RyR2 regulates store-operated Ca2+ entry, phospholipase C activity, and electrical excitability in the insulinoma cell line INS-1.

The ER Ca2+ channel ryanodine receptor 2 (RyR2) is required for maintenance of insulin content and glucose-stimulated insulin secretion, in part, via regulation of the protein IRBIT in the insulinoma cell line INS-1. Here, we examined store-operated and depolarization-dependent Ca2+entry using INS-1 cells in which either RyR2 or IRBIT were deleted. Store-operated Ca2+ entry (SOCE) stimulated with thapsigargin was reduced in RyR2KO cells compared to controls, but was unchanged in IRBITKO cells. STIM1 protein levels were not different between the three cell lines. Basal and stimulated (500 µM carbachol) phospholipase C (PLC) activity was also reduced specifically in RyR2KO cells. Insulin secretion stimulated by tolbutamide was reduced in RyR2KO and IRBITKO cells compared to controls, but was potentiated by an EPAC-selective cAMP analog in all three cell lines. Cellular PIP2 levels were increased and cortical f-actin levels were reduced in RyR2KO cells compared to controls. Whole-cell Cav channel current density was increased in RyR2KO cells compared to controls, and barium current was reduced by acute activation of the lipid phosphatase pseudojanin preferentially in RyR2KO cells over control INS-1 cells. Action potentials stimulated by 18 mM glucose were more frequent in RyR2KO cells compared to controls, and insensitive to the SK channel inhibitor apamin. Taken together, these results suggest that RyR2 plays a critical role in regulating PLC activity and PIP2 levels via regulation of SOCE. RyR2 also regulates ß-cell electrical activity by controlling Cav current density and SK channel activation.

RyR2/IRBIT regulates insulin gene transcript, insulin content, and secretion in the insulinoma cell line INS-1.

The role of ER Ca2+ release via ryanodine receptors (RyR) in pancreatic ß-cell function is not well defined. Deletion of RyR2 from the rat insulinoma INS-1 (RyR2KO) enhanced IP3 receptor activity stimulated by 7.5 mM glucose, coincident with reduced levels of the protein IP3 Receptor Binding protein released with Inositol 1,4,5 Trisphosphate (IRBIT). Insulin content, basal (2.5 mM glucose) and 7.5 mM glucose-stimulated insulin secretion were reduced in RyR2KO and IRBITKO cells compared to controls. INS2 mRNA levels were reduced in both RyR2KO and IRBITKO cells, but INS1 mRNA levels were specifically decreased in RyR2KO cells. Nuclear localization of S-adenosylhomocysteinase (AHCY) was increased in RyR2KO and IRBITKO cells. DNA methylation of the INS1 and INS2 gene promotor regions was very low, and not different among RyR2KO, IRBITKO, and controls, but exon 2 of the INS1 and INS2 genes was more extensively methylated in RyR2KO and IRBITKO cells. Exploratory proteomic analysis revealed that deletion of RyR2 or IRBIT resulted in differential regulation of 314 and 137 proteins, respectively, with 41 in common. These results suggest that RyR2 regulates IRBIT levels and activity in INS-1 cells, and together maintain insulin content and secretion, and regulate the proteome, perhaps via DNA methylation.

The ERG1A K+ Channel Is More Abundant in Rectus abdominis Muscle from Cancer Patients Than that from Healthy Humans.

BACKGROUND: The potassium channel encoded by the ether-a-gogo-related gene 1A (erg1a) has been detected in the atrophying skeletal muscle of mice experiencing either muscle disuse or cancer cachexia and further evidenced to contribute to muscle deterioration by enhancing ubiquitin proteolysis; however, to our knowledge, ERG1A has not been reported in human skeletal muscle. METHODS AND RESULTS: Here, using immunohistochemistry, we detect ERG1A immunofluorescence in human Rectus abdominis skeletal muscle sarcolemma. Further, using single point brightness data, we report the detection of ERG1A immunofluorescence at low levels in the Rectus abdominis muscle sarcolemma of young adult humans and show that it trends toward greater levels (10.6%) in healthy aged adults. Interestingly, we detect ERG1A immunofluorescence at a statistically greater level (53.6%; p < 0.05) in the skeletal muscle of older cancer patients than in age-matched healthy adults. Importantly, using immunoblot, we reveal that lower mass ERG1A protein is 61.5% (p < 0.05) more abundant in the skeletal muscle of cachectic older adults than in healthy age-matched controls. Additionally, we report that the ERG1A protein is detected in a cultured human rhabdomyosarcoma line that may be a good in vitro model for the study of ERG1A in muscle. CONCLUSIONS: The data demonstrate that ERG1A is detected more abundantly in the atrophied skeletal muscle of cancer patients, suggesting it may be related to muscle loss in humans as it has been shown to be in mice experiencing muscle atrophy as a result of malignant tumors.

The ERG1a potassium channel increases basal intracellular calcium concentration and calpain activity in skeletal muscle cells.

BACKGROUND: Skeletal muscle atrophy is the net loss of muscle mass that results from an imbalance in protein synthesis and protein degradation. It occurs in response to several stimuli including disease, injury, starvation, and normal aging. Currently, there is no truly effective pharmacological therapy for atrophy; therefore, exploration of the mechanisms contributing to atrophy is essential because it will eventually lead to discovery of an effective therapeutic target. The ether-a-go-go related gene (ERG1A) K+ channel has been shown to contribute to atrophy by upregulating ubiquitin proteasome proteolysis in cachectic and unweighted mice and has also been implicated in calcium modulation in cancer cells. METHODS: We transduced C2C12 myotubes with either a human ERG1A encoded adenovirus or an appropriate control virus. We used fura-2 calcium indicator to measure intracellular calcium concentration and Calpain-Glo assay kits (ProMega) to measure calpain activity. Quantitative PCR was used to monitor gene expression and immunoblot evaluated protein abundances in cell lysates. Data were analyzed using either a Student's t test or two-way ANOVAs and SAS software as indicated. RESULTS: Expression of human ERG1A in C2C12 myotubes increased basal intracellular calcium concentration 51.7% (p < 0.0001; n = 177). Further, it increased the combined activity of the calcium-activated cysteine proteases, calpain 1 and 2, by 31.9% (p < 0.08; n = 24); these are known to contribute to degradation of myofilaments. The increased calcium levels are likely a contributor to the increased calpain activity; however, the change in calpain activity may also be attributable to increased calpain protein abundance and/or a decrease in levels of the native calpain inhibitor, calpastatin. To explore the enhanced calpain activity further, we evaluated expression of calpain and calpastatin genes and observed no significant differences. There was no change in calpain 1 protein abundance; however, calpain 2 protein abundance decreased 40.7% (p < 0.05; n = 6). These changes do not contribute to an increase in calpain activity; however, we detected a 31.7% decrease (p < 0.05; n = 6) in calpastatin which could contribute to enhanced calpain activity. CONCLUSIONS: Human ERG1A expression increases both intracellular calcium concentration and combined calpain 1 and 2 activity. The increased calpain activity is likely a result of the increased calcium levels and decreased calpastatin abundance.

Regulation of cAMP accumulation and activity by distinct phosphodiesterase subtypes in INS-1 cells and human pancreatic ß-cells.

Pancreatic ß-cells express multiple phosphodiesterase (PDE) subtypes, but the specific roles for each in ß-cell function, particularly in humans, is not clear. We evaluated the cellular role of PDE1, PDE3, and PDE4 activity in the rat insulinoma cell line INS-1 and in primary human ß-cells using subtype-selective PDE inhibitors. Using a genetically encoded, FRET-based cAMP sensor, we found that the PDE1 inhibitor 8MM-IBMX, elevated cAMP levels in the absence of glucose to a greater extent than either the PDE3 inhibitor cilostamide or the PDE4 inhibitor rolipram. In 18 mM glucose, PDE1 inhibition elevated cAMP levels to a greater extent than PDE3 inhibition in INS-1 cells, while PDE4 inhibition was without effect. Inhibition of PDE1 or PDE4, but not PDE3, potentiated glucose-stimulated insulin secretion in INS-1 cells. PDE1 inhibition, but not PDE3 or PDE4 inhibition, reduced palmitate-induced caspase-3/7 activation, and enhanced CREB phosphorylation in INS-1 cells. In human ß-cells, only PDE3 or PDE4 inhibition increased cAMP levels in 1.7 mM glucose, but PDE1, PDE3, or PDE4 inhibition potentiated cAMP levels in 16.7 mM glucose. Inhibition of PDE1 or PDE4 increased cAMP levels to a greater extent in 16.7 mM glucose than in 1.7 mM glucose in human ß-cells. In contrast, elevation of cAMP levels by PDE3 inhibition was not different at these glucose concentrations. PDE1 inhibition also potentiated insulin secretion from human islets, suggesting that the role of PDE1 may be conserved between INS-1 cells and human pancreatic ß-cells. Our results suggest that inhibition of PDE1 may be a useful strategy to potentiate glucose-stimulated insulin secretion, and to protect ß-cells from the toxic effects of excess fatty acids.

Molecular Determinants of the Differential Modulation of Cav1.2 and Cav1.3 by Nifedipine and FPL 64176.

Nifedipine and FPL 64176 (FPL), which block and potentiate L-type voltage-gated Ca2+ channels, respectively, modulate Cav1.2 more potently than Cav1.3. To identify potential strategies for developing subtype-selective inhibitors, we investigated the role of divergent amino acid residues in transmembrane domains IIIS5 and the extracellular IIIS5-3P loop region in modulation of these channels by nifedipine and FPL. Insertion of the extracellular IIIS5-3P loop from Cav1.2 into Cav1.3 (Cav1.3+) reduced the IC50 of nifedipine from 289 to 101 nM, and substitution of S1100 with an A residue, as in Cav1.2, accounted for this difference. Substituting M1030 in IIIS5 to V in Cav1.3+ (Cav1.3+V) further reduced the IC50 of nifedipine to 42 nM. FPL increased current amplitude with an EC50 of 854 nM in Cav1.3, 103 nM in Cav1.2, and 99 nM in Cav1.3+V. In contrast to nifedipine block, substitution of M1030 to V in Cav1.3 had no effect on potency of FPL potentiation of current amplitude, but slowed deactivation in the presence and absence of 10 µM FPL. FPL had no effect on deactivation of Cav1.3/dihydropyridine-insensitive (DHPi), a channel with very low sensitivity to nifedipine block (IC50 ∼93 µM), but did shift the voltage-dependence of activation by ∼-10 mV. We conclude that the M/V variation in IIIS5 and the S/A variation in the IIIS5-3P loop of Cav1.2 and Cav1.3 largely determine the difference in nifedipine potency between these two channels, but the difference in FPL potency is determined by divergent amino acids in the IIIS5-3P loop.

Agonists of the γ-aminobutyric acid type B (GABAB) receptor derived from ß-hydroxy and ß-amino difluoromethyl ketones.

ß-Hydroxy difluoromethyl ketones represent the newest class of agonists of the GABA-B receptor, and they are structurally distinct from all other known agonists at this receptor because they do not display the carboxylic acid or amino group of γ-aminobutyric acid (GABA). In this report, the design, synthesis, and biological evaluation of additional analogues of ß-hydroxy difluoromethyl ketones characterized the critical nature of the substituted aromatic group on the lead compound. The importance of these new data is interpreted by docking studies using the X-ray structure of the GABA-B receptor. Moreover, we also report that the synthesis and biological evaluation of ß-amino difluoromethyl ketones provided the most potent compound across these two series.

The external gate of the human and Drosophila serotonin transporters requires a basic/acidic amino acid pair for 3,4-methylenedioxymethamphetamine (MDMA) translocation and the induction of substrate efflux.

The substituted amphetamine, 3,4-methylenedioxy-methamphetamine (MDMA, ecstasy), is a widely used drug of abuse that induces non-exocytotic release of serotonin, dopamine, and norepinephrine through their cognate transporters as well as blocking the reuptake of neurotransmitter by the same transporters. The resulting dramatic increase in volume transmission and signal duration of neurotransmitters leads to psychotropic, stimulant, and entactogenic effects. The mechanism by which amphetamines drive reverse transport of the monoamines remains largely enigmatic, however, promising outcomes for the therapeutic utility of MDMA for post-traumatic stress disorder and the long-time use of the dopaminergic and noradrenergic-directed amphetamines in treatment of attention-deficit hyperactivity disorder and narcolepsy increases the importance of understanding this phenomenon. Previously, we identified functional differences between the human and Drosophila melanogaster serotonin transporters (hSERT and dSERT, respectively) revealing that MDMA is an effective substrate for hSERT but not dSERT even though serotonin is a potent substrate for both transporters. Chimeric dSERT/hSERT transporters revealed that the molecular components necessary for recognition of MDMA as a substrate was linked to regions of the protein flanking transmembrane domains (TM) V through IX. Here, we performed species-scanning mutagenesis of hSERT, dSERT and C. elegans SERT (ceSERT) along with biochemical and electrophysiological analysis and identified a single amino acid in TM10 (Glu394, hSERT; Asn484, dSERT, Asp517, ceSERT) that is primarily responsible for the differences in MDMA recognition. Our findings reveal that an acidic residue is necessary at this position for MDMA recognition as a substrate and serotonin releaser.

Bimolecular Fluorescence Complementation (BiFC) Analysis of Protein-Protein Interactions and Assessment of Subcellular Localization in Live Cells.

Bimolecular fluorescence complementation (BiFC) is a fluorescence imaging technique used to visualize protein-protein interactions (PPIs) in live cells and animals. One unique application of BiFC is to reveal subcellular localization of PPIs. The superior signal-to-noise ratio of BiFC in comparison with fluorescence resonance energy transfer or bioluminescence resonance energy transfer enables its wide applications. Here, we describe how confocal microscopy can be used to detect and quantify PPIs and their subcellular localization. We use basic leucine zipper transcription factor proteins as an example to provide a step-by-step BiFC protocol using a Nikon A1 confocal microscope and NIS-Elements imaging software. The protocol given below can be readily adapted for use with other confocal microscopes or imaging software.

Ca2+ influx through L-type Ca2+ channels and Ca2+-induced Ca2+ release regulate cAMP accumulation and Epac1-dependent ERK 1/2 activation in INS-1 cells.

We previously reported that INS-1 cells expressing the intracellular II-III loop of the L-type Ca(2+) channel Cav1.2 (Cav1.2/II-III cells) are deficient in Ca(2+)-induced Ca(2+) release (CICR). Here we show that glucose-stimulated ERK 1/2 phosphorylation (GSEP) is slowed and reduced in Cav1.2/II-III cells compared to INS-1 cells. This parallels a decrease in glucose-stimulated cAMP accumulation (GS-cAMP) in Cav1.2/II-III cells. Influx of Ca(2+) via L-type Ca(2+) channels and CICR play roles in both GSEP and GS-cAMP in INS-1 cells since both are inhibited by nicardipine or ryanodine. Further, the Epac1-selective inhibitor CE3F4 abolishes glucose-stimulated ERK activation in INS-1 cells, as measured using the FRET-based sensor EKAR. The non-selective Epac antagonist ESI-09 but not the Epac2-selective antagonist ESI-05 nor the PKA antagonist Rp-cAMPs inhibits GSEP in both INS-1 and Cav1.2/II-III cells. We conclude that L-type Ca(2+) channel-dependent cAMP accumulation, that's amplified by CICR, activates Epac1 and drives GSEP in INS-1 cells.

Activation of the γ-Aminobutyric Acid Type B (GABA(B)) Receptor by Agonists and Positive Allosteric Modulators.

Since the discovery of the GABA(B) agonist and muscle relaxant baclofen, there have been substantial advancements in the development of compounds that activate the GABA(B) receptor as agonists or positive allosteric modulators. For the agonists, most of the existing structure-activity data apply to understanding the role of substituents on the backbone of GABA as well as replacing the carboxylic acid and amine groups. In the cases of the positive allosteric modulators, the allosteric binding site(s) and structure-activity relationships are less well-defined; however, multiple classes of molecules have been discovered. The recent report of the X-ray structure of the GABA(B) receptor with bound agonists and antagonists provides new insights for the development of compounds that bind the orthosteric site of this receptor. From a therapeutic perspective, these data have enabled efforts in drug discovery in areas of addiction-related behavior, the treatment of anxiety, and the control of muscle contractility.

Uncoupling of Cav1.2 from Ca(2+)-induced Ca(2+) release and SK channel regulation in pancreatic ß-cells.

We investigated the role of Cav1.2 in pancreatic ß-cell function by expressing a Cav1.2 II-III loop/green fluorescent protein fusion in INS-1 cells (Cav1.2/II-III cells) to disrupt channel-protein interactions. Neither block of KATP channels nor stimulation of membrane depolarization by tolbutamide was different in INS-1 cells compared with Cav1.2/II-III cells, but whole-cell Cav current density was significantly increased in Cav1.2/II-III cells. Tolbutamide (200 µM) stimulated insulin secretion and Ca(2+) transients in INS-1 cells, and Cav1.2/II-III cells were completely blocked by nicardipine (2 µM), but thapsigargin (1 µM) blocked tolbutamide-stimulated secretion and Ca(2+) transients only in INS-1 cells. Tolbutamide-stimulated endoplasmic reticulum [Ca(2+)] decrease was reduced in Cav1.2/II-III cells compared with INS-1 cells. However, Ca(2+) transients in both INS-1 cells and Cav1.2/II-III cells were significantly potentiated by 8-pCPT-2'-O-Me-cAMP (5 µM), FPL-64176 (0.5 µM), or replacement of extracellular Ca(2+) with Sr(2+). Glucose (10 mM) + glucagon-like peptide-1 (10 nM) stimulated discrete spikes in [Ca(2+)]i in the presence of verapamil at a higher frequency in INS-1 cells than in Cav1.2/II-II cells. Glucose (18 mM) stimulated more frequent action potentials in Cav1.2/II-III cells and primary rat ß-cells expressing the Cav1.2/II-II loop than in control cells. Further, apamin (1 µM) increased glucose-stimulated action potential frequency in INS-1 cells, but not Cav1.2/II-III cells, suggesting that SK channels were not activated under these conditions in Cav1.2/II-III loop-expressing cells. We propose the II-III loop of Cav1.2 as a key molecular determinant that couples the channel to Ca(2+)-induced Ca(2+) release and activation of SK channels in pancreatic ß-cells.

The mERG1a channel modulates skeletal muscle MuRF1, but not MAFbx, expression.

INTRODUCTION: We investigated the mechanism by which the MERG1a K+ channel increases ubiquitin proteasome proteolysis (UPP). METHODS: Hindlimb suspension and electro-transfer of Merg1a cDNA into mouse gastrocnemius muscles induced atrophy. RESULTS: Atrophic gastrocnemius muscles of hindlimb-suspended mice express Merg1a, Murf1, and Mafbx genes. Electrotransfer of Merg1a significantly decreases muscle fiber size (12.6%) and increases UPP E3 ligase Murf1 mRNA (2.1-fold) and protein (23.7%), but does not affect Mafbx E3 ligase expression. Neither Merg1a-induced decreased fiber size nor Merg1a-induced increased Murf1 expression is curtailed significantly by coexpression of inactive HR-Foxo3a, a gene encoding a transcription factor known to induce Mafbx expression. CONCLUSIONS: The MERG1a K+ channel significantly increases expression of Murf1, but not Mafbx. We explored this expression pattern by expressing inactive Foxo3a and showing that it is not involved in MERG1a-mediated expression of Murf1. These findings suggest that MERG1a may not modulate Murf1 expression through the AKT/FOXO pathway.

The Ubr2 Gene is Expressed in Skeletal Muscle Atrophying as a Result of Hind Limb Suspension, but not Merg1a Expression Alone.

Skeletal muscle (SKM) atrophy is a potentially debilitating condition induced by muscle disuse, denervation, many disease states, and aging. The ubiquitin proteasome pathway (UPP) contributes greatly to the protein loss suffered in muscle atrophy. The MERG1a K(+) channel is known to induce UPP activity and atrophy in SKM. It has been further demonstrated that the mouse ether-a-gogo-related gene (Merg)1a channel modulates expression of MURF1, an E3 ligase component of the UPP, while it does not affect expression of the UPP E3 ligase Mafbx/ATROGIN1. Because the UBR2 E3 ligase is known to participate in SKM atrophy, we have investigated the effect of Merg1a expression and hind limb suspension on Ubr2 expression. Here, we report that hind limb suspension results in a significant 25.6% decrease in mouse gastrocnemius muscle fiber cross sectional area (CSA) and that electro-transfer of Merg1a alone into gastrocnemius muscles yields a 15.3% decrease in CSA after 7 days. More interestingly, we discovered that hind limb suspension caused a significant 8-fold increase in Merg1a expression and a significant 4.7-fold increase in Ubr2 transcript after 4 days, while electro-transfer of Merg1a into gastrocnemius muscles resulted in a significant 6.2-fold increase in Merg1a transcript after 4 days but had no effect on Ubr2 expression. In summary, the MERG1a K(+) channel, known to induce atrophy and MURF1 E3 ligase expression, does not affect UBR2 E3 ligase transcript levels. Therefore, to date, the MERG1a channel's contribution to UPP activity appears mainly to be through up-regulation of Murf1 gene expression.

Development of a high-throughput screening paradigm for the discovery of small-molecule modulators of adenylyl cyclase: identification of an adenylyl cyclase 2 inhibitor.

Adenylyl cyclase (AC) isoforms are implicated in several physiologic processes and disease states, but advancements in the therapeutic targeting of AC isoforms have been limited by the lack of potent and isoform-selective small-molecule modulators. The discovery of AC isoform-selective small molecules is expected to facilitate the validation of AC isoforms as therapeutic targets and augment the study of AC isoform function in vivo. Identification of chemical probes for AC2 is particularly important because there are no published genetic deletion studies and few small-molecule modulators. The present report describes the development and implementation of an intact-cell, small-molecule screening approach and subsequent validation paradigm for the discovery of AC2 inhibitors. The NIH clinical collections I and II were screened for inhibitors of AC2 activity using PMA-stimulated cAMP accumulation as a functional readout. Active compounds were subsequently confirmed and validated as direct AC2 inhibitors using orthogonal and counterscreening assays. The screening effort identified SKF-83566 [8-bromo-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepin-7-ol hydrobromide] as a selective AC2 inhibitor with superior pharmacological properties for selective modulation of AC2 compared with currently available AC inhibitors. The utility of SKF-83566 as a small-molecule probe to study the function of endogenous ACs was demonstrated in C2C12 mouse skeletal muscle cells and human bronchial smooth muscle cells.

Evaluation of difluoromethyl ketones as agonists of the γ-aminobutyric acid type B (GABAB) receptor.

The design, synthesis, biological evaluation, and in vivo studies of difluoromethyl ketones as GABAB agonists that are not structurally analogous to known GABAB agonists, such as baclofen or 3-aminopropyl phosphinic acid, are presented. The difluoromethyl ketones were assembled in three synthetic steps using a trifluoroacetate-release aldol reaction. Following evaluation at clinically relevant GABA receptors, we have identified a difluoromethyl ketone that is a potent GABAB agonist, obtained its X-ray structure, and presented preliminary in vivo data in alcohol-preferring mice. The behavioral studies in mice demonstrated that this compound tended to reduce the acoustic startle response, which is consistent with an anxiolytic profile. Structure-activity investigations determined that replacing the fluorines of the difluoromethyl ketone with hydrogens resulted in an inactive analogue. Resolution of the individual enantiomers of the difluoromethyl ketone provided a compound with full biological activity at concentrations less than an order of magnitude greater than the pharmaceutical, baclofen.

Potentiation of sulfonylurea action by an EPAC-selective cAMP analog in INS-1 cells: comparison of tolbutamide and gliclazide and a potential role for EPAC activation of a 2-APB-sensitive Ca2+ influx.

Tolbutamide and gliclazide block the K(ATP) channel K(ir)6.2/Sur1, causing membrane depolarization and stimulating insulin secretion in pancreatic beta cells. We examined the ability of the EPAC-selective cAMP analog 8-pCPT-2'-O-Me-cAMP-AM to potentiate the action of these drugs and the mechanism that might account for it. Insulin secretion stimulated by both 200 µM tolbutamide and 20 µM gliclazide, concentrations that had equivalent effects on membrane potential, was inhibited by thapsigargin (1 µM) or the L-type Ca(2+) channel blocker nicardipine (2 µM) and was potentiated by 8-pCPT-2'-O-Me-cAMP-AM at concentrations ≥2 µM in INS-1 cells. Ca(2+) transients stimulated by either tolbutamide or gliclazide were inhibited by thapsigargin or nicardipine and were significantly potentiated by 8-pCPT-2'-O-Me-cAMP-AM at 5 µM but not 1 µM. Both tolbutamide and gliclazide stimulated phospholipase C activity; however, only gliclazide did so independently of its activity at K(ATP) channels, and this activity was partially inhibited by pertussis toxin. 8-pCPT-2'-O-Me-cAMP-AM alone (5 µM) did not stimulate insulin secretion, but did increase intracellular Ca(2+) concentration significantly, and this activity was inhibited by 25 µM 2-aminoethoxydiphenylborate (2-APB) or the removal of extracellular Ca(2+). 8-pCPT-2'-O-Me-cAMP-AM potentiation of insulin secretion stimulated by tolbutamide was markedly inhibited by 2-APB (25 µM) and enhanced by the PKC inhibitor bisindolylmaleimide I (1 µM). Our data demonstrate that the actions of both tolbutamide and gliclazide are strongly potentiated by 8-pCPT-2'-O-Me-cAMP-AM, that gliclazide can stimulate phospholipase C activity via a partially pertussis toxin-sensitive mechanism, and that 8-pCPT-2'-O-Me-cAMP-AM potentiation of tolbutamide action may involve activation of a 2-APB-sensitive Ca(2+) influx.

Distinct properties of amlodipine and nicardipine block of the voltage-dependent Ca2+ channels Cav1.2 and Cav2.1 and the mutant channels Cav1.2/dihydropyridine insensitive and Cav2.1/dihydropyridine sensitive.

The binding site within the L-type Ca(2+) channel Ca(v)1.2 for neutral dihydropyridines is well characterized. However, the contributions of the alkylamino side chains of charged dihydropyridines such as amlodipine and nicardipine to channel block are not clear. We tested the hypothesis that the distinct locations of the charged side chains on amlodipine and nicardipine would confer distinct properties of channel block by these two drugs. Using whole-cell voltage clamp, we investigated block of wild type Ca(v) 2.1, wild type Ca(v)1.2, and Ca(v)1.2/Dihydropyridine insensitive, a mutant channel insensitive to neutral DHPs, by amlodipine and nicardipine. The potency of nicardipine and amlodipine for block of closed (stimulation frequency of 0.05 Hz) Ca(v)1.2 channels was not different (IC(50) values of 60 nM and 57 nM, respectively), but only nicardipine block was enhanced by increasing the stimulation frequency to 1 Hz. The frequency-dependent block of Ca(v)1.2 by nicardipine is the result of a strong interaction of nicardipine with the inactivated state of Ca(v)1.2. However, nicardipine block of Ca(v)1.2/Dihydropyridine insensitive was much more potent than block by amlodipine (IC(50) values of 2.0 µM and 26 µM, respectively). A mutant Ca(v)2.1 channel containing the neutral DHP binding site (Ca(v)2.1/Dihydropyridine sensitive) was more potently blocked by amlodipine (IC(50)=41 nM) and nicardipine (IC(50)=175 nM) than the parent Ca(v)2.1 channel. These data suggest that the alkylamino group of nicardipine and amlodipine project into distinct regions of Ca(v)1.2 such that the side chain of nicardipine, but not amlodipine, contributes to the potency of closed-channel block, and confers frequency-dependent block.

Cav1.2 and Cav1.3 are differentially coupled to glucagon-like peptide-1 potentiation of glucose-stimulated insulin secretion in the pancreatic beta-cell line INS-1.

The incretin peptides, glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1), potentiate glucose-stimulated insulin secretion (GSIS) and beta-cell proliferation and differentiation. Ca(2+) influx via voltage-gated L-type Ca(2+) channels is required for GLP-1 and GIP potentiation of GSIS. We investigated the role of the L-type Ca(2+) channels Ca(v)1.2 and Ca(v)1.3 in mediating GLP-1- and GIP-stimulated events in INS-1 cells and INS-1 cell lines expressing dihydropyridine-insensitive (DHPi) mutants of either Ca(v)1.2 or Ca(v)1.3. Ca(v)1.3/DHPi channels supported full potentiation of GSIS by GLP-1 (50 nM) compared with untransfected INS-1 cells. However, GLP-1-potentiated GSIS mediated by Ca(v)1.2/DHPi channels was markedly reduced compared with untransfected INS-1 cells. In contrast, GIP (10 nM) potentiation of GSIS mediated by both Ca(v)1.2/DHPi and Ca(v)1.3/DHPi channels was similar to that observed in untransfected INS-1 cells. Disruption of intracellular Ca(2+) release with thapsigargin, ryanodine, or 2-aminoethyldiphenylborate and inhibition of protein kinase A (PKA) or protein kinase C (PKC) significantly reduced GLP-1 potentiation of GSIS by Ca(v)1.3/DHPi channels and by endogenous L-type channels in INS-1 cells, but not by Ca(v)1.2/DHPi channels. Inhibition of glucose-stimulated phospholipase C activity with 1-(6-((17b-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122) did not inhibit potentiation of GSIS by GLP-1 in INS-1 cells. In contrast, wortmannin, an inhibitor of phosphatidylinositol 3-kinase, and 2'-amino-3'-methoxyflavone (PD98059), an inhibitor of mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase, both markedly inhibited GLP-1 potentiation of GSIS by endogenous channels in INS-1 cells and Ca(v)1.3/DHPi channels, but not by Ca(v)1.2/DHPi channels. Thus, Ca(v)1.3 is preferentially coupled to GLP-1 potentiation of GSIS in INS-1 cells via a mechanism that requires intact intracellular Ca(2+) stores, PKA and PKC activity, and activation of ERK1/2.