The effect of external calcium and lanthanum on platelet calcium content and on the release reaction.

Calcium compartments in calf platelets were studied using a lanthanum washout procedure to distinguish between surface-bound calcium and intracellular calcium. The calcium content of calf platelets ranges from 20 to 60 nmol/109. platelets and is sensitive to the calcium concentration of the suspending medium. With 1 mM calcium in the medium, calcium uptake is rapid and reaches steady state within 1-2 min. Results obtained with the lanthanum procedure indicate that it is the surface compartment which is most affected by the extracellular calcium concentration. The surface compartment appears to be saturable and is highly exchangeable. Although the total calcium as well as the calcium content of the surface and internal compartments are variable, the ratio of calcium in either compartment to the total saturated calcium is quite constant. The data indicate that 68-85% of the platelet calcium is located internally. Thrombin produces an immediate release of platelet calcium and labeled serotonin and an increase in the 45Ca2+ uptake of both the surface and internal compartments. The release reaction is not dependent upon exogenous calcium or an influx of exogenous calclium since it occurs even in the presence of ethyleneglycol-bis-(beta-aminoethylether)-N, N'-tetraacetic acid. Lanthanum, however, inhibits the release reaction possibly by blocking surface calcium site and reducing the mobility of endogenous platelet calcium.

Calcium uptake and associated adenosine triphosphatase activity of isolated platelet membranes.

A platelet subcellular fraction, sedimenting between 14,000 and 40,000 g and consisting primarily of membrane vesicles, accumulates up to 200-400 nmoles calcium/mg protein in the presence of ATP and oxalate. Steady-state levels of calcium accumulation are attained in 40-60 min. Calcium uptake requires adenosine triphosphate (ATP), is enhanced by oxalate, and is accompanied by the release of inorganic phosphate. Calcium accumulation and phosphate release require magnesium and are inhibited by Salyrgan (10 microM) and adenosine diphosphate (ADP) (1 mM), but not by ouabain (0.1 mM). The ATPase activity is stimulated by low concentrations of calcium (5-10 microM) and is inhibited by 2 mM EGTA. Electron microscopic histochemistry using lead nitrate to precipitate released phosphate results in lead precipitates localized primarily at the inner surface of membrane vesicles. These results provide evidence for a membrane ATPase that is stimulated by low concentrations of calcium and may be involved in the transport of calcium across the membrane. It is postulated that the observed calcium uptake activity is an in vitro manifestation of a calcium extrusion pump in the intact platelet.

Calcium electrode--a method for the continuous monitoring of the platelet release reaction.

The calcium electrode is a convenient, inexpensive, and non-traumatic method for measuring changes in the extracellular calcium which accompanies a platelet release reaction. With this instrument, the temporal pattern of release by platelets in a buffered-saline medium following thrombin stimulation was observed as follows: an initial time lag phase, followed by a maximum release phase, and finally a slow release phase.

Electrical stimulation with Pt electrodes. V. The effect of protein on Pt dissolution.

The effect of protein on the charge-induced dissolution of Pt stimulation electrodes was studied in an in vitro system. Biphasic pulses (+/- 400 mA, +/- 400 microC cm-2 geom.) were applied to smooth Pt bead electrodes (surface area = 0.03-0.08 cm2) in a buffered saline solution containing 0.075-0.2 mg/ml human serum albumin; pulse solutions were analysed periodically for dissolved Pt by flameless atomic absorption spectrometry. The incorporation of protein in the pulse solution drastically altered the dissolution behaviour of Pt electrodes. Whereas dissolution in organic saline solutions proceeded essentially in a linear fashion, the rate in protein solution decreased with time and asymptotically approached zero. The passage of electric charge was required for the observed inhibition to develop but was not required to maintain it. In contrast, the constant presence of at least 0.15 mg/ml protein in the pulse solution was required for inhibition to develop and continue. Higher concentrations of protein did not enhance the observed inhibition. These findings bear favourably on the prospect for in vivo use of Pt stimulation electrodes: the dissolution process now appears to place no greater restriction on the acceptable pulse parameters than do other irreversible reactions such as H2O electrolysis.

Electrical stimulation with pt electrodes. IV. Factors influencing Pt dissolution in inorganic saline.

Trace analysis has shown that Pt electrodes can suffer appreciable dissolution when used to apply biphasic current pulses of the type used in neural stimulation. The dissolution occurs even under conditions where other irreversible faradaic reactions, e.g., H2O electrolysis, are avoided. In the present study, factors influencing the dissolution of Pt electrodes during biphasic pulsing in neutral inorganic saline have been examined. The findings are consistent with the behaviour of Pt electrodes reported in other inorganic media. In a given test, the quantity of Pt dissolved was found to be a linear function of the aggregate anodic or cathodic charge injected. Therefore, dissolution 'rates' can be conveniently expressed in terms of nanograms of Pt per coulomb injected, e.g., 100 ng C-1. Most of the Pt went into solution as Pt (II) species, so the above rate would correspond approximately to 100 p.p.m. of the anodic charge per pulse. For anodic-first (AF) pulses, charge density was the major factor controlling dissolution, whereas for cathodic-first (CF) pulses, pulse duration had the greater influence. Depending on the polarity (AF or CF), charge density, and duration of the biphasic pulse, the dissolution rate for smooth bead electrodes ranged from 30 to 300 ng C-1. Lower rates were achieved with platinized electrodes but in the absence of organic solutes, it is unlikely that Pt dissolution can be completely suppressed.

Criteria for selecting electrodes for electrical stimulation: theoretical and practical considerations.

Smaller, more charge-intensive electrodes are needed for "safe" stimulation of the nervous system. In this paper we review critical concepts and the state of the art in electrodes. Control of charge density and charge balance are essential to avoid tissue electrolysis. Chemical criteria for "safe" stimulation are reviewed ("safe" is equated with "chemically reversible"). An example of a safe, but generally impractical, charge-injection process is double-layer charging. The limit here is the onset of irreversible faradaic processes. More charge can be safely injected with so-called "capacitor" electrodes, such as porous intermixtures of Ta/Ta2O5. BaTiO3 has excellent dielectric properties and may provide a new generation of capacitor electrodes. Faradaic charge injection is usually partially irreversible since some of the products escape into the solution. With Pt, up to 400 muc/cm2 real area can be absorbed by faradaic reactions of surface-adsorbed species, but a small part is lost due to metal dissolution. The surface of "activated" Ir is covered with a multilayer hydrated oxide. Charge injection occurs via rapid valence change within this oxide. Little or no metal dissolution is observed, and gassing limits are not exceeded even under stringent conditions.

Electrical stimulation with Pt electrodes. VII. Dissolution of Pt electrodes during electrical stimulation of the cat cerebral cortex.

Procedures are described for determining trace quantities of Pt released into brain tissue directly beneath cortical surface stimulation electrodes. Implanted electrodes (1.1 mm Pt discs) were stimulated for 4.5 h, 9 h and 36 h (4 X 9 h/day) with balanced biphasic pulses (20 micro C/cm2 or 100 micro C/cm2 per phase, 50 Hz), following which tissue 0-2 mm beneath stimulation electrodes and the encapsulating tissue adherent to electrodes was excised and analyzed for Pt. A time-dependent increase in Pt concentration was observed between 4.5 h (4-20 ng Pt/stimulation site) and 9 h (50-339 ng Pt/site) of stimulation at 100 micro C/cm2 with nearly all of the Pt located in the encapsulating tissue associated with the electrodes. Somewhat less Pt was observed in the 36 h samples, and it was almost equally distributed between the encapsulating tissue of the electrodes and the first millimeter depth of underlying brain tissue. Little or no Pt was found at electrode sites receiving 20 micro C/cm2 pulses. Control brain tissue samples as well as samples of blood, CSF and kidney were negative for Pt. The findings indicate that the rate of Pt dissolution gradually decreases during in vivo stimulation, and that dissolved Pt may slowly move away from stimulation sites, possibly by diffusion or fluid exchange.

Assessment of capacitor electrodes for intracortical neural stimulation.

Capacitor electrodes offer the potential for the safest method of stimulation of neural tissue because they operate without any faradaic process occurring at the electrode-electrolyte interface. Their use eliminates problems associated with metal dissolution or water electrolysis which may occur with electrodes of noble metals. This paper reviews recent work aimed at increasing the charge storage density of capacitor electrodes to allow their application with the small areas of 10(-4) mm2 required for intracortical stimulation of single neurons. Increased charge storage with electrodes using anodic films such as TiO2 and Ta2O5 has been obtained by increasing the real surface area of microelectrodes. Experiments have also been done with BaTiO3 films which have a much higher dielectric constant than the anodic film dielectrics. State-of-the-art electrodes made with these materials, however, have a charge storage density which at best is comparable to that obtained with Pt and is considerably lower than electrochemically safe charge densities that have been reported for activated Ir. It is concluded that for very small intracortical electrodes, capacitor electrodes will not be competitive with electrodes which operate using surface localized faradaic reactions.

Electrical stimulation with Pt electrodes. VIII. Electrochemically safe charge injection limits with 0.2 ms pulses.

The charge injection limits of a Pt electrode using 0.2 ms charge balanced, biphasic current pulses ranged from 50 to 150 microC/cm2 geometric if the potential excursions of the electrode are kept below those at which H2 or O2 is produced. These charge densities are three to ten times smaller than the currently accepted value based on earlier experiments in which the reversible surface reactions were fully utilized and the pulse widths were longer.

Preparation of etched tantalum semimicro capacitor stimulation electrodes.

The ideal electrode for stimulation of the nervous system is one that will inject charge by purely capacitive processes. One approach is to exploit the type of metal-oxide combination used in electrolytic capacitors, e.g., Ta/Ta2O5. For this purpose, fine tantalum wire (0.25 mm diam) was etched electrolytically at constant current in a methanol solution of NH4Br containing 1.5 wt % H2O. Electrolytic etching produced a conical tip with a length of ca. 0.5 mm and shaft diameters ranging from 0.10 to 0.16 mm. The etched electrodes were anodized to 10 V (vs. SCE) in 0.1 vol % H3PO4. The capacitance values normalized to geometric area of etched electrodes ranged from 0.13 to 0.33 micro F mm-2. Comparison of these values to the capacitance of "smooth" tantalum anodized to 10 V (0.011 micro F mm-2) indicated that the degree of surface enhancement, or etch ratio, was 12-30. The surface roughness was confirmed by scanning electron microscopy studies which revealed an intricate array of irregularly shaped surface projections about 1-2 micrometers wide. The etched electrodes were capable of delivering 0.06-0.1 micro C of charge with 0.1 ms pulses at a pulse repetition rate of 400 Hz when operated at 50% of the anodization voltage. This quantity of charge corresponded to volumetric charge densities of 20-30 micro C mm-3 and area charge densities of 0.55-0.88 micro C mm-2. Charge storage was proportionately higher at higher fractional values of the formation voltage. Leakage currents at 5 V were ca. 2 nA. Neither long-term passive storage (1500 h) nor extended pulsing time (18 h) had a deleterious effect on electrode performance. The trend in electrical stimulation work is toward smaller electrodes. The procedures developed in this study should be particularly well-suited to the fabrication of even smaller electrodes because of the favorable electrical and geometric characteristics of the etched surface.

Serotonin transport by cultured bovine aortic endothelium.

Endothelial cells isolated from bovine aortas without prior treatment with enzymes were cultured in RPMI 1640 medium containing 17% fetal calf serum and antibiotics. The endothelial cells at confluency (7 days) were similar to endothelium in situ or to freshly isolated endothelial cells from blood vessels as seen by light, scanning, and electron microscopy. Cultured and freshly isolated indothelial cells exposed to labeled serotonin, even in the presence of iproniazid (5 x 10-4M), took up approximately 125 and 250 pmoles 14C-serotonin/mg protein, respectively, in 3 hours. Imipramine (10-4M) reduced uptake for both cell groups. Cold (4 degrees C) and metabolic inhibitors sharply reduced serotonin uptake by both freshly isolated and cultured endothelial cells. Ouabain (10-5M) almost completely blocked serotonin transport. Six analogues of serotonin at concentrations ten times above experimental serotonin concentrations did not affect serotonin transport in the cultured endothelial cells but did reduce it in the freshly isolated endothelial cells by 50%. The data on transport suggest that serotonin uptake is not unique to pulmonary endothelium, as has been suggested previously. In addition, using cultured indothelial cells to study serotonin transport is compatible with using other serotonin model systems such as platelets, lung, or brain. Lastly, serotonin uptake by endothelial cells may involve an active transport mechanism similar to that described for the pulmonary circulation, platelets, and insect salivary glands.