Ligand-induced endocytosis and nuclear localization of angiotensin II receptors expressed in CHO cells

A construct (AT1R-NF) containing a "Flag" sequence added to the N-terminus of the rat AT1 receptor was stably expressed in Chinese hamster ovary cells and quantified in the cell membrane by confocal microscopy after reaction with a fluorescein-labeled anti-Flag monoclonal antibody. Angiotensin II bound to AT1R-NF and induced endocytosis with a half-time of 2 min. After 60-90 min, fluorescence accumulated around the cell nucleus, suggesting migration of the ligand-receptor complex to the nuclear membrane. Angiotensin antagonists also induced endocytosis, suggesting that a common step in the transduction signal mechanism occurring after ligand binding may be responsible for the ligand-receptor complex internalization.

In vitro cytotoxicity of the LDE: daunorubicin complex in acute myelogenous leukemia blast cells

Acute myelogenous leukemia (AML) blast cells show high-affinity degradation of low-density lipoprotein (LDL), suggesting an increased expression of cellular LDL receptors. LDE is a lipid microemulsion easily synthesized in vitro which is known to mimic the metabolic pathway of LDL. We used LDE as a carrier for daunorubicin and assayed the cytotoxicity of the complex using AML blast cells since RT-PCR analysis showed that AML cells express LDL receptor mRNA. The LDE:daunorubicin complex killed 46.7% of blast cells and 20.2% of normal bone marrow cells (P<0.001; Student t-test). Moreover, this complex destroyed AML blast cells as efficiently as free daunorubicin. Thus, LDE might be a suitable carrier of chemotherapeutic agents targeting these drugs to neoplastic cells and protecting normal tissues.

Frequent occurrence of nicotinic acetylcholine receptors in GABAergic neurons of the chick visual system

Double-labeling immunohistochemical methods were used to investigate the occurrence of the alpha8 and alpha5 nicotinic receptor subunits in presumptive GABAergic neurons of the chick nervous system. Nicotinic receptor immunoreactivity was often found in cells exhibiting GABA-like immunoreactivity, especially in the visual system. The alpha8 subunit appeared to be present in presumptive GABAergic cells of the ventral lateral geniculate nucleus, nucleus of the basal optic root of the accessory optic system, and the optic tectum, among several other structures. The alpha5 subunit was also found in GABA-positive neurons, as observed in the lentiform nucleus of the mesencephalon and other pretectal nuclei. The numbers of alpha8- and alpha5-positive neurons that were also GABA-positive represented high percentages of the total number of neurons containing nicotinic receptor labeling in several brain areas, which indicates that most of the alpha8 and alpha5 nicotinic receptor subunits are present in GABAergic cells. Taken together with data from other studies, our results indicate an important role of the nicotinic acetylcholine receptors in the functional organization of GABAergic circuits in the visual system.

The influence of fasting/refeeding on the lipoprotein lipase activity of adipose tissue and muscle

Lipoprotein lipase activity in adipose tissue and muscle is modulated by changes in the pattern of food intake. We have measured total lipoprotein lipase activity in adipose tissue and muscle of male Wistar rats (N = 6-10), weighing 200-250 g (~12 weeks), during the refeeding/fasting state following 24 h of fasting. Lipoprotein lipase activity in tissue homogenates was evaluated using a [³H]-triolein-containing substrate, and released [³H]-free fatty acids were extracted and quantified by liquid scintillation. Adipose tissue lipoprotein lipase activity did not completely recover within 2 h of refeeding (60% of refed ad libitum values). Cardiac lipoprotein lipase activity remained increased even 2 h after refeeding (100% of refed ad libitum values), whereas no significant changes were observed in the soleus and diaphragm muscles. Adipose tissue lipoprotein lipase activities were consistently higher than the highest skeletal muscle or heart values. It is therefore likely that adipose tissue, rather than muscle makes the major contribution to triacylglycerol clearance. There was concomitant relatively high lipoprotein lipase activity in both adipose tissue and cardiac muscle during the first few hours of refeeding, therefore cardiac muscle may contribute significantly to triacylglycerol clearance during this period. The results suggest that during fasting, increased lipoprotein lipase activity provides a complementary source of free fatty acids from circulating triacylglycerol, allowing the heart to maintain its continuous, high-energy expenditure.

Estrogen replacement therapy and cardioprotection: mechanisms and controversies

Epidemiological and case-controlled studies suggest that estrogen replacement therapy might be beneficial in terms of primary prevention of coronary heart disease (CHD). This beneficial effect of estrogens was initially considered to be due to the reduction of low density lipoproteins (LDL) and to increases in high density lipoproteins (HDL). Recent studies have shown that estrogens protect against oxidative stress and decrease LDL oxidation. Estrogens have direct effects on the arterial tissue and modulate vascular reactivity through nitric oxide and prostaglandin synthesis. While many of the effects of estrogen on vascular tissue are believed to be mediated by estrogen receptors alpha and ß, there is evidence for `immediate non-genomic' effects. The role of HDL in interacting with 17ß-estradiol including its esterification and transfer of esterified estrogens to LDL is beginning to be elucidated. Despite the suggested positive effects of estrogens, two recent placebo-controlled clinical trials in women with CHD did not detect any beneficial effects on overall coronary events with estrogen therapy. In fact, there was an increase in CHD events in some women. Mutations in thrombogenic genes (factor V Leiden, prothrombin mutation, etc.) in a subset of women may play a role in this unexpected finding. Thus, the cardioprotective effect of estrogens appears to be more complicated than originally thought and requires more research.

Phagocytosis by macrophages mediated by receptors for denatured proteins - dependence on tyrosine protein kinases

Previous studies have demonstrated that some components of the leukocyte cell membrane, CR3 (Mac-1, CD11b/CD18) and p150/95, are able to bind to denatured proteins. Thus, it is of interest to know which effector functions of these cells can be triggered by these receptors when they interact with particles or surfaces covered with denatured proteins. In the present study we analyzed their possible role as mediators of phagocytosis of red cells covered with denatured bovine serum albumin (BSA) by mouse peritoneal macrophages. We observed that a) macrophages are able to recognize (bind to) these red cells, b) this interaction can be inhibited by denatured BSA in the fluid phase, c) there is no phagocytosis of these particles by normal macrophages, d) phagocytosis mediated by denatured BSA can be, however, effectively triggered in inflammatory macrophages induced by glycogen or in macrophages activated in vivo with LPS, and e) this phagocytic capacity is strongly dependent on the activity of tyrosine protein kinases in its signal transduction pathway, as demonstrated by using three kinds of enzyme inhibitors (genistein, quercetin and herbimycin A).

Gender differences in vascular expression of endothelin and ET A/ET B receptors, but not in calcium handling mechanisms, in deoxycorticosterone acetate-salt hypertension

We determined if the increased vascular responsiveness to endothelin-1 (ET-1) observed in male, but not in female, DOCA-salt rats is associated with differential vascular mRNA expression of ET-1 and/or ET A/ET B receptors or with functional differences in Ca2+ handling mechanisms by vascular myocytes. Uninephrectomized male and female Wistar rats received DOCA and drinking water containing NaCl/KCl. Control rats received vehicle and tap water. Blood pressure and contractile responses of endothelium-denuded aortic rings to agents which induce Ca2+ influx and/or its release from internal stores were measured using standard procedures. Expression of mRNA for ET-1 and ET A/ET B receptors was evaluated by RT-PCR after isolation of total cell RNA from both aorta and mesenteric arteries. Systolic blood pressure was higher in male than in female DOCA rats. Contractions induced by Bay K8644 (which activates Ca2+ influx through voltage-operated L-type channels), and by caffeine, serotonin or ET-1 in Ca2+-free buffer (which reflect Ca2+ release from internal stores) were significantly increased in aortas from male and female DOCA-salt compared to control aortas. DOCA-salt treatment of male, but not female, rats statistically increased vascular mRNA expression of ET-1 and ET B receptors, but decreased the expression of ET A receptors. Molecular up-regulation of vascular ET B receptors, rather than differential changes in smooth muscle Ca2+ handling mechanisms, seems to account for the increased vascular reactivity to ET-1/ET B receptor agonists and higher blood pressure levels observed in male DOCA-salt rats.

Dissociation between the circulating renin-angiotensin system and angiotensin II receptors in central losartan-induced hypertension

Losartan, an AT1 angiotensin II (ANG II) receptor non-peptide antagonist, induces an increase in mean arterial pressure (MAP) when injected intracerebroventricularly (icv) into rats. The present study investigated possible effector mechanisms of the increase in MAP induced by icv losartan in unanesthetized rats. Male Holtzman rats (280-300 g, N = 6/group) with a cannula implanted into the anterior ventral third ventricle received an icv injection of losartan (90 µg/2 µl) that induced a typical peak pressor response within 5 min. In one group of animals, this response to icv losartan was completely reduced from 18 ± 1 to 4 ± 2 mmHg by intravenous (iv) injection of losartan (2.5-10 mg/kg), and in another group, it was partially reduced from 18 ± 3 to 11 ± 2 mmHg by iv prazosin (0.1-1.0 mg/kg), an alpha1-adrenergic antagonist (P<0.05). Captopril (10 mg/kg), a converting enzyme inhibitor, injected iv in a third group inhibited the pressor response to icv losartan from 24 ± 3 to 7 ± 2 mmHg (P<0.05). Propranolol (10 mg/kg), a ß-adrenoceptor antagonist, injected iv in a fourth group did not alter the pressor response to icv losartan. Plasma renin activity and serum angiotensin-converting enzyme activity were not altered by icv losartan in other animals. The results suggest that the pressor effect of icv losartan depends on angiotensinergic and alpha1-adrenoceptor activation, but not on increased circulating ANG II.

The role of reelin in the development and evolution of the cerebral cortex

Reelin is an extracellular matrix protein that is defective in reeler mutant mice and plays a key role in the organization of architectonic patterns, particularly in the cerebral cortex. In mammals, a "reelin signal" is activated when reelin, secreted by Cajal-Retzius neurons, binds to receptors of the lipoprotein receptor family on the surface of cortical plate cells, and triggers Dab1 phosphorylation. As reelin is a key component of cortical development in mammals, comparative embryological studies of reelin expression were carried out during cortical development in non-mammalian amniotes (turtles, squamates, birds and crocodiles) in order to assess the putative role of reelin during cortical evolution. The data show that reelin is present in the cortical marginal zone in all amniotes, and suggest that reelin has been implicated in the evolution of the radial organization of the cortical plate in the synapsid lineage leading from stem amniotes to mammals, as well as in the lineage leading to squamates, thus providing an example of homoplastic evolution (evolutionary convergence). The mechanisms by which reelin instructs radial cortical organization in these two lineages seem different: in the synapsid lineage, a drastic amplification of reelin production occurred in Cajal-Retzius cells, whereas in squamates, in addition to reelin-secreting cells in the marginal zone, a second layer of reelin-producing cells developed in the subcortex. Altogether, our results suggest that the reelin-signaling pathway has played a significant role in shaping the evolution of cortical development.

Hormone replacement therapy increases levels of antibodies against heat shock protein 65 and certain species of oxidized low density lipoprotein

Hormone replacement therapy (HRT) reduces cardiovascular risks, although the initiation of therapy may be associated with transient adverse ischemic and thrombotic events. Antibodies against heat shock protein (Hsp) and oxidized low density lipoprotein (LDL) have been found in atherosclerotic lesions and plasma of patients with coronary artery disease and may play an important role in the pathogenesis of atherosclerosis. The aim of the present study was to assess the effects of HRT on the immune response by measuring plasma levels of antibodies against Hsp 65 and LDL with a low and high degree of copper-mediated oxidative modification of 20 postmenopausal women before and 90 days after receiving orally 0.625 mg equine conjugate estrogen plus 2.5 mg medroxyprogesterone acetate per day. HRT significantly increased antibodies against Hsp 65 (0.316 ± 0.03 vs 0.558 ± 0.11) and against LDL with a low degree of oxidative modification (0.100 ± 0.01 vs 0.217 ± 0.02) (P<0.05 and P<0.001, respectively, ANOVA). The hormone-mediated immune response may trigger an inflammatory response within the vessel wall and potentially increase plaque burden. Whether or not this immune response is temporary or sustained and deleterious requires further investigation.

Effect of one-week ethanol treatment on monoamine levels and dopaminergic receptors in rat striatum

We studied the effects of ethanol on the levels of norepinephrine, dopamine, serotonin (5-HT) and their metabolites as well as on D1- and D2-like receptors in the rat striatum. Ethanol (2 or 4 g/kg, po) was administered daily by gavage to male Wistar rats and on the 7th day, 30 min or 48 h after drug administration, the striatum was dissected for biochemical assays. Monoamine and metabolite concentrations were measured by HPLC and D1- and D2-like receptor densities were determined by binding assays. Scatchard analyses showed decreases of 30 and 43%, respectively, in D1- and D2-like receptor densities and no change in dissociation constants (Kd) 48 h after the withdrawal of the dose of 4 g/kg. Ethanol, 2 g/kg, was effective only on the density of D2-like receptors but not on Kd of either receptor. Thirty minutes after the last ethanol injection (4 g/kg), decreases of D2 receptor density (45%) as well as of Kd values (34%) were detected. However, there was no significant effect on D1-like receptor density and a 46% decrease was observed in Kd. An increase in dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC), a decrease in norepinephrine, and no alteration in 5-HT levels were demonstrated after 48-h withdrawal of 4 g/kg ethanol. Similar effects were observed in dopamine and DOPAC levels 30 min after drug administration. No alteration in norepinephrine concentration and a decrease in 5-HT levels were seen 30 min after ethanol (4 g/kg) administration. Our findings indicate the involvement of the monoaminergic system in the responses to ethanol.

The role of chemokines and chemokine receptors in eosinophil activation during inflammatory allergic reactions

Chemokines are important chemotactic cytokines that play a fundamental role in the trafficking of leukocytes to sites of inflammation. They are also potent cell-activating factors, inducing cytokine and histamine release and free radical production, a fact that makes them particularly important in the pathogenesis of allergic inflammation. The action of chemokines is regulated at the level of agonist production and processing as well as at the level of receptor expression and coupling. Therefore, an analysis of the ligands must necessarily consider receptors. Eosinophils are target cells involved in the allergic inflammatory response since they are able to release a wide variety of mediators including CC and CXC chemokines and express their receptors. These mediators could damage the airway epithelial cells and might be important to stimulate other cells inducing an amplification of the allergic response. This review focuses on recently emerging data pertaining to the importance of chemokines and chemokine receptors in promoting eosinophil activation and migration during the allergic inflammatory process. The analysis of the function of eosinophils and their chemokine receptors during allergic inflammation might be a good approach to understanding the determinants of asthma severity and to developing novel therapies.

Estrogen and progesterone receptors in human papilloma virus-related cervical neoplasia

Estrogen (ER) and progesterone (PR) receptors in the normal uterine cervix, cervical intraepithelial neoplasia and invasive carcinoma were studied in consecutive samples from Hospital do Câncer, São Paulo, between 1996 and 1997. Tissue was collected by removing a fragment of the tumoral area using a 5-mm diameter biopsy punch, followed by removal of a macroscopically normal area as close as possible from the tumor. Histopathological confirmation was obtained for all specimens analyzed. A total of 24 normal tissues, 17 cases of cervical intraepithelial neoplasia and 7 of invasive carcinomas were studied. The ER/PR ratio was determined by immunohistochemistry using monoclonal antibodies specific for each receptor. Adjacent tissue slides were submitted to generic PCR for human papillomavirus (HPV) DNA detection followed by typing by dot blot hybridization. About half (45.8%) of the tumors were HPV DNA positive while 29.1% of the patients were also HPV positive in their respective normal tissue. ER was negative in the tumoral epithelium of 11 HPV-positive patients (P = 0.04). There was a trend in the ER distribution in normal tissue that was opposite to that from lesions, but it was not statistically significant (P = 0.069). No difference in ER distribution in stromal tissues was observed between HPV-positive and HPV-negative tissues. PR staining was negative in the epithelium of all cases studied. The results obtained from this small number of cases cannot be considered to be conclusive but do suggest that factors related to viral infection affect the expression of these ER/PR cervix receptors.

Cardiovascular complications and increased levels of circulating modified low density lipoprotein in HIV patients and patients with lipodystrophy

The introduction of highly active antiretroviral therapy (HAART) for patients infected with HIV has significantly prolonged the life expectancy and to some extent has restored a functional immune response. However, the premature introduction of HAART has led to a significant and alarming increase in cardiovascular complications, including myocardial infarction and the appearance of abnormal distribution of body fat seen as lipodystrophy. One key element in the development of ischemic coronary artery disease is the presence of circulating and tissue-fixed modified low density lipoprotein (mLDL) that contributes to the initiation and progression of arterial lesions and to the formation of foam cells. Even though not completely elucidated, the most likely mechanism involves mLDL in the inflammatory response and the induction of a specific immune response against mLDL. Circulating antibodies against mLDL can serve as an indirect marker of the presence of circulating and vessel-fixed mLDL. In the present study, we measured antibodies to mLDL and correlated them with immune status (i.e., number of CD4+ T cells) in 59 HIV patients and with the clinical manifestation of lipodystrophy in 10 patients. We observed a significant reduction in anti-mLDL antibody levels related both to lipodystrophy and to an immunocompromised state in HIV patients. We speculate that these antibodies may explain in part the rapid development of ischemic coronary artery disease in some patients.

Imidazoline receptors in the heart: a novel target and a novel mechanism of action that involves atrial natriuretic peptides

Chronic stimulation of sympathetic nervous activity contributes to the development and maintenance of hypertension, leading to left ventricular hypertrophy (LVH), arrhythmias and cardiac death. Moxonidine, an imidazoline antihypertensive compound that preferentially activates imidazoline receptors in brainstem rostroventrolateral medulla, suppresses sympathetic activation and reverses LVH. We have identified imidazoline receptors in the heart atria and ventricles, and shown that atrial I1-receptors are up-regulated in spontaneously hypertensive rats (SHR), and ventricular I1-receptors are up-regulated in hamster and human heart failure. Furthermore, cardiac I1-receptor binding decreased after chronic in vivo exposure to moxonidine. These studies implied that cardiac I1-receptors are involved in cardiovascular regulation. The presence of I1-receptors in the heart, the primary site of production of natriuretic peptides, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), cardiac hormones implicated in blood pressure control and cardioprotection, led us to propose that ANP may be involved in the actions of moxonidine. In fact, acute iv administration of moxonidine (50 to 150 µg/rat) dose-dependently decreased blood pressure, stimulated diuresis and natriuresis and increased plasma ANP and its second messenger, cGMP. Chronic SHR treatment with moxonidine (0, 60 and 120 µg kg-1 h-1, sc for 4 weeks) dose-dependently decreased blood pressure, resulted in reversal of LVH and decreased ventricular interleukin 1ß concentration after 4 weeks of treatment. These effects were associated with a further increase in already elevated ANP and BNP synthesis and release (after 1 week), and normalization by 4 weeks. In conclusion, cardiac imidazoline receptors and natriuretic peptides may be involved in the acute and chronic effects of moxonidine.