Physiological characterization of two functional states in subpopulations of prolactin cells from lactating rats.
1. Lactotroph cells from lactating female rat pituitary glands were dissociated, separated and enriched on a continuous gradient of bovine serum albumin at unit gravity. Two lactotroph subpopulations were observed in the light (F(3-5)) and the heavy (F(7-9)) fractions of the gradient. Both populations were maintained for at least 6 days in culture before experiments were performed. 2. Patch-clamp recordings, in the whole-cell mode, were performed on both lactotroph subpopulations in order to measure passive membrane properties and Ca2+ currents. Resting membrane potential as well as membrane capacitance values were found to be lower in light fraction cells. The two components of Ca2+ currents, called fast and slow deactivating (FD and SD) currents, were present with different proportions in each subpopulation; the ratio of current amplitudes, SD/FD, was 2.42 +/- 0.41 (n = 18) in light fraction cells and 1.17 +/- 0.27 (n = 17) in heavy fraction cells (P less than 0.02). 3. Reverse haemolytic plaque assay showed that in the light and heavy fractions, 68 and 47% of the lactotroph cells, respectively, were secreting. Population analysis of the plaque areas revealed a bimodal frequency distribution of plaque sizes consisting of small (1500 microns 2) and large plaques (3995 microns 2). A majority of light fraction cells produced large plaques whereas most of the heavy fraction cells produced small plaques. 4. Perifusion experiments performed on enriched prolactin cells showed that (1) basal prolactin (PRL) release was higher in light fraction than in heavy fraction cells, (2) the dopamine (10(-8)M)-induced inhibition of PRL release was greater in light fraction cells (86 +/- 15%) than in heavy fraction cells (41 +/- 21%), and (3) the thyrotrophin-releasing hormone (TRH, 10(-8)M)-induced increase of PRL release was 150 +/- 60% in light fraction versus 330 +/- 82% in heavy fraction cells. 5. Current-clamp recordings were performed using the intracellular technique. Lactotrophs were categorized according to their electrophysiological response following application of dopamine or TRH (both 10(-8)M). In the light fractions, the majority of the cells tested were hyperpolarized by dopamine (68%), whereas only 7% were depolarized by TRH application. In the heavy fractions, most of the cells (63%) responded to TRH application, while only 13% were dopamine sensitive. 6. Cytosolic free Ca2+ concentration ([Ca2+]i) measurements with the fluorescent probe Indo-1 revealed two lactotroph subtypes. Most cells in the light fractions (sixteen of twenty-two tested cells) exhibited an unstable level of [Ca2+]i with values fluctuating between 114.1 +/- 34.3 and 221 +/- 50 nM (mean +/- S.D.). Application of dopamine or of the D2 receptor agonist RU 24213 (10(-8)M) resulted in the disappearance of these fluctuations and in an accompanying decrease in basal [Ca2+]i level.(ABSTRACT TRUNCATED AT 400 WORDS)
Lledo
P M
PM
Laboratoire de Neurobiologie des Comportements, INSERM U. 176, Bordeaux, France.
Guerineau
N
N
Mollard
P
P
Vincent
J D
JD
Israel
J M
JM
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D004298:Dopamine / Q000502:physiology; D004594:Electrophysiology; D005260:Female; D006462:Hemolytic Plaque Technique; D007774:Lactation / Q000502:physiology*; D008564:Membrane Potentials / Q000502:physiology; D010903:Pituitary Gland, Anterior / Q000166:cytology* / Q000378:metabolism; D011247:Pregnancy; D011388:Prolactin / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D013973:Thyrotropin-Releasing Hormone / Q000502:physiology
D013973:Thyrotropin-Releasing Hormone; D011388:Prolactin; D002118:Calcium; D004298:Dopamine