Anandamide-induced mobilization of cytosolic Ca2+ in endothelial cells. (49/2700)

1. Experiments were designed to determine whether anandamide affects cytosolic Ca2+ concentrations in endothelial cells and, if so, whether CB1 cannabinoid receptors are involved. To this effect, human umbilical vein-derived EA.hy926 endothelial cells were loaded with fura-2 to monitor changes in cytosolic Ca2+ using conventional fluorescence spectrometry methods. 2. Anandamide induced an increase in Ca2+ in endothelial cells which, in contrast to histamine, developed slowly and was transient. Anandamide caused a concentration-dependent release of Ca2+ from intracellular stores without triggering capacitative Ca2+ entry, contrary to histamine or the endoplasmic reticulum Ca2+ -ATPase inhibitor thapsigargin. 3. Anandamide pretreatment slightly reduced the mobilization of Ca2+ from intracellular stores that was evoked by histamine. The mobilization of Ca2+ from intracellular stores evoked by anandamide was impaired by 10 mM caffeine. 4. Anandamide and histamine each significantly increased NO synthase activity in EA.hy926 cells, as determined by the enhanced conversion of L-[3H]-arginine to L-[3H]-citruline. 5. The CB1 cannabinoid receptor antagonist SR141716A (1 microM) only produced a marginal reduction of the mobilization of Ca2+ produced by 5 microM anandamide. However, at 5 microM SR141716A elicited the release of Ca2+ from intracellular stores. This concentration strongly impaired the mobilization of cytosolic Ca2+ evoked by either anandamide, histamine or thapsigargin. 6. Pretreatment of the cells with either 200 microM phenylmethylsulphonyl fluoride (to inhibit the conversion of anandamide into arachidonic acid) or 400 ng ml(-1) pertussis toxin (to uncouple CB1 cannabinoid receptors from Gi/o proteins) had no significant effect on the mobilization of cytosolic Ca2+ evoked by either anandamide, or histamine. 7. Taken together the results demonstrate that anandamide mobilizes Ca2+ from a caffeine-sensitive intracellular Ca2+ store that functionally overlaps in part with the internal stores mobilized by histamine. However, a classical CB1 cannabinoid receptor-mediated and pertussis toxin-sensitive mechanism does not mediate this novel effect of anandamide in endothelial cells. 8. The mobilization of cytosolic Ca2+ in endothelial cells may account for the endothelium-dependent and NO-mediated vasodilator actions of anandamide. Due to its non-specific inhibition of Ca2+ signalling in endothelial cells, SR141716A may not be used to assess the physiological involvement of endogenous cannabinoids to endothelium-dependent control of vascular smooth muscle tone.  (+info)

A point mutation inactivating the sulfonylurea receptor causes the severe form of persistent hyperinsulinemic hypoglycemia of infancy in Finland. (50/2700)

Mutations in genes encoding the ATP-regulated potassium (K(ATP)) channels of the pancreatic beta-cell (SUR1 and Kir6.2) are the major known cause of persistent hyperinsulinemic hypoglycemia of infancy (PHHI). We collected all cases of PHHI diagnosed in Finland between 1983 and 1997 (n = 24). The overall incidence was 1:40,400, but in one area of Central Finland it was as high as 1:3,200. Haplotype analysis using polymorphic markers spanning the SUR1/Kir6.2 gene cluster confirmed linkage to the 11p region. Sequence analysis revealed a novel point mutation in exon 4 of SUR1, predicting a valine to aspartic acid change at amino acid 187 (V187D). Of the total cases, 15 affected individuals harbored this mutation in heterozygous or homozygous form, and all of these had severe hyperinsulinemia that responded poorly to medical treatment and required subtotal pancreatectomy. No K(ATP) channel activity was observed in beta-cells isolated from a homozygous patient or after coexpression of recombinant Kir6.2 and SUR1 carrying the V187D mutation. Thus, the mutation produces a nonfunctional channel and, thereby, continuous insulin secretion. This unique SUR1 mutation explains the majority of PHHI cases in Finland and is strongly associated with a severe form of the disease. These findings provide diagnostic and prognostic utility for suspected PHHI patients.  (+info)

Synthesis and characterization of potent and selective agonists of the neuronal cannabinoid receptor (CB1). (51/2700)

Two subtypes of the cannabinoid receptor (CB1 and CB2) are expressed in mammalian tissues. Although selective antagonists are available for each of the subtypes, most of the available cannabinoid agonists bind to both CB1 and CB2 with similar affinities. We have synthesized two analogs of N-arachidonylethanolamine (AEA), arachidonylcyclopropylamide (ACPA) and arachidonyl-2-chloroethylamide (ACEA), that bind to the CB1 receptor with very high affinity (KI values of 2.2 +/- 0.4 nM and 1.4 +/- 0.3 nM, respectively) and to the CB2 receptor with low affinity (KI values of 0.7 +/- 0.01 microM and 3.1 +/- 1.0 microM, respectively). Both ACPA and ACEA have the characteristics of agonists at the CB1 receptor; both inhibit forskolin-induced accumulation of cAMP in Chinese hamster ovary cells expressing the human CB1 receptor, and both analogs increase the binding of [35S]GTPgammaS to cerebellar membranes and inhibit electrically evoked contractions of the mouse vas deferens. ACPA and ACEA produce hypothermia in mice, and this effect is inhibited by coadministration of the CB1 receptor antagonist SR141716A. Therefore, ACPA and ACEA are high-affinity agonists of the CB1 receptor but do not bind the CB2 receptor, suggesting that structural analogs of AEA can be designed with considerable selectivity for the CB1 receptor over the CB2 receptor.  (+info)

Cannabinoid inhibition of the processing of intact lysozyme by macrophages: evidence for CB2 receptor participation. (52/2700)

Delta9-tetrahydrocannabinol (THC) impairs multiple immunological functions. The ability of a macrophage hybridoma to function as an antigen-presenting cell was examined by the stimulation of a soluble protein antigen-specific helper T cell hybridoma to secrete interleukin-2. THC exposure significantly reduced the T cell response to the native form of the antigen after a 24-h pretreatment of the macrophages with nanomolar drug concentrations. However, THC did not affect interleukin-2 production when the macrophages presented a synthetic peptide of the antigen to the T cells, suggesting that the drug may interfere with antigen processing, not peptide presentation. Cannabinoid inhibition of the T cell response to the native antigen was stereoselective consistent with the involvement of a cannabinoid (CB) receptor. Bioactive CP-55,940 diminished T cell activation, whereas the inactive stereoisomer CP-56,667 did not. The macrophage hybridoma expressed mRNA for the CB2 but not the CB1 receptor whereas the T cells expressed an extremely low level of mRNA for the CB2 receptor. The CB1-selective antagonist SR141716A did not reverse the suppression caused by THC, demonstrating that the CB1 receptor was not responsible for the drug's inhibitory effect. In contrast, the CB2-selective antagonist SR144528 completely blocked THC's suppression of the T cell response, implicating the participation of the CB2 receptor. These findings suggest that the CB2 receptor may be involved in CB inhibition of antigen processing by macrophages in this system.  (+info)

Ultrastructural studies on the surface membrane of the mouse egg. (53/2700)

Fertilized and unfertilized mouse eggs were examined by scanning and transmission electron microscopy for evidence of mosaicism in the organization and concanavalin A-binding properties of their surface membranes. No obvious quantitative mosaicism in concanavalin A binding was noted. The egg membrane was microvillous over most of its surface, but was smooth in the region overlying the 2nd metaphase spindle of the unfertilized egg and on the polar body of the fertilized egg.  (+info)

Presynaptically located CB1 cannabinoid receptors regulate GABA release from axon terminals of specific hippocampal interneurons. (54/2700)

To understand the functional significance and mechanisms of action in the CNS of endogenous and exogenous cannabinoids, it is crucial to identify the neural elements that serve as the structural substrate of these actions. We used a recently developed antibody against the CB1 cannabinoid receptor to study this question in hippocampal networks. Interneurons with features typical of basket cells showed a selective, intense staining for CB1 in all hippocampal subfields and layers. Most of them (85.6%) contained cholecystokinin (CCK), which corresponded to 96.9% of all CCK-positive interneurons, whereas only 4.6% of the parvalbumin (PV)-containing basket cells expressed CB1. Accordingly, electron microscopy revealed that CB1-immunoreactive axon terminals of CCK-containing basket cells surrounded the somata and proximal dendrites of pyramidal neurons, whereas PV-positive basket cell terminals in similar locations were negative for CB1. The synthetic cannabinoid agonist WIN 55,212-2 (0.01-3 microM) reduced dose-dependently the electrical field stimulation-induced [3H]GABA release from superfused hippocampal slices, with an EC50 value of 0. 041 microM. Inhibition of GABA release by WIN 55,212-2 was not mediated by inhibition of glutamatergic transmission because the WIN 55,212-2 effect was not reduced by the glutamate blockers AP5 and CNQX. In contrast, the CB1 cannabinoid receptor antagonist SR 141716A (1 microM) prevented this effect, whereas by itself it did not change the outflow of [3H]GABA. These results suggest that cannabinoid-mediated modulation of hippocampal interneuron networks operate largely via presynaptic receptors on CCK-immunoreactive basket cell terminals. Reduction of GABA release from these terminals is the likely mechanism by which both endogenous and exogenous CB1 ligands interfere with hippocampal network oscillations and associated cognitive functions.  (+info)

Identification of the high-affinity tolbutamide site on the SUR1 subunit of the K(ATP) channel. (55/2700)

ATP-sensitive potassium channels (K(ATP)) are formed from four pore-forming Kir6.2 subunits complexed with four regulatory sulfonylurea receptor subunits (SUR1 in pancreatic beta-cells, SUR2A in heart). The sensitivity of the channel to different sulfonylureas depends on the SUR isoform. In particular, Kir6.2-SUR1 but not Kir6.2-SUR2A channels are blocked by tolbutamide with high affinity. We made chimeras between SUR1 and SUR2A to identify the region of the protein involved in high-affinity tolbutamide block. Chimeric SURs were coexpressed with Kir6.2 in Xenopus oocytes, and macroscopic currents were measured in inside-out membrane patches. High-affinity tolbutamide inhibition could be conferred on SUR2A by replacing transmembrane domains (TMs) 14-16 with the corresponding region of SUR1. Conversely, high-affinity tolbutamide inhibition of SUR1 was abolished by replacing TMs 13-16 with the corresponding SUR2A sequence, or by mutating a single serine residue within this region to tyrosine (S1237Y). Binding of [3H]glibenclamide to membranes expressing SUR1 was abolished concomitantly with the loss of high-affinity tolbutamide block. These results suggest that a site in the COOH-terminal set of TMs of the SUR1 subunit of the K(ATP) channel is involved in the binding of tolbutamide and glibenclamide.  (+info)

Stoichiometry of sulfonylurea-induced ATP-sensitive potassium channel closure. (56/2700)

Hypoglycemic sulfonylureas (e.g., glibenclamide, glipizide, and tolbutamide) exert their stimulatory effect on excitatory cells by closure of ATP-sensitive potassium (KATP) channels. These channels are heteromultimers composed with a 4:4 stoichiometry of an inwardly rectifying K+ channel (KIR) subunit 6.x plus a sulfonylurea receptor (SUR). SUR1/KIR6.2 reconstitutes the neuronal/pancreatic beta-cell channel, whereas SUR2A/KIR6.2 and SUR2B/KIR6.1 (or KIR6.2) are proposed to reconstitute the cardiac and the vascular smooth muscle-type KATP channels, respectively. SUR2A and SUR2B are splice variants of a single gene differing only in their C-terminal 42 amino acids. Affinities of sulfonylureas for rat SUR2A, rat or human SUR2B, and a SUR2 chimera containing the C-terminal 42 amino acids of SUR1 did not differ significantly, implying that the C terminus does not form part of the binding pocket. Consistent with these findings, reconstituted SUR2A/KIR6.2 and SUR2B/KIR6.2 channels revealed similar sensitivities for glibenclamide and tolbutamide. Dissociation constants of sulfonylureas for SUR2A and SUR2B were 10- to 400-fold higher than for SUR1, however, amazingly the benzoic acid derivative meglitinide did not show lower affinity for SUR2 isoforms. Potencies of glibenclamide, glipizide, tolbutamide, and meglitinide to inhibit activity of SUR1/KIR6.2 and SUR2B/KIR6.2 channels were 3- to 6-fold higher than binding affinities of these drugs with concentration-inhibition relations being significantly steeper (Hill coefficients 1.23-1.32) than binding curves (Hill coefficients 0.93-1.06). The data establish that the C terminus of SURs does not affect sulfonylurea affinity and sensitivity. We conclude that occupation of one of the four SUR sites per channel complex is sufficient to induce KATP channel closure.  (+info)