Ethics in research.
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Conflict of Interest↗
Biomedical subjects
C D Dunigan
Publications and source records attributed to C D Dunigan.
Detection of beta-adrenergic receptors by radioligand binding.
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Animals↗
Cation requirements of basal and ATP-regulated dopamine transport in rat pheochromocytoma cells.
The transport of dopamine into presynaptic nerve terminals is the primary mechanism for the termination of dopaminergic neurotransmission. This transport process has recently been found to be composed of two components, a basal dopamine transport pathway which exists in the absence of extracellular ATP and an ATP-regulated moiety which comprises approximately 66% of the total transport system [Cao C. J. et al. (1990) Biochem. Pharmac. 39, R9-R14; Cao C. J. et al. (1989) Biochemistry 8, 207-220; Dunigan C. D. and Shamoo A. E. (1995) Neuroscience 65, 1-4; Eshleman A. et al. (1995) Life Sci. 56, 1613-1621]. Using a rat pheochromocytoma cell line and a Krebs bicarbonate buffering system, the present study examined the effect of several cations on both basal and ATP-regulated dopamine transport. In the absence of extracellular ATP, dopamine transport had an absolute dependence on the presence of Na+, but exhibited no requirement for Mg2+. Kinetically, the addition of 120 mM NaCl increased the Vmax of basal dopamine transport by approximately 150%. In contrast, the ATP-regulated dopamine transport pathway displayed a different sensitivity to Na+ and was completely dependent upon the presence of Mg2+. The addition of 1.2 mM MgSO4 increased the Vmax of transport in the presence of 0.7 mM extracellular ATP by 222%. Both basal and ATP-regulated transport were unaffected by the removal of either Ca2+ or K+ from the assay buffer. When the effects of ouabain, a potent inhibitor of Na+, K(+)-ATPase, were tested in the rat pheochromocytoma cell model, it was found that concentrations of ouabain as high as 1 mM were ineffective at inhibiting either the basal or ATP-regulated dopamine transport components. These results imply that the Na+ gradient supplied by Na+, K(+)-ATPase is not the sole provider of energy needed to drive either transport process. The ionic requirements of the basal and ATP-regulated dopamine transport pathways demonstrate the distinction between the two transport processes. In addition, the ionic dependency profile of the ATP-regulated moiety has provided some mechanistic insights into ATP-regulated catecholamine uptake, as the absolute Mg2+ requirement and the ineffectiveness of Ca2+ argues against the involvement of either purinergic receptors or a Ca(2+)-dependent, Mg(2+)-independent ectokinase in the ATP-regulated transport system.
Adenosine Triphosphate↗
Identification of the major transport pathway for the parkinsonism-inducing neurotoxin 1-methyl-4-phenylpyridinium.
1-Methyl-4-phenylpyridinium is a potent parkinsonism-inducing neurotoxin which has become a valuable tool for the examination of the mechanisms and therapeutic treatment strategies for Parkinson's syndrome. Recently, it has been found that physiological levels of extracellular ATP (0.1-1 mM) stimulate dopamine uptake into both rat and bovine brain synaptosomes and rat pheochromocytoma cells in a dose-dependent manner. In this study we report that physiological levels of extracellular ATP (0.1-2 mM) stimulate the transport of 1-methyl-4-phenylpyridinium into the pheochromocytoma cell line by 270% over basal levels. Kinetically, the presence of ATP increases both the K(m) and Vmax of 1-methyl-4-phenylpyridinium transport. In addition, 1-methyl-4-phenylpyridinium is far more effective at inhibiting ATP-stimulated dopamine transport (IC50 = 11 microM) than basal dopamine transport (IC50 100 microM) into pheochromocytoma cells. These data show that the ATP-regulated 1-methyl-4-phenylpyridinium transport pathway is the major component (approximately 95%) of total 1-methyl-4-phenylpyridinium transport, and provide the first evidence for the involvement of extracellular ATP in the bulk transport of 1-methyl-4-phenylpyridinium.
1-Methyl-4-phenylpyridinium↗
Li+ stimulates ATP-regulated dopamine uptake in PC12 cells.
Increased levels of dopamine have been associated with schizophrenia and mania; conversely, decreased levels of dopamine are associated with depression. Since the main mechanism for the termination of dopamine's pharmacological action is by re-uptake into the presynaptic cell, the speed of dopamine transport dramatically influences the concentration of dopamine present in the synaptic cleft, which in turn could determine brain disorders. Our preliminary studies have found that ATP can stimulate dopamine transport in rat synaptosomal preparation. We have also observed this ATP-regulated moiety of the dopamine uptake system when tested in PC12 cells. The large magnitude of ATP stimulation suggests that this dopamine uptake pathway may be important in the etiology and treatment of brain disorders. In order to test the relevance of ATP-stimulated dopamine uptake, we tested the effect of lithium salts on this system. Lithium chloride, one of the first drugs used in the treatment of mania, has become one of the most important agents utilized for the treatment of manic-depression and many schizoeffective disorders. Unfortunately, despite all efforts that have been made to explain lithium's mode of action, a clear cut biochemical mechanism has not been defined. We have found that lithium chloride, at therapeutic levels, is able to stimulate the ATP-regulated component of the dopamine uptake system by 49%. The further enhancement of dopamine re-uptake by lithium ions is consistent with its therapeutic effect. It is suggested that any substance that facilitates dopamine re-uptake could be of great importance in defining a useful treatment for mania and schizophrenia, as well as depression.
Adenosine Triphosphate↗
Uncoupling of occlusion from ATP hydrolysis activity in sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase.
The uncoupling of Ca2+ transport from ATP hydrolysis in the sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase by trypsin digestion was re-investigated by comparing ATPase activity with the ability of the enzyme to occlude Eu3+ (a transport parameter) after various tryptic digests. With this method, re-examination of uncoupling by tryptic digest of the ATPase revealed that TD2 cleavage (Arg-198) had no effect on either occlusion or ATPase activity. Digestion past TD2 in the presence of 5 mM Ca2+ and at 25 degrees C resulted in the loss of about 70% of the ATPase activity, but no loss of occlusion. Digestion past TD2 in the presence of 5 mM Ca2+, 3 mM ATP, and at 25 degrees C resulted in a partially uncoupled enzyme complex which retained about 50% of the ATPase activity, but completely lost the ability to occlude Eu3+. Digest past TD2 in the presence of 5 mM Ca2+ and 3 mM AMP-PNP (a non-hydrolyzable ATP analog) at 25 degrees C resulted in no loss of occlusion, thus revealing the absolute requirement of ATP during the digest to eliminate occlusion. From these findings we conclude that uncoupling of Ca2+ transport from ATPase activity is possible by tryptic digestion of the (Ca2+ + Mg2+)-ATPase. Interestingly, only after phosphorylation of the enzyme do the susceptible bond(s) which lead to the loss of occlusion become exposed to trypsin.
Adenosine Triphosphate↗