Thyroid stimulating hormone directly modulates cardiac electrical activity.

BACKGROUND: The electrocardiogram of hypothyroid patients shows a series of abnormalities of cardiac repolarization due to a reduction of some repolarizing K(+) currents and an increase of the L-type calcium current. Experimental and clinical works call into question the unique role of T3 and T4 in these mechanisms and correlate increased serum TSH levels with the repolarization abnormalities in patients with both subclinical and overt hypothyroidism. In this context, the aim of the present study was to investigate the direct effects of TSH upon cardiac electrical properties. METHODS: The action potential recording and the ion channel subunits mRNA expression were obtained from left ventricle of adult rats. Additionally, the repolarizing K(+) currents and the L-type Ca(2+) current (ICa-L) were recorded in isolated rat adult ventricular myocytes by the patch-clamp technique. RESULTS: 24h exposure to TSH lengthened the action potential and slightly depolarized the resting membrane potential. TSH- receptor activation causes a reduction of the amplitude of Ito and IK1 currents caused by a reduction in channels expression. However, TSH had no effect on ICa-L, IK or IKur. CONCLUSION: These results support the idea that some of the electrical disturbances seen in hypothyroid hearts, such as the Ito and IK1 current reduction, could be caused not by low T3 but by the elevation of circulating TSH.

Basolateral expression of GRP94 in parietal cells of gastric mucosa.

GRP94 is a member of the heat shock protein family normally confined to the endoplasmic reticulum that sometimes escapes the KDEL-mediated retention system. It is overexpressed in some gastric and other gastrointestinal carcinomas, but little is known about the physiological role of GRP94 in gastric mucosa. We investigated the membrane presence of GRP94 in parietal cells, which secrete acid into the gastric lumen, using subcellular fractionation, selective solubilization of membrane proteins, Western blotting, and radio-ligand binding and provided evidence of functional GRP94 expression at the surface of gastric mucosa parietal cells anchored to the basolateral domain. Our results show that GRP94 is not an integral membrane protein since 50 mM Na2CO3 treatment dissociates part of it from the membrane. However, 100 mM Na2CO3 treatment did not extract all GRP94 from the membrane, which indicates that it is strongly associated with it. The presence of GRP94 in isolated plasma membrane was demonstrated by Western blotting and its functionality by radio-ligand binding experiments. Both the K(D) value obtained in saturation experiments with N-ethylcarboxamido-[3H]adenosine at 4°C, at the nanomolar range, and the inhibition constant of its binding by radicicol, the most specific GRP94 inhibitor, indicate that active receptor regions are exposed at the membrane surface. Western blotting of plasma membrane subfractions showed that GRP94 is mainly expressed in the basolateral membrane of gastric parietal cells, while its presence in the apical domain is negligible, thereby inferring a role for GRP94 in processes operating in this membrane domain.

Improvement of the metabolic status recovers cardiac potassium channel synthesis in experimental diabetes.

AIMS: The fast transient outward current, I(to,fast) , is the most extensively studied cardiac K(+) current in diabetic animals. Two hypotheses have been proposed to explain how type-1 diabetes reduces this current in cardiac muscle. The first one is a deficiency in channel expression due to a defect in the trophic effect of insulin. The second one proposes flawed glucose metabolism as the cause of the reduced I(to,fast) . Moreover, little information exists about the effects and possible mechanisms of diabetes on the other repolarizing currents of the human heart: I(to,slow) , I(Kr) , I(Ks) , I(Kur) , I(Kslow) and I(K1) . METHODS: We recorded cardiac action potentials and K(+) currents in ventricular cells isolated from control and streptozotocin- or alloxan-induced diabetic mice and rabbits. Channel protein expression was determined by immunofluorescence. RESULTS: Diabetes reduces the amplitude of I(to,fast) , I(to,slow) and I(Kslow) , in ventricular myocytes from mouse and rabbit, with no effect on I(ss) , I(Kr) or I(K1) . The absence of changes in the biophysical properties of the currents and the immunofluorescence experiments confirmed the reduction in channel protein synthesis. Six-hour incubation of myocytes with insulin or pyruvate recovered current amplitudes and fluorescent staining. The activation of AMP-K reduced the same K(+) currents in healthy myocytes and prevented the pyruvate-induced current recovery. CONCLUSION: Diabetes reduces K(+) current densities in ventricular myocytes due to a defect in channel protein synthesis. Activation of AMP-K secondary to deterioration in the metabolic status of the cells is responsible for K(+) channel reductions.

Mechanisms responsible for the altered cardiac repolarization dispersion in experimental hypothyroidism.

AIMS: To identify the causes for the inhomogeneity of ventricular repolarization and increased QT dispersion in hypothyroid mice. METHODS: We studied the effects of 5-propyl-2-thiouracil-induced hypothyroidism on the ECG, action potential (AP) and current density of the repolarizing potassium currents I(to,fast), I(to,slow), I(K,slow) and I(ss) in enzymatically isolated myocytes from three different regions of mouse heart: right ventricle (RV), epicardium of the left ventricle (Epi-LV) and interventricular septum. K(+) currents were recorded with the patch-clamp technique. Membranes from isolated ventricular myocytes were extracted by centrifugation. Kv4.2, Kv4.3, KChIP and Na/Ca exchanger proteins were visualized by Western blot. RESULTS: The frequency or conduction velocity was not changed by hypothyroidism, but QTc was prolonged. Neither resting membrane potential nor AP amplitude was modified. The action potential duration (APD)(90) increased in the RV and Epi-LV, but not in the septum. Hypothyroid status has no effect either on I(to,slow), I(k,slow) or I(ss) in any of the regions analysed. However, I(to,fast) was significantly reduced in the Epi-LV and in the RV, whereas it was not altered in cells from the septum. Western blot analysis reveals a reduction in Kv4.2 and Kv4.3 protein levels in both the Epi-LV and the RV and an increase in Na/Ca exchanger. CONCLUSION: From these results we suggest that the regional differences in APD lengthening, and thus in repolarization inhomogeneity, induced by experimental hypothyroidism are at least partially explained by the uneven decrease in I(to,fast) and the differences in the relative contribution of the depolarization-activated outward currents to the repolarization process.

Type 1 diabetes alters brain cannabinoid receptor expression and phosphorylation status in rats.

One of the most common symptoms of diabetes is extreme hunger, but the brain mechanism underlying this hyperphagia is unknown. The endocannabinoid system has emerged as one of the main food intake regulators in the brain. However, the effects of type 1 diabetes on the endocannabinoid system are not completely known. Thus, the aim of the present work is to establish the possible alterations induced by type 1 diabetes on the brain endocannabinoid system in rats. Western blot and immunocytochemistry were used to measure CB1 and phosphorylated CB1 receptor expression in several prosencephalic regions in streptozotocin-induced type 1 diabetic rats. Serum leptin levels were measured by ELISA. CB1 receptor expression was increased in striatum and hypothalamus of diabetic animals, with no changes in other brain areas studied. CB1 receptor phosphorylation was also increased in the same brain areas. Type 1 diabetes induced significant weight loss, and serum leptin levels were severely decreased. These results reinforce the possible role of the CB1 receptor as a pharmacological target for the clinical management of appetite in diabetic patients.

Changes in erythrocyte morphology induced by imipramine and chlorpromazine

Chlorpromazine (CPZ), a phenothiazine derivative, is a potent antipsychotic agent and imipramine (IP) is a widely used tricyclic antidepressant. The interaction between these molecules and erythrocyte membranes is of particular interest considering the role of these cells in the transport and release of these drugs at the central nervous system. In the present paper, we intend to study the effects of IP on erythrocyte membranes and to compare these effects with those of CPZ. Erythrocytes from adult Sprague-Dawley rats were incubated separately with different concentrations of IP or CPZ for 1 h at room temperature, fixed and stained by Giemsa. Changes in erythrocyte morphology were quantified by an image analysis system. The interaction of both drugs, CPZ and IP, with the erythrocyte membrane causes similar changes in cell shape. Increasing concentrations of both drugs induces the formation of stomatocytes, spherostomatocytes and spherocytes, because of an irreversible loss of area and volume, probably due to endovesiculation. Our results also show that the CPZ is more potent than IP (AU)

La clorpromazina (CPZ), un derivado de las fenotiazinas, es un potente agente antipsicótico y la imipramina (IP) es un antidepresivo tricíclico extensamente utilizado. La interacción entre estas moléculas y las membranas de los eritrocitos es de particular interés considerando la importancia de estas células en el transporte y liberación de estos fármacos en el sistema nervioso central. En este trabajo, nos proponemos estudiar los efectos de la IP en las membranas de los eritrocitos y comparar esos efectos con los de CPZ. Los eritrocitos de ratas Sprague-Dawley adultas fueron incubados por separado con varias concentraciones de IP o CPZ durante 1h a temperatura ambiente, fijados y teñidos con Giemsa. Los cambios en morfología de los eritrocitos fueron cuantificados mediante un sistema de análisis de imagen. La interacción de ambos productos, CPZ e IP, con la membrana de los eritrocitos provoca cambios similares en forma de la célula. El aumento de la concentración de ambos fármacos induce la formación de estomatocitos, esferoestomatocitos y esferocitos, debido a una pérdida irreversible de área y de volumen, probablemente debido a la endovesiculación, siendo la CPZ más potente que la IP (AU)

Changes in erythrocyte morphology induced by imipramine and chlorpromazine.

Chlorpromazine (CPZ), a phenothiazine derivative, is a potent antipsychotic agent and imipramine (IP) is a widely used tricyclic antidepressant. The interaction between these molecules and erythrocyte membranes is of particular interest considering the role of these cells in the transport and release of these drugs at the central nervous system. In the present paper, we intend to study the effects of IP on erythrocyte membranes and to compare these effects with those of CPZ. Erythrocytes from adult Sprague-Dawley rats were incubated separately with different concentrations of IP or CPZ for lh at room temperature, fixed and stained by Giemsa. Changes in erythrocyte morphology were quantified by an image analysis system. The interaction of both drugs, CPZ and IP, with the erythrocyte membrane causes similar changes in cell shape. Increasing concentrations of both drugs induces the formation of stomatocytes, spherostomatocytes and spherocytes, because of an irreversible loss of area and volume, probably due to endovesiculation. Our results also show that the CPZ is more potent than IP.

Diabetic cardiomyopathy: electromechanical cellular alterations.

Diabetic patients show a higher incidence of cardiac arrhythmias, including ventricular fibrillation and sudden death. However, although diabetic cardiomyopathy is a frequent and important complication of diabetes mellitus, its physiological basis is not completely known. The electrocardiogram of diabetic patients shows several alterations from normal patterns, most of them related to the QT interval and T wave. Recently, different alterations in cardiac ionic currents have been described in myocytes isolated from diabetic hearts, mainly a reduction in potassium repolarizing currents. Three different mechanisms could be involved in these alterations. First, direct metabolic alterations of the cardiac myocyte, such as impaired activity of protein kinases and phosphatases, intracellular pH regulation, intracellular calcium handling, and others. Second, impaired support of extra cardiac factors regulating cardiac activity, such as sympathetic regulation of heart rate and contractility. Thus, diabetic autonomic neuropathy leads to diminished noradrenaline release in cardiac ventricle in response to standing, exercise or cold stress. Besides, diabetic cardiomyopathy reduces cardiac myocyte response to acute noradrenaline exposure and finally, impairs support of different trophic factors responsible for the regulation of ionic channel expression. Thus, basal noradrenaline release in the ventricles, necessary to maintain adequate potassium channel expression, is reduced by sympathetic neuropathy. Moreover, the levels of insulin and other trophic factors required for the maintenance of adequate ionic channel expression are also altered in diabetic patients. Therefore, different physiopathological mechanisms are involved in diabetic cardiomyopathy. Thus, further research is needed in order to prevent the development of this long-term complication, and to improve the pharmacological management of diabetic patients.

Diabetes-induced biochemical changes in central and peripheral catecholaminergic systems.

A great variety of alterations have been described in the nervous system of diabetic animals. They are named as diabetic neuropathy and affect the brain, spinal cord and peripheral nerves. In diabetic animals, plasma and tissue catecholamine levels have been reported to be increased, decreased or unchanged, and these disparities have been explained by differences in the tissues selected, severity or duration of diabetes. Dopamine, norepinephrine and epinephrine from different tissues were extracted by absorption onto alumina, and measured by high performance liquid chromatography with electrochemical detection. We found that diabetes alters catecholaminergic systems in a highly specific manner. The dopamine content is reduced in the dopaminergic nigrostriatal system only. Norepinephrine is differently altered in several areas of the sympathetic nervous system. It is increased in cardiac ventricles, and decreased in stellate ganglia and the blood serum. However, it is not altered in the central nervous system. Finally, epinephrine is only altered in the adrenal gland where it is increased, and in the serum where it is reduced. Our results suggest that diabetes reduces the activity of the nigrostriatal dopaminergic system. Changes found at the sympathoadrenal level could be explained by reduced norepinephrine and epinephrine synthesis, with increased storage due to a reduced release from synaptic vesicles.

Blood pressure responsiveness to sympathetic agonists in anaesthetised diabetic rats.

Chronic diabetes alters sympathetic modulation of the cardiovascular system. In the present work, we examined if the cardiovascular system also demonstrates an impaired responsiveness to sympathetic control. The effects of streptozotocin-induced diabetes on pressure responses to noradrenaline or isoproterenol infusion of diabetic rats in vivo are studied. Systolic and diastolic pressures were recorded through a cannula implanted in the right carotid artery. Increasing doses of noradrenaline or isoproterenol were infused through a catheter implanted in the left jugular vein. The dose-response curves for the effects of noradrenaline on blood pressure were altered in streptozotozin-induced diabetic rats. Noradrenaline induced a statistically significant higher increase of both systolic and diastolic pressure in control than in diabetic rats. The maximum depressor response of systolic pressure to isoproterenol was lower in diabetic than in control animals. Diabetes fully abolishes the effect of beta-adrenoceptor stimulation on diastolic blood pressure. The present results demonstrate that streptozotocin-induced diabetes reduces systolic and diastolic arterial pressure, and diminishes the arterial pressure reactivity to sympathetic stimulation.

Spironolactone and captopril attenuates isoproterenol-induced cardiac remodelling in rats.

The role of renin-angiotensin-aldosterone system in cardiac remodelling was studied in isoproterenol-induced cardiac hypertrophy in rats. The effects of captopril and spironolactone were compared. Isoproterenol treatment increased ventricular to body weight ratio (4.6 vs 3.7) and collagen area (22.6 vs 8.2%), and reduced systolic (89.93 vs 107.5 mm Hg) and diastolic (59.6 vs 70.8 mm Hg) pressure. In these animals, captopril decreased systolic (67.4 mm Hg) and diastolic pressure (31.9 mm Hg), whereas spironolactone regressed systolic pressure to control values (101.2 mm Hg). Captopril and spironolactone prevented cardiac hypertrophy (4.01 and 3.95). However, only spironolactone prevented myocardial fibrosis (11.3%).

Regulation of cardiac transient outward potassium current by norepinephrine in normal and diabetic rats.

BACKGROUND: Alpha-adrenergic stimulation regulates cardiac contractility by reducing repolarising K+ currents. Despite this, no published work exists on the effects of norepinephrine on isolated cardiac transient outward current, responsible for action potential duration in the rat and human. Besides, diabetes alters cardiac inotropic responses to sympathetic innervation, and this can result from altered responsiveness of the transient outward current to norepinephrine. METHODS: Transient outward K+ current was measured using the whole-cell configuration of the patch-clamp technique. Myocytes were isolated from the right ventricle of healthy and streptozotocin-induced diabetic rats. RESULTS: Norepinephrine, through alpha(1)-adrenoceptors, reduces current amplitude in a concentration-dependent way, with no effects on current kinetics or voltage dependence of inactivation. Diabetes reduces current amplitude and accelerates its inactivation process. Norepinephrine also reduces current amplitude in diabetic cells; however diabetes shifts to the right the concentration-response curve and reduces the maximum effect of the neurotransmitter. CONCLUSIONS: Norepinephrine reduces the amplitude of isolated ventricular transient outward K+ current with no effects on current properties in myocytes isolated from either healthy or diabetic hearts. Diabetes shifts the concentration-response curve; thus diabetic myocytes are more resistant to sympathetic regulation than are healthy cells.

Toluene alters mu-opioid receptor expression in the rat brainstem.

Toluene is an ototoxic organic solvent widely used in industry and could be a cause of sleep apnea. Acute toluene administration in rats induces an increase in the number of neural cells immunostained for mu-opioid receptors in several brainstem nuclei, such as the inferior colliculus, dorsal and lateral periaqueductal gray and dorsal raphe, without changes in the superior colliculus and the interpeduncular and lateral reticular nuclei. These data suggest that mu-opioid receptors could be involved in toluene-induced neurotoxic effects on the physiological regulation of breathing during sleep, and auditive function.

Effects of amphetamine on calcium and potassium currents in rat heart.

We used the patch-clamp technique to study the effects of amphetamine on the membrane currents responsible for rat cardiac action-potential duration. Amphetamine has no effect on the slow inward Ca2+ current (I(Ca)-L), the inwardly rectifying K+ current (I(K1) and the outward K+ delayed rectifier (I(K)) and sustained (I(SS)) currents. Amphetamine blocks the transient outward K+ current (I(to)) both in the open and in the rested state. The transient outward K+ current is largely responsible for action-potential repolarization and for the regional differences in action-potential duration in rat ventricle. Therefore, the reduction of the transient outward K+ current (I(to)) caused by amphetamine may facilitate the appearance of ventricular tachycardia and fibrillation, a reported cause of death in amphetamine users.

Effects of diabetic cardiomyopathy on regional electrophysiologic characteristics of rat ventricle.

AIMS/HYPOTHESIS: To identify the possible causes of the lengthening of the action potential duration described in patients affected by diabetes mellitus. METHODS: We studied the effects of streptozotocin-induced diabetes on the current density of the repolarising potassium currents It(o), IK, Iss and IK1 in enzymatically isolated myocytes from three different regions of rat heart: total right ventricle, subepicardium at the apex of the left ventricle and subendocardium at the base of the left ventricle. RESULTS: No changes in IK1 were found due to diabetes, but there was a uniform decrease in It(o) (50%) and Iss (40%) current densities in the three regions. In contrast, IK diminished unevenly, with the greatest decrease in the subendocardium at the base of the left ventricle (48%), followed by the subepicardium at the apex of the left ventricle (32%) and right ventricle (10%). CONCLUSION/INTERPRETATION: These findings suggest the existence of regional differences in ion channel expression associated with diabetes. The decrease of these repolarising currents could account for the lengthening of action potential and the consequent change in the Q-T interval of the ECG observed in diabetic rats.

Restoration of cardiac transient outward potassium current by norepinephrine in diabetic rats.

In cardiac ventricle, the density of the transient outward potassium current, Ito, is clearly related to sympathetic nervous system integrity. This sympathetic regulation of Ito expression may be greatly significant to the genesis of cardiac complications of several diseases such us diabetes mellitus. Autonomic neuropathy, including cardiac neuropathy, is a complication of chronic diabetes. The objective of the present study was to identify the possible role of cardiac sympathetic neuropathy in the reduction of Ito current density in diabetic ventricular myocardium. Thus, we employed the patch-clamp technique to test whether Ito can be restored in diabetic myocytes incubated with norepinephrine. We also measured, using HPLC, the catecholamine content of the stellate ganglion, which is responsible for cardiac sympathetic innervation, in normal and diabetic animals. The main result of the present study was to show that a 24-h incubation of diabetic cells with norepinephrine restores Ito density to control values. The restoration of Ito current density by norepinephrine suggests that the diabetes-induced reduction of Ito is at least partially attributable to a reduced trophic effect of norepinephrine on the expression of Ito.

Effects of fluoxetine administration on mu-opoid receptor immunostaining in the rat forebrain.

Fluoxetine is a selective serotonin reuptake inhibitor. Analysis of mu-opioid receptor immunostaining after chronic fluoxetine administration in rats revealed an increase in the density of cells expressing mu-opioid receptors in the caudatus-putamen, the dentate gyrus, the lateral septum and the frontal, parietal and piriform cortices. These data suggest that mu-opioid receptor expression in the rat forebrain is altered by in vivo chronic fluoxetine treatment.

Imipramine inhibits soluble enkephalin-degrading aminopeptidase activity in vitro.

Considerable evidence has appeared recently connecting the mechanism of action of some antidepressant drugs with the inhibition of the enzymes responsible for enkephalin degradation. Imipramine in vitro inhibits the enkephalin-degrading aminopeptidase MII and interacts with the enzyme in a mixed competitive-noncompetitive manner. The present work shows that imipramine in vitro also inhibits reversibly soluble enkephalin-degrading aminopeptidase activity in rat brain. Kinetic analysis showed that this enzyme has two different binding sites for the drug, and that imipramine interacts with the enzyme in a mixed noncompetitive-acompetitive way.

Disopyramide, imipramine, and amitriptyline bind to a common site on the transient outward K+ channel.

Previous work demonstrated that several antiarrhythmic agents and antidepressive drugs block transient outward K+ current (I(to)) in rat ventricular myocytes. The antiarrhythmic drug, disopyramide, and the tricyclic antidepressants, imipramine and amitriptyline, block the I(to) channel mainly when it is in the open state. The rate of recovery from block induced by disopyramide is so slow that the drug produces a use-dependent block at 1 Hz, whereas the rate of recovery from block in the presence of imipramine and amitriptyline is fast enough so as not to induce any use-dependent block at this frequency. We studied the effects of the combinations of disopyramide-imipramine and disopyramide-amitriptyline on I(to) to detect possible interactions between the drugs on I(to) blockade. The effects of imipramine and amitriptyline on the use-dependent effect induced by disopyramide and on the rate of recovery of the channels blocked by this drug allow us to conclude that there is only one common receptor site in the channel molecule for the three drug molecules.

Mechanism of block of cardiac transient outward K+ current (I(to)) by antidepressant drugs.

Imipramine, amitriptyline, mianserine, maprotiline, and trazodone are five widely used antidepressant drugs with different chemical structures. Imipramine and amitriptyline are tricyclics, mianserine and maprotiline are tetracyclics, and trazodone is a triazolopyridine derivative. We studied the effects of these drugs on the transient outward K+ current (I(to)) and the interaction mechanisms within the drug molecules and the channel-binding site. The transient outward K+ current is mainly responsible for action-potential repolarization in the rat ventricle, and all of the five drugs studied block I(to), but in different manners. Cyclic drugs block I(to) in the open state of the channel with very little block in the rested or inactivated states or both. Trazodone blocks the channel in a state-independent manner. From these results, we suggest that a relation exists between drug structure and preference for the different channel conformations.