Faculty Bibliography

It has been proposed that the activity of the cardiac sarcolemmal Na+/Ca2+ exchanger may be greatest in developing animals before the sarcoplasmic reticulum (SR) reaches functional maturity. Experiments were performed in rabbits, which have a sparse SR at birth, and in newborn guinea pigs, which exhibit a more extensive SR. Whole cell voltage clamp techniques were used to characterize the Ni(2+)-sensitive Na+/Ca2+ exchange current in single freshly isolated cardiac myocytes. Na+/Ca2+ exchange current was measured from a holding potential of -40 mV by using a slow-ramp voltage protocol (-120 to +60 mV, 0.09 V/s) in the presence of Ba2+, Cs+, tetraethylammonia, D-600, and ouabain to block Ca2+, Na+, and K+ currents. Experiments in developing rabbits (1-22 days old) demonstrated that Na+/Ca2+ exchange current density was greatest at 1-4 days and declined rapidly over the first 3 wk of age. In contrast, Na+/Ca2+ exchange current density in newborn guinea pig myocytes did not differ from that recorded in adults. These results confirm that Na+/Ca2+ exchange is functional at birth in both rabbits and guinea pigs. The species-related difference in the ontogeny of Na+/Ca2+ exchange is consistent with the concept that Na+/Ca2+ exchange assumes a relatively greater role in newborn animals with a sparse SR

ATP-sensitive K+ (KATP) channels are thought only to open during conditions of metabolic impairment (e.g., myocardial ischemia). However, the regulation of KATP channel opening during ischemia remains poorly understood. We tested whether thiol (SH) group oxidation, which is known to occur during ischemia, may be involved in KATP channel regulation. Inside-out membrane patches were voltage clamped at a constant potential (O mV) in asymmetrical K+ solutions. The effects of compounds that specifically modify SH groups [p-chloromercuri-phenylsulfonic acid (pCMPS), 5-5'-dithio-bis(2-nitrobenzoic acid) [DTNB], and thimerosal] were tested. The membrane-impermeable compound, pCMPS (> or = 5 microM), caused a quick and irreversible inhibition of KATP channel activity. The reducing agent, dl-dithiothreitol (DTT) (3 mM) was able to reverse this inhibition. DTNB (500 microM) caused a rapid, but spontaneously reversible, block of KATP channel activity. After DTNB, no change was observed in single channel conductance. Oxidized glutathione (GSSG, 3 mM) did not block KATP channel activity. Thimerosal (100-500 microM) induced a DTT-reversible block of partially rundown KATP channels, or channels that underwent complete rundown; these channels were reactivated with trypsin (1 mg/ml). Thimerosal did not block KATP channels that had a high degree of activity. However, the ATP sensitivity was decreased; the concentration of ATP needed to half-maximally inhibit the channel (Ki) was increased from 47 +/- 12 to 221 +/- 35 microM (n = 6, P < 0.05). This was not due to a spontaneous change with time.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of oxidant stress (xanthine oxidase plus hypoxanthine or photoactivation of rose bengal) on the Na(+)-Ca(2+)-exchange current were studied in guinea pig ventricular myocytes with the use of voltage-clamp techniques. Oxidant stress depressed both the Ni(2+)-sensitive and extracellular calcium concentration ([Ca2+]o)-activated current in a time-dependent manner (e.g., xanthine oxidase plus hypoxanthine inhibited the Ni(2+)-sensitive current at +60 mV from 6.81 +/- 3.24 to 5.54 +/- 0.48 pA/pF; n = 6; P < 0.05). This effect was independent of the [Ca2+] of the pipette solution. Diamide, an alkylating agent that modifies protein sulfhydryl groups, also decreased the Ni(2+)-sensitive current (at + 60 mV: from 5.76 +/- 1.55 to 3.43 +/- 0.99 pA/pF; n = 6; P < 0.05). The stoichiometry (n) and partition coefficient (gamma) of the electrogenic Na(+)-Ca(2+)-exchange current seemed unchanged. Our results suggest that oxidant stress causes a direct or indirect sulfhydryl group-mediated decrease of the Na(+)-Ca2+ exchanger

Voltage-clamp studies were performed on guinea pig ventricular myocytes to clarify the action of N-(1-[4-(4-fluorophenoxy)butyl]-4-piperidinyl)-N-methyl-2-benzothiazo lamine (R-56865), an inhibitor of cardiac glycoside-induced arrhythmias. Transient inward current ((Iti)) was induced using low-K+/high-Ca2+ Tyrode solution. R-56865 (1 mM) was found to abolish I(ti). R-56865 had no influence on the peak Ca2+ current, steady-state current during the clamp, holding current, or the Ni(2+)-sensitive electrogenic Na(+)-Ca2+ exchange current. Fluorescence transients after repolarization (temporally related to the I(ti)) were abolished by R-56865 without affecting the fluorescence transients during depolarization. In separate experiments, the threshold of Ca2+ release from sarcoplasmic reticulum (SR) by the Ca2+ current was found to be unchanged, whereas Ca2+ transients (presumably triggered by Ca2+ entry through the Na(+)-Ca2+ exchanger) were depressed. Our results suggest that R-56865 inhibits spontaneous Ca2+ release from the SR when it is mediated by Ca2+ entry through the Na(+)-Ca2+ exchanger but that it has no direct effect on the well-known 'physiological' Ca(2+)-induced Ca(2+)-release mechanism from SR