The role of catecholamines on intercellular coupling,myocardial cell synchronization and self ventricular defibrillation

Ventricular fibrillation (VF) is one of the most life threatening events. Although in humans VF is generally sustained (SVF) requiring artificial defibrillation, in various mammals and in some cases in humans VF terminates by itself, reverting spontaneously into sinus rhythm. Since VF is one of the main causes of sudden death, one of the important clinical problems today is if and how we can transform the fatal SVF into a self limited transient one (TVF).From electrophysiological studies carried out on anaesthetized open chest animals, we have found that TVF requires a high degree of intercellular coupling and synchronization.Cardiac myocytes are electrically coupled with adjacent cells. The intercellular coupling is a focus of low electrical resistance which allows rapid transmission of electrical impulses between cells. Any decrease in intercellular coupling decreases the ability of the heart for self defibrillation. The cell-to-cell coupling decreases with age, ischemia, VF and variations in physiological conditions probably due to an increase in intercellular resistance (Ri), widening in the internexal gaps, decrease in electrotonic space constant () etc. All of these factors are known to be affected by intracellular concentration of free Ca⁺⁺ ([Ca⁺⁺]).On the basis of studies carried out on various mammals at different ages, we hypothesized that the ability of the heart to defibrillate depends on the cardiac catecholamine level [CA], during VF. This hypothesis is supported by the facts, known from the literature, that increase in [CA] decreases intracellular free Ca⁺⁺ concentration, decreases Ri and increases . By these effects, increase in [CA] enhances intercellular coupling and intercellular synchronization, and thereby, according to our hypothesis, leads to spontaneous ventricular defibrillation — TVF.During VF the sympathetic activity is enhanced but in some cases the [CA] does not reach the level needed for TVF. In order to help the heart in its effort to elevate the [CA] during VF, we proposed to treat these cases with drugs which inhibit the reuptake of [CA]. The facts that administration of [CA] reuptake inhibitors, before the induction of VF, and/or intracoronary infusion of adrenaline, during VF, transforms SVF into TVF, emphasized the validity of our hypothesis.     

Presynaptic regulation of dopaminergic neurotransmission

The development of electrochemical recordings with small carbon-fiber electrodes has significantly advanced the understanding of the regulation of catecholamine transmission in various brain areas. Recordings in vivo or in slice preparations monitor diffusion of catecholamine following stimulated synaptic release into the surrounding tissue. This synaptic 'overflow' is defined by the amount of release, by the activity of reuptake, and by the diffusion parameters in brain tissue. Such studies have elucidated the complex regulation of catecholamine release and uptake, and how psychostimulants and anti-psychotic drugs interfere with it. Moreover, recordings with carbon-fiber electrodes from cultured neurons have provided analysis of catecholamine release and its plasticity at the quantal level.     

High affinity dopamine reuptake inhibitors as potential cocaine antagonists: a strategy for drug development

The addictive and euphorogenic effects of cocaine are thought to result primarily from inhibition of dopamine reuptake. Although the potency of cocaine-like drugs as inhibitors of DA reuptake is highly correlated with their potency as reinforcers in animals, several potent DA reuptake blockers (bupropion, nomifensine, benztropine, and mazindol) have not been reported to produce addiction or euphoria in humans. Based on these observations in humans, DA reuptake inhibitors are classified into two groups; type 1 blockers, which produce addiction and euphoria, and type 2 blockers, which do not. Given that type 1 and type 2 blockers act at the same site (the DA transporter), the author suggests that type 2 agents may antagonize the effects of cocaine, and might prove useful in the treatment of cocaine addiction.     

Reversal of the Progression of Fatal Coronavirus Infection in Cats by a Broad-Spectrum Coronavirus Protease Inhibitor

Coronaviruses infect animals and humans causing a wide range of diseases. The diversity of coronaviruses in many mammalian species is contributed by relatively high mutation and recombination rates during replication. This dynamic nature of coronaviruses may facilitate cross-species transmission and shifts in tissue or cell tropism in a host, resulting in substantial change in virulence. Feline enteric coronavirus (FECV) causes inapparent or mild enteritis in cats, but a highly fatal disease, called feline infectious peritonitis (FIP), can arise through mutation of FECV to FIP virus (FIPV). The pathogenesis of FIP is intimately associated with immune responses and involves depletion of T cells, features shared by some other coronaviruses like Severe Acute Respiratory Syndrome Coronavirus. The increasing risks of highly virulent coronavirus infections in humans or animals call for effective antiviral drugs, but no such measures are yet available. Previously, we have reported the inhibitors that target 3C-like protease (3CLpro) with broad-spectrum activity against important human and animal coronaviruses. Here, we evaluated the therapeutic efficacy of our 3CLpro inhibitor in laboratory cats with FIP. Experimental FIP is 100% fatal once certain clinical and laboratory signs become apparent. We found that antiviral treatment led to full recovery of cats when treatment was started at a stage of disease that would be otherwise fatal if left untreated. Antiviral treatment was associated with a rapid improvement in fever, ascites, lymphopenia and gross signs of illness and cats returned to normal health within 20 days or less of treatment. Significant reduction in viral titers was also observed in cats. These results indicate that continuous virus replication is required for progression of immune-mediated inflammatory disease of FIP. These findings may provide important insights into devising therapeutic strategies and selection of antiviral compounds for further development for important coronaviruses in animals and humans.     

Effects of citalopram on cognitive performance in passive avoidance, elevated plus-maze and three-panel runway tasks in naïve rats

Recent studies have shown that learning and memory capacity is disturbed in depressive patients, and it is important to reveal the effects of antidepressant drugs on cognitive function in depressive patients with memory problems. Citalopram, a selective serotonin reuptake inhibitor (SSRI), is one of the most widely used drugs for the treatment of disorders related to serotonergic dysfunction like depression and anxiety. Contradictory findings exist regarding the effects of SSRIs on memory. The aim of this study is to investigate whether citalopram affects memory in various models of learning and memory tasks in rats. Citalopram (at 20 and 50 mg/kg) significantly shortened the retention latency in the passive avoidance test and prolonged the transfer latency on the second day at 10 and 50 mg/kg doses in the elevated plus-maze test. Citalopram also significantly increased the number of errors (at the 10 mg/kg dose) and prolonged the latency values compared to the control group in both reference and working memory trials in the three-panel runway test. Citalopram also impaired reference memory trials of animals at the 20 mg/kg dose. In conclusion, citalopram impaired cognitive performance in passive avoidance, elevated plus-maze and three-panel runway tasks in naive rats. These effects might be related to serotonergic and nitrergic mechanisms, which need to be investigated in further studies.     

THERPA: A small molecule database related to prion protein regulation and prion diseases progression

Prion diseases are fatal neurodegenerative disorders that affect humans and animals. Although various small molecules have been evaluated for application in the treatment of prion diseases, none have been shown to be efficacious. Expanding our knowledge of these molecules is important for understanding of the complex mechanisms of prion diseases. To improve access to the scattered information on small molecules related to prion diseases, we built a database of therapeutic molecules associated with prion diseases (THERPA, therpa.pythonanywhere.com). THERPA includes 119 small molecules and their 283 relationships with prion diseases. THERPA is an interactive visual database and useful for improving search efficiency which can help researchers identify intrinsic small molecules that can be used for developing therapeutics for prion diseases.     

Relation between brain 5-HIAA levels and the release of serotonin into brain synapses

Our findings in experiments using reserpine, an amine releaser, and fluoxetine, a serotonin uptake blocker, indicate that the reuptake of serotonin from brain synapses precedes its transformation to 5-hydroxyindoleacetic acid (5-HIAA). Male rats were injected with reserpine or fluoxetine alone, or with fluoxetine one hour before reserpine; control animals received diluents. Reserpine lowered brain serotonin and raised brain 5-HIAA levels. Fluoxetine alone did not change serotonin levels but lowered 5-HIAA. Fluoxetine completely antagonized the reserpine-induced increase in 5-HIAA, and significantly enhanced its depletion of serotonin. In order to determine whether the ability of fluoxetine to block the rise in 5-HIAA after reserpine resulted from its effect on serotonin reuptake or from suppression of impulse flow along serotoninergic neurons, we also examined the effects of the drugs on serotonin metabolism in distal portions of acutely transected neurons (which, presumably, were no longer able to conduct impulses). No differences were noted between the responses of intact and lesioned serotoninergic neurons, indicating that fluoxetine's blockade of the rise in brain 5-HIAA results from its effect on serotonin reuptake.     

WISE-2005: developing a non-invasive method to monitor cardiovascular deconditioning

We have determined the gain of the cardiopulmonary baroreflex (CPBR) by measuring the central venous pressure (CVP) by venous catheter and calculating the total peripheral resistance (TPR) from mean arterial pressure (MAP) by Finometer and cardiac output (QD) by Doppler ultrasound. We tested the hypothesis that the BeatScope software of the Finometer, which calculates cardiac stroke volume (SVF) and cardiac output (QF) from the arterial pulse wave, could provide data with which to estimate CVP and TPR. The estimate of QF was linearly related to QD with a correction factor. Further, we found linear relationships between CVP and SVF that allowed us to establish a prediction of CVP from the SVF as the new input to the CPBR. To test the ability of this method to monitor changes in CPBR we are testing the subjects of the WISE-2005 study before and after 50-days of bed rest. We conclude that the TPR can be assessed with the Finometer and without any invasive method to record the CVP.     

Extraneuronal Monoamine Transporter Mediates the Permissive Action of Cortisol in the Guinea Pig Trachea: Possible Involvement of Tracheal Chondrocytes

Cortisol, a member of glucocorticoids, could potentiate the action of catecholamine by a non-genomic mechanism. Although this permissive effect has been well appreciated in the anti-asthmatic medication, the underlying signaling pathway has remained mysterious. Here, we show that extraneuronal monoamine transporter (EMT), a membraneous reuptake transporter for circulating catecholamine clearance, is the direct target of cortisol in its permissive effect. We found that BSA-conjugated cortisol, which functions as a cortisol but cannot penetrate cell membrane, enhanced the spasmolytic effect of β-adrenoceptor agonist (isoprenaline) in histamine-sensitized tracheal spirals of guinea pigs, and pharmacological inhibition of EMT with famotidine was powerful enough to imitate the permissive action of cortisol. To our surprise, EMT protein expression was high in the chondrocytes of tracheal cartilage, but was undetectable in tracheal smooth muscle cells. The functionality of EMT was further confirmed with measurement of catecholamine uptake by tracheal chondrocytes. Moreover, cortisol-initiated membrane signaling could activate protein kinase C (PKC), which phosphorylates EMT and induces its internalization via a lipid raft-dependent pathway. Both of the mechanisms slow down the reuptake process by chondrocytes, leading to extracellular catecholamine accumulation and results in a more profound adrenergic signaling activation in tracheal smooth muscle cells. Thus, an EMT-centered pathway was proposed to explain the permissive action of cortisol. Collectively, our results highlight the role of EMT in the crosstalk between glucocorticoid and catecholamine. EMT may represent a promising target for adrenergic signaling modulation.     

Turning off neurotransmitters

The historic discovery that the catecholamine neurotransmitters of the sympathetic nervous system, norepinephrine and epinephrine, are inactivated through their reuptake by presynaptic nerve terminals provided new insights into neurotransmitter action and paved the way for the development of modern antidepressant drugs.     

Citrobacter freundii septicemia in a stranded newborn Cuvier's beaked whale (Ziphius cavirostris)

Citrobacter freundii, a gram-negative enterobacterium, may cause fatal septicemia in humans and animals. Its potential pathogenic role in cetaceans (bottlenose dolphins and beluga whales) has been hypothesized. Here we describe fatal C. freundii septicemia in a stranded newborn Cuvier's beaked whale (Ziphius cavirostris).     

Antagonism of the anticonvulsant action of phenytoin,phenobarbital and acetazolamide by 6-hydroxydopamine

The ability of phenytoin, phenobarbital and acetazolamide to prevent the tonic extensor component of the maximal electroshock seizure was evaluated 30–50 days after treatment with 6-hydroxydopamine (6-OHDA). In the case of all 3 drugs, protection of rats from tonic extension was markedly reduced in the catecholamine amine deficient animals. However, the 6-OHDA-induced antagonism of anticonvulsant action was in all cases surmountable by increasing the dose of the anticonvulsant. These findings suggest a nonspecific antagonism of anticonvulsant action in 6-OHDA treated rats probably resulting from the increase in seizure susceptibility associated with catecholamine depletion.     

The stromal‐vascular fraction of adipose tissue contributes to major differences between subcutaneous and visceral fat depots

Adipose tissue represents a complex tissue both in terms of its cellular composition, as it includes mature adipocytes and the various cell types comprising the stromal‐vascular fraction (SVF), and in relation to the distinct biochemical, morphological and functional characteristics according to its anatomical location. Herein, we have characterized the proteomic profile of both mature adipocyte and SVF from human visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) fat depots in order to unveil differences in the expression of proteins which may underlie the distinct association of VAT and SAT to several pathologies. Specifically, 24 proteins were observed to be differentially expressed between SAT SVF versus VAT SVF from lean individuals. Immunoblotting and RT‐PCR analysis confirmed the differential regulation of the nuclear envelope proteins lamin A/C, the membrane‐cytoskeletal linker ezrin and the enzyme involved in retinoic acid production, aldehyde dehydrogenase 1A2, in the two fat depots. In sum, the observation that proteins with important cell functions are differentially distributed between VAT and SAT and their characterization as components of SVF or mature adipocytes pave the way for future research on the molecular basis underlying diverse adipose tissue‐related pathologies such as metabolic syndrome or lipodystrophy.     

Effects of Handling or Immobilization on Plasma Levels of 3,4-Dihydroxyphenylalanine, Catecholamines, and Metabolites in Rats

In conscious animals, handling and immobilization increase plasma levels of the catecholamines norepinephrine (NE) and epinephrine (EPI). This study examined plasma concentrations of endogenous compounds related to catecholamine synthesis and metabolism during and after exposure to these stressors in conscious rats. Plasma levels of 3,4-dihydroxyphenylalanine (DOPA), NE, EPI, and dopamine (DA), the deaminated catechol metabolites 3,4-dihydroxyphenylglycol (DHPG), and 3,4-dihydroxyphenylacetic acid (DOPAC), and their O-methylated derivatives methoxyhydroxyphenylglycol (MHPG) and homovanillic acid (HVA) were measured using liquid chromatography with electrochemical detection at 1, 3, 5, 20, 60, and 120 min of immobilization. By 1 min of immobilization, plasma NE and EPI levels had already reached peak values, and plasma levels of DOPA, DHPG, DOPAC, and MHPG were increased significantly from baseline, whereas plasma DA and HVA levels were unchanged. During the remainder of the immobilization period, the increased levels of DOPA, NE, and EPI were maintained, whereas levels of the metabolites progressively increased. In animals immobilized briefly (5 min), elevated concentrations of the metabolites persisted after release from the restraint, whereas DOPA and catecholamine levels returned to baseline. Gentle handling for 1 min also significantly increased plasma levels of DOPA, NE, EPI, and the NE metabolites DHPG and MHPG, without increasing levels of DA or HVA. The results show that in conscious rats, immobilization or even gentle handling rapidly increases plasma levels of catecholamines, the catecholamine precursor DOPA, and metabolites of NE and DA, indicating rapid increases in the synthesis, release, reuptake, and metabolism of catecholamines.     

Reduction of infarct size by intravenous injection of uncultured adipose derived stromal cells in a rat model is dependent on the time point of application

Stem cell therapy is a promising tool to improve outcome after acute myocardial infarction (AMI), but needs to be optimized since results from clinical applications remain ambiguous. A potent source of stem cells is the stromal vascular fraction of adipose tissue (SVF), which contains high numbers of adipose derived stem cells (ASC). We hypothesized that: 1) intravenous injection can be used to apply stem cells to the heart. 2) Uncultured SVF cells are easier and safer when cultured ASCs. 3) Transplantation after the acute inflammation period of AMI is favorable over early injection. For this, AMI was induced in rats by 40min of coronary occlusion. One or seven days after AMI, rats were intravenously injected with vehicle, 5×10(6) uncultured rat SVF cells or 1×10(6) rat ASCs. Rats were analyzed 35 days after AMI. Intravenous delivery of both fresh SVF cells and cultured ASCs 7 days after AMI significantly reduced infarct size compared to vehicle. Similar numbers of stem cells were found in the heart, after treatment with fresh SVF cells and cultured ASCs. Importantly, no adverse effects were found after injection of SVF cells. Using cultured ASCs, however, 3 animals had shortness of breath, and one animal died during injection. In contrast to application at 7 days post AMI, injection of SVF cells 1 day post AMI resulted in a small but non-significant infarct reduction (p=0.35). Taken together, intravenous injection of uncultured SVF cells subsequent to the acute inflammation period, is a promising stem cell therapy for AMI.     

Genetic Linkage and Horizontal Gene Transfer,the Roots of the Antibiotic Multi-Resistance Problem

Bacteria carrying resistance genes for many antibiotics are moving beyond the clinic into the community, infecting otherwise healthy people with untreatable and frequently fatal infections. This state of affairs makes it increasingly important that we understand the sources of this problem in terms of bacterial biology and ecology and also that we find some new targets for drugs that will help control this growing epidemic. This brief and eclectic review takes the perspective that we have too long thought about the problem in terms of treatment with or resistance to a single antibiotic at a time, assuming that dissemination of the resistance gene was affected by simple vertical inheritance. In reality antibiotic resistance genes are readily transferred horizontally, even to and from distantly related bacteria. The common agents of bacterial gene transfer are described and also one of the processes whereby nonantibiotic chemicals, specifically toxic metals, in the environment can select for and enrich bacteria with antibiotic multiresistance. Lastly, some speculation is offered on broadening our perspective on this problem to include drugs directed at compromising the ability of the mobile elements themselves to replicate, transfer, and recombine, that is, the three “infrastructure” processes central to the movement of genes among bacteria.     

Genetic linkage and horizontal gene transfer, the roots of the antibiotic multi-resistance problem

Bacteria carrying resistance genes for many antibiotics are moving beyond the clinic into the community, infecting otherwise healthy people with untreatable and frequently fatal infections. This state of affairs makes it increasingly important that we understand the sources of this problem in terms of bacterial biology and ecology and also that we find some new targets for drugs that will help control this growing epidemic. This brief and eclectic review takes the perspective that we have too long thought about the problem in terms of treatment with or resistance to a single antibiotic at a time, assuming that dissemination of the resistance gene was affected by simple vertical inheritance. In reality antibiotic resistance genes are readily transferred horizontally, even to and from distantly related bacteria. The common agents of bacterial gene transfer are described and also one of the processes whereby nonantibiotic chemicals, specifically toxic metals, in the environment can select for and enrich bacteria with antibiotic multiresistance. Lastly, some speculation is offered on broadening our perspective on this problem to include drugs directed at compromising the ability of the mobile elements themselves to replicate, transfer, and recombine, that is, the three "infrastructure" processes central to the movement of genes among bacteria.     

Protection of neonatal mice against herpes simplex virus infection: probable in vivo antibody-dependent cellular cytotoxicity

Infant mice are extremely susceptible to fatal Herpes simplex virus (HSV) infection. They are unable to produce antibody to HSV, and their leukocytes cannot mediate antibody-dependent cellular cytotoxicity (ADCC) to HSV-infected cells. In order to avoid H-2-dependent effector mechanisms and instead analyze possible in vivo ADCC, a murine model employing adoptive transfer of antibody and human leukocytes was developed. Administration of either human immune globulin or leukocytes i.p. from HSV immune or nonimmune humans could not protect infant C57BL/6 mice from fatal HSV infection. In contrast, a combination of a subneutralizing dilution of globulin and leukocytes from nonimmune or immune human donors, given one day before inoculation, was highly protective against lethal HSV infection. The cells involved included lymphocytes or monocyte-macrophages. At least 5 X 10(6) viable leukocytes (or 1 X 10(6) monocyte-macrophages) and immune serum globulin concentrations as low as 10(-8) were protective. Infected cell monolayer adsorption and DEAE column fractionation demonstrated that the protection by globulin was due to specific antiviral IgG antibody. Protection was n ot seen in animals receiving virus before immune transfer. Protection did not involve synergistic viral neutralization by antibody and cells, as shown by in vitro experiments. Animals receiving globulin and cells, unlike normal infant mice, had circulating antiviral antibody and peritoneal leukocytes able to mediate ADCC to HSV-infected cells. This is the first in vivo evidence for the role of human ADCC. This model also allows for the in vivo evaluation of the ability of cells from immunocompromised humans to curb viral infection.     

Distinct temperature-dependent dopamine-releasing effect of drugs of abuse in the olfactory bulb

It was recently shown in the olfactory bulb (OB) that the response to olfactory stimulation might be related to local reinforcement mechanisms involved in discrimination of different odors. Therefore, it seemed interesting to study the effects of several drugs of abuse on the release of dopamine (DA) in the OB. Nicotine, amphetamine, 3,4-methylenedioxymethamphetamine (MDMA, "ecstasy"), and cocaine at 37 degrees C increased the release of [3H] DA from olfactory bulb slice preparations of the rats. While nicotine, amphetamine, and MDMA directly evoked DA release, cocaine, by inhibiting the reuptake processes, enhanced the electrical stimulation-evoked release. At low temperature (17 degrees C), a condition in which the transmitter uptake carriers of the plasma membrane in both the normal and reverse mode of operation are inhibited, the nicotine-evoked [3H] DA release was potentiated, whereas those evoked by amphetamine and MDMA were inhibited. At low temperature the field stimulation-evoked [3H] DA release was potentiated, but under this condition cocaine failed to increase the release. Our results show that low temperature (a) increases the concentration of extracellular DA released by Ca(2+)-dependent vesicular exocytosis elicited by nicotine, (b) inhibits the extracellular Ca(2+)-independent amphetamine- and MDMA-induced release of DA that occurs by the reverse operation of membrane carriers transporting DA from the cytoplasm of presynaptic terminals to the extraneuronal space, and (c) does not alter the inhibitory effect of cocaine on DA uptake that increases the concentration of extracellular DA released by field stimulation. The findings that the drugs of abuse tested all enhanced the release of DA in the olfactory bulb suggest that local reinforcing mechanisms may also exist in this brain area. In addition, we also show that lowering the temperature in in vitro experiments is an easy and straightforward method for separating vesicular and cytoplasmic release of transmitters, and is suitable for studying the mechanism of catecholamine release evoked by drugs of abuse. This technique may be applicable in other neurochemical studies that need inhibition of the uptake carriers without the blockade of the ligand-gated ion channels caused by reuptake inhibitor drugs.     

Role of visceral adipose tissue in aging

Background

Visceral fat (VF) accretion is a hallmark of aging in humans. Epidemiologic studies have implicated abdominal obesity as a major risk factor for insulin resistance, type 2 diabetes, cardiovascular disease, metabolic syndrome and death.

Methods

Studies utilizing novel rodent models of visceral obesity and surgical strategies in humans have been undertaken to determine if subcutaneous (SC) abdominal or VF are causally linked to age-related diseases.

Results

Specific depletion or expansion of the VF depot using genetic or surgical tools in rodents has been shown to have direct effects on disease risk. In contrast, surgically removing large quantities of SC fat does not consistently improve metabolic parameters in humans or rodents, while benefits were observed with SC fat expansion in mice, suggesting that SC fat accrual is not an important contributor to metabolic decline. There is also compelling evidence in humans that abdominal obesity is a stronger risk factor for mortality risk than general obesity. Likewise, we have shown that surgical removal of VF improves mean and maximum lifespan in rats, providing the first causal evidence that VF depletion may be an important underlying cause of improved lifespan with caloric restriction.

General significance

This review provides both corollary and causal evidence for the importance of accounting for body fat distribution, and specifically VF, when assessing disease and mortality risk. Given the hazards of VF accumulation on health, treatment strategies aimed at selectively depleting VF should be considered as a viable tool to effectively reduce disease risk in humans.