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Biomedical subjects

Elmer M Price

Publications and source records attributed to Elmer M Price.

10 recordsLinked to original sources
PubMed2006

Multipotent adult progenitor cell lines originating from the peripheral blood of green fluorescent protein transgenic swine.

Multipotent self-renewing stem cell lines have been established using peripheral blood mononuclear cells from adult green fluorescent protein transgenic swine. These cells proliferate as nonadherent spheroids in primordial-specific culture media and readily differentiate into angiogenic, osteogenic, adipogenic, and neurogenic phenotypes when cultured under the appropriate conditions. These cells are designated peripheral blood-derived multipotent adult progenitor cells (PBD-MAPCs). When differentiated in endothelial-specific media, these cells exhibit a cobblestone morphology and express von Willebrand factor (vWF), take up 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarboxyanine-labeled acetylated low-density lipoprotein DiI-Ac-LDL, and form tubes with lumens when grown on pads of Matrigel. Under different culture conditions, the cells appear whorl-like in appearance and express alpha-actin, indicative of smooth muscle phenotype. In the presence of dexamethasone and ascorbic acid, PBD-MAPCs differentiate into cells that produce Alizarin Red-staining extracellular mineral, consistent with an osteogenic potential. Under different conditions the cells produce Oil Red O-staining lipid vacuoles, suggestive of an adipocyte phenotype. We have also developed conditions that induce PBDMAPCs to differentiate into neural cells, confirmed by the expression of specific neuron- and glial-specific markers. Upon transplantation into rat brain, the neurogenic cells survive and migrate throughout the striatum and corpus callosum. The cells remain brightly fluorescent throughout their time in culture, during in vitro differentiation, and after in vivo transplantation. PBD-MAPCs have been maintained in primordial cell media for more than 100 doublings, yet can be induced to differentiate rapidly and efficiently into distinct cell types. PBD-MAPCs are ideal tools to study the mechanisms of differentiation and may be superior to embryonic stem cells as cellular therapeutics.

Actins↗
PubMed2004

Shear stress induces eNOS mRNA expression and improves endothelium-dependent dilation in senescent soleus muscle feed arteries.

We tested the hypothesis that increased intraluminal shear stress induces endothelial nitric oxide (NO) synthase (eNOS) mRNA expression and improves endothelium-dependent dilation in senescent soleus muscle feed arteries (SFA) by increasing NO production. SFA were isolated from young (4 mo) and old (24 mo) male Fischer 344 rats and cannulated with two resistance-matched glass micropipettes. SFA were exposed to no flow (NF), low flow (LF), intermediate flow (IF), or high flow (HF) for 4 h. Mean intraluminal shear stress ranged from 0 to 82 dyn/cm(2). At the end of the 4-h treatment period, eNOS mRNA expression was assessed in each SFA. eNOS mRNA expression was significantly lower in old NF SFA than in young NF SFA. In old SFA, eNOS mRNA expression was induced by IF (+154%) and HF (+136%), resulting in a level of expression that was not different from that of young SFA. In a separate series of experiments, SFA were pretreated with NF or HF for 4 h, and endothelial function was assessed by examining vasodilator responses to ACh. ACh-induced dilation was less in old NF SFA than young NF SFA. Pretreatment with HF improved ACh-induced dilation in old SFA such that the response was similar to that of young SFA. In the presence of N(omega)-nitro-L-arginine to inhibit NOS, ACh-induced dilation was inhibited in old HF SFA such that the response was no longer greater than that of old NF SFA. These results indicate that increased intraluminal shear stress induces eNOS mRNA expression and improves endothelium-dependent dilation in senescent SFA by increasing NO production.

Aging↗
PubMed2004

A domain mimic increases DeltaF508 CFTR trafficking and restores cAMP-stimulated anion secretion in cystic fibrosis epithelia.

The major disease-causing mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) is deletion of phenylalanine 508 (DeltaF508), which adversely affects processing and plasma membrane targeting of CFTR. Under conditions predicted to stabilize protein folding, DeltaF508 CFTR is capable of trafficking to the plasma membrane and retains cAMP-regulated anion channel activity. Overexpression is one factor that increases CFTR trafficking; therefore, we hypothesized that expression of a domain mimic of the first nucleotide-binding fold (NBF1) of CFTR, i.e., the site of F508, may be sufficient to overwhelm the quality control process or otherwise stabilize DeltaF508 CFTR and thereby restore cAMP-stimulated anion secretion. In epithelial cells expressing recombinant DeltaF508 human (h)CFTR, expression of wild-type NBF1 increased the amount of both core-glycosylated and mature protein to a greater extent than expression of DeltaF508 NBF1. Expression of wild-type NBF1 in the DeltaF508 hCFTR cells increased whole cell Cl(-) current density to approximately 50% of that in cells expressing wild-type hCFTR. Expression of NBF1 in polarized epithelial monolayers from a DeltaF508/DeltaF508 cystic fibrosis mouse (MGEF) restored cAMP-stimulated transepithelial anion secretion but not in monolayers from a CFTR-null mouse (MGEN). Restoration of anion secretion was sustained in NBF1-expressing MGEF for >30 passages, whereas MGEN corrected with hCFTR progressively lost anion secretion capability. We conclude that expression of a NBF1 domain mimic may be useful for correction of the DeltaF508 CFTR protein trafficking defect in cystic fibrosis epithelia.

Animals↗
PubMed2003

Selected Contribution: Aging impairs nitric oxide and prostacyclin mediation of endothelium-dependent dilation in soleus feed arteries.

We tested the hypothesis that endothelium-dependent dilation in soleus muscle feed arteries (SFA) is impaired by aging due to attenuated nitric oxide (NO)-mediated vasodilation. SFA were isolated from young (4 mo) and old (24 mo) male Fischer 344 rats and cannulated with two glass micropipettes for examination of endothelium-dependent [flow or acetylcholine (ACh)] and endothelium-independent [sodium nitroprusside (SNP)] vasodilator function. Flow- and ACh-induced dilation was significantly attenuated by age, whereas dilation to SNP was not compromised. To determine the mechanism(s) by which aging affected dilator responses to flow and ACh, dilation was assessed in the presence of Nomega-nitro-L-arginine (L-NNA; to inhibit NO synthase), indomethacin (Indo; to inhibit cyclooxygenase), and L-NNA + Indo. In the presence of L-NNA, Indo, or L-NNA + Indo, flow-induced dilation was inhibited in young SFA, resulting in a response to flow that was no longer greater than old SFA. In the presence of L-NNA or Indo, ACh-induced dilation was not significantly inhibited in young or old SFA; however, double blockade with L-NNA + Indo inhibited ACh-induced dilation in young SFA such that the response to ACh was no longer greater than old SFA. Collectively, these data indicate that aging impairs vasodilator responses in SFA by attenuating NO- and prostacyclin-mediated, endothelium-dependent, dilation.

Acetylcholine↗
PubMed2003

Regulation of nitric oxide-dependent vasodilation in coronary arteries of estrogen receptor-alpha-deficient mice.

Estrogen has been shown to increase endothelium-dependent vasodilation and expression of endothelial nitric oxide (NO) synthase (eNOS); however, the role of estrogen receptors in mediating estrogen effects on endothelial function remains to be elucidated. The purpose of this study was to test the hypothesis that estrogen modulates NO-dependent vasodilation of coronary arteries through its action on estrogen receptor-alpha (ER-alpha) to increase protein levels of eNOS and Cu/Zn superoxide dismutase (SOD-1). Vasodilation to acetylcholine (ACh) and sodium nitroprusside was assessed in isolated coronary arteries from intact and ovariectomized female wild-type (WT) and ER-alpha knockout (ERalphaKO) mice. Protein levels for eNOS and SOD-1 were also evaluated. Vasodilation to ACh was not significantly altered in ERalphaKO mice compared with WT mice. Ovariectomy reduced responsiveness to ACh in ERalphaKO mice but not WT mice. Responses to sodium nitroprusside were not altered by disruption of ER-alpha or by ovariectomy. Supplementation with estrogen restored ACh-induced vasodilation in ovariectomized ERalphaKO mice. eNOS protein was reduced in ERalphaKO mice compared with WT mice. Ovariectomy caused a further reduction in eNOS protein in ERalphaKO mice, but this reduction was reversed by estrogen treatment. SOD-1 protein levels were increased by disruption of ER-alpha. Ovariectomy reduced SOD-1 protein in ERalphaKO mice, but this reduction was partially reversed by estrogen replacement. These results suggest that estrogen modulation of eNOS protein content is mediated in part through ER-alpha. NO-dependent responses are preserved in ERalphaKO mice, possibly through increased SOD-1 expression and enhanced bioavailability of NO.

Acetylcholine↗
PubMed2003

GABA(A) alpha1 and alpha2 receptor subunit expression in rostral ventrolateral medulla in nonpregnant and pregnant rats.

Pregnancy results in attenuated baroreflex mediated sympathoexcitatory responses which may be due to potentiation of gamma-aminobutyric acid (GABA) inhibition in the rostral ventrolateral medulla (RVLM). The major metabolite of progesterone, 3alpha-hydroxy-dihydroprogesterone (3alpha-OH-DHP), which is elevated in pregnancy, is a potent neurosteroid positive modulator of GABA(A) receptors, and sensitivity of GABA(A) receptors to 3alpha-OH-DHP is dependent on the receptor subunit composition. The purpose of this study was to evaluate the GABA(A) alpha(1) and alpha(2) receptor subunit mRNA and protein expression in the RVLM of nonpregnant and late term pregnant rats. Micropunches of RVLM were collected from nonpregnant and late term pregnant rats and the expression levels of GABA(A) alpha(1) and alpha(2) receptor subunits were analyzed using quantitative competitive reverse transcriptase polymerase chain reaction (RT-PCR) and immunoblot techniques. The competitive RT-PCR analysis allows comparison of expression levels between different mRNA, and the mRNA expression level of GABA(A) alpha(1) was several hundred fold greater than GABA(A) alpha(2) in both groups. However, this relative distribution of GABA(A) alpha(1) and alpha(2) receptor subunits protein or mRNA expression was not altered in late term pregnant compared to nonpregnant rats. These data demonstrate, that within the RVLM of both nonpregnant and late term pregnant rats, the relative expression levels of GABA(A) alpha(1,2) receptor subunits favor GABA(A) receptors susceptible to positive modulation by progesterone metabolites.

Animals↗
PubMed2003

Increased calcium buffering in coronary smooth muscle cells from diabetic dyslipidemic pigs.

No studies exist concerning the ability of the plasma membrane Ca(2+) pump (PMCA), sarcoplasmic reticulum Ca(2+) pump (SERCA) and Na(+)-Ca(2+) exchanger (NCX) to regulate myoplasmic Ca(2+) (Ca(m)) in vascular smooth muscle cells from diabetic individuals with dyslipidemia. We tested the hypothesis that diabetic dyslipidemia would increase vascular smooth muscle cells to buffer Ca(m). Cells were isolated from the coronary artery of male Yucatan pigs treated for 20 weeks with: (1) a low fat diet (control group); (2) a high fat/cholesterol diet (F group); or (3) alloxan-induced diabetic pigs fed the high fat diet (DF group). The maximum Ca(m) response to a depolarizing 80 mM KCl (80 K) solution was evaluated in the absence and presence of thapsigargin (TSG; inhibits SERCA) and low Na (inhibits NCX). In response to 80 K alone, there was no difference in the Ca(m) response between groups. In the presence of TSG, the 80 K response decreased by 43% in the DF group; TSG did not affect the 80 K response in the control and F groups. When exposed to both TSG and low Na, the 80 K response also decreased by 55% in the DF group. This suggests increased Ca(m) buffering by the PMCA and/or mitochondria in the DF group when SERCA and NCX are inhibited. Compared to the control and F groups, low Na alone elicited a 50% lower Ca(m) amplitude in the DF group, which was reversed with TSG treatment; this suggests that SERCA activity is increased in DF pigs. Western blots also indicated a 7-fold increase in the approximately 115 kDa band density of an anti-SERCA2 antibody in DF compared to control pigs. This is the first report to demonstrate increased Ca(2+) buffering, specifically by SERCA, in vascular smooth muscle cells from diabetic individuals with dyslipidemia.

Analysis of Variance↗
PubMed2002

Expressing and purifying membrane transport proteins in high yield.

Structural analysis of native or recombinant membrane transport proteins has been hampered by the lack of effective methodologies to purify sufficient quantities of active protein. We addressed this problem by expressing a polyhistidine tagged construct of the cardiac sodium-calcium exchanger (NCX1) in Trichoplusia ni larvae (caterpillars) from which membrane vesicles were prepared. Larvae vesicles containing recombinant NCX1-his protein supported NCX1 transport activity that was mechanistically not different from activity in native cardiac sarcolemmal vesicles although the specific activity was reduced. SDS-PAGE and Western blot analysis demonstrated the presence of both the 120 and 70 kDa forms of the NCX1 protein. Larvae vesicle proteins were solubilized in sodium cholate detergent and fractionated on a chelated Ni(2+) affinity chromatography column. After extensive washing, eluted fractions were mixed with soybean phospholipids and reconstituted. The resulting proteoliposomes contained NCX1 activity suggesting the protein retained native conformation. SDS-PAGE revealed two major bands at 120 and 70 kDa. Purification of large amounts of active NCX1 via this methodology should facilitate biophysical analysis of the protein. The larva expression system has broad-based application for membrane proteins where expression and purification of quantities required for physical analyses is problematic.

Animals↗
PubMed2002

Aging induces muscle-specific impairment of endothelium-dependent dilation in skeletal muscle feed arteries.

We tested the hypothesis that aging decreases endothelium-dependent vasodilation in feed arteries perfusing rat skeletal muscle. In addition, we tested the hypothesis that attenuated vasodilator responses are associated with decreased endothelial nitric oxide synthase (eNOS) and superoxide dismutase-1 (SOD-1) expression. Soleus feed arteries (SFA) and gastrocnemius feed arteries (GFA) were isolated from young (4 mo) and old (24 mo) male Fischer 344 rats. Feed arteries from the right hindlimb were cannulated with two glass micropipettes for examination of endothelium-dependent [acetylcholine (ACh)] and endothelium-independent [adenosine (Ado) or sodium nitroprusside (SNP)] vasodilator function. Feed arteries from the left hindlimb were frozen and used to assess eNOS and SOD-1 protein and mRNA expression. In SFA, endothelium-dependent dilation to ACh was reduced in old rats (0.9 +/- 0.04 vs. 0.8 +/- 0.03), whereas dilator responses to Ado and SNP were similar in SFA of young and old rats. In GFA, vasodilator responses to ACh, Ado, and SNP were not altered by age. eNOS and SOD-1 protein expression declined with age in SFA (-71 and -54%, respectively) but not in GFA. eNOS and SOD-1 mRNA expression were not altered by age in SFA or GFA. Collectively, these data indicate aging induces muscle-specific impairment of endothelium-dependent vascular function in SFA.

Aging↗