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.

The intracellular II-III loops of Cav1.2 and Cav1.3 uncouple L-type voltage-gated Ca2+ channels from glucagon-like peptide-1 potentiation of insulin secretion in INS-1 cells via displacement from lipid rafts.

L-type Ca(2+) channels play a key role in the integration of physiological signals regulating insulin secretion that probably requires their localization to specific subdomains of the plasma membrane. We investigated the role of the intracellular II-III loop domains of the L-type channels Ca(v)1.2 and 1.3 in coupling of Ca(2+) influx with glucose-stimulated insulin secretion (GSIS) potentiated by the incretin hormone glucagon-like peptide (GLP)-1. In INS-1 cell lines expressing the Ca(v)1.2/II-III or Ca(v)1.3/II-III peptides, GLP-1 potentiation of GSIS was inhibited markedly, coincident with a decrease in GLP-1-stimulated cAMP accumulation and the redistribution of Ca(v)1.2 and Ca(v)1.3 out of lipid rafts. Neither the Ca(v)1.2/II-III nor the Ca(v)1.3/II-III peptide decreased L-type current density compared with untransfected INS-1 cells. GLP-1 potentiation of GSIS was restored by the L-type channel agonist 2,5-dimethyl-4-[2-(phenylmethyl)benzoyl]-1H-pyrrole-3-carboxylic acid methyl ester (FPL-64176). In contrast, potentiation of GSIS by 8-bromo-cAMP (8-Br-cAMP) was inhibited in Ca(v)1.2/II-III but not Ca(v)1.3/II-III cells. These differences may involve unique protein-protein interactions because the Ca(v)1.2/II-III peptide, but not the Ca(v)1.3/II-III peptide, immunoprecipitates Rab3-interacting molecule (RIM) 2 from INS-1 cell lysates. RIM2, and its binding partner Piccolo, localize to lipid rafts, and they may serve as anchors for Ca(v)1.2 localization to lipid rafts in INS-1 cells. These findings suggest that the II-III interdomain loops of Ca(v)1.2, and possibly Ca(v)1.3, direct these channels to membrane microdomains in which the proteins that mediate potentiation of GSIS by GLP-1 and 8-Br-cAMP assemble.

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.

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.

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.