Identification of latent tissue kallikrein, prolactin and growth hormone secretion in GH 3 pituitary cells using modified radioimmunoassays

Abstract

Our studies demonstrate that rat anterior pituitary cells (GH 3) are capable of synthesizing and secreting tissue kallikrein together with prolactin and growth hormone. The secretion of prolactin and growth hormone in GH 3 cells was measured by two newly developed sensitive radioimmunoassays (RIA), using the polyethylene glycol separation technique. In the direct radioimmunoassay for rat tissue kallikrein, using a polyclonal antiserum which recognizes both active and prokallikrein, the GH 3 kallikrein displays parallelism with standard curves of rat urinary kallikrein. The production of immunoreactive kallikrein, prolactin, and growth hormone is time-dependent, and the levels after a 72 h incubation in serum-free media are ~ 12.2 ± 4.4 ng, 272.2 ± 33.0 ng, and 475.6 ± 4.8 ng per 10 6 cells per ml (mean ± SD, n = 3), respectively. In Western blot analyses, a specific monoclonal antibody to tissue kallikrein (V 4D 11) identifies GH 3-secreted kallikrein as a ~ 39000 Da protein, slightly larger than ~ 38000 Da kallikreins of submandibular gland, mouse anterior pituitary cells (AtT 20) or rodent neuroblastoma × glioma hybrid cells (NG108). Kallikrein mRNA in GH 3 cells was identified in Northern blot analyses, using a tissue kallikrein cDNA probe. In a RIA using a kallikrein monoclonal antibody (V 1C 3) recognizing only active kallikrein, kallikrein could not be detected in the media incubated up to 48 h with GH 3 cells. However, after trypsin treatment, a time-dependent increase of immunoreactive kallikrein (using monoclonal antibody V 1C 3), Tos-Arg-OMe esterase, and kinin-releasing activities can be measured in the conditioned média. The activated esterase activity was inhibited by aprotinin and by affinity-purified kallikrein monoclonal antibody (V 4D 11) in a dose-dependent manner. The data indicated that rat anterior pituitary GH 3 cells secrete latent tissue kallikrein, which can be converted to active kallikrein by trypsin. These hormonally responsive cells co-synthesize kallikrein with prolactin and growth hormone and provide a model System for studying the regulation of kallikrein gene expression.