Adenosine 3′,5′‐cyclic monophosphate‐mediated enhancement of calcium‐evoked prolactin release from electrically permeabilised 7315c tumour cells

Abstract

1 The .7315c tumour cell was used as a model system for the investigation of adenosine 3',5'-cyclic monophosphate (cyclic AMP-mediated enhancement of calcium-evoked prolactin release. 2 7315c cells were permeabilised by subjecting the cells to intense electric fields. Studies investigating the penetration of the dye ethidium bromide indicated that the cells were completely permeabilised after 2 discharges of 2000 volts and that the pores remained open for at least 30 min before beginning to reseal. These permeabilisation parameters were consistent with those which gave maximal calcium-stimulated prolactin release. 3 In the absence of calcium and in the presence of EGTA (1 mM), permeabilised 7315c cells secreted prolactin at a rate of 0.23ngmin-1 per 106 cells. When EGTA was replaced by 1.5mM calcium, permeabilised cells secreted prolactin at a rate of 2.20 + 0.30ngmin-1 per 106 cells in the first 5min of exposure. Maximal calcium-dependent prolactin secretion from permeabilised cells occurred at 37°C. 4 The amount of prolactin secreted, in a 5 min incubation at 37°C, from permeabilised cells depended upon the free calcium concentration in the permeabilisation medium. Calcium stimulated prolactin release from permeabilised cells in the concentration range 0.1-10 M (half maximal = 5.8pM). When permeabilised cells were exposed to cyclic AMP (100pM) for 5 min prior to and during a 5min challenge with various concentrations of calcium, the amount of prolactin secreted at each effective concentration of calcium was increased. However, cyclic AMP did not alter the potency of calcium as a stimulant of prolactin secretion. 5 The results suggest that cyclic AMP potentiates calcium-evoked secretion from 7315c cells, not by increasing the entry of calcium into the cytosol, but at a step in the secretory process, distal to calcium entry, which modulates the ability of an increase in cytosolic calcium concentration to stimulate prolactin release.

Adenosine A1 receptors in rat brain synaptosomes: Transductional mechanisms, effects on glutamate release, and preservation after metabolic inhibition

“…Conversely, adenosine analogucs inhibitcd cAhIP formation in synaptosomes, with sinall but significant effects at agonist concentrations that were also effective on dcpolarization-induced glutamate exocytosis. These results are in agreeinelit with the involvement of CAMP in adenosine iiiodulation of exocytosis (Delahunty et al, 1988;Abbracchio et al, 1992) and with other studies demonstrating a direct correlation between CAMP formation and Ca2 ' -depeiiderit transmitter release (Guild et al, 1988;Jones ct al., 1986). Clearly, adenosine analogue inhibition of CAMP formation through A, receptor is only one of multiple mechanisms that conciir to a fine CAMP modulation, and this will justify the small, but significant, effect of CPA alone, The mechanisms by which CAMP modulates release are far from being defined, although CAMP-dependent phosphorylation of synaptic proteins involved in vesicular neurotransmitter release might play a role (Valtorta et al, 1992).…”

Effects of adenosine 3′: 5′‐cyclic monophosphate and guanine nucleotides on calcium‐evoked ACTH release from electrically permeabilized AtT‐20 cells

“…In the present study, the electrical permeabilization technique (Knight & Baker, 1982) was used to test this hypothesis further. This technique has been used previously in this laboratory to investigate the interaction between cyclic AMP and calcium upon hormone secretion from dispersed cells of the intermediate lobe of the rat pituitary (Yammamoto et al, 1987) and the 7315c rat anterior pituitary tumour cell line (Guild et al, 1988). These previous findings strongly suggested a post-calcium site of action in the stimulus-secretion coupling pathway for cyclic AMP-mediated stimulation of hormone secretion.…”

“…To determine the parameters that lead to 100% permeabilization, the cells were subjected to varying numbers of discharges of varying voltages and subsequently stained with ethidium bromide (50#M) as previously described (Guild et al, 1988). Optimum permeabilization parameters were also determined for the ability of calcium, added to the external medium, to stimulate ACTH secretion from permeabilized cells as previously described (Guild et al, 1988). For both indicators of permeabilization, optimum parameters were determined to be 5 discharges of 2500 V cm-' (data not shown) and were subsequently adopted in these experiments.…”

“…For both indicators of permeabilization, optimum parameters were determined to be 5 discharges of 2500 V cm-' (data not shown) and were subsequently adopted in these experiments. The time period for which the cells remained permeabilized was also determined by methods previously described (Guild et al, 1988) and it was found that AtT-20 cells remained permeabilized for 60 min (data not shown).…”

Adenosine receptors in rat basophilic leukaemia cells: transductional mechanisms and effects on 5‐hydroxytryptamine release

“…Since Ali et al (1990) The role of cyclic AMP in regulating secretion is far from defined. Calcium-evoked secretion has been shown to be potentiated by increases in cyclic AMP levels in some secretory systems (Jones et al, 1986;Guild et al, 1988), probably through modulation of the functional state of some components of the exocytotic apparatus. Conversely, in GH3 cells inhibition of cyclic AMP formation subsequent to activation of Al receptors is associated with a reduction of prolactin release (Delahunty et al, 1988).…”