High-affinity σ1 protein agonist reduces clinical and pathological signs of experimental autoimmune encephalomyelitis

Background and Purpose

Selective agonists of the sigma-1 receptor (σ1 protein) are generally reported to protect against neuronal damage and modulate oligodendrocyte differentiation. Human and rodent lymphocytes possess saturable, high-affinity binding sites for compounds binding to the σ1 protein and potential immunomodulatory properties have been described for σ1 protein ligands. Experimental autoimmune encephalomyelitis (EAE) is recognized as a valuable model of the inflammatory aspects of multiple sclerosis (MS). Here, we have assessed the role of a σ1 protein agonist, containing the tetrahydroisoquinoline-hydantoin structure, in EAE.

Experimental Approach

EAE was induced in SJL/J female mice by active immunization with myelin proteolipid protein (PLP)_139–151 peptide. The σ1 protein agonist was injected i.p. at the time of immunization (day 0). Disease severity was assessed clinically and by histopathological evaluation of the CNS. Phenotyping of B-cell subsets and regulatory T-cells were performed by flow cytometry in spleen and cervical lymph nodes.

Key Results

Prophylactic treatment of EAE mice with the σ1 protein agonist prevented mononuclear cell accumulation and demyelination in brain and spinal cord and increased T2 B-cells and regulatory T-cells, resulting in an overall reduction in the clinical progression of EAE.

Conclusions and Implications

This σ1 protein agonist, containing the tetrahydroisoquinoline-hydantoin structure, decreased the magnitude of inflammation in EAE. This effect was associated with increased proportions of B-cell subsets and regulatory T-cells with potential immunoregulatory functions. Targeting of the σ1 protein might thus provide new therapeutic opportunities in MS.     

Functional, morphological and molecular characterization of bladder dysfunction in streptozotocin-induced diabetic mice: evidence of a role for L-type voltage-operated Ca2+ channels

BACKGROUND AND PURPOSE

Diabetic cystopathy is one of the most common and incapacitating complications of diabetes mellitus. This study aimed to evaluate the functional, structural and molecular alterations of detrusor smooth muscle (DSM) in streptozotocin-induced diabetic mice, focusing on the contribution of Ca²⁺ influx through L-type voltage-operated Ca²⁺ channels (L-VOCC).

EXPERIMENTAL APPROACH

Male C57BL/6 mice were injected with streptozotocin (125 mg·kg⁻¹). Four weeks later, contractile responses to carbachol, α,β-methylene ATP, KCl, extracellular Ca²⁺ and electrical-field stimulation were measured in urothelium-intact DSM strips. Cystometry and histomorphometry were performed, and mRNA expression for muscarinic M₂/M₃ receptors, purine P2X1 receptors and L-VOCC in the bladder was determined.

KEY RESULTS

Diabetic mice exhibited higher bladder capacity, frequency, non-void contractions and post-void pressure. Increased bladder weight, wall thickness, bladder volume and neural tissue were observed in diabetic bladders. Carbachol, α,β-methylene ATP, KCl, extracellular Ca²⁺ and electrical-field stimulation all produced greater DSM contractions in diabetic mice. The L-VOCC blocker nifedipine almost completely reversed the enhanced DSM contractions in bladders from diabetic animals. The Rho-kinase inhibitor Y27632 had no effect on the enhanced carbachol contractions in the diabetic group. Expression of mRNA for muscarinic M₃ receptors and L-VOCC were greater in the bladders of diabetic mice, whereas levels of M₂ and P2X1 receptors remained unchanged.

CONCLUSIONS AND IMPLICATIONS

Diabetic mice exhibit features of urinary bladder dysfunction, as characterized by overactive DSM and decreased voiding efficiency. Functional and molecular data suggest that overactive DSM in diabetes is the result of enhanced extracellular Ca²⁺ influx through L-VOCC.     

Symptom-relieving and neuroprotective effects of the phytocannabinoid Δ⁹-THCV in animal models of Parkinson's disease

BACKGROUND AND PURPOSE

Previous findings have indicated that a cannabinoid, such as Δ⁹-THCV, which has antioxidant properties and the ability to activate CB₂ receptors but to block CB₁, might be a promising therapy for alleviating symptoms and delaying neurodegeneration in Parkinson''s disease (PD).

EXPERIMENTAL APPROACH

The ability of Δ⁹-THCV to reduce motor inhibition and provide neuroprotection was investigated in rats lesioned with 6-hydroxydopamine and in mice lesioned with lipopolysaccharide (LPS).

KEY RESULTS

Acute administration of Δ⁹-THCV attenuated the motor inhibition caused by 6-hydroxydopamine, presumably through changes in glutamatergic transmission. Moreover, chronic administration of Δ⁹-THCV attenuated the loss of tyrosine hydroxylase–positive neurones caused by 6-hydroxydopamine in the substantia nigra, through an effect related to its antioxidant properties (it was reproduced by cannabidiol -enriched botanical extract). In addition, CB₂ receptor–deficient mice responded to 6-hydroxydopamine in a similar manner to wild-type animals, and CB₂ receptors were poorly up-regulated in the rat substantia nigra in response to 6-hydroxydopamine. By contrast, the substantia nigra of mice that had been injected with LPS exhibited a greater up-regulation of CB₂ receptors. In these animals, Δ⁹-THCV also caused preservation of tyrosine hydroxylase–positive neurones. This effect probably involved CB₂ receptors as it was also elicited by the selective CB₂ receptor agonist, HU-308, and CB₂ receptor–deficient mice were more vulnerable to LPS lesions.

CONCLUSIONS AND IMPLICATIONS

Given its antioxidant properties and its ability to activate CB₂ but to block CB₁ receptors, Δ⁹-THCV has a promising pharmacological profile for delaying disease progression in PD and also for ameliorating parkinsonian symptoms.

LINKED ARTICLES

This article is part of a themed issue on Cannabinoids in Biology and Medicine. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph.2011.163.issue-7     

Mitochondrially targeted nitro-linoleate: a new tool for the study of cardioprotection

Background and Purpose

Cardiac ischaemia–reperfusion (IR) injury remains a significant clinical problem with limited treatment options available. We previously showed that cardioprotection against IR injury by nitro-fatty acids, such as nitro-linoleate (LNO₂), involves covalent modification of mitochondrial adenine nucleotide translocase 1 (ANT1). Thus, it was hypothesized that conjugation of LNO₂ to the mitochondriotropic triphenylphosphonium (TPP⁺) moiety would enhance its protective properties.

Experimental Approach

TPP⁺-LNO₂ was synthesized from aminopropyl-TPP⁺ and LNO₂, and characterized by direct infusion MS/MS. Its effects were assayed in primary cultures of cardiomyocytes from adult C57BL/6 mice and in mitochondria from these cells, exposed to simulated IR (SIR) conditions (oxygen and metabolite deprivation for 1h followed by normal conditions for 1h) by measuring viability by LDH release and exclusion of Trypan blue. Nitro-alkylated mitochondrial proteins were also measured by Western blots, using antibodies to TPP⁺.

Key Results

TPP⁺-LNO₂ protected cardiomyocytes from SIR injury more potently than the parent compound LNO₂. In addition, TPP⁺-LNO₂ modified mitochondrial proteins, including ANT1, in a manner sensitive to the mitochondrial uncoupler carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP) and the ANT1 inhibitor carboxyatractyloside. Similar protein nitro-alkylation was obtained in cells and in isolated mitochondria, indicating the cell membrane was not a significant barrier to TPP⁺-LNO₂.

Conclusions and Implications

Together, these results emphasize the importance of ANT1 as a target for the protective effects of LNO₂, and suggest that TPP⁺-conjugated electrophilic lipid compounds may yield novel tools for the investigation of cardioprotection.

Linked Articles

This article is part of a themed issue on Mitochondrial Pharmacology: Energy, Injury & Beyond. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph.2014.171.issue-8     

Loss of multidrug and toxin extrusion 1 (MATE1) is associated with metformin-induced lactic acidosis

BACKGROUNDS AND PURPOSE

Lactic acidosis is a fatal adverse effect of metformin, but the risk factor remains unclear. Multidrug and toxin extrusion 1 (MATE1) is expressed in the luminal membrane of the kidney and liver. MATE1 was revealed to be responsible for the tubular and biliary secretion of metformin. Therefore, some MATE polymorphisms, that cause it to function abnormally, are hypothesized to induce lactic acidosis. The purpose of this study is to clarify the association between MATE dysfunction and metformin-induced lactic acidosis.

EXPERIMENTAL APPROACH

Blood lactate, pH and bicarbonate ion (HCO₃^-) levels were evaluated during continuous administration of 3 mg·mL⁻¹ metformin in drinking water using Mate1 knockout (−/−), heterozygous (+/−) and wild-type (+/+) mice. To determine the tissue accumulation of metformin, mice were given 400 mg·kg⁻¹ metformin orally. Furthermore, blood lactate data were obtained from diabetic patients given metformin.

KEY RESULTS

Seven days after metformin administration in drinking water, significantly higher blood lactate, lower pH and HCO₃^- levels were observed in Mate1^−/− mice, but not in Mate1^+/− mice. The blood lactate levels were not affected in patients with the heterozygous MATE variant (MATE1-L125F, MATE1-G64D, MATE2-K-G211V). Sixty minutes after metformin administration (400 mg·kg⁻¹, p.o.) the hepatic concentration of metformin was markedly higher in Mate1^−/− mice than in Mate1^+/+ mice.

CONCLUSION AND IMPLICATIONS

MATE1 dysfunction caused a marked elevation in the metformin concentration in the liver and led to lactic acidosis, suggesting that the homozygous MATE1 variant could be one of the risk factors for metformin-induced lactic acidosis.     

Novel pyrazole compounds for pharmacological discrimination between receptor-operated and store-operated Ca2+ entry pathways

Background and purpose

Pyrazole derivatives have recently been suggested as selective blockers of transient receptor potential cation (TRPC) channels but their ability to distinguish between the TRPC and Orai pore complexes is ill-defined. This study was designed to characterize a series of pyrazole derivatives in terms of TRPC/Orai selectivity and to delineate consequences of selective suppression of these pathways for mast cell activation.

Experimental approach

Pyrazoles were generated by microwave-assisted synthesis and tested for effects on Ca²⁺ entry by Fura-2 imaging and membrane currents by patch-clamp recording. Experiments were performed in HEK293 cells overexpressing TRPC3 and in RBL-2H3 mast cells, which express classical store-operated Ca²⁺ entry mediated by Orai channels. The consequences of inhibitory effects on Ca²⁺ signalling in RBL-2H3 cells were investigated at the level of both degranulation and nuclear factor of activated T-cells activation.

Key Results

Pyr3, a previously suggested selective inhibitor of TRPC3, inhibited Orai1- and TRPC3-mediated Ca²⁺ entry and currents as well as mast cell activation with similar potency. By contrast, Pyr6 exhibited a 37-fold higher potency to inhibit Orai1-mediated Ca²⁺ entry as compared with TRPC3-mediated Ca²⁺ entry and potently suppressed mast cell activation. The novel pyrazole Pyr10 displayed substantial selectivity for TRPC3-mediated responses (18-fold) and the selective block of TRPC3 channels by Pyr10 barely affected mast cell activation.

Conclusions and Implications

The pyrazole derivatives Pyr6 and Pyr10 are able to distinguish between TRPC and Orai-mediated Ca²⁺ entry and may serve as useful tools for the analysis of cellular functions of the underlying Ca²⁺ channels.     

Activation of KCa3.1 by SKA-31 induces arteriolar dilatation and lowers blood pressure in normo- and hypertensive connexin40-deficient mice

BACKGROUND AND PURPOSE

The calcium-activated potassium channel K_Ca3.1 is expressed in the vascular endothelium where its activation causes endothelial hyperpolarization and initiates endothelium-derived hyperpolarization (EDH)-dependent dilatation. Here, we investigated whether pharmacological activation of K_Ca3.1 dilates skeletal muscle arterioles and whether myoendothelial gap junctions formed by connexin40 (Cx40) are required for EDH-type dilatations and pressure depressor responses in vivo.

EXPERIMENTAL APPROACH

We performed intravital microscopy in the cremaster muscle microcirculation and blood pressure telemetry in Cx40-deficient mice.

KEY RESULTS

In wild-type mice, the K_Ca3.1-activator SKA-31 induced pronounced concentration-dependent arteriolar EDH-type dilatations, amounting to ∼40% of maximal dilatation, and enhanced the effects of ACh. These responses were absent in mice devoid of K_Ca3.1 channels. In contrast, SKA-31-induced dilatations were not attenuated in mice with endothelial cells deficient in Cx40 (Cx40^fl/fl:Tie2-Cre). In isolated endothelial cell clusters, SKA-31 induced hyperpolarizations of similar magnitudes (by ∼38 mV) in Cx40^fl/fl:Tie2-Cre, ubiquitous Cx40-deficient mice (Cx40^-/-) and controls (Cx40^fl/fl), which were reversed by the specific K_Ca3.1-blocker TRAM-34. In normotensive wild-type and Cx40^fl/fl:Tie2-Cre as well as in hypertensive Cx40^-/- animals, i.p. injections of SKA-31 (30 and 100 mg·kg⁻¹) decreased arterial pressure by ∼32 mmHg in all genotypes. The depressor response to 100 mg·kg⁻¹ SKA-31 was associated with a decrease in heart rate.

CONCLUSIONS AND IMPLICATIONS

We conclude that endothelial hyperpolarization evoked by pharmacological activation of K_Ca3.1 channels induces EDH-type arteriolar dilatations that are independent of endothelial Cx40 and Cx40-containing myoendothelial gap junctions. As SKA-31 reduced blood pressure in hypertensive Cx40-deficient mice, K_Ca3.1 activators may be useful drugs for severe treatment-resistant hypertension.     

The chemokine receptor antagonist,TAK-779, decreased experimental autoimmune encephalomyelitis by reducing inflammatory cell migration into the central nervous system,without affecting T cell function

Background and purpose:

The C–C chemokine receptor CCR5, and the C–X–C chemokine receptor CXCR3 are involved in the regulation of T cell-mediated immune responses, and in the migration and activation of these cells. To determine whether blockade of these chemokine receptors modulated inflammatory responses in the central nervous sytem (CNS), we investigated the effect of a non-peptide chemokine receptor antagonist, TAK-779, in mice with experimental autoimmune encephalomyelitis (EAE).

Experimental approach:

EAE was induced by immunization of C57BL/6 mice with myelin oligodendrocyte glycoprotein (MOG) 35–55. TAK-779 was injected s.c. once a day after immunization. Disease incidence and severity (over 3 weeks) were monitored by histopathological evaluation and FACS assay of inflammatory cells infiltrating into the spinal cord, polymerase chain reaction quantification of mRNA expression, assay of T cell proliferation, by [³H]-thymidine incorporation and cytokine production by enzyme-linked immunosorbent assay.

Key results:

Treatment with TAK-779 reduced incidence and severity of EAE. It strongly inhibited migration of CXCR3/CCR5 bearing CD4⁺, CD8⁺ and CD11b⁺ leukocytes to the CNS. TAK-779 did not reduce proliferation of anti-MOG T cells, the production of IFN-γ by T cells or CXCR3 expression on T cells. In addition, TAK-779 did not affect production of IL-12 by antigen-presenting cells, CCR5 induction on T cells and the potential of MOG-specific T cells to transfer EAE.

Conclusions and implications:

TAK-779 restricted the development of MOG-induced EAE. This effect involved reduced migration of inflammatory cells into the CNS without affecting responses of anti-MOG T cells or the ability of MOG-specific T cells to transfer EAE.     

Effects of the endogenous cannabinoid anandamide on voltage-dependent sodium and calcium channels in rat ventricular myocytes

BACKGROUND AND PURPOSE

The endocannabinoid anandamide (N-arachidonoyl ethanolamide; AEA) exerts negative inotropic and antiarrhythmic effects in ventricular myocytes.

EXPERIMENTAL APPROACH

Whole-cell patch-clamp technique and radioligand-binding methods were used to analyse the effects of anandamide in rat ventricular myocytes.

KEY RESULTS

In the presence of 1–10 μM AEA, suppression of both Na⁺ and L-type Ca²⁺ channels was observed. Inhibition of Na⁺ channels was voltage and Pertussis toxin (PTX) – independent. Radioligand-binding studies indicated that specific binding of [³H] batrachotoxin (BTX) to ventricular muscle membranes was also inhibited significantly by 10 μM metAEA, a non-metabolized AEA analogue, with a marked decrease in B_max values but no change in K_d. Further studies on L-type Ca²⁺ channels indicated that AEA potently inhibited these channels (IC₅₀ 0.1 μM) in a voltage- and PTX-independent manner. AEA inhibited maximal amplitudes without affecting the kinetics of Ba²⁺ currents. MetAEA also inhibited Na⁺ and L-type Ca²⁺ currents. Radioligand studies indicated that specific binding of [³H]isradipine, was inhibited significantly by metAEA. (10 μM), changing B_max but not K_d.

CONCLUSION AND IMPLICATIONS

Results indicate that AEA inhibited the function of voltage-dependent Na⁺ and L-type Ca²⁺ channels in rat ventricular myocytes, independent of CB₁ and CB₂ receptor activation.     

The TRPC channel blocker SKF 96365 inhibits glioblastoma cell growth by enhancing reverse mode of the Na+/Ca2+ exchanger and increasing intracellular Ca2+

BACKGROUND AND PURPOSE

SKF 96365 is well known for its suppressing effect on human glioblastoma growth by inhibiting pre-activated transient receptor potential canonical (TRPC) channels and Ca²⁺ influx. The effect of SKF 96363 on glioblastoma cells, however, may be multifaceted and this possibility has been largely ignored.

EXPERIMENTAL APPROACH

The effects of SKF 96365 on cell cycle and cell viability of cultured human glioblastoma cells were characterized. Western blot, Ca²⁺ imaging and patch clamp recordings were used to delineate cell death mechanisms. siRNA gene knockdown provided additional evidence.

KEY RESULTS

SKF 96365 repressed glioblastoma cell growth via increasing intracellular Ca²⁺ ([Ca²⁺]_i) irrespective of whether TRPC channels were blocked or not. The effect of SKF 96365 primarily resulted from enhanced reverse operation of the Na⁺/Ca²⁺ exchanger (NCX) with an EC₅₀ of 9.79 μM. SKF 96365 arrested the glioblastoma cells in the S and G₂ phases and activated p38-MAPK and JNK, which were all prevented by the Ca²⁺ chelator BAPTA-AM or EGTA. The expression of NCX in glioblastoma cells was significantly higher than in normal human astrocytes. Knockdown of the NCX1 isoforms diminished the effect of SKF 96365 on glioblastoma cells.

CONCLUSIONS AND IMPLICATIONS

At the same concentration, SKF 96365 blocks TRPC channels and enhances the reverse mode of the NCX causing [Ca²⁺]_i accumulation and cytotoxicity. This finding suggests an alternative pharmacological mechanism of SKF 96365. It also indicates that modulation of the NCX is an effective method to disrupt Ca²⁺ homeostasis and suppress human glioblastoma cells.     

Melatonin MT1 and MT2 receptors display different molecular pharmacologies only in the G-protein coupled state

BACKGROUND AND PURPOSE

Melatonin receptors have been extensively characterized regarding their affinity and pharmacology, mostly using 2-[¹²⁵I]-melatonin as a radioligand. Although [³H]-melatonin has the advantage of corresponding to the endogenous ligand of the receptor, its binding has not been well described.

EXPERIMENTAL APPROACH

We characterized [³H]-melatonin binding to the hMT₁ and hMT₂ receptors expressed in a range of cell lines and obtained new insights into the molecular pharmacology of melatonin receptors.

KEY RESULTS

The binding of [³H]-melatonin to the hMT₁ and hMT₂ receptors displayed two sites on the saturation curves. These two binding sites were observed on cell membranes expressing recombinant receptors from various species as well as on whole cells. Furthermore, our GTPγS/NaCl results suggest that these sites on the saturation curves correspond to the G-protein coupled and uncoupled states of the receptors, whose pharmacology was extensively characterized.

CONCLUSIONS AND IMPLICATIONS

hMT₁ and hMT₂ receptors spontaneously exist in two states when expressed in cell lines; these states can be probed by [³H]-melatonin binding. Overall, our results suggest that physiological regulation of the melatonin receptors may result from complex and subtle mechanisms, a small difference in affinity between the active and inactive states of the receptor, and spontaneous coupling to G-proteins.     

Characterization of histamine H3 receptors in Alzheimer's Disease brain and amyloid over-expressing TASTPM mice

Background and purpose:

Histamine H₃ receptor antagonists are currently being evaluated for their potential use in a number of central nervous system disorders including Alzheimer''s Disease (AD). To date, little is known about the state of H₃ receptors in AD.

Experimental approach:

In the present study we used the radiolabelled H₃ receptor antagonist [³H]GSK189254 to investigate H₃ receptor binding in the amyloid over-expressing double mutant APPswe × PSI.MI46V (TASTPM) transgenic mouse model of AD and in post-mortem human AD brain samples.

Key results:

No significant differences in specific H₃ receptor binding were observed between wild type and TASTPM mice in the cortex, hippocampus or hypothalamus. Specific [³H]GSK189254 binding was detected in sections of human medial frontal cortex from AD brains of varying disease severity (Braak stages I–VI). With more quantitative analysis in a larger cohort, we observed that H₃ receptor densities were not significantly different between AD and age-matched control brains in both frontal and temporal cortical regions. However, within the AD group, [³H]GSK189254 binding density in frontal cortex was higher in individuals with more severe dementia prior to death.

Conclusions and implications:

The maintenance of H₃ receptor integrity observed in the various stages of AD in this study is important, given the potential use of H₃ antagonists as a novel therapeutic approach for the symptomatic treatment of AD.     

AMP-activated protein kinase (AMPK)-dependent and -independent pathways regulate hypoxic inhibition of transepithelial Na(+) transport across human airway epithelial cells

BACKGROUND AND PURPOSE

Pulmonary transepithelial Na⁺ transport is reduced by hypoxia, but in the airway the regulatory mechanisms remain unclear. We investigated the role of AMPK and ROS in the hypoxic regulation of apical amiloride-sensitive Na⁺ channels and basolateral Na⁺K⁺ ATPase activity.

EXPERIMENTAL APPROACH

H441 human airway epithelial cells were used to examine the effects of hypoxia on Na⁺ transport, AMP : ATP ratio and AMPK activity. Lentiviral constructs were used to modify cellular AMPK abundance and activity; pharmacological agents were used to modify cellular ROS.

KEY RESULTS

AMPK was activated by exposure to 3% or 0.2% O₂ for 60 min in cells grown in submerged culture or when fluid (0.1 mL·cm⁻²) was added to the apical surface of cells grown at the air–liquid interface. Only 0.2% O₂ activated AMPK in cells grown at the air–liquid interface. AMPK activation was associated with elevation of cellular AMP : ATP ratio and activity of the upstream kinase LKB1. Hypoxia inhibited basolateral ouabain-sensitive I_sc (I_ouabain) and apical amiloride-sensitive Na⁺ conductance (G_Na+). Modification of AMPK activity prevented the effect of hypoxia on I_ouabain (Na⁺K⁺ ATPase) but not apical G_Na+. Scavenging of superoxide and inhibition of NADPH oxidase prevented the effect of hypoxia on apical G_Na+ (epithelial Na⁺ channels).

CONCLUSIONS AND IMPLICATIONS

Hypoxia activates AMPK-dependent and -independent pathways in airway epithelial cells. Importantly, these pathways differentially regulate apical Na⁺ channels and basolateral Na⁺K⁺ ATPase activity to decrease transepithelial Na⁺ transport. Luminal fluid potentiated the effect of hypoxia and activated AMPK, which could have important consequences in lung disease conditions.     

Acute desipramine restores presynaptic cortical defects in murine experimental autoimmune encephalomyelitis by suppressing central CCL5 overproduction

Background and Purpose

Altered glutamate exocytosis and cAMP production in cortical terminals of experimental autoimmune encephalomyelitis (EAE) mice occur at the early stage of disease (13 days post-immunization, d.p.i.). Neuronal defects were paralleled by overexpression of the central chemokine CCL5 (also known as RANTES), suggesting it has a role in presynaptic impairments. We propose that drugs able to restore CCL5 content to physiological levels could also restore presynaptic defects. Because of its efficacy in controlling CCL5 overexpression, desipramine (DMI) appeared to be a suitable candidate to test our hypothesis.

Experimental Approach

Control and EAE mice at 13 d.p.i. were acutely or chronically administered DMI and monitored for behaviour and clinical scores. Noradrenaline and glutamate release, cAMP, CCL5 and TNF-α production were quantified in cortical synaptosomes and homogenates. Peripheral cytokine production was also determined.

Key Results

Noradrenaline exocytosis and α₂-adrenoeceptor-mediated activity were unmodified in EAE mice at 13 d.p.i. when compared with control. Acute, but not chronic, DMI reduced CCL5 levels in cortical homogenates of EAE mice at 13 d.p.i., but did not affect peripheral IL-17 and TNF-α contents or CCL5 plasma levels. Acute DMI caused a long-lasting restoration of glutamate exocytosis, restored endogenous cAMP production and impeded the shift from inhibition to facilitation of the CCL5-mediated control of glutamate exocytosis. Finally, DMI ameliorated anxiety-related behaviour but not motor activity or severity of clinical signs.

Conclusions

We propose DMI as an add-on therapy to normalize neuropsychiatric symptoms in multiple sclerosis patients at the early stage of the disease.     

Simultaneous oral therapeutic and intravenous 14C‐microdoses to determine the absolute oral bioavailability of saxagliptin and dapagliflozin

Aim

To determine the absolute oral bioavailability (F_p.o.) of saxagliptin and dapagliflozin using simultaneous intravenous ¹⁴C‐microdose/therapeutic oral dosing (i.v.micro + oraltherap).

Methods

The F_p.o. values of saxagliptin and dapagliflozin were determined in healthy subjects (n = 7 and 8, respectively) following the concomitant administration of single i.v. micro doses with unlabelled oraltherap doses. Accelerator mass spectrometry and liquid chromatography‐tandem mass spectrometry were used to quantify the labelled and unlabelled drug, respectively.

Results

The geometric mean point estimates (90% confidence interval) F_p.o. values for saxagliptin and dapagliflozin were 50% (48, 53%) and 78% (73, 83%), respectively. The i.v.micro had similar pharmacokinetics to oraltherap.

Conclusions

Simultaneous i.v.micro + oraltherap dosing is a valuable tool to assess human absolute bioavailability.     

β3-Adrenoceptor activation attenuates atherosclerotic plaque formation in ApoE?/? mice through lowering blood lipids and glucose

Aim:

To examine the effects of β₃-adrenoceptor (β₃-AR) activation on atherosclerotic plaque development in ApoE^−/− mice.

Methods:

Thirty six week-old male ApoE^−/− mice on a high-fat diet were treated with atorvastatin (10 mg·kg^-1·d^-1, po), BRL37344 (β₃-AR agonist, 1.65 or 3.30 μg/kg, ip, twice a week) or SR52390A (β₃-AR antagonist, 50 μg/kg, ip, twice a week) for 12 weeks. Wild-type C57BL/6J mice receiving a normal diet were taken as healthy controls. At the end of the treatments, serum levels of triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), non-high density lipoprotein cholesterol (nHDL-C), glucose and insulin were measured. The thoracic aortas were dissected out, the area of atherosclerotic plaques and extent of fibrosis in the plaques were examined using HE and Masson''s trichome staining, respectively.

Results:

Compared to wild-type mice, ApoE^−/− mice fed on a high-fat diet exhibited prominent hyperlipidemia and insulin resistance, associated with large area of atherosclerotic plaques and great extent of fibrosis in aortas. Atorvastatin significantly decreased the serum levels of TC and nHDL-C, and reduced the plaque area and collagen content in aortas. BRL37344 significantly decreased the serum levels of TG, TC, nHDL-C, glucose and insulin, and increased HDL-C and the insulin sensitivity, and dose-dependently reduced the plaque area and collagen content in aortas. SR52390A treatment did not affect any parameters studied.

Conclusion:

The β₃-AR agonist impedes the progression of atherosclerosis in ApoE^−/− mice, through improvement of the lipid and glucose profiles.     

Phosphatidylinositol 3-kinase-δ up-regulates L-type Ca2+ currents and increases vascular contractility in a mouse model of type 1 diabetes

BACKGROUND AND PURPOSE

Vasculopathies represent the main cause of morbidity and mortality in diabetes. Vascular malfunctioning in diabetes is associated with abnormal vasoconstriction and Ca²⁺ handling by smooth muscle cells (SMC). Phosphatidylinositol 3-kinases (PI3K) are key mediators of insulin action and have been shown to modulate the function of voltage-dependent L-type Ca²⁺ channels (Ca_V1.2). In the present work, we investigated the involvement of PI3K signalling in regulating Ca²⁺ current through Ca_V1.2 (I_Ca,L) and vascular dysfunction in a mouse model of type I diabetes.

EXPERIMENTAL APPROACH

Changes in isometric tension were recorded on myograph. Ca²⁺ currents in freshly dissociated mice aortic SMCs were measured using the whole-cell patch-clamp technique. Antisense techniques were used to knock-down the PI3Kδ isoform.

KEY RESULTS

Contractile responses to phenylephrine and KCl were strongly enhanced in diabetic aorta independent of a functional endothelium. The magnitude of phenylephrine-induced I_Ca,L was also greatly augmented. PI3Kδ expression, but not PI3Kα, PI3Kβ, PI3Kγ, was increased in diabetic aortas and treatment of vessels with a selective PI3Kδ inhibitor normalized I_Ca,L and contractile response of diabetic vessels. Moreover, knock-down of PI3Kδin vivo decreased PI3Kδ expression and normalized I_Ca,L and contractile response of diabetic vessels ex vivo.

CONCLUSIONS AND IMPLICATIONS

Phosphatidylinositol 3-kinase δ was essential to the increased vascular contractile response in our model of type I diabetes. PI3Kδ signalling was up-regulated and most likely accounted for the increased I_Ca,L, leading to increased vascular contractility. Blockade of PI3Kδ may represent a novel therapeutic approach to treat vascular dysfunction in diabetic patients.

LINKED ARTICLE

This article is commented on by Sturek, pp. 1455–1457 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2010.00997.x     

Electrical conduction along endothelial cell tubes from mouse feed arteries: confounding actions of glycyrrhetinic acid derivatives

BACKGROUND AND PURPOSE

Electrical conduction along endothelium of resistance vessels has not been determined independently of the influence of smooth muscle, surrounding tissue or blood. Two interrelated hypotheses were tested: (i) Intercellular conduction of electrical signals is manifest in endothelial cell (EC) tubes; and (ii) Inhibitors of gap junction channels (GJCs) have confounding actions on EC electrical and Ca²⁺ signalling.

EXPERIMENTAL APPROACH

Intact EC tubes were isolated from abdominal muscle feed (superior epigastric) arteries of C57BL/6 mice. Hyperpolarization was initiated with indirect (ACh) and direct (NS309) stimulation of intermediate- and small-conductance Ca²⁺-activated K⁺ channels (IK_Ca/SK_Ca). Remote membrane potential (V_m) responses to intracellular current injection defined the length constant (λ) for electrical conduction. Dye coupling was evaluated following intracellular microinjection of propidium iodide. Intracellular Ca²⁺ dynamics were determined using Fura-2 photometry. Carbenoxolone (CBX) or β-glycyrrhetinic acid (βGA) was used to investigate the role of GJCs.

KEY RESULTS

Steady-state V_m of ECs was −25 mV. ACh and NS309 hyperpolarized ECs by −40 and −60 mV respectively. Electrical conduction decayed monoexponentially with distance (λ∼1.4 mm). Propidium iodide injected into one EC spread into surrounding ECs. CBX or βGA inhibited dye transfer, electrical conduction and EC hyperpolarization reversibly. Both agents elevated resting Ca²⁺ while βGA inhibited responses to ACh.

CONCLUSIONS AND IMPLICATIONS

Individual cells were effectively coupled to each other within EC tubes. Inhibiting GJCs with glycyrrhetinic acid derivatives blocked hyperpolarization mediated by IK_Ca/SK_Ca channels, regardless of Ca²⁺ signalling, obviating use of these agents in distinguishing key determinants of electrical conduction along the endothelium.