Laser-drilled multichannel multidepth extracellular microelectrodes

The paper describes the construction and characteristics of a new multichannel, multidepth microelectrode designed for extracellular biopotential recording. The electrode is constructed from a group of six borosilicate glass capillary tubes which are hot drawn to form 40–50 μm tubes. A CO₂ laser of 400 kW cm⁻² peak power is used to cut side holes in the drawn tubes of 30–40 μm diameter and at intervals of 200 μm in respective tubes, thus creating six electrodes at six different depths. When filled with 9·9M NaCl solution typical electrode resistance is 300 kΩ and the interelectrode resistance 800 kΩ. Delay in publication due to temporary withdrawal of paper during patent application     

Faradic resistance of the electrode/electrolyte interface

A new method is used to measure the direct-current (Faradic) resistance of a single electrode/electrolyte interface. The method employs a constant-current pulse and a potential-sensing electrode. By choosing a sufficiently long pulse duration, the voltage between the test and potential-sensing electrode exhibits a three-phase response. In the steady-state phase, the voltage measured is equal to the current flowing through the electrode Faradic resistance and the resistance of the electrolyte between the test and potential-sensing electrode. By measuring this latter resistance with a high-frequency sinusoidal alternating current, the voltage drop in the electrolyte is calculated and subtracted from the voltage measured between the test and potential-sensing electrode, thereby allowing calculation of the Faradic resistance. By plotting the reciprocal of the Faradic resistance against current density and fitting the data points to a third-order polynomial, it is possible to determine the zero-current density (Faradic) resistance. This technique was used to determine the Faradic resistance of electrodes (0·1 cm²) of stainless-steel, platinum, platinum-iridium and rhodium in 0·9 per cent NaCl at 25°C. The zero current Faradic resistance is lowest for platinum (30·3 kΩ), slightly higher for platinum-iridium (47·6kΩ), much higher for rhodium (111 kΩ) and highest for type 316 stainless-steel (345 kΩ). In all cases, the Faradic resistance decreases dramatically with increasing current density.     

Urinary Ca2+ and the regulation of K+ secretion in toad bladder by neurohypophyseal hormones

We measured net K⁺ fluxes in the isolated toad urinary bladder to determine whether neurohypophyseal hormones control K⁺ secretion in this tissue. To determine if the pathway involved in K⁺ secretion is similar to a Ca²⁺-blockable alkali cation channel in the apical membrane of toad bladder, previously described in electrophysiological studies, we measured K⁺ fluxes in either the presence or absence of Ca²⁺ in the mucosal bathing solution. Oxytocin enhanced K⁺ secretion in both cases; however, the enhancement was markedly greater in the absence of mucosal Ca²⁺. In other experiments the transepithelial voltage was held constant at a value of 120 mV (mucosa negative) to find whether hyperpolarization would enhance K⁺ secretion to the levels seen in Ca²⁺-free solutions. The response to oxytocin was markedly greater in the absence of mucosal Ca²⁺ even when the transepithelial voltage was continuously hyperpolarized. These observations suggest that the properties of the activated pathway are akin to those of the previously described Ca²⁺-blockable alkali cation channel. We also found that toad bladder urine often contained extremely low levels of Ca²⁺; therefore neurohypophyseal hormonal control of K⁺ transport across the bladder may play an important role in amphibian K⁺ balance.     

Intracellular potentials of toad urinary bladder

Short-circuited urinary bladders of Bufo marinus were impaled with microelectrodes. Intracellular potentials in the order of -80 mV were recorded. On inhibition of apical Na entry they hyperpolarized by about 15 mV and the fractional resistance of the apical membrane increased to near 1.0. These patterns are similar to those of other tight Na transporting amphibian epithelia. On two occasions, stable recordings from a single cell were obtained before and after oxytocin. Intracellular potential and fractional resistance of the apical membrane remained constant despite doubling of Na transport and cellular conductance. This finding suggests that oxytocin stimulates conductive pathways in both, apical and basolateral cell membranes of the toad urinary bladder.     

Improved Ag/AgCl pressure electrodes

When electrodes are used to measure slowly varying signals such as the slow wave in the gastrointestinal tract (0·01 to 10 Hz) it is necessary that the impedances of the electrodes are low at these frequencies, and the drift must also be low. We have carried out a programme of (a) electrode surface pretreatment, (b) electrolytic AgCl deposition, (c) impedance measurements, (d) electrode storage methods and (e) noise and drift measurements. Optimal preplating treatment was found to be roughening of the cleaned Ag surface. The electrode impedance could be lowered by about an order of magnitude using 50 μm Al₂O₃ abrasive powder. The impedance measured at fixed frequencies (0·01 to 0·05 Hz etc) plotted against the plating product (mAs cm⁻²) showed a sharp minimum at 3 mAs cm⁻². This mimimum was very sharp at the very low frequencies. This indicates a critical dependence of impedance on the plating product. Storage of the electrodes in saturated AgCl solution was found to be better than any other method tried. Electrodes pretreated as above and optimally plated showed drift voltages of less than 1 μV min⁻¹ and noise voltages of less than 1 μV. The low drift allowed the use of d.c. coupling during slow wave measurement on intestinal smooth muscle. The waveform in this case approximated the intracellular one closely. The electrode impedance could be described accurately in the frequency range from 0·01 Hz to 1 kHz by the relationships R=1851×f^−0·347Ω and |X|=1190×f^−0·356Ω where f=frequency in Hertz.     

Electrophysiological analysis of sodium-transport in the colon of the frog (Rana esculenta)

Sodium transport and apical bioelectrical membrane properties were investigated in frog colonic epithelium in the absence and presence of the antidiuretic hormone arginine-vasotocin (AVT). Apical Na-permeability and intracellular Na-activity were evaluated by analysis of current-voltage relationships in the serosally K-depolarized tissue. Tissue- and apical membrane capacitance were measured by voltages step analysis. The frog colon was found to be a tight epithelium with a transepithelial resistance of 2.63±0.25 k·F (n=17). 85–90% of short circuit current (11.2±1.1 A·F·l^–1;n=17) was related to electrogenic Na-transport from mucosa to serosa. Graded doses of amiloride (<50 mol·l^–1) induced Michaelis-Menten-type inhibition kinetics. Serosal addition of 10^–6 mol·l^–1 AVT induced a significant increase in sodium current (25%), apical sodium permeability (19%) and tissue capacitance (4.3%) whereas intracellular Na-activity remained unchanged. There was a good correlation between increased Na-current and apical Na-permeability. No correlation was found between Na-current and membrane capacitance. Our results demonstrate that in contrast to other species the amphibian colon shows a natriferic reaction to AVT. We suggest that the regulation of Na-transport in frog colon is similar to that in the toad urinary bladder. It is caused by an activation of preexisting apical Na-channels and not by fusion of subapical cytoplasmic vesicles with the apical membrane.     

The effects of adenosine on transepithelial resistance and sodium uptake in the inner medullary collecting duct

It has been previously demonstrated that adenosine induces natriuresis when administered directly into the renal circulation of the rat. It was postulated that the mechanism was inhibition of tubule Na⁺ reabsorption. In the current study, the hypothesis was tested that adenosine inhibits ion reabsorption across the inner medullary collecting duct (IMCD), a tubule segment which is rich in adenosine receptors. IMCD epithelium from rat kidney was grown in primary culture as a confluent monolayer on Costar filters, allowing selective access to the basolateral and apical surfaces of the cells. Transepithelial resistance was taken as a measure of epithelial permeability and ion conductance. Na⁺ uptake was studied using ²²Na⁺ and used to determine the permeability of the epithelial monolayer specifically to Na⁺. Exposure of the basolateral aspect of the monolayer to adenosine (10⁻⁸–10⁻⁷ M) increased transepithelial resistance in a dose- and time-dependent manner; in parallel, adenosine (10⁻⁷–10⁻⁶ M) reduced apical Na⁺ uptake from 20±5 to 10±2 nmol/cm². 1,3-Dipropyl-8-(2-amino-4-chlorophenyl)-xanthine (PACPX, 5×10⁻⁹ M), an adenosine antagonist with selectivity for the A₁ receptor, inhibited the rise in transepithelial resistance and the decrease in Na⁺ uptake following the addition of adenosine. The effects of adenosine on transepithelial resistance were reproduced with the A₁ receptor selective adenosine analogue N ⁶-cyclohexyladenosine (CHA, 10⁻⁸ –10⁻⁷ M) but not with the A₂ selective analogues, 5′-N-ethylcarboxamidoadenosine (NECA) or CGS 21680. CHA (10⁻⁷ M) inhibited apical Na⁺ uptake by 50%, an effect abolished by PACPX. The effects of adenosine on transepithelial resistance and Na⁺ uptake were inhibited, but only in part, by amiloride. These data suggest that adenosine inhibits ion movement, specifically apical Na⁺ uptake, across the IMCD epithelium and that this effect is mediated by A₁ receptors from the basolateral aspect of the cell. The results are consistent with the hypothesis that adenosine inhibits Na⁺ reabsorption across the IMCD.     

cAMP increases the basolateral Cl−-conductance in the isolated perfused medullary thick ascending limb of Henle's loop of the mouse

The effect of cAMP on transepithelial and transmembrane potential differences and resistances was examined in isolated in vitro perfused mouse medullary thick ascending limbs of Henle's loop (mTAL). The effects of furosemide and barium were tested. Stimulation of NaCl transport by ADH 10^–9+dbcAMP 4·10^–4+forskolin 10^–6 mol·l^–1 (paired experiments) resulted in: a) an increase in transepithelial potential difference, referenced to the grounded bath, from +6.7±0.3 mV to +12.0±0.4 mV (n=47); b) a decrease in transepithelial resistance from 25±1 cm² to 20±1 cm² (n=47); c) a depolarization of the basolateral membrane by 12 mV and of the apical membrane by 7 mV (n=36); d) a decrease in the fractional resistance of the basolateral membrane from 0.27±0.005 to 0.15±0.06 (n=12). Furosemide (10^–4 mol·l^–1) abolished the active transepithelial transport potential and hyperpolarized the basolateral membrane potential to values which were similar in both control and cAMP treated mTAL segments. Barium increased the transepithelial resistance and depolarizedPD _bl to similar values in both functional states. An increase in the fractional conductance of the basolateral membrane was also seen, if, prior to the cAMP treatment, the luminal Na⁺2Cl^–K⁺ contransport was inhibited by furosemide. Thus, we propose that stimulation of active NaCl reabsorption in the mTAL segment of the mouse by ADH, mediated via cAMP, increases primarily the basolateral chloride conductance.Supported by Deutsche Forschungsgemeinschaft Gr 480/6-2Parts of this study have been presented at the 59th Meeting of the German Physiological Society in Dortmund 1984 and at the 69th FASEB Meeting in Anaheim 1985     

Cytotoxic effect of choline,abolished by furosemide,in the diluting segment of frog kidney

Previous observations suggest that luminal application of tetra-N-alkylammonium ions may impair ion transport in the amphibian diluting segment. To investigate this question conventional KCl-filled and Cl⁻ sensitive microelectrodes were applied in diluting segments of the isolated perfused kidney ofrana esculenta to evaluate transepithelial electrical and chloride electrochemical (PD_te, E _te ^Cl ) as well as peritubular cell membrane potential difference (PD_pt), measured at static head conditions. After determination of control values the tubule lumen was exposed to choline (95 mmol/l, substituted for Na⁺) both in presence or absence of furosemide (5×10⁻⁵ mol/l). Then, the lumen was again perfused with control solution and the measurements were repeated. Thus, a time course for possible choline induced effects was obtained both in the presence and absence of furosemide. The lumen positive PD_te decreased from 11.2±1.0 mV to 6.3±0.8 mV after 2 min and to 1.9±0.4 mV after 30 min exposure to choline. PD_pt (cell interior negative) decreased from 70±2 mV to 58±3 mV and to 42±5 mV after 2 and 30 min, respectively. Intraluminal Cl⁻ activity increased from its initial steady state value of 20±2 mol/l to 39±2 mmol/l after 30 min exposure to choline. However, if the tubule lumen was exposed to choline. in presence of furosemide (5×10⁻⁵ mol/l), all the above described choline-induced effects did not become apparent. The data are consistent with the hypothesis that choline traverses the cell membrane via the furosemide-sensitive cotransport system, accumulates in the cell cytosol, blocks luminal K⁺ conductance and by this means inhibits transepithelial ion transport. Thus, longterm exposure of furosemide-sensitive epithelia to choline and its derivatives should be avoided.     

Distal colonic Na+ absorption inhibited by luminal P2Y2 receptors

Luminal P2 receptors are ubiquitously expressed in transporting epithelia. In steroid-sensitive epithelia (e.g., lung, distal nephron) epithelial Na⁺ channel (ENaC)-mediated Na⁺ absorption is inhibited via luminal P2 receptors. In distal mouse colon, we have identified that both, a luminal P2Y₂ and a luminal P2Y₄ receptor, stimulate K⁺ secretion. In this study, we investigate the effect of luminal adenosine triphosphate/uridine triphosphate (ATP/UTP) on electrogenic Na⁺ absorption in distal colonic mucosa of mice treated on a low Na⁺ diet for more than 2 weeks. Transepithelial electrical parameters were recorded in an Ussing chamber. Baseline parameters: transepithelial voltage (V _te): −13.7 ± 1.9 mV (lumen negative), transepithelial resistance (R _te): 24.1 ± 1.8 Ω cm², equivalent short circuit current (I _sc): −563.9 ± 63.8 μA/cm² (n = 21). Amiloride completely inhibited I _sc to −0.5 ± 8.5 μA/cm². Luminal ATP induced a slowly on-setting and persistent inhibition of the amiloride-sensitive I _sc by 160.7 ± 29.7 μA/cm² (n = 12, NMRI mice). Luminal ATP and UTP were almost equipotent with IC₅₀ values of 10 μM and 3 μM respectively. In P2Y₂ knock-out (KO) mice, the effect of luminal UTP on amiloride-sensitve Na⁺ absorption was absent. In contrast, in P2Y₄ KO mice the inhibitory effect of luminal UTP on Na⁺ absorption remained present. Semiquantitative polymerase chain reaction did not indicate regulation of the P2Y receptors under low Na⁺ diet, but it revealed a pronounced axial expression of both receptors with highest abundance in surface epithelia. Thus, luminal P2Y₂ and P2Y₄ receptors and ENaC channels co-localize in surface epithelium. Intriguingly, only the stimulation of the P2Y₂ receptor mediates inhibition of electrogenic Na⁺ absorption.     

Dynamic model of the short-term regulation of arterial pressure in the cat

The purpose of this study was to characterise the dynamics of the short-term control of arterial pressure in the cat with the aid of a model consisting of a nonlinear negative-feedback control system. The arterial system was described by a three element windkessel model (peripheral resistance, R, aortic characteristic impedance, R_c, and total arterial compliance, C). The resistance regulation was represented by a second-order system with static gain G_R, a damping factor σ and an undamped natural frequency ω_n. The resistance gain, G_R, and the windkessel parameters were obtained from measurements of aortic and venous pressures and cardiac output in two steady states. The parameters σ and ω_n were estimated from mean pressure and mean flow during the transient from control to the new steady state. Pressure reductions averaged 10 per cent and resistance changes averaged 12 per cent. Average windkessel model parameters in the control condition were: C=(25·9±6·1) 10⁻⁶ g⁻¹ cm⁴ s², R_c=(2·51±0·53) 10³ g cm⁻⁴ s⁻¹, R=(40·9±9·8) 10³ g cm⁻⁴ s⁻¹. Average estimates of parameters of the resistance regulator were: G_R=(4·14±2·38) 10⁻³ min ml⁻¹, ω_n = 1·0 ± 1·0 rad s⁻¹, σ=0·41±0·19. A satisfactory fit was found between model predicted and measured pressure. The results suggest that the dynamic short-term control of pressure is underdamped and oscillatory. The amplitude of these oscillations is affected by arterial compliance, suggesting an interaction between the arterial system and short-term resistance regulation.     

Electrophysiological studies in principal cells of rat cortical collecting tubules ADH increases the apical membrane Na+-conductance

The mechanism of ion transport across principal cells of rat cortical collecting tubules (CCT) and its regulation by vasopressin (ADH) has been studied in the isolated perfused tubule. To amplify the response to ADH rats were treated with 5 mg I. M. desoxycorticosterone 4–9 days prior to the experiments. Addition of 2·10^–10 mol·1^–1 ADH increased the transepithelial voltage from –5.1 ±0.7 mV to –16.1±1.4 mV (n=37) and decreased the transepithelial resistance from 51±4 cm² to 39±2 cm² (n=33). Optical and functional differentiation of impalements of principal and intercalated cells was made and only data of principal cells are presented. ADH depolarized the apical membrane from 79±1 mV to 66±2 mV (n=26) and decreased the fractional resistance of the apical membrane from 0.76±0.04 to 0.70±0.04 (n=13). These ADH effects were prevented by 10^–5 or 10^–4 mol·1^–1 luminal amiloride which hyperpolarized the apical membrane when added in the presence or absence of ADH. Apical and basolateral membranes were dominated by large K⁺ conductances and addition of 3 mmol·1^–1 barium to bath or lumen perfusates increased transepithelial resistance almost two-fold, whereas luminal amiloride increased the transepithelial resistance only by 26–35%. Ouabain (0.5 mmol·1^–1, bath) depolarized the basolateral membrane and decreased its K⁺ conductance. These effects were prevented by the simultaneous presence of apical amiloride suggesting that the only route of Na⁺ entry into the principal cells occurred via the amiloride sensitive Na⁺ conductance. We conclude that ADH stimulates Na⁺ reabsorption and K⁺ secretion in the rat CCT primarily by increasing the Na⁺ conductance in the apical cell membrane.Parts of this study have been presented at the 19th ASN meeting in Washington, DC, USA 1986     

Electrical properties of amphibian urinary bladder epithelia

Summary Necturus urinary bladders were mounted in a two-compartment chamber filled with Ringer's solution and individual cells were punctured with microelectrodes from the serosal surface. By measuring the changes of transepithelial resistance and potential in response to luminal application of amiloride, and measuring the effects of amiloride on the voltage divider ratio and on the serosal cell membrane potential, two independent methods were obtained to calculate the cell membrane resistances and the resistance of the shunt path. The results from both methods agreed favourably. Th mucosal and serosal membrane resistances were found to vary respectively from 9–65 k cm² and from 1–7 k cm² under control conditions. Significant correlations were observed between the luminal membrane resistance as well as the luminal membrane zero current potential and the short circuit current, which indicate that the luminal membrane conductance consists predominantly of Na⁺-selective elements, whose density varies from bladder to bladder and determines the rate of transpithelial Na⁺ transport. Amiloride which blocks these elements increased the luminal resistance to 220 k cm². Increasing short circuit current was also correlated with increasing conductance of the serosal membrane (at fairly constant zero current potential) which points to an electrogenic mechanism of active Na⁺ transport across the serosal cell surface. The paracellular shunt path across the terminal bars plays a minor role in urinary bladder epithelium. Its resistance was estimated to be in the range of 50–125 k cm² or greater.     

Electrolyte transport across the rabbit caecum in vitro

Electrolyte transport across rabbit caecal epithelium was investigated in vitro using conventional shortcircuiting and radioisotope techniques. In standard saline the caecum exhibited a relatively high short-circuit current (I _sc=4.4 μEq · cm⁻² · h⁻¹) and conductance (6.43 mS · cm⁻²). Both sodium and chloride were absorbed (J _net ^Na =6.40 andJ _net ^Cl =3.40 μEq · cm⁻² · h⁻¹) and potassium was secreted (J _net ^K =−0.5 μEq · cm⁻² · h⁻¹). Removal of Na⁺ abolishedI _sc andJ _net ^Cl whereas removal of Cl⁻ reducedJ _net ^Na to 2.92 μEq · cm⁻² · h⁻¹ but did not alterI _sc. In HCO ₃ ⁻ free salines containing 10⁻⁴ M acetazolamideJ _net ^Cl was abolished andJ _net ^Na andI _sc were reduced to 2.3 and 2.5 μEq · cm⁻² · h⁻¹ respectively. A positive residual ion flux (∼ 1 μEq · cm⁻² · h⁻¹) was detected in standard and Cl⁻-free salines but not in Na⁺-free or HCO ₃ ⁻ buffers. Mucosal amiloride (10⁻³ M) decreased net Na⁺ and Cl⁻ absorption but did not decreaseI _sc. Mucosal DIDS (10⁻⁴ M) decreasedJ _net ^Cl while mucosal bumetanide (10⁻⁴ M) did not affect any of the measured parameters. Finally, addition of theophylline (8 mM) stimulated Cl⁻ secretion and increasedI _sc. It is concluded that net sodium absorption by caecal epithelia occurs by both electrogenic and electroneutral mechanisms whereas net chloride absorption occurs only by an electroneutral process. Coupling of the absorptive fluxes of Na⁺ and Cl⁻ may result from Na⁺/H⁺ and Cl⁻/HCO ₃ ⁻ antiport systems in this tissue. Finally, it is proposed that up to half of theI _sc is due to a Na⁺-dependent secretion of bicarbonate ion.     

Vestibular semicircular canal epithelium of the rat in culture on filter support: polarity and barrier properties

 The inner ear of mammals contains the vestibular apparatus which is involved in the maintenance of posture and balance. The tubular structure of the apparatus is bathed by the potassium-rich endolymph and sodium-rich perilymph in the luminal and abluminal compartments, respectively. The luminal compartment is lined by a continuous epithelium with islets of receptor organs, which separates the luminal from the abluminal compartment. The present work focuses on the epithelium, without the receptor organs, and shows that it can be reconstituted in culture. The epithelium from 4-day-old Wistar rats was grown on microporous membranes. High transepithelial electrical resistances (4000–6000 Ω·cm²)were achieved after 4–8 days in culture. The epithelium was characterized by the presence of cytokeratin, ZO-1 protein, occludin, and the presence of tight junctions and kinocilia. The transepithelial resistance of the cell monolayer withstood endolymph/perilymph dual bathing when the apical pole of the cells was in contact with endolymph, but collapsed in the reverse configuration. Weak but statistically highly significant basal to apical rubidium (⁸⁶Rb) transport was observed. These findings show that this epithelium maintains its in vivo polarity and could enhance the potassium composition of endolymph up to maturity. This new culture model, in which dual bathing is possible, should enable further in vitro studies of the sensory vestibular epithelia. Received: 19 July 1998 / Received after revision: 10 December 1998 / Accepted: 18 December 1998     

Effects of prolonged high-altitude exposure on peripheral adrenergic receptors in young healthy volunteers

The regulation of adrenergic receptors during hypoxia is complex, and the results of published reports have not been consistent. In the present study blood cell adrenoceptor characteristics at sea level (SL) before and after prolonged exposure to high altitude (HA) were measured in seven trained young male lowlanders. Sympathoadrenal activity and clinical haemodynamic parameters were also evaluated before departure (SLB), after 1 week (HA1) and 4 weeks (HA4) at HA and 1 week after return to sea level (SLA). As compared to pre-departure sea level values, urinary norepinephrine excretion increased significantly during altitude exposure [SLB: 10.26 (3.04) μg · 3 h⁻¹; HA1: 23.2 (4.19) μg · 3 h⁻¹; HA4: 20.3 (8.68) μg · 3 h⁻¹] and fell to pre-ascent values 1 week after return to sea level [SLA: 9 (2.91) μg · 3 h⁻¹]. In contrast, mean urinary epinephrine levels did not increase over time at HA. Both systolic and diastolic blood pressures, as well as heart rate, were increased during HA exposure. The circadian blood pressure and heart rate rhythms were preserved during all phases of altitude exposure. Mean maximal binding (B _max) of the α₂-adrenoceptor antagonist [³H]rauwolscine to platelet membranes was significantly reduced (P < 0.001) after exposure to chronic hypoxia [SLB: 172.6 (48.5) fmol · mg⁻¹ protein versus SLA: 136.8 (56.1) fmol · mg⁻¹ protein] without change in the dissociation constant (K _D). Neither the lymphomonocyte β₂-adrenoceptor B _max [SLB: 38.5 (13.6) fmol · mg⁻¹ protein, versus SLA: 32.4 (12.1) fmol · mg⁻¹ protein] nor the K _D for [³H]dihydroalprenolol was affected by chronic hypoxia. Cyclic AMP (adenosine 3′,5′-cyclic monophoshate) generation in lymphomonocytes by maximal isoproterenol stimulation was not modified after prolonged HA exposure. In conclusion, the down-regulation of α₂-adrenoceptors appears to be an important component of the adrenergic system response to HA exposure. Accepted: 20 April 2000     

Patch clamp study on primary culture of isolated proximal convoluted tubules

Primary cultures were obtained from microdissected rabbit proximal tubules (S₁ segments). The growing epithelia were maintained in culture for up to 30 days. Electron microscopy study revealed that the cells formed a monolayer and showed a morphological polarity with apical microvilli and tight junctions. An immunofluorescence technique using two monoclonal antibodies raised against two apical brush border enzymes of the proximal tubule (LAP, DPP IV) revealed that these enzymes were expressed in the cultured cells. Membrane associated and cytosolic enzyme activities were measured on 12, 20 and 30-day-old cultures. Cultured epithelia exhibited leucine aminopeptidase, glutamyl transferase and fructose 1–6 biphosphatase activities that remained constant for up to 30 days, whereas alkaline phosphatase activity decreased in the oldest cultures. Hexokinase activity on the other hand, increased after 12 days of culture. Cyclic AMP synthesis was stimulated by parathyroid hormone at 12, 20 and 30 days of culture and was insensitive to arginine vasopressin. After 20 days of culture the epithelia grown on permeable supports developed a transepithelial potential of –0.13 mV (apical negative) and a transepithelial resistance of 37 cm² that increased to –1.13 mV and 60 cm² respectively in 30-day-old cultures. The patch clamp technique was applied to the apical membrane of 12–15-day-old cultures. In the whole cell recording configuration, a cellular potential of –61.5 mV was measured, which was mainly due to K⁺ diffusion. A non-selective cationic channel was present in the apical membrane of the cultured cells. In cell-attached patches the channel carried an inward current and had a conductance of 13 pS. On excised patches the channel discriminated poorly between Na⁺ and K⁺ and was impermeant to Cl^– and its conductance ranged between 20 and 28 pS. The channel activity was not voltage dependent but required a high calcium concentration (1 mM Ca²⁺) on the cytoplasmic face.     

A reappraisal of the Ca<Superscript>2+</Superscript> dependence of fast inactivation of Ca<Superscript>2+</Superscript> release in frog skeletal muscle

Two drugs, 2-APB and SKF-96365, commonly used to block Store Operated Ca²⁺ Entry (SOCE) were found to have inhibitory effects at different levels of the Excitation Contraction Coupling (ECC) process in frog skeletal muscle fibers. Treatment with either drug suppressed Ca²⁺ release from the Sarcoplasmic Reticulum, but this effect was not due to inhibition of SOCE as it occurred in Ca²⁺-free conditions. 2-APB applied extracellularly at 100 μM, the usual concentration to suppress SOCE, reversibly reduced the charge movement elicited by pulses in the range between −45 and −35 mV from 7.99 ± 0.73 nC/μF (N = 17) before drug application to 6.27 ± 0.68 nC/μF in the presence of 2-APB. This effect was mostly on the delayed Q_γ component. In fibers treated with the SERCA ATPase inhibitor CPA the Q_γ component disappeared, under this condition the application of 2-APB did not suppress the remaining charge movement. Thus the effect of 2-APB on charge movement currents seemed to be secondary to the suppression of Ca²⁺ release, likely occurring directly on the release channels. No significant suppression of ECC was observed for concentration below 20 μM. 2-APB also inhibited the L-type Ca²⁺ current (20 ± 4%, N = 8). On the other hand SKF-96365 had a direct effect on the voltage sensor promoting its voltage dependent inactivation. Applied at 20 μM, a typical concentration used for inhibiting SOCE, to fibers held at −80 mV inhibited the charge moved in response to pulses ranging −45 to −30 mV from 7.95 ± 2.59 nC/μF to 3.48 ± 0.9 nC/μF (N = 12). A parallel reduction of Ca²⁺ release was observed. Wash out was drastically increased by hyperpolarization of the holding potential to −100 mV. SKF-96365 also inhibited the L-type Ca²⁺ current (41 ± 8%, N = 4) and increased its rate of inactivation.     

Electrophysiological characterization of rabbit distal convoluted tubule cell

The distal convoluted tubule (DCT) from rabbit kidney were perfused in vitro to study the conductive properties of the cell membranes by using electrophysiological methods. When the lumen and the bath were perfused with a biearbonate free solution buffered with HEPES, the transepithelial voltage (V _T) averaged –2.8±0.6 mV (n=20), lumen negative. The basolateral membrane voltage (V _B) averaged –77.8±1.1 mV (n=33) obtained by intracellular impalement of microelectrodes. Cable analysis performed by injecting a current from perfusion pipette revealed that the transepithelial resistance was 21.8±1.7 ·cm² and the fractional resistance of the luminal membrane was 0.78±0.03 (n=8), indicating the existence of ionic conductances in the luminal membrane. Addition of amiloride (10^–5 mol/l) to the luminal perfusate or Na⁺ removal from the lumen abolished the lumen negativeV _T and hyperpolarized the apical membrane. An increase in luminal K⁺ concentration from 5 to 50 mmol/l reduced the apical membrane potential (V _A) by 37.5±2.6 mV (n=7), whereas a reduction of Cl^– in the luminal perfusate did not changeV _A significantly (0.5±0.5 mV,n=4). Addition of Ba²⁺ to the lumen reducedV _A by 42.6±1.0 mV (n=4). When the bathing fluid was perfused with 50 mmol/l K⁺ solution, the basolateral membrane voltage (V _B) fell from –76.8±1.5 to –31.0±1.3 mV (n=18), and addition of Ba²⁺ to the bath reducedV _B by 18.3±4.8 mV (n=7). Although a reduction of Cl^– in the bathing fluid from 143 to 5 mmol/l did not cause any significant fast initial depolarization (1.8±1.7 mV,n=8), a spike like depolarization (14.0±2.5 mV,n=4) was observed, upon Cl^– reduction in the presence of Ba²⁺ in the bath. From these results, we conclude that the apical membrane of DCT has both K⁺ and Na⁺ conductances and the basolateral membrane has a K⁺ conductance and a small Cl^– conductance.     

Effects of antidiuretic hormone,parathyroid hormone and glucagon on transepithelial voltage and resistance of the cortical and medullary thick ascending limb of Henle's loop of the mouse nephron

The effect of antidiuretic hormone (arginine vasopressin, AVP, 10⁻¹⁰mol.l⁻¹), parathyroid hormone (PTH, 10⁻⁸ mol.l⁻⁸) and glucagon (10⁻⁸ mol.l⁻¹) on the transepithelial potential difference (PD_te) and the transepithelial resistance (R_te) were tested in in vitro perfused cortical (cTAL) and medullary (mTAL) thick ascending limbs of Henle's loop of the mouse nephron. When compared with mTAL segments (PD_te: 8.5±0.4 mV,n=16), cTAL segments displayed a high PD_te of 15.7±0.9 mV (n=11) at the beginning of perfusion experiments which reached a value of 9.4±0.6 mV (n =11) after 38±4 min perfusion. Simultaneously R_te increased significantly from 24±3 to 28±1 Ω cm² (n=11). When PTH, AVP or glucagon were added to the bath solution, PD_te increased with PTH from 10.3±0.8 to 15.2±0.8 mV (n=13), with AVP from 10.2±0.5 to 15.0±0.7 mV (n=24) and with glucagon from 11.3±1.9 to 15.3±2.1 mV (n=8). At the same time R_te decreased from 30±3 to 23±2 Ω cm², from 28±1 to 23±1 Ω cm² and from 23±2 to 18±2 Ω cm², respectively. In mTAL segments, AVP and glucagon increased PD_te from 8.4+0.5 to 13.5±0.9 mV (n=11) and from 8.8±0.6 to 12.8±0.6 mV (n=8) respectively, while R_te decreased significantly from 23±1 to 20±1 Ω cm² and from 27±3 to 21±3 Ω cm². PTH, on the other hand, had no effect on PD_te and R_te. Since the response to PTH appeared to be specific to cTAL segments, paired experiments were performed, in which AVP or glucagon were successively tested with PTH on cTAL and mTAL segments, to ascertain the specificity of the hormonal response. In cTAL segments, PTH and AVP increased the equivalent short-circuit current (I_sc=PD_te/R_te) by 82% and 86% respectively, while PTH and glucagon, in another series, increased I_sc by 95% and 81% respectively. In mTAL segments, I_sc was increased in the presence of AVP and glucagon by 88%, and 93% respectively, whereas PTH had no effect. These results indicate that Nacl reabsorption in cTAL segments is stimulated by AVP, PTH and glucagon and in mTAL segments by AVP and glucagon. The amplitude of the response to the hormones is similar in the two segments. The residual stimulation in cTAL segments, however, persists longer than in mTAL segments.