Glucocorticoid receptor dimerization is required for proper recovery of LPS-induced inflammation, sickness behavior and metabolism in mice.

Endogenous glucocorticoids are essential for mobilizing energy resources, restraining inflammatory responses and coordinating behavior to an immune challenge. Impaired glucocorticoid receptor (GR) function has been associated with impaired metabolic processes, enhanced inflammation and exaggerated sickness and depressive-like behaviors. To discern the molecular mechanisms underlying GR regulation of physiologic and behavioral responses to a systemic immune challenge, GR(dim) mice, in which absent GR dimerization leads to impaired GR-DNA-binding-dependent mechanisms but intact GR protein-protein interactions, were administered low-dose lipopolysaccharide (LPS). GR(dim)-LPS mice exhibited elevated and prolonged levels of plasma corticosterone (CORT), interleukin (IL)-6 and IL-10 (but not plasma tumor necrosis factor-α (TNFα)), enhanced early expression of brain TNFα, IL-1ß and IL-6 mRNA levels, and impaired later central TNFα mRNA expression. Exaggerated sickness behavior (lethargy, piloerection, ptosis) in the GR(dim)-LPS mice was associated with increased early brain proinflammatory cytokine expression and late plasma CORT levels, but decreased late brain TNFα expression. GR(dim)-LPS mice also exhibited sustained locomotor impairment in the open field, body weight loss and metabolic alterations measured by indirect calorimetry, as well as impaired thermoregulation. Taken together, these data indicate that GR dimerization-dependent DNA-binding mechanisms differentially regulate systemic and central cytokine expression in a cytokine- and time-specific manner, and are essential for the proper regulation and recovery of multiple physiologic responses to low-dose endotoxin. Moreover, these results support the concept that GR protein-protein interactions are not sufficient for glucocorticoids to exert their full anti-inflammatory effects and suggest that glucocorticoid responses limited to GR monomer-mediated transcriptional effects could predispose individuals to prolonged behavioral and metabolic sequelae of an enhanced inflammatory state.

Cannabinoid 1 receptor activation contributes to vascular inflammation and cell death in a mouse model of diabetic retinopathy and a human retinal cell line.

AIMS/HYPOTHESIS: Recent studies have demonstrated that cannabinoid-1 (CB(1)) receptor blockade ameliorated inflammation, endothelial and/or cardiac dysfunction, and cell death in models of nephropathy, atherosclerosis and cardiomyopathy. However the role of CB(1) receptor signalling in diabetic retinopathy remains unexplored. Using genetic deletion or pharmacological inhibition of the CB(1) receptor with SR141716 (rimonabant) in a rodent model of diabetic retinopathy or in human primary retinal endothelial cells (HREC) exposed to high glucose, we explored the role of CB(1) receptors in the pathogenesis of diabetic retinopathy. METHODS: Diabetes was induced using streptozotocin in C57BL/6J Cb(1) (also known as Cnr1)(+/+) and Cb(1)(-/-) mice aged 8 to 12 weeks. Samples from mice retina or HREC were used to determine: (1) apoptosis; (2) activity of nuclear factor kappa B, intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), poly (ADP-ribose) polymerase and caspase-3; (3) content of 3-nitrotyrosine and reactive oxygen species; and (4) activation of p38/Jun N-terminal kinase/mitogen-activated protein kinase (MAPK). RESULTS: Deletion of CB(1) receptor or treatment of diabetic mice with CB(1) receptor antagonist SR141716 prevented retinal cell death. Treatment of diabetic mice or HREC cells exposed to high glucose with SR141716 attenuated the oxidative and nitrative stress, and reduced levels of nuclear factor κB, ICAM-1 and VCAM-1. In addition, SR141716 attenuated the diabetes- or high glucose-induced pro-apoptotic activation of MAPK and retinal vascular cell death. CONCLUSIONS/INTERPRETATION: Activation of CB(1) receptors may play an important role in the pathogenesis of diabetic retinopathy by facilitating MAPK activation, oxidative stress and inflammatory signalling. Conversely, CB(1) receptor inhibition may be beneficial in the treatment of this devastating complication of diabetes.

Endocannabinoids and cannabinoid receptors in ischaemia-reperfusion injury and preconditioning.

Ischaemia-reperfusion (I/R) is a pivotal mechanism of organ injury during stroke, myocardial infarction, organ transplantation and vascular surgeries. Ischaemic preconditioning (IPC) is a potent endogenous form of tissue protection against I/R injury. On the one hand, endocannabinoids have been implicated in the protective effects of IPC through cannabinoid CB1/CB2 receptor-dependent and -independent mechanisms. However, there is evidence suggesting that endocannabinoids are overproduced during various forms of I/R, such as myocardial infarction or whole body I/R associated with circulatory shock, and may contribute to the cardiovascular depressive state associated with these pathologies. Previous studies using synthetic CB1 receptor agonists or knockout mice demonstrated CB1 receptor-dependent protection against cerebral I/R injury in various animal models. In contrast, several follow-up reports have shown protection afforded by CB1 receptor antagonists, but not agonists. Excitedly, emerging studies using potent CB2 receptor agonists and/or knockout mice have provided compelling evidence that CB2 receptor activation is protective against myocardial, cerebral and hepatic I/R injuries by decreasing the endothelial cell activation/inflammatory response (for example, expression of adhesion molecules, secretion of chemokines, and so on), and by attenuating the leukocyte chemotaxis, rolling, adhesion to endothelium, activation and transendothelial migration, and interrelated oxidative/nitrosative damage. This review is aimed to discuss the role of endocannabinoids and CB receptors in various forms of I/R injury (myocardial, cerebral, hepatic and circulatory shock) and preconditioning, and to delineate the evidence supporting the therapeutic utility of selective CB2 receptor agonists, which are devoid of psychoactive effects, as a promising new approach to limit I/R-induced tissue damage.

CB2 cannabinoid receptor agonists attenuate TNF-alpha-induced human vascular smooth muscle cell proliferation and migration.

BACKGROUND AND PURPOSE: Vascular smooth muscle proliferation and migration triggered by inflammatory stimuli are involved in the development and progression of atherosclerosis and restenosis. Cannabinoids may modulate cell proliferation in various cell types through cannabinoid 2 (CB2) receptors. Here, we investigated the effects of CB2 receptor agonists on TNF-alpha-induced proliferation, migration and signal transduction in human coronary artery smooth muscle cells (HCASMCs). EXPERIMENTAL APPROACH: HCASMCs were stimulated with TNF-alpha. Smooth muscle proliferation was determined by the extent of BrdU incorporation and the migration was assayed by modified Boyden chamber. CB2 and/or CB1 receptor expressions were determined by immunofluorescence staining, western blotting, RT-PCR, real-time PCR and flow cytometry. KEY RESULTS: Low levels of CB2 and CB1 receptors were detectable in HCASMCs compared to the high levels of CB2 receptors expressed in THP-1 monocytes. TNF-alpha triggered up to approximately 80% increase (depending on the method used) in CB2 receptor mRNA and/or protein expression in HCASMCs, and induced Ras, p38 MAPK, ERK 1/2, SAPK/JNK and Akt activation, while increasing proliferation and migration. The CB2 agonists, JWH-133 and HU-308, dose-dependently attenuated these effects of TNF-alpha. CONCLUSIONS AND IMPLICATIONS: Since the above-mentioned TNF-alpha-induced phenotypic changes are critical in the initiation and progression of atherosclerosis and restenosis, our findings suggest that CB2 agonists may offer a novel approach in the treatment of these pathologies by decreasing vascular smooth muscle proliferation and migration.

Rapid reversal of the diabetic endothelial dysfunction by pharmacological inhibition of poly(ADP-ribose) polymerase.

Oxygen- and nitrogen-derived free radicals and oxidants play an important role in the pathogenesis of diabetic endothelial dysfunction. Recently we proposed the importance of oxidant-induced DNA strand breakage and activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) in the pathogenesis of diabetic endothelial dysfunction. In this study, we tested whether established diabetic endothelial dysfunction is reversible by PARP inhibition. The novel PARP inhibitor PJ34 (10 mg/kg per day PO) was given at various lengths (4 weeks or 3 days) for established streptozotocin-diabetic animals. In addition, we also tested whether incubation of the aortic rings with PJ34 (3 micromol/L) or a variety of other PARP inhibitors for 1 hour affects the diabetic vascular changes. Both 4-week and 3-day PARP-inhibitor treatment of streptozotocin-diabetic mice with established endothelial dysfunction fully reversed the acetylcholine-induced endothelium-dependent relaxations in vitro. Furthermore, 1-hour in vitro incubation of aortae from streptozotocin-diabetic mice with various PARP inhibitors was able to reverse the endothelial dysfunction. ATP, NAD(+), and NADPH levels were markedly reduced in diabetic animals, and PARP-inhibitor treatment was able to restore these alterations. Unexpectedly, pharmacological inhibition of PARP not only prevents the development of the endothelial dysfunction but is also able to rapidly reverse it. Thus, PARP activation and the associated metabolic compromise represent an ongoing process in diabetic blood vessels. Pharmacological inhibition of this process is able to reverse diabetic endothelial dysfunction.

Role of the endocannabinoid system in diabetes and diabetic complications.

UNLABELLED: Increasing evidence suggests that an overactive endocannabinoid system (ECS) may contribute to the development of diabetes by promoting energy intake and storage, impairing both glucose and lipid metabolism, by exerting pro-apoptotic effects in pancreatic beta cells and by facilitating inflammation in pancreatic islets. Furthermore, hyperglycaemia associated with diabetes has also been implicated in triggering perturbations of the ECS amplifying the pathological processes mentioned above, eventually culminating in a vicious circle. Compelling evidence from preclinical studies indicates that the ECS also influences diabetes-induced oxidative stress, inflammation, fibrosis and subsequent tissue injury in target organs for diabetic complications. In this review, we provide an update on the contribution of the ECS to the pathogenesis of diabetes and diabetic microvascular (retinopathy, nephropathy and neuropathy) and cardiovascular complications. The therapeutic potential of targeting the ECS is also discussed. LINKED ARTICLES: This article is part of a themed section on Endocannabinoids. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v173.7/issuetoc.

Cardiovascular pharmacology of cannabinoids.

Cannabinoids and their synthetic and endogenous analogs affect a broad range of physiological functions, including cardiovascular variables, the most important component of their effect being profound hypotension. The mechanisms of the cardiovascular effects of cannabinoids in vivo are complex and may involve modulation of autonomic outflow in both the central and peripheral nervous systems as well as direct effects on the myocardium and vasculature. Although several lines of evidence indicate that the cardiovascular depressive effects of cannabinoids are mediated by peripherally localized CB1 receptors, recent studies provide strong support for the existence of as-yet-undefined endothelial and cardiac receptor(s) that mediate certain endocannabinoid-induced cardiovascular effects. The endogenous cannabinoid system has been recently implicated in the mechanism of hypotension associated with hemorrhagic, endotoxic, and cardiogenic shock, and advanced liver cirrhosis. Furthermore, cannabinoids have been considered as novel antihypertensive agents. A protective role of endocannabinoids in myocardial ischemia has also been documented. In this chapter, we summarize current information on the cardiovascular effects of cannabinoids and highlight the importance of these effects in a variety of pathophysiological conditions.

Increased myogenic tone in skeletal muscle arterioles of diabetic rats. Possible role of increased activity of smooth muscle Ca2+ channels and protein kinase C.

OBJECTIVE: The diabetes mellitus-induced microangiopathy is still not clearly characterized. In this study we aimed to elucidate the effect of streptozotocin (STZ)-induced diabetes on myogenic response of isolated rat skeletal muscle arterioles and the mechanisms responsible for its alterations. METHODS: Male rats were divided into two groups: (1) control rats (C, plasma glucose: 6.4 +/- 0.5 mmol/l, n = 40) (2) diabetic rats (DM, 65 mg/kg STZ i.v., plasma glucose: 25.7 +/- 0.7 mmol/l, n = 40). Changes in diameter of isolated, cannulated gracilis skeletal muscle arterioles (approximately 130 microns in diameter) were measured by video-microscopy. RESULTS: Step increases in perfusion pressure (PP; from 10 to 140 mmHg) elicited significantly greater constrictions in DM than in C gracilis arterioles, in the presence of the endothelium (E). Also, a step increase in PP (from 40 to 100 mmHg) elicited greater and faster constrictions in DM vs. C arterioles. There were no significant differences in the pressure-passive diameter (in Ca2+ free solution) curves of arterioles. Dilations to acetylcholine were impaired in arterioles of DM as compared to those of C rats (EC50, C: 4.0 +/- 0.9 x 10(-9) mol/l, DM: 4.8 +/- 2.0 x 10(-8) mol/l (p < 0.01), and unaffected by inhibition of nitric oxide synthesis with L-NNA (10(-4) mol/l). Arteriolar constrictions to norepinephrine (NE) were significantly greater in DM compared to those of C rats (EC50, C: 6.2 +/- 0.6 x 10(-7) mol/l, DM: 8.0 +/- 2.0 x 10(-8) mol/l, p < 0.01) both in the presence and absence of E. In the absence of the E, constrictions to increases in pressure, or Ca2+ (0.25-7.5 mmol/l), or the voltage-dependent Ca(2+)-channel agonist Bay K 8644 (EC50; DM: 4.2 +/- 1.5 x 10(-10) mol/l, C: 1.7 +/- 0.8 x 10(-9) mol/l, p < 0.05) or the protein kinase C activator phorbol 12-myristate 13-acetate (PMA, EC50; DM: 6 +/- 2 x 10(-9) mol/l, C: 2 +/- 1 x 10(-8) mol/l, p < 0.05) were significantly greater in arterioles of DM compared to those of C rats. CONCLUSION: The novel findings of our study are that in diabetes mellitus the myogenic response of rat skeletal muscle arterioles is enhanced, which seems to be independent from the impaired endothelial function present simultaneously, and likely due to the increased activity of voltage-dependent Ca2+ channels and/or upregulation of protein kinase C in arteriolar smooth muscle.

Cardiac NO signalling in the metabolic syndrome.

It is well documented that metabolic syndrome (i.e. a group of risk factors, such as abdominal obesity, elevated blood pressure, elevated fasting plasma glucose, high serum triglycerides and low cholesterol level in high-density lipoprotein), which raises the risk for heart disease and diabetes, is associated with increased reactive oxygen and nitrogen species (ROS/RNS) generation. ROS/RNS can modulate cardiac NO signalling and trigger various adaptive changes in NOS and antioxidant enzyme expressions/activities. While initially these changes may represent protective mechanisms in metabolic syndrome, later with more prolonged oxidative, nitrosative and nitrative stress, these are often exhausted, eventually favouring myocardial RNS generation and decreased NO bioavailability. The increased oxidative and nitrative stress also impairs the NO-soluble guanylate cyclase (sGC) signalling pathway, limiting the ability of NO to exert its fundamental signalling roles in the heart. Enhanced ROS/RNS generation in the presence of risk factors also facilitates activation of redox-dependent transcriptional factors such as NF-κB, promoting myocardial expression of various pro-inflammatory mediators, and eventually the development of cardiac dysfunction and remodelling. While the dysregulation of NO signalling may interfere with the therapeutic efficacy of conventional drugs used in the management of metabolic syndrome, the modulation of NO signalling may also be responsible for the therapeutic benefits of already proven or recently developed treatment approaches, such as ACE inhibitors, certain ß-blockers, and sGC activators. Better understanding of the above-mentioned pathological processes may ultimately lead to more successful therapeutic approaches to overcome metabolic syndrome and its pathological consequences in cardiac NO signalling.

Control of apoptosis by IP(3) and ryanodine receptor driven calcium signals.

Intracellular calcium signals mediated by IP(3)and ryanodine receptors (IP(3)R/RyR) play a central role in cell survival, but emerging evidence suggests that IP(3)R/RyR are also important in apoptotic cell death. Switch from the life program to the death program may involve coincident detection of proapoptotic stimuli and calcium signals or changes in the spatiotemporal pattern of the calcium signal or changes at the level of effectors activated by the calcium signal (e.g. calpain, calcineurin). The fate of the cell is often determined in the mitochondria, where calcium spikes may support cell survival through stimulation of ATP production or initiate apoptosis v ia opening of the permeability transition pore and release of apoptotic factors such as cytochrome c. The functional importance of these mitochondrial calcium signalling pathways has been underscored by the elucidation of a highly effective, local Ca(2+)coupling between IP(3)R/RyR and mitochondrial Ca(2+)uptake sites. This article will focus on the IP(3)R/RyR-dependent pathways to apoptosis, particularly on the mitochondrial phase of the death cascade.

Current trends in the development of new antidepressants.

Early antidepressant medications e.g. tricyclic antidepressants (TCAs) and monoamine oxidase inhibitors (MAOIs) are effective because they enhance either noradrenergic or serotonergic mechanisms, or both. Unfortunately, these compounds block cholinergic, histaminergic and alpha-1-adrenergic receptor sites, interact with a number of other medications and bring about numerous undesirable side effects. Several chemically unrelated agents have been developed and introduced in the past decade to supplement the early antidepressants. These include selective inhibitors of the reuptake of serotonin (the selective serotonin reuptake inhibitors (SSRIs)) or noradrenaline (reboxetine) or both (SNRIs: milnacipran and venlafaxine), as well as drugs with distinct neurochemical profiles such as mirtazapine, nefazodone, moclobemide and tianeptine. All these newer compounds are the results of rational developmental strategies to find drugs that were as effective as the TCAs but of higher safety and tolerability profile. In spite of the remarkable structural diversity, most currently introduced antidepressants are monoamin based and modulating monoamine activity as a therapeutic strategy continues to dominate antidepressant research. It must be emphasised, however, that these newer antidepressants are far from the ideal ones, also resulting in undesirable side effects and requiring 2-6 weeks of treatment to produce therapeutic effect. Furthermore, approximately 30% of the population do not respond to current therapies. An important new development has been the emergence of potential novel mechanisms of action beyond the monoaminergic synapse. The results of recent novel developmental approaches have suggested that modulation of N-methyl-D-aspartate (NMDA), neuropeptide (substance P and corticotrophin-releasing factor) receptors and the intracellular messenger system may provide an entirely new set of potential therapeutic targets. This paper discusses the advances from monoamine-based treatment strategies and looks at the future developments in the treatment of depression.

Review of cardiovascular effects of fluoxetine, a selective serotonin reuptake inhibitor, compared to tricyclic antidepressants.

Fluoxetine is an antidepressant drug, a potent and specific inhibitor of serotonin reuptake (SSRI). Evidence suggests that being compared with tricyclic antidepressants, fluoxetine may cause significantly fewer anticholinergic, antihistaminergic and cardiotoxic side effects in the treatment of major depressive disorders. Chronic treatment with fluoxetine was not reported to affect the electrocardiogram (ECG). There is no clinical evidence of conduction delay and very little evidence of orthostatic hypotension. In the overdosed patients fewer cardiac symptoms were reported than with tricyclic antidepressants. However, dysrhythmia (atrial fibrillation and bradycardia) and syncope associated with fluoxetine treatment and overdose were reported. Although such reports have not been common, they do raise concerns. Thus we investigated the direct cardiovascular effects of the fluoxetine in isolated heart preparations and vessels of rats and rabbits. From 10(-6)M to 10(-4)M concentrations fluoxetine showed cardiodepressant and vasodilatory effects. These effects were similar to those of previously reported on tricyclic compounds. This review is a brief summary of possible cardiovascular effects of fluoxetine and other new SSRIs antidepressants from the literature based on experience of clinical studies and our experiments with fluoxetine on isolated rat and rabbit cardiac preparations and vessels. Possible explanations of the lower incidence of cardiovascular complications with fluoxetine in humans and cardiodepressant effects in vitro are discussed.

Speculations on difference between tricyclic and selective serotonin reuptake inhibitor antidepressants on their cardiac effects. Is there any?

The cardiovascular effects and toxicity of tricyclic antidepressants (TCAs) have been well documented in medical literature. The most common manifestation of such effects is slowing of intraventricular conduction, manifested by prolonged PR, QRS and QT intervals on the standard electrocardiogram (ECG) and postural hypotension. In contrast to TCAs, selective serotonin reuptake inhibitors (SSRIs), including fluoxetine and citalopram, are considered to cause less effect on cardiac impulse conduction. In addition, these compounds induced significantly less anticholinergic, antihistaminergic and cardiotoxic side-effects than TCAs. However, there is an increasing number of case reports on dysrhythmias, like atrial fibrillation or bradycardia and syncope associated with fluoxetine and another SSRI treatment and overdose. Although such reports have not been common, they do raise concerns. In cardiac tissues isolated from canine, rabbit, rat and guinea pig hearts we have found that fluoxetine and citalopram inhibited cardiac Na+ and Ca2+ channels. These direct cardiac electrophysiological effects were similar to those of observed for tricyclic antidepressants clomipramine and imipramine. The inhibition of cardiac Ca2+ and Na+ channels by fluoxetine may explain most cardiac side-effects observed occasionally with the drug and mild but significant bradycardia reported during chronic treatment. Our results suggest that fluoxetine and citalopram may have antiarrhythmic (class I + IV type), as well as proarrhythmic properties (due to impairment of atrioventricular or intraventricular conduction and shortening of repolarization). Taking all these into consideration, in depressed patients having also severe cardiac disorders, ECG control may be suggested during fluoxetine and probable another SSRI therapy. The primary goal of this review is to compare these direct cardiac effects of fluoxetine and citalopram to those of previously reported for TCAs. This paper also summarizes the recently observed effects of fluoxetine apparently not related to the blockage of 5-HT transporter based on literature.

Inhibition of voltage-gated calcium channels by fluoxetine in rat hippocampal pyramidal cells.

Fluoxetine, an antidepressant which is used world-wide, is a prominent member of the class of selective serotonin re-uptake inhibitors. Recently, inhibition of voltage-gated Na(+) and K(+) channels by fluoxetine has also been reported. We examined the effect of fluoxetine on voltage-gated calcium channels using the patch-clamp technique in the whole-cell configuration. In hippocampal pyramidal cells, fluoxetine inhibited the low-voltage-activated (T-type) calcium current with an IC(50) of 6.8 microM. Fluoxetine decreased the high-voltage-activated (HVA) calcium current with an IC(50) between 1 and 2 microM. Nifedipine and omega-conotoxin GVIA inhibited the HVA current by 24% and 43%, respectively. Fluoxetine (3 microM), applied in addition to nifedipine or omega-conotoxin, further reduced the current. When fluoxetine (3 microM) was applied first neither nifedipine nor omega-conotoxin attenuated the remaining component of the HVA current. This observation indicates that fluoxetine inhibits both L- and N-type currents. In addition, fluoxetine inhibited the HVA calcium current in carotid body type I chemoreceptor cells and pyramidal neurons prepared from prefrontal cortex. In hippocampal pyramidal cells high K(+)-induced seizure-like activity was inhibited by 1 microM fluoxetine; the mean burst duration was shortened by an average of 44%. These results provide evidence for inhibition of T-, N- and L-type voltage-gated calcium channels by fluoxetine at therapeutically relevant concentrations.

Serotonin reuptake inhibitor, fluoxetine, dilates isolated skeletal muscle arterioles. Possible role of altered Ca2+ sensitivity.

1. Inhibitors of serotonin reuptake in the central nervous system, such as fluoxetine, may also affect the function of vascular tissues. Thus, we investigated the effect of fluoxetine on the vasomotor responses of isolated, pressurized arterioles of rat gracilis muscle (98 +/- 4 microns in diameter at 80 mmHg perfusion pressure). 2. We have found that increasing concentrations of fluoxetine dilated arterioles up to 155 +/- 5 microns with an EC50 of 2.5 +/- 0.5 x 10(-6) M. 3. Removal of the endothelium, application of 4-aminopyridine (4-AP, an inhibitor of aminopyridine sensitive K+ channels), or use of glibenclamide (an inhibitor of ATP-sensitive K+ channels) did not affect the vasodilator response to fluoxetine. 4. In the presence of 10(-6), 2 x 10(-6) or 10(-5) M fluoxetine noradrenaline (NA, 10(-9)-10(-5) M) and 5-hydroxytryptamine (5-HT, 10(-9)-10(-5)M)-induced constrictions were significantly attenuated resulting in concentration-dependent parallel rightward shifts of their dose-response curves (pA2 = 6.1 +/- 0.1 and 6.9 +/- 0.1, respectively). 5. Increasing concentrations of Ca2+ (10(-4) 3 x 10(-2) M) elicited arteriolar constrictions (up to approximately 30%), which were markedly reduced by 2 x 10(-6)M fluoxetine, whereas 10(-5)M fluoxetine practically abolished these responses. 6. In conclusion, fluoxetine, elicits substantial dilations of isolated skeletal muscle arterioles, a response which is not mediated by 4-AP- and ATP-sensitive K+ channels or endothelium-derived dilator factors. The findings that fluoxetine had a greater inhibitory effect on Ca2+ elicited constrictions than on responses to NA and 5-HT suggest that fluoxetine may inhibit Ca2+ channel(s) or interfere with the signal transduction by Ca2+ in the vascular smooth muscle cells.

Oxidative stress-induced isoprostane formation may contribute to aspirin resistance in platelets.

Recent studies suggest that aggregation of platelets from patients with coronary artery and cerebrovascular disease may be resistant to low-dose aspirin (ASA) treatment, which may promote plaque-associated thrombus formation. However, the underlying mechanisms of platelet ASA resistance are poorly understood. ASA is thought to inhibit platelet aggregation primarily by inactivating the cyclooxygenase (COX), thus decreasing the synthesis of the pro-aggregatory arachidonic acid metabolite thromboxane A(2) (TxA(2)). However, recent studies also identified a non-enzymatic, oxidation-dependent pathway for the synthesis of the arachidonic acid derivative isoprostanes, which exhibit potent vasoconstrictor and pro-aggregatory effects similar to that of TxA(2). Because the pathophysiological conditions that promote arteriosclerotic vascular diseases (e.g. hypercholesterolemia, diabetes, hyperhomocysteinemia) are thought to be associated with an increased formation of reactive oxygen species and increased plasma isoprostane levels, it can be hypothesized that increased COX-independent isoprostane formation in platelets contribute to ASA resistance.

Topical administration of a novel nitric oxide donor, linear polyethylenimine-nitric oxide/nucleophile adduct (DS1), selectively increases vaginal blood flow in anesthetized rats.

The aim of the present study was to test the effects of a topical administration of a novel nitric oxide donor, linear polyethylenimine-nitric oxide/nucleophile adduct (DS1), on vaginal blood flow and hemodynamics in rats. Laser Doppler flowmetry was used to measure blood flow changes following topical application of DS1 (0.3 or 1.5 mg in 0.15 ml saline) into the vagina of anesthetized Wistar rats. In vivo hemodynamic parameters were measured with Millar-tip-catheter placed in the left ventricle. DS1 (1.5 mg) increased vaginal blood flow by 191+/-24, 226+/-22 and 166+/-23% of the baseline value (at 5, 15 and 30 min, respectively, after application) without affecting systemic blood pressure, heart rate and cardiac function. The increased vaginal blood flow following DS1 application returned to baseline between 45 and 60 min. Thus, topical application of nitric oxide donors such as DS1 may be useful for the treatment of female sexual dysfunction that develops due to an impairment of local blood flow supply to the vaginal tissue.

Electrophysiological effects of fluoxetine in mammalian cardiac tissues.

Fluoxetine is a widely used antidepressant compound having selective serotonin reuptake inhibitor properties. In this study, the actions of fluoxetine were analyzed in guinea pig, rat, rabbit and canine ventricular myocardiac preparations using conventional microelectrode and whole cell voltage clamp techniques. Low concentrations of fluoxetine (1-10 micromol/l) caused significant shortening of action potential duration (APD) and depression of the plateau potential in guinea pig and rabbit papillary muscles and single canine ventricular myocytes. In rat papillary muscle, APD was not affected by fluoxetine (up to 100 micromol/l), however, the drug decreased the force of contraction with EC50 of 10 micromol/l. Fluoxetine (10 micromol/l) also decreased the maximum velocity of depolarization and action potential overshoot in each species studied. At this concentration no effect was observed on the resting membrane potential; high concentration (100 micromol/l), however, caused depolarization. In voltage clamped canine ventricular myocytes, fluoxetine caused concentration-dependent block of the peak Ca2+ current at 0 mV with EC50 of 5.4+/-0.94 micromol/l and Hill coefficient of 1.1+/-0.14 (n=6). In addition, 10 micromol/l fluoxetine shifted the midpoint of the steady-state inactivation curve of the Ca2+ current from -20.7+/-0.65 to -26.7+/-1 mV (P<0.001, n=8) without changing its slope factor. These effects of fluoxetine developed rapidly and were fully reversible. Fluoxetine did not alter voltage-dependence of activation or time constant for inactivation of I(Ca). Fluoxetine had no effect on the amplitude of K+ currents (I(K1) and I(to)). The inhibition of cardiac Ca2+ and Na+ channels by fluoxetine may explain most cardiac side effects observed occasionally with the drug. Our results suggest that fluoxetine may have antiarrhythmic (class I + IV type), as well as proarrhythmic properties (due to impairment of atrioventricular or intraventricular conduction and shortening of repolarization). Therefore, in depressed patients with cardiac disorders, ECG control may be suggested during fluoxetine therapy.

Anti-inflammatory effects of inosine in human monocytes, neutrophils and epithelial cells in vitro.

Inosine is an endogenous purine, which has been recently shown to exert immunomodulatory, anti-inflammatory and anti-shock effects in rodent experimental systems. Some of these actions may be related to partial adenosine receptor agonistic effects. It has not been investigated previously whether inosine exerts similar immunomodulatory or anti-inflammatory effects in human cells or enzymes. Here we investigated the effects of inosine on the activation of human monocytes, neutrophils and epithelial cells in vitro. Furthermore, using a human inosine-5'-monophosphate dehydrogenase (IMPDH) enzyme, we examined the potential effects of inosine on the activity of IMPDH, an enzyme involved in the regulation of certain inflammatory/immune processes. Tumor necrosis factor alpha (TNF-alpha) production of bacterial lipopolysaccharide (LPS) stimulated whole blood was used as an indicator of human monocyte activation. The response was dose-dependently, partially suppressed in the presence of inosine. Inosine exerted a dose-dependent and, at the highest dose (3 mM), complete inhibition of the ability of human neutrophils activated with N-formyl-methionyl-leucyl-phenylalanine (fMLP) to induce cytochrome C reduction in vitro. In the human colon cancer cell line HT-29, inosine dose-dependently attenuated the production of IL-8. Inosine failed to affect the activity of IMPDH. Taken together, we conclude that inosine exerts anti-inflammatory effects in many human cell types. Further studies need to establish whether inosine supplementation exerts anti-inflammatory effects in human beings.

Selective serotonin-reuptake inhibitor antidepressants increase the risk of falls and hip fractures in elderly people by inhibiting cardiovascular ion channels.

Surprising results from recently published retrospective studies show that the use of new selective serotonin-reuptake inhibitor antidepressants (SSRIs), similarly to the older tricyclic antidepressants (TCAs), increases the risk of falls and hip fractures among elderly people.The mechanism whereby antidepressants increase this risk is complex and may include orthostatic hypotension, arrhythmias, sedation and confusion. The increased risk of falls and hip fractures with the use of TCAs is not surprising considering their well-known cardiovascular, anticholinergic and antihistaminergic side-effects. But the increased risk of falls with SSRIs is highly unexpected since these drugs are believed to be free from the disadvantages of TCAs. We hypothesized that the new SSRI antidepressants may also have cardiovascular effects similarly to the older TCA compounds, which may be an explanation for the increased rate of falls and hip fractures. The experimental and clinical evidence in support of this hypothesis are discussed.