D4 dopamine receptor-mediated phospholipid methylation and its implications for mental illnesses such as schizophrenia.

Previous studies have shown D2-like dopamine receptor involvement in the regulation of phospholipid methylation (PLM), while others have documented impaired methionine and folate metabolism in schizophrenia. Utilizing [14C]formate labeling in cultured neuroblastoma cell lines, we now show that D4 dopamine receptors (D4R) mediate the stimulatory effect of dopamine (DA) on PLM. The effect of DA was potently blocked by highly D4R-selective antagonists and stimulated by the D4R-selective agonist CP-226269. DA-stimulated PLM was dependent upon the activity of methionine cycle enzymes, but DA failed to increase PLM in [3H]methionine labeling studies, indicating that a methionine residue in the D4R might be involved in mediating PLM. A direct role for MET313, located on transmembrane helix No. 6 immediately adjacent to phospholipid headgroups, was further suggested from adenosylation, site-directed mutagenesis and GTP-binding results. A comparison of PLM in lymphocytes from schizophrenia patients vs control samples showed a four-fold lower activity in the schizophrenia group. These findings reveal a novel mechanism by which the D4R can regulate membrane composition. Abnormalities in D4R-mediated PLM may be important in psychiatric illnesses such as schizophrenia.

Punctate appearance of dopamine-beta-hydroxylase on the chromaffin cell surface reflects the fusion of individual chromaffin granules upon exocytosis.

A secretion from cultured bovine chromaffin cells was stimulated to examine the pattern of exocytotic fusion on the plasma membrane. Confocal microscopy revealed that dopamine-beta-hydroxylase immunofluorescence in intact cells stimulated for 20s with the nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium was almost entirely punctate and evenly distributed on the cell surface. The basis for the fine, punctate appearance of dopamine-beta-hydroxylase was investigated. Dopamine-beta-hydroxylase presentation on the surface of permeabilized cells stimulated with 1-30 microM Ca2+ was punctate and similar to that on the plasma membrane of intact cells. The fluorescence intensities of both surface dopamine-beta-hydroxylase sites and internal chromaffin granules were estimated by computerized digital image analysis. The surface area of punctate surface dopamine-beta-hydroxylase (0.218 +/- 0.013 microm2, mean +/- S.E.M.) is similar to the surface area of a 0.28 microm diameter chromaffin granule (0.25 microm2). The average fluorescence intensity integrated over the area of the surface spots was 25-30% of the average chromaffin granule intensity, a fraction that is similar to the published values of 40-50% of the dopamine-beta-hydroxylase in the chromaffin granule being membrane bound. The surface density of the spots is consistent with the number of granules undergoing exocytosis. The spots do not tend to be clumped. The key conclusions from this work are that each individual punctate site of dopamine-beta-hydroxylase represents the fusion of a single chromaffin granule and that the distribution of dopamine-beta-hydroxylase spots over the cell surface is extensive and random, suggesting that each individual granule associates with its own release site.

Effects of expression of a mouse brain L-type calcium channel alpha 1 subunit on secretion from bovine adrenal chromaffin cells.

Regulated exocytosis from bovine chromaffin cells is stimulated by the influx of Ca2+ through plasma membrane ion channels that are opened by nicotinic stimulation and/or depolarization. Recently, we developed a novel method that enabled us to investigate the function of a cloned Ca2+ channel type C alpha 1 subunit in forming channels that stimulate exocytosis. In the present study, we demonstrate by immunocytochemistry that bovine chromaffin cells normally express an epitope specific for the type C alpha 1 subunit. We investigated the effects of expression of additional class C alpha 1 subunits (mouse brain clone) on various aspects of secretory function in bovine chromaffin cells by measuring secretion of cotransfected human growth hormone (GH, a reporter for the regulated secretory pathway in the transfected cells). New channels were activated in response to depolarization by both elevated K+ and nicotinic cholinergic agonist. The new channels had their greatest effects when secretion was stimulated suboptimally. Secretion was enhanced only after the first 30 sec of stimulation, and the enhancement extended beyond 5 min of continuous stimulation. In contrast to the endogenous L-type Ca2+ channels, the latency was not decreased by the dihydropyridine L-type Ca2+ channel agonist, Bay K 8644. The findings suggest that (i) the Ca(2+)-sensitive mechanism for triggering or maintaining exocytosis is capable of being saturated by high levels of Ca2+, (ii) secretion caused by nicotinic agonist stimulation can be significantly enhanced by activation of voltage-sensitive Ca2+ channels, and (iii) the effects on secretion of the L-type Ca2+ channels formed on expression of the mouse brain class C alpha 1 subunit are distinct from those of endogenous L-type Ca2+ channels.

Transient transfection studies of secretion in bovine chromaffin cells and PC12 cells. Generation of kainate-sensitive chromaffin cells.

We have developed a transient transfection method to measure protein secretion from non-dividing, primary bovine chromaffin cells and from the continuous cell line, PC12. A plasmid coding human growth hormone (GH) was expressed in sufficient amounts in bovine chromaffin and PC12 cells to allow precise measurements of secretion from the small fraction (less than 1%) of transfected cells in a dish. GH was secreted in a similar proportion to endogenous catecholamine upon nicotinic stimulation, depolarization with elevated K+, and upon permeabilization with digitonin and subsequent stimulation with micromolar Ca2+. GH in homogenates from GH-transfected chromaffin cells cosedimented with catecholamine on discontinuous sucrose gradients. The data indicate that transiently expressed human GH in chromaffin and PC12 cells is localized predominantly in secretory vesicles in the regulated secretory pathway. With transient transfection there is a high probability of coexpression in the same cell of two plasmids which are cotransfected. Coexpression of a plasmid for GH and a plasmid for the non-N-methyl-D-aspartate glutamate receptor, GluR1, created chromaffin cells in which Ca(2+)-dependent GH secretion could be stimulated by the glutamatergic agonist kainate. The ability to coexpress a plasmid of interest with a plasmid for GH will allow the investigation of the role of other cloned proteins in the regulated secretory pathway in differentiated, non-dividing cells.

Alpha-1 adrenergic coupling events induced by full and partial agonists in rabbit aorta.

Differences in the ability of full vs. partial agonists to initiate alpha-1 adrenergic receptor-mediated coupling events were studied in isolated segments of rabbit aorta. Mono- and dimethoxysubstituted tolazolines produced contractile responses which, at their maximum, were 27 to 100% of the response produced by the full agonist phenylephrine. In addition to differences in maximum response, contraction kinetics varied between full and partial agonists. Responses to partial agonists displayed a slower approach to peak tension and loss of the rapid phase of tension development which is associated with release of intracellular Ca++. Among the tolazoline series 3,5 dimethoxy-, 3 methoxy-, and 2 methoxy derivatives were compared further with phenylephrine for their ability to cause phosphatidylinositol cycle turnover, intracellular Ca++ release and Ca++ influx. For each of these coupling events, a rank of phenylephrine greater than or equal to 3, 5 greater than 3 greater than 2 was observed. However, a higher percentage of Ca++ influx vs. Ca++ release was observed for the partial agonists, suggesting that their contractile responses may be more dependent upon extracellular Ca++ than intracellular Ca++. Our results indicate that partial agonists initiate the same coupling events as full agonists; however, the relative proportion of Ca++ release and influx may be different for partial agonists because of the reduced rate of second messenger production.