Outward currents in rabbit pulmonary artery cells dissociated with a new technique

Abstract

SUMMARYSingle cells from the rabbit pulmonary artery were isolated using a new and convenient procedure. Strips of muscle were incubated overnight in papain at 6°C and dispersed the following morning after warming the tissue for 10 min. This method consistently produced a high yield of relaxed cells, which reversibly responded to vasoconstrictors and remained viable for many hours. The electrophysiological properties of these cells were studied using the patchclamp technique in the whole-cell configuration. In physiological Ca2+ solution with K+-filled pipettes, cells had a high input resistance (-17 GfQ) and an average resting potential of -55 mV. In voltage clamp, several components of outward current could be identified. Depolarizing voltage steps revealed a prominent, transient current (Iran), having extremely rapid activation (< S ms) and inactivation (< 15 ms) kinetics. 4ran was followed by a more slowly activating current ('KSO) that was sustained over 100 ms. Both currents were essentially abolished by a 4-aminopyridine (4-AP) and sensitive to Ca2+ influx. IKSO' but not Itran, was blocked by tetraethylammonium (TEA) and had the properties of a Ca2+-activated K+ current. Holding the membrane potential at -40 mV completely inactivated I4an and unmasked a timeindependent, background current superimposed on IKSO The background current was also blocked by 4-AP. In addition, when adenosine triphosphate (ATP), but not guanosine triphosphate (GTP), was omitted from the patch-pipette, spontaneous bursts of outward current (SOCs) were superimposed on the voltage-activated currents. However, since SOCs were rarely observed when ATP and GTP were present together, they are unlikely to be active under physiological conditions. Thus at least four types of outward current can be distinguished in isolated rabbit pulmonary artery cells. These include a novel transient current which could be activated from the resting potential. It activates much more rapidly than outward currents previously reported in vascular muscle, and would rapidly oppose action potential firing. This current could therefore be responsible for the inability of large elastic arteries to fire action potentials.

The pharmacological properties of K+ currents from rabbit isolated aortic smooth muscle cells

“…A voltage-gated K+ current with rapid activation and inactivation kinetics similar to the neuronal fast 'A' current is found in pulmonary artery (Clapp & Gurney, 1991) and portal vein (Beech & Bolton, 1989;Hume & Leblanc, 1989). In large, quiescent arteries such as the aorta, this current may act as a 'brake' to counteract quickly any depolarizing influence.…”

“…Cells were enzymatically isolated by the method of Clapp and Gurney (1991). Cell preparation with this technique is extremely reproducible, and has been used for other vascular smooth muscle cells.…”

“…Whether they exist in spontaneously active or quiescent tissue, the role of K+ channels is to stabilize the membrane by drawing the membrane potential closer to the K+ equilibrium potential and further from firing threshold. K+ currents have been characterized in isolated cells from a variety of vascular smooth muscles, including rabbit pulmonary artery (Clapp & Gurney, 1991), mesenteric artery (Smirnov & Aaronson, 1992), portal vein (Beech & Bolton, 1989;Hume & Leblanc, 1989), and guinea-pig mesenteric artery (Bolton et al, 1985). From these and other studies, it is apparent that several different types of voltage-sensitive K+ channel exist in smooth muscle.…”

“…These channels serve to terminate periods of Ca2" entry by repolarizing or hyperpolarizing the cell. They appear to be the most abundant K+ channel in several vascular smooth muscles (Beech & Bolton, 1989; Clapp & Gurney, 1991;Neylon et al, 1994;Zhang & Cook, 1994).…”

Magnesium lithospermate B dilates mesenteric arteries by activating BKCa currents and contracts arteries by inhibiting KV currents

“…Single mesenteric artery cells were isolated using an enzymatic dissociation method as described previously, with slight modifications [13] . The mesenteric artery was dissected and placed in dissociation medium containing a low concentration of Ca 2+ (160 μmol/L).…”