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Pancreatic islet β‐cells undergo apoptosis (APO) following culture with cytokines (CTK) in an in vitro model for type 1 diabetes mellitus. The aim was to determine if sphingosine 1‐phosphate (S1P) modulates the biochemical pathways that mediate the β‐cell APO response. Isolated rat islets or cells were pretreated with S1P (400 nM) for 5 h prior to and during CTK stimulation for 24‐48 h. APO was determined by TUNEL staining. CTK (1 ng/ml interleukin‐1β and 5 ng/ml interferon‐γ ) induced 28±2% APO in islet cells after 48 h. S1P alone did not affect islet cell APO, however, S1P together with CTK reduced APO to 13±3% (P<0.001 vs. CTK) of total cells. Caspase 3 activity in INS‐1e cells was also a measure of APO. CTK increased caspase 3 activity after 24 h to 193±33% of control (C) cells without CTK treatment (P<0.001). The presence of S1P or dihydro‐S1P (400 nM) with CTK reduced INS‐1e cell caspase 3 activity to 127±15% C (P>0.05) and 105±14% C (P>0.05), respectively; the occurrence of APO cells with S1P alone was 94±7% C (P>0.05). Forskolin (50 nM) reduced CTK‐induced caspase 3 activity to 121±5% C (P>0.05). Analysis by real‐time PCR showed that the mRNA expression for the anti‐APO gene Bcl‐xL increased by 337±34% C (P<0.001) in CTK‐treated islets, and that S1P augmented the expression to 570±63% C (P<0.01); with S1P alone Bcl‐xL mRNA levels were 108±15% C. In conclusion, S1P protects the islet β‐cell from cytokine‐induced APO to a similar extent as forskolin, and increased expression of Bcl‐xL and reduced activity of caspase 3 play a role in the antiapoptotic effects. (Supported by Juvenile Diabetes Research Foundation grant 1‐2002‐613)

The endothelial differentiation gene (EDG) receptors are a class of G protein-coupled receptors. EDG-1, -3, -5, -6, and -8 bind the bioactive lipid sphingosine-1-phosphate (SPP) as the primary signaling ligand. EDG-2, -4, and -7 bind the ligand lysophosphatidic acid. EDG-1, -2, -3, -5, -6, and -7, but not -8, mRNAs were expressed in isolated rat pancreatic islets, whereas INS-1 insulinoma cells expressed only EDG-1, -2, -3, and -5 mRNAs. EDG-4 mRNA was expressed in mouse islets. EDG-1 mRNA but not EDG-3 mRNA was rapidly induced relative to 18S rRNA after stimulation of isolated islets with phorbol 12-myristate 13-acetate (PMA) or cholecystokinin-8S for 2 h. The protein kinase C inhibitor GF 109203X blocked the EDG-1 induction by PMA. Similarly, in islets stimulated for 2 h with 17 mmol/l glucose, the relative EDG-1 mRNA levels increased almost twofold compared with levels in control islets at 5.5 mmol/l glucose. In contrast, after 11 mmol/l glucose stimulation for 7 days, the relative levels of rat islet EDG-1 mRNA were significantly reduced to 54% below that of islets cultured at 5.5 mmol/l glucose. There was no change in relative EDG-3 mRNA levels. Stimulation of EDG receptors in islets and INS-1 cells with SPP inhibited glucagon-like peptide 1 (GLP-1)-stimulated cAMP production and insulin secretion in a concentration-dependent manner. Pertussis toxin antagonized the SPP effects on insulin release. Thus, EDG receptors are expressed in pancreatic islet ␤-cells and G i seems to mediate the inhibition by SPP of adenylyl cyclase and cAMP formation and inhibition of the stimulation of insulin secretion by GLP-1. Diabetes

Functional and molecular biological evidence exists for the expression of ryanodine receptors in non-muscle cells. In the present study, RT-PCR and 5'-rapid amplification of cDNA 5'-end (5'-RACE analysis) provided evidence for the presence of a type 1 ryanodine receptor/Ca2+ channel (RyR1) in diverse cell types. In parotid gland-derived 3-9 (epithelial) cells, the 3'-end 1589 nucleotide sequence for a rat RyR shared 99% homology with rat brain RyR1. Expression of this RyR mRNA sequence in exocrine acinar cells, endocrine cells, and liver in addition to skeletal muscle and cardiac muscle, suggests wide tissue distribution of the RyR1. Positive identification of a 5'-end sequence was made for RyR1 mRNA in rat skeletal muscle and brain, but not in parotid cells, pancreatic islets, insulinoma cells, or liver. These data suggest that a modified RyR1 is present in exocrine and endocrine cells, and liver. Western blot analysis showed L-type Ca2+ channel-related proteins in parotid acinar cells, which were of comparable size to those identified in skeletal and cardiac muscle, and in brain. Immunocytochemistry carried out on intact parotid acini demonstrated that the dihydropyridine receptor was preferentially co-localized with the IP3 receptor in the apical membranes. From these data we conclude that certain non-muscle cells express a modified RyR1 and L-type Ca2+ channel proteins. These receptor/channels may play a role in Ca2+ signaling involving store-operated Ca2+ influx via receptor-mediated channels.

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