GTPγS and phorbol ester act synergistically to stimulate both Ca2+ -independent secretion and phospholipase D activity in permeabilized human platelets : Inhibition by BAPTA and analogues

A new family of high-affinity buffers and optical indicators for Ca2+ is rationally designed and synthesized. The parent compound is 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), a relative of the well-known chelator EGTA [ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid] in which methylene links between oxygen and nitrogen are replaced by benzene rings. BAPTA and its derivatives share the high (greater than 10(5)) selectivity for Ca2+ over Mg2+ of EGTA but are very much less affected by pH changes and are faster at taking up and releasing Ca2+. The affinity of the parent compound for Ca2+ (dissociation constant 1.1 x 10(-7) M in 0.1 M KCl) may be strengthened or weakened by electron-releasing or -withdrawing substituents on the aromatic rings. The Ca2+ and Mg2+ affinities may further be altered by replacing the ether oxygens by heterocyclic nitrogen atoms. The compounds described are fluorescent Ca2+ indicators absorbing in the ultraviolet region; the very large spectral shifts observed on binding Ca2+ fit the prediction that complexation should hinder the conjugation of the nitrogen lone-pair electrons with the aromatic rings. Derivatives with quinoline nuclei are notable for their high sensitivity of fluorescent quantum yield to the binding of Ca2+ but not of Mg2+. Preliminary biological tests have so far revealed little or no binding to membranes or toxic effects following intracellular microinjection.

A number of calcium buffers were examined for their ability to reduce evoked transmitter release when injected into the presynaptic terminal of the squid giant synapse. Injection of EGTA was virtually ineffective at reducing transmitter release, even at estimated intracellular concentrations up to 80 mM. Conversely, the buffer 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), which has an equilibrium affinity for calcium similar to that of EGTA at pH 7.2, produced a substantial reduction in transmitter release when injected presynaptically. This effect of BAPTA was reversible, presumably because the buffer diffused out of the terminal and into uninjected regions of the presynaptic axon. BAPTA derivatives with estimated intracellular calcium dissociation constants (Kd) ranging from 0.18 to 4.9 microM were effective at reducing transmitter release at similar estimated concentrations. A BAPTA derivative with an estimated intracellular Kd of 31 mM was less effective. BAPTA did not affect presynaptic action potentials or calcium spikes in ways that could explain its ability to reduce transmitter release. The relative effects of presynaptic injections of BAPTA and derivatives are consistent with the calcium-buffering capability of these compounds if the presynaptic calcium transient that triggers release is hundreds of microM or larger. The superior potency of BAPTA compared to EGTA apparently results from the faster calcium-binding kinetics of BAPTA and suggests that the calcium-binding molecule that triggers release binds calcium in considerably less than 200 microsec and is located very close to calcium channels.

We have determined or redetermined the calcium dissociation constants of seven BAPTA-type buffers with KD's in the range from 0.4 microM to about 20 mM in 300 mM KCl. These include four newly synthesized ones: 5-nitro BAPTA; 5,5'-dinitro BAPTA; 5-methyl-5'-nitro BAPTA; and 5-methyl-5'-formyl BAPTA. Moreover, we tabulate dissociation constants or KD's for BAPTA and eleven BAPTA-type buffers, compare most of them with an empirical curve based upon so-called Hammett values, and predict KD's for several still unsynthesized but potentially valuable buffers.