Salient features of polyamine/Ca-activated MEND using nucleotide-free standard cytoplasmic solutions. (A) In contrast to other MEND-promoting agents, spermidine does not cause MEND after a Ca transient has occurred; it must be present in the cytoplasm during the Ca transient. (B) Ca-activated MEND with spermidine is often small at the first Ca influx episode but large and rapid at a second Ca transient. Thus, MEND undergoes long-term facilitation by a mechanism that does not involve phosphorylation. (C) The occurrence of MEND at the first Ca influx episode is more reliable using 2 mM EDA as polyamine in both intracellular and extracellular solutions (n = 6). (D) Whereas 0.5 mM GTPγS blocks ATP-dependent MEND (Fig. 5 A), EDA/Ca-activated MEND is unaffected (n = 7). Results in C and D are paired experiments from one batch of BHK cells.