[3H]-verapamil binding to rat cardiac sarcolemmal membrane fragments; an effect of ischaemia.
The [3H]-verapamil binding activity of rat cardiac sarcolemmal fragments was studied, using membranes harvested from non-perfused, aerobically-perfused and ischaemic hearts. Glass-fibre filters were found to contain specific, high affinity--(KD 38 +/- 3.1 nM) [3H]-verapamil binding sites--making them unsuitable for use in [3H]-verapamil binding studies. Incubation of membranes from non-perfused hearts in a medium containing 150 mM NaCl, 1 mM CaCl2 and 50 mM Tris revealed two populations of [3H]-verapamil binding sites. When centrifugation instead of filtration was used to separate bound and free [3H]-verapamil, high affinity sites with a KD of 0.57 +/- 0.19 microM and a Bmax of 38 +/- 5.2 pmol mg-1 protein, and low affinity sites with a KD of 78 +/- 27.5 microM and a Bmax of 2.9 +/- 1.3 nmol mg-1 protein were detected. However, only low affinity binding sites could be detected in membranes which had been incubated in a cation-free medium containing 50 mM Tris. [3H]-verapamil binding to the low and high affinity sites was saturable, reversible, stereospecific and displaceable by D600 greater than diltiazem greater than Ca2+ but not by nifedipine, nitrendipine, nisoldipine or prazosin. The two populations of binding sites survived aerobic perfusion and 60 min ischaemia at 37 degrees C. Ischaemia reduced the Bmax and KD but selectivity was maintained.
D000818:Animals; D015220:Calcium Channels; D003326:Coronary Circulation; D066298:In Vitro Techniques; D008297:Male; D009206:Myocardium / Q000378:metabolism*; D009568:Nitrendipine / Q000378:metabolism; D011869:Radioligand Assay; D051381:Rats; D011919:Rats, Inbred Strains; D011978:Receptors, Nicotinic / Q000378:metabolism*; D012508:Sarcolemma / Q000378:metabolism*; D014700:Verapamil / Q000378:metabolism*
D015220:Calcium Channels; D011978:Receptors, Nicotinic; D009568:Nitrendipine; D014700:Verapamil