Application of an optimized immunostaining technique to evaluate the heterogeneous secretory response from porcine somatotropes by cell blotting.
The objective of the present study was to quantify the absolute hormone release from individual porcine pituitary cells incubated on polyvinylidene difluoride (PVDF) transfer membranes (cell-blot assay). After immunoperoxidase staining, growth hormone (GH) release from isolated somatotrope cells appeared like a colored zone of secretion surrounding the cell. Optical densities of these secretion zones were quantitated by computerized image analysis and translated into picograms by means of an appropriate standard curve. As a prior step, the staining method and the optimal immunocytochemical conditions were selected by applying purified porcine growth hormone (pGH) to the transfer membranes. The avidin-biotin-peroxidase nickel-intensified (ABC-Ni) method produced a better resolution than the peroxide-anti-peroxidase (PAP) method, although both techniques were similar with regard to background, sensitivity, and range of quantitation. The amount of GH released from single porcine somatotropes was highly heterogeneous, although the cells were treated under the same conditions. Moreover, this fact was consistent with the stimulation of the average release of GH by GH-releasing factor (GHRF) but not by GHRF+somatostatin (SRIF). Our results confirm the availability of the recently developed cell-blot assay and support the concept of functional heterogeneity in anterior pituitary cell populations.
Dobado-Berrios
P M
PM
Department of Cell Biology, University of Córdoba, Spain.
Ruiz-Navarro
A
A
Torronteras
R
R
Gracia-Navarro
F
F
D000818:Animals; D002478:Cells, Cultured; D005260:Female; D013007:Growth Hormone-Releasing Hormone / Q000378:metabolism; D015151:Immunoblotting; D007124:Immunoenzyme Techniques; D008567:Membranes, Artificial; D010902:Pituitary Gland / Q000166:cytology / Q000378:metabolism*; D011145:Polyvinyls; D013004:Somatostatin / Q000378:metabolism; D013552:Swine; D005465:Fluorocarbon Polymers
D013007:Growth Hormone-Releasing Hormone; D008567:Membranes, Artificial; D011145:Polyvinyls; D013004:Somatostatin; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers