scholarly journals CaV1.2 rather than CaV1.3 is coupled to glucose-stimulated insulin secretion in INS-1 832/13 cells

2008 ◽  
Vol 41 (1) ◽  
pp. 1-11 ◽  
Author(s):  
Marloes Dekker Nitert ◽  
Cecilia L F Nagorny ◽  
Anna Wendt ◽  
Lena Eliasson ◽  
Hindrik Mulder
Keyword(s):  
Insulin Secretion ◽  
Calcium Channels ◽  
Intracellular Calcium ◽  
Calcium Influx ◽  
Mrna Level ◽  
Calcium Currents ◽  
Protein Levels ◽  
Whole Cell Patch Clamp ◽  
Glucose Stimulated Insulin Secretion ◽  
Cell Capacitance

In clonal β-cell lines and islets from different species, a variety of calcium channels are coupled to glucose-stimulated insulin secretion. The aim of this study was to identify the voltage-gated calcium channels that control insulin secretion in insulinoma (INS)-1 832/13 cells. The mRNA level of CaV1.2 exceeded that of CaV1.3 and CaV2.3 two-fold. Insulin secretion, which rose tenfold in response to 16.7 mM glucose, was completely abolished by 5 μM isradipine that blocks CaV1.2 and CaV1.3. Similarly, the increase in intracellular calcium in response to 15 mM glucose was decreased in the presence of 5 μM isradipine, and the frequency of calcium spikes was decreased to the level seen at 2.8 mM glucose. By contrast, inhibition of CaV2.3 with 100 nM SNX-482 did not significantly affect insulin secretion or intracellular calcium. Using RNA interference, CaV1.2 mRNA and protein levels were knocked down by ∼65% and ∼34% respectively, which reduced insulin secretion in response to 16.7 mM glucose by 50%. Similar reductions in calcium currents and cell capacitance were seen in standard whole-cell patch-clamp experiments. The remaining secretion of insulin could be reduced to the basal level by 5 μM isradipine. Calcium influx underlying this residual insulin secretion could result from persisting CaV1.2 expression in transfected cells since knock-down of CaV1.3 did not affect glucose-stimulated insulin secretion. In summary, our results suggest that CaV1.2 is critical for insulin secretion in INS-1 832/13 cells.

Role of high-voltage-activated calcium channels in glucose-regulated β-cell calcium homeostasis and insulin release

2005 ◽  
Vol 289 (5) ◽  
pp. E900-E908 ◽  
Author(s):  
James T. Taylor ◽  
Luping Huang ◽  
Brian M. Keyser ◽  
Hean Zhuang ◽  
Craig W. Clarkson ◽  
...  
Keyword(s):  
Insulin Secretion ◽  
Calcium Channels ◽  
High Voltage ◽  
Calcium Influx ◽  
Islet Cells ◽  
Primary Source ◽  
Elevated Glucose ◽  
Glucose Stimulated Insulin Secretion ◽  
Insulinoma Cells

High-voltage-activated (HVA) calcium channels are known to be the primary source of calcium for glucose-stimulated insulin secretion. However, few studies have investigated how these channels can be regulated by chronically elevated levels of glucose. In the present study, we determined the level of expression of the four major HVA calcium channels (N-type, P/Q-type, LC-type, and LD-type) in rat pancreatic β-cells. Using quantitative real-time PCR (QRT-PCR), we found the expression of all four HVA genes in rat insulinoma cells (INS-1) and in primary isolated rat islet cells. We then determined the role of each channel in insulin secretion by using channel-selective antagonists. Insulin secretion analysis revealed that N- and L-type channels are both involved in immediate glucose-induced insulin secretion. However, L-type was preferentially coupled to secretion at later time points. P/Q-type channels were not found to play a role in insulin secretion at any stage. It was also found that long-term exposure to elevated glucose increases basal calcium in these cells. Interestingly, chronically elevated glucose decreased the mRNA expression of the channels involved with insulin secretion and diminished the level of stimulated calcium influx in these cells. Using whole cell patch clamp, we found that N- and L-type channel currents increase gradually subsequent to lower intracellular calcium perfusion, suggesting that these channels may be regulated by glucose-induced changes in calcium.

Ca2+ influx via T-type channels modulates PDGF-induced replication of mouse fibroblasts

1993 ◽  
Vol 265 (5) ◽  
pp. C1239-C1246 ◽  
Author(s):  
Z. Wang ◽  
M. Estacion ◽  
L. J. Mordan
Keyword(s):  
Cell Proliferation ◽  
Intracellular Calcium ◽  
Cell Cycle Progression ◽  
Calcium Influx ◽  
Extracellular Calcium ◽  
Calcium Currents ◽  
Patch Clamp Technique ◽  
Mouse Fibroblasts ◽  
Whole Cell Patch Clamp ◽  
Low Threshold

The role of low-threshold voltage-gated calcium channels (VGCC) in modulating extracellular calcium influx and proliferation was investigated in platelet-derived growth factor (PDGF)-stimulated C3H/10T1/2 mouse fibroblasts. Previous studies demonstrated that cell cycle progression after PDGF stimulation was dependent on extracellular calcium influx producing a sustained increase in the intracellular calcium concentration. In this study, PDGF-induced calcium influx, the sustained intracellular calcium increase, and progression to S phase were inhibited by nordihydroguariaretic acid (NDGA), an inhibitor of calcium influx through VGCC. With the use of the whole cell patch-clamp technique to measure calcium currents, NDGA inhibited inward calcium current through low-threshold VGCC, the only VGCC expressed in C3H/10T1/2 fibroblasts. The inhibitory effects of NDGA on calcium influx and cell proliferation each had a mean inhibitory dose of 2-3 microM. Although NDGA also effectively inhibits cyclooxygenase and lipoxygenase, the addition of prostaglandins or leukotrienes could not reverse this inhibition nor could it be replicated by other antioxidants. These data support the hypothesis that low-threshold VGCC can mediate extracellular calcium influx on the stimulation of cell proliferation by PDGF.

Effects of relaxin on rat atrial myocytes. II. Increased calcium influx derived from action potential prolongation

1997 ◽  
Vol 272 (4) ◽  
pp. H1798-H1803 ◽  
Author(s):  
E. S. Piedras-Renteria ◽  
O. D. Sherwood ◽  
P. M. Best
Keyword(s):  
Action Potential ◽  
Calcium Influx ◽  
Calcium Currents ◽  
Atrial Myocytes ◽  
Inotropic Effects ◽  
Whole Cell Patch Clamp ◽  
Rat Hearts ◽  
Action Potential Prolongation ◽  
Dose Dependent ◽  
Rat Atrial Cells

Relaxin produces positive inotropic and chronotropic effects in rat hearts. The effect of relaxin on the action potential duration (APD) of single quiescent rat atrial cells was investigated with a whole cell patch clamp. Relaxin induced a significant, dose-dependent prolongation of the APD. This effect was maximal at 200 ng/ml (nominal concentration of 33.6 nM), which caused, on average, a 57% increase in the time taken to reach 90% repolarization. The effect of relaxin was blocked by the protein kinase A inhibitor 5-24 amide, indicating that its effect is mediated by an adenosine 3',5'-cyclic monophosphate-dependent mechanism. The increased APD induced by relaxin caused an enhanced entrance of calcium, with the charge carried through voltage-activated calcium channels increased by approximately 25%. This increase was not due to a direct modulation of calcium currents (20); rather, it was a consequence of the longer period of cellular depolarization. Our findings that relaxin increased the APD and therefore increased the calcium influx in atrial myocytes could explain the positive inotropic effects induced by relaxin in atrial preparations.

scholarly journals CLOCK-BMAL1 regulate the cardiac L-type calcium channel subunit CACNA1C through PI3K-Akt signaling pathway

2016 ◽  
Vol 94 (9) ◽  
pp. 1023-1032 ◽  
Author(s):  
Yanhong Chen ◽  
Didi Zhu ◽  
Jiamin Yuan ◽  
Zhonglin Han ◽  
Yao Wang ◽  
...  
Keyword(s):  
Circadian Rhythms ◽  
Calcium Channels ◽  
Calcium Currents ◽  
Patch Clamping ◽  
Akt Signaling Pathway ◽  
Inhibitory Effects ◽  
Akt Inhibitor ◽  
Α Subunit ◽  
Protein Levels ◽  
Phosphorylated Akt

The heterodimerized transcription factors CLOCK-BMAL1 regulate the cardiomyocyte circadian rhythms. The L-type calcium currents play important role in the cardiac electrogenesis and arrhythmogenesis. Whether and how the CLOCK-BMAL1 regulate the cardiac L-type calcium channels are yet to be determined. The functions of the L-type calcium channels were evaluated with patch clamping techniques. Recombinant adenoviruses of CLOCK and BMAL1 were used in the expression experiments. We reported that the expressions and functions of CACNA1C (the α-subunit of the L-type calcium channels) showed circadian rhythms, with the peak at zeitgeber time 3 (ZT3). The endocardial action potential durations 90 (APD90) were correspondingly longer at ZT3. The protein levels of the phosphorylated Akt at threonine 308 (pAkt T308) also showed circadian rhythms. Overexpressions of CLOCK-BMAL1 significantly reduced the levels of CACNA1C while increasing the levels of pAkt T308 and pik3r1. Furthermore, the inhibitory effects of CLOCK-BMAL1 on CACNA1C could be abolished by the Akt inhibitor MK2206 or the PDK1 inhibitor GSK2334470. Collectively, our findings suggested that the expressions of the cardiac CACNA1C were under the CLOCK-BMAL1 regulation, probably through the PI3K-Akt signal pathway.

scholarly journals First Phase of Glucose-Stimulated Insulin Secretion From MIN 6 Cells Does Not Always Require Extracellular Calcium Influx

2006 ◽  
Vol 101 (4) ◽  
pp. 293-302 ◽  
Author(s):  
Makoto Shigeto ◽  
Masashi Katsura ◽  
Masafumi Matsuda ◽  
Seitaro Ohkuma ◽  
Kohei Kaku
Keyword(s):  
Insulin Secretion ◽  
Calcium Influx ◽  
Extracellular Calcium ◽  
Glucose Stimulated Insulin Secretion

scholarly journals Exocytosis proteins as novel targets for diabetes prevention and/or remediation?

2017 ◽  
Vol 312 (5) ◽  
pp. R739-R752 ◽  
Author(s):  
Arianne Aslamy ◽  
Debbie C. Thurmond
Keyword(s):  
Insulin Secretion ◽  
Glucose Uptake ◽  
Cell Mass ◽  
Β Cell ◽  
Protein Levels ◽  
Receptor Complexes ◽  
Metabolic Dysregulation ◽  
Protein Receptor ◽  
The Us ◽  
Glucose Stimulated Insulin Secretion

Diabetes remains one of the leading causes of morbidity and mortality worldwide, affecting an estimated 422 million adults. In the US, it is predicted that one in every three children born as of 2000 will suffer from diabetes in their lifetime. Type 2 diabetes results from combinatorial defects in pancreatic β-cell glucose-stimulated insulin secretion and in peripheral glucose uptake. Both processes, insulin secretion and glucose uptake, are mediated by exocytosis proteins, SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complexes, Sec1/Munc18 (SM), and double C2-domain protein B (DOC2B). Increasing evidence links deficiencies in these exocytosis proteins to diabetes in rodents and humans. Given this, emerging studies aimed at restoring and/or enhancing cellular levels of certain exocytosis proteins point to promising outcomes in maintaining functional β-cell mass and enhancing insulin sensitivity. In doing so, new evidence also shows that enhancing exocytosis protein levels may promote health span and longevity and may also harbor anti-cancer and anti-Alzheimer’s disease capabilities. Herein, we present a comprehensive review of the described capabilities of certain exocytosis proteins and how these might be targeted for improving metabolic dysregulation.

Development of functional calcium channels in cultured avian photoreceptors

1992 ◽  
Vol 8 (4) ◽  
pp. 315-327 ◽  
Author(s):  
Evanna Gleason ◽  
Peter Mobbs ◽  
Richard Nuccitelli ◽  
Martin Wilson
Keyword(s):  
Calcium Channels ◽  
Time Course ◽  
Lectin Binding ◽  
Cytosolic Calcium ◽  
Calcium Currents ◽  
Oil Droplet ◽  
Whole Cell Patch Clamp ◽  
Cone Cells ◽  
Internal Calcium ◽  
Patch Clamp Method

AbstractVertebrate photoreceptors are unusual neurons in that they are capable of continuous calcium-mediated release of neurotransmitter (Trifonov, 1968; Hagins et al., 1970). In this study, we have examined the development and characteristics of calcium currents in chick cone cells placed in culture on embryonic day 8. Cone cells were identified by their lectin-binding properties, rhodopsin-like immunoreactivity, and the presence of an oil droplet. Using the whole-cell patch-clamp method, we have seen calcium currents in these cells after three days in culture, slightly before the appearance of synapses (Gleason & Wilson, 1989). Because cone calcium currents are blocked by cadmium and nifedipine but are enhanced by Bay K 8644, they most closely resemble L-type current (Nowycky et al., 1985). An unexpected feature of these currents is that their gating ranges varied widely between cells so that some cells showed the foot of their activation range at —70 mV and others as positive as —25 mV. Calcium imaging of fura-2 loaded cells was used to confirm the time course of calcium current development and describe the distribution of cytosolic calcium. As expected, depolarization of young cells failed to increase cytosolic calcium but in older cells an increase of threefold to fourfold was usually observed. Both at rest and during depolarization, most cone cells showed regional differences in internal calcium concentration. In the most mature cones, depolarization strongly elevated cytosolic calcium at the terminal end of the cell while producing a lesser change around the oil droplet and the ellipsoid region, suggesting that calcium channels are localized to the terminal.

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