Date of Award

1984

Embargo Period

8-1-2024

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Pharmacology

College

College of Graduate Studies

First Advisor

Jerry G. Webb

Abstract

The objective of this study was to evaluate the hypothesis that nerve terminals may serve as a site for storage and release of beta adrenergic receptor blocking drugs. Experiments were conducted with two different in vitro neural preparations, cultured superior cervical ganglia from adult rats and synaptosomes prepared from rat cerebral cortex. The release of propranolol by depolarizing stimuli was examined and compared with the release of the more hydrophilic, cardioselective beta receptor antagonist atenolol. In both neuronal models, norepinephrine release was monitored as an index of exocytosis. Cultured superior cervical ganglia of the rat represent an in vitro model for adrenergic neurons which contains no post junctional effector tissue. Ganglia excised from adult rats and maintained in a chemically defined media for 48 hours develop axonal sprouts that have uptake, storage and release mechanisms characteristic of sympathetic nerve endings. Electrical field stimulation of 48-hour cultured ganglia preloaded with either 3H-propranolol or 3H-norepinephrine was found to promote the release of each molecule in a frequency-dependent manner. Stimulation for 30 seconds at a frequency of 15 Hz released 3.00% of the norepinephrine and 0.15% of the propranolol accumulated by the ganglia. Removal of extracellular calcium and elevation of magnesium reduced the stimulation-induced release of norepinephrine (control = 161 ± 31 fmol/30 sec/ganglion) by 60% but did not affect the stimulation-induced release of propranolol (control = 17 ± 5 fmol/30 sec/ ganglion). Norepinephrine release was also inhibited by bretylium and enhanced by phenoxybenzamine, whereas neither agent altered the stimulation-induced release of propranolol. Moreover, reserpine pretreatment almost abolished norepinephrine accumulation and release without affecting the accumulation or release of propranolol. These results demonstrate that propranolol is released from neural tissue in response to electrical stimulation. In addition, the data indicate that propranolol release from cultured ganglia occurs by a different mechanism than that observed for norepinephrine and appears to result from a calcium-independent, presumably nonexocytotic, process. Synaptosomes were prepared by discontinuous sucrose density gradient centrifugation and used as a model of isolated nerve terminals. When incubated at 30°C with radiolabelled drugs (0.1 µM), synaptosomes accumulated 20 pmoles propranolol and 0.6 pmoles atenolol/mg protein. Exposure of 3H-propranolol-loaded synaptosomes to elevated potassium, rubidium or cesium was found to evoke a concentration-dependent increase in drug efflux. The action of these ions to release propranolol was highly correlated with their ability to produce synaptosomal membrane depolarization as estimated with the voltage-sensitive dye diS-C3-(5). When external potassium was increased to 75 mM, 25% of the propranolol accumulated by the synaptosomes was released within 15 seconds. Elevated external potassium also promoted the efflux of 3H-atenolol from preloaded synaptosomes, with 75 mM potassium releasing in three minutes 25% of the atenolol taken up. 3H-Norepinephrine was released by potassium with a time course similar to that found for atenolol. Depolarization of synaptosomes with veratridine was also observed to stimulate the release of both propranolol and atenolol and these effects were antagonized by tetrodotoxin. Under calcium-free conditions, potassium-induced release of propranolol was reduced 37% while atenolol release was decreased 68% and norepinephrine release 82%. Pretreatment of rats with 6-hydroxydopamine to destroy adrenergic nerve terminals failed to alter the steady state accumulation or the potassium-induced release of either propranolol or atenolol from synaptosomes. The results from these studies demonstrate that both polar and nonpolar beta receptor antagonists are accumulated by nerve endings and released in response to membrane depolarization. The data also raise the possibility that these drugs may be released from nonadrenergic as well as adrenergic neurons. The depolarization-induced release of propranolol from cultured sympathetic ganglia and synaptosomes is consistent with previous findings of propranolol release in association with sympathetic nerve stimulation in situ. The release of atenolol extends these observations to include polar beta receptor antagonists as well as the lipophilic propranolol molecule and suggests that this phenomenon may apply to the entire class of these drugs. While atenolol release has the characteristics of an exocytotic event, propranolol release from these preparations appears to be complex and may involve a combination of exocytotic and nonexocytotic mechanisms. The results of the present study support the concept that neural storage and release of beta adrenergic receptor blocking drugs may contribute to their pharmacological actions by modulating drug concentration at localized sites of action.

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