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Adenosine stimulates connexin 43 expression and gap-junctional communication in pituitary folliculostellate cells

B. Mary Lewis^*, Annette Pexa, Karen Francis^*, Vandana Verma, Anne M. McNicol, Maurice Scanlon^*, Andreas Deussen, W. Howard Evans, D. Aled Rees^* and Jack Ham^*,1

^* Centre for Endocrine and Diabetes Sciences,

Department of Medical Biochemistry and Immunology, School of Medicine, Cardiff University, Cardiff, UK;

Institut fuer Physiologie, Medizinische Fakultaet Carl Gustav Carus, Dresden, Germany; and

University of Glasgow, Department of Pathology, Royal Infirmary, Glasgow, UK

¹Correspondence: Centre for Endocrine and Diabetes Sciences, School of Medicine, Cardiff University, Cardiff CF14 4XN, UK. E-mail: wmdjh{at}cardiff.ac.uk

SPECIFIC AIMS

Adenosine receptors are present in the anterior pituitary gland where they mediate the secretion of interleukin (IL)-6 and vascular endothelial growth factor (VEGF) from folliculostellate (FS) cells. Adenosine is produced during the dephosphorylation of AMP by the enzyme ecto-5^/-nucleotidase (CD73). Within the cell, adenosine is recycled back to AMP by adenosine kinase (AK), whereas excess extracellular ATP is degraded to inosine by adenosine deaminase (AD). The aim of this study was to investigate if GH3, MMQ, and TtT/GF pituitary cells possess functional CD73 and whether such cells produce extracellular adenosine under basal conditions. CD73 and AD expression were determined by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunocytochemistry, and CD73 enzyme activity was determined by HPLC analysis of the conversion of exogenously added fluorescent 1,N⁶-ethenoAMP to ethenoadenosine. Basal adenosine secretion in the presence of the AD inhibitor erythro-9-(2-hydroxy-3-nonyl)-adenine (EHNA; 5 µM) and the AK inhibitor iodotubericidin (10 µM) was also determined by HPLC.

As ATP has been shown to decrease intercellular gap-junction communication in astrocytes, and down-regulation of connexin 43 (Cx43) leads to decreased expression of ATP receptors, we asked whether adenosine plays a related role in regulating Cx43 activity. We therefore investigated the effects of adenosine on Cx43 expression by comparative QRT-PCR, Western blotting, and direct intercellular communication via dye transfer of Alexa Fluor 488 in murine TtT/GF FS cells.

To determine whether endocrine cells, influence the functional activity of FS cells in the setting of the anterior pituitary gland, cocultures of MMQ and TtT/GF cells were prepared and expression of Cx43 monitored.

PRINCIPAL FINDINGS

1. Anterior pituitary cells express CD73 and produce extracellular adenosine
CD73 and AD were detected by RT-PCR in rodent anterior pituitary tissue and in all pituitary cell lines tested, including those of growth-hormone (GH3), prolactin (MMQ), adrenocorticotropin (AtT20DV16), and FS (TtT/GF) lineages. Fluorescence immunostaining of rat anterior pituitary for CD73 protein showed its expression was less widespread and confined to only 20% of the cells; these cells were clustered around sinusoids and exhibited a similar pattern of staining to that of a subpopulation of prolactin cells. In colocalization experiments, CD73 immunostained with prolactin and growth hormone in normal human pituitary and CD73 and AD were both present in the MMQ cells.

Extracellular adenosine production was demonstrated in MMQ (3 µM/h) and GH3 (1 µM/h) but not in TtT/GF cells. CD73 enzyme activity was demonstrated in GH3, MMQ and TtT/GF cells with an ED₅₀ of 1 h. Confirmation that this enzyme in GH3 cells was CD73 was demonstrated by its inhibition with ßbeta;-methylene-adenosine diphosphate (AOPCP). On the other hand, the identity of the enzyme involved in the breakdown of ethenoAMP in MMQ and TtT/GF cells remains unclear but it is neither CD73 nor alkaline phosphatase owing to its lack of inhibition by levamisole (Fig. 1 ).

View larger version (20K): [in this window] [in a new window]   Figure 1. Adenosine production (A) and adenosine forming enzyme activity (B) in rodent pituitary cell lines. A) Cultures were incubated in serum-free media (in the presence of EHNA and ITU) for 30, 60, 120, and 180 min.; the conditioned media were then analyzed for adenosine using HPLC. For GH3 and MMQ cells, values at each time point were all significantly different from each other with P values between <0.05 and <0.001; for TtT/GF cells there was no difference in values at any of the time points. B) Cultures were incubated with fluorescent 1,N6-ethenoAMP for 5, 15, 30, 60, and 120 min in the absence or presence of AOPCP (CD73 inhibitor). Production of ethenoadenosine was quantified using HPLC. All values at 15 min and later time points were significantly different from the zero time point and each other (P<0.01–0.001) except in GH3 + AOPCP cells, where only the 60 (P<0.05) and 120 (P<0.001) minute time points were significantly different from 0. All values are mean ± SE of 3 replicates; 2–3 experiments were carried out.

2. Adenosine stimulates Cx43 expression and gap-junctional intercellular communication in TtT/GF cells
Adenosine and the universal adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA) stimulated Cx43 protein expression in TtT/GF cells as shown by Western blot analysis with respectively EC₅₀ values of 0.5 and 0.1 µM. Both the nonphosphorylated and phosphorylated Cx43 forms were detected in these cells and both increased after adenosine receptor stimulation; changes were detectable within 0.5 h and were stable over 2–8 h when a 3-fold increase was observed. In parallel experiments, comparative QRT-PCR showed that 10 µM NECA stimulated increases in Cx43 mRNA; 4- to 5-fold (P<0.001) and 2- to 3-fold (P<0.01) increases were observed at 1 and 2–4 h when related to either ßbeta;-actin or PGK-1 (phosphoglycerate kinase 1) as invariant genes. By 8 h, the levels of Cx43 had returned to normal.

Intercellular communication in TtT/GF cells was studied by microinjecting the dye Alexa Fluor 488 into individual cells and assessing the transfer of dye into adjacent cells after 10 min using fluorescence microscopy. Prior treatment of the cells with either 10 µM adenosine or NECA for 4 h resulted in marked increases in the number of cells that contained the dye, respectively, 24 ± 5.7 (P<0.001) and 33 ± 7.8 (P<0.001) when compared with untreated cells (4±1.7; Fig. 2 A–C). The gap-junction inhibitor octanol (1 mM for 30 min) completely blocked the stimulatory effects of adenosine or NECA (Fig. 2D-E ). These data are shown in a graphical format in Fig. 2F .

View larger version (12K): [in this window] [in a new window]   Figure 2. Alexa 488 dye transfer in TtT/GF cells after NECA and adenosine stimulation. Cultures were incubated with and without 10 µM NECA or adenosine for 4 h and 20 individual cells in each culture were microinjected with dye, and cell fluorescence was observed after 10 min. In some experiments, 1 mM octanol was added before addition of dye. Each experiment was performed 3–6 times. A) untreated cells; B) 10 µM adenosine; C) 10 µM NECA; D) 10 µM adenosine + octanol; E) 10 µM NECA + octanol; F) Mean ± SE values for the numbers of cells that contained dye after each treatment from 3–6 experiments. *P values are <0.05 and ***P < 0.001 when compared with untreated cells. Magnification = x400.

3. MMQ cells influence the expression of Cx43 in TtT/GF cells
MMQ and TtT/GF cells, cocultured for 24 h, resulted in stimulated expression of non- and phosphorylated forms of Cx43 in the TtT/GF cells. The degree of Cx43 expression was directly related to the number of MMQ cells plated out. Adenosine levels in the culture medium surrounding the TtT/GF cells however showed only very small increases with increasing MMQ cell number (0.6 µM over 24 h) compared with the 3µM/hr seen with MMQ cells alone (Fig. 1) .

CONCLUSIONS AND SIGNIFICANCE

These studies show that anterior pituitary cells produce extracellular adenosine under basal conditions and they possess the enzyme machinery, i.e., CD73 and AD, to generate and inactivate adenosine via the ATP metabolic pathway. We also showed that some of these cells are likely to be derived from prolactin or growth hormone lineages. In addition to CD73, there may be other unidentified ecto-nucleotidases that have the capacity to convert AMP to adenosine in the anterior pituitary gland. Our data also show, for the first time, that adenosine increases Cx 43 expression at the mRNA and protein levels and stimulates intercellular communication in TtT/GF cells. Although both nonphosphorylated and phosphorylated forms of Cx43 were increased by adenosine, the functional consequences, in the context of gap-junction channel gating, is unclear.

The role of FS cells, in the pituitary gland, is ill-defined, since the cells do not produce classical "hormones" as is indicative of other cell types. FS cells produce cytokines and growth factors that are thought to act in a paracrine or autocrine rather than an endocrine fashion. We previously showed that adenosine can play such a role in TtT/GF cells by stimulating cytokine secretion. The present studies also support the view that adenosine can modulate intercellular communication across gap junctions by facilitating the passage of small molecules such as amino acids, glucose, nucleotides, and ions into adjoining cells. Our data also confirm that lactotrophs can, via the secretion of adenosine, potentially influence the functional activity of FS cells in the setting of the anterior pituitary gland. In cocultures of MMQ and TtT/GF cells, the apparent levels of adenosine were much lower than that secreted from MMQ cells when cultured alone. The reason behind this is unclear but may be indicative of adenosine uptake by the TtT/GF cells. A schematic diagram of postulated lactotroph and FS cell interactions is shown in Fig. 3 .

View larger version (20K): [in this window] [in a new window]   Figure 3. A schematic diagram showing postulated effects of adenosine on FS and lactotroph function in anterior pituitary gland. Adenosine, produced by lactotrophs and FS cells, has autocrine and paracrine effects that modulate prolactin, IL-6 and VEGF secretion as well as influencing gap junction communication.

Although our experiments were carried out in pituitary cells, it is likely that the modulatory action of adenosine on intercellular communication is applicable to a variety of cells and tissues. There is an increased presence of extracellular adenosine during cell "stress" and in diseased states adenosine may, for example, contribute to tumor growth and stimulate angiogenesis.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.06-6121fje

This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.06-6121fjev1 20/14/2585    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Lewis, B. M. Articles by Ham, J. Search for Related Content PubMed PubMed Citation Articles by Lewis, B. M. Articles by Ham, J.