Biomedical subjects

J F García-Díaz

Publications and source records attributed to J F García-Díaz.

8 recordsLinked to original sources

PubMed1999

Development of a fast transient potassium current in chick cochlear ganglion neurons.

Neurons of the cochlear ganglion are endowed with a set of voltage-gated ion channels that enable them to encode and transmit sound information from the cochlear receptors to the brain. The temporal expression pattern of the K+ currents in chick cochlear ganglion neurons during embryonic development was analyzed using whole-cell voltage clamp techniques. In acutely isolated neurons, slowly activating delayed rectifier K+ currents appear at embryonic day 7 (E7) and increase in amplitude during development. A fast activating, fast inactivating K+ current of the A type is first expressed at E10, increasing in amplitude thereafter. To investigate the possible role of neurotrophins in the induction of these K+ channels, neurons were grown in culture in the presence or absence of brain-derived neurotrophic factor (BDNF) or neurotrophin-3 (NT-3). Neurons isolated at E8 and grown in culture for 1 day exhibit a high expression of A-current, together with the outgrowth of neurites. A-currents are not seen in acutely dissociated neurons from age-matched embryos (E9) which lack neurites, cut off by the isolation procedure. This suggests a preferential neuritic location of the channels carrying the A-current. However, the level of expression of the K+ currents was independent of BDNF or NT-3 application. Similarly, neurons isolated at E10 and grown in culture for up to 4 days maintain the amplitude of the K+ currents independently of the presence of the neurotrophins. These results indicate that BDNF and NT-3 may not directly regulate the expression of K+ channels in chick cochlear ganglion neurons. The notable expression of the fast inactivating A-current suggests that it plays a significant role in the modulation of synaptic efficacy and the encoding of auditory stimuli.

4-Aminopyridine↗

PubMed1997

Calcium currents in dissociated cochlear neurons from the chick embryo and their modification by neurotrophin-3.

Calcium entry through voltage-dependent channels play a critical role in neuronal development. Using patch-clamp techniques we have identified the components of the macroscopic Ca2+ current in acutely-isolated chick cochlear ganglion neurons and analysed their functional expression throughout embryonic development. With Ba2+ as a charge carrier, the currents exhibited two main components, both with a high activation threshold but differing in their inactivation kinetics. One component showed inactivation with a time constant around 100 ms (transient) whereas the other hardly inactivated (sustained). The currents were sensitive to omega-Conotoxin GVIA and dihydropyridines, blocked by 20 microM Cd2+, but unaffected by omega-Agatoxin IVA. In a few cases, only with Ca2+ as a charge carrier, an additional component with low activation threshold and fast inactivation (time constant of 20 ms), was observed. Currents were first detected at day 7 of embryonic development. Current density (amplitude/cell capacitance) increased through embryonic day 9, when early synaptic contacts are established, and decreased thereafter to lower steady values. The effect of neurotrophin-3, a neurotrophic factor required for survival and differentiation of cochlear ganglion neurons, was also examined. Neurons isolated at embryonic day 7 or day 11 and maintained two days in culture with 2 ng/ml neurotrophin-3 showed a substantial increase in Ca2+ current density, particularly in the transient component. These findings indicate that the expression of neuronal Ca2+ channels is predominant at the time of synapse formation between transducing hair cells and their primary afferents. Besides its effects on survival and neuritogenesis, neurotrophin-3 enhances the expression of Ca2+ channels in cultured neurons. Taken together these results suggest that the functional expression of Ca2+ channels is regulated during embryonic development of cochlear neurons by the release of neurotrophin-3 from the differentiating sensory epithelium of the cochlea.

Animals↗

PubMed1996

Altered contractility of rabbit penile corpus cavernosum smooth muscle by hypoxia.

PURPOSE: To investigate the effects of severe hypoxia on trabecular smooth muscle contractility. MATERIALS AND METHODS: Strips of rabbit corpus cavernosum were mounted in organ chambers to measure isometric tension. In some experiments intracellular free Ca2+ concentration and tension were measured by the intracellular fluorescent dye FURA-2 and isometric tension recording simultaneously. RESULTS: Contractions elicited by norepinephrine, endothelin-1 or potassium were attenuated under hypoxic conditions (pO2 approximately 10 mm. Hg). Strips contracted with 20 mM. K+ under normoxic conditions and then exposed to hypoxia consistently lost the potassium-induced tone. The hypoxia-induced relaxation was not affected by the removal of the endothelium, by treatment with the cyclooxygenase blocker, indomethacin, or with the guanylate cyclase blocker, methylene blue. The potassium channel opener, cromakalim, and adenosine relaxed potassium contracted strips; however, the potassium channel blockers glibenclamide, apamin, barium chloride and charybdotoxin or the adenosine receptor antagonist 8-(p-sulfonyl)theophylline were unable to prevent hypoxia-induced relaxation. Tetraethylammonium, a potassium channel blocker, and the depolarizing agents ouabain and high potassium (80 mM.), partially prevented hypoxia-induced relaxation. The calcium ionophore, ionomycin, had no effect on hypoxia-induced relaxations. Hypoxia, within 2 to 6 minutes, caused a large accumulation in intracellular calcium, concomitant with a loss of trabecular smooth muscle tone. Both these effects could be reproduced by inhibiting oxidative phosphorylation with 2,4,dinitrophenol. Reoxygenation resulted in an immediate recovery of both tone and intracellular calcium levels. Tissues under hypoxic conditions for 30 minutes had a 24% and 67% decrease in the ATP/ADP ratio and in creatine phosphate concentrations, respectively. CONCLUSION: Hypoxia causes a simultaneous increase in intracellular calcium and relaxation which, we propose, is the consequence of inhibition of oxidative phosphorylation with loss of high energy phosphates, necessary for the homeostasis of Ca2+ and the contractile mechanism of the cavernosal smooth muscle.

Adenosine Diphosphate↗

PubMed1991

Whole-cell and single channel K+ and Cl- currents in epithelial cells of frog skin.

Whole-cell and single channel currents were studied in cells from frog (R. pipiens and R. catesbiana) skin epithelium, isolated by collagenase and trypsin treatment, and kept in primary cultures up to three days. Whole-cell currents did not exhibit any significant time-dependent kinetics under any ionic conditions used. With an external K gluconate Ringer solution the currents showed slight inward rectification with a reversal potential near zero and an average conductance of 5 nS at reversal. Ionic substitution of the external medium showed that most of the cell conductance was due to K and that very little, if any, Na conductance was present. This confirmed that most cells originate from inner epithelial layers and contain membranes with basolateral properties. At voltages more positive than 20 mV outward currents were larger with K in the medium than with Na or N-methyl-D-glucamine. Such behavior is indicative of a multi-ion transport mechanism. Whole-cell K current was inhibited by external Ba and quinidine. Blockade by Ba was strongly voltage dependent, while that by quinidine was not. In the presence of high external Cl, a component of outward current that was inhibited by the anion channel blocker diphenylamine-2-carboxylate (DPC) appeared in 70% of the cells. This component was strongly outwardly rectifying and reversed at a potential expected for a Cl current. At the single channel level the event most frequently observed in the cell-attached configuration was a K channel with the following characteristics: inward-rectifying I-V relation with a conductance (with 112.5 mM K in the pipette) of 44 pS at the reversal potential, one open and at least two closed states, and open probability that increased with depolarization. Quinidine blocked by binding in the open state and decreasing mean open time. Several observations suggest that this channel is responsible for most of the whole-cell current observed in high external K, and for the K conductance of the basolateral membrane of the intact epithelium. On a few occasions a Cl channel was observed that activated upon excision and brief strong depolarization. The I-V relation exhibited strong outward rectification with a single channel conductance of 48 pS at 0 mV in symmetrical 112 mM Cl solutions. Kinetic analysis showed the presence of two open and at least two closed states. Open time constants and open probability increased markedly with depolarization.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1988

Voltage dependence of cellular current and conductances in frog skin.

Knowledge of the voltage dependencies of apical and basolateral conductances is important in determining the factors that regulate transcellular transport. To gain this knowledge it is necessary to distinguish between cellular and paracellular currents and conductances. This is generally done by sequentially measuring transepithelial current/voltage (It/Vt) and conductance/voltage (gt/Vt) relationships before and after the abolition of cellular sodium transport with amiloride. Often, however, there are variable time-dependent and voltage-dependent responses to voltage perturbation both in the absence and presence of amiloride, pointing to effects on the paracellular pathway. We have here investigated these phenomena systematically and found that the difficulties were significantly lessened by the use of an intermittent technique, measuring It and gt before and after brief (less than 10 sec) exposure to amiloride at each setting of Vt. I/V relationships were characterized by these means in frog skins (Rana pipiens, Northern variety, and Rana temporaria). Cellular current, Ic, decreased with hyperpolarization (larger serosa positive clamps) of Vt. Derived Ic/Vt relationships between Vt = 0 and 175 mV (serosa positive) were slightly concave upwards. Because values of cell conductance, gc, remained finite, it was possible to demonstrate reversal of Ic. Values of the reversal potential Vr averaged 156 +/- 14 (SD, n = 18) mV. Simultaneous microelectrode measurements permitted also the calculation of apical and basolateral conductances, ga and gb. The apical conductance decreased monotonically with increasing positivity of Vt (and Va). In contrast, in the range in which the basolateral conductance could be evaluated adequately (Vt less than 125 mV), gb increased with more positive values of Vt (and Vb). That is, there was an inverse relation between gb and cellular current at the quasi-steady state, 10-30 sec after the transepithelial voltage step.

Amiloride↗

PubMed1986

Cell sodium activity and sodium pump function in frog skin.

Cell Na activity, acNa, was measured in the short-circuited frog skin by simultaneous cell punctures from the apical surface with open-tip and Na-selective microelectrodes. Skins were bathed on the serosal surface with NaCl Ringer and, to reduce paracellular conductance, with NaNO3, Ringer on the apical surface. Under control conditions acNa averaged 8 +/- 2 mM (n = 9, SD). Apical addition of amiloride (20 microM) or Na replacement reduced acNa to 3 mM in 6-15 min. Sequential decreases in apical [Na] induced parallel reductions in acNa and cell current, Ic. On restoring Na after several minutes of exposure to apical Na-free solution Ic rose rapidly (approximately less than 30 sec) to a stable value while acNa increased exponentially, with a time constant of 1.8 +/- 0.7 min (n = 8). Analysis of the time course of acNa indicates that the pump Na flux is linearly related to acNa in the range 2-12 mM. These results indicate that acNa plays an important role in relating apical Na entry to basolateral active Na flux.

Animals↗

PubMed1985

Cell K activity in frog skin in the presence and absence of cell current.

Cell K activity, acK, was measured in the short-circuited frog skin by simultaneous cell punctures from the apical surface with open-tip and K-selective microelectrodes. Strict criteria for acceptance of impalements included constancy of the open-tip microelectrode resistance, agreement within 3% of the fractional apical voltage measured with open-tip and K-selective microelectrodes, and constancy of the differential voltage recorded between the open-tip and the K microelectrodes 30-60 sec after application of amiloride or substitution of apical Na. Skins were bathed on the serosal surface with NaCl Ringer and, to reduce paracellular Cl conductance and effects of amiloride on paracellular conductance, with NaNO3 Ringer on the apical surface. Under control conditions acK was nearly constant among skins (mean +/- SD = 92 +/- 8 mM, 14 skins) in spite of a wide range of cellular currents (5 to 70 microA/cm2). Cell current (and transcellular Na transport) was inhibited by either apical addition of amiloride or substitution of Na by other cations. Although in some experiments the expected small increase in acK after inhibition of cell current was observed, on the average the change was not significant (98 +/- 11 mM after amiloride, 101 +/- 12 mM after Na substitution), even 30 min after the inhibition of cell current. The membrane potential, which in the control state ranged from -42 to -77 mV, hyperpolarized after inhibition of cell current, initially to -109 +/- 5 mV, then depolarizing to a stable value (-88 +/- 5 mV) after 15-25 min. At this time K was above equilibrium (EK = 98 +/- 2 mV), indicating that the active pump mechanism is still operating after inhibition of transcellular Na transport. The measurement of acK permitted the calculation of the passive K current and pump current under control conditions, assuming a "constant current source" with almost all of the basolateral conductance attributable to K. We found a significant correlation between pump current and cell current with a slope of 0.31, indicating that about one-third of the cell current is carried by the pump, i.e., a pump stoichiometry of 3Na/2K.

Amiloride↗

PubMed1983

Voltage-dependent K conductance at the apical membrane of Necturus gallbladder.

The epithelial and cellular effects of clamping the transepithelial potential (Vt, mucosa reference) have been investigated in the Necturus gallbladder. Following initial equilibration at short circuit, tissue conductance gt was 4.1 +/- 1.2 (SD) mS/cm2, the apical potential Va was -76 +/- 8 mV, and the apical fractional voltage on brief voltage perturbation (fa = delta Va/delta Vt, reflecting the ratio of apical membrane to transcellular resistance) was 0.72 +/- 0.11 (21 gallbladders, 34 impalements). On clamping Vt at positive values, Va depolarized and fa decreased; at the same time gt decreased. Clamping Vt at negative values produced converse effects. All of the above changes were related directly to the magnitude of the clamping potential Vt and were reversed on return to the short circuit state. Effects of Vt on fa are not due to changes in the extracellular pathway resistances (which, however, contribute to gt). Furthermore, the effects of Vt on fa were abolished by the mucosal application of TEA or Ba, or acidification of the mucosal solution. Thus, these experiments disclose the presence of a voltage-dependent apical K conductance that increases with apical membrane depolarization. The calculated dose-response curve of TEA inhibition of apical conductance and the values of the apparent dissociation constant were in good agreement with those found for K channels in excitable tissues. Mucosal application of the Ca ionophore A23187 shifted the voltage dependence curve of fa to more negative values of Va without altering its shape. The effect of A23187 suggests a possible role of intracellular Ca in the modulation of the apical K channels.

Animals↗