Stability of Bovine Adrenal Medulla Cells in Culture

Abstract

The functional stability of primary cultures of adrenal medulla cells was investigated. Isolated cells were prepared by treatment of bovine adrenal glands with collagenase followed by purification on Percoll density gradients and were maintained in Dulbecco's medium containing 10% fetal calf serum. Within 12 h after plating on plastic culture dishes, the cells became firmly attached and exhibited good survival for periods of time up to 3 weeks, as indicated by their morphology using light and electron microscopy, by maintenance of their content of catecholamines, tyrosine hydroxylase, dopamine-beta-hydroxylase, and phenylethanolamine N-methyltransferase, and their ability to respond to secretagogues. During the first 10 days to 2 weeks in culture there was little or no change in any of these parameters. During the 3rd week there were progressive losses of catecholamine and enzyme activities and increased vacuolization of medullary cells. The cells synthesized protein and RNA with no apparent loss in activities over the period studied, but did not incorporate [3H]thymidine into PCA-precipitable material. The cells responded to secretagogues and secretory antagonists similarly to isolated perfused adrenal glands. The studies described here demonstrate that primary cultures of adrenal medulla cells provide an excellent experimental system for obtaining more detailed information on stimulus-secretion coupling and other functional aspects of the adrenal medulla.

Muscarinic receptor modulation of release of [Met5]enkephalin immunoreactive material and catecholamines from the bovine adrenal gland

“…These findings confirm data published earlier (Kilpatrick, et al, 1980;Barron & Hexum, 1984). In these experiments, C-6 did not completely abolish the response of the gland to stimulation, despite the fact that the concentration of C-6 was greater than the concentration of stimulating drug.…”

Presynaptic Inhibition by Concanavalin A: Are α‐Latrotoxin Receptors Involved in Action Potential‐Dependent Transmitter Release?

“…This procedure accurately reflects the release of endogenous catecholamines (Kilpatrick et al, 1980). After loading (2 h at 36°C)wmth 0.01~M [3H]NA and subsequent to three washes, the cultures were incubated for 2 h in culture medium at 36°C.All subsequent steps were performed at room temperature (20-24°C)with cultures submerged in the buffer used for superfusion experiments (see above).…”

Pertussis toxin stimulates delayed‐onset, Ca2+‐dependent catecholamine release and the ADP‐ribosylation of a 40 kDa protein in bovine adrenal chromaffin cells

“…Bovine adrenal chromaffin cells were isolated from adrenal medullae by collagenase digestion [8] and purified on a Percoll gradient [9]. The cells were maintained as monolayer cultures in Dulbecco's modified Eagle's medium and Ham's nutrient mixture F-12 (DME/F12) with 15 mM Hepes, L-glutamine and phenol red (Sigma, MO), supplemented with 26 mM NaHCOS, 5% (v/v) heat-inactivated fetal calf serum (Gibco, NY) 100 pg streptomycin/ml, 100 U penicillin/ml, 250 ng Fungizone/ml (Gibco) and adjusted to pH 7.3.…”