T-type calcium channel inhibition underlies the analgesic effects of the endogenous lipoamino acids.

Lipoamino acids are anandamide-related endogenous molecules that induce analgesia via unresolved mechanisms. Here, we provide evidence that the T-type/Cav3 calcium channels are important pharmacological targets underlying their physiological effects. Various lipoamino acids, including N-arachidonoyl glycine (NAGly), reversibly inhibited Cav3.1, Cav3.2, and Cav3.3 currents, with potent effects on Cav3.2 [EC(50) approximately 200 nm for N-arachidonoyl 3-OH-gamma-aminobutyric acid (NAGABA-OH)]. This inhibition involved a large shift in the Cav3.2 steady-state inactivation and persisted during fatty acid amide hydrolase (FAAH) inhibition as well as in cell-free outside-out patch. In contrast, lipoamino acids had weak effects on high-voltage-activated (HVA) Cav1.2 and Cav2.2 calcium currents, on Nav1.7 and Nav1.8 sodium currents, and on anandamide-sensitive TRPV1 and TASK1 currents. Accordingly, lipoamino acids strongly inhibited native Cav3.2 currents in sensory neurons with small effects on sodium and HVA calcium currents. In addition, we demonstrate here that lipoamino acids NAGly and NAGABA-OH produced a strong thermal analgesia and that these effects (but not those of morphine) were abolished in Cav3.2 knock-out mice. Collectively, our data revealed lipoamino acids as a family of endogenous T-type channel inhibitors, suggesting that these ligands can modulate multiple cell functions via this newly evidenced regulation.

Regulation of T-type calcium channels: signalling pathways and functional implications.

T-type calcium channels (T-channels) contribute to a wide variety of physiological functions, especially in the cardiovascular and nervous systems. Recent studies using knock-out mouse models have been instrumental in documenting further the role of T-channels in sleep, heartbeat, pain and epilepsy. Importantly, several novel aspects of the regulation of these channels have been identified over the last few years, providing new insights into their physiological and pathophysiological roles. Here, we review recent evidence supporting that the Cav3 subunits of T-channels are modulated by endogenous ligands such as anandamide, zinc, redox and oxidizing agents, as well as G-protein and protein kinases pathways. The study of T-channel mutations associated with childhood absence epilepsy has also revealed new aspects of Cav3 subunit trafficking. Collectively, these findings identify novel regulatory mechanisms involved in the fine tuning of T-channel expression and activity, and offer new directions for the design of novel therapeutic strategies targeting these channels.

Homomeric RDL and heteromeric RDL/LCCH3 GABA receptors in the honeybee antennal lobes: two candidates for inhibitory transmission in olfactory processing.

gamma-Aminobutyric acid (GABA)-gated chloride channel receptors are abundant in the CNS, where their physiological role is to mediate fast inhibitory neurotransmission. In insects, this inhibitory transmission plays a crucial role in olfactory information processing. In an effort to understand the nature and properties of the ionotropic receptors involved in these processes in the honeybee Apis mellifera, we performed a pharmacological and molecular characterization of GABA-gated channels in the primary olfactory neuropile of the honeybee brain-the antennal lobe (AL)-using whole cell patch-clamp recordings coupled with single-cell RT-PCR. Application of GABA onto AL cells at -110 mV elicited fast inward currents, demonstrating the existence of ionotropic GABA-gated chloride channels. Molecular analysis of the GABA-responding cells revealed that both subunits RDL and LCCH3 were expressed out of the three orthologs of Drosophila melanogaster GABA-receptor subunits encoded within the honeybee genome (RDL, resistant to dieldrin; GRD, GABA/glycine-like receptor of Drosophila; LCCH3, ligand-gated chloride channel homologue 3), opening the door to possible homo- and/or heteromeric associations. The resulting receptors were activated by insect GABA-receptor agonists muscimol and CACA and blocked by antagonists fipronil, dieldrin, and picrotoxin, but not bicuculline, displaying a typical RDL-like pharmacology. Interestingly, increasing the intracellular calcium concentration potentiated GABA-elicited currents, suggesting a modulating effect of calcium on GABA receptors possibly through phosphorylation processes that remain to be determined. These results indicate that adult honeybee AL cells express typical RDL-like GABA receptors whose properties support a major role in synaptic inhibitory transmission during olfactory information processing.

Study of nicotinic acetylcholine receptors on cultured antennal lobe neurones from adult honeybee brains.

In insects, acetylcholine (ACh) is the main neurotransmitter, and nicotinic acetylcholine receptors (nAChRs) mediate fast cholinergic synaptic transmission. In the honeybee, nAChRs are expressed in diverse structures including the primary olfactory centres of the brain, the antennal lobes (AL) and the mushroom bodies. Whole-cell, voltage-clamp recordings were used to characterize the nAChRs present on cultured AL cells from adult honeybee, Apis mellifera. In 90% of the cells, applications of ACh induced fast inward currents that desensitized slowly. The classical nicotinic agonists nicotine and imidacloprid elicited respectively 45 and 43% of the maximum ACh-induced currents. The ACh-elicited currents were blocked by nicotinic antagonists methyllycaconitine, dihydroxy-beta-erythroidine and alpha-bungarotoxin. The nAChRs on adult AL cells are cation permeable channels. Our data indicate the existence of functional nAChRs on adult AL cells that differ from nAChRs on pupal Kenyon cells from mushroom bodies by their pharmacological profile and ionic permeability, suggesting that these receptors could be implicated in different functions.

Functional outcome of patients with prolonged hypoglycemic encephalopathy.

BACKGROUND: Little is known about the causes, clinical course and long-term outcome of comatose patients with prolonged hypoglycemic encephalopathy. METHODS: In a multicenter retrospective study conducted in patients hospitalized from July 1, 2004, to July 1, 2014, we investigated functional long-term prognosis and identified prognosis factors of patients admitted in an intensive care unit (ICU) with prolonged neurological manifestations related to hypoglycemia. Eligible patients were adults admitted to the ICU with a Glasgow Coma Score <8 due to hypoglycemia and persistent consciousness disorders after normalizing blood glucose levels. Patients with possible other causes of consciousness disorders, previous cognitive disorders, hypothermia <35 °C or circulatory arrest within 24 h after ICU admission, were excluded. Follow-up phone call was used to determine patients' functional outcome using modified Rankin Scale (mRS) at a minimum of 1-year follow-up with mRS 0-3 defining good and mRS 4-6 poor outcomes. RESULTS: Forty-nine patients were included. Causes of hypoglycemia were various, mainly including insulin or oral antidiabetic drugs abuse (65%) and neuroendocrine carcinoma (16%). Twenty (41%) patients died in the ICU, two (4%) patients further died and nine (18%) patients had a poor outcome at long-term follow-up. Five patients discharged from the ICU with mRS > 3 improved enough to be in the good outcome group 1 year later. Twenty-two (45%) patients underwent therapeutic limitation, mainly related to no expected hope for improvement. On multivariate analysis, only low mRS prior to ICU admission (OR 2.6; 95% CI 1.1-6.3; P = 0.03) and normal brain imaging (OR 7.1; 95% CI 1.1-44; P = 0.03) were significantly predictive of a good outcome. All patients (n = 15) who remained hypoglycemic >480 min had a poor outcome. CONCLUSION: Poor outcome was observed in about 60% of this population of hypoglycemic encephalopathy. However, some patients can recover satisfactorily over time.

Akt regulates L-type Ca2+ channel activity by modulating Cavalpha1 protein stability.

The insulin IGF-1-PI3K-Akt signaling pathway has been suggested to improve cardiac inotropism and increase Ca(2+) handling through the effects of the protein kinase Akt. However, the underlying molecular mechanisms remain largely unknown. In this study, we provide evidence for an unanticipated regulatory function of Akt controlling L-type Ca(2+) channel (LTCC) protein density. The pore-forming channel subunit Ca(v)alpha1 contains highly conserved PEST sequences (signals for rapid protein degradation), and in-frame deletion of these PEST sequences results in increased Ca(v)alpha1 protein levels. Our findings show that Akt-dependent phosphorylation of Ca(v)beta2, the LTCC chaperone for Ca(v)alpha1, antagonizes Ca(v)alpha1 protein degradation by preventing Ca(v)alpha1 PEST sequence recognition, leading to increased LTCC density and the consequent modulation of Ca(2+) channel function. This novel mechanism by which Akt modulates LTCC stability could profoundly influence cardiac myocyte Ca(2+) entry, Ca(2+) handling, and contractility.

Acetylcholine, GABA and glutamate induce ionic currents in cultured antennal lobe neurons of the honeybee, Apis mellifera.

The honeybee, Apis mellifera, is a valuable model system for the study of olfactory coding and its learning and memory capabilities. In order to understand the synaptic organisation of olfactory information processing, the transmitter receptors of the antennal lobe need to be characterized. Using whole-cell patch-clamp recordings, we analysed the ligand-gated ionic currents of antennal lobe neurons in primary cell culture. Pressure applications of acetylcholine (ACh), gamma-amino butyric acid (GABA) or glutamate induced rapidly activating ionic currents. The ACh-induced current flows through a cation-selective ionotropic receptor with a nicotinic profile. The ACh-induced current is partially blocked by alpha-bungarotoxin. Epibatidine and imidacloprid are partial agonists. Our data indicate the existence of an ionotropic GABA receptor which is permeable to chloride ions and sensitive to picrotoxin (PTX) and the insecticide fipronil. We also identified the existence of a chloride current activated by pressure applications of glutamate. The glutamate-induced current is sensitive to PTX. Thus, within the honeybee antennal lobe, an excitatory cholinergic transmitter system and two inhibitory networks that use GABA or glutamate as their neurotransmitter were identified.