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Biomedical subjects

J Whittembury

Publications and source records attributed to J Whittembury.

At least 19 recordsLinked to original sources
PubMed1991

Intracellular ionized calcium changes in squid giant axons monitored by Fura-2 and aequorin.

Squid giant axons were injected simultaneously with Ca indicators Fura-2 and aequorin. Fura-2 was calibrated in situ by measuring fluorescence at 510 nm upon UV excitation at 340 nm, 360 nm, and 380 nm with a time-sharing multiple wavelength spectrofluorimeter. Limiting values for dye fluorescence were obtained by allowing a massive load of Ca to enter the axon with the aid of procedures such as prolonged depolarization in the presence of CN (for saturation) and by sequestration of all Ca present in the axoplasm accomplished with injection of EGTA into the axon (for a zero-Ca signal). The average intracellular Ca concentration obtained with Fura-2 was 184 nM. The sensitivity of Fura-2 to intracellular Ca is at least as great as that of aequorin, thus permitting its use in the characterization of Ca homeostasis mechanisms such as Na-Ca exchange. It was found, however, that for voltage-clamp experiments requiring an internal current electrode, Fura-2 is not a convenient Ca probe because electrode reactions in the axoplasm denature the dye, thereby restricting its use in characterization of Ca movements associated with electrically induced changes in membrane potential. A comparison of aequorin luminescence with Fura-2 fluorescence demonstrated that light output by aequorin is linear with intracellular Ca concentrations up to values of 750 nM, changing to a square law relationship from 750 nM up to 10 microM Ca.

Aequorin↗
PubMed1986

Increases in internal Ca2+ and decreases in internal H+ are induced by general anesthetics in squid axons.

Squid axons were injected with arsenazo III and treated with sea water containing compounds usually classified as general anesthetics, (pentanol-decanol and a variety of hydrocarbons and their derivatives). Such treatment led to an increase in absorbance by arsenazo III at wavelengths sensitive to [Ca]i. The effect was independent of the presence or absence of Ca++ in sea water and it was not modified by substances that release Ca from internal stores. The effect was easily reversible. In axons injected with phenol red or impaled with a glass electrode sensitive to H+, a similar treatment led to an alkalinization that was also readily reversible. Both Ca release and the change to an alkaline pH had identical time courses. The dose required for action by all of the chemical agents studied could be predicted from a knowledge of their fractional saturation in sea water, i.e. from their thermodynamic activity. For compounds with 8-10 carbon atoms, Ca-release effects can occur at concentration less than those necessary to block either conduction or Na/Ca exchange. A special chemical agent was octylamine, which induced a marked rise in pHi and in addition its nonionic form produced the typical Ca release associated with general anesthetics.

Anesthetics↗
PubMed1986

Dependence of ionized and total Ca in squid axons on Nao-free or high-Ko conditions.

The level of intracellular Ca in squid axons (both ionized and total Ca) was studied as a function of the experimental variables [Na]i, [Na]o, pHi, cyanide, and depolarization. Ionized Ca was measured by following the light emission of aequorin while total Ca was measured by the atomic absorption analysis of samples of axoplasm. Aequorin glow is known to be increased either by the application of Nao-free solutions or by depolarization produced by external solutions containing greater than normal K concentrations. The present results show that if [Na]i is low, the depolarization that is brought about by solutions with elevated [K] leads to a resting light emission that is decreased rather than increased, as is the case when [Na]i is high. In axons where [Na]i is varied, a comparison of the increments in light emission produced by the application first of Na-free and then of high-K solutions shows that they have an identical dependence on [Na]i, with a half-activation of Ca entry produced by an [Na]i of 25-30 mM. Changes in pHi affect the aequorin signal produced by depolarization, with acidification reducing and alkanization increasing the response. Cyanide did not greatly affect the size of the signal resulting from either Nao removal or that from depolarization.

Animals↗
PubMed1985

Acute mountain sickness: critical appraisal of the Pariacaca story and on-site study.

The physiological and medical literature on the description of acute mountain sickness by Father Acosta in the Peruvian Andes shows many historical misconceptions and clinical misinterpretations. A recent paper by Gilbert (1983) not only contains these traditional misinterpretations but also adds geographical errors in the description of the area where Acosta described his sufferings. In view of these facts the authors have made a review of the old and modern writings on the so called Pariacaca story and during an on-site visit to the area of Pariacaca have taken actual measurements of distances, altitudes and geographical locations which they hope will put this story in the context of historical and scientific objectivity.

Acute Disease↗
PubMed1985

Normal whole blood Bohr effect in Peruvian natives of high altitude.

Measurements of whole blood O2 affinity were made with an automatic technique that allowed recording of O2 saturation continuously over a range of PO2 of 1 to 150 torr at constant pH and PCO2 with fresh samples of blood obtained from 5 natives of high altitude at Morococha, Peru (altitude, 4540 m). The results were not significantly different from those obtained with controls living at sea level with regard to the dlog P50/dpH slope at constant PCO2 or at constant base excess. Both results also agree with previous studies with normal sea-level blood. Thus earlier claims of an increased Bohr in Peruvian high-altitude natives is not supported.

Adult↗
PubMed1985

Ca2+ entry in squid axons during voltage-clamp pulses is mainly Na+/Ca2+ exchange.

Intact squid axons were injected with aequorin and bathed in 3 mM Ca seawater (a concentration close to that of squid blood). Sodium and potassium currents were pharmacologically blocked and repetitive voltage-clamp pulses of a duration of 1.5 ms were applied (to simulate the duration of an action potential) at amplitudes of +30 to +90 mV and at frequencies of 100/s. In a very fresh axon (low internal Na concentration) no detectable change in aequorin glow resulted from this treatment, whether the axons were in Na-containing or in Na-free seawater. In axons subjected to modest Na loading, repetitive voltage-clamp pulsing did not result in an increased aequorin glow when the pulses were delivered in Na seawater, whereas in Na-free seawater there was an easily measurable increase in aequorin light emission during repetitive pulsing. The increase in aequorin photons emitted per voltage-clamp pulse was e-fold for 22 mV of depolarization, and the process showed no signs of saturating at pulse amplitudes of +180 mV (i.e., at a membrane potential close to ECa). The aequorin light emission per voltage-clamp pulse increased linearly with pulse duration (at constant amplitude).

Action Potentials↗
PubMed1985

The influence of chemical agents on the level of ionized [Ca2+] in squid axons.

Squid giant axons injected with either aequorin or arsenazo III and bathed in 3 mM Ca (Na) seawater were transferred to 3 mM Ca (K) seawater and the response of the aequorin light or the change in the absorbance of arsenazo III was followed. These experimental conditions were chosen because they measure the change in the rate of Na/Ca exchange in introducing Ca into the axon upon depolarization; [Ca]o is too low to effect a channel-based system of Ca entry. This procedure was applied to axons treated with a variety of compounds that have been implicated as inhibitors of Na/Ca exchange. The result obtained was that the substances tested could be placed in three groups. (a) Substances that were without effect on Ca entry effected by Na/Ca exchange were: D600 at 10-100 microM, nitrendipine at 1-5 microM, Ba2+ and Mg2+ at concentrations of 10-50 mM, lidocaine at 0.1-10 mM, cyanide at 2 mM, adriamycin at a concentration of 3 microM, chloradenosine at 35 microM, 2,4-diaminopyridine at 1 mM, Cs+ at 45-90 mM, and tetrodotoxin at 10(-7). (b) Substances that had a significant inhibitory effect on Na/Ca exchange were: Mn2+, Cd2+, and La3+ at 1-50 mM, and quinidine at 50 microM. (c) There were also blocking agents and biochemical inhibitors whose action appeared to be the inhibition of nonmitochondrial Ca buffering in axoplasm rather than an inhibition of Na/Ca exchange. These were the general anesthetic l-octanol at 0.1 mM and 1 mM orthovanadate plus apyrase.

Aequorin↗
PubMed1984

A comparison of measurements of intracellular Ca by Ca electrode and optical indicators.

Squid giant axons were injected with aequorin or arsenazo III and impaled with a Ca-sensing electrode. The light output of aequorin or the spectrophotometer output when measuring arsenazo was compared with the voltage output of the electrode when the squid axon was depolarized with high-K solutions, when the seawater was made Na-free, or when the axon was tetanized for several minutes. The results from these treatments were that the optical response rose (as much as 50-fold) with all treatments known to increase Ca entry, while the electrode remained unaffected by these treatments. If axons previously subjected to Ca load are treated with electron-transport poisons such as CN, it is known that [Ca]i rises after a time necessary to deplete ATP stores. In such axons one expects a rise of [Ca]i in axoplasm which does not necessarily have to be uniform although the source of such Ca is the mitochondria and these are uniformly distributed in axoplasm. Under conditions of CN application, the optical signals from aequorin or arsenazo and Ca electrode output do rise together when [Ca]i is high, but there is a region of [Ca]i concentration where aequorin light output or arsenazo absorbance rises while electrode output does not. Axons not loaded with Ca but injected with apyrase and vanadate have mitochondria that still retain some Ca and this can be released by CN in a truly uniform manner. The results show that such a release (which is small) can be readily measured with aequorin, but again the Ca electrode is insensitive to such [Ca]i change.

Aequorin↗
PubMed1983

Effects of internal sodium and hydrogen ions and of external calcium ions and membrane potential on calcium entry in squid axons.

Squid giant axons were impaled with electrodes to measure pNai, pHi, Em, and were injected with either aequorin or arsenazo III to measure [Ca]i or with phenol red to measure [H]i. Depolarization of such axons with elevated [K] in sea water leads to a Ca entry that is a function of [Ca]o, [Na]i, and [H]i. With saturating [Na]i half-maximal Ca entry is produced by a [Ca]o of 0.58 mM. With saturating [Ca]o, depolarization produced by 450 mM-K+ leads to half-maximal Ca entry when [Na]i is 25 mM; entry is virtually undetectable if [Na]i is 18 mM. If [Ca]o is 50 mM, Ca entry upon depolarization as measured with aequorin is phasic with a rapid phase of light emission and a plateau; Ca entry as measured with arsenazo III shows no such phasic behaviour, absorbance vs. time is a square wave that closely follows the depolarization vs. time trace. Both detectors of [Ca]i show a square-wave response if [Ca]o is 3 mM. The introduction of 2 mM-CN into the sea water bathing the axon does not affect the response to depolarization nor does the destruction of most of the ATP in the axon following the injection of apyrase. If axons are microinjected with phenol red rather than arsenazo, the entry of Ca produces an acidification in the peripheral parts of the axoplasm. Other experiments measuring [Ca]i show that Ca entry is strongly inhibited by a decrease in pHi. Making sea water alkaline with pH buffers scarcely affects the Ca entry induced by depolarization; making axoplasm alkaline by adding NH4+ to sea water greatly enhances Ca entry by Na/Ca exchange and also enhances the ability of axoplasmic buffers to absorb Ca.

Aequorin↗
PubMed1981

Variability of oxygen affinity of blood: human subjects native to high altitude.

Whole blood O2 equilibrium curves (OEC) were measured in 46 Peruvians native to high altitude (4,540 m) and in 25 sea-level controls. A method was employed that records the entire OEC from 0 to 150 Torr with constant pH and PCO2. The data were analyzed by fitting the Adair equation describing the successive oxygenation of hemoglobin. At pH 7.4 the PO2 at which hemoglobin is half-saturated with O2 (P50) was significantly higher in the high-altitude population (31.2 +/- 1.9 Torr) than in controls (29.2 +/- 1.8 Torr, P less than 0.001). The acid-base status of the high-altitude subjects, however, was that of compensated respiratory alkalosis (plasma pH 7.439 +/- 0.065), and when the P50's were corrected to the subjects' plasma pH the values (30.1 +/- 2.2 Torr) could no longer be distinguished from the controls. We conclude that, on the average, increased P50 resulting from increased red cell 2,3-diphosphyoglycerate concentration at high altitude is offset by compensated respiratory alkalosis with the net result that the position of the OEC more closely approaches that of sea-level humans than has hitherto been thought. Considerable variation exists in P50, both at sea level and high altitude. This variation might have important consequences for acclimatization and survival under adverse environmental conditions.

2,3-Diphosphoglycerate↗
PubMed1976

Chronic mountain sickness.

Chronic mountain sickness was first described in the Peruvian Andes. It consists of an excesive polycythemia for the altitude of residence. Its main symptoms are of cerebral congestion and occasionally of right heart insufficiency. The authors postulate that it is caused by a decay of the ventilatory rate with age with the corresponding increase in hematocrit.

Adolescent↗