After perfusion with enzyme, the heart was taken down from the cannula, pinned out right side uppermost and the AVN area excised as described previously . The excised tissue was chopped up and shaken gently with enzyme to release cells, which were then stored in a high potassium, low calcium KB storage solution . The composition of the KB solution was as follows (in mmol/L): L-glutamate 100, KCl 30, sodium pyruvate 5, taurine 20, creatine 5, succinic acid 5, Na2ATP 2, P-OH butyrate 5, glucose 20, MgCl2 5, EGTA 1 and HEPES 10, titrated to a pH of 7.2 by addition of KOH.
For recording, AVN cells were placed in a chamber mounted on an inverted microscope (Nikon Diaphot, Tokyo, Japan) and superfused with Tyrode’s solution containing (in mmol/L) NaCl 130, KCl 2.7, CaCl2 2 , MgCl2 1, glucose 10 and HEPES 5, titrated to a pH of 7.4 by addition of NaOH. For protocols investigating the steady state inactivation of Ica,L, N-methyl-D-glucamine was substituted for sodium to block the sodium current (INa), and 40 |iM Ni was added to block T-type calcium current . AVN cells were placed in the chamber as a cell suspension in KB storage medium and allowed to settle on the glass bottom for 20 s. Perfusion was then started at a slow rate and gradually increased (to 2 to 4 mL/min). In this way, flow rate could be increased without cells becoming dislodged, and as the calcium-containing bath solution replaced KB, the cells were slowly re-exposed to calcium. It’s time to spend less every time you need your treatment with fast delivery: you can now purchase the amount of zoloft generic you need for the treatment within a very short period, at the best pharmacy that deserves your trust.