Alpha-methyltyrosine reduces the acute cardiovascular and behavioral sequelae in a murine model of traumatic brain injury.

BACKGROUND: Increased catecholamines contribute to heightened cardiovascular reactivity and behavioral deficits after traumatic brain injury (TBI); adrenergic receptor blockade has limited success in reducing adverse sequelae of TBI. Injury-induced increases in the synthesis of catecholamines could contribute to adverse outcomes in TBI. Inhibition of catecholamine synthesis with alpha-methyltyrosine (αMT) could offer a benefit after TBI. METHODS: Original research trial in mice randomized to αMT (50 mg·kg -1 ·d -1 ) or vehicle for 1 week after TBI induced by controlled cortical impact. Primary outcomes of cardiovascular reactivity and behavioral deficits were assessed after 1 week. Secondary outcomes included blood brain barrier permeability and quantification of gene transcription whose products determine intraneuronal chloride concentrations, the release of catecholamines, and activation of the sympathetic nervous system. These genes were the alpha-2 adrenergic receptor ("Adra2c"), the sodium-potassium-chloride cotransporter ("Nkcc1"), and the potassium chloride cotransporter ("Kcc2"). We also assessed the effect of TBI and αMT on the neuronal chloride/bicarbonate exchanger ("Ae3"). RESULTS: Traumatic brain injury-induced increases in blood pressure and cardiac reactivity were blocked by αMT. Inhibition of catecholamine synthesis decreased blood brain barrier leakage and improved behavioral outcomes after TBI. Traumatic brain injury diminished the transcription of Adra2c and enhanced expression of Nkcc1 while reducing Kcc2 transcription; αMT prevented the induction of the Nkcc1 by TBI without reversing the effects of TBI on Kcc2 expression; αMT also diminished Ae3 transcription. CONCLUSION: Traumatic brain injury acutely increases cardiovascular reactivity and induces behavioral deficits in an αMT-sensitive manner, most likely by inducing Nkcc1 gene transcription. Alpha-methyltyrosine may prove salutary in the treatment of TBI by attenuating the enhanced expression of Nkcc1, minimizing blood brain barrier leakage, and diminishing central catecholamine and sympathetic output. We also found an unreported relationship between Kcc2 and the chloride/bicarbonate exchanger, which should be considered in the design of trials planned to manipulate central intraneuronal chloride concentrations following acute brain injury.

Ivabradine-Induced Bradycardia is Accompanied by Reduced Stress-Related Anxiety.

BACKGROUND: Hypertensive individuals with higher heart rates and anxiety have greater cardiovascular morbidity and mortality. Despite the correlation between hypertension, heart rate, and anxiety, scant attention has been paid to the effect of hypertension drug therapy on behavioral outcomes in cardiovascular disease. Ivabradine, an inhibitor of hyperpolarization-activated, cyclic nucleotide-gated funny channels (HCNs), has been used clinically to reduce heart rates and has been shown to improve quality of life in patients with angina and heart failure. We postulated that in addition to lowering heart rate, ivabradine could reduce anxiety in mice exposed to a significant stress paradigm. METHODS: Mice underwent a stress induction protocol, subsequently they received either vehicle or ivabradine (10 mg/kg) via osmotic minipumps. Blood pressure and heart rates were measured with tail cuff photoplethysmography. Anxiety was assessed quantitatively through the open field test (OFT) and the elevated plus maze (EPM). Cognition was assessed with an object recognition test (ORT). Pain tolerance was measured by the hot plate test or subcutaneous injection of formalin. HCN gene expression was measured with RT-PCR. RESULTS: Ivabradine reduced resting heart rate in the stressed mice by 22%. Stressed mice treated with ivabradine displayed significantly greater exploratory behavior in the OFT, EPM, and ORT. The expression of central HCN channels was significantly reduced following stress. CONCLUSION: It is suggested from our findings that ivabradine can reduce anxiety following significant psychological stress. Reductions in heart rate may directly improve quality of life by reducing anxiety in patients with hypertension and high heart rates.

Acetazolamide increases locomotion, exploratory behavior, and weight loss following social stress: A treatment for emotional eating?

Sympathomimetics are effective, centrally acting drugs that induce weight loss through their potent anorexic and locomotor properties. We reported that sympathomimetics antagonize catecholamine-dependent, alpha-2 adrenergic receptor-dependent signal transduction mediated by chloride/bicarbonate transport. We posit that other drugs that target cellular chloride/bicarbonate antiport would similarly demonstrate anorectic properties, induce locomotion, and diminish weight gain. Male and female inbred mice were housed in groups or stressed by prolonged social isolation. Mice consumed either normal chow or a high fat, high fructose corn syrup, (i.e. "Western") diet. To inhibit chloride/bicarbonate transport, acetazolamide (ACT, 3 mM) was added to the drinking water. Rodents underwent evaluations of exploratory locomotion and learning with the object recognition test. Mice consuming a "Western" diet gain more weight compared to mice given a normal diet. When placed on a "Western" diet, stressed mice gained weight more rapidly than unstressed. The body weight of mice fed a normal diet with ACT was significantly reduced compared to control mice not given ACT (weight, g ± SEM), 23.7 ± 0.8 v. 21.0 ± 0.5, p = 0.02. ACT did not reduce weight gain in animals chronically maintained on a "Western" diet. Compared to unstressed mice, living in social isolation reduced spontaneous exploratory locomotion time, an indicator of anxiety, in male mice (sec +SEM) from 22.8 ± 3.5 to 12.2 ± 2.1 (p < 0.001), and in female mice, from 47 ± 5.7 to 19.6 ± 2.3 (p < 0.001). ACT had no effect on exploration time in unstressed mice, but ACT completely restored the diminished exploratory locomotion time found in stressed mice compared to unstressed mice. The ratio of time spent exploring new objects compared to familiar items (discrimination ratio [DR]) was reduced following social isolation in males from 2.6 ± 0.5 to 1.2 ± 0.2 (p < 0.05) and in females from 3.8 ± 0.6 to 1.5 ± 0.2 (p < 0.01). ACT normalized the DR ratio of the stressed mice. Decreased food consumption and greater locomotor activity induced by ACT may contribute to acute weight loss; this effect is diminished when rodents were maintained on an unhealthful Western diet. Inhibition of chloride/bicarbonate transport through agents such as acetazolamide could offer a safe, new approach to achieving weight loss.

Sensitive and specific discrimination of pathogenic and nonpathogenic Escherichia coli using Raman spectroscopy-a comparison of two multivariate analysis techniques.

The determination of bacterial identity at the strain level is still a complex and time-consuming endeavor. In this study, visible wavelength spontaneous Raman spectroscopy has been used for the discrimination of four closely related Escherichia coli strains: pathogenic enterohemorrhagic E. coli O157:H7 and non-pathogenic E. coli C, E. coli Hfr K-12, and E. coli HF4714. Raman spectra from 600 to 2000 cm(-1) were analyzed with two multivariate chemometric techniques, principal component-discriminant function analysis and partial least squares-discriminant analysis, to determine optimal parameters for the discrimination of pathogenic E. coli from the non-pathogenic strains. Spectral preprocessing techniques such as smoothing with windows of various sizes and differentiation were investigated. The sensitivity and specificity of both techniques was in excess of 95%, determined by external testing of the chemometric models. This study suggests that spontaneous Raman spectroscopy with visible wavelength excitation is potentially useful for the rapid identification and classification of clinically-relevant bacteria at the strain level.

Chlorthalidone decreases platelet aggregation and vascular permeability and promotes angiogenesis.

Variations in diuretic-mediated inhibition of carbonic anhydrase-dependent chloride transport in platelets and vascular smooth muscle could account for the contrasting efficacy of the thiazide and thiazide-like diuretics in reducing cardiovascular morbidity in patients with hypertension. We assessed platelet carbonic anhydrase activity and catecholamine-induced platelet aggregation in the presence of a thiazide and a "thiazide-like" inhibitor of the sodium-chloride cotransporter. Individual variation in platelet carbonic anhydrase activity correlated with contrasting sensitivity to epinephrine-mediated platelet aggregation. Both chlorthalidone, which potently inhibits platelet carbonic anhydrase, and bendroflumethiazide, which has much less effect on this enzyme, increased the amount of epinephrine needed to induce platelet aggregation when compared with the absence of a diuretic. However, chlorthalidone was significantly more effective than bendroflumethiazide in reducing epinephrine-mediated platelet aggregation. Chlorthalidone also induced marked changes in the number of gene transcripts for two proteins that mediate angiogenesis and vascular permeability, vascular endothelial growth factor C and transforming growth factor-beta3; chlorthalidone and bendroflumethiazide had contrasting effects on the expression of vascular endothelial growth factor C. Chlorthalidone and bendroflumethiazide reduced vascular permeability to albumin, but only chlorthalidone increased angiogenesis. Thiazides and thiazide-like diuretics can comparably reduce blood pressure, but the drugs in this class are not all alike. It can be suggested from our findings that thiazide and thiazide-like diuretics vary in their pleiotropic effects on platelets and in the vasculature, and these differences could explain the contrasting ability of these drugs to reduce cardiovascular morbidity despite comparable reduction in blood pressure.

Ephedra alkaloids inhibit platelet aggregation.

Sympathomimetics, such as Ephedra alkaloids, are associated with an increased incidence of intracerebral hemorrhage believed to be secondary to concomitant elevations in blood pressure. We hypothesized that sympathomimetics decrease platelet aggregation. Reductions in epinephrine-mediated platelet aggregation by ephedrine, phenylpropanolamine, and racemic amphetamine were determined by measuring the changes that these sympathomimetics induced in the optical density of platelet-rich plasma from healthy individuals. Intracellular signal transduction was followed ex vivo by assaying the release of intracellular cyclic AMP and the ligand for the cytokine chemoreceptor 5 (RANTES) into platelet rich plasma. The effect of ephedrine on epinephrine-mediated increases in platelet selectin (CD62p) activation was assessed with flow cytometry. Data were analyzed with repeated-measures analyses of variance. Aggregation responses to epinephrine were greatly reduced in the presence of commonly used sympathomimetics such as ephedrine, phenylpropanolamine, and racemic amphetamine that have been found in cold remedies, appetite suppressants, or used in the treatment of attention-deficit hyperactivity disorder, respectively. Ephedrine diminished aggregation responses to ADP and gamma-thrombin, and this sympathomimetic reduced RANTES exocytosis, basal CD62p expression, and aggregation in platelets exposed to caffeine. Caffeine enhanced the effect of ephedrine on platelet function, and phenylpropanolamine amplified the inhibitory effect of aspirin on platelet aggregation. Sympathomimetics significantly alter platelet function, and they may increase the potential for bleeding independently of their effects on blood pressure. Despite restrictions imposed on their use, the consumption of sympathomimetics should be considered when any patient presents with findings of cerebral hemorrhage.

Alpha-methyltyrosine inhibits formation of reactive oxygen species and diminishes apoptosis in PC12 cells.

OBJECTIVE: Circulating catecholamines and adrenal steroids are significantly increased following traumatic brain injury, and elevations in plasma catecholamines and cortisol portend a poor outcome. We hypothesize that an increase in the generation of reactive oxygen species from the synthesis of adrenal steroids and catecholamines is responsible for neuronal injury following traumatic brain injury. As a first step in testing this hypothesis, we sought to determine whether or not inhibition of catecholamine synthesis would decrease neuronal damage. METHODS AND RESULTS: Using PC12 cells as a model of catecholamine synthesizing neurons, and serum deprivation as a method to induce neuronal damage, we show (1) adrenal corticosteroids increase reactive oxygen species formation and apoptosis induced by serum deprivation; (2) the inhibitor of catecholamine synthesis, alpha-methyltyrosine, reduces reactive oxygen species formation and apoptosis in PC12 cells; and (3) that acetazolamide, chlorthalidone, and the neurosteroid, allopregnanolone, which inhibits chloride transport, protect PC12 cells from apoptosis. CONCLUSIONS: It may be possible to protect catecholaminergic neurons from reactive oxygen species-induced apoptotic death by not only blocking catecholamine synthesis, but also, by inhibiting carbonic anhydrase-dependent chloride/bicarbonate exchange with acetazolamide or chlorthalidone. These agents may prove salutary in reducing cell death in patients with traumatic brain injury or stroke.

Preserving cardiac output with beta-adrenergic receptor blockade and inhibiting the Bezold-Jarisch reflex during resuscitation from hemorrhage.

BACKGROUND: Successful fluid resuscitation after severe hemorrhage may be limited by activation of the Bezold-Jarisch reflex. We postulated that pharmacologic inhibition of this reflex would restore cardiovascular hemodynamics more effectively than would volume repletion alone during resuscitation for hemorrhagic shock. METHODS: We measured mean arterial pressure (MAP), heart rate (HR), and cardiac output (CO) during fluid resuscitation after hemorrhaging laboratory rats until their CO had decreased by 90% to 95%. To block distinct components of the Bezold-Jarisch reflex, animals received capsazepine, yohimbine, or propranolol before iso-osmotic volume repletion. RESULTS: Hemorrhage decreased MAP and CO; despite an initial tachycardia, HR fell significantly in response to this large volume blood loss. The degree of hemorrhage-induced bradycardia mediated by the Bezold-Jarisch reflex predicted resuscitation MAP. Capsazepine-treated animals had greater resuscitation-induced increases in MAP (values in mm Hg +/- SEM), 130 +/- 12, when compared with the saline-only animals, 90 +/- 7 (p = 0.004). The capsazepine group also had a greater increase in systemic vascular resistance over baseline values during resuscitation (86% +/- 19%) compared with vehicle-treated animals (26% +/- 14%, p = 0.02). Capsazepine had no effect on cardiac dynamics. On the other hand, yohimbine increased HR and diminished CO, and propranolol dramatically increased stroke volume by 30%. CONCLUSION: Inhibition of the Bezold-Jarisch reflex may aid fluid resuscitation after hemorrhage only if stroke volume is restored. Beta-adrenergic receptor antagonists such as propranolol may prove the most salutary of these agents in enhancing fluid resuscitation in patients with severe hemorrhage.