1 microM external Ca2+, [Ca2+]cyt is approx. 50 nM and [Ca2+]dt is very low. When 2 mM external Ca2+ is added, [Ca2+]cyt rises to approx. 100 nM and the process of dense tubular Ca2+ uptake can be resolved. Forskolin (10 microM) and Bt2-cAMP increase the rate of dense tubular Ca2+ uptake (V) to 2.1 +/- 0.60 and 1.70 +/- 40 times control values (respectively). The agents also increase the final [Ca2+]dt to 1.70 +/- 0.21 and 1.72 +/- 0.60 times control values (respectively). Titrations with ionomycin (Iono) showed that the increase was due to an increase in the Vm of the dense tubular Ca2+ pump. With [Iono] = 500 nM, [Ca2+]cyt was raised to greater than or equal to 1.0 microM and Vm of the dense tubular pump was elicited. (At [Iono] = 1.0 microM, the final [Ca2+]dt values were degraded 15% due to shunting of Ca2+ uptake.) Analysis showed that forskolin (10 microM) and Bt2-cAMP (1 mM) increase the Vm by a factors of 1.56 +/- 40 and 1.56 +/- 40, respectively. Analysis showed that neither agent changed the Km of the pump significantly from its control value of 180 nM. Neither agent changed the rate constant for passive leakage of Ca2+ across the DT membrane (1.7 min-1).

Cyclic GMP increases the rate of the calcium extrusion pump in intact human platelets but has no direct effect on the dense tubular calcium accumulation system.

Sodium nitroprusside (SNP) and other agents that elevate cGMP levels are known to inhibit the aggregation of human platelets. Published data suggest that cGMP attenuation of agonist-induced Ca2+ transients is involved in this effect. The present study shows that elevation of cGMP increases the rate of the Ca2+ extrusion pump located in the plasma membrane (PM) but does not have a direct effect on the Ca2+ accumulating pump of the dense tubules (DT). The study verifies that SNP can specifically elevate the cGMP level in the platelet. The kinetics of the Ca2+ extrusion system were studied in situ in platelets overloaded with the cytoplasmic Ca2+ indicator quin2 according to a published protocol developed in this laboratory. Elevation of cGMP by means of (10 microM) SNP increased the Vm of the Ca(2+)-ATPase pump by 63%, without affecting its Km (66-80 nM) or Hill coefficient (1.6-1.8). Dibutyryl-cGMP (Bt2-cGMP), preincubated for 45 min at 1 mM, increased the Vm by a factor of 2.2 +/- 0.4. The experiments did not give any indication that SNP or Bt2-cGMP change the rate of the Na+/Ca2+ exchanger which makes a minor contribution to Ca2+ extrusion in the studied [Ca2+]cyt range. The rate constant for passive leakage of Ca2+ across the PM was increased by 32 +/- 4% by SNP and 90 +/- 34% by Bt2-cGMP. The net result is that the free Ca2+ in the cytoplasm ([Ca2+]cyt) at 'rest' is lowered from control values of 112 nM to 89 nM or 80 nM, respectively. The kinetics of Ca2+ uptake by the dense tubules were determined in situ using the fluorescence of chlorotetracycline (CTC) according to protocols developed in this laboratory. Analysis showed that SNP and Bt2-cGMP had no effect on the Vm or Km of the dense tubular pump, and did not affect the rate constant for passive leakage. The agents did decrease resting [Ca2+]dt by 25% or 30%, respectively, but this result can be explained purely in terms of the reduced [Ca2+]cyt. The effects of cGMP (vs. cAMP) on the PM and DT pumps are closely correlated with reported effects of cGMP/cAMP induced phosphorylation of a protein of the molecular weight of the PM pump and a 22 kDa activator of the DT pump. Cyclic AMP increases the rate of both the PM and the DT pumps, whereas cGMP increases the rate of the PM pump only.(ABSTRACT TRUNCATED AT 400 WORDS)

Calibration methods and avoidance of errors in measurement of intracellular pH (pHcyt) using the indicator bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) in human platelets.

Determinations of pHcyt in suspensions of human platelets using BCECF [bis(carboxyethyl)-5(6)-carboxyfluorescein] can be seriously biased by leakage of the fluorescent indicator. Two methods ("pH jump" and "Mn(2+)") are presented for determining the fraction of external indicator (B ext) and eliminating this error. Both methods rely on rapid perturbations (pH jump or Mn(2+) addition), which affect the fluorescence of the external dye immediately and the intracellular dye more slowly. Identical values ofB ext are reported. Failure to correct for dye leakage can result in overestimation of pHcyt by as much as 0.4 unit at physiological external pH (pHext). Two methods of calibration of the cytoplasmic signal were compared after correcting forB ext: the "digitonin lysis" method and the "nigericin calibration" method. In the digitonin method the dye is released at the end of the experiment and the dependence of its fluorescence is determined as a function of pH. The method assumes that the fluorescence and titration characteristics of the dye in the cytoplasm are not different from those in solution. It gives pHcyt=6.75±0.07 for pHext=7.3. In the nigericin method, 150 mM external K(+) and 10 µM nigericin are used for the purpose of setting pHcyt=pHext to accomplish anin situ calibration. The method was complicated by extra leakage induced by nigericin. Assuming that the ionophore could equilibrate pH in the alkaline range, the fluorescence of the anionic form of BCECF in the cytoplasm would be 15% lower than in solution and pHcyt would be 0.3 unit higher than presented above. A number of observations favor the digitonin lysis method of calibration. The fluorescence polarization of BCECF in platelets is small and indistinguishable from that in solution (0.000±0.022). The spectrofluorimetric characteristics of the intracellular dye are identical to those in solution (150 mM NaCl or KCl). There was no evidence for self-quenching or binding to cellular elements for cytoplasmic BCECF concentrations up to 1.8 mM. The following agents are capable of introducing error: (1) the Na(+) substituteN-methyl-D-glucamine doubles theK d and decreases by 13% the ΔF max of BCECF; (2) the Na(+)/H(+) exchange inhibitor amiloride quenches BCECF fluorescence and is intrinsically fluorescent; and (3) bovine serum albumin (used to remove nigericin) quenches external BCECF with kinetics mimicking acidification of the cytoplasm.

Long duration local anesthesia with lecithin-coated microdroplets of methoxyflurane: studies with rat skin.

Lecithin-coated microdroplets of methoxyflurane (MOF) are shown to produce local anesthesia of three- to six-day duration in the skin with a single intradermal injection in rats. Anesthesia was quantitated by elevation of the threshold (milliampere) for shock vocalization with intradermal electrodes. Intradermal injection of 0.1 ml 0.5% MOF gave moderate (2.1 mA) anesthesia of approximately three-day duration for a 10-12-mm diameter area, with no damage to the tissue. Higher concentrations gave six-day duration anesthesia at very high level (7 to 12 mA) anesthesia of three- to five-day duration with some damage to the tissue. At 4.4% MOF, ulcers formed in the center of the injection site with maximal dimensions of 1.3 mm (11-13% of the site diameter). Phenol, a widely used neurolytic agent, was tested as a control in the same concentration range. Phenol at 4.4% gave very high level (8 mA) anesthesia for longer than seven-day duration and caused formation of ulcers with maximal dimensions of 3.8 mm (31-38% of the site diameter). Analysis showed that MOF produced less damage than phenol for any given degree of anesthesia. Systemic toxicity and pharmacokinetic data are also presented. Phenol produced a hypothermic reaction and behavioral changes, whereas MOF was without systemic effect. The plasma concentrations of phenol were four to five times greater than those of MOF. These results suggest that MOF may have clinical advantages over phenol.

Long duration local anesthesia with lecithin-coated microdroplets of methoxyflurane: studies with human skin.

Lecithin-coated microdroplets of methoxyflurane (MOF) have been reported to produce local anesthesia of long duration in rats. The present study was conducted in two phases. The first phase was open label studies in two human volunteers aimed at determining the effective concentration of MOF in human skin. Over the concentration range of 0.3-2.4%, MOF produced local anesthesia to pinprick and cold stimuli within 15 seconds. The duration of the anesthesia effect of 2.4% MOF in the skin of the buttock, forearm and leg was five to eight days. Microdroplets containing isoflurane, a more volatile agent, gave an anesthetic effect that reversed within two to five hours. In the second phase of the study, the safety and efficacy of MOF were compared to phenol in placebo-controlled and blinded studies using indwelling stimulating electrodes. Phenol was destructive to skin at a concentration necessary to obtain a degree of local anesthesia comparable to MOF. The greater part of the anesthetic effect produced by phenol at this "toxic" concentration was transient (approximately one hour). In contrast to phenol, MOF produced an anesthetic effect lasting four to seven days without producing visible damage to skin. These results suggest that MOF is safer and more efficacious than phenol for producing long-lasting local anesthesia of human skin.

The pH dependence of the cardiac sarcolemmal Ca2(+)-transporting ATPase: evidence that the Ca2+ translocator bears a doubly negative charge.

The pH dependence of the Ca2(+)-transporting ATPase of bovine cardiac sarcolemma was determined in a membrane vesicle preparation. The maximal velocity (Vmax) at saturating external Ca2+ showed a sigmoidal pH dependence with maximal values in the 6.0-6.5 range, a half-maximal value at 7.2 and minimal (less than or equal to 15%) values at pH greater than or equal to 8.0. The apparent affinity for Ca2+ (1/Km) varied over 10(4)-fold for 6.0 less than or equal to pH less than or equal to 8.5, increasing with increasing pH. Plots of log(1/Km) vs. pH were biphasic. In the acid range (6.0 less than or equal to pH less than or equal to 7.2), a slope of 2.6 was observed for the calmodulin-activated form of the pump. For 7.2 less than or equal to pH less than or equal to 8.5, a slope of 0.5 was observed. At pH 7.4, the Km is approx. 48 +/- 19 nM. The Ca2+ pump of cardiac sarcoplasmic reticulum in the same preparation had a Km of 304 +/- 115 nM and showed a similar pH dependence except that the slope in the acid range was 1.7. When calmodulin was removed from the sarcolemmal pump, its Km was raised to approx. 1.0 microM, the slope in the acid range was reduced to 1.7 and the Vmax was markedly reduced. The results are explicable in terms of a model in which each of the two Ca2+ binding sites on the pump contains two buried COO- groups responsible for high affinity. The Km effect is explained by 2 H+ vs. 1 Ca2+ competition for occupation of each of the two cytoplasmically-oriented translocators (4 H+ vs. 2 Ca2+). The Vmax effect is explained by counter-transport of H+. The findings are considered in terms of the published amino acid sequence of the cardiac sarcolemmal pump and recent site-directed mutagenesis vs. function studies identifying the Ca2+ binding site in the skeletal sarcoplasmic reticulum pump. The kinetic data are also applied to pump behavior under conditions of ischemia and acidosis.

The calmodulin-activated form of the Ca2(+)-pumping ATPase of the cardiac sarcolemmal membrane produces Ca2+ gradients with a thermodynamic efficiency of 100%.

The thermodynamic efficiency of the calmodulin-activated form of the Ca2+-pumping ATPase of the bovine cardiac sarcolemma (SL) was evaluated in sealed vesicles under reversible conditions. The free internal Ca2+ concentration ([Ca2+]i) established in the SL vesicle lumen by action of the ATPase was determined as a function of the [ATP]/([ADP][Pi]) ratio for the following experimental conditions: 250 mM sucrose, 100 mM KCl, 0.1 mM Mg2+, 25 mM HEPES, 25 mM Tris, pH 7.40, at 37 degrees C, [Ca2+]o = 50 nM (1 mM Ca/EGTA buffer), 0.75 mM Mg-ATP, 0.1 mM Pi, variable [ADP]. Under these conditions, with the pump working near its Km of 64 nM, the [Ca2+]i achieved was less than or equal to 18 mM, decreasing with increasing [ADP] for [ADP] greater than or equal to 0.84 mM. A plot of the square of the [Ca2+]i/[Ca2+]o ratio against [ATP]/([ADP][Pi]) gave a straight line with a slope of 1.5 x 10(7) M. This was in agreement, within the experimental error, with the equilibrium constant for ATP hydrolysis under these conditions (1.09 x 10(7) M). These results demonstrate (1) tight coupling between Ca2+ transport and ATP hydrolysis with a stoichiometry of 2 Ca2+ moved per ATP split and (2) a low degree of passive leakage. Analysis at low [ADP] (less than 0.83 mM) showed the unexpected result that ADP increases the rate of the forward reaction of the pump. The maximal effect on the initial rate is a 96 +/- 5% increase, with an EC50 of approximately 0.4 mM (ADP). Similar but lesser stimulation was observed with CDP. The implications of the above results for the energetics of the pump and for its physiological function in the beating heart are discussed.

Ca2+ pumping ATPase of cardiac sarcolemma is insensitive to membrane potential produced by K+ and Cl- gradients but requires a source of counter-transportable H+.

The sensitivity of the Ca2+ pumping ATPase of bovine cardiac sarcolemma (SL) to changes in membrane potential was studied in a preparation of sealed SL vesicles. Membrane potential was imposed by preincubating the vesicles in media of defined ion composition (K+, Cl-, choline+ and gluconate-) and diluting into media of differing ion composition. The durations of the ion gradients and relative ion permeabilities were determined in separate experiments by the dependence of the half time for net K+ (or choline+) movement coupled with these anions (Cl- or gluconate-), registered by the fluorescence of 1-anilino-8-naphthalene sulfonate (Chiu, V.C.K., Haynes, D.H. 1980. J. Membrane Biol. 56:203-218). Relative permeabilities were: 1.0, K+; greater than or equal to 10.0, 1 microM valinomycin-K+; 4.0, Cl-; 0.66, choline+; 0.38, gluconate-. Durations of the gradients ranged between 17 sec (KCl, valinomycin) to 195 sec (K(+)-gluconate-). In separate experiments, active Ca2+ uptake was monitored using chlorotetracycline (CTC) fluorescence, a technique validated by 45-Ca2+ measurements (Dixon, D., Brandt, N., Haynes, D.H. 1984. J. Biol. Chem. 259:13737-13741). Active Ca2+ uptake was initiated in the presence of monovalent ion gradients. The values of the membrane potentials (Em) imposed by the monovalent ion gradients were calculated using the ion concentrations, their relative permeabilities and the Goldman-Hodgkin-Katz equation. No effect of membrane potential on transport rate was observed (less than or equal to 4%, for 5-7% SD) for imposed potentials as extreme as greater than or equal to +71 and less than or equal to -67 mV. Formal analysis shows that the above observations are not compatible with models in which the Ca2+ pumping ATPase functions in an electrogenic or charge-uncompensated fashion. Further experimentation showed that the pump rate is slowed when uptake is measured at less-than-adequate concentrations of buffer (5 vs. 25 mM HEPES/Tris). This, together with further control experiments using nigericin and FCCP, gave evidence that the pump requires a source of counter-transportable H+ in the vesicle lumen. The above experimentation also underlines the need for control of internal pH to obviate erroneous interpretation of ion perturbation experiments. The results are compared with results obtained with the Ca2+ ATPase pump of skeletal sarcoplasmic reticulum.