atrial myocardium and is elevated in the plasma of patients with congestive heart failure. Additionally, Lainchbury et al. (2002) proposed that, although the molecular mechanism for the effects of DNP on the cardiac function is poorly defined, DNP may have a role as a cardiac unloading and natriuretic peptide in the dog. Based on our results, DNP inhibits the cardiac L-type Ca2+ channel, thereby regulating cardiac function including the cardiac contractility and action potential duration. This is supported by our additional findings that DNP (0.1 μM) caused shortening of the action potential duration in rabbit ventricular myocytes (Figure 6) and inhibited ventricular contractility in isolated rabbit ventricular tissues (data not shown). It is well known that the inhibition of cardiac Ca2+ channel results in shortening the action potential duration. Therefore, our study will be helpful when investigating the therapeutic uses or pathophysiological role of DNP in various cardiovascular diseases. Methods Action potential recording in rabbit ventricular tissues All experiments were approved by the Experimental Animal Care and Ethics Committee of Chonbuk National University. Male New Zealand White rabbit (Han I, Jeonju, Korea) were housed under 12:12 light:dark cycles with free access to food and water. Male New Zealand White rabbits weigh- ing about 1.8 to 2 kg were anesthetized by injection of pen- tobarbital into the marginal ear vein and were euthanized by cervical dislocation at between 10:00 and 12:00 h. Hearts were rapidly excised and transferred to a dissection bath filled with Tyrode’s solution oxygenated with 97% O2 and 3% CO2 gas mixture. Ventricular muscles were care- fully dissected free from the left ventricular wall. Each dis- sected tissue was mounted horizontally in a narrow chan- nel of a tissue chamber and continuously superfused with oxygenated Tyrode’s solution at 36-37oC. The size of dis- sected tissues ranged from 0.5 to 1 mm in width and was about 2 to 3 mm in length. The one end of each tissue was fixed by an insect pin to the bottom of the chamber coated with Sylgard. The tissue next to the insect pin was pressed against the floor by stimulating electrodes, which were used to elicit action potentials and contractions. The action potentials were elicited by stimulating the cardiac cells with square pulses (2 Hz, 1 ms duration, 20-30% above thresh- old voltage) by a stimulator via a stimulus isolation unit (WPI, Sarasota, FL). Action potentials were recorded with a 3 M KCl-filled microelectrode (10-20 megaohm) con- nected to an amplifier (KS-700, WPI), and were displayed on an oscilloscope (Dual beam storage 5113, Tektronix, Beaverton, OR). Tracings on the oscilloscope screen were photographed on a 35-mm film and also recorded on a chart recorder (RS 3400, Gould, Cleveland, OH). Rabbit cardiac tissue was superfused with a Tyrode’s solution at a constant rate (5 ml/min). Tyrode’s solution contained 137 mM NaCl, 5.4 mM KCl, 1.05 mM MgCl2, 0.45 mM Inhibition of calcium channel by DNP 367 NaH2PO4, 11.9 mM NaHCO3, 1.8 mM CaCl2 and 5 mM dextrose. The solution was gassed with 97% O2 + 3% CO2 (pH 7.3-7.4). Cell isolation Single cardiac myocytes of male New Zealand White rab- bits weighing 1.8 to 2 kg were prepared by enzymatic di- gestion as described previously (Kwak et al., 1995). Rabbits were anesthetized by injection of pentobarbital into the marginal ear vein and were euthanized by cervical dislocation. After opening the chest cavity, the heart was excised and immersed in Krebs-Henseleit (KH) buffer sol- ution (pH 7.35). Hearts were perfused retrogradely via the aorta in a Langendorff apparatus with KH solution for 5 min to clear visible blood. Hearts were then perfused with Ca2+-free KH solution until hearts stopped beating, and then with Ca2+-free KH solution containing 0.075% collage- nase (CLS2, Worthington, Freehold, NJ) for 30 min. After enzymatic digestion, ventricular muscles were carefully dissected free from the left ventricular wall. The dissected tissues were placed in Ca2+-free KH solution containing 1% bovine serum albumin, cut into small pieces and then me- chanically dissociated into single cells. All cells used for experiments were rod-shaped with clear striations. The KH solution contained (in mM) 118 NaCl, 4.7 KCl, 1.2 MgSO4, 1.2 KH2PO4, 10 HEPES, 25 NaHCO3, 10 pyruvate, 11 dex- trose and 1 CaCl2. Conventional whole cell patch clamp techniques Conventional (ruptured patch) recording techniques were used to measure the voltage-gated L-type Ca2+ currents, ICa,L. The pipette solution contained (in mM) CsCl 125, tet- raethylammonium chloride (TEA-Cl) 20, MgATP 5, creatine phosphate 3.6, EGTA 10, and HEPES 10 (pH 7.2) with CsOH. The bath solution contained (in mM) TEA-Cl 157, CaCl2 1, MgCl2 0.5, and HEPES 10 (pH 7.4) with CsOH. The junction potential with these solutions was 7.5 mV (calculated using Axoscope, version 1.1, Axon Instruments, Foster City, CA) and was not corrected. Data were ac- quired using a Pentium computer that controlled data ac- quisition hardware and software (pClamp 6.03, Axon Instruments) connected to Axopatch 200B amplifiers (Axon Instruments). Currents were filtered at 2 kHz and sampled at 4 to 10 kHz. A holding potential of -80 mV was used to inactivate Na+ current. ICa,L was elicited with step depolari- zation protocols, using test potentials in the range of -40 to +30 mV. ICa,L densities were computed by dividing current amplitudes by the whole-cell capacitance. Peak Ca2+ cur- rent density refers to ICa,L density at the peak of the cur- rent-voltage (I-V) curve. Phosphorylation of cardiac voltage-sensitive Ca2+ channel protein, α1c by DNP Rabbit ventricular myocytes were washed twice with phos- phate free Dulbeco’s modified Eagle’s medium (DMEM) containing 0.5% dialysed fetal calf serum. Cells were then incubated in 2 ml of phosphate free DMEM containing 1 mCi of [32P]orthophosphate (Sigma-Aldrich, St. Louis, MO) for 4 h at 37°C. The cells were treated with DNP (Phoenix