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At least 181 records · Page 10Linked to original sources
PubMed2003

Dopamine as a novel anti-migration factor of vascular smooth muscle cells through D1A and D1B receptors.

To elucidate the roles of rat vascular dopamine D1A and D1B receptors in vascular smooth muscle cell migration, the effect of antisense oligonucleotides to D1A receptors (+1 to +21 of rat D1A receptors) and to D1B receptors (-12 to +6 of rat D1B receptors) on dopamine-mediated suppression of platelet-derived growth factor BB-mediated vascular smooth muscle cell migration, evaluated by the Boyden's chamber method, was studied. Increased vascular smooth muscle cell migration by platelet-derived growth factor BB (5 ng/ml) was suppressed significantly by co-incubation with dopamine (0.025-10 micromol/l) (by 15-59%). This suppression by 10 micromol/l dopamine was reversed by D1A antisense oligonucleotides (46%) and D1B antisense oligonucleotides (51%), but by neither the sense nor the random sense oligodeoxynucleotides to these receptors. The suppression by antisense oligodeoxynucleotides (21-51%) is dose dependent (1-10 micromol/l) and time dependent (0-4 h). Dopamine (10 micromol/l)-induced cyclic AMP formation is also suppressed by D1A antisense oligonucleotides (50%) and D1B antisense oligonucleotides (58%), but by neither the sense nor the random sense oligodeoxynucleotides to these receptors. The platelet-derived growth factor BB (5 ng/ml)-mediated activation of phospholipase D and protein kinase C activities were significantly suppressed by co-incubation with 10 micromol/l dopamine, which was reversed by D1A antisense oligonucleotides (45%) and D1B antisense oligonucleotides (50%) but by neither the sense nor the random sense oligodeoxynucleotides to these receptors. These results suggest that vascular D1A and D1B receptors inhibit migration of vascular smooth muscle cells, possibly through cyclic AMP activation and the suppression of phospholipase D and protein kinase C activities.

Animals↗
PubMed1997

Effect of high glucose on permeability of retinal capillary endothelium in vitro.

PURPOSE: To study the effect of high glucose on the permeability of bovine retinal capillary endothelial cell (BRCEC) monolayers. METHODS: The paracellular permeability of second-passage BRCEC cultured on millipore filters in two chamber transwell inserts was assayed by measuring the peak trans-monolayer electrical resistance and percent equilibration of 14C-inulin 48 hours after it had been added to the luminal chamber. RESULTS: High glucose increased the paracellular permeability of BRCEC monolayers independently of its hypertonic action (5 mM glucose: 154.2 +/- 21.2 and 19.5 +/- 2.4; 30 mM glucose: 134.2 +/- 5.1 [P = 0.01] and 23.5 +/- 2.1 [P = 0.01]; 5 mM glucose + 25 mM mannitol: 168.7 +/- 13.7 ohm.cm2 [P = 0.04] and 19.3% +/- 1.2% 48-hour equilibration of inulin [P = 0.008]). In a separate series of experiments, the authors were unable to show that either aminoguanidine or ponalrestat prevented the effect of high glucose on permeability (30 mM glucose 95.1 +/- 16.7 and 45.4 +/- 5.6; 5 mM glucose: 122.9 +/- 14.2 [P = 0.02] and 36.6 +/- 5.6 [P = 0.001]; 30 mM glucose + aminoguanidine 87.9 +/- 17.5 [P = 0.04] and 75.3 +/- 14.9 [P = 0.6]; 30 mM glucose + ponalrestat 79.9 +/- 12.7 ohm.cm2 [P = 0.1] and 48.2 +/- 2.5% 48-hour equilibration of inulin [P = 0.15]). Ponalrestat did not abrogate the effect of high glucose despite its ability to reduce a high glucose-induced increase in BRCEC intracellular sorbitol levels. CONCLUSIONS: The data are consistent with a role for increased paracellular permeability in breakdown of the blood-retinal barrier in diabetic retinopathy, which appears to be independent of both nonenzymatic glycosylation and the polyol pathway.

Animals↗
PubMed2006

Altered ion transport and responsiveness to methacholine and hyperosmolarity in air interface-cultured guinea-pig tracheal epithelium.

INTRODUCTION: Challenge of guinea-pig tracheal epithelium with hyperosmolar solution alters ion transport and evokes the release of epithelium-derived relaxing factor (EpDRF). Cultured tracheal epithelial cells (CE) offer the potential to examine biochemical pathways related to EpDRF release, but whether the bioelectric properties and responses of fresh, adherent epithelial cells (FE) are modeled by CE has not been established. METHODS: Tracheal epithelial cells grown in air-interface culture and fresh tracheal segments were mounted in Ussing chambers to determine short circuit current (I(sc)) and transepithelial resistance (R(t)) and to compare responses to transport inhibitors, methacholine and hyperosmolarity. RESULTS: Significant differences in basal I(sc) and R(t) between FE and CE were observed (I(sc), 41.3+/-3.5 and 8.5+/-0.8 microA/cm(2), P<0.05; R(t), 106+/-7 and 422+/-4 Omega cm(2), P<0.05; respectively); basal spontaneous potential difference values were not different (4.2+/-0.3 and 3.4+/-0.3 mV, respectively). Amiloride (mucosal, 3 x 10(-5) M), bumetanide (basolateral, 10(-5) M) and ouabain (basolateral, 10(-5) M) reduced I(sc) equally in FE and CE. In contrast, NPPB (10(-5) M) in the presence of amiloride had a differential effect, decreasing I(sc) by 11% in FE and 71% in CE (P<0.05). Iberiotoxin (basolateral, 10(-7) M) was without effect in either preparation. In FE, serosal methacholine (3x10(-5) M) elicited an NPPB-insensitive monotonic increase in I(sc), but in CE caused a large, transient, NPPB-inhibitable increase which was followed by an NPPB-resistant plateau. Addition of apical D-mannitol (0.3-267 mosM) to increase osmolarity decreased I(sc) in FE, whereas in CE d-mannitol initially increased (0.3-84.3 mosM) and then decreased (84.3-267 mosM) I(sc). DISCUSSION: Cell culture causes substantial changes in the bioelectric and pharmacological properties of respiratory epithelium. Caution should be exercised when using CE as a substitute for FE in studies of ion transport- and cell volume-dependent processes.

Amiloride↗
PubMed2003

Cotransport of H+, lactate, and H2O in porcine retinal pigment epithelial cells.

The retinal pigment epithelium (RPE) of the eye transports water and lactate ions in the direction from retina to choroid. The water transport is important in maintenance of retinal adhesion and the transport of lactate ions serves to regulate the lactate levels and pH of the subretinal space. This study investigates by means of a non-invasive technique the mechanism of coupling between transport of H(+), lactate ion, and water in the monocarboxylate transporter (MCT1) located in the apical (retinal) membrane of a mammalian RPE. Primary cultures of porcine RPE cells were grown to confluence and placed in a perfusion chamber in which the solution facing the retinal membrane could be changed rapidly. Two types of experiments were performed: Changes in cell water volume were measured by self-quenching of the fluorescent dye Calcein, and changes in intracellular pH were measured ratiometrically using the fluorescent dye BCECF. In lactate-free solutions, mannitol addition to the retinal bath caused intracellular acidification and cell shrinkage, given by a single osmotic water permeability of 1.2+/-0.1 x 10(-4)cmsec(-1) (osmoll(-1))(-1). In solutions containing 50 mmoll(-1) lactate, however, the mannitol-induced cell shrinkage was faster and the cells alkalinized. These effects were not linear functions of the magnitude of the imposed osmotic gradients: Both volume effects and changes in intracellular pH showed apparent saturation with increasing gradients. Abrupt isosmotic replacement of Cl(-) with lactate in the concentration range from 3 to 50 mmoll(-1) caused an immediate cell swelling as well as an immediate intracellular acidification; both effects showed apparent saturation with increasing lactate concentration. The K(m) values were: 11+/-2 mmoll(-1) for the water fluxes and 13+/-4 mmoll(-1) for the H(+) and lactate fluxes. The data suggest that H(2)O is cotransported along with H(+) and lactate ions in MCT1 localized to the retinal membrane. The study emphasizes the importance of this cotransporter in the maintenance of water homeostasis and pH in the subretinal space of a mammalian tissue and supports our previous study performed by an invasive technique in an amphibian tissue.

Animals↗
PubMed1997

Direct visualization and characterization of stable microtubules from the neurites of cultured dorsal root ganglion cells.

We tested the stability of microtubules (MTs) in the neurites of cultured dorsal root ganglion cells by dissolving the cytoplasmic membrane with detergent and exposing them to defined extracellular medium under observation with a video-enhanced differential interference contrast (DIC) microscope. Smooth cytoplasmic filaments visualized after membrane removal were suggested to be MTs by the preservation of all of the filaments in the presence but not in the absence of taxol. They were further confirmed to be MTs by specific immunostaining with anti-tubulin antibody. A significant number of MTs in the established neurites of 6-day-old cultures remained longer than 10 min after membrane removal while MTs in the Schwann cell processes or in the distal regions of the growth cone-bearing neurites of 3-day-old cultures disappeared within 2 min. A population of very stable MTs persisting longer than 30 min was also found specifically in the 6-day-old cultures. Association with other structures or bundling seemed to stabilize the MTs to some degree. The most stable MTs, however, were not associated with some structure along the length but were mainly anchored at points, suggesting that specific point attachments may be another important mechanism operating in MT stabilization. The present method is thus capable of directly demonstrating the unusual stability of neuritic MTs, and provides a new system for further investigation on the mechanism of stabilization.

Animals↗
PubMedDate not supplied

Effects of methyl paraben on skin keratinocytes.

Some ingredients of dermatological formulations result in skin irritation and allergy. In particular, preservatives have been reported extensively as a cause of allergic contact dermatitis. The study focused on parabens which have been used extensively as antimicrobial preservatives in foods, drugs and cosmetics. The aim of this study was to clarify the effects of the daily use of methyl paraben (MP) on human skin. The concentrations of MP in the stratum corneum (SC) of the human forearm were measured using the cup method and GC-MS after daily applications of MP containing formulations. The study also investigated the effects of long-term exposure to MP on keratinocytes in vitro. Normal human keratinocytes and the skin equivalents were cultured in the medium containing MP. The following changes were analysed: proliferating ability, apoptotic cells, morphological changes, mRNA and protein expressions. After 1 month of daily applications of MP containing formulations, MP remained unmetabolized and persisted slightly in the SC. MP decreased the proliferating ability of keratinocytes and changed the cell morphology. MP also decreased the expressions of hyaluronan synthase 1 and 2 mRNAs and type IV collagen. In contrast, it increased the expressions of involucrin and HSP27. Furthermore, MP influenced the epidermal differentiation of the skin equivalent. These results suggest that MP exposure through application of dermatological formulations results in MP persistence and accumulation in the SC, and that MP might influence the aging and differentiation of keratinocytes.

Administration, Topical↗
PubMed1987

A tissue-specific protein in rat osteogenic tissues.

A tissue-specific protein fraction has been detected in rat osteogenic tissue. Dissociative extraction of adult rat bone matrix with 4 M guanidinium chloride solution was followed sequentially by gel chromatography and polyacrylamide gel electrophoresis. By the latter procedure a prominent protein component of molecular weight 19,000 was isolated from the low molecular weight fraction, and antibodies directed against this protein were raised in rabbits. The antibodies were mainly against antigenic sites on this protein, as shown by protein blotting techniques. By embedding rat tissues in hydrophilic plastic and by using immunohistochemical procedures the presence of this protein was demonstrated specifically in bone matrix in vivo, in osteogenic tissue developing in diffusion chamber culture, and in a malignant osteoblast cell line (UMR 106). Soft tissues (liver, kidney, spleen, gut, skin, thymus, eye) showed no reactivity with the antiserum and in vitro a further malignant osteoblast cell line (ROS 17/2.8) did not synthesize the 19,000 molecular weight protein. This protein appears to be expressed solely by osteogenic tissue and may be used as a biochemical criterion of osteogenic differentiation.

Animals↗
PubMed2005

Comparison of human tracheal/bronchial epithelial cell culture and bovine nasal respiratory explants for nasal drug transport studies.

Ten drug compounds with varying physicochemical properties and transporter substrate specificities were investigated to compare their in vitro permeabilities across bovine nasal respiratory explants and the EpiAirway system, both established models for the assessment of nasal drug absorption. Permeability across the bovine explants and EpiAirway correlated well with the partitioning behavior of compounds whose clogDC values were greater than 0. The permeabilities of all ten compounds were well-correlated between the two tissue models, with the permeability values through the EpiAirway tissues being approximately 10-fold higher than through the bovine explants due to the thickness differences between the models. For more lipophilic compounds, the in vitro permeabilities measured with both tissue systems were also predictive of the reported in vivo nasal bioavailabilities. Deviations from these correlations were observed for compounds reported to be substrates of p-glycoprotein or OCT transporters, and differences were also seen between the permeabilities measured in the tissue models for these compounds. Both models can be used to estimate the systemic bioavailability of moderately lipophilic compounds administered intranasally, while each may have particular advantages or disadvantages in estimating the bioavailability of drug compounds that are subject to local mucosal metabolism or to carrier-mediated uptake or efflux.

Animals↗
PubMed1993

A perifusion loop-gap resonator NMR probe for aerobic cell suspensions.

NMR studies of aerobic cell metabolism require that spectral data of high sensitivity and resolution be acquired from samples maintained under suitable conditions. The loop-gap resonator 31P NMR probe described here was designed for investigations of highly aerobic-dependent renal proximal tubules in an environment similar to those of conventional incubation flasks. The inherently higher sensitivity of the loop-gap resonator design and associated circuitry made it possible to obtain adequate spectra in short periods of time with small amounts of sample. Cellular physiological properties of perifused aerobic renal proximal tubule cell suspensions in the loop-gap resonator probe were found to be similar to equivalent tissue samples incubated in conventional flasks. Furthermore, the loop-gap perifusion probe was found to provide useful 31P NMR spectra of the kidney tubule system after acquisition times as short as 3 min.

Aerobiosis↗
PubMed2004

Glioblastoma cells release factors that disrupt blood-brain barrier features.

The blood-brain barrier (BBB), mediated by endothelial tight junctions, is defective in malignant gliomas such as glioblastoma, resulting in cerebral edema and contrast enhancement upon neuroradiological examination. The mechanisms underlying BBB breakdown are essentially unknown. Since non-neoplastic astrocytes are required to induce BBB features of cerebral endothelial cells, it is conceivable that malignant astrocytes have lost this ability due to dedifferentiation. Alternatively, glioma cells might actively degrade previously intact BBB tight junctions. To examine the latter hypothesis, we have employed a transepithelial electrical resistance breakdown assay using monolayers of the C7 subclone of Madin-Darby canine kidney (MDCK-C7) cells forming tight junctions similar to those of BBB endothelial cells. We found that glioblastoma primary cells co-cultured with the MDCK-C7 monolayer (without direct contact of the two cell types) resulted in marked breakdown of electrical resistance, whereas primary cultures derived from low-grade gliomas (fibrillary astrocytoma, oligoastrocytoma) showed delayed or no effects. These results suggest that malignant gliomas have acquired the ability to actively degrade tight junctions by secreting soluble factors, eventually leading to BBB disruption within invaded brain tissue.

Animals↗
PubMed1997

Culture of embryonic renal collecting duct epithelia in a gradient container.

During organogenesis the ampullar epithelium of the renal collecting duct acts as an inducer which generates all of the nephron anlagen. As development proceeds, one part of the collecting duct cells in the ampullar tip retain their inducer capability, while others develop into the functional epithelium consisting of principal and intercalated (IC) cells. The events leading from the embryonic inducer to the mature tissue are unknown. We investigated the maturation of embryonic collecting duct epithelium derived from neonatal rabbit kidney under in vitro conditions. To prevent dedifferentiation the epithelia were cultured on kidney-specific support material within a tissue carrier. Apical and basal compartments of the epithelia were simulated in a gradient culture container. The two sides of the epithelium were each constantly perfused with a different medium. During the 14-day incubation the tissue was not subcultured. The development of collecting duct cell features was investigated with morphological and immunohistochemical methods. Both light and electron microscopy revealed morphologically intact epithelia following gradient culture. The polarized cells rested on a uniformly developed basement membrane. The continuous application of aldosterone during the culture modulated the development of collecting duct cell characteristics. Both basal and luminal administration of aldosterone initiated differentiation in the embryonic epithelia. Using the sodium (Na) channel blocker amiloride, it was demonstrated that Na channels are involved in the differentiation of the IC cell phenotype.

Aldosterone↗
PubMed1991

Vectorial transcytosis of immunoglobulin G by human term trophoblast cells in culture.

Trophoblast cells were grown on filters that allow access to apical and basal surfaces of cells. Using this experimental system, IgG transport was shown to be specific and to occur primarily in the apical to basal direction. This transport was time- and temperature-dependent, with approximately 10% of added IgG appearing on the basal side within a 60-min incubation at 37 degrees C. Other substances such as heparin were transported only minimally, whereas horseradish peroxidase was transported to the same degree in both directions. Greater than 90% of the transported IgG was precipitable by trichloroacetic acid and 81% was capable of binding to protein G. Such a rapid transport of large amounts of IgG in trophoblast cells is consistent with a receptor-mediated process of transcytosis.

Biological Transport↗
PubMed2006

CaMKII inactivation by extracellular Ca(2+) depletion in dorsal root ganglion neurons.

A mechanism by which Ca(2+)/CaM-dependent protein kinase (CaMKII) is autophosphorylated by changes in extracellular calcium in the absence of detectable changes in cytoplasmic [Ca(2+)] has been identified. We find that when the external Ca(2+) concentration ([Ca(2+)](O)) is lowered, Ca(2+) is released from intracellular stores to maintain a constant cytoplasmic Ca(2+) level, gradually depleting the endoplasmic Ca(2+) stores. Accompanying the store-depletion is a rapid decrease in CaMKII activity. Approximately 25% of the measured CaMKII autophosphorylation in DRG neurons in culture can be regulated by Ca(2+) flux from intracellular stores caused by manipulating [Ca(2+)](O), as shown by blocking refilling of store-operated Ca(2+)-channels with SK&F 96365, Ruthenium Red, and a partial block with Ni(2+). Blocking voltage-gated Ca(2+)-channels with either isradipine or SR 33805, had no effect on CaMKII autophosphorylation induced by restoring Ca(2+)(O) to normal after depleting the intracellular Ca(2+) stores. These results show that removal of Ca(2+)(O) has profound effects on intracellular Ca(2+) signaling and CaMKII autophosphorylation, in the absence of measurable changes in intracellular Ca(2+). These findings have wide-ranging significance, because [Ca(2+)](O) is manipulated in many experimental studies. Moreover, this explanation for the paradoxical changes in CaMKII phosphorylation in response to manipulating [Ca(2+)](O) provides a possible mechanism linking activity-dependent depletion of Ca(2+) from the synaptic cleft to a protein kinase regulating many neuronal properties.

Animals↗
PubMed1998

Reoxygenation injury affects isolated islet response to fatty acid stimulation.

Hyperlipidemia is frequently associated with hyperinsulinemia, but because the effects of fatty acids on insulin secretion in in vitro studies using isolated perifused islets have mostly been described with supraphysiological concentrations of fatty acids, it has remained uncertain whether elevated lipid levels contribute to hyperinsulinemia by their direct stimulation of insulin secretion. In the present study, we have identified reoxygenation injury in isolated islet function as a contributing factor in the failure of physiological concentrations of free fatty acids to stimulate insulin secretion in isolated perifused islets. Reoxygenation of isolated islets is associated with the production of reactive oxygen species, which impair islet function. We have found that pretreatment of freshly isolated islets with the antioxidant glutathione (GSH), as well as a 24-hour preculture of isolated islets under appropriate conditions, enhanced their sensitivity to fatty acid stimulation.

Animals↗
PubMed2003

Working with Xenopus spinal neurons in live cell culture.

Neurons from the Xenopus spinal cord are highly versatile and easily manipulated, making them an ideal model system to answer questions regarding the cellular and molecular basis of early neural development and function. Xenopus has been a productive model system in studies ranging from axon growth and guidance to synaptic plasticity. Exogenous molecules, such as proteins, fluorescent tracers, and nucleic acids, can be injected into early blastomeres to load tracers in all neurons or into late blastomeres to target specific classes of neurons based on established lineage maps. Xenopus spinal neurons also provide an excellent culture system, as neurons extend processes on a variety of substrata and develop at room temperature in minimal salt solutions. Live fluorescent neurons can be imaged for hours with fluorescence microscopy at room temperature in static cultures without neurotrophic support or serum. This highly reduced culture system minimizes variables that can confound interpretation of results. Cultures can be prepared at various stages of development as dissociated neurons or as spinal cord explants. Both excitatory and inhibitory neurons develop in culture, and synaptic contacts among neurons and between neurons and nonneuronal targets form naturally. The simple anatomy and rapid rostral-to-caudal development of the Xenopus spinal cord also make this an excellent in vivo model system to analyze axon guidance by identifiable classes of neurons. This chapter focuses on techniques that exploit both in vitro and in vivo qualities of this system.

Animals↗
PubMed2001

Sustained delivery of GDNF: towards a treatment for Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disease characterized by the progressive loss of nigral dopaminergic neurons. Although symptomatic therapies to substitute for the missing neurotransmitter dopamine are efficient at the early stages of the disease, the goal is to find alternative therapies which could protect dopaminergic neurons from the degenerative process. We have used two distinct gene therapy approaches to deliver the neuroprotective molecule glial cell line-derived neurotrophic factor (GDNF) in animal models of the disease: (i) an encapsulated genetically engineered cell line releasing GDNF (ex vivo gene therapy); and (ii) a lentiviral vector encoding the GDNF gene (in vivo gene therapy). Both approaches allowed protection of nigral dopaminergic neurons against lesion-induced cell death in rodent as well as monkey models of PD. Behavioral symptoms were also ameliorated in these animals. In addition, co-transplantation of embryonic dopaminergic neuronal grafts and a GDNF-releasing capsule allowed improvement of graft survival and differentiation, thereby accelerating behavioral recovery. These results should lead to clinical application in the near future.

Animals↗
PubMed2000

Migration of CD18-deficient neutrophils in vitro: evidence for a CD18-independent pathway induced by IL-8.

Neutrophils isolated from a child with severe leukocyte adhesion deficiency 1 (LAD1) had a complete absence of expression of the CD11/CD18 beta2 integrin family of adhesion molecules, and were shown to be deficient in the in vitro adhesion and migration properties. However, we found that interleukin-8 (IL8), a potent chemoattractant for neutrophils, and sputum sol phase induced these LAD1 neutrophils to migrate through an endothelial cell layer in vitro, and confirmed that this migration was CD18-independent. These findings add to evidence of CD18-independent mechanisms of neutrophil recruitment, in particular neutrophil infiltration into the lungs, where IL8 may be an important recruitment factor.

Antibodies↗