Biomedical subjects

Thomas J Abbruscato

Publications and source records attributed to Thomas J Abbruscato.

11 recordsLinked to original sources

PubMed2006

Angiotensin II induces phosphorylation of glucose-regulated protein-75 in WB rat liver cells.

Studies in vascular smooth muscle cells suggest that, angiotensin II (Ang II)-mediated cellular response requires transactivation of epidermal growth factor receptor (EGF-R), and involves tyrosine phosphorylation of caveolin-1. Here we demonstrate that, exposure of WB rat liver cells to Ang II does not cause transactivation of EGF-R, but did rapidly activate p42/p44 mitogen-activated protein (MAP) kinases suggesting that it activates MAP kinases independent of EGF-R transactivation. We observed that the phospho-specific anti-caveolin-1 antibody detected a tyrosine phosphorylated, 75kDa protein in Ang II-treated cells which we identified as glucose regulated protein-75 (GRP-75). Phosphoamino acid analysis showed that Ang II induced its phosphorylation at tyrosine, serine and threonine residues and was localized to the cytoplasm. The ability of Ang-II to induce GRP-75 phosphorylation suggests that it may play a role in the protection of cytoplasmic proteins from the damaging effect of oxidative stress known to be produced during Ang-II induced signaling.

Amino Acid Sequence↗

PubMed2006

Nicotine exposure does not alter plasma to brain choline transfer.

Acute and chronic nicotine exposure in rats is associated with an increase in brain acetylcholine (ACh) transmission. The acquisition of choline for neuronal ACh synthesis occurs primarily via two pathways; first, free choline is transported from the blood across the blood-brain barrier (BBB) and/or second, from synaptic choline generated by either hydrolysis of non-bound ACh or membrane phosphatidylcholine catabolism. To determine if nicotine-induced cholinergic demand is associated with increased choline transport rates into brain, we measured BBB choline transport in naïve and S-(-) nicotine exposed rats (acute and chronic, 4.5 mg/kg/d for 1, 14, 21 and 28 d; osmotic minipumps) using the in situ rat brain perfusion technique. No significant changes in choline uptake after acute or chronic nicotine exposure were observed in whole brain or cortex. Of considerable interest was a significant decrease in regional brain choline uptake measured in the hippocampus after chronic nicotine exposure (28 d). Our data suggest that the increased ACh transmission observed after nicotine exposure does not correlate with increased blood-to-brain transfer of choline. Considering these data and previous literature reports, we propose that the additional free choline required under conditions of nicotine exposure (for ACh synthesis) is primarily recruited from membrane phospholipid metabolism.

Animals↗

PubMed2006

Protein kinase C family members as a target for regulation of blood-brain barrier Na,K,2Cl-cotransporter during in vitro stroke conditions and nicotine exposure.

PURPOSE: The aim of the study is to identify specific protein kinase C (PKC) isoforms involvement in K(+) transport mediated at altered blood-brain barrier (BBB) response to stroke conditions with prior nicotine exposure, which provides ways to intervene pharmacologically in PKC-mediated molecular pathways that could lead to effective treatment for smoking stroke patients. METHODS: Changes in PKC isoform levels were studied in the cytosolic and membrane fractions of bovine brain microvessel endothelial cells subjected to stroke conditions as well as nicotine/cotinine exposure. Furthermore, abluminal Na,K,2Cl-cotransporter (NKCC) activity regulated by specific conventional PKC isoform activators and inhibitors was investigated using rubidium ((86)Rb) uptake studies. RESULTS: Membrane-bound PKCalpha, PKCbetaI, and PKCepsilon levels were increased after 6 h hypoxia/aglycemia, and this was attenuated by 24-h nicotine/cotinine exposure. Interestingly, membrane-bound PKCgamma protein level was decreased after 6 h hypoxia/aglycemia and increased by 24-h nicotine/cotinine exposure. (86)Rb uptake studies showed that basolateral NKCC activity was down-regulated by both a conventional PKC inhibitor and specific inhibitors for PKCalpha, PKCbeta, and PKCvarepsilon and was up-regulated by an activator of conventional PKCs during 6-h hypoxia/aglycemia treatment. CONCLUSION: Specific PKC inhibitors or activators might be designed to individualize stroke therapies and improve health outcome for smokers by rebalancing ion transport into and out of the brain.

Animals↗

PubMed2005

Tobacco smoke chemicals attenuate brain-to-blood potassium transport mediated by the Na,K,2Cl-cotransporter during hypoxia-reoxygenation.

Smoking tobacco, including cigarettes, has been associated with an increased incidence and relative risk for cerebral infarction in both men and women. Recently, we have shown that nicotine and cotinine attenuate abluminal (brain facing) K(+) uptake mediated by the Na,K,2Cl-cotransporter (NKCC) in bovine brain microvessel endothelial cells (BBMECs) after hypoxic/aglycemic exposure (stroke conditions). The purpose of the current study was to explore the effects of nicotine and tobacco smoke chemicals on K(+) movement through the blood-brain barrier during both hypoxia/aglycemia and reoxygenation. BBMECs were exposed to nicotine/cotinine, nicotine-containing cigarette smoke extract (N-CSE), or nicotine-free cigarette smoke extract (NF-CSE) in quantities designed to mimic plasma concentrations of smokers. Stroke conditions were mimicked in vitro in BBMECs through 6 h of hypoxia/aglycemia with or without 12 h of reoxygenation, after which NKCC-mediated K(+) uptake and paracellular integrity were measured with (86)Rb and [(14)C]sucrose, respectively. In addition, K(+) concentrations in brain extracellular fluid were estimated in (86)Rb-injected rats that were administered nicotine, N-CSE, or NF-CSE and on whom global ischemia/reperfusion by in vivo four-vessel occlusion was performed. Both in vitro and in vivo paradigms showed nicotine, the major alkaloid present in tobacco smoke, to be the determining factor of an inhibited response of abluminal NKCC in BBMECs during and after stroke conditions. This was measured as a decrease in abluminal brain endothelial cell NKCC activity and as an increase in brain extracellular K(+) concentration measured as the brain extracellular fluid (86)Rb/plasma ratio after in vivo four-vessel occlusion with reperfusion.

Animals↗

PubMed2005

NGP1-01, a lipophilic polycyclic cage amine, is neuroprotective in focal ischemia.

NGP1-01, a member of the pentacycloundecylamine cage compound family, was recently shown to exhibit both NMDA receptor channel blocking and L-type calcium channel antagonism activity. In the present study, focal ischemia was induced in mice by permanent middle cerebral artery occlusion (MCAO) to test for potential neuroprotective properties of the compound. In female CD-1 mice injected 30 min before MCAO, NGP1-01 (20 mg/kg) reduced infarct area by 42.6% (P < 0.05) compared to vehicle-treated controls as visualized by 2,3,5-triphenyltetrazolium chloride (TTC) staining. Concomitantly, NGP1-01 reduced brain swelling by 78.3% (P < 0.001), compared to vehicle (DMSO) treated controls. These data identify NGP1-01 and related compounds as potential lead structures to develop neuroprotective compounds based on a dual mechanism of action.

Animals↗

PubMed2005

Methods for assessing drug-related anticholinergic activity.

The geriatric population is a large consumer of both prescription and over-the-counter drugs. Positive outcomes from drugs depend on the delicate interplay between therapeutic and adverse effects. This relationship becomes tortuous with simultaneous administration of several drugs. Numerous concomitant drug therapies may be essential for providing quality patient care but may also increase the possibility of an adverse drug event. Increasing sensitivity to drug effects in the geriatric population also creates concern over adverse effects. Drugs that possess anticholinergic properties are especially worrisome, as these properties may manifest as hazardous physiologic and psychological adverse drug events. Consequently, clinicians strive to minimize total drug exposure to agents possessing anticholinergic properties in elderly patients. A review of the literature revealed four methods that might help clinicians systematically reduce or eliminate potentially offending anticholinergic drugs. Each of the four has merits and limitations, with no ideal evidence-based approach used. Three of the four methods described have research utility; however, only one of the methods is clinically useful.

Cholinergic Antagonists↗

PubMed2004

Nicotine increases in vivo blood-brain barrier permeability and alters cerebral microvascular tight junction protein distribution.

The blood-brain barrier (BBB) is critical to the health of the central nervous system. The BBB is formed primarily by the presence of tight junctions (TJ) between cerebral microvessel endothelial cells. In light of the known effects of nicotine on endothelial cell biology, the specific effects of nicotine on the in vivo BBB were examined. Using in situ brain perfusion, it was found that continuous administration of nicotine (4.5 mg free base x kg(-1) x day(-1)) for 1 and 7 days led to increased permeability of the BBB to [14C]-sucrose without significant changes in its initial volume of distribution. The expression and distribution of the TJ-associated proteins actin, occludin, claudin-1, -3, and -5, and ZO-1 and -2 were analyzed by Western blot and immunofluorescence microscopy. Though no changes in total protein expression were observed, nicotine treatment was associated with altered cellular distribution of ZO-1 and diminished junctional immunoreactivity of claudin-3. It is proposed that nicotine leads to changes in BBB permeability via the modulation of TJ proteins.

Animals↗

PubMed2004

Regulation of blood-brain barrier Na,K,2Cl-cotransporter through phosphorylation during in vitro stroke conditions and nicotine exposure.

Nicotine, a major constituent of tobacco smoke, has important effects on brain recovery after focal ischemia (Wang et al., 1997). The purpose of this work is to systematically test the effects of nicotine during stroke conditions on blood-brain barrier (BBB) potassium transport, protein expression of the Na,K,2Cl-cotransporter (NKCC), and cell signaling pathways that control NKCC activity at the BBB. Confluent bovine brain microvessel endothelial cells (BBMECs) were exposed to both a hypoxic/aglycemic (H/A) environment to model BBB function during stroke conditions and nicotine and cotinine (N/C) to model plasma levels seen in smokers. BBMECs exhibit both Na,K-ATPase and NKCC activity (60 and 34 nmol/min/g, respectively) that contribute to 98% of the K(+) uptake in cultured endothelial cells. An adaptive up-regulation of NKCC activity was identified to occur on the basolateral surface of the BBB after in vitro stroke conditions. Twenty-four hours of N/C exposure, at doses equivalent to plasma levels of smokers, combined with 6 h of H/A, reduced NKCC protein expression and total NKCC activity (shown by bumetanide-sensitive (86)RB uptake) compared with 6 h of H/A alone (P < 0.01). Basolateral K(+) transport was found to be modulated by nicotinic acetylcholine receptors expressed at the BBB. NKCC activity on the basolateral surface of the BBB is controlled by an ongoing phosphorylation/dephosphorylation processes. We have identified a potential mechanism in altered BBB response to stroke conditions with prior N/C exposure directly implicating damage to brain-to-blood K(+) transport mediated at the BBB and perhaps neuronal recovery after stroke.

Animals↗

PubMed2004

Cyclodextrins in nasal delivery of low-molecular-weight heparins: in vivo and in vitro studies.

PURPOSE: To test the hypothesis that cyclodextrins reversibly enhance nasal absorption of low-molecular-weight heparins (LMWHs) and to investigate the mechanisms by which cyclodextrins enhance LMWH absorption via the nose. METHODS: Absorption of LMWHs was studied by measuring plasma anti-factor Xa activity after nasal administration of various LMWH formulations to anesthetized rats. In vivo reversibility studies were performed to investigate if the effects of cyclodextrins are reversible and diminish with time. The absorption-enhancing mechanisms of cyclodextrins were investigated in cell culture model. The transport of enoxaparin and mannitol, changes in transepithelial electrical resistance (TEER), and distribution of tight junction protein ZO-1 were investigated. RESULTS: Formulations containing 5% dimethyl-beta-cyclodextrin (DMbetaCD) produced the highest increase in the bioavailability of LMWH preparations tested. In vivo reversibility studies with 5% DMbetaCD showed that the effect of the absorption enhancer at the site of administration diminished with time. Transport studies using 16HBE14o(-) cells demonstrated that the increase in the permeability of enoxaparin and mannitol, reduction in TEER, and the changes in the tight junction protein ZO-1 distribution produced by 5% DMbetaCD were much greater than those produced by beta-cyclodextrin (betaCD) or hydroxyl-propyl-beta-cyclodextrin (HPbetaCD). CONCLUSIONS: Of the cyclodextrins tested, DMbetaCD was the most efficacious in enhancing absorption of LMWHs both in vivo and in vitro. The study also suggests that cyclodextrins enhance nasal drug absorption by opening of cell-cell tight junctions.

Administration, Intranasal↗

PubMed2003

In vivo and in vitro assessment of baseline blood-brain barrier parameters in the presence of novel nanoparticles.

PURPOSE: Nanoparticles have advantage as CNS drug delivery vehicles given they disguise drug permeation limiting characteristics. Conflicting toxicological data, however, is published with regard to blood-brain barrier integrity and gross mortality. METHODS: To address this issue two novel nanoparticle types: "emulsifying wax/Brij 78" and "Brij 72/Tween 80 nanoparticles were evaluated in vivo for effect on cerebral perfusion flow, barrier integrity, and permeability using the in situ brain perfusion technique. Additional evaluation was completed in vitro using bovine brain microvessel endothelial cells for effect on integrity, permeability, cationic transport interactions, and tight junction protein expression. RESULTS: In the presence of either nanoparticle formulation, no overall significant differences were observed for cerebral perfusion flow in vivo. Furthermore, observed in vitro and in vivo data showed no statistical changes in barrier integrity, membrane permeability, or facilitated choline transport. Western blot analyses of occludin and claudin-1 confirmed no protein expression changes with incubation of either nanoparticle. CONCLUSIONS: The nanoparticle formulations appear to have no effect on primary BBB parameters in established in vitro and in vivo blood-brain barrier models.

Animals↗

PubMed2002

Nicotine and cotinine modulate cerebral microvascular permeability and protein expression of ZO-1 through nicotinic acetylcholine receptors expressed on brain endothelial cells.

The blood-brain barrier (BBB) adapts to a variety of pathological processes. Little is known about the effects of nicotine exposure on BBB function and the ability to adapt to stroke conditions. We have demonstrated, using a well-characterized in vitro BBB model, bovine brain microvessel endothelial cells (BBMEC) model, that nicotine and its major metabolite, cotinine, modulate BBB integrity by opening the paracellular route of solute entry into the brain. Additionally, nicotine and cotinine together increase the permeability change observed after 6 h of hypoxia/aglycemia, an in vitro model of stroke. This has important implications for how the BBB initially adapts to stroke in an environment that is previously exposed to nicotine. Nicotine and cotinine exposure also resulted in reduced ZO-1 immunoreactivity (tight junctional protein) that occurred in a time-dependent manner. Interestingly, attenuation of bovine brain microvessel endothelial cell (BBMEC) ZO-1 protein expression was reversed using 10 nM BGT, an alpha7 nicotinic acetycholine receptor (nAChR) antagonist, suggesting that the effects of nicotine on BBMEC protein expression of ZO-1 protein are mediated by nAChR expressed on brain endothelial cells. In addition to alpha7, we found that BBMEC also contain positive immunoreactivity for the alpha3, alpha5, beta2, beta3 nAChR subunit. Both alpha7 and beta2 nAChR subunit protein levels decreased with prior nicotine and cotinine exposure. These data provide evidence that nicotine and cotinine alter BBB permeability and tight junctional protein expression of ZO-1, thereby altering the BBB response to stroke conditions. These changes in brain endothelial cell paracellular permeability are believed to be associated with nicotine binding to nAChRs present at the BBB.

Animals↗