Adjunctive β2-agonists reverse neuromuscular involvement in murine Pompe disease.

Pompe disease has resisted enzyme replacement therapy with acid α-glucosidase (GAA), which has been attributed to inefficient cation-independent mannose-6-phosphate receptor (CI-MPR) mediated uptake. We evaluated β2-agonist drugs, which increased CI-MPR expression in GAA knockout (KO) mice. Clenbuterol along with a low-dose adeno-associated virus vector increased Rotarod latency by 75% at 4 wk, in comparison with vector alone (P<2×10(-5)). Glycogen content was lower in skeletal muscles, including soleus (P<0.01), extensor digitorum longus (EDL; P<0.001), and tibialis anterior (P<0.05) following combination therapy, in comparison with vector alone. Glycogen remained elevated in the muscles following clenbuterol alone, indicating an adjunctive effect with gene therapy. Elderly GAA-KO mice treated with combination therapy demonstrated 2-fold increased wirehang latency, in comparison with vector or clenbuterol alone (P<0.001). The glycogen content of skeletal muscle decreased following combination therapy in elderly mice (P<0.05). Finally, CI-MPR-KO/GAA-KO mice did not respond to combination therapy, indicating that clenbuterol's effect depended on CI-MPR expression. In summary, adjunctive β2-agonist treatment increased CI-MPR expression and enhanced efficacy from gene therapy in Pompe disease, which has implications for other lysosomal storage disorders that involve primarily the brain.

Alpha-2 agonists for sedation of mechanically ventilated adults in intensive care units: a systematic review

BACKGROUND: Care of critically ill patients in intensive care units (ICUs) often requires potentially invasive or uncomfortable procedures, such as mechanical ventilation (MV). Sedation can alleviate pain and discomfort, provide protection from stressful or harmful events, prevent anxiety and promote sleep. Various sedative agents are available for use in ICUs. In the UK, the most commonly used sedatives are propofol (Diprivan(®), AstraZeneca), benzodiazepines [e.g. midazolam (Hypnovel(®), Roche) and lorazepam (Ativan(®), Pfizer)] and alpha-2 adrenergic receptor agonists [e.g. dexmedetomidine (Dexdor(®), Orion Corporation) and clonidine (Catapres(®), Boehringer Ingelheim)]. Sedative agents vary in onset/duration of effects and in their side effects. The pattern of sedation of alpha-2 agonists is quite different from that of other sedatives in that patients can be aroused readily and their cognitive performance on psychometric tests is usually preserved. Moreover, respiratory depression is less frequent after alpha-2 agonists than after other sedative agents.

OBJECTIVES: To conduct a systematic review to evaluate the comparative effects of alpha-2 agonists (dexmedetomidine and clonidine) and propofol or benzodiazepines (midazolam and lorazepam) in mechanically ventilated adults admitted to ICUs.

DATA SOURCES: We searched major electronic databases (e.g. MEDLINE without revisions, MEDLINE In-Process & Other Non-Indexed Citations, EMBASE and Cochrane Central Register of Controlled Trials) from 1999 to 2014.

METHODS: Evidence was considered from randomised controlled trials (RCTs) comparing dexmedetomidine with clonidine or dexmedetomidine or clonidine with propofol or benzodiazepines such as midazolam, lorazepam and diazepam (Diazemuls(®), Actavis UK Limited). Primary outcomes included mortality, duration of MV, length of ICU stay and adverse events. One reviewer extracted data and assessed the risk of bias of included trials. A second reviewer cross-checked all the data extracted. Random-effects meta-analyses were used for data synthesis.

RESULTS: Eighteen RCTs (2489 adult patients) were included. One trial at unclear risk of bias compared dexmedetomidine with clonidine and found that target sedation was achieved in a higher number of patients treated with dexmedetomidine with lesser need for additional sedation. The remaining 17 trials compared dexmedetomidine with propofol or benzodiazepines (midazolam or lorazepam). Trials varied considerably with regard to clinical population, type of comparators, dose of sedative agents, outcome measures and length of follow-up. Overall, risk of bias was generally high or unclear. In particular, few trials blinded outcome assessors. Compared with propofol or benzodiazepines (midazolam or lorazepam), dexmedetomidine had no significant effects on mortality [risk ratio (RR) 1.03, 95% confidence interval (CI) 0.85 to 1.24, I (2) = 0%; p = 0.78]. Length of ICU stay (mean difference -1.26 days, 95% CI -1.96 to -0.55 days, I (2) = 31%; p = 0.0004) and time to extubation (mean difference -1.85 days, 95% CI -2.61 to -1.09 days, I (2) = 0%; p < 0.00001) were significantly shorter among patients who received dexmedetomidine. No difference in time to target sedation range was observed between sedative interventions (I (2) = 0%; p = 0.14). Dexmedetomidine was associated with a higher risk of bradycardia (RR 1.88, 95% CI 1.28 to 2.77, I (2) = 46%; p = 0.001).

LIMITATIONS: Trials varied considerably with regard to participants, type of comparators, dose of sedative agents, outcome measures and length of follow-up. Overall, risk of bias was generally high or unclear. In particular, few trials blinded assessors.

CONCLUSIONS: Evidence on the use of clonidine in ICUs is very limited. Dexmedetomidine may be effective in reducing ICU length of stay and time to extubation in critically ill ICU patients. Risk of bradycardia but not of overall mortality is higher among patients treated with dexmedetomidine. Well-designed RCTs are needed to assess the use of clonidine in ICUs and identify subgroups of patients that are more likely to benefit from the use of dexmedetomidine.

STUDY REGISTRATION: This study is registered as PROSPERO CRD42014014101.

FUNDING: The National Institute for Health Research Health Technology Assessment programme. The Health Services Research Unit is core funded by the Chief Scientist Office of the Scottish Government Health and Social Care Directorates.

beta(2)-Agonists and cAMP inhibit protein degradation in isolated chick (Gallus domesticus) skeletal muscle

1. The role of beta(2)-agonist and of cAMP in chick skeletal muscle proteolytic pathways and protein synthesis was investigated using an in vitro preparation that maintains tissue glycogen stores and metabolic activity for several hours.2. In extensor digitorum longus (EDL) muscle total proteolysis decreased by 15 to 20% in the presence of equimolar concentrations of epinephrine, clenbuterol, a selective beta(2)-agonist, or dibutyryl-cAMP. Rates of protein synthesis were not altered by clenbuterol or dibutyryl-cAMP.3. The decrease in the rate of total protein degradation induced by 10(-5) M clenbuterol was paralleled by a 44% reduction in Ca2+-dependent proteolysis, which was prevented by 10(-5) M ICI 118.551, a selective beta(2)-antagonist.4. No change was observed in the activity of the lysosomal, ATP-dependent, and ATP-independent proteolytic systems. Ca2+-dependent proteolytic activity was also reduced by 58% in the presence of 10(-4) M dibutyryl-cAMP or isobutylmethylxanthine.5. The data suggest that catecholamines exert an inhibitory control of Ca2+-dependent proteolysis in chick skeletal muscle, probably mediated by beta(2)-adrenoceptors, with the participation of a cAMP-dependent pathway.

Cannabinoid receptor 1 and 2 agonists increase lipid accumulation in hepatocytes

Cannabinoid receptors CB1 and CB2 are expressed in the liver, but their regulation in fatty hepatocytes is poorly documented. The aim of this study was to investigate the effects of selective CB1 or CB2 agonists on the expression of key regulators of lipid metabolism.

Comparison of the effects of the alpha-2 agonists detomidine, romifidine and xylazine on nociceptive withdrawal reflex and temporal summation in horses

OBJECTIVE: To evaluate and compare the antinociceptive effects of the three alpha-2 agonists, detomidine, romifidine and xylazine at doses considered equipotent for sedation, using the nociceptive withdrawal reflex (NWR) and temporal summation model in standing horses. STUDY DESIGN: Prospective, blinded, randomized cross-over study. ANIMALS: Ten healthy adult horses weighing 527-645 kg and aged 11-21 years old. METHODS: Electrical stimulation was applied to the digital nerves to evoke NWR and temporal summation in the left thoracic limb and pelvic limb of each horse. Electromyographic reflex activity was recorded from the common digital extensor and the cranial tibial muscles. After baseline measurements a single bolus dose of detomidine, 0.02 mg kg(-1), romifidine 0.08 mg kg(-1), or xylazine, 1 mg kg(-1), was administered intravenously (IV). Determinations of NWR and temporal summation thresholds were repeated at 10, 20, 30, 40, 60, 70, 90, 100, 120 and 130 minutes after test-drug administration alternating the thoracic limb and the pelvic limb. Depth of sedation was assessed before measurements at each time point. Behavioural reaction was observed and recorded following each stimulation. RESULTS: The administration of detomidine, romifidine and xylazine significantly increased the current intensities necessary to evoke NWR and temporal summation in thoracic limbs and pelvic limbs of all horses compared with baseline. Xylazine increased NWR thresholds over baseline values for 60 minutes, while detomidine and romifidine increased NWR thresholds over baseline for 100 and 120 minutes, respectively. Temporal summation thresholds were significantly increased for 40, 70 and 130 minutes after xylazine, detomidine and romifidine, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: Detomidine, romifidine and xylazine, administered IV at doses considered equipotent for sedation, significantly increased NWR and temporal summation thresholds, used as a measure of antinociceptive activity. The extent of maximal increase of NWR and temporal summation thresholds was comparable, while the duration of action was drug-specific.

Should inhaled anticholinergics be added to β2 agonists for treating acute childhood and adolescent asthma? A systematic review

Objectives To estimate the therapeutic and adverse effects of addition of inhaled anticholinergics to β2 agonists in acute asthma in children and adolescents.

Protein kinase D isoforms are expressed in rat and mouse primary sensory neurons and are activated by agonists of protease-activated receptor 2

Serine proteases generated during injury and inflammation cleave protease-activated receptor 2 (PAR(2)) on primary sensory neurons to induce neurogenic inflammation and hyperalgesia. Hyperalgesia requires sensitization of transient receptor potential vanilloid (TRPV) ion channels by mechanisms involving phospholipase C and protein kinase C (PKC). The protein kinase D (PKD) serine/threonine kinases are activated by diacylglycerol and PKCs and can phosphorylate TRPV1. Thus, PKDs may participate in novel signal transduction pathways triggered by serine proteases during inflammation and pain. However, it is not known whether PAR(2) activates PKD, and the expression of PKD isoforms by nociceptive neurons is poorly characterized. By using HEK293 cells transfected with PKDs, we found that PAR(2) stimulation promoted plasma membrane translocation and phosphorylation of PKD1, PKD2, and PKD3, indicating activation. This effect was partially dependent on PKCepsilon. By immunofluorescence and confocal microscopy, with antibodies against PKD1/PKD2 and PKD3 and neuronal markers, we found that PKDs were expressed in rat and mouse dorsal root ganglia (DRG) neurons, including nociceptive neurons that expressed TRPV1, PAR(2), and neuropeptides. PAR(2) agonist induced phosphorylation of PKD in cultured DRG neurons, indicating PKD activation. Intraplantar injection of PAR(2) agonist also caused phosphorylation of PKD in neurons of lumbar DRG, confirming activation in vivo. Thus, PKD1, PKD2, and PKD3 are expressed in primary sensory neurons that mediate neurogenic inflammation and pain transmission, and PAR(2) agonists activate PKDs in HEK293 cells and DRG neurons in culture and in intact animals. PKD may be a novel component of a signal transduction pathway for protease-induced activation of nociceptive neurons and an important new target for antiinflammatory and analgesic therapies.

Epidural anesthesia and postoperatory analgesia with alpha-2 adrenergic agonists and lidocaine for ovariohysterectomy in bitches

The aim of this study was to determine the viability and cardiorespiratory effects of the association of epidural alpha-2 adrenergic agonists and lidocaine for ovariohysterectomy (OH) in bitches. Forty-two bitches were spayed under epidural anesthesia with 2.5 mg/kg body weight (BW) of 1% lidocaine with adrenaline (CON) or in association with 0.25 mg/kg BW of xylazine (XYL), 10 mu g/kg BW of romifidine (ROM), 30 mu g/kg BW of detomidine (DET), 2 mu g/kg BW of dexmedetomidine (DEX), or 5 mu g/kg BW of clonidine (CLO). Heart rate (HR), respiratory rate (f(R)) and arterial pressures were monitored immediately before and every 10 min after the epidural procedure. Blood gas and pH analysis were done before, and at 30 and 60 min after the epidural procedure. Animals were submitted to isoflurane anesthesia if they presented a slightest sign of discomfort during the procedure. Time of sensory epidural block and postoperative analgesia were evaluated. All animals in CON and DEX, 5 animals in ROM and CLO, 4 animals in XYL, and 3 in DET required supplementary isoflurane. All groups, except CLO, showed a decrease in HR. There was an increase in arterial pressures in all groups. Postoperative analgesia lasted the longest in XYL. None of the protocols were totally efficient to perform the complete procedure of OH; however, xylazine provided longer postoperative analgesia than the others.

Exchange protein directly activated by cyclic AMP (EPAC) activation reverses neutrophil dysfunction induced by β2-agonists, corticosteroids, and critical illness

Background Neutrophils play a role in the pathogenesis of asthma, chronic obstructive pulmonary disease, and pulmonary infection. Impaired neutrophil phagocytosis predicts hospital-acquired infection. Despite this, remarkably few neutrophil-specific treatments exist. 

Objectives We sought to identify novel pathways for the restoration of effective neutrophil phagocytosis and to activate such pathways effectively in neutrophils from patients with impaired neutrophil phagocytosis. 

Methods Blood neutrophils were isolated from healthy volunteers and patients with impaired neutrophil function. In healthy neutrophils phagocytic impairment was induced experimentally by using β₂-agonists. Inhibitors and activators of cyclic AMP (cAMP)-dependent pathways were used to assess the influence on neutrophil phagocytosis in vitro. 

Results β₂-Agonists and corticosteroids inhibited neutrophil phagocytosis. Impairment of neutrophil phagocytosis by β₂-agonists was associated with significantly reduced RhoA activity. Inhibition of protein kinase A (PKA) restored phagocytosis and RhoA activity, suggesting that cAMP signals through PKA to drive phagocytic impairment. However, cAMP can signal through effectors other than PKA, such as exchange protein directly activated by cyclic AMP (EPAC). An EPAC-activating analog of cAMP (8CPT-2Me-cAMP) reversed neutrophil dysfunction induced by β₂-agonists or corticosteroids but did not increase RhoA activity. 8CPT-2Me-cAMP reversed phagocytic impairment induced by Rho kinase inhibition but was ineffective in the presence of Rap-1 GTPase inhibitors. 8CPT-2Me-cAMP restored function to neutrophils from patients with known acquired impairment of neutrophil phagocytosis. 

Conclusions EPAC activation consistently reverses clinical and experimental impairment of neutrophil phagocytosis. EPAC signals through Rap-1 and bypasses RhoA. EPAC activation represents a novel potential means by which to reverse impaired neutrophil phagocytosis.

Beta2-agonists use during pregnancy and the risk of congenital malformations

Selon les lignes directrices de traitement de l'asthme pendant la grossesse, les beta2-agonistes inhalés à courte durée d’action (SABA) sont les médicaments de choix pour tous les types d’asthme [intermittent, persistant, léger, modéré et sévère] comme médicaments de secours rapide et dans la gestion des exacerbations aiguës. D’autre part, les beta2-agonistes inhalés à longue durée d’action (LABA) sont utilisés pour les patients atteints d'asthme persistant, modéré à sévère, qui ne sont pas entièrement contrôlés par des corticostéroïdes inhalés seuls. Malgré que plusieurs études aient examinées l’association entre les LABA, les SABA et les malformations congénitales chez les nouveau-nés, les risques réels restent controversés en raison de résultats contradictoires et des difficultés inhérentes à la réalisation d'études épidémiologiques chez les femmes enceintes. L'objectif de cette étude était d'évaluer l'association entre l'exposition maternelle aux SABA et LABA pendant le premier trimestre de grossesse et le risque de malformations congénitales chez les nouveau-nés de femmes asthmatiques. Une cohorte de grossesses de femmes asthmatiques ayant accouchées entre le 1er janvier 1990 et le 31 décembre 2002 a été formée en croisant trois banques de données administratives de la province de Québec (Canada). Les issues principales de cette étude étaient les malformations congénitales majeures de touts types. Comme issues secondaires, nous avons considéré des malformations congénitales spécifiques. L'exposition principale était la prise de SABA et/ou de LABA au cours du premier trimestre de grossesse. L'exposition secondaire étudiée était le nombre moyen de doses de SABA par semaine au cours du premier trimestre. L'association entre les malformations congénitales et la prise de SABA et de LABA a été évaluée en utilisant des modèles d’équations généralisées (GEE) en ajustant pour plusieurs variables confondantes reliées à la grossesse, l’asthme de la mère et la santé de la mère et du foetus. Dans la cohorte formée de 13 117 grossesses de femmes asthmatiques, nous avons identifié 1 242 enfants avec une malformation congénitale (9,5%), dont 762 avaient une malformation majeure (5,8%). Cinquante-cinq pour cent des femmes ont utilisé des SABA et 1,3% ont utilisé des LABA pendant le premier trimestre. Les rapports de cotes ajustées (IC à 95%) pour une malformation congénitale associée à l'utilisation des SABA et des LABA étaient de 1,0 (0,9-1,2) et 1,3 (0,9-2,1), respectivement. Les résultats correspondants étaient de 0,9 (0,8-1,1) et 1,3 (0,8-2,4) pour les malformations majeures. Concernant le nombre moyen de doses de SABA par semaine, les rapports de cotes ajustées (IC à 95%) pour une malformation congénitale était de 1.1 (1.0-1.3), 1.1 (0.9-1.3), et 0.9 (0.7-1.1) pour les doses >0-3, >3-10, and >10 respectivement. Les résultats correspondants étaient de 1.0 (0.8-1.2), 0.8 (0.7-1.1), et 0.7 (0.5-1.0) pour les malformations majeures. D'autre part, des rapports de cotes (IC à 95%) statistiquement significatifs ont été observés pour les malformations cardiaques (2.4 (1.1-5.1)), les malformations d'organes génitaux (6.8 (2.6-18.1)), et d'autres malformations congénitales (3.4 (1.4 à 8.5)), en association avec les LABA pris pendant le premier trimestre. Notre étude procure des données rassurantes pour l’utilisation des SABA pendant la grossesse, ce qui est en accord avec les lignes directrices de traitement de l’asthme. Toutefois, d'autres études sont nécessaires avant de pouvoir se prononcer sur l’innocuité des LABA pendant la grossesse.

Mechanisms of protease-activated receptor 2-evoked hyperexcitability of nociceptive neurons innervating the mouse colon

Agonists of protease-activated receptor 2 (PAR(2)) evoke hyperexcitability of dorsal root ganglia (DRG) neurons by unknown mechanisms. We examined the cellular mechanisms underlying PAR(2)-evoked hyperexcitability of mouse colonic DRG neurons to determine their potential role in pain syndromes such as visceral hyperalgesia. Colonic DRG neurons were identified by injecting Fast Blue and DiI retrograde tracers into the mouse colon. Using immunofluorescence, we found that DiI-labelled neurons contained PAR(2) immunoreactivity, confirming the presence of receptors on colonic neurons. Whole-cell current-clamp recordings of acutely dissociated neurons demonstrated that PAR(2) activation with a brief application (3 min) of PAR(2) agonists, SLIGRL-NH(2) and trypsin, evoked sustained depolarizations (up to 60 min) which were associated with increased input resistance and a marked reduction in rheobase (50% at 30 min). In voltage clamp, SLIGRL-NH(2) markedly suppressed delayed rectifier I(K) currents (55% at 10 min), but had no effect on the transient I(A) current or TTX-resistant Na(+) currents. In whole-cell current-clamp recordings, the sustained excitability evoked by PAR(2) activation was blocked by the PKC inhibitor, calphostin, and the ERK(1/2) inhibitor PD98059. Studies of ERK(1/2) phosphorylation using confocal microscopy demonstrated that SLIGRL-NH(2) increased levels of immunoreactive pERK(1/2) in DRG neurons, particularly in proximity to the plasma membrane. Thus, activation of PAR(2) receptors on colonic nociceptive neurons causes sustained hyperexcitability that is related, at least in part, to suppression of delayed rectifier I(K) currents. Both PKC and ERK(1/2) mediate the PAR(2)-induced hyperexcitability. These studies describe a novel mechanism of sensitization of colonic nociceptive neurons that may be implicated in conditions of visceral hyperalgesia such as irritable bowel syndrome.

Presence and bronchomotor activity of protease-activated receptor-2 in guinea pig airways

The protease activated receptor-2 (PAR-2) belongs to a family of G-protein-coupled receptors that are activated by proteolysis. Trypsin cleaves PAR-2, exposing an N-terminal tethered ligand (SLIGRL) that activates the receptor. Messenger RNA (mRNA) for PAR-2 was found in guinea pig airway tissue by reverse transcription-polymerase chain reaction, and PAR-2 was found by immunohistochemistry in airway epithelial and smooth-muscle cells. In anesthetized guinea pigs, trypsin and SLIGRL-NH(2) (given intratracheally or intravenously) caused a bronchoconstriction that was inhibited by the combination of tachykinin-NK(1) and -NK(2) receptor antagonists and was potentiated by inhibition of nitric oxide synthase (NOS). Trypsin and SLIGRL-NH(2) relaxed isolated trachea and main bronchi, and contracted intrapulmonary bronchi. Relaxation of main bronchi was abolished or reversed to contraction by removal of epithelium, administration of indomethacin, and NOS inhibition. PAR-1, PAR-3, and PAR-4 were not involved in the bronchomotor action of either trypsin or SLIGRL-NH(2), because ligands of these receptors were inactive either in vitro or in vivo, and because thrombin (a PAR-1 and PAR-3 agonist) did not show cross-desensitization with PAR-2 agonists in vivo. Thus, we have localized PAR-2 to the guinea-pig airways, and have shown that activation of PAR-2 causes multiple motor effects in these airways, including in vivo bronchoconstriction, which is in part mediated by a neural mechanism.

PAR(2) and Temporomandibular Joint Inflammation in the Rat

The proteinase-activated receptor 2 (PAR(2)) is a putative therapeutic target for arthritis. We hypothesized that the early pro-inflammatory effects secondary to its activation in the temporomandibular joint (TMJ) are mediated by neurogenic mechanisms. Immunofluorescence analysis revealed a high degree of neurons expressing PAR(2) in retrogradely labeled trigeminal ganglion neurons. Furthermore, PAR(2) immunoreactivity was observed in the lining layer of the TMJ, co-localizing with the neuronal marker PGP9.5 and substance-P-containing peripheral sensory nerve fibers. The intra-articular injection of PAR(2) agonists into the TMJ triggered a dose-dependent increase in plasma extravasation, neutrophil influx, and induction of mechanical allodynia. The pharmacological blockade of natural killer 1 (NK(1)) receptors abolished PAR(2)-induced plasma extravasation and inhibited neutrophil influx and mechanical allodynia. We conclude that PAR(2) activation is proinflammatory in the TMJ, through a neurogenic mechanism involving NK(1) receptors. This suggests that PAR(2) is an important component of innate neuro-immune response in the rat TMJ.

PAR(2) and Temporomandibular Joint Inflammation in the Rat

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)