Electrophysiological responses to dopamine of rat hypophysial cells in lactotroph‐enriched primary cultures.

Abstract

1. Cells from 14-day-old and lactating female rat pituitary glands were dissociated, separated and enriched on a continuous gradient of bovine serum albumin at unit gravity. They were maintained for at least 6 days in culture before perifusion and electrophysiological experiments were performed. 2. Immunofluorescent staining of the resulting gradient fractions (numbered F2 to F9) from both groups of animals indicated that the majority of lactotrophs were located in the light fractions (F3-F4). However, a second population of lactotrophs was observed in the heavy fractions (F7-F9) isolated from lactating females. 3. Basal secretion rates of prolactin were in the order of 2-40 ng 2 min-1 106 cells-1 and were inhibited by dopamine in a dose-dependent manner. 4. According to their electrophysiological properties, cells from 14-day-old females (first group) were categorized as follows: (1) inexcitable cells, which displayed a low resting potential of about -35 mV (39% of cells tested, n = 118); and (2) excitable cells, which displayed either triggered or spontaneous action potentials and resting membrane potentials higher than -50 mV (61% of cells tested, n = 185). 5. In the light fraction from lactating females (second group), the majority of the cells were excitable (70%) and showed high resting membrane potentials (-50 to -55 mV) and 15% of these cells displayed spontaneous action potentials. 6. Heavy fractions (third group) contained a high percentage of non-spontaneous but excitable cells (80% of the cells tested, n = 65). These cells were able to elicit action potentials after the cession of hyperpolarizing current pulses (''off'' potentials). 7. Action potentials were insensitive to the sodium channel blocker, tetrodotoxin (TTX; 5 .times. 10-6 M) but were reversibly blocked by calcium channel blockers such as cobalt, manganese and cadmium (10 mM). 8. In excitable cells from the three groups, dopamine (10-7 M) induced a hyperpolarizing response due to an increase of the membrane conductance. During this response, action potentials were inhibited. It was shown that this was not a direct effect of dopamine. The reversal potential of the dopamine-induced response in these cells was found to be at -100 mV. This value was shifted to more positive potentials (-50 mV) when high-potassium medium was used (56 mM). 9. In non-excitable cells (first group), dopamine (10-7 M) induced a hyperpolarizing response due to a decrease of the membrane conductance. The amplitude of this response was dependent upon the membrane potential and the reversal potential was found to be near 0 mV. 10. Some of the non-spontaneous cells from the third group showed no membrane responses to dopamine but the ''off'' spike was suppressed. 11. Because of the apparent implication of potassium currents in the dopamine response found in excitable cells, we attempted to characterize this response by using potassium current blockers. Cobalt and tetraethylammonium had no effect, whereas 4-aminopyridine induced a weak modification of the dopamine-induced response. 12. The dopamine response due to the increase of the membrane conductance was mimicked by a specific agonist of the D2 receptors (RU24926) and was blocked by specific dopaminergic antagonists (haloperidol and domperidone). 13. The physiological significance of the inhibition of electrical activity by dopamine is interpreted in terms of the stimulus-secretion coupling hypothesis.