Microinjection of calmodulin antibodies into cultured chromaffin cells blocks catecholamine release in response to...

Polyclonal monospecific antibodies raised in sheep against rat testis calmodulin demonstrated cross-reactivity with bovine adrenal medullary chromaffin cell calmodulin. This antibody immunoprecipitated a [35S]methionine-labelled protein from chromaffin cell extracts prepared from [35S]methionine prelabelled cells that comigrated on a sodium dodecylsulfate gel electrophoresis system with calmodulin. In addition, an excess of non-radioactive exogenous calmodulin was shown to readily compete with this labelled endogenous protein for the antibodies' binding sites. Erythrocyte ghosts were used as vehicles for microinjecting either preimmune immunoglobulin G or anti-calmodulin immunoglobulin G into chromaffin cells following a polyethylene glycol-induced cell fusion procedure. The efficiency of ghost cell fusion was monitored and found to be 43.6 +/- 1% (n = 33). Cell morbidity subsequent to fusion and microinjection was negligible (87.8 +/- 0.6% of the total cell population were viable cells; n = 33) as determined by the Trypan Blue exclusion test. The delivery of intact antibodies raised against calmodulin directly into the cytoplasm of cultured chromaffin cells by erythrocyte ghost-mediated microinjection, inhibited catecholamine output in response to stimulation by either acetylcholine (10(-4) M) or a depolarizing concentration of potassium (56 mM). However, under these conditions, the chromaffin cell's ability to accumulate exogenous catecholamines through a high affinity uptake system, as well as the kinetic parameters that characterize this uptake mechanism remained unaltered. Furthermore, microinjection of preimmune immunoglobulin G did not modify either catecholamine uptake or stimulation-induced amine release from chromaffin cells. It therefore appears that calmodulin may play a role in the process of stimulus-secretion coupling in the chromaffin cell in culture while it is of little significance to the high affinity amine uptake mechanism.