Biomedical subjects

W A Varanda

Publications and source records attributed to W A Varanda.

24 records · Page 2Linked to original sources

PubMed1985

The acetylcholine receptor of the neuromuscular junction recognizes mecamylamine as a noncompetitive antagonist.

The secondary amine, mecamylamine, interacts with the nicotinic receptor ionic channel complex as a noncompetitive antagonist. Mecamylamine (1-10 microM) blocked indirect muscle twitches with no discernible effect on the membrane potential, overshoot, or amplitude of the action potential. It also produced a voltage- and concentration-dependent depression of the peak amplitude of the endplate currents (EPC) and induced nonlinearity in the current-voltage relationship. The decay time constant of the EPC (TEPC) was significantly shortened. The linear relationship between the reciprocal of TEPC and the drug concentration suggested an open channel blockade. Patch-clamp studies, in agreement with the noise analysis results, revealed that mecamylamine (1-8 microM) shortened the lifetime of the open channels. Further, the single channel studies showed that at high concentrations mecamylamine reduced the double exponential nature of the distribution of open times characteristic of channels recorded from myoballs. Closed times had a complex distribution that could not be fitted to a single exponential function because of the presence of short closures or "flickers" during the open state. Although the frequency of channel openings progressively decreased with increasing drug concentration, the single channel conductance remained unchanged at all the concentrations tested. Biochemical studies showed that mecamylamine (up to 100 microM) did not block [3H]acetylcholine binding to the nicotinic receptor of the Torpedo electroplax, but inhibited the binding of [3H]perhydrohistionicotoxin to its channel site, both in the resting and the activated state. These results suggested that, at the nicotinic receptors of the neuromuscular junction, mecamylamine acted as a noncompetitive blocker, binding primarily to the receptor's open channel conformation. Most of the alterations of EPCs were consistent with the predictions of a sequential model for open channel blockade. Biochemical and patch-clamp results, however, could not be fully explained by this model and provided some evidence of the existence of additional blocked states most likely through pathways into desensitized species. In contrast to a competitive antagonism of acetylcholine receptors reported at autonomic ganglia, there was no such action of the drug at the neuromuscular junction; thus, mecamylamine is a useful tool to characterize the nicotinic receptors from different synapses.

Amphibian Venoms↗

PubMed1984

Interactions of gephyrotoxin with the acetylcholine receptor-ionic channel complex. I. Blockade of the ionic channel.

The novel tricyclic alkaloid, gephyrotoxin ( GyTX ), found in the skin secretions of the frog Dendrobates histrionicus , potentiates and blocks the indirectly elicited muscle twitch in a concentration-dependent manner. GyTX prolongs the falling phase of the muscle action potential and decreases delayed rectification, supporting the idea that the alkaloid blocks the voltage-sensitive potassium conductance of the electrically excitable membrane. The peak amplitude of the end-plate currents (EPC) and miniature end-plate currents ( MEPC ) were depressed, but no significant deviation from linearity relative to control was seen in the current-voltage relationship. The decay time constant of the EPC (tau EPC) was markedly shortened by GyTX , the effect being greater at 10 degrees than at 22 degrees. The relationship between the log of tau EPC and membrane potential disclosed a linear relationship at all concentrations tested, but a progressive loss of voltage sensitivity of tau EPC was seen when GyTX concentrations were increased. Also, the plot of 1/tau EPC against GyTX concentration revealed a linear relationship. The lack of voltage and time dependence suggests that GyTX has little effect on the ACh receptor-ionic channel complex in the closed conformation. Single-channel conductance studied by means of fluctuation analysis did not change after GyTX application, but the channel lifetime decreased by about 40% at clamp potentials of -105 mV and at a toxin concentration of 7.5 microM. Repetitive nerve stimulation led to a pronounced " rundown " in the EPCs which was frequency-dependent. These findings were taken as evidence that GyTX interacts with the acetylcholine receptor complex, causing a blockade of its channel mainly in the open conformation.

Action Potentials↗

PubMed1984

Interactions of gephyrotoxin with the acetylcholine receptor-ionic channel complex. II. Enhancement of desensitization.

The actions of the tricyclic alkaloid gephyrotoxin ( GyTX ) on the extrajunctional and junctional acetylcholine (ACh) sensitivity and desensitization were studied in the chronically denervated rat soleus muscle and cutaneous pectoris muscle of the frog. At low concentrations, GyTX greatly depressed the extrajunctional ACh sensitivity of the chronically denervated soleus muscles. In addition, GyTX produced a strong inhibition of junctional end-plate potentials evoked by ACh. Junctional and extrajunctional desensitizations induced by microiontophoretically applied ACh were greatly enhanced by the alkaloid in a frequency-dependent manner. These effects were readily reversible. The interaction of GyTX with binding sites on the acetylcholine receptor-channel (AChR) complex was studied on electroplax membranes from Torpedo californica. GyTX binds to the AChR complex at a site distinct from the ACh binding site, as revealed by its lack of inhibition of [125I]alpha-bungarotoxin ( [125I]BGT) binding. On the other hand, GyTX at a concentration range between 1 microM and 100 microM significantly increased the potency of the agonist carbamylcholine as an antagonist of binding of [125I]BGT. At low micromolar concentrations, GyTX inhibited the binding of [3H]perhydrohistrionicotoxin and [3H] phencyclidine to sites associated with the ionic channel of the AChR complex. The affinity of GyTX for these sites was increased 3- to 5-fold by carbamylcholine. Results of electrophysiological and binding studies indicate that GyTX not only blocks the open channel of the AChR but also enhances desensitization of the AChR complex by increasing receptor affinity for agonists.

Acetylcholine↗

PubMed1982

A quartz cell for studying planar lipid bilayer membranes.

A quartz chamber is proposed for use in experiments with planar lipid bilayer membranes. Membranes are formed in a hole made on the lateral wall of a fused quartz test tube, immersed in an electrolyte solution. The quartz cell is easy to clean, chemically inert and easily made. Membranes formed in this chamber had specific resistances higher than 10(8) omega.cm2 and excellent mechanical stability.

Lipid Bilayers↗

PubMed1979

Transient potassium fluxes in toad skin.

Experiments were carried out in the isolated short-circuited skin of the toad Bufo marinus ictericus. 42K influx and efflux experiments were carried out with skins bathed on both sides by NaCl-Ringer's solution. Those fluxes showed very similar kinetics of equilibration with time and the results could be fitted by equations of a model of two intraepithelial compartments and the bathing solutions. In the steady state K influx is 3.99 +/- 0.36 nmol cm-2 hr-1 (n = 7) and efflux 3.62 +/- 0.38 nmol cm-2 hr-1 (n = 7) and are not statistically different, indicating that no net K flux is present across the epithelium. Different kinds of perturbations affecting the rates of 42K discharge into the bathing solutions were studied. Immediately after addition of amiloride (10(-4) M) to the outer solution, a sharp decline is observed in the rate of 42K discharge into the bathing solution, JK21, which falls from 3.62 +/- 0.38 nmol cm-2 hr-1 to 2.02 +/- 0.04 nmol cm-2 hr-1 (n = 7) 2 min after addition of the drug, followed by a partial recuperation with time. A complete Na by K substitution in the outer bathing solution induces a prompt and marked decline in JK21 which is similar to that induced by amiloride. Increase in the outer bathing solution Na concentration from zero Na concentration induces a nonlinear increase in JK21 and a linear relationship was observed between JK21 and short-circuit current in the range of 0 to 115 mM external Na concentration. The decline in JK21 induced by amiloride or by lowering external Na concentration was interpreted as being caused by electrical hyperpolarization of the external barrier of the epithelium induced by these procedures. Depolarization of the epithelial barriers by inner Na by K substitution in the short-circuited state (when the potential barriers are equal) drastically interfere with the rate of 42K discharge from the epithelium into the bathing solutions. Thus, transient increases are observed both in the rate of 42K discharge to the outer and to the inner bathing solutions upon depolarization of the barriers. These results indicate that at least the most important component of transepithelial K unidirectional fluxes goes through a transcellular route with a negligible paracellular component. Addition of ouabain (10(-3) M) to the inner bathing solution induces a transient rise in the rate of 42K discharge to the outer bathing solution with a peak on the order of 200% of the stationary value previous to the action of the inhibitor, followed by a return to new stationary values not statistically different from those observed previously to the effect of ouabain. The behavior of JK21 upon the effect of ouabain, as suggested by comparison with predictions from computer simulation, strongly supports the notion of a rheogenic Na pump in the inner barrier of the epithelium against the notion of a nonrheogenic 1:1 Na--K pump.

Amiloride↗

PubMed1978

Transients in toad skin: short circuit current and ionic fluxes related to inner sodium substitution by monovalent cations.

When the Na electrochemical potential difference across the skin (delta muNa) is altered by perturbing the transmembrane electrical potential difference or the external Na concentration, effects on transport and associated oxygen consumption can be described by the formalism of linear nonequilibrium thermodynamics (Vieira, Caplan & Essig, 1972, J. Gen. Physiol. 59:77; Danisi & Lacaz-Vieira, 1974, J. Gen. Physiol. 64:372; Procópio and Lacaz-Vieira, 1977, J. Membrane Biol. 35:219). We now show that with modifications of delta muNa by substitution of Li or choline for Na in the inner bathing solution, this formalism is no longer applicable. Inner Na by K substitution ((Na X K)i) causes profound alterations in short-circuit current (SCC), JinNa, K efflux (JeffK) and PD. SCC drops transiently after (Na X K)i in Cl and in SO4 media, increasing subsequently. In Cl medium, following the initial transient, there is a late decline in SCC toward a steady state. The rate of SCC decline in Cl medium is more pronounced than that observed in SO4 medium. (Na X K)i causes a transient increase in JinNa with a peak synchronous to the minimum in SCC, both in Cl and in SO4 media. This was interpreted as due to depolarization of the inner membrane. In SO4 medium, following the peak observed after (Na X K)i, JimNa drops, to increase again toward a steady state in which SCC and JinNa are not statistically different, resembling the control condition before (Na X K)i. In Cl medium, however, the JinNa steady state is approximately 100% higher than SCC. This difference is due to an important K efflux (JeffK), which builds up progressively after the substitution. The apparent K permeability [JeffK/(Ki)] is of comparable magnitude in Cl and in SO4 media before (Na X K)i and also in SO4 medium after (Na X K)i. However, in Cl medium, after (Na X K)i, the apparent K permeability increases one order of magnitude as compared to the control condition before the ionic substitution. In Cl medium, the high levels of JinNa and of Jeff(K) observed in the steady state after (Na X K)i were interpreted as being a consequence of cell swelling. SCC and PD follow very different temporal patterns after (Na X K)i which are characterized by transients in SCC and a simple fall in PD. Reasons for these differences are discussed.

Animals↗