Biomedical subjects

D Hockett

Publications and source records attributed to D Hockett.

5 recordsLinked to original sources

PubMed1996

Elemental composition of Na pump inhibited rabbit aorta VSM cells by electron probe X-ray microanalysis.

The Na pump in vascular smooth muscle (VSM) is likely to influence not only intracellular Na content but also the content and distribution of other cations and anions measured by electron probe X-ray microanalysis (EPXMA). The hypothesis we tested was that EPXMA of pump-inhibited VSM would yield a characteristic cellular elemental profile, providing insight into the contribution of the Na pump to the intracellular milieu and an approach to identifying when VSM operates under the constraints of pump inhibition. We assessed the contractile state and elemental EPXMA profile of rabbit aorta that was either quiescent or contracted by serotonin (10(-6) M) or ouabain (10(-6) M). VSM cytoplasm showed the anticipated low Na (28 +/- 2 mM) and high K (182 +/- 5 mM) content. With ouabain, Na rose and K fell to reverse the Na-to-K ratio (0.15 +/- 0.01 vs. 6.6 +/- 0.3; P < 0.01). With serotonin, the ratio rose slightly (0.28 +/- 0.2; P < 0.05). Nuclei and mitochondria showed a similar pattern. CI showed a small increase (56 +/- 2 to 102 +/- 4 mM) with ouabain, a shift that could not be accounted for on the basis of charge redistribution to maintain neutrality because the change in Na and K were essentially offsetting. EPXMA measures total and not ionized Ca. If changes in cytoplasmic Ca occurred, they were too small to be measured by the imaging methods employed. The sustained, myogenic contractile response of VSM to Na pump inhibition shows a characteristic elemental profile that could prove useful in its identification. Direct measurement of membrane potential during the myogenic response to Na pump inhibition should have a high priority.

Animals↗

PubMed1994

Mitochondrial localization and characterization of 99Tc-SESTAMIBI in heart cells by electron probe X-ray microanalysis and 99Tc-NMR spectroscopy.

As the development of targeted intracellular magnetic resonance contrast agents proceeds, techniques for the quantitative analysis of the subcellular compartmentation and characterization of metallopharmaceuticals must also advance. To this end, the subcellular distribution and chemical state of hexakis (2-methoxyisobutyl isonitrile) technetium-99 (99Tc-SESTAMIBI), the ground state of the organotechnetium radiopharmaceutical used for the noninvasive evaluation of myocardial perfusion and viability by scintigraphy, has been determined by a novel application of electron probe X-ray microanalysis (EPXMA) and 99Tc-NMR spectroscopy. In cryopreserved cultured chick heart cells equilibrated in 36 microM 99Tc-SESTAMIBI, EPXMA imaging of mitochondria yielded a respiratory uncoupler-sensitive characteristic 99Tc X-ray peak representing 32.0 +/- 2.9 nmoles Tc/mg dry weight, while EPXMA of cytoplasm or nucleus showed no peak significantly greater than the threshold detectability limit of approximately 1 nmole/mg dry weight. Technetium-99 NMR spectroscopy of heart cells equilibrated with 99Tc-SESTAMIBI showed a single peak at -45.5 ppm with no evidence of significant line broadening or chemical shift compared to aqueous chemical standards, indicating that the majority of the complex exists unbound within the mitochondrial matrix. These data quantitatively demonstrate the localization of this lipophilic cationic organometallic complex within mitochondria in situ, consistent with a sequestration mechanism dependent on membrane potentials. Furthermore, this study establishes the general feasibility of combined EPXMA and NMR spectroscopy for the direct subcellular localization and characterization of metallopharmaceuticals, techniques that are readily applicable to MR contrast agents.

Animals↗

PubMed1993

Microprobe analysis of Tc-MIBI in heart cells: calculation of mitochondrial membrane potential.

Hexakis (2-methoxyisobutylisonitrile) technetium-99m (99mTc-MIBI) is a gamma-emitting radiopharmaceutical probe currently in clinical use to evaluate myocardial perfusion. Biochemical and cellular pharmacological studies have suggested that Tc-MIBI, a lipophilic cation, is sequestered in mitochondria in response to transmembrane potentials. To assess directly the subcellular distribution of the probe in heart tissue, cultured chick heart cells were analyzed by electron-probe X-ray microanalysis (EPXMA) following equilibration in micromolar concentrations of carrier-added 99Tc-MIBI, the ground-state radiopharmaceutical. Quantitation of the physiological elements Na, Ca, Mg, K, S, P, and Cl was correlated with exposure to increasing concentrations of 99Tc-MIBI. EPXMA signals indicated that 99Tc-MIBI was concentrated up to 1,000 times into mitochondria in a dose-dependent fashion based on measured Tc content in the mitochondria. Inner membrane potential (delta psi) of individual mitochondria was calculated as -117 mV using the Nernst equation. Concentrations of 99Tc-MIBI > 36 microM caused a significant efflux of K and Mg from the cell, as well as an increase in Cl in the mitochondria. Comparison of cell ultrastructure with conventional electron microscopy at extracellular 99Tc-MIBI concentrations of 36-72 microM showed no changes compared with control. 99Tc-MIBI allows valuable in situ investigation of cellular bioenergetics with EPXMA by quantitation of delta psi.

Animals↗

PubMed1982

Ejection fraction derived using dye dilution and angiographic methods.

Ventriculographically derived ejection fraction (EF-V) is the most frequently used method to measure left ventricular (LV) function, However, significant error may result in the measurement of end-systolic volume (ESV), which is used to calculate EF-V. This error is ascribed to the variable, irregular, nonellipsoidal geometry of the end-systolic ventricular chamber. Since stroke volume (SV) is determined more accurately by dilution methods than by ventriculography, an improved measure of ESV can be calculated by subtracting green dye determined SV from the ventriculographic determined end-diastolic volume (EDV). The purpose of this study was to measure a correlated ejection fraction (EF-C) using EDV by ventriculography and SV derived using green dye. In eight anesthesized dogs cardiac outputs (COs) were calculated by green dye and left ventriculography. CO determined by ventriculography was greater than that measured by green dye (p less than 0.005). EF-V (55 +/- 15%) was always greater than EF-C (32 +/- 12%) (p less than 0.005). These studies (1) may partially explain the discrepancy in CO calculated from the use of dilution methods and ventriculography and (2) present a method to improve the calculation of LV ejection fraction.

Animals↗

PubMedDate not supplied

In situ cryofixation of kidney for electron probe X-ray microanalysis.

Cell physiological and pathophysiological studies often require information about the elemental composition of intracellular organelles in situ. Electron probe X-ray microanalysis (EPXMA) is one of the few methods by which intracellular elemental content and distribution can be measured simultaneously. While several cryofixation techniques for EPXMA have been utilized on isolated cells, few have been applied successfully to whole tissue in vivo or in situ. A recently developed, commercial, portable, metal-mirror device was used for preserving kidney in situ to determine the intracellular element distribution in proximal tubule cells. Kidneys of male rats were exposed, cryofixed, and analyzed for organelle elemental contents by EPXMA imaging. In addition, some portions of the frozen tissue were prepared for conventional transmission electron microscopy. Proximal tubules were preserved with intact brush borders and open lumens. The quality of preservation of tubule cell organelles varied inversely as a function of depth from the point of first contact with the mirror surface; the best preservation was within 15 microns, while the poorest preservation was deeper than 30 microns. Analysis of EPXMA images from the best-preserved regions revealed that proximal tubule cell cytoplasmic K/Na was approximately 6, cytoplasmic Cl was low relative to other subcellular compartments, and mitochondrial Ca levels were 1.8 nmole/mg dry weight; these observations indicate that the cells were physiologically viable at the time of cryofixation. The advantages of in situ cryofixation by this metal-mirror method include acquisition of organelle elemental content data in vivo, ease of use, reproducibility, portability, applicability to other tissues, and suitability for pathophysiological studies.

Animals↗