Development of low-affinity, membrane-targeted Ca2+ sensors suitable for measuring presynaptic Ca2+.

1. Our aim is to measure near-membrane Ca(2+) flux within the presynaptic terminals of central neurons by modifying new genetically encoded Ca(2+) sensors to develop tools capable of measuring localized Ca(2+) signals. 2. We used standard recombinant DNA technologies to generate the DNA coding for a fusion construct of a modified fluorescent 'pericam' Ca(2+) biosensor with a presynaptic P2X7 receptor (P2X7R). The Ca(2+) sensitivity of the biosensor was modified by rational site-directed mutagenesis of the calmodulin portion of the pericam. 3. Biosensor-receptor fusions were transfected into expression systems for evaluation. Expression studies in HEK-293 cells showed that biosensor-receptor fusion construct-delivered protein was localized exclusively to the plasma membrane, confirming that fusion did not affect the ability of the receptor to undergo normal protein synthesis and trafficking. 4. The Ca(2+)-dependent fluorescence of the pericam portion of the fusion protein was also retained. Site-direct mutagenesis within the calmodulin moiety of the pericam significantly reduced the Ca(2+) affinity of the complex. The dynamic range of the sensor following this modification is better matched to the higher Ca(2+) levels expected within presynaptic Ca(2+) microdomains.

[New methods for noninvasive and continuous determination of dynamic compliance of the carotid artery]. / Neues Verfahren zur nichtinvasiven und kontinuierlichen Bestimmung der dynamischen Compliance der A. carotis.

A new method for the noninvasive, continuous determination of the compliance of the carotid artery wall has been developed and, in an initial study, validated. Measurements of pulsatile changes in the diameter of the carotid artery are accomplished with the 4-electrode impedance method, and the intravascular blood pressure is measured using an applanation tonometer developed during this project. The method has been employed for measurements in 12 individuals with no vascular disease, and in one patient with carotid artery stenosis before, during and after successful dilatation. With the pressure-volume curves recorded during the cardiac cycle, it is possible to calculate dynamic compliance and the non-elastic deformation work. While initial results are very promising, further validation by a large-scale clinical study is required.

Effect of postural changes on the reliability of volume estimations from bioimpedance spectroscopy data.

Bioimpedance spectroscopy (BIS) has been suggested for the assessment of fluid shifts between intracellular (ICV) and extracellular volume (ECV) during dialysis. The electrical tissue parameters are estimated by fitting a Cole-Cole model to the impedance data. Those parameters are used for the calculation of ICV and ECV with a fluid distribution model (FDM). We investigated whether postural changes cause artifacts in the volume data measured with a commercial BIS system. This is of importance at the beginning of dialysis, when the patient lies down for treatment. Volume estimations were performed during tilt table experiments with 11 healthy volunteers. Impedance spectra (5 to 500 kHz) were recorded for the total body as well as for body segments (leg and arm) during three phases: (1) 30 minutes resting in a supine position after standing; (2) 30 minutes 70 degrees head up tilt; and (3) a 30-minute resting period in a supine position. ECV and ICV were estimated with a commercially utilized FDM which is based on Hanai's mixture theory. A monoexponential function was fitted to the data for extracting the time constants and the extrapolated steady state values of the volume changes. The ECV and ICV data changed significantly during all three periods, that is, a steady state could not be reached within 30 minutes. During phase 1 the ECV decreased by 1.8 +/- 0.7%, in the tilt phase it increased by 3.8 +/- 1.1%, and in phase 3 it decreased again by 2.9 +/- 1%. The ICV increased by 3.6 +/- 2.4% during phase 1 and decreased by 6.8 +/- 5.1% during tilting; in phase 3 it increased by 4.6 +/- 1.7%. The time constants were 36.4 +/- 12.7 minutes (ECV) and 10.8 +/- 5.4 minutes (ICV) during phase 3. Segmental measurements revealed that the legs contribute significantly to the measured volume changes. The absolute volume changes in ICV and ECV differed significantly in all phases, and the same was found for the time constants during phases 1 and 3. From this discrepancy it is concluded that the measured volume changes are artifacts that are caused by extracellular fluid redistribution. Furthermore, it appears unlikely that the measured fluid shifts actually occur between ECV and ICV in the absence of osmotic changes in the body fluids. The validity of the method for a reliable assessment of volume changes during dialysis appears questionable, as dialysis-induced volume changes lie in the same range as the orthostatically-induced spurious volume changes.