Characterisation of P2X receptors within the anterior pituitary and their role in modulating pituitary function

Abstract

Extracellular ATP acts in an autocrine/paracrine manner influencing cellular responses via two classes of purinoreceptors: metabotropic P2Y and inotropic P2X receptors. Recent observations have demonstrated a role for P2X receptors (P2X1-7) in calcium mediated hormone secretion from the pituitary. In the present study we used immortalised cell lines and primary pituitary cultures to investigate the effects of P2X receptors within the anterior pituitary. We used Reverse Transcriptase Polymerase Chain reaction (RT-PCR) to determine the expression of P2XR sub-types within pituitary cell lines and rat pituitary tissue. Our studies demonstrated similar expression of P2X receptors between rat pituitary tissue and cell lines. The expression of P2X1-7 receptors was identified in rat primary pituitary tissue. In the lactotroph cell lines, MMQ and GH3 we found P2X2, 3, 4, 5, 7 and P2X3, 4, 7 receptors, respectively. In LβT2 gonadotroph type cells, P2X2, 3, 4, 7 were identified whereas the corticotroph cell line AtT20:D16 expressed P2X1,2. Finally the murine cell line TtT/GF, which represent an in vitro model of the non-endocrine folliculostellate were profiled for P2X receptor expression. To our knowledge this is the first time this particular cell type has been examined for P2X receptor expression. Weak expression for P2X2, 4 was identified. The distribution pattern of P2X1-7 receptors within the rat pituitary was determined using double labelling immunoflourecscence and western blot. Protein expression for P2X4 and P2X7 was confirmed by Western blot of rat pituitary lysates, but no signal for P2X3 could be determined. Double immunofluorescence found P2X4 to be localised in corticotrophs, gonadotrophs, sommaotrophs, lactotrophs and thyrotrophs within a small group of cells. P2X7 was only found localised in a small group of somaotrophs. We then undertook a more thorough characterisation of the functional significance of P2X receptor expression within the different mammalian pituitary subtypes. We began our investigation by establishing a rat primary pituitary culture model and utilised multiplexing technology to assess the effects of ATP on the secretion of multiple pituitary hormones (Adrenocorticotropic hormone (ACTH), Growth Hormone (GH), Prolactin (PRL) and Thyroid Stimulating Hormone (TSH) ) simultaneously. ACTH and PRL were the most responsive to ATP stimulation. TSH and GH showed little response to ATP stimulation. We then went on to examine the effects of ATP on hormone secretion within our various cell lines. In AtT20:D16 cells, ATP had a significant effect on ACTH release when applied alone and in combination with CRH following incubation for 7 and 12 hours. Using the lactotroph model GH3, we assessed the effects of ATP and various ATP analogues on PRL release in vitro. All concentrations of ATP tested (0.001-1000µM) had a significant effect of PRL release from cells. On the other hand both analogues, the non- hydrolysable form of ATP adenosine 5'-O-(3-thio)triphosphate (ATP--S) and the P2X selective analogue, 2',3’-O-(4-benzoyl-benzoyl) (BzATP) had no significant effect on PRL release. We then attempted to characterise P2XR function in the pituitary using highly specific P2 and P2XR antagonists. The P2 receptor antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS) significantly decreased both basal release of PRL and stimulated PRL secretion in response to TRH (1nM), ATP, (1 and 100µM) and TRH and ATP in combination. A similar profile was observed after treatment with the selective P2X7R inhibitor, A438079. The selective P2X3R inhibitor failed to influence basal PRL secretion but did significantly attenuate the effect of TRH and ATP stimulated PRL release. Our results confirm previous investigations that ATP interactions with purinergic receptors expressed within the pituitary may play a role in modulating pituitary function. Our data has also shown through the use of specific receptor antagonists, that P2X receptors, particularly P2X3 and P2X7 may be involved in regulating hormone secretion from lactotrophs.