Cytosolic free Ca2+ in insulin secreting cells and its regulation by isolated organelles

The role of Ca2+ in secretagogue-induced insulin release is documented not only by the measurements of 45Ca fluxes in pancreatic islets, but also, by direct monitoring of cytosolic free Ca2+, [Ca2+]i. As demonstrated, using the fluorescent indicator quin 2, glyceraldehyde, carbamylcholine and alanine raise [Ca2+]i in the insulin secreting cell line RINm5F, whereas glucose has a similar effect in pancreatic islet cells. The regulation of cellular Ca2+ homeostasis by organelles from a rat insulinoma, was investigated with a Ca2+ selective electrode. The results suggest that both the endoplasmic reticulum and the mitochondria participate in this regulation, albeit at different Ca2+ concentrations. By contrast, the secretory granules do not appear to be involved in the short-term regulation of [Ca2+]i. Evidence is presented that inositol 1,4,5-trisphosphate, which is shown to mobilize Ca2+ from the endoplasmic reticulum, is acting as an intracellular mediator in the stimulation of insulin release.     

Cytosolic free Ca2+ in insulin secreting cells and its regulation by isolated organelles

Summary The role of Ca²⁺ in secretagogue-induced insulin release is documented not only by the measurements of⁴⁵Ca fluxes in pancreatic islets, but also, by direct monitoring of cytosolic free Ca²⁺, [Ca²⁺]_i. As demonstrated, using the fluorescent indicator quin 2, glyceraldehyde, carbamylcholine and alanine raise [Ca²⁺]_i in the insulin secreting cell line RINm5F, whereas glucose has a similar effect in pancreatic islet cells. The regulation of cellular Ca²⁺ homeostasis by organelles from a rat insulinoma, was investigated with a Ca²⁺ selective electrode. The results suggest that both the endoplasmic reticulum and the mitochondria participate in this regulation, albeit at different Ca²⁺ concentrations. By contrast, the secretory granules do not appear to be involved in the short-term regulation of [Ca²⁺]_i. Evidence is presented that inositol 1,4,5-trisphosphate, which is shown to mobilize Ca²⁺ from the endoplasmic reticulum, is acting as an intracellular mediator in the stimulation of insulin release.     

ERK activation by Ca2+ ionophores depends on Ca2+ entry in lymphocytes but not in platelets, and does not conduct membrane scrambling

Rapid Ca²⁺-dependent phospholipid (PL) reorganization (scrambling) at the plasma membrane is a mechanism common to hematopoietic cells exposing procoagulant phosphatidylserine (PS). The aim of this research was to determine whether activation of the extracellular signal-regulated kinase (ERK) pathway was required for PL scrambling, based on a single report analyzing both responses induced by Ca²⁺ ionophores in megakaryoblastic HEL cells. Ca²⁺ ionophore-stimulated ERK phosphorylation was induced in platelets without external Ca²⁺, whereas exogenous Ca²⁺ entry was crucial for ERK activation in Jurkat T cells. In both cells, membrane scrambling only occurred following Ca²⁺ entry and was not blocked by inhibiting ERK phosphorylation. Furthermore, ERK proteins are strongly phosphorylated in transformed B lymphoblastic cell lines, which do not expose PS in their resting state. Overall, the data demonstrated that ERK activation and membrane scrambling are independent mechanisms. A. Arachiche, I. Badirou: These authors contributed equally to this work. Received 18 June 2008; received after revision 24 September 2008; accepted 1 October 2008     

Proinsulin C-peptide and its analogues induce intracellular Ca2+ increases in human renal tubular cells

Based on the findings that proinsulin C-peptide binds specifically to cell membranes, we investigated the effects of C-peptide and related molecules on the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in human renal tubular cells using the indicator fura-2/AM. The results show that human C-peptide and its C-terminal pentapeptide (positions 27–31, EGSLQ), but not the des (27–31) C-peptide or randomly scrambled C-peptide, elicit a transient increase in [Ca²⁺]_i. Rat C-peptide and rat C-terminal pentapeptide also induce a [Ca²⁺]_i response in human tubular cells, while a human pentapeptide analogue with Ala at position 1 gives no [Ca²⁺]_i response, and those with Ala at positions 2–5 induce responses with different amplitudes. These results define a species cross-reactivity for C-peptide and demonstrate the importance of Glu at position 1 of the pentapeptide. Preincubation of cells with pertussis toxin abolishes the effect on [Ca²⁺]_i by both C-peptide and the pentapeptide. These results are compatible with previous data on C-peptide binding to cells and activation of Na⁺,K⁺ATPase. Combined, all data show that C-peptide is a bioactive peptide and suggest that it elicits changes in [Ca²⁺]_i via G-protein-coupled pathways, giving downstream enzyme effects. Received 13 May 2002; accepted 16 May 2002     

Role of protein kinase C and Ca2+ in glucose-induced sensitization/desensitization of insulin secretion

The role of protein kinase C and Ca²⁺ in glucose-induced sensitization/desensitization of insulin secretion was studied. A 22–24h exposure of mouse pancreatic islets to glucose (16.7 mmol/l) in TCM 199 culture medium, with 0.26 mmol/l or 1.26 mmol/l Ca²⁺, reduced total islet protein kinase C activity to approx. 85% and 60% of control values, respectively. At 0.26 mmol/l Ca²⁺ in TCM 199 medium, exposure to glucose (16.7 mmol/l) led to a potentiation of both phase 1 and phase 2 of glucose-induced insulin secretion, and caused a shift in the dose-response curve with 10 mmol/l and 16.7 mmol/l glucose exhibiting equipotent effects in stimulation of insulin secretion. In glucose-sensitized islets, the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (0.16 μmol/l) did not further potentiate induction of secretion by 10 mmol/l or 16.7 mmol/l glucose. At 3.3 mmol/l glucose, however, phorbol ester-induced secretion was augmented, and was characterized by a faster onset of secretion in glucose-sensitized islets relative to control islets. In contrast, a partial reduction in arachidonic acid (100 μmol/l)-induced insulin release was observed in glucose-sensitized islets in the absence of extracellular Ca²⁺. Increasing the Ca²⁺ concentration to 1.26 mmol/l in TCM 199 during the 22–24h exposure to glucose (16.8 mmol/l) led to inhibition of phase 1 and abolition of phase 2 of glucose (10 mmol/l, 16.7 mmol/l)-induced insulin secretion. In addition, this treatment abolished phorbol ester-induced and arachidonic acid-induced insulin secretion at 3.3 mmol/l glucose. Altogether, these data suggest that sensitization of insulin secretion is caused by a preferential down-regulation of the inhibitory effects of protein kinase C, leading to an increased first phase, and an increased coupling of glucose to the stimulatory effects of protein kinase C during the second phase of glucose-induced insulin secretion. Desensitization of insulin secretion appears to be a consequence of sustained Ca²⁺ influx, inducing extensive down-regulation of protein kinase C and also causing deleterious effects on islet cell function in protein kinase C-deprived islets.     

Sr2+ can become incorporated into an agonist-sensitive,cytoplasmic Ca2+ store in a cell line derived from the equine sweat gland epithelium

We have explored the properties of a Ca²⁺-dependent cell-signalling pathway that becomes active when cultured equine sweat gland cells are stimulated with ATP. The ATP-regulated, Ca²⁺-influx pathway allowed Sr²⁺ to enter the cytoplasm but permitted only a minimal influx of Ba²⁺. Experiments in which cells were repeatedly stimulated with ATP suggested that Sr²⁺, but not Ba²⁺, could become incorporated into the agonist-sensitive, cytoplasmic Ca²⁺ store. Further evidence for this was provided by experiments using ionomycin, a Ca²⁺ ionophore which has no affinity for Sr²⁺.     

Role of protein kinase C and Ca2+ in glucose-induced sensitization/desensitization of insulin secretion.

The role of protein kinase C and Ca2+ in glucose-induced sensitization/desensitization of insulin secretion was studied. A 22-24 h exposure of mouse pancreatic islets to glucose (16.7 mmol/l) in TCM 199 culture medium, with 0.26 mmol/l or 1.26 mmol/l Ca2+, reduced total islet protein kinase C activity to approx. 85% and 60% of control values, respectively. At 0.26 mmol/l Ca2+ in TCM 199 medium, exposure to glucose (16.7 mmol/l) led to a potentiation of both phase 1 and phase 2 of glucose-induced insulin secretion, and caused a shift in the dose-response curve with 10 mmol/l and 16.7 mmol/l glucose exhibiting equipotent effects in stimulation of insulin secretion. In glucose-sensitized islets, the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (0.16 mumol/l) did not further potentiate induction of secretion by 10 mmol/l or 16.7 mmol/l glucose. At 3.3 mmol/l glucose, however, phorbol ester-induced secretion was augmented, and was characterized by a faster onset of secretion in glucose-sensitized islets relative to control islets. In contrast, a partial reduction in arachidonic acid (100 mumol/l)-induced insulin release was observed in glucose-sensitized islets in the absence of extracellular Ca2+. Increasing the Ca2+ concentration to 1.26 mmol/l in TCM 199 during the 22-24 h exposure to glucose (16.7 mmol/l) led to inhibition of phase 1 and abolition of phase 2 of glucose (10 mmol/l, 16.7 mmol/l)-induced insulin secretion. In addition, this treatment abolished phorbol ester-induced and arachidonic acid-induced insulin secretion at 3.3 mmol/l glucose. Altogether, these data suggest that sensitization of insulin secretion is caused by a preferential down-regulation of the inhibitory effects of protein kinase C, leading to an increased first phase, and an increased coupling of glucose to the stimulatory effects of protein kinase C during the second phase of glucose-induced insulin secretion. Desensitization of insulin secretion appears to be a consequence of sustained Ca2+ influx, inducing extensive down-regulation of protein kinase C and also causing deleterious effects on islet cell function in protein kinase C-deprived islets.     

Relationship between action potential, contraction-relaxation pattern, and intracellular Ca2+ transient in cardiomyocytes of dogs with chronic heart failure

Abnormalities of contractile function have been identified in cardiomyocytes isolated from failed human hearts and from hearts of animals with experimentally induced heart failure (HF). The mechanism(s) responsible for these functional abnormalities are not fully understood. In the present study, we examined the relationship between action potential duration, pattern of contraction and relaxation, and associated intracellular Ca²⁺ transients in single cardiomyocytes isolated from the left ventricle (LV) of dogs (n = 7) with HF produced by multiple sequential intracoronary microembolizations. Comparisons were made with LV cardiomyocytes isolated from normal dogs. Action potentials were measured in isolated LV cardiomyocytes by perforated patch clamp, Ca²⁺ transients by fluo 3 probe fluorescence, and cardiomyocyte contraction and relaxation by edge movement detector. HF cardiomyocytes exhibited an abnormal pattern of contraction and relaxation characterized by an attenuated initial twitch (spike) followed by a sustained contracture ('dome') of 1 to 8 s in duration and subsequent delayed relaxation. This pattern was more prominent at low stimulation rates (58% at 0.2 Hz, n = 211, 21% at 0.5 Hz, n = 185). Measurements of Ca²⁺ transients in HF cardiomyocytes at 0.2 Hz manifested a similar spike and dome configuration. The dome phase of both the contraction/relaxation pattern and Ca²⁺ transients seen in HF cardiomyocytes coincided with a sustained plateau of the action potential. Shortening of the action potential duration by administration of saxitoxin (100 nM) or lidocaine (30 μM) reduced the duration of the dome phase of both the contraction/relaxation profile as well as that of the Ca²⁺ transient profile. An increase of stimulation rate up to 1 Hz caused shortening of the action potential and disappearance of the spike-dome profile in the majority of HF cardiomyocytes. In HF cardiomyocytes, the action potential and Ca²⁺ transient duration were not significantly different from those measured in normal cells. However, the contraction-relaxation cycle was significantly longer in HF cells (314 ± 67 ms, n = 21, vs. 221 ± 38 ms, n = 46, mean ± SD), indicating impaired excitation-contraction uncou pling in HF cardiomyocytes. The results show that, in cardiomyocytes isolated from dogs with HF, contractile abnormalities and abnormalities of intracellular Ca²⁺ transients at low stimulation rates are characterized by a spike-dome configuration. This abnormal pattern appears to result from prolongation of the action potential. Received 22 January 1998; received after revision 16 March 1998; accepted 27 March 1998     

Induction, by 5-bromo-2'-deoxyuridine, of a "foamy" virus previously undetected in hamster cells transformed by SV40]

TSV5 clone 2 cells in normal conditions of culture contain only an expressed RNA virus (R-type virus). However, exposure of the cells to 5-bromo-2'-deoxyuridine with dexamethasone, induced synthesis of a syncitium-forming ("Foamy") virus. In other hamster cell lines, the same treatment fails to induce a "foamy" virus. The origin of this "foamy" virus is discussed.     

Cyclin A1 is a p53-induced gene that mediates apoptosis, G2/ M arrest, and mitotic catastrophe in renal, ovarian, and lung carcinoma cells

We were the first to identify cyclin A1 as a p53-induced gene by cDNA expression profiling of p53-sensitive and -resistant tumor cells [Maxwell S. A. and Davis G. E. (2000) Proc. Natl. Acad. Sci. USA 97, 13009–13014]. We show here that cyclin A1 can induce G2 cell cycle arrest, polyploidy, apoptosis, and mitotic catastrophe in H1299 non-small cell lung, TOV-21G ovarian, or 786-0 renal carcinoma cells. More cdk1 protein and kinase activities were observed in cyclin A1-induced cells than in GFP control-induced cells. Thus, cyclin A1 might mediate apoptosis and mitotic catastrophe through an unscheduled or inappropriate activation of cdk1. Two primary renal cell carcinomas expressing mutated p53 exhibited reduced or absent expression of cyclin A1 relative to the corresponding normal tissue. Moreover, renal carcinoma-derived mutant p53s were deficient in inducing cyclin A1 expression in p53-null cells. Cyclin A1 but not cyclin A2 was upregulated in etoposide-treated tumor cells undergoing p53-dependent apoptosis and mitotic catastrophe. Forced upregulation of cyclin A2 did not induce apoptosis. The data implicate cyclin A1 as a downstream player in p53-dependent apoptosis and G2 arrest. Received 1 November 2005; received after revision 17 February 2006; accepted 13 April 2006     

Disruption of clc-5 leads to a redistribution of annexin A2 and promotes calcium crystal agglomeration in collecting duct epithelial cells

Mutations in CLCN5, which encodes the voltage-dependent Cl⁻/H⁺antiporter, CLC-5, cause Dent’s disease. This disorder is characterized by low molecularweight proteinuria, hypercalciuria, nephrocalcinosis and nephrolithiasis. Using a collecting duct cell model (mIMCD-3) in which endogenous clc-5 is disrupted by antisense clc-5 or overexpression of truncated clc-5, we demonstrate altered expression of the crystal adhesion molecule, annexin A2. Endogenously expressed annexin A2 is intracellular with limited plasma membrane localization. Following clc-5 disruption, there is both a marked increase in plasma membrane annexin A2 and an increase in cell surface crystal retention and agglomeration, which may be attenuated using pretreatment with anti-annexin A2 antibodies or wheat germ agglutinin lectin but not by concanavalin A. We hypothesize that in Dent’s disease, endocytic failure leads to an accumulation at the plasma membrane of crystal-binding molecules that include annexin A2 leading to retention of calcium crystals and ultimately nephrocalcinosis and nephrolithiasis. Received 22 October 2005; received after revision 26 November 2005; accepted 2 December 2005