Hormone Secretion by Cells Dissociated From Rat Anterior Pituitaries

Abstract

A new procedure has been developed for dissociating anterior pituitary tissue and producing a viable suspension of single cells . The procedure involves incubation of small tissue blocks in 1 mg/ml trypsin (15 min), followed by incubation in 8µg/ml neuraminidase and 1 mM EDTA (15 min), followed by mechanical dispersion . Cell yields are -55%, based on recovered DNA. By electron microscopy five types of secretory cells (somatotrophs, mammotrophs, thyrotrophs, gonadotrophs, and corticotrophs) plus endothelial and follicular cells can be identified and are morphologically well preserved up to 20 h after dissociation . Throughout this period, the cells incorporate linearly [ 3 H]leucine into protein for up to 4 h at a rate 90% greater than hemipituitaries, and they synthesize, transport intracellularly, and release the two major pituitary secretory products, growth hormone and prolactin . Immediately after dissociation the cells' ability to respond to secretogogues (high K+ and dibutyryl cyclic AMP) is impaired, but after a 6-12-h culture period, the cells apparently recover and discharge 24% and 52%, respectively, of their content of prelabeled growth hormone over a 3-h period in response to these two secretogogues . This represents a stimulation of 109% and 470% over that released by cells incubated in control medium . The results demonstrate that function and morphologic integrity are preserved in this cell system . Therefore it is suitable for the study of various aspects of pituitary secretion and its control .

Dopaminergic regulation of avian prolactin gene transcription

“…The posterior lobes of the pituitary glands were removed, and the anterior lobes were dissociated using a modification of the trypsin/ neuraminidase procedure described by Hopkins & Farquhar (1973). They were minced and replaced in Krebs-Ringer bicarbonate buffer solution (KRB, pH 7·4) supplemented with amino acids (MEM Eagle essential amino acids; BioWhittaker, Walkersville, MD, USA), 0·3 µg/ml glutamine sulfate (Sigma Chemical Co., St Louis, MO, USA), 2·5 mg/ml glucose (Sigma), 3 mg/ml BSA (fraction V, Sigma), and 100 µg/ml gentamycin sulfate (Sigma).…”

Cellular in vivo imaging reveals coordinated regulation of pituitary microcirculation and GH cell network function

“…Oxygen supply by portal vessels is essential for pituitary function (3,6); however, practically nothing is known about how incoming oxygen is distributed within the gland and whether oxygen intake and consumption are temporally regulated during hormone secretion. The distribution of incoming signaling molecules by the pituitary microcirculation is also a long-standing question: how and to what extent secretagogues can be regionally distributed to their target pituitary cells (4,5,7,8)?…”

“…To understand how these hormone pulses are built up in vivo, one must take into account the close link between blood supply, metabolic requirements, and functional activities of pituitary cells (2)(3)(4)(5). Oxygen supply by portal vessels is essential for pituitary function (3,6); however, practically nothing is known about how incoming oxygen is distributed within the gland and whether oxygen intake and consumption are temporally regulated during hormone secretion.…”

Temperature and Protein Kinase C Modulation of Gonadotropin‐Releasing Hormone Receptors in Pituitary Cells from Intact or Castrated Male Rats

“…Anterior pituitary glands were removed and dispersed as previously described (38,39). Briefly, the anterior pituitaries were cut into small cubes and, after enzymatic digestion (consecutive incubation with trypsin (Sigma, St. Louis, MO, USA; 5 mg/ml for 15 min) and DNase (Sigma; 2 pg/ml for 1 min)), were washed in a CaZf/MgZ'-free medium and mechanically dispersed with a Pasteur Pipette.…”