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.

Large conductance Ca(2+)-dependent K+ channels were studied in smooth muscle cells enzymatically dissociated from rabbit pulmonary artery. The current-voltage relationship of single channels recorded in cell-attached patches revealed strong inward rectification, which disappeared after patch excision. Cell permeabilization with saponin, beta-escin or equinatoxin II also removed rectification. These observations imply the existence of fast open channel block by an intracellular substance(s). Application to the cytosolic side of inside-out patches of Na+ ions, mono- di- and trinucleotides, taurine, reduced and oxidized forms of glutathione, or peptides extracted from pulmonary artery smooth muscle, did not reproduce the inward rectification. Patch treatment with either alkaline phosphatase or protein kinase A alpha-subunit, which strongly affected open state probability, was also incapable of reducing the outward single channel current. Mg2+ ions applied from the cytosolic side induced concentration- and voltage-dependent block of the outward single channel currents with a Kd of 7.9 +/- 2.3 mM, resulting in inward rectification qualitatively similar to that observed in cell-attached patches. An increase in the Mg2+ concentration of the intracellular solution induced a significant decrease in the outward whole-cell current at depolarized potentials. Another putative endogenous channel blocker, the polyamine putrescine, was not effective. However, its metabolites spermidine and spermine reduced the amplitude of the outward single channel current with Kd values of 4.9 +/- 0.6 and 1.4 +/- 0.4 mM, respectively. Pre-incubation of the cells with the irreversible inhibitor of polyamine synthesis difluoromethylornithine abolished the rectification in the cell-attached patches. These results suggest that intracellular polyamines may underlie at least part of the inward rectification of the Ca2+ activated K+ channel in this tissue, but that intracellular Mg2+ is unlikely to play a major role.

The effects of dihydropyridine calcium antagonists on whole-cell Ca2+ and K+ currents in the neurosecretory bag cells of the marine mollusc Aplysia californica have been investigated. Nifedipine and nisoldipine blocked bag cell Ca2+ currents with effects similar to those seen previously on Ca2+ currents in cardiac muscle: both compounds appeared to interact with Ca2+ channels when they were closed, open, and inactivated. Also, as seen in cardiac cells, nifedipine apparently binds with higher affinity to Ca2+ channels when they are inactivated than when they are either closed or open. Nifedipine and nisoldipine also inhibited 2 outward K+ currents in bag cells: the “delayed rectifier” (IK) and the “A” (IA) currents. Half-maximal blockade of Ca2+ currents occurred at approximately 1.4 microM nifedipine, compared to approximately 3–5 microM for half-maximal blockade of IK and IA. The effects of these compounds on bag cell Ca2+ and K+ currents are interpreted and discussed here in terms of the “modulated receptor” model of drug action. In contrast, however, no measurable effects of nifedipine or nisoldipine were seen on Ca2+ (and/or K+) currents in several vertebrate neuronal cell types. Our results suggest that there are likely to be structural and/or conformational variations in Ca2+ channels in different cells, tissues, and/or species and also that, in some cells, Ca2+ and K+ channels might be structurally similar. These findings also suggest, therefore, that if dihydropyridine binding is used to identify Ca2+ channels, care should be taken to ensure that binding correlates closely with the Ca2+ channels of interest.

The response of pulmonary arteries to hypoxia varies as a function of vessel diameter. Small intrapulmonary resistance arteries are thought to be the main site of hypoxic pulmonary vasoconstriction (HPV), with hypoxia causing minimal contraction or even dilatation in large, conduit vessels. This has been proposed to reflect a differential distribution of morphologically and electrophysiologically distinct pulmonary artery smooth muscle (PASM) cells. We investigated longitudinal heterogeneity in smooth muscle cells isolated from five regions of the rabbit pulmonary vasculature and could find no evidence of morphological heterogeneity at the level of the light microscope. PASM cells from main (8 mm outer diameter) and branch (5 mm) arteries and large ( 400 m) intrapulmonary arteries (IPA) were similar in shape and size, as indicated by cell capacitance (25 pF). PASM cells from medium (200-400 m) and small ( 200 m) IPA were significantly smaller (15 pF), but had the same classical spindle shape. Cells from all five regions also had similar resting membrane potentials and displayed voltage-activated K+ currents of similar amplitude when recorded in standard physiological solution. Longitudinal heterogeneity in K+ current became apparent when tetraethylammonium ions (TEA; 10 mM) and glibenclamide (10 M) were added. The remaining delayed rectifier current (IK(V)) doubled in amplitude upon moving down the pulmonary arterial tree from the main artery (9 pA pF-1 at 40 mV) to the large IPA (17 pA pF-1), but remained constant throughout the intrapulmonary vasculature. The O2-sensitive, non-inactivating K+ current (IK(N)) showed a similar trend, but was significantly reduced in the smallest IPA, where its amplitude was comparable with the main artery. Thus the IK(N)/IK(V) ratio was relatively constant, at around 0.14, from the main pulmonary artery to medium IPA, but fell by 50% in the smallest vessels. The amplitude of the TEA-sensitive K+ current was similar (16 pA pF-1 at 40 mV) at all levels of the pulmonary arterial tree, except in the medium sized vessels where it was 50% smaller. These variations in K+ current expression correlate with reported variations in sensitivity to hypoxia and may contribute to the regional heterogeneity of HPV in the rabbit lung.

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