Achilles Pappano

Education

B.S., St. Joseph's College
Ph.D., University of Pennsylvania

Research interests

My laboratory's research interest is excitation-contraction coupling in mammalian heart cells and its regulation by autonomic neurotransmitters. The theme for this involves evaluation of signal transduction pathways in single ventricular myocytes by simultaneous measurements of ionic currents, intracellular calcium transients and contractions. Ionic currents are measured under voltage clamp conditions with the whole cell patch electrode technique. Single cell contractions are recorded with a sensitive (0.1µm) and relatively rapid (16.7 ms) video edge detector system. Intracellular calcium transients are detected with the fluorescent indicator, fura-2. With these techniques, we are evaluating a novel action of the vagal transmitter, acetylcholine, which increases the amplitude of isotonic contractions. This effect is associated with an increased magnitude of the intracellular calcium transient without a change in the amplitude of the L-type calcium current, the usual trigger for the release of calcium from the sarcoplasmic reticulum. For these reasons, we propose the hypothesis that acetylcholine stimulates heart contractions by increasing the store of calcium in the sarcoplasmic reticulum. This can be achieved by augmenting the sodium-calcium exchange current. To this end, measurements of the sodium-calcium exchange current (forward mode) have been carried out. The working hypothesis is supported by tests that show acetylcholine increases the sodium-calcium exchange current in single ventricular myocytes.

The contraction of heart cells is initiated by the entry of calcium through L-type channels in the plasma membrane. This raises the calcium concentration at the site of intracellular calcium storage, the sarcoplasmic reticulum, and serves to trigger the release of stored calcium through another calcium permeant channel in the sarcoplasmic reticulum membrane that has a receptor for the alkaloid, ryanodine. The ryanodine receptor binds the alkaloid tightly and interferes with the process of calcium-induced calcium release.This highly selective chemical interaction has allowed the isolation, purification and cloning of the calcium release channel from the sarcoplasmic reticulum. The calcium-induced calcium release amplifies the trigger signal and disinhibits the actin and myosin filaments to permit shortening and tension development. Another source of calcium entry is the sodium/calcium exchanger in the plasma membrane. The direction of ion movement is governed by electrochemical forces that allow movement of calcium into and out of the cell. Recent evidence indicates that the sodium/calcium exchange not only contributes to changing the load of calcium in the sarcoplasmic reticulum but also may trigger the release of stored calcium. This hypothesis is also being tested in our laboratory in experiments using rapid superfusion of drugs that block the L-type calcium channel to isolate the possible contribution of calcium entry through the exchanger on intracellular calcium release and contraction in individual myocytes.

We also characterize pharmacologically the muscarinic and ß-adrenoceptors involved in autonomic neurotransmitter signaling in single cardiac myocytes. Rapid superfusion (t1/2<200 ms) of agonist drugs that mimic the actions of acetylcholine and norepinephrine provides a sensitive test of the activation of intracellular signaling for these transmitters. The whole cell voltage clamp technique together with cell contraction measurements are used to unravel the modulation of excitation-contraction coupling by agonist drugs. Intracellular dialysis of second messengers including cyclic AMP, cyclic GMP and inositol (1,4,5)-trisphosphate complements the experiments with rapid extracellular application of agonist drugs to allow systematic determination of the signal transduction paths and mechanisms for regulation of muscle cell function.

Recent publications

Department of Cell Biology Faculty