Regulation of transmitter release by muscle length in frog motor nerve terminals. Dynamics of the effect and the role of integrin-ECM interactions.

Changes in muscle length cause large changes in the probability of transmitter release from frog motor nerve terminals. A 5% to 10% stretch from rest length can increase EPP amplitude or mEPP frequency by more than 100%. The phenomenon is fully reversible and extremely rapid. Within 7-10 milliseconds of the stretch, the enhancement is complete, and it is maintained essentially constant at the new level for as long as the stretch is sustained. Given these properties, the length modulation of release is unquestionably of functional importance, strongly amplifying the spinal stretch reflex. The stretch-induced enhancement of transmitter release persists at a reduced level in a 0 Ca++, 2 mM Mg++ Ringer. This finding indicates a lack of dependence on Ca++ influx from outside the terminal. Release of Ca++ from intracellular stores close to release sites cannot be ruled out as a contributing factor. Our results, however, suggest a mechanism involving physical connections between the extracellular matrix and the nerve terminal that can alter release probability directly. Morphological evidence for connections that might be responsible can be demonstrated in micrographs of deep-etched freeze fractures through neuromuscular junctions. Hypothesizing that the ECM-nerve terminal connections responsible for the stretch effect involve proteins from the integrin family and knowing that many of the integrin-ECM binding interactions occur at sites on the ECM proteins containing the amino acid sequence RGD, we treated preparations with 0 Ca++, 2 mM Mg++ Ringer to reduce integrin binding and then returned the muscle to normal Ringer containing 0.1-0.2 mM of a six-amino-acid peptide containing the RGD sequence. This peptide strongly suppressed the stretch effect, while a control peptide (RGE) had no effect. A 50 microM Ca++/50 microM Mg++ Ringer had little effect on stretch enhancement but permitted a strong inhibition of enhancement when RGD was present. The identity of the ECM molecule(s), the integrin(s), and the mechanism of enhancement of release are unknown. However, our findings imply that much or all of the length-dependent modulation of release probability is mediated by an RGD-sensitive integrin-ECM interaction that depends more on external Ca++ than on Mg++.