Polyphosphate (poly-P) is an important metabolite and signaling molecule in prokaryotes and eukaryotes. DAPI (4',6-diamidino-2-phenylindole), a widely used fluorescent label for DNA, also interacts with polyphosphate. Binding of poly-P to DAPI, shifts its peak emission wavelength from 475 to 525 nm (excitation at 360 nm), allowing use of DAPI for detection of poly-P in vitro, and in live poly-P accumulating organisms. This approach, which relies on detection of a shift in fluorescence emission, allows use of DAPI only for qualitative detection of relatively high concentrations of poly-P, in the microg/ml range. Here, we report that long-wavelength excitation (> or = 400 nm) of the DAPI-poly-P complex provides a dramatic increase in the sensitivity of poly-P detection. Using excitation at 415 nm, fluorescence of the DAPI-poly-P complex can be detected at a higher wavelength (550 nm) for as little as 25 ng/ml of poly-P. Fluorescence emission from free DAPI and DAPI-DNA are minimal at this wavelength, making the DAPI-poly-P signal highly specific and essentially independent of the presence of DNA. In addition, we demonstrate the use of this protocol to measure the activity of poly-P hydrolyzing enzyme, polyphosphatase and demonstrate a similar signal from the mitochondrial region of cultured neurons.

Despite the fact that Ca2+ transport into the sarcoplasmic reticulum (SR) of muscle cells is electrogenic, a potential difference is not maintained across the SR membrane. To achieve electroneutrality, compensatory charge movement must occur during Ca2+ uptake. To examine the role of Cl- in this charge movement in smooth muscle cells, Ca2+ transport into the SR of saponin-permeabilized smooth muscle cells was measured in the presence of various Cl- channel blockers or when I-, Br-, or SO42- was substituted for Cl-. Calcium uptake was inhibited in a dose-dependent manner by 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and by indanyloxyacetic acid 94 (R(+)-IAA-94), but not by niflumic acid or 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS). Smooth muscle SR Ca2+ uptake was also partially inhibited by the substitution of SO42- for Cl-, but not when Cl- was replaced by I- or Br-. Neither NPPB nor R(+)-IAA-94 inhibited Ca2+ uptake into cardiac muscle SR vesicles at concentrations that maximally inhibited uptake in smooth muscle cells. These results indicate that Cl- movement is important for charge compensation in smooth muscle cells and that the Cl- channel or channels involved are different in smooth and cardiac muscle cells.

Ca2+ transients in isolated cardiac ventricular myocytes and the amount of Ca2+ that could be released from the sarcoplasmic reticulum (SR) in these cells by caffeine were reduced in the presence of tamoxifen. To examine the effects of tamoxifen on the cardiac muscle SR directly, isolated SR vesicles and fluorimetry methods were used to measure the uptake of Ca2+ by the SR and the ATPase activity of the SR Ca2+ pump. SR Ca2+ uptake was inhibited by tamoxifen at concentrations greater than 2.4 microM. Half-maximal inhibition was seen at approximately 5 microM. Inhibition of uptake was not due to the development of a substantial tamoxifen-dependent leak of Ca2+ from the SR or to a direct inhibitory effect of tamoxifen on the ATPase activity of the SR Ca2+ pump. In addition to its effect on SR Ca2+ uptake, tamoxifen also reduced the rate at which stored Ca2+ could be released from the SR by the Ca2+ ionophore 4-bromo A23187. Our results are consistent with the hypothesis that tamoxifen inhibits an ion current that accompanies Ca2+ movement across the SR membrane. This possibility is also consistent with the known inhibitory action of tamoxifen on some types of Cl- and K+ channels.

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We explored the use of four methods for analyzing real and simulated fura 2 measurements of Ca2+ uptake by membrane vesicles derived from the sarcoplasmic reticulum (SR) of cardiac muscle. Uptake velocity was calculated 1) directly from the raw data, 2) after segmenting the raw data and averaging the data points in each segment, 3) after smoothing of the raw data by moving-window averaging, and 4) by Savitsky-Golay convolution. Methods 2, 3, and 4 could be used to determine maximum uptake velocity, the Hill coefficient, and the Ca2+ concentration at half-maximal pump velocity from Ca2+ concentration vs. time and velocity curves that were too noisy to analyze directly. Data analysis using these methods should have general applicability to biological experiments, especially those in which large numbers of measurements are made. The fluorometric method we describe for measuring Ca2+ uptake by cardiac SR vesicles opens up the possibility of studying SR function from very small starting tissue samples.