Single nisoldipine-sensitive calcium channels in smooth muscle cells isolated from rabbit mesenteric artery.

Abstract

Single smooth muscle cells were enzymatically isolated from the rabbit mesenteric artery. At physiological levels of external Ca, these cells were relaxed and contracted on exposure to norepinephrine, caffeine, or high levels of potassium. The patch-clamp technique was used to measure unitary currents through single channels in the isolated cells. Single channels were selective for divalent cations and exhibited two conductance levels, 8 pS and 15 pS. Both types ofchannels were voltage-dependent, and channel activity occurred at potentials positive to -40 mV. The activity of both channel types was almost completely inhibited by 50 nM nisoldipine. These channels appear to be the pathways for voltage-dependent Ca influx in vascular smooth muscle and may be the targets of the clinically used dihydropyridines.Calcium ions that enter cells through voltage-dependent pathways, Ca channels, regulate a wide variety of physiological functions, including contraction of vascular smooth muscle (VSM) (1-3); the degree of contraction of VSM cells determines the arterial resistance to blood flow. VSM appears to be the site of action of the dihydropyridines, an important class of therapeutic drugs (4,5). Because these Ca antagonists are potent vasodilators, the dihydropyridines are prescribed for certain types of cardiovascular disorders, such as angina and hypertension. The selectivity of these drugs in relaxing VSM seems to be related to the greater affinity of these agents for Ca-influx pathways in VSM than similar pathways in such other tissue types as heart and nerve (5, 6).Voltage-dependent Ca-influx pathways in VSM have been studied primarily by the technique of depolarizing the cell with high external potassium concentration to measure Ca isotope influx and muscle contraction (7-9). However, difficulties in isolating viable single cells from VSM have impeded investigation of the electrophysiological properties of these important Ca-influx pathways.We report a procedure for isolating physiologically responsive single smooth muscle cells from the rabbit mesenteric artery. Single Ca channels from these cells were identified by measuring unitary current fluctuations in excised membrane patches using the patch-clamp technique. Single Ca channels exhibited two different conductance levels, -8 pS and 15 pS (in 80 mM barium). The activity of both channel types was reduced by the dihydropyridine nisoldipine (37). METHODS AND MATERIALSCell Isolation. Single smooth muscle cells were enzymatically isolated from the rabbit (New Zealand White) mesenteric artery (200-to 300-,um diameter). Following dissection, the artery was placed in Hanks' solution (137 mM NaCl/5.4 mM KCl/0.44 mM KH2PO4/0.42 mM NaH2PO4/4.17 mM NaCO3/5.55 mM glucose/0.02 mM EGTA/2 mM MgCl2/0.2 mM CaCl2/10 mM Hepes/NaOH, pH 7.4), and cleared of connective and fat tissue. A 5-mm segment of the artery was next pulled over a glass cannula to allow a perfusion of Hanks' solution containing collagenase at 1.0 mg/ml (CLS-II, Cooper Biomedicals, Malvern, PA), elastase...

Purified skeletal muscle 1,4-dihydropyridine receptor forms phosphorylation-dependent oligomeric calcium channels in planar bilayers.

“…These values more than cover the range of reported conductances, and the often-seen 7.5-and 15-pS events match the values found in smooth muscle cells (34). Most remarkably, oligochannels showed marked voltage dependence in contrast to monochannels, closely matching the expectation from incorporated T-tubule membranes, whereas Ba2+ selectivity remained unchanged during the transition from monomeric to oligomeric channels.…”

Low‐voltage‐activated calcium current in rat aorta smooth muscle cells in primary culture.

“…On the contrary, ICa, HVA of this preparation seems to be less sensitive to the dihydropyridine Ca2+ antagonist compared with that of vascular SMC in other portions such as rat portal vein (Loirand et al 1986), canine saphenous vein (Yatani et al 1987) and rabbit mesenteric artery (Worley, Deitmer & Nelson, 1986), in which preparations the IC50 for the blocking effect of the dihydropyridine Ca2+ antagonist was of the order of 10-8 M.…”

Effects of pinaverium on voltage‐activated calcium channel currents of single smooth muscle cells isolated from the longitudinal muscle of the rabbit jejunum

“…Worley et al, 1986;Benham et al, 1987;Yatani et al, 1987). In some smooth muscles it has been inferred that these separate populations contribute components to the voltage-dependent whole-cell calcium current which can be identified by various voltage protocols.…”