Daniel C. Marcus

Direct measurement of longitudinal endolymph flow rate in the guinea pig cochlea

The rate of longitudinal endolymph flow in the guinea pig cochlea has been measured with a novel tracer technique. The tracer we utilized was the tetramethylammonium (TMA) ion, the movement of which was monitored by ion-sensitive microelectrodes. Extremely small amounts of tracer were required as the electrodes could readily detect TMA concentrations in endolymph as low as 10 microM. TMA was introduced into scala media in the form of a small bolus, varying from 2-20 nl in volume. To examine whether longitudinal flow affects the dispersion of TMA in endolymph, we compared the characteristics of TMA spread to turn I following injection into turn II, with those of TMA spread to turn II following injection into turn I. The comparison of these data with an analytical model combining the processes of diffusion and volume flow demonstrates that the spread of tracer is dominated by passive diffusion processes with very little contribution from longitudinal endolymph flow. The rate of longitudinal endolymph flow between turn I and turn II was estimated to be less than 0.01 mm/min directed towards the basal turn. This value is considerably lower than recently published estimates using other techniques.

Vascular perfusion of the cochlea: Effect of potassium-free and rubidium-substituted media

SummaryBy means of vascular perfusion of the cochlea with K+-free synthetic blood, it is possible to maintain the endocochlear potential (EP) at normal or supernormal levels for periods of 20–42 min. The subsequent decline of the EP is comparatively slow. This is in contrast to the immediate onset and rapid decline of the EP during perilymphatic perfusion with K+-free media. Possible implications of these data concerning the mode of generation of the EP are briefly discussed. It is further shown, that synthetic blood, in which K+ is replaced by Rb+, maintains the EP at normal or near normal levels for 2 h.

Sidedness of action of loop diuretics and ouabain on nonsensory cells of utricle: A micro-Ussing chamber for inner ear tissues

It is known that nonsensory tissues of the utricle produce a lumen-positive transepithelial electrical potential difference (VT). This potential has been shown previously to be inhibited by ouabain and bumetanide applied to the bathing medium in vitro. In order to more fully characterize the origin of this potential we mounted the utricle as a flat sheet in a newly designed Ussing chamber and measured the VT and transepithelial resistance (RT) while perfusing the endolymphatic and perilymphatic surfaces independently with identical solutions. The aperture of the chamber was 1.5 X 10(-4) cm2. VT averaged 5.6 +/- 0.46 mV and RT was 24.0 +/- 2.43 omega X cm2 (n = 45). Ouabain and loop diuretics of the furosemide family were found to inhibit the VT only from the serosal side. The KI for ouabain was 7.63 X 10(-5) M. The loop diuretics tested inhibited the VT in the same order as in other tissues known to contain a Na/2 Cl/K cotransporter (KI: 2-benzylamino-4-cyclohexylsulfonyl-5-sulfamoylbenzolsulfonate++ + (BCSB), 1.72 X 10(-7) M; bumetanide, 1.10 X 10(-6) M; piretanide, 5.67 X 10(-6) M; furosemide, 4.14 X 10(-5) M). It is concluded that this tissue produces a lumen-positive VT (i) in the absence of a transepithelial chemical gradient; the generation of which is dependent upon the activity of (ii) Na,K-ATPase and (iii) a Na/2 Cl/K cotransporter; (iv) in the basolateral membranes of the nonsensory cells; (v) which is not depressed by luminal application of inhibitors of these transporters.

Changes in cation contents of stria vascularis with ouabain and potassium-free perfusion.

Perfusion of the perilymphatic space of guinea pig cochleae with K-free medium leads to a gradual decline of the endocochlear potential (EP) over 30-50 min to a negative value (mean: -12 mV). The input resistance of scala media does not decrease during this time. The ATP and K content of the stria vascularis are reduced by similar amounts (26 and 34%, respectively) during this period. Perfusion of 1 mM ouabain produces a different pattern of response: strial ATP remains normal while strial K content is strongly reduced (by 77%). Strial Na rises in a complementary way to the K loss. These results demonstrate that a reduction of the K concentration of the perilymph leads to an inhibition of the generator of the positive component of the EP rather than to a general increase of cochlear duct membrane conductance. In addition, they suggest, in concert with other considerations (such as the slower rate of decline of the EP during K-free vascular perfusion (Wada, J., Kambayashi, J., Marcus, D.C. and Thalmann, R (1979): Arch. Otorhinolaryngol. 225, 79-81)), that the mode of action may be different from that of ouabain. In spite of the lack of teleological support, we offer the hypothesis that the strial generator of the EP may primarily utilize K from perilymph and that vascular K may not have access to the generator.

Respiratory rate and ATP content of stria vascularis of guinea pig in vitro.

Stria vascularis from guinea pig cochleae was incubated in vitro to determine its metabolic response to variations in substrate and ion composition of the incubation medium. The respiratory rate at 37° in a medium containing glucose and pyruvate as substrate was 17.3 ± 1.33 (SEM, n = 51) μI O2/mg dry weight‐hour. The stria could not maintain constant respiration by relying solely upon endogenous fuel stores. With substrate supplied, the ATP level could be maintained at about 73% of that existing in vivo. Glucose appears to be an adequate substrate for stria in vitro since glutamate, pyruvate, and fumarate did not increase the respiratory rate. Succinate increased respiration markedly but did not increase the ATP level. Ouabain (10−4 M) caused a 48% decrease in the respiratory rate. Incubation in Na+‐free and K+‐free medium, each resulted in irreversible decrease of respiratory rate comparable to (or greater than) that caused by ouabain. These data are in accord with the high activity of Na+‐K+‐ATPase in the stria and the pronounced sensitivity of the endolymphatic potential to ouabain.

Response of cochlear potentials to presumed alterations of ionic conductance: Endolymphatic perfusion of barium, valinomycin and nystatin

Two models (single-pump and two-pump) of transepithelial potassium movement by the marginal cells of the stria vascularis have been proposed in the literature. Their validity was considered by exposing the endolymphatic (luminal) surface to agents (barium, valinomycin and nystatin) which are known to alter specific cellular membrane conductances in other tissues. This was accomplished by the use either of injections or of a relatively satisfactory technique for perfusion of scala media, which is described. Injection of barium caused the endocochlear potential (EP) to increase in normal animals and had no effect on the EP of deaf, Waltzing guinea pigs. Perfusion of the ionophores caused a decline in the EP in both normal and Waltzing guinea pigs. Only the two-pump model (Na/K-ATPase-mediated cation pump on the basolateral membrane and rheogenic K transporter at the luminal membrane) is consistent with the results. The cellular heterogeneity of the cochlear duct, however, introduces a measure of uncertainty into this interpretation.

Membrane transport parameters in frog corneal epithelium measured using impedance analysis techniques

SummaryActive Cl− transport in bullfrog corneal epithelium was studied using transepithelial impendance analysis methods, and direct-current (DC) measurements of membrane voltages and resistance ratios. The technique allows the estimation of the apical and basolateral membrane conductances, and the paracellular conductance, and does not rely on the use of membrane conductance-altering agents to obtain these measurements as was requisite in earlier DC equivalent-circuit analysis studies. In addition, the analysis results in estimates of the apical and basolateral membrane capacitances, and allows resolution of the paracellular conductance into properties of the tight junctions and lateral spaces. Membrane capacitances (proportional to areas) were used to estimate the specific conductances of the apical and basolateral membranes, as well as to evaluate coupling between the cell layers. We confirm results obtained from earlier studies: (1) apical membrane conductance is proportional to the rate of active Cl− transport and is, highly Cl− selective; (2) intracellular Cl− activity is above electrochemical equilibrium, thereby providing a net driving force for apical membrane Cl− exit; (3) the paracellular conductance is comparable to the transcellular conductance. We also found that: (1) the paracellular conductance is composed of the series combination of the junctional conductance and a nonnegligible lateral space resistance; (2) a small K+ conductance reported in the apical membrane may result from Cl− channels possessing a finite permeability to K+; (3) the basolateral membrane areas is 36 times greater than the apical membrane area which is consistent with the notion of electrical coupling between the five to six cell layers of the epithelium; (4) the specific conductance of the basolateral membrane is many times lower than that of the apical membrane; (5) the net transport of Cl− is modulated primarily by changes in the conductance of the apical membrane and not by changes in the net electrochemical gradient resulting from opposite changes in the electrical and chemical gradients; (6) the conductance of the basolateral membrane does not change with transport which implies that the net driving force for K+ exit increases with transport, possibly due to an increase in the intracellular K+ activity.

Comparison of the non-adrenergic action of phentolamine with that of vanadate on cochlear function.

Two drugs, which upon superficial examination appeared to be acting on common processes, have been found upon closer investigation to act by quite different means. Both act primarily at the organ of Corti, causing a pronounced increase of the endocochlear potential and a depression of the cochlear microphonic (CM). These effects are accompanied by the elimination of a negative component of the EP; however, it was found that these three effects are produced by phentolamine in scala media (or, more slowly, in scala tympani) but by vanadate only in scala tympani. This difference in locus of action isd manifested further by different changes of the summating potential (SP): phentolamine has little effect on the magnitude of SP-, while vanadate leads to an elevated SP-. In spite of this difference in the zeroth order harmonic, the second harmonic of the CM is depressed by both agents. It is argued that phentolamine may act either by blocking the acoustically-modulated ion channels in the luminal membranes of the hair cells or by inducing a large, non-selective, paracellular conductance in the organ of Corti. The present results, in conjunction with our previous results (Marcus, D.C., DeMott, J.E., Kobayashi, T., Ge, X.-X. and Thalmann, R. (1981): Hearing Res. 5, 231-243), are further interpreted as suggesting that vanadate may initially act by depolarizing the hair cells.

Specificity of action of vanadate to the organ of corti

Although vanadate strongly inhibits Na/K-ATPase activity of the stria vascularis in vitro, it initially causes no depression of the ouabain-sensitive endocochlear potential (EP) when perfused perilymphatically or via the vasculature. However, when the perilymph of scala tympani is replaced with artificial media containing 0.1 to 1 mM vanadate, there is a large (about 17 mV) increase in the EP of the second cochlear turn. Further experiments showed that the cochlear microphonics declined during the time in which the EP increased, and that the response of these two potentials to vanadate is greater in the second turn than in the first. Injection of 50 n1 of 1 mM vanadate (in artificial endolymph) into the endolymphatic space of the second turn caused no increase in the EP. These results support the notion that the early effects of vanadate are on the contra-luminal membranes of cells of the organ of Corti rather than on the stria vascularis. By superimposing anoxia or furosemide (i.v.) upon vanadate intoxication, we determined that the initial increase of the compound EP due to vanadate alone was due to a reduction in magnitude of the negative component of the EP. It is argued that of the three prevalent theories concerning the generation of the negative EP, the data tend to support the hypothesis that the intracellular potential of the hair cells gives rise to the negative EP.

Prolonged maintenance of endocochlear potential by vascular perfusion with media devoid of oxygen carriers.

SummaryA method is described for maintaining the cochlear potentials of the guinea pig via arterial perfusion of the surviving inner ear with an artificial medium devoid of oxygen carriers or oncotic agents. The endocochlear potential (EP) can be maintained at a normal level for periods in excess of 5 h; the responses of the EP to anoxia and to furosemide closely approximate those seen in the intact animal. This preparation may represent a simplified method for carrying out selected arterial perfusion experiments in the surviving inner ear.