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Biomedical subjects

M L Weber

Publications and source records attributed to M L Weber.

At least 19 recordsLinked to original sources
PubMed2005

Mediation of highly concentrative uptake of pregabalin by L-type amino acid transport in Chinese hamster ovary and Caco-2 cells.

Pregabalin (PGB) is a novel drug under development for the treatment of epilepsy, neuropathic pain, fibromyalgia, and generalized anxiety disorder. In this study, we investigated PGB transport in rats, mammalian cell lines, and Xenopus laevis oocytes. In contrast to gabapentin (GBP), PGB absorption in rats showed unique linear pharmacokinetics. PGB entered CHO and Caco-2 cells predominately via Na(+)-independent processes. Uptake of PGB was mutually exclusive with leucine, GBP and 2-aminobicyclo(2,2,1)heptane-2-carboxylic acid, the substrates preferential for system L. The preloaded PGB in CHO cells was exchangeable with leucine, but at a lower exchange rate than that of leucine and GBP. Dixon plots showed competitive inhibition of leucine uptake by PGB, with a K(i) value very close to the K(m) value for PGB uptake (377 versus 363 microM). At an extracellular concentration of 300 microM, the intracellular PGB concentration in CHO cells reached 1.5- and 23-fold higher than that of GBP and leucine, respectively. In contrast, at clinically relevant concentrations, PGB seemed not to interact with GABA transport in GAT1, GAT2, and GAT3 cell lines, system y(+), b(0,+), B(0,+), and B(0) transport activities in Caco-2 and NBL-1 cells, and the b(0,+)-like transport activity in rBAT cRNA-injected X. laevis oocytes. Taken together, these results suggest that L-type transport is the major transport route for PGB and GBP uptake in mammalian cells. The differential affinity of PGB and GBP at L-type system leads to more concentrative accumulation of PGB than GBP, which may facilitate PGB transmembrane absorption in vivo.

Amino Acid Transport System y+↗
PubMed2001

Effects of ion channel blockade on the distribution of Na, K, Ca and other elements in oxygen-glucose deprived CA1 hippocampal neurons.

The pathophysiology of brain ischemia and reperfusion injury involves perturbation of intraneuronal ion homeostasis. To identify relevant routes of ion flux, rat hippocampal slices were perfused with selective voltage- or ligand-gated ion channel blockers during experimental oxygen-glucose deprivation and subsequent reperfusion. Electron probe X-ray microanalysis was used to quantitate water content and concentrations of Na, K, Ca and other elements in morphological compartments (cytoplasm, mitochondria and nuclei) of individual CA1 pyramidal cell bodies. Blockade of voltage-gated channel-mediated Na+ entry with tetrodotoxin (1 microM) or lidocaine (200 microM) significantly reduced excess intraneuronal Na and Ca accumulation in all compartments and decreased respective K loss. Voltage-gated Ca2+ channel blockade with the L-type antagonist nitrendipine (10 microM) decreased Ca entry and modestly preserved CA1 cell elemental composition and water content. However, a lower concentration of nitrendipine (1 microM) and the N-, P-subtype Ca2+ channel blocker omega-conotoxin MVIIC (3 microM) were ineffective. Glutamate receptor blockade with the N-methyl-D-aspartate (NMDA) receptor-subtype antagonist 3-(2-carboxypiperazin-4-yl) propyl-1-phosphonic acid (CPP; 100 microM) or the alpha-amino-3-hydroxy-5-methyl-4-isoazole propionic acid (AMPA) receptor subtype blocker 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 microM/100 microM glycine) completely prevented Na and Ca accumulation and partially preserved intraneuronal K concentrations. Finally, the increase in neuronal water content normally associated with oxygen-glucose deprivation/reperfusion was prevented by Na+ channel or glutamate receptor blockade. Results of the present study demonstrate that antagonism of either postsynaptic NMDA or AMPA glutaminergic receptor subtypes provided nearly complete protection against ion and water deregulation in nerve cells subjected to experimental ischemia followed by reperfusion. This suggests activation of ionophoric glutaminergic receptors is involved in loss of neuronal osmoregulation and ion homeostasis. Na+ channel blockade also effectively diminished neuronal ion and water derangement during oxygen-glucose deprivation and reperfusion. Prevention of elevated Nai+ levels is likely to provide neuroprotection by decreasing presynaptic glutamate release and by improving cellular osmoregulation, adenosine triphosphate utilization and Ca2+ clearance. Thus, we suggest that voltage-gated tetrodotoxin-sensitive Na+ channels and glutamate-gated ionotropic NMDA or AMPA receptors are important routes of ion flux during nerve cell injury induced by oxygen-glucose deprivation/reperfusion.

Animals↗
PubMed1999

Synthesis of a series of 4-benzyloxyaniline analogues as neuronal N-type calcium channel blockers with improved anticonvulsant and analgesic properties.

In this article, the rationale for the design, synthesis, and biological evaluation of a series of N-type voltage-sensitive calcium channel (VSCC) blockers is described. N-Type VSCC blockers, such as ziconotide, have shown utility in several models of stroke and pain. Modification of the previously reported lead, 1a, led to several 4-(4-benzyloxylphenyl)piperidine structures with potent in vitro and in vivo activities. In this series, the most interesting compound, (S)-2-amino-1-{4-[(4-benzyloxy-phenyl)-(3-methyl-but-2-enyl)-amino]-p iperidin-1-yl}-4-methyl-pentan-1-one (11), blocked N-type calcium channels (IC(50) = 0.67 microM in the IMR32 assay) and was efficacious in the audiogenic DBA/2 seizure mouse model (ED(50) = 6 mg/kg, iv) as well as the antiwrithing model (ED(50) = 6 mg/kg, iv). Whole-cell voltage-clamp electrophysiology experiments demonstrated that compound 11 blocked N-type Ca(2+) channels and Na(+) channels in superior cervical ganglion neurons at similar concentrations. Compound 11, which showed superior in vivo efficacy, stands out as an interesting lead for further development of neurotherapeutic agents in this series.

Acoustic Stimulation↗
PubMed1999

N,N-dialkyl-dipeptidylamines as novel N-type calcium channel blockers.

Selective N-type voltage sensitive calcium channel (VSCC) blockers have shown utility in several models of stroke and pain. We are especially interested in small molecule N-type calcium channel blockers for therapeutic use. Herein, we report a series of N,N-dialkyl-dipeptidylamines with potent functional activity at N-type VSCCs and in vivo efficacy. The synthesis, SAR, and pharmacological evaluation of this series are discussed.

Animals↗
PubMed1999

Intercellular adhesion molecule-1 expression in endothelial cells is activated by cytomegalovirus immediate early proteins.

BACKGROUND: Human cytomegalovirus (HCMV) infection is associated with allograft vasculopathy and rejection. One potential mechanism is vascular injury from immunologically mediated processes. HCMV infection has been shown to increase the constitutive expression of intercellular adhesion molecule-1 (ICAM-1). The objective of this study was to determine the molecular basis of HCMV enhanced ICAM-1 gene expression in endothelial cells using human umbilical vein endothelial cells (HUVECs) as a model. METHODS: HUVECS were infected with HCMV virus and the level of ICAM-1 mRNA determined over time. HUVECS were then transiently transfected with plasmid constructs containing ICAM-1 and HCMV immediate early (IE) gene sequences and the effect of IE proteins on ICAM-1 promoter expression determined. Antibodies to cytokines known to be affected by HCMV IE proteins or to modulate ICAM-1 expression were added to determine if cytokines were mediating ICAM-1 expression. RESULTS: Infection of HUVECs with HCMV resulted in a rapid rise in ICAM-1 mRNA levels, suggesting that the viral IE proteins were involved in gene activation. The HCMV IE1 and IE2 proteins synergistically activated both transfected and endogenous ICAM-1 gene expression. The addition of antibodies to interleukin-1, tumor necrosis factor-a, transforming growth factor-beta, or interleukin-6 had no effect on the IE protein-mediated increase in ICAM-1 expression. Deletion analysis of the ICAM-1 gene promoter revealed that a minimum of 370 base pairs of 5' flanking sequences was required for maximal transactivation by IE proteins; mutation analysis showed that an NFkappaB site at base pairs -187 to -178 was not required for promoter activation. CONCLUSIONS: These results demonstrate that HCMV regulates the heterologous ICAM-1 gene promoter in endothelial cells not via cellular cytokine production, but rather by a direct effect of IE proteins, and supports a model in which HCMV IE gene products interact with ICAM-1 promoter elements to increase gene expression.

Base Sequence↗
PubMed1999

Oxygen/glucose deprivation in hippocampal slices: altered intraneuronal elemental composition predicts structural and functional damage.

Effects of oxygen/glucose deprivation (OGD) on subcellular elemental composition and water content were determined in nerve cell bodies from CA1 areas of rat hippocampal slices. Electron probe x-ray microanalysis was used to measure percentage water and concentrations of Na, P, K, Cl, Mg, and Ca in cytoplasm, nucleus, and mitochondria of cells exposed to normal and oxygen/glucose deficient medium. As an early (2 min) consequence of OGD, evoked synaptic potentials were lost, and K, Cl, P, and Mg concentrations decreased significantly in all morphological compartments. As exposure to in vitro OGD continued, a negative DC shift in interstitial voltage occurred ( approximately 5 min), whereas general elemental disruption worsened in cytoplasm and nucleus (5-42 min). Similar elemental changes were noted in mitochondria, except that Ca levels increased during the first 5 min of OGD and then decreased over the remaining experimental period (12-42 min). Compartmental water content decreased early (2 min), returned to control after 12 min of OGD, and then exceeded control levels at 42 min. After OGD (12 min), perfusion of hippocampal slices with control oxygenated solutions (reoxygenation) for 30 min did not restore synaptic function or improve disrupted elemental composition. Notably, reoxygenated CA1 cell compartments exhibited significantly elevated Ca levels relative to those associated with 42 min of OGD. When slices were incubated at 31 degreesC (hypothermia) during OGD/reoxygenation, neuronal dysfunction and elemental deregulation were minimal. Results show that in vitro OGD causes loss of transmembrane Na, K, and Ca gradients in CA1 neurons of hippocampal slices and that hypothermia can obtund this damaging process and preserve neuronal function.

Animals↗
PubMed1997

Micellar electrokinetic capillary chromatography with laser-induced fluorimetric detection of amines in beer.

Capillary electrophoresis with simultaneous ultraviolet absorbance and laser-induced fluorescence detection is applied to identify and quantify selected amines in beer following derivatization with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. Quantitation was performed using the method of standard addition in order to avoid pH-dependent variations in the reactivity of the derivatizing agent with the added benefit of verifying peak identity. An inexpensive and "easy-to-use" on-column fiber optic fluorescence detection cell is described and implemented in the analysis.

Amines↗
PubMed1996

Cytoplasmic sequestration of p53 in cytomegalovirus-infected human endothelial cells.

Human umbilical vein endothelial cells were infected with human cytomegalovirus (HCMV) at a multiplicity of infection of 0.1 plaque-forming unit/cell and remained > 95% viable even after 10 days of infection. To induce apoptosis, control human umbilical vein endothelial cells and cells infected with HCMV for 3, 5, and 7 days were serum starved for 48 hours. Almost one-half of the uninfected cells lost viability after 48 hours of serum starvation whereas HCMV-infected cells were virtually unaffected (< 20% death, P < 0.05). Uninfected cells showed typical hallmarks of apoptosis, including unique morphological changes and DNA laddering. HCMV-infected cells, concomitant with their resistance to serum-starvation-induced death, displayed almost none of these characteristics. Active replication of HCMV was necessary for the anti-apoptotic effect, as cells treated with ultraviolet light-inactivated virus were not protected. p53, the G1/S phase cell cycle brake protein, was elevated in HCMV-infected cells. However, rather than accumulating in the nucleus, immunofluorescent and Western blot studies demonstrated remarkable and predominant cytoplasmic sequestration of p53 in HCMV-infected endothelial cells. Although HCMV proteins have already been shown to block apoptosis, we suggest that the aberrant subcellular pattern of p53 is the disturbed cellular mechanism that may be responsible for the anti-apototic properties of HCMV-infected cells. The selective resistance to apoptosis can be important during HCMV replication and may explain the oncogenic potential of HCMV as well as its pathogenic role in intimal-proliferation-mediated vascular diseases.

Cells, Cultured↗
PubMed1995

Hippocampal slices: glutamate overflow and cellular damage from ischemia are reduced by sodium-channel blockade.

We evaluated concentrations of excitatory amino acids released from slices into the superfusing solution and also evaluated extracellular field potential recordings and histological appearance of slice tissues to evaluate several sodium-channel modulating drugs as potential treatments for ischemia. The selective sodium-channel blocker tetrodotoxin (TTX, 1 microM) reduced glutamate release from deprivation of oxygen and D-glucose, while calcium-channel blockade was ineffective. Thus, during ischemia, we propose that glutamate may be released from the free cytosolic pool ('metabolic' glutamate) rather than by exocytosis. TTX (100-500 nM) and voltage-dependent sodium-channel blockers (phenytoin, 20-100 microM; lidocaine, 2-200 microM) each prevented damage to slices without blocking action potentials. The reduction of cellular depolarization and sodium loading during ischemia may explain the neuroprotective action of several sodium-channel modulating drugs in our in vitro studies and also in animal models.

Animals↗
PubMed1994

Damage from oxygen and glucose deprivation in hippocampal slices is prevented by tetrodotoxin, lidocaine and phenytoin without blockade of action potentials.

In vitro ischemia (IVI) was simulated with rat hippocampal slices in medium lacking D-glucose, equilibrated with 95% nitrogen, 5% carbon dioxide. Within 5-8 min, synaptic potentials disappeared and a DC negative shift (5-15 mV) occurred. Prolonged application of 95% oxygen and D-glucose 12 min later did not allow synaptic potentials to recover. Slices pretreated with sodium channel blocking drugs allowed synaptic potentials to recover after IVI. Tetrodotoxin (TTX, 100-600 nM), the anticonvulsant phenytoin (5.0 to 100 microM) and the local anesthetic lidocaine (2.0 to 200 microM) each delayed or prevented negative DC shifts from IVI. Histological examination showed that drug treatments also prevented CA1 pyramidal cell damage from IVI. Neuroprotection occurred without blocking synaptic potentials or presynaptic fiber volleys, suggesting relevance for treatment of brain ischemia.

Action Potentials↗
PubMed1993

Hypothalamic dysfunction in a child: a distinct syndrome? Report of a case and review of the literature.

We report the case of a 9-year-old girl with multiple problems due to hypothalamic dysfunction of obscure origin: apnoeic spells, behavioural problems, developmental delay, hypodipsia with bouts of hypernatraemia, episodes of spontaneous hypothermia, obesity, petit-mal seizures, non-progressive precocious puberty, absence of respiratory response to CO2 and probably insensitivity of hyposensitivity to pain. She also had hyperprolactinaemia and decreased human growth hormone secretion. Hypothyroidism of central origin and hyposecretion of cortisol were also present. Multiple brain CT-scans failed to reveal any tumour or other anatomical abnormality. Her clinical course was improved initially by treatment with clomipramine, but she died suddenly, and the autopsy failed to disclose any anatomical lesion. We compare this case with three similar previously reported cases.

Child↗
PubMed1993

Effect of temperature on synaptic function after reduced oxygen and glucose in hippocampal slices.

We performed experiments in vitro to observe electrophysiological events that may relate to the protective effect of decreased temperature during cerebral ischemia in vivo. Extracellular field potentials were recorded from area CA1 of rat hippocampal slices with reduced oxygen and 2.0 mM D-glucose, producing irreversible changes within c. 10 min (more slowly than with complete deprivation of oxygen and glucose but more rapidly than with hypoxia alone). At 36 degrees C, synaptic potentials rapidly disappeared, followed by a d.c. negative shift similar to spreading depression. Elevated oxygen and glucose were reapplied within 5 min of each negative shift (duration of hypoxia ranged from 15 to 21 min). Application of normal medium for up to 45 min after negative shifts did not allow synaptic potentials to recover. At 33 degrees C negative shifts from reduced oxygen were delayed and excitatory postsynaptic potentials recovered in one experiment. At 31 degrees C negative shifts were usually absent and synaptic potentials always recovered, even with > 50 min of reduced oxygen and glucose. At both 33 degrees C and 31 degrees C, excitatory postsynaptic potential amplitude oscillated one or more times, whether or not a negative shift occurred. Our results show that negative shifts and irreversible loss of synaptic activity from hypoxia in vitro are delayed or prevented by decreased temperature.

Animals↗
PubMed1991

[Continuing medical education in pediatrics: maintaining of professional competence with different interactive models].

The forming of professional competence constitutes the primary mission of the university. It follows that the maintenance of professional competence is part of that same mission. Physicians will always remain the first motor of their own competence, but the university will play a major role in organizing and providing continuing medical education activities. At the University of Montreal, many CME programs are regularly offered to practising physicians concerned by pediatric patients (400 pediatricians and 7,000 family physicians). These programs include the following collective activities: 1), biannual convention on pediatric updates; 2), one-day meeting on selected topics; 3), advanced pediatric life support courses; 4), regional conference touring programs by university staff. We have also developed the following individual CME activities: 1), CME letter; 2), self-evaluation questionnaires; 3), miniresidency programs; 4), CME TV series; 5), problem-solving slide series. All these activities are planned according to a need assessment basis and an on-going evaluation process. Priority is given to interactive methods of student-teacher relationship.

Education, Medical, Continuing↗
PubMed1990

Treatment with conotoxin, an 'N-type' calcium channel blocker, in neuronal hypoxic-ischemic injury.

Therapeutic efficacy of calcium channel blockers in stroke remains controversial, but previously used agents bind almost exclusively to L-type calcium channels. The newly-discovered N-type calcium channel is specific to neurons, and therapy involving blockade of this site has not been previously attempted. We assessed the neuroprotective effect of omega-conotoxin GVIA (CgTx), a blocker of N-type calcium channels, using both in vitro hypoxic injury to rat cortical neurons and an in vivo model of reversible spinal cord ischemia in the rabbit. In cell cultures, CgTx inhibited hypoxia-induced 45Ca accumulation and neuronal injury minimally, compared to the NMDA antagonist ketamine. In vivo, the duration of spinal cord ischemia which produced permanent paraplegia in 50% of control animals (ET50) was 24.0 +/- 2.6 min. Animals treated 2 h prior to ischemia with 0.5 nmol CgTx in the subarachnoid space had an ET50 of 26.9 +/- 1.8 min (P = 0.36). Animals treated 24 h prior to ischemia (all had persistent systemic tremor) had a ET50 of 28.9 +/- 1.8 min (P = 0.13). We conclude that pharmacologic modulation of the N-type calcium channel does not provide a significant protective effect against neuronal hypoxic-ischemic injury.

Animals↗