Biomedical subjects

W Geng

Publications and source records attributed to W Geng.

At least 19 recordsLinked to original sources

PubMed2001

Concentration-dependent phorbol stimulation of PKCalpha localization at the nucleus or subplasmalemma in A7r5 cells.

The subcellular translocation of PKCalpha was studied in A7r5 cells by confocal microscopy through use of standard immunohistologic staining and PKCalpha-enhanced green fluorescent protein (PKCalpha-EGFP) fusion protein expression. The results from both methods were consistent in indicating that PKCalpha, observed to be diffusely distributed in the unstimulated cell, was translocated primarily to either the perinuclear region of the cell or to subplasmalemmal sites depending on the concentration of phorbol 12, 13 dibutyrate (PDBu) used to activate the response. Translocation of PKCalpha to the perinucleus but not the plasmalemma was blocked by the use of colchicine to disrupt cell microtubules. However, there was little evidence of significant colocalization of PKCalpha with the microtubular cytoskeleton during the interval of translocation. By comparison, cytochalasin B disruption of actin microfilaments had no significant effect on PKCalpha translocation to either the plasmalemma or the perinucleus. The results indicate that the target site of PKCalpha translocation may vary with activating stimulus strength in A7r5 cells and that the translocation of the isoform to perinuclear target loci depends on an intact microtubular cytoskeleton. This suggests that multiple pathways are available for the redistribution of PKCalpha that may employ different mechanisms to regulate the movement and/or docking of the isoform at specific target sites.

Animals↗

PubMed2001

The furry gene of Drosophila is important for maintaining the integrity of cellular extensions during morphogenesis.

The Drosophila imaginal cells that produce epidermal hairs, the shafts of sensory bristles and the lateral extensions of the arista are attractive model systems for studying the morphogenesis of polarized cell extensions. We now report the identification and characterization of furry, an essential Drosophila gene that is involved in maintaining the integrity of these cellular extensions during morphogenesis. Mutations in furry result in the formation of branched arista laterals, branched bristles and a strong multiple hair cell phenotype that consists of clusters of epidermal hairs and branched hairs. By following the morphogenesis of arista laterals in pupae, we have determined that the branched laterals are due to the splitting of individual laterals during elongation. In genetic mosaics furry was found to act cell autonomously in the wing. The phenotypes of double mutant cells argue that furry functions independently of the frizzled planar polarity pathway and that it probably functions in the same pathway as the tricornered gene. We used a P-element insertion allele as a tag to clone the furry gene and found it to be a large and complicated gene that encodes a pair of large conserved proteins of unknown biochemical function.

Amino Acid Sequence↗

PubMed2001

Skeletal sensitivity to dietary calcium deficiency is increased in the female compared with the male rat.

We investigated potential sex differences in bone resorption and the conservation of whole body bone mass in 24-week-old Sprague-Dawley rats maintained on a 1.0% calcium diet and then fed diets containing 0.02, 0.5, 1.0, or 1.75% calcium for 31 days. Lowering dietary calcium from 1.00% to 0.02% doubled whole skeleton bone resorption (urinary 3H-tetracycline loss). Female rats were more sensitive to calcium stress, exhibiting the maximal resorptive response when fed the 0.5% calcium diet, whereas the 0.02% calcium diet was required to elicit this response in males. Despite the evidence of increased bone resorption, whole skeleton mass was unchanged in females and was significantly increased in males, indicating that switching to even the 0.02% calcium diet did not result in an overt loss of total body bone mass. Compared with controls, the skeleton mass of females (97+/-1.4%) maintained on the 0.02% calcium diet was significantly lower than males (107+/-2.4%), again suggesting a greater impact of calcium deficiency in females. The calculation of the average percentage growth of selected individual bones in male rats indicated a proportional increase in bone mass between the axial and appendicular skeleton of approximately +4% and +18% in animals maintained on 0.02 and 1.75% diets, respectively. By comparison, female rats consuming the 0.02% calcium diet showed an average 14% loss in axial bone and 7.5% gain in appendicular bone mass. The results indicate increased sensitivity to dietary calcium deficiency in female rats which involves a significant loss in axial bone mass not observed in male rats maintained under similar dietary conditions.

Animals↗

PubMed2000

Analysis of skeletal development on the basis of body mass and bone resorption.

Regression analysis was utilized to define the relationships among body weight, whole skeleton bone resorption (3H-tetracycline method), and the development of skeletal mass. The results indicated that skeletal development (% body weight) of slowly growing, 24-week-old rats consisted of two major components, one directly (r = 0.985, P<0.001) and one inversely (r = 0.977, P<0.001) related to body weight. It was further noted that the skeletal resorption rate was inversely correlated to body weight (r = 0.879, P<0.05) and directly related to skeletal development (r = 0.865, P<0.05), suggesting that whole skeleton bone resorption and formation were highly correlated in the slowly growing animal. A third small component of skeletal development identified in the analysis of data from 24-week-old animals, showed no direct relationship to either body weight or resorptive activity. The model presented enables the separation of skeleton mass into major components which may represent mechanically and metabolically driven bone formation.

Animals↗

PubMed2000

Effect of calcium stress on the skeleton mass of intact and ovariectomized rats.

Female rats were ovariectomized (Ovx) or sham-operated (control) at 18 weeks and the entire skeleton obtained at 24 weeks (baseline) or after an additional 31 day (28 week) interval on a normal (1.0%) or deficient (0.02%) calcium diet. Ovx rats showed a 42% increase in whole body bone resorption (3H-tetracycline loss) in the absence of calcium stress (1.0% calcium diet) and a 70% increase in resorption with morphological evidence of dramatic loss of cancellous bone mass when placed on calcium-deficient (0.02%) diets. Ovx rats kept on the 1.0% calcium diet showed a significant increase in both their body weight (30.2%) and total bone mass (11.6%) compared to baseline sham-operated controls. However, the total skeleton mass of these animals was significantly reduced (-20%) from that predicted by calculations based on body weight. Maintaining animals on calcium-deficient diets had no significant effect on the total skeleton mass of either control or Ovx rats in comparison with age-matched controls on 1.0% diets. It was further determined that an increase in bone mass between 24 and 28 weeks in rats receiving 1.0% dietary calcium occurred in both the axial and appendicular skeleton and was proportionately similar between control and Ovx groups. However, in animals subjected to dietary calcium stress during this interval, the decreased skeletal growth noted was confined primarily to the axial skeleton. The data indicate that ovariectomy or ovariectomy plus calcium stress does not result in loss of total bone mass during the interval of dramatically increased resorption and rapid loss of cancellous bone. The results suggest that the deterioration in individual bone structural and mechanical integrity due to ovariectomy or dietary calcium deficiency may not be attributed to overt loss in total bone mass but may involve a redistribution of bone mass.

Animals↗

PubMed2000

Remodeling of the actin cytoskeleton in the contracting A7r5 smooth muscle cell.

It has been proposed that the reorganization of components of the actin cytomatrix could contribute to force development and the low energy cost of sustained contraction in contractile cells which lack a structured sarcomere (A.S. Battistella-Patterson, S. Wang and G.L. Wright (1997) Can J Physiol Pharmacol 75: 1287-1299). However, there has been no direct evidence of an apropos actin reorganization specifically linked to the contractile response in cells of this type. Remodeling of the alpha- and beta-actin domains was studied in A7r5 smooth muscle cells during phorbol 12,13 dibutyrate (PDB)-induced contraction using immunohistologic staining and beta-actin-green fluorescent protein (beta-actin-GFP) fusion protein expression. Cell stained with phalloidin as well as cells expressing beta-actin-GFP showed densely packed actin stress cables, arranged in parallel and extending across the cell body. PDB caused approximately 85% of cells to contract with evidence of forcible detachment from peripheral adhesion sites seen in many cells. The contraction of the cell body was not uniform but occurred along a principal axis parallel to the system of densely packed beta-actin cables. During the interval of contraction, the beta-actin cables shortened without evidence of disassembly or new cable formation. The use of cytochalasin to inhibit actin polymerization resulted in the dissolution of the actin cables at the central region of the cell and caused the elongation of precontracted cells. In unstimulated cells, alpha-actin formed cables similar in arrangement to the cell spanning beta-actin cables. Within a short interval after PDB addition; however, the majority of alpha-actin cables disassembled and reformed into intensely fluorescing column-like structures extending vertically from the cell base at the center of clusters of alpha-actin filaments. The alpha-actin columns of contracting cells showed strong colocalization of alpha-actinin suggesting they could be structurally analogous to the dense bodies of highly differentiated smooth muscle cells. The results indicate that the alpha- and beta-actin domains of A7r5 cells undergo a highly structured reorganization during PDB-induced contraction. The extent and nature of this restructuring suggest that remodeling could play a role in contractile function.

Actins↗

PubMed2000

PKCalpha translocation is microtubule-dependent in passaged smooth muscle cells.

The translocation of protein kinase C (PKC) isozymes from their inactive cell locus to a variety of cytoskeletal, organelle, and plasmalemmal sites is thought to play an important role in their activation and substrate specificity. We have utilized confocal microscopy to compare phorbol 12, 13 dibutyrate (PDB) - stimulated translocation of PKCalpha in cultured cells derived from rat vascular smooth muscle. In enzymatically dispersed, passaged smooth muscle cells, PKCalpha was uniformly distributed throughout the unstimulated cell. PDB stimulation resulted in extensive association of the PKCalpha into filamentous strands with subsequent accumulation of the isoform in the peri-nuclear region of the cell. Dual immunostaining indicated that PKCalpha was extensively colocalized with microtubules in the interval immediately following PDB stimulation but was largely disassociated from microtubules at 10 min, at which time the translocation of PKCalpha to the peri-nucleus/nucleus was nearly complete. It was further found that the use of colchicine to disrupt the microtubules caused the loss of PKCalpha translocation to the peri-nuclear region. By comparison, cytochalasin B disruption of actin microfilaments had no significant effect on this parameter. The data suggest that PDB stimulation results in a transient association of PKCalpha with cell microtubules and that the microtubules play an important role in the translocation of PKCalpha from the cytosol in passaged cells derived from rat aortic smooth muscle.

Actin Cytoskeleton↗

PubMed2000

The tricornered gene, which is required for the integrity of epidermal cell extensions, encodes the Drosophila nuclear DBF2-related kinase.

During their differentiation epidermal cells of Drosophila form a rich variety of polarized structures. These include the epidermal hairs that decorate much of the adult cuticular surface, the shafts of the bristle sense organs, the lateral extensions of the arista, and the larval denticles. These cuticular structures are produced by cytoskeletal-mediated outgrowths of epidermal cells. Mutations in the tricornered gene result in the splitting or branching of all of these structures. Thus, tricornered function appears to be important for maintaining the integrity of the outgrowths. tricornered mutations however do not have major effects on the growth or shape of these cellular extensions. Inhibiting actin polymerization in differentiating cells by cytochalasin D or latrunculin A treatment also induces the splitting of hairs and bristles, suggesting that the actin cytoskeleton might be a target of tricornered. However, the drugs also result in short, fat, and occasionally malformed hairs and bristles. The data suggest that the function of the actin cytoskeleton is important for maintaining the integrity of cellular extensions as well as their growth and shape. Thus, if tricornered causes the splitting of cellular extensions by interacting with the actin cytoskeleton it likely does so in a subtle way. Consistent with this possibility we found that a weak tricornered mutant is hypersensitive to cytochalasin D. We have cloned the tricornered gene and found that it encodes the Drosophila NDR kinase. This is a conserved ser/thr protein kinase found in Caenorhabditis elegans and humans that is related to a number of kinases that have been found to be important in controlling cell structure and proliferation.

Actins↗

PubMed2000

Short-term exposure to homocysteine depresses rat aortic contractility by an endothelium-dependent mechanism.

The effect of short-term exposure to homocysteine (Hcy) on the contractile characteristics of rat aortic tissue was assessed both in vitro and in vivo. The contractile response of Hcy-treated aortic rings in culture for 1 or 4 days was unchanged from control responses. By comparison, aortic rings from animals injected with Hcy showed marked attenuation of response compared with controls injected with saline, cysteine or methionine. The contractile response to K+ was decreased within 24 hours of Hcy injection, whereas the response to both K+ (-27%) and noradrenaline (-56%) was significantly decreased by 4 days. In contrast, the contractile response to phorbol-12,13-dibutyrate was not different between Hcy and control groups. Intimal rubbing completely restored the responsiveness of Hcy-treated tissue to K+ and noradrenaline. By comparison, L-NAME only partially restored contractile responsiveness, while the cyclooxygenase inhibitor indomethacin had no effect on contractile attenuation induced by Hcy. Western blot analysis showed a 2-fold increase of endothelial nitric oxide synthase (eNOS) and a 3-fold increase in inducible nitric oxide synthase (iNOS) protein expression in the aortic endothelial cells from Hcy-injected rats. The results indicate an early detectable effect of Hcy on the in vivo contractile properties of vascular smooth muscle. The effect is endothelium-mediated and may vary depending on the agonist studied. The mechanism is uncertain but appears to involve increased nitric oxide (NO) production. Finally, the data suggest that attenuation of contraction may not be due to a direct effect of Hcy but that the compound is modified or acts indirectly in vivo.

Animals↗

PubMed1999

Psychometric functions for gap detection in a yes-no procedure.

To examine models of temporal resolution and to investigate the decision processes underlying the detection of a brief pause in a bandpass noise, psychometric functions for gap detection were measured at octave frequencies from 0.25 to 8 kHz. Three normal listeners were tested using a constant-stimulus procedure with a cued Yes-No paradigm. The Minimum Detectable Gap (MDG) estimated from the midpoint of the psychometric functions decreased systematically with increasing frequency. The slopes of the psychometric functions generally increased as the test frequency increased up to 2 kHz, but remained constant at the higher frequencies. Two models were investigated: an energy-detector model and a loudness-detector model. Both consisted of auditory filtering, a nonlinearity, and short-term integration. In the energy-detector model, the nonlinearity was a square law. In the loudness-detector model, it was a compressive power law. Using the usual Gaussian approximations, the energy-detector model fails at low frequencies because the probability distributions of short-term energy differ from Gaussian distributions. The probability distributions of short-term loudness closely follow Gaussian distributions. The loudness-detector model predicts the frequency dependence of the MDG quite accurately, except at 0.25 kHz. It also predicts psychometric functions that resemble the data at low frequencies, but the predicted slopes increase much less with frequency than the measured slopes. This result may indicate that the onset response to the trailing marker of the gap provides an important cue for detection of gaps with durations exceeding the MDG.

Adult↗

PubMed1999

[Recovery of gastrointestinal motility following laparoscopic versus open cholecystectomy].

OBJECTIVE: To investigate the recovery of gastrointestinal motility after laparoscopic cholecystectomy (LC) and open cholecystectomy (OC). METHODS: Motilin (MOT), gastrin (GAS), electrogastrogram (EGG), the peristaltic sound recovery timing (PSRT) and the anorectum exhaust timing (AET) were determined in 30 patients having LC and 18 patients having OC. RESULTS: MOT, GAS, EGG frequency and EGG amplitude on the first, second and third day after operation in LC group were not significantly different in comparison with preoperative data (P > 0.05), but significant difference on the first and second postoperative day compared with preoperative data and data of the the LC group (P < 0.05). The PSRT and AET in the LC group were much shorter in comparison with the OC group (P < 0.05). CONCLUSIONS: The recovery of gastrointestinal motility after LC is earlier than OC, which justifies early feeding for patients after LC.

Cholecystectomy↗

PubMed1999

Differences in excretion of hippurate, as a metabolite of benzoate and as an administered species, in the single-pass isolated perfused rat kidney explained.

The extents of excretion of [14C]hippurate and [3H]hippurate were compared in the single-pass, constant flow (8 ml/min) isolated rat kidney which was perfused simultaneously with tracer concentrations of [14C]benzoate and [3H]hippurate. The steady-state renal extraction ratio of [14C]benzoate was 0.26 +/- 0.04 and was associated with a renal clearance of 1.13 +/- 0.17 ml/min/g. The urinary clearance of [14C]benzoic acid was low (0.011 +/- 0.01 ml/min/g), yielding a low fractional excretion [unbound urinary clearance/glomerular filtration rate (GFR)] value of 0.27 +/- 0.19 and suggesting that glycination of [14C]benzoate to [14C]hippurate accounted almost completely for the total renal clearance. Fractional excretion for preformed [3H]hippurate was eight times that of GFR, but the steady-state renal extraction ratio of preformed [3H]hippurate, E(pmi) (0.24 +/- 0.05) was much lower than the apparent extraction ratio of the renally formed [14C]hippuric acid [E(mi) = 0.39 +/- 0.09] (p <.05). The theoretical basis for the discrepancy was explored with mathematical formulations developed from a physiologically based model of the kidney. It was found that parent drug kinetic parameters (transport and metabolic intrinsic clearance of benzoate) were unimportant for E(mi) or E(pmi). Rather, the value of EK(mi) exceeded EK(pmi) because of the ratio of efflux clearances at the basolateral and luminal membranes for hippurate [corrected] was less than 26.089, a value determined by the GFR, plasma renal flow, and the unbound fraction of hippurate of the system that would render E(mi) to equal E(pmi) in the system. The influx clearance for hippurate to enter from plasma to cell at the basolateral membrane and the reabsorption clearance of hippurate to enter from tubular urine to cell at the luminal membrane failed to alter the ratio of EK(pmi)/EK(mi).

Animals↗

PubMed1998

Application of the dispersion model for description of the outflow dilution profiles of noneliminated reference indicators in rat liver perfusion studies.

The dispersion model (DM) is a stochastic model describing the distribution of blood-borne substances within organ vascular beds. It is based on assumptions of concurrent convective and random-walk (pseudodiffusive) movements in the direction of flow, and is characterized by the mean transit time (t) and the dispersion number (inverse Peclet number), DN. The model is used with either closed (reflective) boundary conditions at the inflow and the outflow point (Danckwerts conditions) or a closed condition at the inflow and an open (transparent) condition at the outflow (mixed conditions). The appropriateness of DM was assessed with outflow data from single-pass perfused rat liver multiple indicator dilution (MID) experiments, with varying lengths of the inflow and outflow catheters. The studies were performed by injection, of bolus doses of 51Crlabeled red blood cells (vascular indicator), 125I-labeled albumin and [14C] sucrose (interstitual indicators), and [3H]2O (whole tissue indicator) into the portal vein at a perfusion rate of 12 ml/ min. The outflow profiles based on the DM were convolved with the transport function of the catheters, then fitted to the data. A fairly good fit was obtained for most of the MID curve, with the exception of the late-in-time data (prolonged tail) beyond 3 x [symbol: see text]. The fitted DNS were found to differ among the indicators, and not with the length of the inflow and outflow catheters. But the differences disappeared when a delay parameter, t0 = 4.1 +/- 0.7 sec (x +/- SD), was included as an additional fitted parameter for all of the indicators except water. Using the short catheters, the average DN for the model with delay was 0.31 +/- 0.13 for closed and 0.22 +/- 0.07 for mixed boundary conditions, for all reference indicators. Mean transit times and the variances of the fitted distributions were always smaller than the experimental ones (on average, by 6.8 +/- 3.7% and 58 +/- 19%, respectively). In conclusion, the DM is a reasonable descriptor of dispersion for the early-in-time data and not the late-in-time data. The existence of a common DN for all noneliminated reference indicators suggests that intrahepatic dispersion depends only on the geometry of the vasculature rather than the diffusional processes. The role of the nonsinusoidal ("large") vessels can be partly represented by a simple delay.

Animals↗

PubMed1998

Hepatic uptake of bromosulfophthalein-glutathione in perfused Eisai hyperbilirubinemic mutant rat liver: a multiple-indicator dilution study.

The hepatocellular uptake of the glutathione conjugate of bromosulfophthalein (BSPGSH) was examined in Eisai hyperbilirubinemic rats (EHBR; originating from Sprague-Dawley rats), which lacked the ATP-dependent canalicular transport for non-bile acid organic anions, a trend common to other mutant rat strains (TR- and GY, originating from Wistar rats). Single-pass perfused rat liver experiments were conducted with BSPGSH (26-257 microM) using the multiple indicator dilution technique. The steady-state extraction ratio of BSPGSH was close to zero due to lack of biliary excretion. After the introduction of a bolus dose containing vascular (51Cr-labeled red blood cells), interstitial (125I-labeled albumin and [14C]sucrose) and cellular space (D2O) indicators and [3H]BSPGSH into the portal vein, the outflow dilution profile of [3H]BSPGSH was found to display a protracted declining profile (tailing) at low input BSPGSH concentrations; the tail disappeared at higher BSPGSH concentrations. When data were fitted with the barrier-limited model of Goresky as used previously for BSPGSH for the Sprague-Dawley rat (SDR), model fitting was found to evoke an additional "deep pool" within the hepatocyte to account for the "tail" component. The deep pool became evident for the EHBR because biliary excretion of BSPGSH was absent and the rate of return from the deep pool was slow. The concentration of BSPGSH within the deep pool was estimated to be 12 +/- 8 times that in the cytosol. The binding of BSPGSH to EHBR S9 (effective binding concentration of 53 microM and a binding association constant KA of 2.4 x 10(4) M-1), however, was found to be lower than that of SDR S9 and could not account for the late-in-time data. The influx permeability-surface area product was concentration dependent and decreased from 0.27 to 0.01 ml.sec-1.g-1 with increasing BSPGSH concentration; the throughput component, or the portion of the dose that goes through the liver without entering the hepatocyte, increased with increasing concentration. The trends were characteristic of carrier-mediated transport and were similar to those found for the uptake of BSPGSH in SDR.

Animals↗

PubMed1998

Hepatic clearance models: comparison of the dispersion and Goresky models in outflow profiles from multiple indicator dilution rat liver studies.

The multiple indicator dilution (MID) technique is often used for investigation of the kinetic behavior of substrates and metabolites in eliminating organs. The present study was a systematic comparison of the utility of the Goresky model (GM) (a structural model) and the mixed-boundary dispersion model (DM) (a stochastic model) in the interpretation of influx, efflux, and removal (sequestration) coefficients, with data generated from rat liver-perfusion/MID studies. Although the GM and the DM are equivalent in their descriptions of membrane transport, they differ in their classifications of the dispersion of blood-borne elements. For the DM, the dispersion is an inverse Gaussian distribution of vascular transit times; for the GM, it is accounted for by the dispersion observed among noneliminated reference indicators (e.g. labeled red blood cells, albumin, sucrose, and H2O) or the derived reference. In this study, previously published rat liver-perfusion/MID data obtained for the glutathione conjugate of bromosulfophthalein and hippuric acid, compounds that exhibit saturable carrier-mediated transport, with the GM were reanalyzed with the two-compartment DM. When the fitted values for volume and transfer coefficients were compared, good correlation was found between the fitted vascular volume for the DM and the vascular volume for the reference indicator for the GM. The influx coefficients were generally similar between the models, but improved correspondence was observed when the DM was modified to include the large-vessel transit time. In contrast, the efflux and sequestration coefficients obtained for the DM did not correspond well to those from the GM. The disagreement was due, in part, to differences in the interpretation of the late-in-time component of the reference transit time distribution curve, which was not described well by the DM. Consequently, the residence time distribution and the relative dispersion were underestimated by the DM.

Animals↗

PubMed1998

Scale effect in bioequivalence evaluations.

OBJECTIVE: The objective of the study was to evaluate the relationship between the magnitude of the sample means for a bioavailability measure and the ratio of the sample means in bioequivalence evaluations. METHODS: We assumed that bioavailability data were obtained from crossover trials on a test and a reference drug products. The true test-reference ratio was constructed as a function of dosage strength under certain dose-response relationship assumptions, and was used for deriving analytical results of assessing the scale effect due to dosage strength. In addition, the test-reference ratio of sample means was constructed as a function of the true test-reference ratio and the correlated sampling deviation in the sample means, and was used in a deterministic simulation for quantifying the scale effect due to the correlated sampling deviation. RESULTS: The major systematic factors that influence the magnitude of the sample means include the dosage strength, dose-response relationship, and the positively correlated sampling deviation in the sample means. RESULTS of this study showed that the true test-reference ratios can be improved with increasing dosage strength in most of the situations studied, but at a diminishing rate. The correlated sampling deviation can alter the test-reference ratio of the sample means through changing magnitude of the sample means, especially for drug products with low bioavailability. CONCLUSIONS: For extended release drug products with multiple dosage strengths, the development efforts should focus on the lowest strength if there is little or no formulation by dose interaction effect. Pilot studies should be conducted for both the highest and the lowest strength instead for possible formulation by dose interaction effect. The magnitude of observed means for bioavailability measures on the reference drug product can indicate the degree of systematic variation in the ratio of means. The expected mean values of the reference drug product can be derived from previous trial(s) and/or from other information sources. If the sample mean for the reference drug product in a failed bioequivalence trial is lower than expected, the investigator should consider repeating the trial.

Algorithms↗

PubMed1997

Retinol influences contractile function and exerts an anti-proliferative effect on vascular smooth muscle cells through an endothelium-dependent mechanism.

Rat aortic rings maintained in organ culture for as little as 24 h show significant loss of contractile responsiveness to different agonists. Smooth muscle cells (SMC) in culture quickly de differentiate into a non-contractile phenotype with a marked capacity for proliferation. Rat aortic ring segments cultured in retinol supplemented (10(-6) M) medium showed significantly increased active tension development in response to 80 mM K+ depolarization compared with 7-day cultured control rings. The improvement of contractile performance of the cultured aorta segments in retinol-supplemented media was lost when rings were denuded of endothelium prior to culture, suggesting the endothelial cell layer as the mediator of this effect. Retinol at concentrations up to 10(-5) M was found to have no direct effect on proliferation of cells of the A7r5 SMC. However, retinol was found to augment significantly the growth inhibition of A7r5 cells grown in co-culture with bovine aortic endothelial cells (BAEC). It was further observed that media conditioned with BAEC treated with 10(-6) M retinol expressed SMC growth inhibitory properties compared with media conditioned by untreated BAEC cells or unconditioned media. Examination of cultured rat aortic ring segments by electron microscopy and BAEC cells with phase contrast microscopy revealed that retinol had obvious effects on endothelial cell morphology and ultrastructure. These results indicate that retinol exerts its effects primarily on the endothelium, which in turn secretes stable factors that directly affect SMC proliferation and contractility.

Animals↗

PubMed1996

Uptake of sulfate conjugates by isolated rat hepatocytes.

The uptake of estrone sulfate (E1S; 1 to 400 microM), harmol sulfate (HS; 5 to 900 microM), and 4-methylumbelliferyl sulfate (4MUS; 5 to 1000 microM) was investigated in isolated rat hepatocytes in the presence or absence of inhibitors. Uptake of all of the sulfate conjugates was rapid and exhibited saturation kinetics, best characterized by saturable and nonsaturable (linear transmembrane clearance) transport systems. The KM's were: 16 +/- 6, 123 +/- 28, and 64 +/- 6 microM for E1S, HS, and 4MUS, respectively, with corresponding Vmax's of 0.85 +/- 0.56, 0.48 +/- 14, and 0.42 +/- 0.07 nmol/min/10(6) cells. The nonsaturable uptake clearances, which displayed concentration-independent uptake, were 3 +/- 2, 1 +/- 0.1, 0.5 +/- 0.1 microliter/min/10(6) cells, respectively. Uptake of E1S was inhibited by ouabain (1 mM) and replacement of sodium by choline, whereas HS was insensitive to the addition or substitution. Uptake of both E1S and HS was significantly reduced by metabolic inhibitors (antimycin A, 2.7 microM, rotenone, 30 microM, and KCN, 2 mM) and temperature reduction (from 37 to 27 degrees C). 4,4'-Diisothiocyanostilbene-2-2-'disulfonic acid (2 mM), an inhibitor of anion transport, reduced E1S and HS uptake; E1S uptake was also reduced by HS. HS uptake by both saturable and nonsaturable transport components was depressed by 4MUS (300 microM); the apparent KM was increased by 83% while the Vmax remained unaltered, and the nonsaturable component was decreased by 48%. The data strongly suggest that multiple pathways exist for the uptake of E1S, HS, and 4MUS. E1S uptake is sodium-dependent, requires energy, and is inhibited by anions such as 4,4'diisothiocyanostilbene-2-2'-disulfonic acid and other sulfate conjugates. HS uptake, while being energy dependent, is not sodium dependent, and is inhibited by 4MUS in a competitive fashion. At least one of these pathways is shared.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗