Continuous On-Line Monitoring of Secretion from Rodent Pituitary Endocrine Cells Using Fluorescent Protein Surrogate Markers

Abstract

We have developed a system to use secreted fluorescent proteins (FPs) as surrogate markers for the continuous on-line monitoring of hormone release from perfused tissue slices. We have tested this system using GH-GFP transgenic rats with green fluorescent protein (GFP) targeted to the secretory vesicles (SVs) of pituitary growth hormone (GH) cells. Brief exposures of vibratome slices to GH secretagogues [GH-releasing hormone (GHRH), GH-releasing peptide-6 (GHRP-6)] or somatostatin caused changes in FP output that correlate with hormone secretion, subsequently measured in fractions of perfusate by radioimmunoassay. The temporal resolution of this method was capable of revealing differences in the kinetics of response to GHRH and GHRP-6 between wild-type and dwarf (dw/dw) rats harbouring the GH-GFP transgene. We further tested the utility of the system by generating transgenic mice with red FPs targeted to secretory vesicles (PRL-mRFP(sv)) and to the cytoplasm (PRL-DsRed(cyto)) of lactotrophs. Dopamine had no effect on the FP output from pituitary slices of PRL-DsRed(cyto) mice but inhibited output from those of PRL-mRFP(sv) animals, with a rebound increase of release after removal, which again correlated with hormone output measured in the perfusate by radioimmunoassay. The inhibition of monomeric RFP secretion by dopamine was dose-dependent, as was stimulation by low concentrations of oxytocin. The temporal resolution afforded by this method provides useful insight into the release kinetics from large populations of pituitary cells, and fills a temporo-spatial gap between single vesicle and single cell monitoring of exocytosis in milliseconds, and in vivo sampling studies of release into the bloodstream on a time scale of minutes.

Existence of long-lasting experience-dependent plasticity in endocrine cell networks

“…To provide an activity index of both population function81920 and calcium (Ca 2+ )-dependent hormone exocytosis21 (the major pituitary output), we measured multi-cellular Ca 2+ signals emanating from lactotrophs in situ within pituitary slices derived from PRL-DsRed mice22. Population functional connectivity (FC) maps were constructed based on the location of significantly correlated cell pairs (that is, cells with related activity profiles).…”

“…Transgenic mice expressing DsRed under the control of the PRL promoter were used in all experiments2231. In studies involving nulliparas (virgin animals), only animals displaying metestrous/diestrous or oestrous were used (vaginal cytology followed for at least two consecutive cycles).…”

Plasticity of the Prolactin (PRL) Axis: Mechanisms Underlying Regulation of Output in Female Mice

“…This neuropeptide may also act as a physiological regulator of PRL release itself [39][40][41]. Pertinently, infusion of a PRL antagonist into the brain of pregnant rats disrupts parturition [42], whilst in lactating animals PRL stimulates both oxytocin gene expression [43] and secretion [44], suggesting a role for PRL in regulating oxytocin, and possibly its own secretion, in both of these processes.…”

Nonsocial Functions of Hypothalamic Oxytocin

“…Thus, OXT can modulate ACTH and cortisol secretion to mobilize body function but curbs its potentially harmful consequence. In addition, OXT is also found to increase the release of prolactin [58, 59], α -MSH [60], LH [61], and FSH [62] as well as its own release [4, 63]. On the contrary, OXT inhibits TSH [64] and GH [65] release.…”