Chris Clausen

A low-cost method for rapid transfer function measurements with direct application to biological impedance analysis

The measurement of the linear transfer function of a biological system has found wide use in the characterization of the systems input/output properties, and in the separation and measurement of the properties of the different subclements that make up the system. Unfortunately sophisticated and expensive instrumentation has traditionally been required to make these measurements. In this paper, we present detailed design specifications of a low-cost instrument that is capable of yielding transfer function measurements with a high degree of accuracy and speed.The instrument is comprised of a pseudo-random binary sequence signal generator with precise data acquisition synchronization circuits, interfaced to a general-purpose mini-or microcomputer system common to many laboratory environments. The instrument is capable of measuring the transfer function of an arbitrary biological system up to a bandwidth of 8.3 KHz, with a frequency resolution of 425 points. In cases where the biological measurements are not contaminated with experimental noise, the transfer function can be determined in as little as 47 ms of data collection. In the case where experimental noise is present in the biological measurements, a simple signal averaging method is described which results in an effective increase in the signal-to-noise ratio, thereby yielding accurate transfer function estimates.The instrument is especially well suited to the measurement of transfer functions of biological systems, where experimental noise is a problem and where only limited time is available to acquire stable measurements.

Urinary proteases degrade epithelial sodium channels

SummaryThe mammalian urinary bladder epithelium accommodates volume changes by the insertion and withdrawal of cytoplasmic vesicles. Both apical membrane (which is entirely composed of fused vesicles) and the cytoplasmic vesicles contain three types of ionic conductances, one amiloride sensitive, an-other a cation-selective conductance and the third a cation conductance which seems to partition between the apical membrane and the mucosal solution. The transport properties of the apical membrane (which has been exposed to urine in vivo) differ from the cytoplasmic vesicles by possessing a lower density of amiloride-sensitive channels and a variable level of leak conductance. It was previously shown that glandular kallikrein was able to hydrolyze epithelial sodium channels into the leak conductance and that this leak conductance was further degraded into a channel which partitioned between the apical membrane and the mucosal solution. This report investigates whether kallikrein is the only urinary constituent capable of altering the apical membrane ionic permeability or whether other proteases or ionic conditions also irreversible modify apical membrane permeability.Alterations of mucosal pH, urea concentrations, calcium concentrations or osmolarity did not irreversible affect the apical membrane ionic conductances. However, urokinase and plasmin (both serine proteases found in mammalian urine) were found to cause an irreversible loss of amiloride-sensitive current, a variable change in the leak current as well as the appearance of a third conductance which was unstable in the apical membrane and appears to partition between the apical membrane and the mucosal solution. Amiloride protects the amiloride-sensitive conductance from hydrolysis but does not protect the leak pathway. Neither channel is protected by sodium. Fluctuation analysis demonstrated that the loss of amiloride-sensitive current was due to a decrease in the sodium-channel density and not a change in the single-channel current. Assuming a simple model of sequential degradation, estimates of single-channel currents and conductances for both the leak channel and unstable leak channel are determined.

Lithium Pharmacokinetics: Single-Dose Experiments and Analysis Using a Physiological Model

The kinetics of lithium (Li+) distribution after a single dose was studied in healthy human subjects. Experiments were performed by simultaneously following changes of Li+ concentration in plasma, erythrocytes (RBC), and urine. The data were fitted by a simple but physiologically realistic model, so that extracted rate constants could be assigned to real body compartments and compared with independent measurements of cellular transport characteristics. The extracted rate constants were used to calculate steady-state cell-to-plasma Li+ ratios for RBC and for inaccessible cells (mainly muscle). In both cell types, the intracellular Li+ concentration is far below electrochemical equilibrium. This finding suggests that the Li+ countertransport efflux mechanism of RBC may be shared with muscle. We also present evidence for a circadian rhythm in Li+ excretion that parallels the daily cycle of Na+ and K+ excretion.

Impedance analysis of epithelia

In view of the importance of epithelial organization in vectorial ion transport, it is clear that a complete understanding of regulation of this process requires an understanding of the role of the structural features involved. For example, although ion channels are the basis of much of the electrical activity and the electrochemical driving forces for ion transport across epithelial cell membranes, we can never truly understand this process without understanding the role of the lateral intercellular spaces or other structures such as microvilli and crypts in regulating extracellular ion concentrations. Similarly, in attempting to understand how the density of conducting channels is regulated, it is important to have knowledge concerning the role of membrane insertion or removal in regulating membrane transport properties.

Changes in membrane conductances and areas associated with bicarbonate secretion in turtle bladder

SummaryTransepithelial impedance-analysis studies were performed in turtle bladder epithelium in order to measure changes in the different epithelial membranes resulting from stimulation of electrogenic bicarbonate secretion. Changes in membrane conductance relate to changes in ionic permeability, whereas changes in membrane capacitance relate to changes in membrane area, since most biological membranes exhibit a specific capacitance of ∼1 μF/cm2. The results of this investigation are summarized as follows: (i) cAMP and carbachol, agents which have been shown previously to stimulate electrogenic bicarbonate secretion, result in increases in apical-membrane conductance and capacitance; (ii) these changes occur concomitantly with the observed change in transport (measured using the short-circuit-current technique), thereby suggesting that bicarbonate secretion may be regulated in part by changes in the chloride conductance of the apical membrane; (iii) the increase in conductance does not reflect an increase in the membranes specific conductance, thereby indicating that it results from the addition of membrane possessing similar ionic permeability as the existing apical membrane; (iv) the magnitude of the changes in capacitance indicate that a minor cell population (β-type carbonic-anhydrase-rich cells) increase their apical-membrane area by several-fold; (v) a lack of transport-associated changes in the basolateral-membrane parameters suggest that transport is not regulated by alterations in basolateral-membrane ionic conductance or area; (vi) a lack of colchicine sensitivity, coupled with the magnitude of the changes in apical-membrane capacitance, indicate that the membrane remodeling processes are different from those involved in the regulation of proton secretion in a different cell population (α-type carbonic-anhydrase-rich cells).

Constitutive and transport-related endocytotic pathways in turtle bladder epithelium

SummaryProton secretion in the urinary bladder of the fresh-water turtle is mediated by a proton pump located in the apical membrane of a population of cells characteristically rich in carbonic anhydrase. Earlier studies have demonstrated that these cells exhibit apical-membrane endocytotic and exocytotic processes which are thought to be involved in the regulation of the rate of proton transport via alterations in the number of pumps within the apical membrane. In this study, we sought to characterize these processes using two different methods. Analysis of transepithelial impedance yielded estimates of membrane capacitance which could be related to membrane area, thereby allowing one to monitor net changes in apical-membrane area resulting from changes in the net rates of endo-and exocytosis. Uptake of the fluid-phase marker FITC-dextran provided a measure of net extracellular volume uptake which was related to net rates of endocytosis. Our major conclusions are summarized as follows. The bladder cells exhibit a high baseline rate of endocytosis which appears to be a constitutive process similar to pinocytosis. This process is completely inhibited when ambient temperature is reduced to 15°C. In addition, serosal application of 0.5mm acetazolamide causes a transient increase in the rate of endocytosis, concomitant with a decrease in the rate of transport. Reduction of ambient temperature to 15°C reduces the rate of acetazolamide-induced endocytosis, but does not abolish it. Addition of 1mm serosal azide not only prevents the acetazolamide-induced increase in endocytosis, but also prevents the decrease in transport caused by acetazolamide. Azide has no effect on the baseline rate of endocytosis, nor does it prevent inhibition of carbonic anhydrase by acetazolamide. The specificity of azide, coupled with the different temperature sensitivities, demonstrate that the constitutive and transport-dependent endocytotic pathways are distinct processes. The observation that azide prevents both the acetazolamide-induced increase in endocytosis and the decrease in transport strongly supports the notion that endocytosis of proton-pump-containing membrane is requisite for the inhibition of transport by acetazolamide. Finally, the results also demonstrate that acetazolamide does not inhibit proton secretion simply by inhibiting carbonic anhydrase.

Transport-related modulation of the membrane properties of toad urinary bladder epithelium

Impedance analysis and transepithelial electrical measurements were used to assess the effects of the apical membrane Na+ channel blocker amiloride and anion replacement on the apical and basolateral membrane conductances and areas of the toad urinary bladder (Bufo marinus). Mucosal amiloride addition decreased both apical and basolateral membrane conductances (Ga and Gbl, respectively) with no change in membrane capacitances (Ca and Cbl). Consequently, the specific conductances of these membranes decreased without significant changes in membrane area. Following amiloride removal, an increase was obtained in the steady-state rate of sodium transport compared to values before amiloride addition. This increase was independent of the initial transport rate, suggesting activation of a quiescent pool of apical sodium channels. Chloride replacement by acetate or gluconate had no significant effects on apical or basolateral membrane capacitances. The effects of these replacements on membrane conductances depended on the anion species. Gluconate (which induces cell shrinkage) decreased both membrane conductances. In contrast, acetate (which induces cell swelling) increased Ga and had no effect on Gbl. The increase in the apical membrane conductance was due to an increase in the amiloride-sensitive Na+ conductance of this membrane. In summary, mucosal amiloride addition or chloride replacements led to changes in membrane conductances without significant effects on net membrane areas.

Electrogenic bicarbonate secretion in the turtle bladder: Apical membrane conductance characteristics

SummaryWe have recently shown that stimulation of electrogenic HCO3− secretion is accompanied by a simultaneous increase in short-circuit current (Isc, equivalent to HCO3− secretion rate under these conditions), apical membrane capacitance (Ca, proportional to membrane area), and apical membrane conductance (Ga, proportional to membrane ionic permeability). The current experiments were undertaken to explore the ionic basis for the increase inGa and the possibility that the rate of electrogenic HCO3− secretion is regulated by changes inGa. Membrane electrical parameters were measured using impedance-analysis techniques before and after stimulation of electrogenic HCO3− secretion with cAMP in three solutions which contained different chloride concentrations. In another series of experiments, the effects of an anion channel blocker, anthracene-9-carboxylic acid (9-AA), were measured after stimulation of electrogenic HCO3− secretion with cAMP. The major conclusions are: (i) a measurable apical Cl− conductance exists in control hemibladders; (ii) the transport-associated increase inGa includes a Cl−-conductive component; (iii)Ga also appears to reflect a HCO3− conductance; (iv) the relative magnitudes of the apical membrane conductances to Cl− and HCO3− are similar; (v) 9-AA reducesGa andIsc appear cAMP-stimulated hemibladders; and (vi) alterations inIsc appear to be mediated by changes inGa.

Lithium concentration in the muscle compartment of manic-depressive patients during lithium therapy

Pharmacokinetic (PK) techniques were used to study the effect of lithium (Li+) on Li+ fluxes and concentrations in body compartments of manic-depressives. Patients not yet on Li+ therapy were similar to normal controls in all parameters. Comparison of patients before and during chronic Li+ therapy showed no effect of Li+ therapy on intestinal absorption and renal excretion of Li+. The calculated erythrocyte (RBC)-to-plasma Li+ concentration ratio increased with Li+ therapy, as already known from direct measurements. The calculated muscle-to-plasma Li+ concentration ratio was 6-8 times higher than the RBC ratio, and increased from 1.8 to 4.2 with Li+ therapy. The higher Li+ concentration in human muscle compared to RBC is attributed to muscles higher inside-negative resting potential, and may underlie side effects that arise in muscle from Li+ therapy. The discrepancy between the observed muscle-to-plasma ratio and that predicted for a passively distributed ion is attributed to extrusion by a countertransport process, and the increase in the observed ratio with Li+ therapy is attributed to inhibition of countertransport, as already established for RBC. Since muscle resembles nerve as an excitable cell, muscle Li+ warrants evaluation as a predictor of therapeutic response and side effects during Li+ therapy.

Changes in the Cell Membranes of the Bullfrog Gastric Mucosa with Acid Secretion

The effective area, resistance, and configuration of the apical and basolateral cell membranes of the bullfrog gastric mucosa were studied as a function of acid secretion rate, by alternating-current impedance methods. The drop in transepithelial resistance with acid secretion is attributed to the great increase in apical membrane area (hence conductance) associated with tubulovesicles. There is no evidence of a change in basolateral membrane resistance or of apical membrane premeability per unit area.