Biomedical subjects

M A Valverde

Publications and source records attributed to M A Valverde.

41 records · Page 3Linked to original sources

PubMed1992

Membrane conductance and cell volume changes evoked by vasoactive intestinal polypeptide and carbachol in small intestinal crypts.

We have used the perforated-patch whole-cell recording mode of the patch-clamp technique to monitor membrane potential and measured cell volume changes by image analysis, to determine the nature of the response to secretagogues of isolated whole guinea-pig small-intestinal crypts. Vasoactive intestinal polypeptide (VIP) produced a dose-dependent depolarisation (EC50 = 30 nM) and an increase in membrane conductance that could be potentiated by carbachol. Similar depolarisations were observed with forskolin. The depolarisation induced by 100 nM VIP was smaller when pipette [Cl-] was 60 mM than when it was 145 mM, suggesting an effect through Cl- conductance activation. Carbachol alone produced a hyperpolarisation (EC50 = 2 microM). The Cl- channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) produced a small hyperpolarization. When VIP was added in the presence of NPPB, the depolarisation was observed instead, consistent with the parallel activation of a K+ conductance. Both carbachol (100 microM) and VIP (100 nM) induced a 25%-30% shrinkage of crypts, which was maximal 8 min after addition of the secretagogue. The induced shrinkage was sustained in the continued presence of agonist and was reversed upon washout. Shrinkage induced by the agonists was abolished by increasing extracellular K+ from 6 mM to 20 mM and was inhibited partially in the presence of 100 microM anthracene-9-carboxylic acid in the bath. The decrease in volume induced by 100 nM VIP was totally abolished in the presence of 100 microM NPPB. The results are consistent with the view that both VIP and carbachol induce secretion in small-intestinal crypts.

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PubMed1992

Development of Na(+)- and K(+)-currents in the cochlear ganglion of the chick embryo.

The development of Na(+)- and K(+)-currents in the primary afferent neurons of the cochlear ganglion was studied using the patch-clamp technique. Cells were dissociated between days 6 and 17 of development and membrane currents recorded within the following 24 h. Outward currents were the first to appear between days 6 and 7 of embryonic development and their magnitude increased throughout development from 200 pA on day 7 to 900 pA on days 14-16. Threshold for activation decreased by 20 mV between days 8 and 14. Outward currents were absent when Cs+ replaced K+ in the pipette and were partially blocked by external tetraethylammonium. Outward currents contained at least three components: (i) a non-inactivating outward current, similar to the delayed-rectifier, predominating in mature neurons; (ii) a slowly inactivating current (tau about 200 ms), most evident in early and intermediate stages (days 7-10); and (iii) a rapidly inactivating outward current (tau about 20 ms) similar to the A-current (IA) described in other neurons, which was distinctly expressed in mature neurons. Sodium currents were identified as fast transient inward currents, sensitive to tetrodotoxin and extracellular Na(+)-removal. They appeared later than K(+)-currents and increased in size from about 100 pA between days 9-11 to 600 pA by days 13-16. The development of membrane currents in cochlear ganglion neurons corresponded to defined stages of the innervation pattern of the chick cochlea [Whitehead and Morest (1985) Neuroscience 14, 255-276]. These currents could be functionally related to the establishment of synaptic connections between transducing cells and primary afferent neurons.

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PubMed1992

Transient outward currents in cochlear ganglion neurons of the chick embryo.

Cochlear ganglion neurons were isolated from chick embryos and membrane currents recorded using the patch-clamp technique. Depolarizing voltage steps elicited transient outward currents whose inactivation was best fitted by a double-exponential function with time constants < 30 ms and > 100 ms. The fast inactivating transient outward current (Ito,f) had a threshold for activation of -61 +/- 5.5 mV; steady-state inactivation was voltage-dependent between -90 and -60 mV, with half-inactivation near -75 mV. The slowly inactivating outward current (Ito,s) showed an activation threshold of 34 +/- 4 mV. Half-inactivation was at -67 +/- 3 mV. Ito,f was blocked by 4-aminopyridine which did not affect Ito,s. The effect was concentration- and voltage-dependent. Tetraethylammonium had no effect on either fast or slow transient currents but reduced the amplitude of the non-inactivating outward current in a dose-dependent manner. Ito,f was strongly inhibited by removing Ca2+ from the extracellular bathing solution. Cobalt ions inhibited Ito,f in a dose-dependent manner between 2 and 20 mM. The inhibitory effect of Co2+ was voltage-dependent, displaying a bell-shaped inhibition curve as a function of membrane voltage, maximal inhibition occurring between -20 and 0 mV. Ca2+ removal did not affect Ito,s and partially reduced the amplitude of the steady-state current. These results provide kinetic and pharmacological evidence for the presence of two distinct transient outward currents in cochlear neurons. These currents may play a role in the first synaptic relay of sound transmission.

4-Aminopyridine↗

PubMed1991

Two types of potassium currents seen in isolated Necturus enterocytes with the single-electrode voltage-clamp technique.

1. The ionic permeability of Necturus maculosus small intestine epithelial cells was investigated using intracellular microelectrodes to measure membrane potential in intact tissue or by the single-electrode voltage-clamp technique in isolated cells. 2. The basolateral membrane of enterocytes appears to be K+ selective as demonstrated by the dependence of membrane potential and fractional serosal resistance measured in the intact epithelium on serosal K+ concentration. 3. Isolated cells had membrane potentials similar to those measured in the intact tissue. Voltage-clamp experiments in a physiological Ringer solution showed the presence of both large outward and inward currents. 4. Removal of Cl- from the bathing medium, linear subtraction or the use of a Cl- channel blocker revealed outwardly rectifying currents. The quasi-linear component was also revealed following K+ channel inhibition; it reversed near ECl, suggesting that the charge carrier was Cl-. 5. Outwardly rectifying currents could be kinetically resolved into two components. A fast component (tau for activation < 4 ms) accounted for 60-80% of the total current at positive potentials. A slowly activating component appeared at voltages positive to 50 mV with tau for activation of > 25 ms. 6. The slow outward current showed strong voltage dependence of both activation and relaxation, which were faster at more depolarized potentials. 7. Both fast and slow outward currents seem to be carried by K+ as they were blocked by Ba2+ and tetraethylammonium (TEA). Tail current analysis of the slow component indicated a reversal potential very similar to EK. 8. Fast outward currents were half-activated at about -40 mV whereas slow outward currents were only apparent at more positive potentials. It is proposed that the fast outward K+ current plays a role, together with Cl- currents, in determining the resting membrane potential of Necturus enterocytes.

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PubMed1991

Potassium currents of isolated Necturus enterocytes: a whole-cell patch-clamp study.

1. The whole-cell recording mode of the patch-clamp technique was used to investigate the ionic currents of enterocytes isolated from the small intestine epithelium of Necturus maculosus. 2. When enterocytes were bathed in a physiological Ringer solution and dialysed with a K(+)-rich intracellular solution containing 1.5 x 10(-7) M intracellular Ca2+, strongly outwardly rectifying currents were observed. These currents were absent from enterocytes internally dialysed with K(+)-free solutions. 3. The kinetics of activation of the outwardly rectifying current was monoexponential with rate constants decreasing with depolarization from 160 ms at 20 mV to 40 ms at 60 mV. Similar voltage dependence of the relaxations after activation were observed. 4. Strongly buffering intracellular Ca2+ with EGTA inhibited outward currents, while increasing Ca2+ increased both their magnitude and rate of activation. 5. Bath application of the K+ channel inhibitors Ba2+ and TEA greatly attenuated outwardly rectifying currents. This observation plus the fact that tail currents reverse near EK points to K+ as the charge carrier in these currents. 6. Outside-out patches showed maxi K+ channels and lower conductance K+ channels. Averaging fluctuations of the maxi K+ channels gave a kinetic behaviour similar to the whole-cell currents.

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