Cyclic 3',5' AMP relay in Dictyostelium discoideum. II. Requirements for the initiation and termination of the response.

The secretion of 3H-cyclic adenosine 3',5'-monophosphate (cAMP) by prelabeled and suitably differentiated Dictyostelium discoideum amoebae was elicited in a perfusion apparatus by 10(-10) to 10(-5) M [14C]cAMP stimuli of defined magnitude and duration. Exogenous stimuli evoked an immediate increase in the rate of [3H]cAMP secretion which accelerated continuously to reach a peak of up to 100 times the unstimulated rate after 2--3 min of stimulation. Withdrawal of the stimulus at any time during the response led to a rapid decline to basal levels.

Cyclic 3',5' AMP relay in Dictyostelium discoideum. I. A technique to monitor responses to controlled stimuli.

A perfusion technique was developed to deliver [14C]adenosine 3',5'-cyclic monophosphate (cAMP) stimuli of well-defined magnitude and duration to tritium-labeled Dictyostelium discoideum amoebae and simultaneously monitor the elicited secretion of [3H]cAMP (i.e., the relay response).

Metabolism of acetylcholine receptors in skeletal muscle.

The acetylcholine receptor in skeletal muscle is an integral plasma membrane glycoprotein. Its biosynthesis and incorporation into plasma membrane and its degradation are being studied with the use of biochemical, biophysical, and microscopic techniques. In this report, previously published data are combined with new information to yield a consistent and fairly detailed description ofthe mechanisms involved in receptor metabolism.

Newly synthesized acetylcholine receptors are located in the Golgi apparatus.

Chick skeletal muscle cells in tissue culture were fixed and treated with saponin to allow [125I]alpha-bungarotoxin access into the cells while preserving ultrastructure. The kinetics of binding of iodinated alpha-bungarotoxin to intracellular acetylcholine (ACh) receptors and to surface A Ch receptors were comparable. About half of the intracellular ACh receptors are newly synthesized and in the pathway leading to incorporation into the plasma membrane.

Kinetics of biosynthesis of acetylcholine receptor and subsequent incorporation into plasma membrane of cultured chick skeletal muscle.

20% of the acetylcholine receptors in cultured chick skeletal muscle remain unbound following long-term growth of muscle in medium containing a potent, essentially irreversible receptor-blocking agent, alpha-bungarotoxin. About half the receptors which are unavailable for interaction with extracellular alpha-bungarotoxin are newly synthesized molecules which presumably are being processed and transported to the plasma membrane. When the muscle cultures are switched to a medium containing 2H, 13C, 15N-amino acids, these receptors are rapidly labeled, the fraction of labeled molecules beginning to plateau at 3 hr.

Synthesis of acetylcholine receptors by cultured chick myotubes and denervated mouse extensor digitorum longus muscles.

Mono-[125I]iodo- alpha - bungarotoxin - receptor complexes extracted from chick myotube cultures as well as from adult denervated extensor digitorum longus muscles of the mouse have been banded at their buoyant density in gradients of metrizamide-deuterium oxide. When cultures or denervated adult muscles are preincubated in media containing 2H- or 13C-substituted amino acids under conditions of active receptor accumulation, the mono-[125I]iodo-alpha-bungarotoxin-receptor complexes have an increased buoyant density and band in a position of higher density in the gradient relative to a marker of mono-[131I]iodo-alpha-bungarotoxin-receptor complexes extracted from cells preincubated in normal media. It is concluded that the accumulation of receptors on the surfaces of cultured chick myotubes and on the non-synaptic surfaces of extensor digitorum longus muscles following denervation are the result of de novo synthesis.

Acetylcholine receptor turnover in membranes of developing muscle fibers.

[125I mono-iodo-alpha-bungarotoxin is used as a specific marker in a description of acetylcholine receptor metabolism. It is concluded that acetylcholine receptors in the surface membranes of chick and rat myotubes developing in cell cultures have a half-life of 22-24 h. Alpha-bungarotoxin (bound to a receptor which is removed from the membrane) is degraded to monoiodotyrosine which appears in the medium.