When measuring cell membrane electrical capacitance in whole cell configuration using alternating currents, the resolution decreases with increasing membrane conductance and pipette resistance. Improved resolution was attained by the dual-frequency method which was modified as to control the voltage amplitude of one of the measuring frequencies. A model circuit was developed for the verification of the method. This circuit allows measurement of calibrated capacitance changes even in the range of 5 to 20 fF. Moreover, the method was applied to capacitance measurements on pancreatic exocrine acinar cells. The results of measurements on the model as well as on pancreatic acinar cells are presented. The principle can also be applied to other hardware and software methods for measuring electrical cell membrane parameters.
Transcellular trafficking in which various molecules are
transported across the interior of a cell, is commonly classified as
transcytosis. However, historically this term has been used
synonymously with transudation. In both cases transcellular
trafficking starts with the internalization of proteins or other
compounds on the basal or basolateral side of a cell and
continues by their transport across the interior to the apical pole
(or vice versa) where they are subsequently released. This allows
a cell to release products which are synthesized elsewhere. Here,
we discuss the common features of both transcytosis and
transudation, and that which differentiates them. It appears that
transcytosis and transudation are identical in terms of vesicular
import and endosomal sorting of cargo, but completely differ in
the re-secretion process. Specialized epithelial cells re-release
substantial quantities of the endocytosed material, and often also
a great variety. Some recent studies indicate that this is achieved
by non-canonical apocrine secretion rather than by the regular
vesicular mechanism of exocytosis, and takes place only on the
apical pole. This massive re-release of endocytosed proteins, and
potentially other compounds via the apocrine mechanism should
be considered as transudation, distinct from transcytosis.
Exocytotic machinery in neuronal and endocrine tissues is sensitive to changes in intracellular Ca2+ concentration. Endocrine cell models, that are most frequently used to study the mechanisms of regulated exocytosis, are pancreatic beta cells, adrenal chromaffin cells and pituitary cells. To reliably study the Ca2+ sensitivity in endocrine cells, accurate and fast determination of Ca2+ dependence in each tested cell is required. With slow photo-release it is possible to induce ramp-like increase in intracellular Ca2+ concentration ([Ca2+]i) that leads to a robust exocytotic activity. Slow increases in the [Ca2+] i revealed exocytotic phases with different Ca2+ sensitivities that have been largely masked in step-like flash photo-release experiments. Strikingly, in the cells of the three described model endocrine tissues (beta, chromaffin and melanotroph cells), distinct Ca2+ sensitivity ‘classes’ of secretory vesicles have been observed: a highly Ca2+ -sensitive, a medium Ca2+ -sensitive and a low Ca2+ - sensitive kinetic phase of secretory vesicle exocytosis. We discuss that a physiological modulation of a cellular activity, e.g. by activating cAMP/PKA transduction pathway, can switch the secretory vesicles between Ca2+ sensitivity classes. This significantly alters late steps in the secretory release of hormones even without utilization of an additional Ca2+ sensor protein., J. Dolenšek, M. Skelin, M. S. Rupnik., and Obsahuje bibliografii a bibliografické odkazy
A method for the measurement of the membrane capacitance and resistance with two simultaneous sinusoidal frequencies is described. This method combines the advantages of measurements with a single sinusoidal frequency (i.e. low noise - high resolution) and those with rectangular waveform or polyfrequent methods. The mathematical analysis of the impedance as well as the admittance are presented for the evaluation with synchronous detection. Preliminary results are given.