Membrane potential measurements of transitional cells from the crista ampullaris of the Gerbil

Transitional cells of the crista ampullaris were impaled with microelectrodes in order to record the membrane potential (PD) and to investigate membrane properties. In control solution the PD was –87±1 mV (n=103). This value is not significantly different from –83±2 mV (n=24) measured in Cl^– free solution. [Cl^–] steps from 150 to 15 mmol/l (n=24) depolarized the membrane by about 2 mV, indicating a minor Cl^– conductance. The transference number for K⁺ was 0.75±0.01 (n=79) obtained from the PD responses to K⁺ steps from 3.6 to 25 mmol/l. The cell membrane depolarized and the amplitude of PD responses to [K⁺] steps was reduced by Ba²⁺ (2·10^–6 to 10^–3 mol/l), quinidine (10^–3 mol/l), quinine (10^–3 mol/l), Rb⁺ (20 mmol/l), Cs⁺ (20 mmol/l), NH₄ ⁺ (20 mmol/l) and Tl⁺ (0.5 mmol/l), whereas tetraethylammonium (TEA, 20 mmol/l) had no effect. The dose-response curve for Ba²⁺ in the presence of 3.6 mmol/l K⁺ was shifted to the right by approximately three decades in the presence of 25 mmol/l K⁺ and by a factor of about 4 in the presence of 135 mmol/l gluconate as a substitute for Cl^–. Transitional cells were depolarized by ouabain, suggesting the presence of (Na⁺+K⁺-ATPase.This work was supported by grants from the Deafness Research Foundation to PhW and the National Institute of Health (NS 19490) to DCM     

Mechanisms of calcium transport across the basolateral membrane of the rabbit cortical thick ascending limb of Henle's loop

Although net Ca²⁺ absorption takes place in the thick ascending limb of Henle's loop, detailed mechanisms are unknown. Because it has been reported that the Ca²⁺ entry step across the luminal membrane is mediated by Ca²⁺ channels inserted by stimulation with parathyroid hormone, we studied the mechanism of Ca²⁺ transport across the basolateral membrane of rabbit cortical thick ascending limb (CTAL) perfused in vitro by using microscopic fluorometry of cytosolic Ca²⁺ ([Ca²⁺]_i) with fura-2. The resting [Ca²⁺]_i in this segment was 49.8±4.5 nmol/l. Neither Na⁺ removal from the bathing solution nor addition of ouabain (0.1 mmol/l) to the bath increased [Ca²⁺]_i, indicating that a Na⁺/Ca²⁺ exchanger in the basolateral membrane may not contribute in any major way to [Ca²⁺]_i of CTAL. To confirm our technical accuracy, similar protocols were conducted in the connecting tubule, where the existence of a Na⁺/Ca²⁺ exchanger has been reported. In this segment, Na⁺ removal from the bath increased cell Ca²⁺ from 148.6 ±6.4 nmol/l to 647.6±132.0 nmol/l, confirming the documented fact. [Ca²⁺]_i in the CTAL was markedly increased when 1 mmol/l NaCN was added to the bath in the absence of glucose. Calmodulin inhibitors (trifluoperazine or W-7) increased [Ca²⁺]_i. When the bath pH was made alkaline, [Ca²⁺]_i was also increased. This response was abolished when Ca²⁺ was eliminated from the bath, indicating that the Ca²⁺ entry across the basolateral membrane is dependent on bath pH. Increase in [Ca²⁺]_i induced by an alkaline bath was inhibited by increased the bath K⁺ from 5 nmol/l to 50 mmol/l, suggesting that the Ca²⁺ entry system is voltage-dependent. However, the pH-dependent [Ca²⁺]_i increase was unaffected by 0.1–10 mol/l nicardipine in the bath. We conclude that Ca²⁺ transport across the basolateral membrane of CTAL is mediated by a pump-and-leak system of Ca²⁺ rather than a Na⁺/Ca²⁺ exchanger secondarily linked to a Na⁺, K⁺ pump.     

A kinetic analysis of the facilitatory action of adrenaline

The²²Na⁺-efflux from skeletal muscle cells of frog (Rana nigromaculata) was measured in Ringer solutions containing different concentrations of K⁺ (0.1 to 30 mM). The effects of adrenaline (30 M) and ouabain (10 M) on the²²Na⁺-efflux were investigated for the purpose to clarify the mechanism of the facilitatory effect of adrenaline on Na⁺–K⁺ pump. The rate coefficient for the ouabain-sensitive²²Na⁺-efflux increases with increasing extracellular K⁺ concentrations and adrenaline potently facilitates these rate coefficients. On the basis of Michaelis-Menten type kinetics assumed for the reaction between pump site and extracellular K⁺, it is concluded that adrenaline decreases the dissociation constant (K_m), and increases the maximum Na⁺-efflux.This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.     

Potassium channels in the basolateral membrane of the rectal gland ofSqualus acanthias

The present study examines the influences of pH and Ca²⁺ and several putative inhibitors on the basolateral K⁺ channel of the rectal gland ofSqualus acanthias. Excised membrane patches were examined using the patch clamp technique. It is shown that reduction of the calcium activity on the cytosolic side to less than 10^–9 mol/l has no detectable inhibitory effect on this channel. Conversely, increase in calcium activity to some 10^–3 mol/l reduced the activity of this channel. Variations in cytosolic pH had only a moderate effect on the current amplitude: alkalosis by one pH unit increased and acidosis reduced the single current amplitude by some 15%. Several inhibitors were tested in excised patches when added to the cytosolic side. Ba²⁺ (5·10^–3 mol/l), quinine (10^–3 mol/l), quinidine (10^–4 mol/l), lidocaine (1 mmol/l), tetraethylammonium (10 mmol/l), Cs⁺ (10 mmol/l), and Rb⁺ (20 mmol/l) all blocked this K⁺ channel reversibly. We conclude that the basolateral K⁺ channel of the rectal gland is distinct from other epithelial K⁺ channels inasmuch as it is not stimulated by Ca²⁺ directly, but that it is qualitatively similar to many other known K⁺ channels with respect to its sensitivity towards blockers.This study was supported by Deutsche Forschungsgemeinschaft Gr 480/8 and by NSF and NIH grants to the Mount Desert Island Biological Laboratory     

Extracellular free calcium and potassium during paroxysmal activity in the cerebral cortex of the cat

Summary Extracellular calcium and potassium activities (a_Ca and a_K) as well as neuronal activity were simultaneously recorded with ion-sensitive electrodes in the somatosensory cortex of cats. Baseline a_Ca was 1.2–1.5 mM/1, baseline a _k 2.7–3.2 mM/1. Transient decreases in a_Ca and simultaneous increases in a_K were evoked by repetitive stimulation of the contralateral forepaw, the nucleus ventroposterolateralis thalami and the cortical surface. Considerable decreases in a_Ca (by up to 0.7 mM/1) were found during seizure activity. A fall in a_Ca preceded the onset of paroxysmal discharges and the rise in a_K after injection of pentylene tetrazol. The decrease in a_Ca led also the rise in a_K during cyclical spike driving in a penicillin focus. It is concluded that alterations of Ca⁺⁺ dependent mechanisms participate in the generation of epileptic activity.     

Mechanisms of chloride influx during KCl-induced swelling in the chicken retina

An increase in extracellular KCl ([KCl]_o) occurs under various pathological conditions in the retina, leading to retinal swelling and possible neuronal damage. The mechanisms of this KCl_o-induced retinal swelling were investigated in the present study, with emphasis on the Cl^– transport mechanisms. Increasing [KCl]_o (from 5 to 70 mM) led to concentration-dependent swelling in chicken retinas. The curve relating the degree of swelling to [KCl]_o was biphasic, with one component from 5 to 35 mM [KCl]_o and another from 35 to 100 mM. As Cl^– omission prevented swelling in all conditions, the effect of cotransporter or Cl^– channel blockers was examined to investigate the mechanisms of Cl^– influx. The cotransporter blockers bumetanide and DIOA reduced swelling by 68% and 76%, respectively at [KCl]_o 25 mM (K25), and by 14–17% at [KCl]_o 54 mM (K54). The Cl^– channel blockers NPPB and niflumic acid did not affect swelling at K25 but reduced it by 90–95% at K54 (NPPB IC₅₀ 60.7 µM). Furosemide showed an atypical effect, decreasing swelling by 14% at K25 and by 95% at K54 (IC₅₀ 173.9 µM). Na⁺ omission decreased swelling at K25 but not at K54. These results suggest the contribution of cotransporters to Cl^– influx at K25 and of Cl^– channels at K54. At K25, swelling was found in the ganglion cell layer and in the lower half of the inner nuclear layer. With K54, swelling occurred in all inner retinal layers. The ganglion cell layer swelling was due to both Müller cell end-foot and ganglion cell soma swelling. K54 also induced ganglion cell damage as shown by disorganized, pyknotic and refringent nuclei.     

Molecular origin of Na/Li exchanger: Evidence against the involvement of major cloned erythrocyte transporters

Numerous studies have demonstrated heightened Na⁺/Li⁺ countertransport (NLCT) activity in erythrocytes of patients with essential hypertension or diabetic nephropathy. The same carrier also contributes to the therapeutic action of lithium salt, widely used in the treatment of psychiatric disorders. However, the molecular origin of NLCT remains unknown. This study examined the role of major ion transporters in NLCT by comparative analysis of its activity and that of ion transporters providing inwardly directed ⁸⁶Rb, ²²Na and ³²P fluxes. NLCT was below the detection limit in rat erythrocytes and ∼50-fold higher in rabbits compared to humans. Unlike NLCT, the activities of Na⁺,K⁺-ATPase, Na⁺,K⁺,2Cl⁻ cotransporter and anion exchanger were somewhat similar in the erythrocytes of these species, whereas Na⁺,P_i cotransport was in 1:2:6 proportion in rats, humans and rabbits, respectively. Loading of erythrocytes with Li⁺ for NLCT measurement did not affect the activity of Na⁺,P_i cotransporter. Keeping in mind that NLCT is much higher in rabbits vs humans and rats, we compared the set of membrane proteins in these species using 2-dimensional gel electrophoresis. This approach revealed 174 common spots, whereas 132 proteins were detected only in human and rabbit erythrocyte membranes. Among these proteins, we found 17 spots whose expression was higher by more than 5-fold in rabbit compared to human erythrocytes. Thus, our results argue against the involvement of major ion transporters in NLCT. They also show that comparative proteomics is a potent tool to identify the molecular origin of this carrier.     

Electron microscopic radioautographic study on the syntheses of DNA, RNA and protein in the livers of aging mice

The syntheses of DNA, RNA and protein in the livers of aging mice was studied by electron microscopic radioautography using radioactive precursors. Labeling with³H-thymidine was observed in the nuclei of some hepatocytes at various prenatal and postnatal ages. The percentage of labeled cells decreased after birth, then slowly fell to the lowest value at 2 years. The silver grains with³H-uridine labeling were observed in both the nuclei and cytoplasm of hepatocytes at various ages. There was a peak in uridine labeling at 14 days, and then it slowly decreased until old age. The number of silver grains with³H-leucine labeling in the cytoplasm of hepatocytes was small. It increased after birth, reached the maximum at 1 month, and continued to decrease with aging.     

Electron-microscopic characteristics of the dynamics of thymidine-H3 and uridine-H3 in the mouse epidermis

The dynamics of distribution of uridine-H³ and thymidine-H³ in the epidermis of C57BL mice was studied electron-microscopically between 2 and 24 h after their separate intraperitoneal injection. A high rate of incorporation of both labeled nucleosides was discovered and their localization was found to depend on the time of injection. Depending on the character of their distribution and the rate of incorporation into the epidermis three zones of active protein synthesis can be distinguished: the boundary zone between the epidermis and dermis, the layer of basal cells, and the granular layer. The character of distribution of the label in the late period after injection confirms electron-microscopic data on the active role of the nucleus and the cytoplasmic ribosomes in fibril synthesis in the basal and prickle cells and in keratohyalin formation in the cells of the granular layer. By determining the hourly changes in the distribution of the labels the stages of transport of the injected nucleosides or their metabolites could be followed from the subepidermal region into the cells of the epidermis.Scientific-Research Laboratory, Ministry of Health of the USSR, Moscow. Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 78, No. 9, pp. 111–115, September, 1974.     

Effect of high NaCl intake on Na+ and K+ transport in the rabbit distal convoluted tubule

Morphological studies have demonstrated that a chronic increase in distal Na⁺ delivery causes hypertrophy of the distal convoluted tubule (DCT). To examine whether high NaCl-intake also causes functional changes in the well defined DCT, we measured transmural voltage (V _T), lumen-to-bath Na⁺ flux (J _Na(LB)), and net K⁺ secretion (J _K(net)) in DCTs obtained from control rabbits and those on high NaCl-intake diets. The lumen negativeV _T was significantly greater in the high NaCl group than in the control group. The net K⁺ secretion (pmol mm^–1 min^–1) was greater in the high NaCl-intake group (54.1±13.0 vs 14.7±5.6). The K⁺ permeabïlities in both luminal and basolateral DCT membranes, as assessed by the K⁺-induced transepithelial voltage deflection inhibitable with Ba²⁺, were increased in the experimental group. The lumen-to-bath²²Na flux (pmol mm^–1 min^–1) was also greater in the experimental group (726±119 vs 396±65). TheV _T component inhibitable with amiloride was also elevated in the high NaCl-intake group. Furthermore, Na⁺–K⁺-ATPase activity of the DCT was higher in the experimental than in the control group. We conclude that high NaCl intake increases both Na⁺ reabsorption and K⁺ secretion by the DCT. This phenomenon is associated with an increased Na⁺–K⁺-ATPase activity along with increased Na⁺ and K⁺ permeabilities of the luminal membrane, and an increase in the K⁺ permeability of the basolateral membrane. Cellular mechanisms underlying these functional changes remain to be established.     

Effect of Cs+, Li+ and Na+ on the potassium conductance and gating kinetics in the frog node of Ranvier

The effects of monovalent internal cations Cs⁺, Li⁺ and Na⁺ on potassium channel conductance in the frog node of Ranvier were studied by means of the voltage clamp. As previously reported, when 10–80% of the internal K⁺ was replaced by one of the above cations, the steady-state current-voltage relationship was significantly modified. The main effect was a voltage-dependent attenuation of the currents. We demonstrate that the current attenuation is associated with a change in the channel gating kinetics. For small depolarizations the kinetics can be described by the usual potassium conductance activation time constant, τ _n . However, under certain experimental conditions (e.g. substitution of the intracellular K⁺ with 10% Cs⁺), during larger depolarizations, stepping the membrane potential to values above 40–60 mV, the conductance develops with two time constants: τ _n and a new, slower time constant that, in contrast to τ _n , grows with membrane potential. These results can be explained by assuming that the catins may occupy two different sites in the channel; when the first site is occupied the channel is blocked, while occupation of the second site results in slowing of the gating kinetics in the affected channels.     

Morphofunctional ontogeny of the urinary system of the European sea bass Dicentrarchus labrax

European sea bass (Dicentrarchus labrax) are euryhaline fish that tolerate wide salinity fluctuations owing to several morphofunctional adaptations. Among the osmoregulatory sites (tegument, branchial chambers, digestive tract, urinary system), little is known about the kidney and the urinary bladder. The present study describes the ontogeny of the urinary system (kidney and urinary bladder) and focuses on the progressive expression of the Na⁺/K⁺-ATPase in the cells of these ion-transporting epithelia. A structural approach has shown that two pronephric urinary tubules are already present at hatching while the urinary bladder starts to differentiate. The glomus, an ultrafiltration site, occurs at day 5 (D5). The opisthonephros differentiates at D19/25 from the pronephric collecting tubules, then it rapidly grows longer and becomes folded. Na⁺/K⁺-ATPase immunolocalization and transmission electron microscopy show that ionocyte-like cells line the urinary tubules and the dorsal wall of the urinary bladder from D2/D5 on. Tubule ionocytes present a basolateral-localized fluorescence. Ionocytes of the collecting ducts and of the dorsal wall of the bladder present a fluorescence distributed in the whole cytoplasm. Fluorescence becomes stronger in later stages, suggesting a progressively increasing functionality of the urinary system in active ion transports. This observation is closely correlated with the ontogeny of osmoregulatory abilities. In juvenile and preadult fish kept in seawater, osmolality measurements demonstrate that urine is isotonic to blood. At low salinity, urine is hypotonic to blood in both stages. The capacity to produce hypotonic urine increases during ontogeny, a fact that suggests an increasing involvement of the urinary system in osmoregulation. The occurrence and the progressive functionality of the urinary system during the ontogeny, along with those of other osmoregulatory sites, are major adaptations allowing the sea bass to live in habitats of variable salinity such as lagoons and estuaries.     

Aldosterone-regulated ion transporters in the kidney

Summary Madin-Darby canine kidney (MDCK) cells resemble intercalated cells of the renal collecting duct. In these cultured epithelial cells aldosterone activates apical Na⁺/H⁺ exchange, initiating a cascade of intracellular events such as cell growth, epithelial cell polarity, and stimulation of transepithelial ion transport. Transepithelial K⁺ secretion is triggered by the insertion of new ion channels and the activation of previously quiescent channels with increasing cytoplasmic pH. Aldosterone supplies the cell with ion transporters necessary for adequate function of the renal collecting duct when the organism is metabolically challenged.     

Ca2+ channels in the apical membrane of the toad urinary bladder

Previously (Van Driessche et al. 1987) we showed that small inward (mucosa towards serosa) oriented shortcircuit currents (I _sc) were recorded through the toad urinary bladder when the mucosal side was exposed to Ca²⁺ free solutions containing K⁺, Na⁺ (+amiloride), Cs⁺ or Rb⁺ as main cation. This current component is inhibitable by micromolar concentrations of mucosal La³⁺ and divalent cations (Ca²⁺, Cd²⁺) and is considerably elevated by oxytocin (0.1 U/ml). The present study demonstrates that the addition of 50 nmol/l Ag⁺ to the mucosal medium during oxytocin treatment caused an additional large increase of the La³⁺-sensitiveI _sc component. The power density spectrum of the fluctuation in current contained a Lorentzian component which was enhanced by oxytocin treatment. The Lorentzian component disappeared as a consequence of the administration of mucosal Ag⁺. In experiments with Ca²⁺, Ba²⁺ or Mg²⁺ as principal mucosal cation, the La³⁺-sensitiveI _sc component was negligible under control conditions and during oxytocin treatment. Mucosal Ag⁺ (40 nmol/l) elicited a large inward oriented current which was blockable by the calcium channel blockers, La³⁺ and Cd²⁺. Also the organic calcium entry blockers, nicardipine and verapamil (10 mol/l) depressed the inward current considerably. Noise analysis of the currents carried by divalent cations showed a La³⁺-sensitive noise component. Oxytocin-Ag⁺ activated currents could not be recorded in the absence of the divalent cations or small inorganic cations, e.g. with solutions which contained N-methyld-glucamine (NMDG) as main mucosal cation.     

Distribution of labeled amino acids and delta sleep-inducing peptide in the body after instillation into the rabbit conjunctiva

P. K. Anokhin Research Institute of Normal Physiology, Academy of Medical Sciences of the USSR. Institute of Molecular Genetics, Academy of Sciences of the USSR. I. M. Sechenov First Moscow Medical Institute. Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 110, No. 9, pp. 236–237, September, 1990.     

Mechanism of hydrogen ion transport in the diluting segment of frog kidney

Transepithelial H⁺ transport was studied in diluting segments of the isolated-perfused kidney ofrana esculenta. The experiments were performed in controls as well as in K⁺-adapted and Na⁺-adapted animals (exposed to 50 mmol/l KCl or NaCl, resp. for at least 3 days). Conventional and single-barreled, liquid ion-exchanger H⁺-sensitive microelectrodes were applied in the tubule lumen to evaluate transepithelial H⁺ net flux (J _te ^H ) as well as limiting transepithelial electrical and H⁺ electrochemical potential differences (PD _te ,E _te ^H ) and luminal pH at zero net flux conditions. The measurements were made in absence (control) and presence of furosemide (5·10^–5 mol/l) or amiloride (10^–3 mol/l). E _te ^H (lumen positive vs ground) was 19±3 mV in controls, 43±3 mV in K⁺ adapted but about zero in Na⁺ adapted animals. Using the correspondingPD _te -values, steady state luminal pH of 7.63±0.05, 7.13±0.05 and 8.02±0.02 was calculated for the respective groups of animals (peritubular pH 7.80). In parallel, significant secretoryJ _te ^H (from blood to lumen) was found in controls (14±2 pmol·cm^–2·S^–1) which was stimulated by K⁺ adaptation (61±8 pmol·cm^–2·s^–1) but reversed in direction by Na⁺-adaptation (–8±1 pmol·cm^–2·s^–1). Amiloride inhibited secretoryJ _te ^H . Elimination of the lumen positivePD _te by furosemide did not affect significantlyE _te ^H andJ _te ^H in control and K⁺ adapted animals but abolished reabsorptiveJ _te ^H in Na⁺ adapted animals.We conclude that in frog diluting segment H⁺ secretion is an active, amiloride-sensitive, furosemide-insensitive transport process. The data are consistent with luminal Na⁺/H⁺ exchange. The activity of this system depends critically on the metabolic state of the animal.Parts of the data were presented at the 16th Ann. Meeting of the Am. Soc. Nephrol., Washington (1983)This work was supported by österr. Forschungsrat, Proj. No.: 4366 and by Dr. Legerlotz Stiftung     

Glucagon inhibits water and NaCl transports in the proximal convoluted tubule of the rat kidney

The effects of glucagon on water and electrolyte transport in the kidney were investigated on hormone-deprived rats, i.e. thyroparathyroidectomized diabetes insipidus Brattleboro rats infused with somatostatin. Glucagon consistently inhibited the reabsorption of water and Na⁺, Cl^–, K⁺ and Ca²⁺ along the proximal tubule accessible to micropuncture, leaving the reabsorption of inorganic phosphate (P_i) untouched. In the loop, besides its previously described stimulatory effects on Na⁺, Cl^–, K⁺, Ca²⁺ and Mg²⁺ reabsorption, glucagon strongly inhibited P_i reabsorption, very probably in the proximal straight tubule. These effects resulted in a significant phosphaturia and considerable reductions of Mg²⁺ and Ca²⁺ excretions. The effects of glucagon at both the whole kidney and the nephron levels are very similar to those previously described for calcitonin. In the absence of an adenylate cyclase system sensitive to glucagon and calcitonin in the rat proximal tubule, and from the analogy of their physiological effects with those elicited by parathyroid hormone, it is suggested that glucagon and calcitonin exert their inhibitory effects on Na and P_i reabsorption in the proximal tubule through another pathway, which could be the phosphoinositide regulatory cascade.     

Effect of parathyroid hormone on the connecting tubule from the rabbit kidney: biphasic response of transmural voltage

The effect of parathyroid hormone (PTH) on ion transport was examined by observing transmural (V _T) and basolateral membrane voltage (V _B) in the in vitro perfused rabbit connecting tubule. Addition of 10 nmol/l PTH to the bath induced a biphasic response of V _T, with hyperpolarization followed by depolarization. Chlorophenylthioadenosine cyclic 3,5-monophosphate mimicked the effect of PTH, which did not change the V _B in the connecting tubule cell, but mainly caused changes in the apical membrane voltage. The V _T of distal convoluted tubule and the cortical collecting duct were not affected by PTH. Elimination of Na⁺ from the lumen abolished the PTH-induced V _T responses in the connecting tubule. In the presence of 10 mol/l amiloride, PTH caused an initial hyperpolarization but did not induce the late depolarization. The same was seen in the absence of luminal Ca²⁺. Either addition of 0.1 mmol/l ouabain to the bath or elimination of bath Na⁺ completely abolished the PTH-induced V _T changes. The presence of 5 mmol/l Ba²⁺ in the lumen did not affect the response to PTH. These findings indicate that the initial hyperpolarization may be caused by an increase in Na⁺ influx across the luminal membrane through an amiloride-insensitive Na⁺ conductive pathway and that the late depolarization may be caused by the decrease in Na⁺ influx through the amiloride-sensitive Na⁺ conductive pathway. Luminal Ca²⁺ is necessary for the late depolarization caused by PTH. On the basis of these observations, we suggest that PTH initially increase influxes of both Na⁺ and Ca²⁺ across the luminal membrane and that an increase in intracellular Ca²⁺ in turn suppresses Na⁺ entry through the luminal amiloride-sensitive Na⁺ channel.