Use of newly designed microperifusion system with amperometric sensors for near-continuous on-line monitoring of hormone secretion. I. correlation with the luteinizing hormone secretory response to gonadotropin-releasing hormone.
LH is rapidly secreted from the anterior pituitary gland in response to episodic release of GnRH. To understand better the detailed dynamics of this secretory process and related time- and dose-dependent effects, we developed a microperifusion system able to preserve resolution of dynamic secretory responses by minimizing dispersion and with capabilities for automation and continuous on-line monitoring. To monitor secretion at a frequency greater than that feasible with discrete samples, we determined whether LH or another molecule cosecreted by anterior pituitary cells could be monitored on a near-continuous basis using amperometric electrochemical sensors operated under conditions of cyclic voltammetry. The developed culture system incorporated a 32-mu l cell chamber in a controllable constant environment. Miniature sensors were positioned immediately adjacent to the cells to permit differential measurements of the input and effluent streams of medium. With the system, square wave pulses of electrochemically active, biologically inert molecules, e.g. ascorbate and phenol red, showed similar redox profiles before and after the cells, with minimal dispersion. Enzymatically dispersed ovine anterior pituitary cells were cultured on SoloHill glass beads for 4 days, loaded into the chamber, and perifused with DMEM containing 10% FCS for 2 h. After stabilization, the medium was switched to protein-free, HEPES-buffered HBSS with L-glutamine and allowed to flow for a 1-h washout period, followed by GnRH challenges of varying concentrations in a random order. Perifusate was collected in six-drop fractions at approximately 30-sec intervals. Although LH, its individual subunits, and FSH could not be detected amperometrically, GnRH stimulation of the cells simultaneously induced a several log order dose-dependent secretion of both an electrochemically detectable molecule and LH, as measured by RIA. The secretory profiles of the amperometrically detected signal and immunoactive LH were very similar. Dose-response relationships of the amperometric signal and LH to a wide range of GnRH were similar. The responses of both secreted LH and the amperometric signaling molecule(s) to GnRH were triphasic; an initial peak of activity was observed within 20-40 sec, a lower plateau level was observed for the duration of the 4-min GnRH stimulation, and a gradual return to baseline followed. The cells then maintained a constant level of GnRH-independent basal secretion. These results indicate that it is feasible to monitor the complex dynamics of endocrine and cellular responsiveness to secretagogues from endocrine cells/tissues continuously in real-time.
Cantor
H C
HC
Reproductive Sciences Program, University of Michigan, Ann Arbor, 48109-0404, USA.
Padmanabhan
V
V
Favreau
P A
PA
Midgley
A R
AR
D000818:Animals; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004563:Electrochemistry / Q000295:instrumentation* / Q000379:methods; D007987:Gonadotropin-Releasing Hormone / Q000494:pharmacology*; D007986:Luteinizing Hormone / Q000378:metabolism*; D008904:Miniaturization; D010477:Perfusion / Q000295:instrumentation / Q000379:methods; D010903:Pituitary Gland, Anterior / Q000187:drug effects / Q000378:metabolism*; D011863:Radioimmunoassay / Q000379:methods; D012756:Sheep; D013997:Time Factors
D007987:Gonadotropin-Releasing Hormone; D007986:Luteinizing Hormone