Regulation of exocytosis in electrically permeabilized insulin-secreting cells. Evidence for Ca2+ dependent and independent secretion

The regulation of insulin secretion from RINm5F cells exposed to high voltage discharge has been investigated. Electron microscopy revealed that the overall structure of the cells was preserved after permeabilization. In this preparation insulin release was stimulated by Ca2+ (EC50=2.4 μM). The stable GTP analogue GTPγS enhanced secretion both at intermediate (nano- to micromolar) and vanishingly low (<10 pM) Ca2+ concentrations. At optimal Ca2+ (10 μM) the effect of GTPγS was greatly reduced. We investigated whether the secretory response to GTP analogues was mediated by any of three enzyme systems regulated by GTP-binding proteins, i.e. generation of cyclic AMP by adenylate cyclase, of diacylglycerol by phospholipase C and of arachidonic acid by phospholipase A2. The involvement of these messenger systems could be excluded as (i) cyclic AMP only had minor, Ca2+ dependent effects, (ii) phospholipase C was not activated in the absence of Ca2+ and insulin secretion due to the phorbol ester TPA displayed a different Ca2+ dependency, (iii) arachidonic acid did not elicit Ca2+ independent insulin secretion. These results, taken together with the finding that insulin secretion due to Ca2+ or TPA is attenuated by the inhibitory guanine nucleotide GDPβS, suggest the existence of a regulatory site in exocytosis which is sensitive to guanine nucleotides.

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Inhibition by cyclic AMP of guanine nucleotide-induced activation of phosphoinositide-specific phospholipase C in human platelets

FEBS Letters ◽

10.1016/0014-5793(89)80503-4 ◽

1989 ◽

Vol 242 (2) ◽

pp. 368-372 ◽

Cited By ~ 44

Author(s):

Yukihiro Yada ◽

Seiji Nagao ◽

Yukio Okano ◽

Yoshinori Nozawa

Keyword(s):

Cyclic Amp ◽

Phospholipase C ◽

Human Platelets ◽

Guanine Nucleotide

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Interaction between insulin-storage granules and F-actin in vitro

Biochemical Journal ◽

10.1042/bj1780367 ◽

1979 ◽

Vol 178 (2) ◽

pp. 367-371 ◽

Cited By ~ 22

Author(s):

S L Howell ◽

M Tyhurst

Keyword(s):

Insulin Secretion ◽

Cyclic Amp ◽

Phospholipase C ◽

Islets Of Langerhans ◽

Actin Filaments ◽

Sedimentation Rates ◽

Storage Granules ◽

Rat Islets Of Langerhans ◽

Insulin Storage

Possible interactions between polymerized (F-) actin and insulin-storage granules from rat islets of Langerhans were examined in vitro by comparing the sedimentation of the granules in the presence of various actin concentrations. Actin in the concentration range 0.1–0.5 mg/ml produced a retardation in granule-sedimentation rates consistent with binding of the granules to the actin filaments. The interaction was increased by addition of ATP (2mM), but was decreased by CaCl2 (0.1 mM). Binding of granules to actin was unaffected by cyclic AMP or by preincubation of the granules with phospholipase C. Specificity of the interaction was confirmed by the use of depolymerized (G-) actin and of myosin to provide a solution of comparable viscosity; neither of these caused any alteration of granule sedimentation. Possible implications of this interaction of insulin-storage granules with actin for the mechanism of insulin secretion are briefly discussed.

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GTP-dependent inhibition of insulin secretion by epinephrine in permeabilized RINm5F cells. Lack of correlation between insulin secretion and cyclic AMP levels.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)68349-8 ◽

1988 ◽

Vol 263 (18) ◽

pp. 8615-8620 ◽

Cited By ~ 5

Author(s):

S Ullrich ◽

C B Wollheim

Keyword(s):

Insulin Secretion ◽

Cyclic Amp ◽

Rinm5f Cells ◽

Dependent Inhibition

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Extracellular ATP causes Ca2(+)-dependent and -independent insulin secretion in RINm5F cells. Phospholipase C mediates Ca2+ mobilization but not Ca2+ influx and membrane depolarization

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)67816-6 ◽

1991 ◽

Vol 266 (6) ◽

pp. 3449-3457

Author(s):

G D Li ◽

D Milani ◽

M J Dunne ◽

W F Pralong ◽

J M Theler ◽

...

Keyword(s):

Insulin Secretion ◽

Phospholipase C ◽

Membrane Depolarization ◽

Extracellular Atp ◽

Rinm5f Cells

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Potentiation of stimulus-induced insulin secretion in protein kinase C-deficient RINm5F cells

Biochemical Journal ◽

10.1042/bj2720637 ◽

1990 ◽

Vol 272 (3) ◽

pp. 637-645 ◽

Cited By ~ 18

Author(s):

G D Li ◽

R Regazzi ◽

S Ullrich ◽

W F Pralong ◽

C B Wollheim

Keyword(s):

Protein Kinase C ◽

Insulin Secretion ◽

Protein Kinase ◽

Phospholipase C ◽

Membrane Depolarization ◽

Rinm5f Cells ◽

Permeabilized Cells ◽

Kinase C ◽

The Difference

The role of protein kinase C (PKC) in stimulus recognition and insulin secretion was investigated after long-term (24 h) treatment of RINm5F cells with phorbol 12-myristate 13-acetate (PMA). Three methods revealed that PKC was no longer detectable, and PMA-induced insulin secretion was abolished. Such PKC-deficient cells displayed enhanced insulin secretion (2-6-fold) in response to vasopressin and carbachol (activating phospholipase C) as well as to D-glyceraldehyde and alanine (promoting membrane depolarization and voltage-gated Ca2+ influx). Insulin release stimulated by 1-oleoyl-2-acetylglycerol (OAG) was also greater in PKC-deficient cells. OAG caused membrane depolarization and raised the cytosolic Ca2+ concentration ([Ca2+]i), both of which were unaffected by PKC down-regulation. Except for that caused by vasopressin, the secretagogue-induced [Ca2+]i elevations were similar in control and PKC-depleted cells. The [Ca2+]i rise evoked by vasopressin was enhanced during the early phase (observed both in cell suspensions and at the single cell level) and the stimulation of diacylglycerol production was also augmented. These findings suggest more efficient activation of phospholipase C by vasopressin after PKC depletion. Electrically permeabilized cells were used to test whether the release process is facilitated after long-term PMA treatment. PKC deficiency was associated with only slightly increased responsiveness to half-maximally (2 microM) but not to maximally stimulatory Ca2+ concentrations. At 2 microM-Ca2+ vasopressin caused secretion, which was also augmented by PMA pretreatment. The difference between intact and permeabilized cells could indicate the loss in the latter of soluble factors which mediate the enhanced secretory responses. However, changes in cyclic AMP production could not explain the difference. These results demonstrate that PKC not only exerts inhibitory influences on the coupling of receptors to phospholipase C but also interferes with more distal steps implicated in insulin secretion.

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Regulation of Ca2+ homeostasis by islet endoplasmic reticulum and its role in insulin secretion

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1988.254.2.e121 ◽

1988 ◽

Vol 254 (2) ◽

pp. E121-E136 ◽

Cited By ~ 5

Author(s):

B. A. Wolf ◽

J. R. Colca ◽

J. Turk ◽

J. Florholmen ◽

M. L. McDaniel

Keyword(s):

Endoplasmic Reticulum ◽

Arachidonic Acid ◽

Insulin Secretion ◽

Beta Cells ◽

Second Messengers ◽

Inositol Trisphosphate ◽

Biochemical Properties ◽

Guanine Nucleotide ◽

Islet Beta Cells ◽

Biochemical Mechanisms

Changes in intracellular Ca2+ concentrations have a major role in the regulation of insulin secretion by islet beta-cells. It has recently become apparent that the endoplasmic reticulum plays a prominent role in the regulation of intracellular Ca2+ concentrations under basal conditions and during insulin secretion. This review describes biochemical properties of the endoplasmic reticulum that contribute to intracellular Ca2+ homeostasis including 1) an ATP-dependent Ca2+ uptake pump associated with a Ca2+-ATPase located in the endoplasmic reticulum; 2) Ca2+ release from the endoplasmic reticulum induced by the second messengers inositol trisphosphate and arachidonic acid as well as the guanine nucleotide GTP; and 3) a Ca2+ sequestration mechanism localized to the endoplasmic reticulum that is regulated by glucose 6-phosphate and glucose-6-phosphatase. The hypothesis is developed that these biochemical mechanisms participate in the regulation of intracellular Ca2+ concentrations and represent central intracellular events involved in the first phase of glucose-induced insulin secretion.

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Stimulation of Ca(2+)-dependent exocytosis of the sperm acrosome by cAMP acting downstream of phospholipase A2

Reproduction ◽

10.1530/reprod/118.1.57 ◽

2000 ◽

pp. 57-68 ◽

Cited By ~ 11

Author(s):

J Garde ◽

ER Roldan

Keyword(s):

Arachidonic Acid ◽

Phospholipase A2 ◽

Phospholipase C ◽

Phosphodiesterase Inhibitors ◽

Dibutyryl Camp ◽

Camp Phosphodiesterase ◽

Ram Sperm ◽

Camp Formation ◽

Stimulation Of

Spermatozoa undergo exocytosis in response to agonists that induce Ca2+ influx and, in turn, activation of phosphoinositidase C, phospholipase C, phospholipase A2, and cAMP formation. Since the role of cAMP downstream of Ca2+ influx is unknown, this study investigated whether cAMP modulates phospholipase C or phospholipase A2 using a ram sperm model stimulated with A23187 and Ca2+. Exposure to dibutyryl-cAMP, phosphodiesterase inhibitors or forskolin resulted in enhancement of exocytosis. However, the effect was not due to stimulation of phospholipase C or phospholipase A2: in spermatozoa prelabelled with [3H]palmitic acid or [14C]arachidonic acid, these reagents did not enhance [3H]diacylglycerol formation or [14C]arachidonic acid release. Spermatozoa were treated with the phospholipase A2 inhibitor aristolochic acid, and dibutyryl-cAMP to test whether cAMP acts downstream of phospholipase A2. Under these conditions, exocytosis did not occur in response to A23187 and Ca2+. However, inclusion of dibutyryl-cAMP and the phospholipase A2 metabolite lysophosphatidylcholine did result in exocytosis (at an extent similar to that seen when cells were treated with A23187/Ca2+ and without the inhibitor). Inclusion of lysophosphatidylcholine alone, without dibutyryl-cAMP, enhanced exocytosis to a lesser extent, demonstrating that cAMP requires a phospholipase A2 metabolite to stimulate the final stages of exocytosis. These results indicate that cAMP may act downstream of phospholipase A2, exerting a regulatory role in the exocytosis triggered by physiological agonists.

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