Desensitization of Superfused Isolated Ovine Anterior Pituitary Cells to Human Corticotropin‐Releasing Factor
Authors: Thomson Murray , Chan Eng‐Cheng , Falconer John , Madsen Gemma , Geraghty Simon , Curryert Neil , Boulton John , Smith Roger 
Published: January 2007

We have employed an in vitro system to study the time‐course of β‐endorphin (β‐EP) immunoreactivity release from anterior pituitary cells stimulated with corticotropin‐releasing factor (CRF) and whether exposure to CRF desensitizes the cells to subsequent stimulation. Ovine anterior pituitaries were enzymatically disrupted into single cells, mixed with Siegel P2 and superfused in mini‐columns with carbogen‐gassed medium at 37 °C. Superfusate fractions were collected at 5‐min intervals and β‐EP immunoreactivity in the eluate was measured by radioimmunoassay. Peaks of β‐EP release that rose significantly above baseline noise were detected using the PULSAR algorithm.

Unstimulated cell columns did not display any spontaneous peaks of β‐EP discharge detectable by PULSAR whereas peaks were identified in the output of columns exposed to 1 nM CRF for 100 min. β‐EP release increased after 10 min of stimulation and maximum stimulated output was achieved after 20 min of continuous CRF exposure. Between 20 and 60 min of CRF stimulation the rate of β‐EP release declined progressively but stabilized in the last 40 min of the exposure at a level significantly above controls for baseline secretion. Peak duration did not depend on the inclusion of calcium in the superfusion medium while peak amplitude and area were significantly reduced when cells were denied extracellular calcium. Following a 100‐min exposure to 1 nM CRF, pituitary cell columns were given a 30‐min rest period then restimulated with either 1 nM CRF or 50 mM KCI for 20 min. The columns given prior exposure to CRF did not mount a response (detectable by PULSAR) to a subsequent dose of 1 nM CRF whereas PULSAR detected a clear response in all members of a control group that had not received prior CRF challenge. Both CRF exposed and control columns responded to 50 mM KCI although the response was significantly attenuated in the cells that had received prior CRF treatment.

These results indicate that unstimulated superfused isolated ovine anterior pituitary cells do not possess an inherent rhythmicity of β‐EP release that can be detected by the PULSAR algorithm while treatment of the cells with CRF results in detectable discharge. The rapid response of β‐EP discharge to CRF treatment suggests the presence of intracellular β‐EP stores available for rapid mobilization. Continuous exposure to 1 nM CRF can tonically amplify corticotrope output for the duration of its presence in the environment of the corticotrope, but the maximum rate of release cannot be maintained. An inrush of extracellular calcium is not essential for the corticotrope to mount a detectable response to continuous CRF exposure but the release of a maximum amount of β‐EP relies on calcium entry. Long‐term treatment with CRF prevents the corticotrope releasing a detectable peak of β‐EP on subsequent CRF stimulation and therefore CRF exposure leaves a lasting impression on the physiological machinery of the corticotrope. The attenuation of responsiveness to 50 mM KCI after long‐term CRF treatment indicates that depletion of β‐EP stores may play a part in corticotrope desensitization although a reduction in CRF receptor number and an alteration in the intracellular mechanisms controlling β‐EP release may also be a factor.