Patch Clamp Recording from Anterior Pituitary Cells Identified by Reverse Hemolytic Plaque Assay

Abstract Epidermal growth factor (EGF) is synthesized and secreted by mammalian anterior pituitary cells. It stimulates GH and prolactin (PRL) secretion, but the cellular origin of EGF is relatively unexplored. The objective of this study was to characterize the cells that secrete EGF in the anterior pituitary of lactating rats. An EGF reverse haemolytic plaque assay (RHPA) was used to identify EGF-secreting cells and this RHPA was combined with immunofluorescence using antibodies to the six major adenohypophysial hormones (i.e. PRL, GH, LH, FSH, TSH and ACTH). Approximately 20% (20·33 ± 2·96%) of the cells in the pituitary of lactating rats secrete EGF. The EGF-secreting cell population was composed of the following labelled cells: PRL (27%), GH (20%), LH (18%), FSH (14%), TSH (14%) and ACTH (5%). The present study showed that EGF is released by a subpopulation of anterior pituitary cells composed of all the classic hormone-containing cells. Journal of Endocrinology (1996) 148, 319–324

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Kallidin stimulates prolactin secretion from individual rat anterior pituitary cells

Journal of Endocrinology ◽

10.1677/joe.0.1280315 ◽

1991 ◽

Vol 128 (2) ◽

pp. 315-320 ◽

Cited By ~ 1

Author(s):

J. A. Stirland ◽

B. L. Brown ◽

P. R. M. Dobson

Keyword(s):

Anterior Pituitary ◽

Time Course ◽

Hormone Secretion ◽

Plaque Assay ◽

Prolactin Secretion ◽

Male Rat ◽

Pituitary Cells ◽

Kinin System ◽

Plaque Area ◽

Anterior Pituitary Cells

ABSTRACT Kinins are localized within the adenohypophysis where they have been shown to stimulate the release of pituitary hormones. In the present study we have investigated the effect of [Lys]-bradykinin (kallidin) on prolactin secretion at the single cell level from cultured male rat anterior pituitary cells. This was assessed by use of a reverse haemolytic plaque assay which permits quantitative evaluation of the proportion of all pituitary cells which are secreting prolactin, and the amount of prolactin secreted per lactotroph (plaque area). The rate of plaque development was used as an index of the rate of hormone secretion in time-course studies. Kallidin induced a dose-dependent increase in both the percentage of plaque-forming cells and the median plaque area during the first 2 and 3 h of incubation respectively. The threshold concentration of kallidin was 10 nmol/l. After 4 h of kallidin stimulation there was no difference between treated and control mono-layers with respect to median plaque area and the total secretion index. Although recruitment of additional cells into the secretory pool cannot be excluded, this seems unlikely since at 3 and 4 h little or no difference was observed in the number of plaque-forming cells. The data suggest that initially kallidin accelerated the rate of prolactin secretion primarily by inducing an increase in the number of cells secreting prolactin, and subsequently by increasing the amount of hormone secreted per lactotroph. The results presented here are consistent with the proposed role of the kallikrein–kinin system in the paracrine or autocrine control of prolactin release from the pituitary gland. Journal of Endocrinology (1991) 128, 315–320

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Estrogen increases the release of epidermal growth factor from individual pituitary cells in female rats

Journal of Endocrinology ◽

10.1677/joe.0.1460495 ◽

1995 ◽

Vol 146 (3) ◽

pp. 495-500 ◽

Cited By ~ 12

Author(s):

A Mouihate ◽

J Lestage

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Anterior Pituitary ◽

Plaque Assay ◽

Female Rats ◽

Pituitary Cells ◽

Anterior Pituitary Cells ◽

Epidermal Growth ◽

The Mean ◽

Estradiol 17Β

Abstract Rat mixed anterior pituitary cells from cycling females were microscopically demonstrated to produce epidermal growth factor (EGF), in culture, using a reverse hemolytic plaque assay. Anterior pituitary cells of proestrous female rats secrete more EGF than those of metestrous rats, as evaluated by the mean area and the percentage of EGF plaque-forming cells. Estradiol-17β (10 nm) treatment of metestrous culture for 24 h increased significantly the size of EGF plaque-forming cells (from 1290±58 to 2566 ± 57 μm2, P<0·01) and the percentage of EGF plaque-forming cells (from 20·57 to 28·19%; P<0·01) to a level as high as observed in proestrous cultures (basal mean area 2171 ± 157 μm2, and basal percentage of EGF plaque-forming cells 22·71%). These results suggest that the hormonal status of female rats influences EGF secretion in the anterior pituitary gland and that estradiol is implicated in this phenomenon. Journal of Endocrinology (1995) 146, 495–500

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Measurement of spontaneous neuronal membrane activity by time lapse confocal microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100141238 ◽

1995 ◽

Vol 53 ◽

pp. 972-973

Author(s):

R H. Selinfreund ◽

A. H. Cornell-Bell

Keyword(s):

Membrane Potential ◽

Confocal Microscopy ◽

Patch Clamp ◽

Electrical Activity ◽

Time Lapse ◽

Neuronal Firing ◽

Neuronal Membrane ◽

Pituitary Cells ◽

Patch Clamp Recording ◽

Spontaneous Electrical Activity

Cellular electrophysiological properties are normally monitored by standard patch clamp techniques . The combination of membrane potential dyes with time-lapse laser confocal microscopy provides a more direct, least destructive rapid method for monitoring changes in neuronal electrical activity. Using membrane potential dyes we found that spontaneous action potential firing can be detected using time-lapse confocal microscopy. Initially, patch clamp recording techniques were used to verify spontaneous electrical activity in GH4\C1 pituitary cells. It was found that serum depleted cells had reduced spontaneous electrical activity. Brief exposure to the serum derived growth factor, IGF-1, reconstituted electrical activity. We have examined the possibility of developing a rapid fluorescent assay to measure neuronal activity using membrane potential dyes. This neuronal regeneration assay has been adapted to run on a confocal microscope. Quantitative fluorescence is then used to measure a compounds ability to regenerate neuronal firing.The membrane potential dye di-8-ANEPPS was selected for these experiments. Di-8- ANEPPS is internalized slowly, has a high signal to noise ratio (40:1), has a linear fluorescent response to change in voltage.

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