Does cyclic AMP mediate rat urinary bladder relaxation by isoproterenol?

Cyclic AMP is the prototypical second messenger of beta-adrenergic receptors, but recent findings have questioned its role in mediating smooth muscle relaxation upon beta-adrenergic receptor stimulation. We have investigated the signaling mechanisms underlying beta-adrenergic receptor-mediated relaxation of rat urinary bladder. Concentration-response curves for isoproterenol-induced bladder relaxation were generated in the presence or absence of inhibitors, with concomitant experiments using passive tension and KCl-induced precontraction. The adenylyl cyclase inhibitor 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ 22,536; 1 microM), the protein kinase A inhibitors 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7; 10 microM), N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H89; 1 microM), and Rp-adenosine 3',5'-cyclic monophosphorothioate (Rp-cAMPS; 30 microM), and the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ; 3 microM) produced only minor if any inhibition of relaxation against passive tension or KCl-induced precontraction. Among various potassium channel inhibitors, BaCl2 (10 microM), tetraethylammonium (3 microM), apamin (300 nM), and glibenclamide (10 microM) did not inhibit isoproterenol-induced relaxation. Some inhibition of the isoproterenol effects against KCl-induced tone but not against passive tension was seen with inhibitors of calcium-dependent potassium channels such as charybdotoxin and iberiotoxin (30 nM each). A combination of SQ 22,536 and ODQ significantly inhibited relaxation against passive tension by about half, but not that against KCl-induced tone. Moreover, the combination failed to enhance inhibition by charybdotoxin against KCl-induced tone. We conclude that cAMP and cGMP each play a minor role in beta-adrenergic receptor-mediated relaxation against passive tension, and calcium-dependent potassium channels play a minor role against active tension.     

High isoproterenol doses are required to activate beta3-adrenoceptor-mediated functions in dogs

The "in vivo" conditions for beta3-adrenoceptors (beta-AR) activation by isoproterenol were investigated in dog. Experiments were carried out in anesthetized dogs using isoproterenol as a nonselective beta-AR agonist. Intravenous infusion of isoproterenol (0.4 nmol/kg/min) induced arterial hypotension and tachycardia with a slight decrease in cutaneous blood flow. At this dose, isoproterenol increased glucose, glycerol, and nonesterified fatty acid plasma levels. The changes in cardiovascular and endocrine-metabolic parameters, induced by the low dose of isoproterenol, were suppressed by pretreatment with nadolol (1 mg/kg, i.v.). After nadolol administration, however, a 10-fold higher dose (4 nmol/kg/min) of isoproterenol was able to induce a decrease in arterial blood pressure with a slight tachycardia and an increase in cutaneous blood flow. This high dose of isoproterenol increased nonesterified fatty acid and glycerol plasma levels but failed to change glucose plasma levels. All these effects were abolished by a pretreatment with nadolol (1 mg/kg, i.v.) plus SR59230A [a selective beta3-adrenoceptor antagonist; (3-(2-ethylphenoxy)-1(1S)-1,2,3,4-tetrahydronaphth-1-ylaminol-(2S)2-propanol oxalate); 1 mg/kg, i.v.]. Moreover, as observed with the high dose of isoproterenol under nadolol pretreatment, an infusion of SR58611A [a selective beta3-adrenoceptor agonist; ((N2S)-7-carbethoxymethoxy-1,2,3,4-tetrahydronaphth-2-yl-(2R)-2-hydroxy-2-chlorophenyl) ethanamine hydrochloride] induces a decrease in mean arterial blood pressure associated with an increase in heart rate, cutaneous blood flow, and nonesterified fatty acid and glycerol plasma levels. These results demonstrate that the in vivo activation of beta3-adrenoceptors requires higher doses of catecholamine than those necessary for beta1- and/or beta2-adrenoceptor stimulation. These results also argue for the lack of a beta3-AR involvement in the control of heart rate and glycogenolysis in dogs.     

An instrument to assess patient perceptions of satisfaction with acute migraine treatment (EXPERT Study)

(Headache 2011;51:590‐601) Objective.— The objective of the nationwide EXPERT survey carried out in France in 2005 was to compare satisfaction with treatment with treatment effectiveness in migraine patients consulting general practitioners (GPs) for migraine, and to establish an instrument to easily evaluate the adequacy of acute treatment of migraine. Background.— Many migraine patients feel satisfied with their current acute treatment of migraine whereas objective evaluation reveals poor treatment effectiveness. Methods.— A total of 2108 GPs included 11,274 migraine patients. Satisfaction with treatment was evaluated using a 4‐point verbal scale and a 10‐cm visual analog scale (VAS). Treatment effectiveness was assessed by the 4‐item questionnaire designed by the French Medico‐Economic Evaluation Service (ANAES) and the French Society for the Study of Migraine Headache (SFEMC). Results.— In total, 5224 patients (49.8%) stated that they were satisfied with their treatment. Mean VAS score was 5.1. Only 17% of patients (1789/10,539) gave positive responses at the 4 questions of the ANAES/SFEMC questionnaire. VAS score was high for patients satisfied with their treatment and with good treatment effectiveness. Two VAS thresholds were determined using receiver operating characteristic curves that allowed easy identification, with high sensitivity and specificity, of patients satisfied/dissatisfied with their current treatment and with good/poor treatment effectiveness. Based on EXPERT data, this instrument showed that only 16% of patients using triptans (597/3719) were dissatisfied and reported poor treatment effectiveness, whereas treatment was inadequate for 63% of those using aspirin or nonsteroidal anti‐inflammatory drugs (1882/2992), 74% of those using paracetamol or other analgesics (2229/2998), and 53% of those using ergotamine (253/474). Conclusions.— The new instrument should allow easy identification in general practice of the patients receiving an effective or ineffective acute treatment of migraine and thus facilitate the implementation of treatment guidelines for migraine.     

Hydrolysis of Clavulanate by Mycobacterium tuberculosis β-Lactamase BlaC Harboring a Canonical SDN Motif

Combinations of β-lactams with clavulanate are currently being investigated for tuberculosis treatment. Since Mycobacterium tuberculosis produces a broad spectrum β-lactamase, BlaC, the success of this approach could be compromised by the emergence of clavulanate-resistant variants, as observed for inhibitor-resistant TEM variants in enterobacteria. Previous analyses based on site-directed mutagenesis of BlaC have led to the conclusion that this risk was limited. Here, we used a different approach based on determination of the crystal structure of β-lactamase Bla_MAb of Mycobacterium abscessus, which efficiently hydrolyzes clavulanate. Comparison of Bla_MAb and BlaC allowed for structure-assisted site-directed mutagenesis of BlaC and identification of the G¹³²N substitution that was sufficient to switch the interaction of BlaC with clavulanate from irreversible inactivation to efficient hydrolysis. The substitution, which restored the canonical SDN motif (SDG→SDN), allowed for efficient hydrolysis of clavulanate, with a more than 10⁴-fold increase in k_cat (0.41 s⁻¹), without affecting the hydrolysis of other β-lactams. Mass spectrometry revealed that acylation of BlaC and of its G¹³²N variant by clavulanate follows similar paths, involving sequential formation of two acylenzymes. Decarboxylation of the first acylenzyme results in a stable secondary acylenzyme in BlaC, whereas hydrolysis occurs in the G¹³²N variant. The SDN/SDG polymorphism defines two mycobacterial lineages comprising rapidly and slowly growing species, respectively. Together, these results suggest that the efficacy of β-lactam–clavulanate combinations may be limited by the emergence of resistance. β-Lactams active without clavulanate, such as faropenem, should be prioritized for the development of new therapies.     

Invited review: Frontotemporal dementia caused by microtubule‐associated protein tau gene (MAPT) mutations: a chameleon for neuropathology and neuroimaging

Hereditary frontotemporal dementia associated with mutations in the microtubule‐associated protein tau gene (MAPT) is a protean disorder. Three neuropathologic subtypes can be recognized, based on the presence of inclusions made of tau isoforms with three and four repeats, predominantly three repeats and mostly four repeats. This is relevant for establishing a correlation between structural magnetic resonance imaging and positron emission tomography using tracers specific for aggregated tau. Longitudinal studies will be essential to determine the evolution of anatomical alterations from the asymptomatic stage to the various phases of disease following the onset of symptoms.     

Chlorotoxin: A Helpful Natural Scorpion Peptide to Diagnose Glioma and Fight Tumor Invasion

Chlorotoxin is a small 36 amino-acid peptide identified from the venom of the scorpion Leiurus quinquestriatus. Initially, chlorotoxin was used as a pharmacological tool to characterize chloride channels. While studying glioma-specific chloride currents, it was soon discovered that chlorotoxin possesses targeting properties towards cancer cells including glioma, melanoma, small cell lung carcinoma, neuroblastoma and medulloblastoma. The investigation of the mechanism of action of chlorotoxin has been challenging because its cell surface receptor target remains under questioning since two other receptors have been claimed besides chloride channels. Efforts on chlorotoxin-based applications focused on producing analogues helpful for glioma diagnosis, imaging and treatment. These efforts are welcome since gliomas are very aggressive brain cancers, close to impossible to cure with the current therapeutic arsenal. Among all the chlorotoxin-based strategies, the most promising one to enhance patient mean survival time appears to be the use of chlorotoxin as a targeting agent for the delivery of anti-tumor agents. Finally, the discovery of chlorotoxin has led to the screening of other scorpion venoms to identify chlorotoxin-like peptides. So far several new candidates have been identified. Only detailed research and clinical investigations will tell us if they share the same anti-tumor potential as chlorotoxin.