Extracellular adenosine 5'-triphosphate modulates insulin secretion via functionally active purinergic receptors of X and Y subtype.

Extracellular nucleotides modulate several cell functions via specific receptors, P2X and P2Y. We explored the differential role of these receptors in the control of insulin secretion (InSec). In INS-1e cells grown in 11 mm glucose and then acutely exposed to 3.3, 7.5, 11, or 20 mm, coincubation with ATP, the global agonist of both P2X and P2Y receptors, induced a dose-dependent (P < 0.0001) reduction in insulin release (P < 0.0001) that was more marked at higher glucose concentrations (P < 0.0001 for the interaction). This effect was fully prevented (P < 0.0001) by incubating ATP-treated cells in the presence of apyrase, an ecto-ATP/ADPase. Uridine 5'-triphosphate (UTP), preferential agonist of P2Y receptors, significantly stimulated InSec at all glucose concentrations tested, whereas benzoyl-benzoyl ATP (BzATP), a strong and highly selective P2X(7) agonist, did not influence InSec. Oxidized ATP, which completely suppresses P2X activity, abolished the inhibitory effect of ATP on InSec. Similar results were obtained in MIN-6 cells. Stimulation with ATP, BzATP, and UTP dose-dependently increased Intracellular free Ca(2+) concentrations. By small interfering RNA we show P2X(3) and P2Y(4) as the main responsible inhibitory and promoting effect on InSec, respectively. Because P2X(7) is not directly involved in InSec, we tested whether the effect of ATP on hormone synthesis might be mediated by apoptosis. However, neither ATP nor BzATP induced either early or late apoptosis. We conclude that: 1) INS-1e cells express multiple purinergic receptors, 2) ATP reduces glucose-induced InSec as a net effect of inhibition through P2X and stimulation through P2Y receptors, and 3) P2X-mediated apoptosis is not involved in the inhibition of InSec.

Effect of type I interferon(s) on cell viability and apoptosis in primary human thyrocyte cultures.

BACKGROUND: Interferon (IFN) therapy may induce a generalized activation of the immune system, hence triggering or exacerbating autoimmune disease. Apoptosis contributes to the development of hypothyroidism in autoimmune thyroiditis. IFN can affect all phases of the cell cycle and may induce apoptosis in several cell lines from varied histologies. To date, no data exist on the possible effect of type I IFN(s) on FAS/FASL system and cell apoptosis of human thyroid follicles. Therefore, we evaluated the effect of both IFN-alpha and -beta on apoptosis in primary human thyrocyte cultures and the potential role of the FAS/FASL pathway. METHODS: Thyrocytes were cultured in monolayers and FAS, FASL, and Bcl-2 mRNA expression was determined by reverse transcriptase polymerase chain reaction after exposure to 10 mIU/mL bovine thyroid-stimulating hormone alone or in combination with increasing doses of IFN-alpha or -beta for 24, 48, and 72 hours. The percentage of apoptotic hypodiploid cells was evaluated by flow cytometry. RESULTS: Thyroid-stimulating hormone significantly decreased FAS and increased Bcl-2 mRNA expression while reducing the percentage of hypodiploid cells. The concomitant addition of either IFN-alpha or -beta reduced cell viability and increased the number of hypodiloid cells, but only IFN-beta modulated the expression of FAS and Bcl-2 mRNA expression in a proapoptotic sense. CONCLUSIONS: Both type I IFN(s) increase apoptosis in primary thyrocyte cultures, but only IFN-beta modulates FAS and Bcl-2 gene expression toward a proapoptotic pathway. Because apoptosis plays an important role in thyroid homeostasis and disease, this mechanism may contribute to the development and progression of type I IFN(s) therapy-associated thyroid disease.

Rosiglitazone increases matrix production and quenches inflammation: studies in human cells.

BACKGROUND: Type 2 diabetes (T2D) is characterized by an accelerated atherogenesis, a process to which both proliferative and inflammatory responses contribute. Peroxisome proliferator-activated receptors-gamma (PPARgamma) agonists have both anti-proliferative and anti-inflammatory properties. We tested the effect of therapeutic doses of rosiglitazone on proliferative and inflammatory pathways in fibroblasts (HF) from five controls (C) and five T2D patients, and in aortic smooth muscle cells (hSMC). METHODS: Transforming growth factor-beta (TGFbeta) and interleukin-6 (IL-6) expression, and IL-6, laminin and fibronectin release were measured. To identify the involved intracellular signalling, extracellular signal-regulated kinases (ERK)1/2 phosphorylation and p38 activation were evaluated. RESULTS: Both phorbol 12-myristate 13-acetate (PMA) [a protein kinase C (PKC) activator] and rosiglitazone increased TGFbeta expression and fibronectin and laminin release in C and T2D patients. Rosiglitazone effect was reversed by its specific inhibitor Sr202. The combination PMA + rosiglitazone was additive in C, but not in T2D patients. IL-6 production was stimulated by PMA in both C and T2D patients; this effect was prevented by rosiglitazone in a Sr202-inhibitable manner. Experiments performed in hSMC yielded the same results. Rosiglitazone increased p38 activation more in C than in T2D patients; PMA-induced phosphorylation of ERK1/2 was similarly reduced in both cells. CONCLUSIONS: In HF and hSMC, rosiglitazone stimulates the synthesis of matrix components via enhanced TGFbeta expression; when combined with PMA, the resulting PKC activation is mediated by enhanced p38 phosphorylation. On the other hand, rosiglitazone quenches inflammation in both cell types, by counteracting PMA-induced phosphorylation of ERK1/2.

Increased P2X7 receptor expression and function in thyroid papillary cancer: a new potential marker of the disease?

Nucleotides are increasingly recognized as nonredundant extracellular signals for chemotaxis, cell growth, and cytokine release. Effects of extracellular nucleotides are mediated by P2 receptors, among which the P2X(7) subtype is attracting increasing attention for its involvement in apoptosis, cell growth, and cytokine release. Recent studies showed that P2X(7) is overexpressed in chronic lymphocytic leukemia and breast and prostate cancer. The aim of the present study was to better understand the clinical significance of P2X(7) receptor expression in normal and cancer human thyroid tissues. P2X(7) receptor message and protein expression and functional activity were tested in two cell lines (FB1 and FB2) established from either anaplastic or papillary primary thyroid cancer and in several histological samples of human papillary cancer. We show here that human thyroid papillary carcinoma, whether of the classical or follicular variant, expresses the P2X(7) receptor (P2X(7)R) to a much higher level than normal thyroid tissue. The P2X(7)R was similarly up-regulated in FB1 and FB2 cell lines. In contrast to normal thyroid cells, both cell lines responded to extracellular nucleotide stimulation with a large increase in intracellular Ca(2+) and secretion of IL-6. Ca(2+) increase was attenuated and release of IL-6 was fully blocked by P2X(7)R inhibitors. Finally, the thyroid carcinoma cell lines had at least a 3-fold higher intracellular ATP concentration and maintained at least a 3-fold higher extracellular ATP level, compared with control cells. These data suggest that an enhanced P2X(7)R function might be a feature of human thyroid cancer.

Multiple P2X receptors are involved in the modulation of apoptosis in human mesangial cells: evidence for a role of P2X4.

Apoptosis, a normal event in renal tissue homeostasis, has been considered as a major mechanism for either resolution of glomerular hypercellularity in glomerulonephritis or loss of cellularity and progression to glomerulosclerosis in chronic renal disease. This study was aimed at investigating the role of extracellular ATP (eATP) in mediating apoptosis in human mesangial cells (HMC) and identifying the subtype(s) of purinergic receptors involved. eATP, but not uridin-5'-triphosphate (UTP), caused dose-dependent modifications of cellular morphology, as assessed by contrast-phase microscopy, and late apoptosis, as measured by Annexin V/propidium iodide-based flow cytometry and caspase-3 activation. Both phenomena were prevented by the P2X antagonist oxidized-ATP. 2', 3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (BzATP) was less effective than ATP, whereas 1[N,O-bis (5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl] -4-phenylpiperazine (KN62), a selective inhibitor of human P2X(7), prevented morphological changes but potentiated apoptosis induced by BzATP. P2X(7) was barely expressed in HMC and showed a relatively scarce functional activity, as assessed by monitoring nucleotide-induced intracellular calcium surge and plasma membrane depolarization by Fura-2/AM and bis[1,3-diethylthiobarbiturate]trimethineoxonal uptake, respectively. These data indicated a negligible role of P2X(7) in eATP-mediated apoptosis and pointed to the involvement of other P2X receptor(s). Molecular and inhibitor studies suggested a main role for P2X(4) receptor in nucleotide-induced apoptosis in HMC, indicating a relevant role for purinergic signaling in regulating death rate in these cells.

Effects of endothelin-1 on fibroblasts from type 2 diabetic patients: Possible role in wound healing and tissue repair.

Endothelin-1 (ET-1) promotes the contractile ability of fibroblasts, essential for wound closure and reconstitution of the dermis. Wound healing is impaired in type 2 diabetic patients (D). We compared the effect of ET-1 on proliferative transforming growth factor (TGFbeta(1)) expression, fibronectin and laminin release), differentiative [alpha-smooth muscle actin (alpha-SMA) expression] and inflammatory [monocyte chemo-attractant protein (MCP-1) and interleukin-6 (IL-6) expression] responses in skin fibroblasts of healthy subjects (C) and D, testing the relative role of ET(A) and ET(B) receptors in mediating these responses. ET-1 did not influence TGFbeta(1), fibronectin or laminin production. alpha-SMA was more abundant and more stimulated in D, as well as MCP-1 and IL-6 expression and release. These effects were prevented by BMS-182874, selective antagonist of ET(A), more abundant than ET(B) in both cell strains and whose expression rose more in D than C upon stimulation with ET-1. This peculiar pattern of responses to ET-1, presumably acquired during the chronic in vivo exposure to hyperglycemia along the natural history of the disease, may partially explain the increased susceptibility of D to chronic ulcerations.

Extracellular adenosine 5'-triphosphate modulates interleukin-6 production by human thyrocytes through functional purinergic P2 receptors.

We investigated the presence of P2 receptors (P2Rs) in human thyrocytes and their possible involvement in the modulation of cytokine release. P2Rs expression was assessed by RT-PCR and, when possible, by immunoblotting. Human primary thyrocytes express the mRNA for the following P2X and P2Y subtypes: P2X(3), P2X(5), P2X(6), P2X(7), and P2Y(1), P2Y(2), P2Y(4), and P2Y(11). Stimulation with extracellular nucleotides of fura-2-loaded thyrocytes triggered an intracellular Ca(2+) signal, suggesting expression of functional receptors. Thyrocytes spontaneously released the proinflammatory cytokine IL-6. The ATP-hydrolyzing enzyme apyrase reduced basal IL-6 release, whereas extracellular ATP dose-dependently increased IL-6 secretion. Uridine 5'-triphosphate was also an effective stimulus, whereas benzoyl-ATP was ineffective, suggesting a P2Y- rather than P2X-modulated response. Finally, TSH reduced both the intracellular Ca(2+) ([Ca(2+)](i)) rise and IL-6 release triggered by P2Rs stimulation. In conclusion, we provide functional, pharmacological, and biochemical evidence that human primary thyrocytes express P2YR and P2XR subtypes, coupled to increases in ([Ca(2+)](i)) and secretion of IL-6. P2R-dependent modulation of IL-6 release from human thyrocytes suggests a novel mechanism whereby an inflammatory and/or immune-mediated damage can be initiated and amplified in the thyroid.