Using the whole-cell patch clamp techniques and tension measurements in isolated pulmonary arterial rings in an organ bath, we found that NO and cGMP activate calcium-dependent potassium channel (K_Ca) in isolated pulmonary myocytes (Figure 1) and that a selective inhibitor of the K_Ca channel, charybdotoxin, blocks NO and cGMP-induced relaxation of pulmonary arterial rings (Figure 2). Inhibition of cGMP synthesis and action, as well as inhibition of the cGMP-dependent protein kinase, prevents the effect of NO on the K_Ca current. These data show that NO causes pulmonary vasodilation by a cGMP-dependent kinase-mediated activation of the K_Ca channel.

Figure 1: Nitric oxide (NO, 2x10-6M) increases KCa current in the isolated pulmonary arterial myocytes.

The current is identified as KCa by its size, spiky shape, and sensitivity to the KCa channel blocker, charybdotoxin (CTX, 200 nM). An example of currents recorded upon increasing membrane potential from -70 to +70 mV is shown at left and the average (+/-SEM, n=4 cells) voltage/current curves are at right. 4-AP = 4-aminopyridine (5 mM), a non-selective inhibitor of the delayed rectifier potassium channels. *P<0.05

Figure 2: Charybdotoxin (CTX), a selective inhibitor of the KCa channels, prevents relaxation of pulmonary arterial rings in response to all but the highest doses of nitric oxide (NO). In this particular ring, the degree of precontraction by norepinephrine (NE) differed between the control and CTX run. That was not always the case, though.

The electrophysiological data in this study were acquired by Dr. Jimmy Huang. The pulmonary arterial rings data were acquired by Dan Nelson.

We tested the hypothesis that pharmacological stimulation of the cGMP-dependent protein kinase will simulate the activating effect of NO on potassium channels. (Sp)-guanosine cyclic 3',5'-phosphorothioate (1 µM), a selective activator of the cGMP-dependent protein kinase, significantly potentiated potassium currents in pulmonary artery smooth muscle cells (Figure) in a manner similar to that seen with NO or cGMP. The current was inhibited by the inhibitor of calcium-gated potassium channels, charybdotoxin. This finding supports the hypothesis that the effect of NO on potassium channels is mediated by the cGMP-dependent protein kinase.

(Sp)-guanosine cyclic 3',5'-phosphorothioate, a selective activator of the cGMP-dependent protein kinase, activates potassium current (IK) in pulmonary artery smooth muscle cells. (Sp)-cGMP[S], which is poorly cell membrane permeable, was added to the patch pipette in the whole-cell configuration, so that after obtaining a seal, (Sp)-cGMP[S] gradually diffused into the cell. *P<0.05 vs. control

The electrophysiological data in this study were acquired by Dr. Jimmy Huang.

We found that isolated pulmonary arterial myocytes form three distinct populations characterized by size, shape, and electrophysiological properties.Based on the predominant type of potassium (K) channel, we call them K_Ca cells, K_DR cells, and mixed cells (Figure 1). The calcium-dependent K channels, predominant in the K_Ca cells, respond to the physiologically and therapeutically important nitric oxide (NO) by an increase in their open probability (Figure 2), leading to an increased inward K current, membrane hyperpolarization, and vasorelaxation. As a result, the large pulmonary arteries, replete with the K_Ca cells, respond to nitric oxide with greater relaxation than the small pulmonary arteries (Figure 3). On the other hand, another important regulator of the pulmonary vascular tone, hypoxia, acts predominantly on the K_DR channels (predominant in the K_DR cells), causing their closure, membrane depolarization and vasoconstriction.

Figure 1: Three types of pulmonary arterial smooth muscle cells.

The large, elongated cells with K current dominated by the calcium-dependent K channel (KCa) have relatively small, spiky K currents and are more common in the large than in the small pulmonary arteries (the right-hand graph). The smaller cells with K currents dominated by the delayed rectifier K channel (KDR) have large, smooth K currents, a perinuclear bulge, and are prevalent in the small pulmonary arteries. The mixed cells are a distinct population with intermediary features.

Figure 2: Nitric oxide increases the open probability of a single charybdotoxin-sensitive K channel of a pulmonary arterial myocyte with the KCa phenotype.

The patch pipette was attached to the membrane of a vesicle removed from the myocyte so that one single K channel was in the pipette's mouth. Tiny perforations were made in the membrane of the vesicle by amphotericin to allow diffusion of the internal pipette solution into the vesicle. Opening of the channel caused the recorded current to suddently rise from the level marked c to the level marked o. Charybdotoxin (CTX) is a selective inhibitor of the KCa channel. NO = nitric oxide, Po = open probability.

Figure 3: Nitric oxide (NO) induces greater vasorelaxation in the isolated main (conduit) pulmonary artery than in rings isolated from small preripheral (resistance) pulmonary artery. Tension changes were recorded in rings isolated from pulmonary arteries in an organ bath. Before NO administration, the rings were precontracted with norepinephrine. The data are the means +/- SEM. *P<0.05

The electrophysiological data in this study were acquired by Drs. Jimmy Huang, Helen Reeve and Evangelos Michelakis. The pulmonary arterial rings data were acquired by Dr. Simona Tolarova.