title: Ryanodine receptors in human pancreatic beta cells: localization and effects on insulin secretion.
abstract: It is clear that pancreatic beta-cell dysfunction, including basal hyperinsulinemia and reduced insulin release in response to glucose, is a key determinant of disease progression in type 2 diabetes, but the underlying molecular defects are not known. In diabetes, the expression and function of ryanodine receptor (RyR) Ca2+ release channels are reduced. The present studies were undertaken to define the subcellular location and role of RyR in the control of stimulated and basal insulin release from human pancreatic beta cells. Using confocal microscopy, we observed RyR immunoreactivity in a vesicular pattern. RyRs did not colocalize with insulin secretory granules but partially colocalized with endosomes. Direct activation with nanomolar concentrations of ryanodine evoked increases in cytosolic Ca2+ that were coupled to transient insulin release. Insulin release stimulated by 1 nM ryanodine was sensitive to BAPTA-AM preincubation but independent of thapsigargin-sensitive endoplasmic reticulum (ER) Ca2+ pools. Blocking RyRs with micromolar concentrations of ryanodine led to BAPTA-resistant insulin release that was not associated with an increase in cytosolic Ca2+, which implicated alterations in luminal Ca2+. However, neither Ca2+ signals nor insulin release stimulated by glucose was blocked by 10-50 microM ryanodine, which suggests that the CD38/cyclic ADP-ribose/RyR pathway is not a primary mechanism of glucose action in nontransformed beta cells. We provide the first evidence that RyRs directly control insulin secretion in primary beta cells. Unexpectedly, stimulation of insulin secretion by ryanodine occurs independently of glucose and by two mechanisms, including a novel cytosolic Ca2+-independent mechanism likely involving changes in Ca2+ within the lumens of non-ER organelles, such as endosomes.
authors.affiliation: Division of Metabolism, Department of Internal Medicine, Washington University School of Medicine, Box 8126, 8831 Wohl Clinic, 660 S. Euclid, St. Louis, MO 63110, USA. jim@jimjohnson.ca
authors.forename: James D
authors.initials: JD
authors.lastname: Johnson
authors.forename: Shihuan
authors.initials: S
authors.lastname: Kuang
authors.forename: Stanley
authors.initials: S
authors.lastname: Misler
authors.forename: Kenneth S
authors.initials: KS
authors.lastname: Polonsky
mesh_terms: D036541:ADP-ribosyl Cyclase / Q000502:physiology; D051997:ADP-ribosyl Cyclase 1; D000818:Animals; D015703:Antigens, CD / Q000502:physiology; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured / Q000187:drug effects / Q000378:metabolism; D003620:Dantrolene / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D011992:Endosomes / Q000378:metabolism; D005089:Exocytosis / Q000187:drug effects*; D005947:Glucose / Q000494:pharmacology; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000187:drug effects / Q000378:metabolism*; D047028:Macrocyclic Compounds; D008562:Membrane Glycoproteins; D051379:Mice; D010080:Oxazoles / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D011189:Potassium Chloride / Q000494:pharmacology; D012433:Ryanodine / Q000494:pharmacology; D019837:Ryanodine Receptor Calcium Release Channel / Q000187:drug effects / Q000502:physiology*; D019284:Thapsigargin / Q000494:pharmacology
chemical_list: D015703:Antigens, CD; D007328:Insulin; D047028:Macrocyclic Compounds; D008562:Membrane Glycoproteins; D010080:Oxazoles; D019837:Ryanodine Receptor Calcium Release Channel; C108644:xestospongin A; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D012433:Ryanodine; D004533:Egtazic Acid; D011189:Potassium Chloride; D019284:Thapsigargin; D036541:ADP-ribosyl Cyclase; C498154:CD38 protein, human; C498155:Cd38 protein, mouse; D051997:ADP-ribosyl Cyclase 1; D003620:Dantrolene; D005947:Glucose; D002118:Calcium