Developmental and reproductive toxicity hazard characterization of 2-amino-2-methyl-1-propanol (AMP).

2-Amino-2-methyl-1-propanol (AMP™) is a widely used pH stabilizer in personal care products (PCPs); thus, the safety implications of dermal AMP exposure remain of interest. We have previously reported that exposure to AMP in PCPs when used as intended is not anticipated to result in an increased risk of hepatotoxicity (primarily steatosis and altered phospholipid homeostasis). The current study focuses on AMP in PCP's potential for developmental and reproductive toxicity (DART) in humans, based on data from animal studies. Animal studies suggest that exposure to AMP can result in post-implantation loss. However, such effects occur at maternally toxic doses, posing a challenge for determining appropriate hazard classifications in the context of relevant consumer use scenarios. Our assessment concluded that human exposure to AMP in PCPs is not anticipated to result in DART at non-maternally toxic doses. Further, mode of action (MOA) analysis elucidated the potential biological pathways underlying DART effects observed in high-dose animal studies, such that perturbation of uterine choline synthesis was the most well-supported MOA hypothesis. Downstream uterine effects might reflect choline-dependent changes in epigenetic control of pathways important for implantation maintenance and uterine cell energetics. Since AMP-induced post-implantation loss occurs at doses higher than pathology related to liver toxicity, maintaining AMP exposures from exceeding the onset dose for maternal liver effects will also be protective of DART effects. Furthermore, dermal exposure to AMP expected from the use of PCPs is highly unlikely to result in toxicologically significant systemic AMP concentrations; thus, DART is not anticipated.

Repurposing Nucleoside Analogs for Human Coronaviruses.

Coronavirus disease 2019 (COVID-19) is a serious illness caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 or CoV-2). Some reports claimed certain nucleoside analogs to be active against CoV-2 and thus needed confirmation. Here, we evaluated a panel of compounds and identified novel nucleoside analogs with antiviral activity against CoV-2 and HCoV-OC43 while ruling out others. Of significance, sofosbuvir demonstrated no antiviral effect against CoV-2, and its triphosphate did not inhibit CoV-2 RNA polymerase.

Retinoid isomerase inhibitors impair but do not block mammalian cone photoreceptor function.

Visual function in vertebrates critically depends on the continuous regeneration of visual pigments in rod and cone photoreceptors. RPE65 is a well-established retinoid isomerase in the pigment epithelium that regenerates rhodopsin during the rod visual cycle; however, its contribution to the regeneration of cone pigments remains obscure. In this study, we use potent and selective RPE65 inhibitors in rod- and cone-dominant animal models to discern the role of this enzyme in cone-mediated vision. We confirm that retinylamine and emixustat-family compounds selectively inhibit RPE65 over DES1, the putative retinoid isomerase of the intraretinal visual cycle. In vivo and ex vivo electroretinography experiments in Gnat1-/- mice demonstrate that acute administration of RPE65 inhibitors after a bleach suppresses the late, slow phase of cone dark adaptation without affecting the initial rapid portion, which reflects intraretinal visual cycle function. Acute administration of these compounds does not affect the light sensitivity of cone photoreceptors in mice during extended exposure to background light, but does slow all phases of subsequent dark recovery. We also show that cone function is only partially suppressed in cone-dominant ground squirrels and wild-type mice by multiday administration of an RPE65 inhibitor despite profound blockade of RPE65 activity. Complementary experiments in these animal models using the DES1 inhibitor fenretinide show more modest effects on cone recovery. Collectively, these studies demonstrate a role for continuous RPE65 activity in mammalian cone pigment regeneration and provide further evidence for RPE65-independent regeneration mechanisms.

Novel hybrids of natural ß-elemene bearing isopropanolamine moieties: Synthesis, enhanced anticancer profile, and improved aqueous solubility.

A series of novel ß-elemene isopropanolamine derivatives were synthesized and evaluated for their antitumor activity. The results indicated that all of the compounds showed stronger antiproliferative activities than ß-elemene as well as improved aqueous solubility. In particular dimer 6q showed the strongest cytotoxicity against four tumor cell lines (SGC-7901, HeLa, U87 and A549) with IC50 values ranging from 4.37 to 10.20µM. Moreover, combination of 6q with cisplatin exhibited a synergistic effect on these cell lines with IC50 values ranging from 1.21 to 2.94µM, and reversed the resistance of A549/DPP cells with an IC50 value of 2.52µM. The mechanism study revealed that 6q caused cell cycle arrest at the G2 phase and induced apoptosis of SGC-7901 cells through a mitochondrial-dependent apoptotic pathway. Further in vivo study in H22 liver cancer xenograft mouse model validated the antitumor activity of 6q with a tumor inhibitory ratio (TIR) of 60.3%, which was higher than that of ß-elemene (TIR, 49.1%) at a dose of 60mg/kg. Altogether, the potent antitumor activity of 6qin vitro and in vivo warranted further preclinical investigation for potential anticancer chemotherapy.

CNS ß3-adrenergic receptor activation regulates feeding behavior, white fat browning, and body weight.

Pharmacological ß3-adrenergic receptor (ß3AR) activation leads to increased mitochondrial biogenesis and activity in white adipose tissue (WAT), a process commonly referred to as "browning", and transiently increased insulin release. These effects are associated with improved metabolic function and weight loss. It is assumed that this impact of ß3AR agonists is mediated solely through activation of ß3ARs in adipose tissue. However, ß3ARs are also found in the brain, in areas such as the brain stem and the hypothalamus, which provide multisynaptic innervation to brown and white adipose depots. Thus, contrary to the current adipocentric view, the central nervous system (CNS) may also have the ability to regulate energy balance and metabolism through actions on central ß3ARs. Therefore, this study aimed to elucidate whether CNS ß3ARs can regulate browning of WAT and other aspects of metabolic regulation, such as food intake control and insulin release. We found that acute central injection of ß3AR agonist potently reduced food intake, body weight, and increased hypothalamic neuronal activity in rats. Acute central ß3AR stimulation was also accompanied by a transient increase in circulating insulin levels. Moreover, subchronic central ß3AR agonist treatment led to a browning response in both inguinal (IWAT) and gonadal WAT (GWAT), along with reduced GWAT and increased BAT mass. In high-fat, high-sugar-fed rats, subchronic central ß3AR stimulation reduced body weight, chow, lard, and sucrose water intake, in addition to increasing browning of IWAT and GWAT. Collectively, our results identify the brain as a new site of action for the anorexic and browning impact of ß3AR activation.

Carvedilol-Enriched Cold Oxygenated Blood Cardioplegia Improves Left Ventricular Diastolic Function After Weaning From Cardiopulmonary Bypass.

OBJECTIVES: To investigate whether adding carvedilol, a nonselective ß- and selective α1-receptor blocking agent with antioxidant properties, to oxygenated blood cardioplegia improves myocardial function after weaning from bypass. DESIGN: A randomized controlled study. SETTING: A university laboratory. PARTICIPANTS: Twenty anesthetized pigs, Norwegian Landrace. INTERVENTIONS: On cardiopulmonary bypass, cardiac arrest was induced with cold (12°C), oxygenated blood cardioplegia, enriched with carvedilol or vehicle, and repeated every 20 minutes. After 100 minutes, the heart was reperfused and weaned. MEASUREMENTS AND MAIN RESULTS: Left ventricular function was evaluated with pressure-volume loops, local myocardial systolic strain, and strain rate from Speckle tracking analysis and multilayer short-axis tissue Doppler Imaging. In the carvedilol group, the load-independent logarithmic end-diastolic pressure volume relationship, ß, decreased from 1 to 3 hours of reperfusion and was low, 0.028±0.004 v 0.042±0.007 (p<0.05) in controls at 3 hours, demonstrating improved left ventricular compliance. The diastolic relaxation constant τ was decreased, 28.9±0.6 ms v 34.6±1.3 ms (pg<0.035), and dP/dtmin was more negative,-1,462±145 mmHg/s v-1,105±105 mmHg/s (pg = 0.024), for carvedilol v control group. The systolic variables, preload recruitable stroke work and end-systolic pressure-volume relationship, did not differ between groups, neither did left ventricular systolic strain and strain rate. Myocardial oxidative stress, measured as tissue levels of malondialdehyde, was reduced by carvedilol, 0.19±0.01 compared to 0.24±0.01 nmol/mg (p = 0.004) in controls. CONCLUSIONS: Carvedilol added to blood cardioplegia improved diastolic cardiac function and reduced oxidative stress during the first 3 hours after reperfusion in a porcine model, with 100 minutes of cardioplegic arrest.

ß-arrestin-biased signaling through the ß2-adrenergic receptor promotes cardiomyocyte contraction.

ß-adrenergic receptors (ßARs) are critical regulators of acute cardiovascular physiology. In response to elevated catecholamine stimulation during development of congestive heart failure (CHF), chronic activation of Gs-dependent ß1AR and Gi-dependent ß2AR pathways leads to enhanced cardiomyocyte death, reduced ß1AR expression, and decreased inotropic reserve. ß-blockers act to block excessive catecholamine stimulation of ßARs to decrease cellular apoptotic signaling and normalize ß1AR expression and inotropy. Whereas these actions reduce cardiac remodeling and mortality outcomes, the effects are not sustained. Converse to G-protein-dependent signaling, ß-arrestin-dependent signaling promotes cardiomyocyte survival. Given that ß2AR expression is unaltered in CHF, a ß-arrestin-biased agonist that operates through the ß2AR represents a potentially useful therapeutic approach. Carvedilol, a currently prescribed nonselective ß-blocker, has been classified as a ß-arrestin-biased agonist that can inhibit basal signaling from ßARs and also stimulate cell survival signaling pathways. To understand the relative contribution of ß-arrestin bias to the efficacy of select ß-blockers, a specific ß-arrestin-biased pepducin for the ß2AR, intracellular loop (ICL)1-9, was used to decouple ß-arrestin-biased signaling from occupation of the orthosteric ligand-binding pocket. With similar efficacy to carvedilol, ICL1-9 was able to promote ß2AR phosphorylation, ß-arrestin recruitment, ß2AR internalization, and ß-arrestin-biased signaling. Interestingly, ICL1-9 was also able to induce ß2AR- and ß-arrestin-dependent and Ca(2+)-independent contractility in primary adult murine cardiomyocytes, whereas carvedilol had no efficacy. Thus, ICL1-9 is an effective tool to access a pharmacological profile stimulating cardioprotective signaling and inotropic effects through the ß2AR and serves as a model for the next generation of cardiovascular drug development.

Glucosylceramide synthase inhibitors D-PDMP and D-EtDO-P4 decrease the GM3 ganglioside level, differ in their effects on insulin receptor autophosphorylation but increase Akt1 kinase phosphorylation in human hepatoma HepG2 cells.

Gangliosides function as modulators of several cell growth related receptors. It was shown for caveolin-rich adipocytes, that GM3 ganglioside binds to insulin receptor (IR), dissociates its complex with caveolin, and thus lowers IR autophosphorylation following insulin treatment. We extended those studies into human hepatocyte-derived HepG2 cells, characterized by a high level of IR but low of caveolin. To lower the glycosphingolipid content, estimated by GM3 concentration, two glucosylceramide synthase inhibitors d-threo-1-pheny-2-decanoylamino-3-morpholino-1-propanol (d-PDMP) and d-threo-1-(3,4,-ethylenedioxy)phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (d-EtDO-P4) were used. d-PDMP at 40 µM or d-EtDO-P4 at 1 µM concentrations in culture medium decreased the GM3 content to 22.3% (17.8-26.1%) and 18.1% (13.7-24.4%), respectively, of the control value. The reduction of GM3 obtained with d-PDMP was accompanied by a 185.1% (153.5-423.8%) significant increase in the level of IR autophosphorylation following cell stimulation with 100 nM insulin. The effect of d-EtDO-P4 on IR autophosphorylation was smaller amounting to an increase by 134.8% (111.3-167.8%) of the control level and statistically non-significant. The effects of d-PDMP and d-EtDO-P4 could also be detected at the level of Akt1 kinase. In cells grown in the presence of d-PDMP the level of phosphorylated Akt1 was 286.0% (151.4%-621.1%) of that in the control. In this case the effect of d-EtDO-P4 was similar: 223.0% (181.4-315.4%) significant increase in phosphorylated Akt1. We assume that glycosphingolipid depletion in HepG2 cells may affect not only IR autophosphorylation but also, independently, the phosphorylation of Akt1, by modifying the membrane microenvironment of this kinase.

Passive Synaptic Normalization and Input Synchrony-Dependent Amplification of Cortical Feedback in Thalamocortical Neuron Dendrites.

Thalamocortical neurons have thousands of synaptic connections from layer VI corticothalamic neurons distributed across their dendritic trees. Although corticothalamic synapses provide significant excitatory input, it remains unknown how different spatial and temporal input patterns are integrated by thalamocortical neurons. Using dendritic recording, 2-photon glutamate uncaging, and computational modeling, we investigated how rat dorsal lateral geniculate nucleus thalamocortical neurons integrate excitatory corticothalamic feedback. We find that unitary corticothalamic inputs produce small somatic EPSPs whose amplitudes are passively normalized and virtually independent of the site of origin within the dendritic tree. Furthermore, uncaging of MNI glutamate reveals that thalamocortical neurons have postsynaptic voltage-dependent mechanisms that can amplify integrated corticothalamic input. These mechanisms, involving NMDA receptors and T-type Ca(2+)channels, require temporally synchronous synaptic activation but not spatially coincident input patterns. In hyperpolarized thalamocortical neurons, T-type Ca(2+)channels produce nonlinear amplification of temporally synchronous inputs, whereas asynchronous inputs are not amplified. At depolarized potentials, the input-output function for synchronous synaptic input is linear but shows enhanced gain due to activity-dependent recruitment of NMDA receptors. Computer simulations reveal that EPSP amplification by T-type Ca(2+)channels and NMDA receptors occurs when synaptic inputs are either clustered onto individual dendrites or when they are distributed throughout the dendritic tree. Consequently, postsynaptic EPSP amplification mechanisms limit the "modulatory" effects of corticothalamic synaptic inputs on thalamocortical neuron membrane potential and allow these synapses to act as synchrony-dependent "drivers" of thalamocortical neuron firing. These complex thalamocortical input-output transformations significantly increase the influence of corticothalamic feedback on sensory information transfer. SIGNIFICANCE STATEMENT: Neurons in first-order thalamic nuclei transmit sensory information from the periphery to the cortex. However, the numerically dominant synaptic input to thalamocortical neurons comes from the cortex, which provides a strong, activity-dependent modulatory feedback influence on information flow through the thalamus. Here, we reveal how individual quantal-sized corticothalamic EPSPs propagate within thalamocortical neuron dendrites and how different spatial and temporal input patterns are integrated by these cells. We find that thalamocortical neurons have voltage- and synchrony-dependent postsynaptic mechanisms, involving NMDA receptors and T-type Ca(2+)channels that allow nonlinear amplification of integrated corticothalamic EPSPs. These mechanisms significantly increase the responsiveness of thalamocortical neurons to cortical excitatory input and broaden the "modulatory" influence exerted by corticothalamic synapses.

Yogurt Containing the Probacteria Lactobacillus acidophilus Combined with Natural Antioxidants Mitigates Doxorubicin-Induced Cardiomyopathy in Rats.

Probiotics and antioxidants have a definite improving effect in cardiovascular diseases. This study aims at mitigating doxorubicin toxicity on cardiac function through consuming a functional food. Five groups of adult male Sprague-Dawley rats were used along 22 weeks. Group I received 30 g/kg/day food enriched with yogurt, green tea extract, and carrots (80, 0.84, and 100 g/kg diet, respectively) from the first week, group II received carvedilol 30 mg/kg/day orally from week 17, group III received both carvedilol and tested food, and groups IV and V were +ve and -ve control groups, respectively. In week 17, cardiomyopathy was induced by i.p. injection of 2.5 mg/kg doxorubicin every 48 h for 2 weeks. Histopathological and electrophysiological examinations and biochemical analysis were done. Lipid peroxidation, antioxidant effect, heart failure compensatory mediators, and proinflammatory cytokines were assessed. Tested food normalized time between the start of Q wave and the end of T wave on electrocardiogram (QT interval) and heart rate compared to the doxorubicin group (P<.05). It also improved hypertrophy indicated by a significant (P<.05) decrease in heart/body weight ratio, angiotensin-II (Ang-II), and atrial natriuretic peptide (ANP) serum levels. Histopathological examination of cardiac sections from the tested food group revealed less marked vacuolization and low perivascular fibrosis percentage (0.7803 ± 0.04). A significant (P<.001) decrease in serum creatine kinase-membrane bound, lactate dehydrogenase, triglycerides, cholesterol, low-density lipoprotein cholesterol, and tissue malondialdehyde (MDA) levels was observed in addition to an increase in serum Na(+)/K(+) ATP1A1 and cardiac reduced glutathione (GSH) levels. Tested food also lowered the inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) serum levels significantly (P<.01). Probiotic food containing Lactobacillus acidophilus, green tea, and carrots can improve membrane integrity and cardiac contractility in doxorubicin-induced cardiomyopathy by decreasing TNF-α, IL-6, MDA, increasing GSH, and modulating compensatory mediators such as Ang-II and ANP.

Local airway anesthesia attenuates hemodynamic responses to intubation and extubation in hypertensive surgical patients.

BACKGROUND: The aim of this study was to evaluate the effects of topical ropivacaine anesthesia on hemodynamic responses during intubation and extubation of hypertensive patients. MATERIAL AND METHODS: One hundred fifty patients with hypertension ASA II-III were scheduled for noncardiac operations. Patients were divided into 3 groups: a control group receiving 5 ml saline, and 2 groups receiving topical anesthesia with 100 mg lidocaine or 37.5 mg ropivacaine. Hemodynamic responses, including blood pressure and heart rate (HR), were recorded at baseline (T0), before intubation (T1), during tracheal intubation (T2), 2 min after intubation (T3), upon eye opening on verbal commands (T4), during tracheal extubation (T5), and 2 min after extubation (T6). Patients were injected with urapidil 5 mg during intubation and extubation if their systolic blood pressure (SBP) was ≥160 mmHg or diastolic blood pressure (DBP) was ³90 mmHg, and esmolol 10 mg when HR was ≥90 bpm. RESULTS: During extubation, the total dosages of urapidil and esmolol were significantly higher in the saline than in the lidocaine or ropivacaine groups, and were significantly lower in the ropivacaine than in the lidocaine group. At T2, SBP, SBP, MAP, and HR were lower in the lidocaine and ropivacaine groups than in the saline group, but the differences were not significant. From T4 to T6, SBP, DBP, MAP, and HR were significantly lower in the ropivacaine group than in the other 2 groups (P<0.05 each). CONCLUSIONS: Topical lidocaine and ropivacaine anesthesia can effectively reduce hemodynamic responses during intubation, with ropivacaine better at inhibiting hemodynamic changes at emergence in hypertensive patients.

Blood lipids affect rat islet blood flow regulation through ß3-adrenoceptors.

Pancreatic islet blood perfusion varies according to the needs for insulin secretion. We examined the effects of blood lipids on pancreatic islet blood flow in anesthetized rats. Acute administration of Intralipid to anesthetized rats increased both triglycerides and free fatty acids, associated with a simultaneous increase in total pancreatic and islet blood flow. A preceding abdominal vagotomy markedly potentiated this and led acutely to a 10-fold increase in islet blood flow associated with a similar increase in serum insulin concentrations. The islet blood flow and serum insulin response could be largely prevented by pretreatment with propranolol and the selective ß3-adrenergic inhibitor SR-59230A. The nitric oxide synthase inhibitor N(G)-nitro-l-arginine methyl ester prevented the blood flow increase but was less effective in reducing serum insulin. Increased islet blood flow after Intralipid administration was also seen in islet and whole pancreas transplanted rats, i.e., models with different degrees of chronic islet denervation, but the effect was not as pronounced. In isolated vascularly perfused single islets Intralipid dilated islet arterioles, but this was not affected by SR-59230A. Both the sympathetic and parasympathetic nervous system are important for the coordination of islet blood flow and insulin release during hyperlipidemia, with a previously unknown role for ß3-adrenoceptors.

Carvedilol protects against iron-induced microparticle generation and apoptosis of endothelial cells.

BACKGROUND: Increased circulating endothelial microparticles (EMPs) have been shown to associate with endothelial dysfunction. We explored the effect of iron on EMP generation by human umbilical vein endothelial cells (HUVECs) and the potential protective effect of carvedilol. METHODS: FeCl 3 was added to HUVEC culture. Iron entry into cells was monitored using fluorescent microscopic imaging, while the quantity of EMPs that was released was determined by flow cytometry. The apoptosis of HUVECs was assessed by annexin V/propidium iodide assay and caspase-3 expression. Membrane bleb formation was visualized using electron microscopy. Intracellular production of reactive oxygen species (ROS) was also monitored. The effects of beta-blockers, carvedilol and propranolol on these processes were determined by co-incubation in a dose-dependent manner. Iron entry into HUVECs was not blocked by either beta-blocker. Iron induced the generation of EMPs, the formation of membrane blebs, the apoptosis of HUVECs and the production of ROS, each in a dose-dependent manner. Carvedilol, but not propranolol, ameliorated all of these processes. RESULTS: Our result indicates that iron induces EMP generation and apoptosis of endothelial cells in association with increased oxidative stress. CONCLUSION: The protective effects of carvedilol, via its antioxidant effect, may have therapeutic potential in patients with iron overload.

The effects of carvedilol on ischemia-reperfusion injury in the rat testis

Objective: To analyze the oxidative damage and histopathological alterations caused by ischemia-reperfusion (I/R) injury and ameliorative effects of carvedilol (CVD) in the rat testis. Materials and Methods: Twenty-one male rats were randomized into 3 groups as follows: Group I (n = 7); control (sham) group, Group II (n = 7); I/R group, in which I/R injury was performed by torsing the left testis 720º clockwise for 2 hours and detorsing for 2 hours. Group III (n = 7); CVD treatment group; in addition to I/R process, one-dose of CVD was administered (2mg/kg, i.p) 30 min. before detorsion. Levels of antioxidant enzymes, superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) and levels of malondialdehyde (MDA) and protein carbonyl (PC) were determined in testicular tissues and serum of rats. Testicular tissues were also examined histopathologically and Johnsen scores were determined. Results: Activities of SOD and GSH-Px in serum and testicular tissues were increased by I/R, but administration of CVD decreased these levels (p < 0.001 and p = 0.001). Significantly increased MDA levels in serum and testicular tissues were decreased by CVD treatment (p < 0.001 and p = 0.001). Concerning PC levels in serum and testicular tissues, there was no statistically significant difference between the groups (p = 0.989 and p = 0.428). There was not a statistically significant difference in terms of mean Johnsen scores between the groups (p = 0.161). Conclusions: Administration of CVD decreased oxidative damage biochemically in the rat testis caused by I/R injury, but histopathologically no change was observed between all of the groups. .

The action of p-synephrine on hepatic carbohydrate metabolism and respiration occurs via both Ca(2+)-mobilization and cAMP production.

Citrus aurantium extracts, which contain large amounts of p-synephrine, are widely used for weight loss purposes and as appetite suppressants. In the liver, C. aurantium (bitter orange) extracts affect hemodynamics, carbohydrate metabolism, and oxygen uptake. The purpose of the present work was to quantify the action of p-synephrine and also to obtain indications about its mechanism of action, a task that would be difficult to accomplish with C. aurantium extracts due to their rather complex composition. The experimental system was the isolated perfused rat liver. p-Synephrine significantly stimulated glycogenolysis, glycolysis, gluconeogenesis, and oxygen uptake. The compound also increased the portal perfusion pressure and the redox state of the cytosolic NAD(+)/NADH couple. A Ca(2+)-dependency for both the hemodynamic and the metabolic effects of p-synephrine was found. p-Synephrine stimulated both cAMP overflow and the initial Ca(2+) release from the cellular stores previously labeled with (45)Ca(2+). The metabolic and hemodynamic actions of p-synephrine were strongly inhibited by α-adrenergic antagonists and moderately affected by ß-adrenergic antagonists. The results allow to conclude that p-synephrine presents important metabolic and hemodynamic effects in the liver. These effects can be considered as both catabolic (glycogenolysis) and anabolic (gluconeogenesis), they are mediated by both α- and ß-adrenergic signaling, require the simultaneous participation of both Ca(2+) and cAMP, and could be contributing to the overall stimulation of metabolism that usually occurs during weight loss periods.

Activity-dependent hyperpolarization of EGABA is absent in cutaneous DRG neurons from inflamed rats.

A shift in GABA(A) signaling from inhibition to excitation in primary afferent neurons appears to contribute to the inflammation-induced increase in afferent input to the CNS. An activity-dependent depolarization of the GABA(A) current equilibrium potential (E(GABA)) has been described in CNS neurons which drives a shift in GABA(A) signaling from inhibition to excitation. The purpose of the present study was to determine if such an activity-dependent depolarization of E(GABA) occurs in primary afferents and whether the depolarization is amplified with persistent inflammation. Acutely dissociated retrogradely labeled cutaneous dorsal root ganglion (DRG) neurons from naïve and inflamed rats were studied with gramicidin perforated patch recording. Rather than a depolarization, 200 action potentials delivered at 2 Hz resulted in a ∼10 mV hyperpolarization of E(GABA) in cutaneous neurons from naïve rats. No such hyperpolarization was observed in neurons from inflamed rats. The shift in E(GABA) was not blocked by 10 µM bumetanide. Furthermore, because activity-dependent hyperpolarization of E(GABA) was fully manifest in the absence of HCO3⁻ in the bath solution, this shift was not dependent on a change in HCO3⁻-Cl⁻ exchanger activity, despite evidence of HCO3⁻-Cl⁻ exchangers in DRG neurons that may contribute to the establishment of E(GABA) in the presence of HCO3⁻. While the mechanism underlying the activity-dependent hyperpolarization of E(GABA) has yet to be identified, because this mechanism appears to function as a form of feedback inhibition, facilitating GABA-mediated inhibition of afferent activity, it may serve as a novel target for the treatment of inflammatory pain.

Esmolol activates endogenous neurokinin activity inhibiting infarction-induced arrhythmias in rats: novel mechanisms of anti-arrhythmia.

Endogenous neurokinin and adrenergic mechanisms might co-participate in the pathology of acute myocardial infarction (MI). This study sought to investigate the role of endogenous neurokinin and its relationship with ß1-adrenergic mechanism in the infarction induced arrhythmias. In 60min of MI in rats, the contents of substance P (SP), a native agonist of neurokinin 1 receptor (NK1-R), norepinephrine (NE), NK1-R and ß1-adrenergic receptor in the myocardium at risk of ischemia were examined and the ventricular arrhythmias were analyzed. The effects of pretreatment with D-SP (152ng/kg), a specific antagonist of NK1-R, esmolol (10mg/kg), a specific blocker of ß1-adrenergic receptor, and a combination of the two blockers were studied. The results showed that the overlaps of up-regulation of NE, SP and the increase of ventricular arrhythmias were observed. D-SP exacerbated the episodes and duration of VT & VF by 54% and 104%, respectively (all P<0.05). Esmolol inhibited the morbidity rate, the episodes and the duration of VT & VF by 66%, 92% and 95%, respectively. Surprisingly, esmolol significantly attenuated the arrhythmogenic effect of D-SP throughout the MI, beyond the time span of esmolol action, during which a significant up-regulation of the NK1-R (by 19%, P<0.05) was detected. In conclusion, the findings of this study may indicate an anti-arrhythmic effect of endogenous neurokinin mechanism, through the activation of which, via up-regulation of NK1 receptor, esmolol may exert its anti-arrhythmic action at the early time of acute myocardial infarction.

Cationic ceramides and analogues, LCL30 and LCL85, as adjuvants to photodynamic therapy of tumors.

Photodynamic therapy (PDT) is known to alter the expression of various genes in treated cells. This prompted us to examine the activity of genes encoding two important enzymes in sphingolipid (SL) metabolism, dihydroceramide desaturase (DES) and sphingosine kinase (SPHK), in mouse SCCVII tumor cells treated by PDT using either the porphyrin-based photosensitizer Photofrin or silicon phthalocyanine Pc4. The results revealed that PDT induced an upregulation in the expression of two major isoforms of both genes (DES1 and DES2 as well as SPHK1 and SPHK2). While the changes were generally moderate (2-3-fold gains), the increase in DES2 expression was more pronounced and it was much greater with Photofrin-PDT than with Pc4-PDT (over 23-fold vs. less than 5-fold). Combining either Photofrin-PDT or Pc4-PDT with the cationic C16-ceramide LCL30 (20mg/kg i.p.) for treatment of subcutaneously growing SCCVII tumors rendered important differences in the therapy outcome. Photofrin-PDT, used at a dose that attained good initial response but no tumor cures, produced 50% cures when combined with a single LCL30 treatment. In contrast, the same LCL30 treatment combined with Pc4-PDT had no significant effect on tumor response. The optimal timing of LCL30 injection was immediately after Photofrin-PDT. The therapeutic benefit was lost when LCL30 was given in two 20mg/kg injections encompassing intervals before and after PDT. LCL85, the cationic B13 ceramide analogue and SL-modulating agent, also increased cure rates of Photofrin-PDT treated tumors, but the therapeutic benefit was less pronounced than with LCL30. These results with LCL30 and LCL85, and our previous findings for LCL29 (another SL analogue), assert the potential of SLs for use as adjuvants to augment the efficacy of PDT-mediated tumor destruction.

Integrative effect of carvedilol and aerobic exercise training therapies on improving cardiac contractility and remodeling in heart failure mice.

The use of ß-blockers is mandatory for counteracting heart failure (HF)-induced chronic sympathetic hyperactivity, cardiac dysfunction and remodeling. Importantly, aerobic exercise training, an efficient nonpharmacological therapy to HF, also counteracts sympathetic hyperactivity in HF and improves exercise tolerance and cardiac contractility; the latter associated with changes in cardiac Ca(2+) handling. This study was undertaken to test whether combined ß-blocker and aerobic exercise training would integrate the beneficial effects of isolated therapies on cardiac structure, contractility and cardiomyocyte Ca(2+) handling in a genetic model of sympathetic hyperactivity-induced HF (α2A/α2C- adrenergic receptor knockout mice, KO). We used a cohort of 5-7 mo male wild-type (WT) and congenic mice (KO) with C57Bl6/J genetic background randomly assigned into 5 groups: control (WT), saline-treated KO (KOS), exercise trained KO (KOT), carvedilol-treated KO (KOC) and, combined carvedilol-treated and exercise-trained KO (KOCT). Isolated and combined therapies reduced mortality compared with KOS mice. Both KOT and KOCT groups had increased exercise tolerance, while groups receiving carvedilol had increased left ventricular fractional shortening and reduced cardiac collagen volume fraction compared with KOS group. Cellular data confirmed that cardiomyocytes from KOS mice displayed abnormal Ca(2+) handling. KOT group had increased intracellular peak of Ca(2+) transient and reduced diastolic Ca(2+) decay compared with KOS group, while KOC had increased Ca(2+) decay compared with KOS group. Notably, combined therapies re-established cardiomyocyte Ca(2+) transient paralleled by increased SERCA2 expression and SERCA2:PLN ratio toward WT levels. Aerobic exercise trained increased the phosphorylation of PLN at Ser(16) and Thr(17) residues in both KOT and KOCT groups, but carvedilol treatment reduced lipid peroxidation in KOC and KOCT groups compared with KOS group. The present findings provide evidence that the combination of carvedilol and aerobic exercise training therapies lead to a better integrative outcome than carvedilol or exercise training used in isolation.

Experimental model of focal atrial tachycardia: clinical correlates.

BACKGROUND: The mechanisms underlying focal atrial tachycardia (AT) are unclear. METHODS: In 14 pentobarbital anesthetized dogs, a right thoracotomy allowed electrical stimulation (ES) of the anterior right ganglionated plexi (ARGP). After ES was applied to the ARGP at baseline, atropine, 1 mg/cc, was injected into the ARGP and repeat stimulation applied. After a left thoracotomy (n = 8), a similar procedure was followed by atropine injected into the superior left (SL) GP. RESULTS: ES (0.6-3.2 V) applied to the ARGP and SLGP caused an average reduction in sinus rate from 151 ± 14/min to 60 ± 11/min. At ≥4.5 V atrial fibrillation (AF) was induced (duration 48 ± 14 seconds). After injection of atropine into the ARGP or SLGP, ES applied to these GP induced no slowing of the sinus rate. Runs of AT were induced at an average voltage of 10 ± 2 V in 14 experiments (duration ≥4 minutes). AT was localized by ice mapping or by 3D noncontact mapping to the crista terminalis (n = 6), AV junction (n = 2) or a focal site at the left superior pulmonary vein (6). In AT lasting <4 minutes (n = 2), epinephrine injected into the GP significantly increased the AT duration. In 4/4 experiments, sustained AT could be terminated by intravenous esmolol. CONCLUSIONS: Atropine injected into the ARGP or SLGP promotes ES-induced AT whose duration is increased by adrenergic agonists and terminated by beta blockade. Presumably cholinergic blockade and accentuated release of adrenergic neurotransmitters provide the AT mechanism. The induced AT was found to be localized at sites similar to those reported clinically.