ATP autocrine/paracrine signaling induces calcium oscillations and NFAT activation in human mesenchymal stem cells

Human bone marrow-derived mesenchymal stem cells (hMSCs) have the potential to differentiate into several types of cells. Calcium ions (Ca(2+)) play an important role in the differentiation and proliferation of hMSCs. We have demonstrated that spontaneous [Ca(2+)](i) oscillations occur without agonist stimulation in hMSCs. However, the precise mechanism of its generation remains unclear. In this study, we investigated the mechanism and role of spontaneous [Ca(2+)](i) oscillations in hMSCs and found that IP(3)-induced Ca(2+) release is essential for spontaneous [Ca(2+)](i) oscillations. We also found that an ATP autocrine/paracrine signaling pathway is involved in the oscillations. In this pathway, an ATP is secreted via a hemi-gap-junction channel; it stimulates the P(2)Y(1) receptors, resulting in the activation of PLC-beta to produce IP(3). We were able to pharmacologically block this pathway, and thereby to completely halt the [Ca(2+)](i) oscillations. Furthermore, we found that [Ca(2+)](i) oscillations were associated with NFAT translocation into the nucleus in undifferentiated hMSCs. Once the ATP autocrine/paracrine signaling pathway was blocked, it was not possible to detect the nuclear translocation of NFAT, indicating that the activation of NFAT is closely linked to [Ca(2+)](i) oscillations. As the hMSCs differentiated to adipocytes, the [Ca(2+)](i) oscillations disappeared and the translocation of NFAT ceased. These results provide new insight into the molecular and physiological mechanism of [Ca(2+)](i) oscillations in undifferentiated hMSCs.     

Notch signaling regulates bile duct morphogenesis in mice

Background

Alagille syndrome is a developmental disorder caused predominantly by mutations in the Jagged1 (JAG1) gene, which encodes a ligand for Notch family receptors. A characteristic feature of Alagille syndrome is intrahepatic bile duct paucity. We described previously that mice doubly heterozygous for Jag1 and Notch2 mutations are an excellent model for Alagille syndrome. However, our previous study did not establish whether bile duct paucity in Jag1/Notch2 double heterozygous mice resulted from impaired differentiation of bile duct precursor cells, or from defects in bile duct morphogenesis.

Methodology/Principal Findings

Here we characterize embryonic biliary tract formation in our previously described Jag1/Notch2 double heterozygous Alagille syndrome model, and describe another mouse model of bile duct paucity resulting from liver-specific deletion of the Notch2 gene.

Conclusions/Significance

Our data support a model in which bile duct paucity in Notch pathway loss of function mutant mice results from defects in bile duct morphogenesis rather than cell fate specification.     

Endocrine and paracrine control of sperm production in stallions.

The specific nature and relative contribution of the major hormones involved in regulation of reproductive function of the stallion are not well defined nor have paracrine or autocrine factors been identified. Over the last 12 years, our laboratory has been engaged in characterizing the hypothalamic-pituitary-testicular axis (HPT) in stallions. A number of endocrine factors and mechanisms important for normal reproductive function have been investigated. Studies investigating poor fertility in stallions suggest that a closer look at the testicular level is warranted. For a complete understanding of intratesticular control mechanisms including cell-to-cell interactions in the stallion, studies on the actions of paracrine/autocrine factors such as growth factors, inhibin, activin, and oxytocin are needed. In other species, paracrine/autocrine systems appear to be important in modulating endocrine control of testicular function and spermatogenesis.     

Endocrine,paracrine and autocrine actions of prolactin on immune cells

The immune response is regulated by locally released factors, collectively referred to as cytokines. Data on the human immune system have convincingly demonstrated that the hormone prolactin (PRL), in addition to exerting its endocrine control on the immune system, acts as a cytokine in that it is released within the immune system and regulates the lymphocyte response by paracrine and autocrine mechanisms. Both lymphocyte and pituitary PRLs are under the control of immune factors. Synthesis of human PRL by lymphocytes is induced by T-cell stimuli, while increased release of PRL by the pituitary, observed in vivo after immune challenge, may be mediated by cytokines produced by monocyte-macrophages. Since hyperprolactinemia and hypoprolactinemia are both immunosuppressive, physiological levels of circulating PRL must be necessary to maintain basal immunocompetence. The effects of Cyclosporin (CsA) on IL-2 and PRL gene activation and the analysis of the intracellular signaling events downstream IL-2 and PRL receptors suggest coordinate actions of these two cytokines during T cell activation.     

Mast cells in human bile duct obstruction

Surgical biopsy specimens obtained from 50 patients with secondary cholangitis caused by obstruction of the common bile duct were studied immunohistochemically. Data on the number and ultrastructural appearances of mast cells positive for tryptase, chymase, vasointestinal polypeptide (VIP), and substance P (SP) were obtained. The bile ducts from patients presenting combined chronic exacerbated cholangitis and chronic sclerotic cholangitis showed significantly higher numbers of mast cell types compared to the controls (P < 0.0001). Cases with sclerotic cholangitis alone had significantly lower number of cells than patients with chronic exacerbated cholangitis alone (P 0.0001). Morphometric measurements of electron micrographs showed that mast cell granules containing VIP, SP and chymase were commensurable in size. Electron-lucent granules without reaction product (altered granules) and granules with focal distribution of the reaction product were observed in all types of mast cells. Furthermore, some nerve fibers positive for SP and VIP and serotonin-positive endocrine cells were observed in close proximity to the mast cells. In conclusion, the results of our study demonstrate the existence of different populations of mast cells, nerve structures and endocrine cells in the lower part of the human large bile duct, and suggest their participation in the development of pathological processes.     

Calcium signaling and the secretory activity of bile duct epithelia

Cytosolic calcium (Ca_i²⁺) is a second messenger that is important for the regulation of secretion in many types of tissues. Bile duct epithelial cells, or cholangiocytes, are polarized epithelia that line the biliary tree in liver and are responsible for secretion of bicarbonate and other solutes into bile. Ca_i²⁺ signaling plays an important role in the regulation of secretion by cholangiocytes, and this review discusses the machinery involved in the formation of Ca²⁺ signals in cholangiocytes, along with the evidence that these signals regulate ductular secretion. Finally, this review discusses the evidence that impairments in cholangiocyte Ca²⁺ signaling play a primary role in the pathogenesis of cholestatic disorders, in which hepatic bile secretion is impaired.     

Regulation of bile secretion by calcium signaling in health and disease

Calcium (Ca²⁺) signaling controls secretion in many types of cells and tissues. In the liver, Ca²⁺ regulates secretion in both hepatocytes, which are responsible for primary formation of bile, and cholangiocytes, which line the biliary tree and further condition the bile before it is secreted. Cholestatic liver diseases, which are characterized by impaired bile secretion, may result from impaired Ca²⁺ signaling mechanisms in either hepatocytes or cholangiocytes. This review will discuss the Ca²⁺ signaling machinery and mechanisms responsible for regulation of secretion in both hepatocytes and cholangiocytes, and the pathophysiological changes in Ca²⁺ signaling that can occur in each of these cell types to result in cholestasis.     

Endocrine, paracrine and autocrine control of follicular development

Development of a single follicle during the menstrual cycle is under control of hormones stimulating follicular maturation, ovulation and luteogenesis. Several factors intervene locally to prevent other follicles from developing at the same time as dominant follicle. These other follicles remain quiescent or evaluate to atresia. Atresia results from the action of several endocrine, paracrine and autocrine mechanisms which synergistically inhibit aromatase activity. The subsequent lack of estrogens reduces granulosa cell multiplication. The oocyte will not become fertilizable before the preovulatory peak of LH, after the resumption of meiosis and after reaching metaphase of the second meiotic division. Several factors are involved in the inhibition of spontaneous resumption of meiosis: cyclic nucleotides, sex steroids, somatostatin and oocyte maturation inhibitor(s) (OMI). Ovulation is related to breakdown of connective tissue synthesized by granulosa cells under the influence of FSH. Connective tissue lysis is dependent on proteolytic enzymes which are released and activated by FSH, LH and relaxin. A paracrine control could be involved in ovulation: LH induces the production of prostaglandin and relaxin by theca cells which, in turn, stimulate collagenase and proteoglycanase secretion by granulosa cells.     

Agonist-stimulated pathways of calcium signaling in pancreatic acinar cells.

In pancreatic acinar cells stimulation of different intracellular pathways leads to different patterns of Ca2+ signaling. Bombesin induces activation of both phosphatidylinositol 4,5-bisphosphate (PIP2)-specific phospholipase C (PLC) and phospholipase D (PLD). The latter leads to generation of diacylglycerol (DAG) in addition to that produced by activation of PIP2-PLC. Strong activation of protein kinase C (PKC) results in inhibition of Ca(2+)-induced Ca2+ release from Ca2+ pools arranged in sequence to the luminally located IP3-sensitive Ca2+ pools. Consequently the Ca2+ wave which starts in the luminal cell pole is slower in the presence of bombesin (5 microm/s) as compared to that in the presence of acetylcholine (17 microm/s) which activates PIP2-PLC but not PLD. Activation of high-affinity CCK-receptors triggers a Ca2+ wave with slow propagation (5 microm/s) due to stimulation of phospholipase A2 (PLA2) and generation of arachidonic acid, which in turn leads to inhibition of Ca(2+)-induced Ca2+ release. Low-affinity CCK-receptors are coupled to both PIP2-PLC and PLD.     

Serotonin paracrine signaling in tissue fibrosis

The molecule serotonin (5-hydroxytryptamine or 5-HT) is involved in numerous biological processes both inside and outside of the central nervous system. 5-HT signals through 5-HT receptors and it is the diversity of these receptors and their subtypes that give rise to the varied physiological responses. It is clear that platelet derived serotonin is critical for normal wound healing in multiple organs including, liver, lung heart and skin. 5-HT stimulates both vasoconstriction and vasodilation, influences inflammatory responses and promotes formation of a temporary scar which acts as a scaffold for normal tissue to be restored. However, in situations of chronic injury or damage 5-HT signaling can have deleterious effects and promote aberrant wound healing resulting in tissue fibrosis and impaired organ regeneration. This review highlights the diverse actions of serotonin signaling in the pathogenesis of fibrotic disease and explores how modulating the activity of specific 5-HT receptors, in particular the 5-HT2 subclass could have the potential to limit fibrosis and restore tissue regeneration. This article is part of a Special Issue entitled: Fibrosis: Translation of basic research to human disease.     

Endoplasmic reticulum calcium signaling in nerve cells

The endoplasmic reticulum (ER) is an important organelle involved in various types of signaling in nerve cells. The ER serves as a dynamic Ca2+ pool being thus involved in rapid signaling events associated with cell stimulation by either electrical (action potential) or chemical (neurotransmitters) signals. This function is supported by Ca2+ release channels (InsP3 and ryanodine receptors) and SERCA Ca2+ pumps residing in the endomembrane. In addition the ER provides a specific environment for the posttranslational protein processing and transport of various molecules towards their final destination. In parallel, the ER acts as a "calcium tunnel," which facilitates Ca2+ movements within the cell by avoiding cytoplasmic routes. Finally the ER appears as a source of numerous signals aimed at the nucleus and involved in long-lasting adaptive cellular responses. All these important functions are controlled by intra-ER free Ca2+ which integrates various signaling events and establishes a link between fast signaling, associated with ER Ca2+ release/uptake, and long-lasting adaptive responses relying primarily on the regulation of protein synthesis. Disruption of ER Ca2+ homeostasis triggers several forms of cellular stress response and is intimately involved in neurodegeneration and neuronal cell death.     

Ryanodine receptors contribute to bile acid-induced pathological calcium signaling and pancreatitis in mice

Biliary pancreatitis is the most common etiology for acute pancreatitis, yet its pathophysiological mechanism remains unclear. Ca(2+) signals generated within the pancreatic acinar cell initiate the early phase of pancreatitis, and bile acids can elicit anomalous acinar cell intracellular Ca(2+) release. We previously demonstrated that Ca(2+) released via the intracellular Ca(2+) channel, the ryanodine receptor (RyR), contributes to the aberrant Ca(2+) signal. In this study, we examined whether RyR inhibition protects against pathological Ca(2+) signals, acinar cell injury, and pancreatitis from bile acid exposure. The bile acid tauro-lithocholic acid-3-sulfate (TLCS) induced intracellular Ca(2+) oscillations at 50 μM and a peak-plateau signal at 500 μM, and only the latter induced acinar cell injury, as determined by lactate dehydrogenase (LDH) leakage. Pretreatment with the RyR inhibitors dantrolene or ryanodine converted the peak-plateau signal to a mostly oscillatory pattern (P < 0.05). They also reduced acinar cell LDH leakage, basolateral blebbing, and propidium iodide uptake (P < 0.05). In vivo, a single dose of dantrolene (5 mg/kg), given either 1 h before or 2 h after intraductal TLCS infusion, reduced the severity of pancreatitis down to the level of the control (P < 0.05). These results suggest that the severity of biliary pancreatitis may be ameliorated by the clinical use of RyR inhibitors.     

Chronic bile duct cannulation in laboratory rats.

To our knowledge this is the first report of rat bile duct cannulations in which the distal cannula is hemisected but extends to the sphincter of Oddi. It is minimally invasive and requires only about 45 minutes preparation time. In contrast to studies described in the literature, enterohepatic recirculation remains intact but bile can always be separated from pancreatic secretions at investigator discretion in the model. In addition, biliary flow and pressure can be measured without compromise. Acute biliary secretory pressure, under anesthesia, was 17 cm water. Bile flow, averaging 9.6 microliters/min/100 g was measured in unanesthetized rats surviving for 2 weeks (60% of animals monitored). Gross necropsy findings indicated that animals dying in less than 7 days usually suffered bile peritonitis subsequent to catheter rupture of the bile duct or loss from the ligature restraint. Deaths after 2 weeks were usually related to cholestasis due to blockage of the catheter with mineral debris and/or duct tissue. A detailed literature review of bile duct cannulation in rats has been made.     

Early changes in pancreatic acinar cell calcium signaling after pancreatic duct obstruction

Intracellular Ca(2+)-changes not only participate in important signaling pathways but have also been implicated in a number of disease states including acute pancreatitis. To investigate the underlying mechanisms in an experimental model mimicking human gallstone-induced pancreatitis, we ligated the pancreatic duct of Sprague-Dawley rats and NMRI mice for up to 6 h and studied intrapancreatic changes including the dynamics of [Ca(2+)](i) in isolated acini. In contrast to bile duct ligation, pancreatic duct obstruction induced intra-pancreatic trypsinogen activation, leukocytosis, hyperamylasemia, and pancreatic edema and increased lung myeloperoxidase activity. Although resting [Ca(2+)](i) in isolated acini rose by 45% to 205 +/- 7 nmol, the acetylcholine- and cholecystokinin (CCK)-stimulated calcium peaks as well as the amylase secretion declined, but neither the [Ca(2+)](i)-signaling pattern nor the amylase output in response to the Ca(2+)-ATPase inhibitor thapsigargin nor the secretin-stimulated amylase release were impaired by pancreatic duct ligation. On the single cell level pancreatic duct ligation reduced the percentage of cells in which submaximal secretagogue stimulation was followed by a physiological response (i.e. Ca(2+) oscillations) and increased the percentage of cells with a pathological response (i.e. peak plateau or absent Ca(2+) signal). Moreover, it reduced the frequency and amplitude of Ca(2+) oscillation as well as the capacitative Ca(2+) influx in response to secretagogue stimulation. Serum pancreatic enzyme elevation as well as trypsinogen activation was significantly reduced by pretreatment of animals with the calcium chelator BAPTA-AM. These experiments suggest that pancreatic duct obstruction rapidly changes the physiological response of the exocrine pancreas to a Ca(2+)-signaling pattern that has been associated with premature digestive enzyme activation and the onset of pancreatitis, both of which can be prevented by administration of an intracellular calcium chelator.