adenosine-5--(n-ethylcarboxamide) and calmidazolium

PC12 cells habituate during repetitive stimulation with acetylcholine, bradykinin, or high potassium. Interspersing these stimulants did not affect the rate of habituation of the others, but it could modulate the amplitude of the norepinephrine secretion each could achieve. Stimulation with acetylcholine inhibited norepinephrine secretion caused by high potassium and bradykinin stimulation, while high potassium had no effect on acetylcholine or bradykinin, and bradykinin increased secretion caused by acetylcholine. Changes in norepinephrine secretion resulting from any of these stimulants correlated with changes in internal calcium levels. Cyclic AMP-, protein kinase C-, and calmodulin-dependent second messenger pathways all modulated norepinephrine secretion caused by acetylcholine and high potassium and showed a distinct hierarchy in their effectiveness. These data demonstrate that different receptor pathways can change the norepinephrine response of one another while not changing the levels of the molecules responsible for habituation.

Topics: Acetylcholine; Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Bradykinin; Calcium; Calmodulin; Cyclic AMP; Cytosol; Habituation, Psychophysiologic; Imidazoles; Kinetics; Membrane Potentials; Norepinephrine; PC12 Cells; Potassium; Protein Kinase C; Rats; Second Messenger Systems; Tetradecanoylphorbol Acetate; Vasodilator Agents

Many antidepressants inhibit 5-hydroxytryptamine (5HT) transport resulting in increased 5HT levels in the synapse. However, physiological regulation of neurotransmitter uptake has not been demonstrated. We have examined the effect of receptor-activated second messengers on the 5HT transporter in rat basophilic leukemia cells (RBL 2H3). Here, we show that activation of an A3 adenosine receptor results in an increase of 5HT uptake in RBL cells, due to an increase in maximum velocity (Vmax). The A3 adenosine receptor-stimulated increase in transport is blocked by inhibitors of nitric oxide synthase and by a cGMP-dependent kinase inhibitor. In fact, compounds that generate nitric oxide (NO) and the cGMP analog 8-bromo-cGMP mimicked the effect of A3 receptor stimulation, suggesting that the elevation in transport occurs through the generation of the gaseous second messenger NO and a subsequent elevation in cGMP. Additionally, the 5HT transporter is differentially regulated by second messengers since direct activation of protein kinase C by phorbol esters decreases 5HT uptake by decreasing Vmax. Our results suggest that the changes in transport are due to a direct modification of the 5HT transporter, possibly by phosphorylation, which appears to alter the rate at which transport occurs. As the 5HT transporter in RBL cells is identical to that in neurons, our results suggest that analogous mechanisms may operate in the brain.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Amino Acid Oxidoreductases; Amino Acid Sequence; Aminoquinolines; Animals; Biological Transport; Carrier Proteins; Cell Membrane; Cyclic GMP; GTP-Binding Proteins; Imidazoles; In Vitro Techniques; Membrane Glycoproteins; Membrane Transport Proteins; Methylene Blue; Molecular Sequence Data; Nerve Tissue Proteins; Nitric Oxide; Nitric Oxide Synthase; Paroxetine; Protein Kinase C; Rats; Receptors, Purinergic P1; Second Messenger Systems; Serotonin; Serotonin Plasma Membrane Transport Proteins; Signal Transduction; Tumor Cells, Cultured