Binding and effects of KATP channel openers in the vascular smooth muscle cell line, A10.
1. The ATP-sensitive K+ channel (KATP channel) in A10 cells, a cell line derived from rat thoracic aorta, was characterized by binding studies with the tritiated KATP channel opener, [3H]-P1075, and by electrophysiological techniques. 2. Saturation binding experiments gave a KD value of 9.2 +/- 5.2 nM and a binding capacity (BMax) of 140 +/- 40 fmol mg-1 protein for [3H]-P1075 binding to A10 cells; from the BMax value a density of binding sites of 5-10 per microns2 plasmalemma was estimated. 3. KATP channel modulators such as the openers P1075, pinacidil, levcromakalim and minoxidil sulphate and the blocker glibenclamide inhibited [3H]-P1075 binding. The extent of inhibition at saturation depended on the compound, levcromakalim inhibiting specific [3H]-P1075 binding by 85%, minoxidil sulphate and glibenclamide by 70%. The inhibition constants were similar to those determined in strips of rat aorta. 4. Resting membrane potential, recorded with microelectrodes, was -51 +/- 1 mV. P1075 and levcromakalim produced a concentration-dependent hyperpolarization by up to -25 mV with EC50 values of 170 +/- 40 nM and 870 +/- 190 nM, respectively. The hyperpolarization induced by levcromakalim (3 microM) was completely reversed by glibenclamide with an IC50 value of 86 +/- 17 nM. 5. Voltage clamp experiments were performed in the whole cell configuration under a physiological K+ gradient. Levcromakalim (10 microM) induced a current which reversed around -80 mV; the current-voltage relationship showed considerable outward rectification. Glibenclamide (3 microM) abolished the effect of levcromakalim. 6. Analysis of the noise of the levcromakalim (10 microM)-induced current at -40 and -20 mV yielded estimates of the channel density, the single channel conductance and the probability of the channel to be open of 0.14 micron-2, 8.8 pS and 0.39, respectively. 7. The experiments showed that A10 cells are endowed with functional KATP channels which resemble those in vascular tissue; hence, these cells provide an easily accessible source of channels for biochemical and pharmacological studies. The density of binding sites for [3H]-P1075 was estimated to be one order of magnitude higher than the density of functional KATP channels; assuming a plasmalemmal localization of the binding sites this suggests a large receptor reserve for the openers in A10 cells.
Russ
U
U
Department of Pharmacology, Medical Faculty, University of Tübingen, Germany.
Metzger
F
F
Kickenweiz
E
E
Hambrock
A
A
Krippeit-Drews
P
P
Quast
U
U
D000818:Animals; D001013:Aorta, Thoracic / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D001667:Binding, Competitive; D002460:Cell Line; D019806:Cromakalim / Q000378:metabolism / Q000494:pharmacology; D005905:Glyburide / Q000378:metabolism / Q000494:pharmacology; D006146:Guanidines / Q000378:metabolism / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects; D008914:Minoxidil / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D018408:Patch-Clamp Techniques; D020110:Pinacidil; D026902:Potassium Channel Blockers; D015221:Potassium Channels / Q000819:agonists*; D011725:Pyridines / Q000378:metabolism / Q000494:pharmacology; D011869:Radioligand Assay; D051381:Rats; D014665:Vasodilator Agents / Q000378:metabolism / Q000494:pharmacology
D006146:Guanidines; D026902:Potassium Channel Blockers; D015221:Potassium Channels; D011725:Pyridines; D014665:Vasodilator Agents; D019806:Cromakalim; C039516:minoxidil sulfate ester; D008914:Minoxidil; C073896:N-cyano-N'-(1,1-dimethylpropyl)-N''-(3-pyridinyl)guanidine; D020110:Pinacidil; D005905:Glyburide