Predictors of pharmacological treatment outcomes with atomoxetine or methylphenidate in patients with attention-deficit/hyperactivity disorder from China, Egypt, Lebanon, Russian Federation, Taiwan, and United Arab Emirates.

BACKGROUND: The reduced availability of data from non-Western countries limits our ability to understand attention-deficit/hyperactivity disorder (ADHD) treatment outcomes, specifically, adherence and persistence of ADHD in children and adolescents. This analysis assessed predictors of treatment outcomes in a non-Western cohort of patients with ADHD treated with atomoxetine or methylphenidate. METHODS: Data from a 12-month, prospective, observational study in outpatients aged 6-17 years treated with atomoxetine (N = 234) or methylphenidate (N = 221) were analysed post hoc to determine potential predictors of treatment outcomes. Participating countries included the Russian Federation, China, Taiwan, Egypt, United Arab Emirates and Lebanon. Factors associated with remission were analysed with stepwise multiple logistic regression and classification and regression trees (CART). Cox proportional hazards models with propensity score adjustment assessed differences in atomoxetine persistence among initial-dose cohorts. RESULTS: In patients treated with atomoxetine who had available dosing information (N = 134), Cox proportional hazards revealed lower (< 0.5 mg/kg) initial dose was significantly associated with shorter medication persistence (p < 0.01). multiple logistic regression analysis revealed greater rates of remission for atomoxetine-treated patients were associated with age (older), country (United Arab Emirates) and gender (female) (all p < 0.05). CART analysis confirmed older age and lack of specific phobias were associated with greater remission rates. For methylphenidate, greater baseline weight (highly correlated with the age factor found for atomoxetine) and prior atomoxetine use were associated with greater remission rates. CONCLUSIONS: These findings may help clinicians assess factors upon initiation of ADHD treatment to improve course prediction, proper dosing and treatment adherence and persistence. TRIAL REGISTRATION: Observational study, therefore no registration.

Structural effects of raloxifene on the proximal femur: results from the multiple outcomes of raloxifene evaluation trial.

INTRODUCTION: Raloxifene improves spine bone mineral density (BMD), and its ability to reduce vertebral fractures by 40-50% suggests that it increases vertebral strength. Positive effects on hip BMD suggest a similar strengthening of the hip, but dimensional ambiguities in BMD by dual energy x-ray absorptiometry (DXA) make it difficult to infer strength effects directly. Hip fractures may be too infrequent to evaluate in practical clinical trials; even the Multiple Outcomes of Raloxifene Evaluation (MORE) study with 7,705 subjects was insufficiently powered to show a comparable reduction in hip fractures. METHODS: An alternative evaluation of hip DXA data in structural terms should provide more direct evidence of treatment effects on hip strength. Hip scans from a subset of the MORE study, including 4,806 postmenopausal women with osteoporosis randomized to daily oral doses of placebo, 60 mg, or 120 mg of raloxifene were reanalyzed by the hip structure analysis (HSA) method. Scans acquired at baseline, 1, 2, and 3 years were evaluated to extract BMD and cross-sectional geometry across the narrowest point on the neck (NN), the intertrochanteric region (IT), and the proximal shaft 1.5 times the minimum neck width distal to the intersection of the neck and shaft axes. RESULTS: While femur outer diameter expanded during follow-up at all three regions, there were no differences in expansion between groups; treatment influenced mainly the amount and distribution of bone within cross-sections. Effects were similar at the two dose levels at the NN region although the 120 mg dose produced a greater effect on section modulus (SM) at the IT region and on BMD, bone cross-sectional area (CSA), SM, average cortical thickness (CT), and buckling ratio (BR) at the shaft region. Compared with placebo after 3 years, treatment groups showed 0.4-2% higher BMD, CSA, SM, and CT and 1-2% lower BR. The smallest treatment effects were evident at the shaft at 60 mg. CONCLUSIONS: We conclude that raloxifene does not influence periosteal apposition in the proximal femur but it nevertheless produces small but significant improvement in resistance to axial and bending stresses (CSA and SM, respectively) at all analyzed regions. The significant reductions in buckling ratio suggest that additional strength loss due to cortical instability is also ameliorated by treatment.

Fire ant venom alkaloid, isosolenopsin A, a potent and selective inhibitor of neuronal nitric oxide synthase.

Massive, multiple fire ant, Solenopsis invicta, stings are often treated aggressively, particularly in the elderly, despite limited evidence of systemic toxicity due to the venom. Over 95% of the S. invicta venom is composed of piperidine alkaloid components, whose toxicity, if any, is unknown. To assess a possible pharmacological basis for systemic toxicity, an alkaloid-rich, protein-free methanol extract of the venom from whole ants was assayed for inhibitory activity on the following nitric oxide synthase (NOS) isoforms, rat cerebellar neuronal (nNOS), bovine recombinant endothelial (eNOS), and murine recombinant immunologic (iNOS). Cytosolic NOS activity was determined by measuring the conversion of [(3)H]arginine to [(3)H]citrulline in vitro. Rat nNOS activity was inhibited significantly and in a concentration-dependent manner by the alkaloid-rich venom extract. For nNOS, enzyme activity was inhibited by approximately 50% with 0.33 +/- 0.06 microg of this venom extract, and over 95% inhibition of the three isoforms, nNOS, eNOS, and iNOS, was found with doses of 60 microg in 60 microl reaction mixture. These results indicate that the alkaloid components of S. invicta venom can produce potent inhibition of all three major NOS isoforms. Isosolenopsin A (cis-2-methyl-6-undecylpiperidine), a naturally occurring fire ant piperidine alkaloid, was synthesized and tested for inhibitory activity against the three NOS isoforms. Enzyme activities for nNOS and eNOS were over 95% inhibited with 1000 microM of isosolenopsin A, whereas the activity of iNOS was inhibited by only about 20% at the same concentration. The IC(50) for each of three NOS isoforms was approximately 18 +/- 3.9 microM for nNOS, 156 +/- 10 microM for eNOS, and >1000 microM for iNOS, respectively. Kinetic studies showed isosolenopsin A inhibition to be noncompetitive with L-arginine (K(i) = 19 +/- 2 microM). The potency of isosolenopsin A as an inhibitor of nNOS compares favorably with the inhibitory potency of widely used nNOS inhibitors. Inhibition of NOS isoforms by isosolenopsin A and structurally similar compounds may have toxicological significance with respect to adverse reactions to fire ant stings.

Mechanisms of increased liver tissue repair and survival in diet-restricted rats treated with equitoxic doses of thioacetamide.

Moderate dietary or caloric restriction (DR) modulates animal physiology in a beneficial fashion. Previously, we have reported an equitoxic dose experiment where liver injury in DR male Sprague-Dawley rats exposed to a low dose of thioacetamide (TA, 50 mg/kg) was similar to that observed in ad libitum fed (AL) rats exposed to a 12-fold higher dose (600 mg/kg). Paradoxically, the AL rats experienced 90% mortality while all of the DR rats, with the same amount of initial bioactivation-mediated liver injury, survived. The protection observed in the DR rats was due to efficient compensatory liver tissue repair, which was delayed and attenuated in the AL rats, leading to progression of liver injury. The objective of the present study was to investigate the molecular mechanisms of the enhanced tissue repair in the DR rats upon equitoxic challenge with TA. Promitogenic mechanisms and mediators such as proinflammatory cytokines (TNF-alpha and IL-6), growth factors (TGF-alpha and HGF), and inducible nitric oxide synthase (iNOS) were estimated over a time course after equitoxic challenge (50 mg/kg to DR vs. 600 mg/kg to AL rats). Except for TNF-alpha, all other molecules were expressed earlier and in greater amount in the DR rats. IL-6 was 10-fold greater and peaked 12 h earlier; HGF also peaked 12 h sooner in the DR rats, when it was 2.5-fold greater than the value in the AL rats. TGF-alpha expression in livers of DR rats increased after TA administration and peaked at 24 h. In the AL rats, it was lower and peaked at 36 h. Diet restriction alone induced iNOS 2-fold in the DR rats and remained elevated until 12 h after TA administration, then declined thereafter. The lower iNOS activity in the AL rats further decreased after TA injection. DR rats exhibited higher apoptosis after thioacetamide administration, which further increased the efficiency of tissue repair. Taken together, these data indicate that even though the liver injury is near equal in AL and DR rats, sluggish signal transduction leads to delayed liver regeneration, progression of liver injury, and death in the AL rats. The equitoxic dose experiment indicates that stimulation of tissue repair is independent of the extent of initial liver injury and is governed by physiology of diet restriction. DR stimulates promitogenic signaling leading to a quick and timely response upon liver injury, arrest of progressive injury on one hand, and recovery from injury on the other, paving the way for survival of the DR rats.

Perinatal lead exposure alters the expression of neuronal nitric oxide synthase in rat brain.

Environmental exposure to lead (Pb) is known to affect the developing nervous system causing cognitive deficits in children. The diffusible nitric oxide (NO) is a biological messenger known to be involved in brain development. We examined the developmental changes of neuronal nitric oxide synthase (nNOS) in cerebellum and hippocampus of developing rat brain by radiometric assay, Western blot analysis and immunohistochemistry. Pb-exposure (0.2% Pb acetate) was initiated on gestation day 6 through the drinking water of the dam and continued through birth and postnatal days (PNDs) 1 to 21. The pups were never exposed to Pb directly. Pb exposure was stopped on weaning of pups from mothers on PND 21. The changes in nNOS were measured in the offspring on PNDs 7, 14, 21, and 35. The nNOS activity was increased gradually from PNDs 7 to 35 in both cerebellum and hippocampus of control rats when the enzyme activity was determined in the presence of either 0.5 or 6 microM calcium (Ca2+) in the reaction mixture. However, Pb exposure decreased the nNOS activity significantly at PNDs 21 to 35 as compared to respective controls when the enzyme activity was determined in the presence of 6 microM Ca2+. The decrease of nNOS was even greater and evident at all PNDs tested when the enzyme activity was assayed in the presence of physiological concentration of Ca2+ (0.5 microM). These findings were further strengthened by the in vitro studies. The cerebellar nNOS activity was inhibited much more at low Ca2+ (0.5 microM) as compared to 6 microM Ca2+, with IC50 values of 35 and 50 nM Pb, respectively. The nNOS protein levels and immunoreactivity in the cerebellum and hippocampus of rats perinatally exposed to Pb were decreased as compared to controls at PNDs 21 and 35. These data suggest perinatal Pb exposure decreases the nNOS in the developing brain. The decrease of nNOS activity and protein may explain the Pb-mediated cognitive deficits because NO regulates long-term potentiation (LTP) and other neurophysiological events in the developing nervous system.

Effects of manganese on inositol polyphosphate receptors and nitric oxide synthase activity in rat brain.

The neurotoxic effects of excessive exposure to manganese (Mn) include degeneration of dopaminergic neurons, impairment of energy metabolism, and perturbations in phosphoinositide (PI) hydrolysis leading to altered calcium (Ca2+) homeostasis. This study is designed to assess the in vitro and in vivo effects of Mn on Ca2+/calmodulin-dependent neuronal nitric oxide synthase (nNOS) activity and on the regulation of inositol 1,4,5-trisphosphate (InsP3) and inositol 1,3,4,5-tetrakisphosphate (InsP4) receptors involved in intracellular and extracellular mobilization of Ca2+. In vivo Mn exposure significantly increased 3H-InsP3 and 3H-InsP4 binding in the cerebellum and the cerebral cortex in a dose-dependent manner. However, in vitro Mn decreased 3H-InsP3 binding and increased 3H-InsP4 binding. In vitro and in vivo exposure of Mn inhibited nNOS activity in the cerebellum and the cerebral cortex. Immunohistochemical studies also showed a notable decrease in nNOS immunoreactivity in the granule cell layer of the cerebellum, whereas no significant changes were observed in the cerebral cortex. These data suggest that Mn neurotoxicity may be due to altered calcium homeostasis by its modulation of inositol polyphosphate receptors. Further, the inhibition of nNOS by Mn is of considerable importance because NO regulates a number of neurotransmitter functions.

Inhibition of calcium ATPase by phencyclidine in rat brain.

Phencyclidine (PCP) is a potent psychotomimetic drug of abuse and has profound effect on the functioning of the central nervous system (CNS). Many of the CNS functions are known to be mediated by calcium (Ca2+). In the present study we have investigated the effects of PCP on Ca2+ ATPase activity in rat brain both in vitro and in vivo. For in vitro studies, synaptic membrane fractions prepared from normal rat brain were incubated with PCP at different concentrations (25-100 microM) before the addition of substrate. For in vivo studies, rats were treated with a single moderate dose of PCP (10 mg/kg, i.p.) and animals were sacrificed at 1,2, 6 and 12 h after treatment. Ca2+ ATPase activity in synaptic membrane fractions was assayed by estimation of inorganic phosphate. PCP inhibited the Ca2+ ATPase in vitro in a concentration dependent manner with significant effect at 50 and 100 microM. A significant time-dependent reduction of the Ca2+ ATPase activity was evident in vivo. As early as 2 h after the treatment of rats with PCP the ATPase activity was significantly reduced. The reduction of Ca2+ ATPase observed even at 12 h after treatment suggesting a prolonged presence of the drug in the brain tissue. Further, kinetic studies in vitro indicated PCP to be a competitive inhibitor of Ca2+ ATPase with respect to the substrate, ATP. The present findings indicate that PCP inhibits synaptic membrane Ca2+ ATPase thus altering cellular Ca2+ homeostasis in CNS which may partially explain the pharmacological effects of the drug and/or its neurotoxicity.

Phencyclidine block of Ca2+ ATPase in rat heart sarcoplasmic reticulum.

Phencyclidine hydrochloride (PCP) also known as Angel Dust is a very potent psychotomimetic drug of abuse. Besides its central nervous system (CNS) effects PCP produces a number of adverse effects in a variety of tissues including the cardiovascular system. Since PCP is known to alter the cellular calcium homeostasis the present studies were initiated to determine the changes in cardiac Ca2+ ATPase activity in rats treated with PCP. For in vitro studies the cardiac sarcoplasmic reticulum (SR) fractions prepared from normal rats were incubated with 25, 50 and 100 microM PCP and the enzyme activities were estimated. Whereas, for in vivo studies the cardiac SR fractions prepared from rats treated with PCP (10 mg/kg body wt. single dose, intra-peritoneally (i.p.)) and sacrificed at different time intervals were used. PCP reduced the Ca2+ ATPase activity significantly both in vitro and in vivo. A 50% inhibition of the enzyme activity was obtained with 100 microM PCP in vitro. A significant reduction of SR Ca2+ ATPase was also evident as early as 1 h after treatment of rats with PCP. The reduction of Ca2+ ATPase activity in SR was irreversible even at 12 h after treatment. The in vitro kinetic studies revealed that PCP was found to be a competitive inhibitor of Ca2+ ATPase with respect to the substrate, ATP, and non-competitive with respect to Ca2+ activation. These results indicate that PCP alters the myocardial Ca2+ homeostasis by inhibiting the Ca2+ ATPase in cardiac SR in rats. Inhibition of SR Ca2+ ATPase may result in the impairment of contraction and relaxation coupling processes in the myocardium.

Marine biomolecules inhibit rat brain nitric oxide synthase activity.

A large number of substances of medical importance have been isolated from marine flora and fauna and their chemical structures were elucidated. Among the many compounds isolated in our laboratories only two compounds were identified as neurotoxins as they produced depolarizing effects in nerve fibers. The Xestospongin D and Araguspongin C, isolated and purified to 100% from sponge, Haliclona exigua were tested for their effects on rat brain nitric oxide synthase (NOS) activity in vitro. The results showed that NOS activity was significantly inhibited in a concentration and time dependent manner with an estimated IC50 of 31.5 and 46.5 microM for Xestospongin D and Araguspongin C, respectively, and the maximum inhibition occurred within 3 min of incubation. To explore the mechanism of action of these compounds on NOS, we have conducted kinetic studies with L-arginine, NADPH and Ca2+ in the presence of IC50 concentrations of these two compounds. The maximum velocity (Vmax) and enzyme constant (Km) were calculated using the Michaelis Menten equation. The results show that both compounds are competitive inhibitors of NOS with the substrate, L-arginine and uncompetitive with NADPH and free Ca2+. The NOS inhibition by these two compounds was similar to N omega-nitro-L-arginine methylester (L-NAME), a known inhibitor of NOS. These results suggest that the marine biomolecules Xestospongin D and Araguspongin C are in vitro modulators of neuronal NOS.

Reduced immunoreactivity to calcium-binding proteins in Purkinje cells precedes onset of ataxia in spinocerebellar ataxia-1 transgenic mice.

Earlier we have shown alterations in immunoreactivity (IR) to the calcium-binding proteins parvalbumin (PV) and calbindin D-28k (CaB) in surviving Purkinje cells of patients with spinocerebellar ataxia-1 (SCA-1). In the present study we determined PV and CaB expression (by immunohistochemical and immunoblot analyses) in Purkinje cells of transgenic mice (TM) expressing the human SCA-1 gene with an expanded (line B05) and normal (line A02) CAG tract, as well as in age-matched nontransgenic mice (nTM). Heterozygotes in the B05 line develop progressive ataxia beginning around 12 weeks of age. A02 animals are phenotypically indistinguishable from wild-type (nontransgenic) animals. In the cerebella of 8-, 9-, and 12-week-old TM-B05 there was a progressive decrease in PV IR in Purkinje cells compared with nTM and TM-A02. Parvalbumin immunostaining in interneurons was well preserved in all groups. A progressive decrease was also observed in CaB IR in Purkinje cells of 8-, 9-, and 12-week-old TM-B05. Cerebellar Purkinje cells of 6-week-old TM-B05, which exhibit no ataxia and even lack demonstrable Purkinje cell loss, also revealed reduction in PV IR. This change was matched by a significant decrease in the amount of cerebellar PV in 6-week-old TM-B05 as determined by Western blot analysis. Calbindin D-28K immunohistochemistry did not detect any marked changes in CaB IR within Purkinje cells at 4 weeks. However, at 6 weeks immunostaining and immunoblot analysis revealed a significant decrease in CaB in TM-B05 compared with controls. These data suggest that decreased levels of calcium-binding proteins in Purkinje cells in SCA-1 transgenic mice may cause alteration in Ca2+ homeostasis.

Decreased parvalbumin immunoreactivity in surviving Purkinje cells of patients with spinocerebellar ataxia-1.

The distribution of two calcium-binding proteins, calbindin D28k (CaBP) and parvalbumin (PV), was investigated by immunohistochemistry in the brains of three individuals dying of nonneurologic illness and three patients with spinocerebellar ataxia-1 (SCA-1). SCA-1 has recently been proven to be due to an unstable CAG repeat mutation on chromosome 6. In the cerebellum of control individuals the Purkinje cells showed strong immunoreactivity to CaBP. Other cells were CaBP-negative. Parvalbumin was highly localized to Purkinje, basket, stellate, and Golgi cells. All surviving Purkinje cells in SCA-1 were strongly immunoreactive to CaBP. The number of PV-immunoreactive Purkinje cells was markedly reduced in SCA-1. In addition, there was a significant decrease in the intensity of PV immunostaining within the individual Purkinje cells compared with controls. However, in the hippocampus, temporal cortex, and lateral geniculate scattered PV-positive neurons were seen in SCA-1 patients, similar to those in controls. The present results suggest that the decreased PV-immunoreactivity in the surviving Purkinje cells in SCA-1 may reflect biochemical alterations preceding Purkinje cell degeneration.

Increase in brain nitric oxide synthase activity in daunorubicin-treated rats.

Anthracyclines such as daunorubicin are very effective anticancer agents. These drugs are known to cause side effects including cardiotoxicity. Anthracyclines are neurotoxic to laboratory animals. Nitric oxide is a novel and very important chemical messenger in the brain. However, at higher levels, nitric oxide causes well defined neurotoxicity. Therefore, we determined nitric oxide synthase activity in rat brain after daunorubicin treatment in an effort to explain the neurotoxicity produced by anthracyclines. Male Sprague-Dawley rats were treated with different subcutaneous doses of daunorubicin (0.1-4.0 mg/kg/week for five weeks) while control animals were injected with phosphate buffered saline. There was a significant increase (80%) of nitric oxide synthase activity in daunorubicin-treated animals as compared to controls. This activity was inhibited by N-monomethyl-L-arginine (NMMA), nitroarginine, N-6-aminohexyl-5-chloro-1-napthalene sulfonamide (W-7), a calmodulin antagonist, suggesting that the nitric oxide synthase activity is calmodulin dependent. Further, our in vitro studies demonstrated that daunorubicin interacted with calmodulin as measured by N-phenyl-1-napthylamine (NPN) fluorescence. These results indicate that daunorubicin increases nitric oxide synthase activity in rat brain which may increase the levels of nitric oxide. The increased levels of nitric oxide may cause neurotoxicity. Our results further indicate that daunorubicin interacts with calmodulin and enhances nitric oxide synthase activity which is dependent on calmodulin.

Developmental changes in cerebellar endothelin-1 receptors in the neurologic mouse lurcher mutant.

In order to elucidate the role of neuromodulator endothelin-1 (ET-1) in cerebellar degeneration we determined ET-1 receptor binding in the control and lurcher mice cerebella at different postnatal (P) ages. The lurcher mutant is a suggested animal model for human cerebellar ataxias. The cerebellar membranes were prepared from 5 to 20 day old lurcher mice and littermate controls. ET-1 receptor binding was determined using radioligand assay and affinity cross-linking. There was a significant decrease in [125I]ET-1 receptor binding with increasing postnatal age in the control and lurcher mice cerebella. Lurcher mice showed a significant increase in [125I]ET-1 receptor binding at P 9 to P 20 as compared with the controls. Whereas, at P5 to P7 no significant change was observed. By autoradiography, ET-1 receptors were localized in tile granule cell layer of 15 day old control and lurcher mice cerebella. Whereas, the lUrcher mice showed an increase in [125I]ET-1 binding in the cerebellum as compared with the littermate controls. The results of the present study suggest that the increase in ET-1 receptor binding as early as P9 (when neuronal loss is minimal) in the lurcher mutant may reflect upregulation of ET-1 receptors in the degenerating neurons. At P 15 to P 20, when about 50% to 75% neurons are lost respectively, these changes may also reflect astrogliosis.

Daunomycin inhibits insulin-like growth factor I-dependent protein tyrosine phosphorylation.

The effect of the antitumor antibiotic daunomycin (DN) was studied on insulin-like growth factor I (IGF-I)-dependent protein tyrosine phosphorylation. DN was found to inhibit IGF-I-dependent phosphorylation of the artificial substrate poly(Glu:Tyr)4:1 by intrinsic IGF-I receptor kinase either from mouse cerebellum or from rat spinal cord. IGF-I-dependent autophosphorylation of the IGF-I receptor was also inhibited as a function of DN concentration (10-100 microM). However, DN at 200 microM concentration had minimal effect on protein kinase C dependent phosphorylation. The IGF-I-dependent protein tyrosine phosphorylation of endogenous proteins of the rat spinal cord was also inhibited by 50 microM DN. The altered IGF-I-dependent protein tyrosine phosphorylation by DN may partially explain its mechanism of action as an antitumor agent. These observations may also explain the neurotoxic effects of DN.

Effects of high cholesterol and n-3 polyunsaturated fish oil diets on tissue and serum lipid composition in male rats.

The correlation between dietary cholesterol, high plasma lipids and cardiovascular disease is well recognized in many species. The purpose of the present study was to examine the effects of high cholesterol and moderately high fat intake of n-3 polyunsaturated fish oil diets on serum lipids in male rats. Male rats were fed either 21% menhaden oil (Control) or 21% menhaden oil with high (2%) cholesterol (MOC) for eight weeks. Whole blood was collected, and analyzed spectrophotometrically for serum cholesterol, triglycerides and lipoproteins. The selected tissues were carefully removed, weighed and analyzed for lipid profiles. The aortas were removed and lipogenesis determined. The results showed that except for spleen the total percent lipid content of heart, lung, liver, adrenal, kidney and brain was not affected in the MOC group. The percent fat content of spleen but not the weight was elevated by 4 fold compared to control. The hematocrit values in the MOC group were unaltered. Serum cholesterol was elevated by 62%, whereas the serum triglycerides and HDL cholesterol were unaltered in MOC group when compared to the MO control. High cholesterol feeding did not affect aortic lipogenesis in the MOC group compared to the control. These results suggest that cholesterol feeding along with n-3 polyunsaturated fish oil diet did not attenuate the anti-atherosclerotic effects of fish oil with the exception of serum cholesterol.

Very low density and low density lipoproteins induce nitric oxide synthesis in macrophages.

The effect of lipoproteins on rat peritoneal macrophage nitric oxide (NO) production was studied. Very low density (VLDL) and low density (LDL) but not high density lipoprotein (HDL) stimulated NO production at 12, 24 and 48 hr of incubation with macrophages. Forty-eight hour incubation of macrophages with VLDL or LDL increased NO production from 10.8 nmoles/10(6) cells in control to 249 and 60 nmoles/10(6) cells, respectively. VLDL-induced nitric oxide production was 825 nmoles/10(6) after 12 days of incubation. VLDL by itself or in the presence of LDL and HDL induced similar levels of NO in 24 hrs. Lipoproteins were without effect on NO, when incubated with macrophages in the absence of L-arginine and in the presence of nitric oxide synthase inhibitor monomethyl-L-arginine (L-NMMA). Oxidized VLDL was as effective as native form in the induction of macrophage NO, whereas oxidized LDL did not induce NO production. Further, the presence of 100 microM vitamin E in the incubation had no effect on VLDL- and LDL-induced NO production. Preincubation of macrophages with VLDL and LDL for 3hr or 6hrs activated macrophages to generate NO. These effects of lipoproteins could be of interest in the pathophysiology of lipoproteins and NO mediated diseases.

Lead alters inositol polyphosphate receptor activities: protection by ATP.

Receptor-mediated phosphoinositide signaling pathway which generates a variety of second messengers is regulated by intracellular free Ca2+ concentrations. Since toxic metal cations like Pb2+ are known to alter Ca(2+)-dependent processes, the present study was initiated to study the effects of Pb2+ on inositol 1,4,5-trisphosphate (InsP3) and inositol 1,3,4,5-tetrakisphosphate (InsP4) receptor binding and InsP3-mediated Ca(2+)-release. Rat cerebellar membrane and microsomal fractions were incubated with various concentrations of Pb2+ (0.01-100 microM). Pb2+ significantly stimulated [3H]-InsP3 and [3H]-InsP4 receptor binding (EC50 22.7 and 13.5 microM respectively) as a function of metal concentrations. However, InsP3-mediated Ca2+ release, determined by measuring the changes in fluorescence intensity of Fura-2, was significantly inhibited by varying concentrations of Pb2+. Re-uptake of Ca2+ into the microsomes was also inhibited by Pb2+. A significant inhibition of microsomal Ca(2+)-pump by micromolar concentration of Pb2+ was also observed. ATP at 5-1000 microM concentration range inhibited [3H]-InsP3 and [3H]-InsP4 binding to the specific receptors. [3H]-InsP4 receptor binding was more sensitive to ATP inhibition as compared to [3H]-InsP3 receptor binding. Furthermore, varying concentrations of ATP also inhibited Pb(2+)-mediated increase in [3H]-InsP3 and [3H]-InsP4 receptor binding. The kinetic analysis of ATP effect on Pb(2+)-stimulated [3H]-InsP4 receptor binding revealed non-competitive type of interaction. The results of the present study suggest that Pb2+ may be increasing the binding of [3H]-InsP3 and [3H]-InsP4 to the specific receptors by modulating the conformation of the receptor sites. ATP may be playing a protective role in Pb2+ induced alteration of the receptor sites.

Effect of carbon tetrachloride on inositol 1,4,5-trisphosphate dependent and independent regulation of rat brain microsomal Ca2+ flux.

Carbon tetrachloride (CCl4) is a highly toxic industrial solvent with pronounced effects on the liver and brain. CCl4 is enzymatically cleaved to produce free radicals which attack membrane components, including proteins. Earlier reports indicated that CCl4 affects Ca(2+)-regulated events in the brain. Hence, the present study was initiated to determine whether CCl4 affects inositol 1,4,5-trisphosphate (IP3) receptor binding, free-Ca2+ movements across the microsomal membrane and protein kinase C (PKC) activity in rat brain, since IP3, Ca2+ and PKC are known to be involved in signal transduction. [3H]IP3 binding, free-Ca2+ movements and 45Ca2+ uptake were determined using rat brain microsomes and PKC activity was determined in the cytosolic fraction. CCl4 in vitro decreased [3H]IP3 binding to microsomes. IP3 mediated Ca2+ release from microsomes was inhibited and also the reuptake of IP3-released Ca2+ into microsomes was decreased in the presence of CCl4. CCl4 at concentrations < 2 microM independently released Ca2+ from microsomes. Uptake of total Ca2+ into microsomes was inhibited by CCl4 as observed with 45Ca(2+)-uptake studies. CCl4 at 1 microM inhibited PKC activity by 50%. Thus, perturbations in the binding of IP3 to its receptor sites, changes in the Ca2+ flux across the microsomal membrane and modulation of PKC activity by CCl4 in vitro suggested that CCl4 may exert neurotoxicity by altering signal transduction pathways.

Decreased insulin-like growth factor I-mediated protein tyrosine phosphorylation in human olivopontocerebellar atrophy and lurcher mutant mouse.

We examined insulin-like growth factor I (IGF-I)-dependent phosphorylation and protein tyrosine kinase (PTK) activity in cerebellar cortex of normal humans, patients with olivopontocerebellar atrophy (OPCA) ("C" kindred) and in lurcher mutant mouse, a suggested animal model for OPCA. PTK activity and IGF-I-dependent protein tyrosine phosphorylation was significantly reduced in cerebellar cortex of human OPCA patients as compared to the normal controls. Immunoblot analysis also demonstrated a decrease in cerebellar 80 kDa phosphotyrosine protein in these patients. By autoradiography, IGF-I receptors were localized in the molecular layer of 30-day-old control and lurcher mutant mice cerebella. However, the lurcher mutant mice showed a decrease in [125I]-IGF-I binding in the molecular layer as compared to the littermate controls. The IGF-I receptor autophosphorylation was also markedly reduced in 15-day- and 22-day-old lurcher cerebella. These results suggest that the process of cerebellar degeneration in human OPCA and lurcher mutant mouse may be associated with altered IGF-I receptor binding and protein tyrosine phosphorylation.