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C W Taylor

Publications and source records attributed to C W Taylor.

At least 91 records · Page 5Linked to original sources
PubMed1995

Vasopressin stimulation of Ca2+ mobilization, two bivalent cation entry pathways and Ca2+ efflux in A7r5 rat smooth muscle cells.

1. Arg8-vasopressin (AVP)-regulated Ca2+ transport were investigated in fura-2-loaded A7r5 cells using both single cell and population measurements. 2. AVP evokes an initial concentration-dependent rise in cytosolic free Ca2+ concentration ([Ca2+ ]i) to a peak which is independent of extracellular Ca2+, and a sustained Ca2+ signal that results from a balance between stimulation of Ca2+ entry and efflux. 3. Depletion of intracellular Ca2+ stores with thapsigargin, ionomycin, or prior treatment with AVP in Ca2(+)-free medium activates 'capacitative' entry of Ca2+, Ba2+ or Mn2+. Capacitative Mn2+ entry is inhibited by refilling stores with Ca2+; neither Sr2+ nor Ba2+ substitute for Ca2+ to give this effect. 4. In cells with empty stores, AVP stimulates further bivalent cation entry, and the effect persists when extracellular Na+ is replaced by N-methyl-D-glucamine or under depolarizing condition (extracellular KCl concentration ([KCl]o), 135 mM). This effect of AVP is not therefore merely a consequence of AVP causing membrane hyperpolarization or stimulation of Na(+)-Ca2+ exchange, but results from opening of a bivalent cation influx pathway. 5. Several lines of evidence indicate that AVP-stimulated bivalent cation entry is not a consequence of more complete emptying of the intracellular stores and consequent further activation of the capacitative pathway. AVP stimulates Ba2+ entry when the intracellular Ca2+ stores have been both emptied by ionomycin and prevented from refilling by thapsigargin. Mn2+ permeates the capacitative pathway, but AVP does not further increase Mn2+ entry, confirming that AVP does not further activate the capacitative pathway and that the two pathways differ in their permeability to Mn2+. When the extracellular [Sr2+] is low, empty stores do not stimulate detectable Sr2+ entry, but addition of AVP causes substantial Sr2+ entry. 6. A decrease in [Ca2+]i occurs when 50 nM AVP is added during a sustained elevation of [Ca2+]i evoked by thapsigargin. Since AVP does not inhibit the capacitative pathway, this result suggests that AVP stimulates Ca2+ extrusion. 7. We conclude that stimulation of Ca2+ mobilization, two modes of bivalent cation entry, and Ca2+ efflux all contribute to the complex concentration-dependent effects of AVP in A7r5 smooth muscle cells.

Animals↗
PubMed1995

Inhibition of cell-mediated cytolysis and P-glycoprotein function in natural killer cells by verapamil isomers and cyclosporine A analogs.

We have previously shown that among normal leukocytes, CD56+ and CD8+ cells express relatively high levels of P-glycoprotein (P-gp), a transmembrane efflux pump. While the physiologic significance of P-gp expression in leukocytes is unknown, the relatively high levels of P-gp in CD56+ and CD8+ cells suggest that P-gp may function in cell-mediated cytolysis. To explore this possibility we examined the effect of four inhibitors of P-gp efflux [(R)-verapamil (R-ver), (S)-verapamil (S-ver), cyclosporine A (CsA), and PSC833 (PSC)] on both the inhibition of natural killer cell (NK) function and on P-gp efflux. NK function was assayed by measuring the lysis of 51Cr-labeled K562 target cells in the presence and absence of inhibitors. All four P-gp efflux inhibitors inhibited NK-mediated cytolysis in a dose-dependent manner. The stereoisomers of verapamil were more potent inhibitors of cell-mediated cytolysis than the cyclosporines CsA and PSC. In contrast, CsA and PSC were more potent as inhibitors of P-gp-mediated rhodamine 123 dye efflux than the verapamil isomers. Both CsA and PSC maximally inhibited P-gp efflux at 3 microM, but only minimally inhibited cell-mediated cytolysis. The verapamil compounds demonstrated closer correlation between efflux inhibition of NK-mediated cytolysis. The data support a role for P-gp in NK-mediated cytolysis; however, these studies also suggest that the NK cytolytic process is multifaceted and that inhibition of the P-gp-mediated efflux mechanism only partially abrogates this process.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Functional domains of nucleolar phosphoprotein p120.

Nucleolar phosphoprotein p120 is a low abundance, proliferation-associated protein. Several functional domains have been characterized and are discussed here such as the antigenic domain recognized by a monoclonal antibody, the nuclear/nucleolar localization domain, phosphorylation domains of casein kinase II (CKII) and protein kinase C, a putative methylation domain and an RNA binding region. By sucrose gradient sedimentation analyses, protein p120 was shown to rapidly sediment with 60-80 S pre-rRNP particles but sedimented more slowly when treated with RNAse or salt suggesting binding to RNA. Nucleolar protein p120 differed from other nucleolar proteins such as C23 (nucleolin) and B23 (nucleophosmin) which sedimented more slowly near the top of the gradient.

Amino Acid Sequence↗
PubMed1994

Two calcium-binding sites mediate the interconversion of liver inositol 1,4,5-trisphosphate receptors between three conformational states.

Cytosolic Ca2+ biphasically regulates Ins(1,4,5)P3-stimulated Ca2+ mobilization in liver [Marshall and Taylor (1993) J. Biol. Chem. 268, 13214-13220]. We have investigated the mechanisms underlying this biphasic control of Ca2+ mobilization in permeabilized hepatocytes by comparing the effects of Sr2+, Ba2+ and Ca2+ on the liver Ins(1,4,5)P3 receptor. Both Ca2+ and Sr2+ increased the binding of [3H]Ins(1,4,5)P3 to liver membranes by converting receptors from a low-affinity (KD approximately 35 nM) to a high-affinity (KD approximately 5 nM) state. Ba2+ (< or = 20 microM) did not affect [3H]Ins(1,4,5)P3 binding. At concentrations similar to those that caused an enhancement of [3H]Ins(1,4,5)P3 binding, Sr2+ (EC50 = 570 nM) and Ca2+ (EC50 = 200 nM) increased the sensitivity of the intracellular Ca2+ stores to Ins(1,4,5)P3. Further modest elevations in [Ca2+] (EC50 = 1.5 microM) inhibited Ins(1,4,5)P3-stimulated Ca2+ mobilization, whereas Sr2+ caused inhibition only when its concentration was very substantially increased (EC50 approximately 900 microM). Sr2+ is therefore only 3-fold less potent than Ca2+ in causing sensitization of Ins(1,4,5)P3-stimulated Ca2+ release, but 600-fold less potent in causing inhibition. Ba2+ neither sensitized ([Ba2+] < or = 20 microM) nor inhibited ([Ba2+] < or = 1 mM) Ins(1,4,5)P3-stimulated Ca2+ release, and did not inhibit either the sensitization of Ca2+ release evoked by Sr2+ or the inhibition of Ca2+ release evoked by Ca2+. Our results suggest that two distinct Ca(2+)-binding sites, which differ in their selectivities for bivalent cations, mediate the interconversion of Ins(1,4,5)P3 receptors between at least three different conformational states. These two Ca(2+)-binding sites, which may reside either on the Ins(1,4,5)P3 receptor itself or on distinct regulatory proteins, can be distinguished by their different selectivities for bivalent cations.

Animals↗
PubMed1994

Interactions between Ca(2+)-mobilizing receptors and their G proteins in hepatocytes.

In hepatocytes, different receptors that share an ability to stimulate inositol 1,4,5-trisphosphate formation by activating guanine nucleotide-binding proteins (G proteins) evoke Ca2+ signals that are characteristic of the receptor that evoked them. High affinity, guanine nucleotide-sensitive binding of agonists to their receptors provides a convenient means of assessing receptor-G protein interactions. We used these methods to examine the extent to which different Ca(2+)-mobilizing receptors share G protein pools. Although our earlier results (Dasso, L. L. T., and Taylor, C.W. (1992) Mol. Pharmacol. 42, 453-457) showed that activated V1-vasopressin receptors prevented alpha 1-adrenoreceptors from associating with G proteins, our present results show that neither alpha 1-adrenergic agonists nor angiotensin II influences the interaction between V1 receptors and their G proteins. This asymmetric scavenging of G proteins by alpha 1-adrenoreceptors and V1 receptors is not a consequence of there being more of the latter and may result either from V1 receptors binding more tightly to G proteins or from their ability to interact with a second G protein pool. We speculate that the different interactions among V1 receptors, alpha 1-adrenoreceptors, and their G proteins contribute to the differently shaped Ca2+ spikes evoked by these receptors.

Amino Acid Sequence↗
PubMed1994

Methylated A-type proanthocyanidins and related metabolites from Cassipourea gummiflua.

The range of natural dimeric A-type proanthocyanidins is extended by identification of two new dimeric afzelechins in the form of 7-OMe-epiafzelechin-(4 beta-->8,2 beta-->0-->7)- epiafzelechin and 7-OMe-epiafzelechin-(4 beta-->8, 2 beta-->0-->7)-ent-afzelechin. These are accompanied in the stem bark of Cassipourea gummiflua by the related metabolites afzelechin, kaempferol-3-0-alpha-L-rhamnopyranoside, afzelechin-3-0- alpha-L-rhamnopyranoside, epiafzelechin (4 beta-->8,2 beta-->0-->7)-epiafzelechin and epiafzelechin-(4 beta-->8, 2 beta-->0-->7)-ent-afzelechin, the latter three compounds being reported from a natural source for the second time.

Anthocyanins↗
PubMed1994

Ca2+ permeability of cloned and native 5-hydroxytryptamine type 3 receptors.

We have used single-cell imaging of fura-2-loaded cells to examine the Ca2+ signals evoked by activation of 5-hydroxytryptamine type 3 (5-HT3) receptors in undifferentiated N1E-115 neuroblastoma cells and in human embryonic kidney (HEK) 293 cells transfected with either of the two cloned 5-HT3 receptor subunits. The selective 5-HT3 receptor agonist 1-(m-chlorophenyl)-biguanide (mCPBG) caused a concentration-dependent increase in the cytoplasmic Ca2+ concentration ([Ca2+]i) in N1E-115 cells and in HEK 293 cells transfected with either the 5-HT3 A subunit or the 5-HT3 As subunit. In each case, the [Ca2+]i rise was steeply dependent on the mCPBG concentration (nH = 2-4) and abolished by removal of extracellular Ca2+ or addition of ondansetron. Pretreatment of N1E-115 cells with thapsigargin, caffeine, and ryanodine to deplete intracellular Ca2+ stores had no effect on the mCPBG-evoked Ca2+ signals, indicating that they result entirely from stimulated Ca2+ entry. The steep concentration-effect curves therefore are not a consequence of amplification of Ca2+ influx by Ca(2+)-induced Ca2+ release from intracellular stores and probably reflect cooperative activation of 5-HT3 receptors by mCPBG. Depolarization of transfected HEK 293 cells with medium containing increased K+ concentrations invariably failed to evoke an increase in [Ca2+]i, confirming the absence of voltage-gated Ca2+ channels and indicating that the mCPBG-evoked rise in [Ca2+]i results from Ca2+ permeation of 5-HT3 receptors. However, in N1E-115 cells and transfected HEK 293 cells, both extracellular Na+ and K+ substantially inhibited the Ca2+ influx evoked by activation of 5-HT3 receptors, possibly by inhibition of agonist binding or by competition with Ca2+ for permeation of the channel. We conclude that 5-HT3 receptors are Ca2+ permeant, that the Ca2+ influx is sufficient to generate a significant rise in [Ca2+]i, and that, because the A and As subunits behave similarly, conflicting electrophysiological analyses of Ca2+ currents cannot be explained by differences between these two subunits.

Biguanides↗
PubMed1993

Characterization of the receptor responsible for thrombin-induced intracellular calcium responses in osteoblast-like cells.

The receptor responsible for the increase in intracellular calcium concentration ([Ca2+]i) after the addition of thrombin to the human osteoblast-like cell line Saos-2 has been characterized. Thrombin caused a dose-dependent increase in [Ca2+]i; a half-maximal stimulation was observed with 3.2 +/- 1.1 nM thrombin. The human platelet thrombin receptor is activated by thrombin cleavage to create a new NH2 terminus that acts as a tethered ligand, and peptides based on the tethered ligand can activate the receptor independently of thrombin. Northern analysis indicated the presence of mRNA encoding the platelet receptor in Saos-2 cells, and surface expression of the receptor was demonstrated by immunocytochemistry. A tethered ligand peptide (SFLLRNPNDKYEPF, single-letter amino acid code) was found to increase [Ca2+]i. The maximal response to the peptide was similar to that observed with thrombin, and a half-maximal response was observed with 22 +/- 6 microM peptide. The time course of the increase in [Ca2+]i with the peptide was different than that observed with thrombin; a pronounced shoulder was observed after an initial sharp rise. The phenylalanine in the second position of the agonist peptide and the arginine in the fifth position were shown to be essential for its activity. The requirement for proteolysis of the receptor for the thrombin-dependent increase in [Ca2+]i was demonstrated by two methods. Antibodies that reacted with the cleavage site of the receptor abolished the effect of thrombin on [Ca2+]i. In addition, a mutant of thrombin without catalytic activity as well as chemically inactivated thrombin failed to cause an increase in [Ca2+]i. Similar results were obtained with the rat osteoblast-like cell line UMR-106; a tethered ligand peptide based on the rat sequence induced an increase in [Ca2+]i, and antibodies to the cleavage site of the rat receptor inhibited the effect of thrombin.

Amino Acid Sequence↗
PubMed1993

Biphasic effects of cytosolic Ca2+ on Ins(1,4,5)P3-stimulated Ca2+ mobilization in hepatocytes.

The increase in cytosolic free Ca2+ concentration that follows mobilization of intracellular Ca2+ stores by inositol 1,4,5-trisphosphate Ins(1,4,5)P3 has been reported to modulate the sensitivity of Ins(1,4,5)P3 receptors. We have examined the effects of cytosolic Ca2+ on Ins(1,4,5)P3-stimulated Ca2+ mobilization in permeabilized hepatocytes. Increasing the free Ca2+ concentration in the medium ([Ca2+]m) caused a concentration-dependent increase in the sensitivity of the stores to Ins(1,4,5)P3; the concentration of Ins(1,4,5)P3 that caused half-maximal Ca2+ mobilization (EC50) decreased from 261 +/- 11 nM (n = 3) to 50 +/- 4 nM (n = 8) as [Ca2+]m was increased from approximately 7 nM to 1.6 microM. The EC50 for this effect of Ca2+ was approximately 250 nM. In addition, higher [Ca2+]m (> 600 nM) reduced the extent of Ca2+ release induced by a maximal concentration of Ins(1,4,5)P3; elevating [Ca2+]m to 2.6 microM reduced the proportion of Ca2+ releasable by Ins(1,4,5)P3 by 94 +/- 8% (n = 3). Both effects of Ca2+ were independent of Ca2(+)-stimulated Ins(1,4,5)P3 formation. When elevated [Ca2+]m was returned to control levels, the sensitization of Ins(1,4,5)P3-mediated Ca2+ mobilization reversed completely, whereas the reduction in the size of the Ins(1,4,5)P3-sensitive Ca2+ pool was reversed by only 59 +/- 12% (n = 5) after 20 s and was not further reversed after 100 s. The two distinct effects of Ca2+ on Ins(1,4,5)P3-mediated Ca2+ release combined to control the amount of Ca2+ released by a submaximal concentration of Ins(1,4,5)P3 in a biphasic manner.

Animals↗
PubMed1993

Luminal Ca2+ increases the affinity of inositol 1,4,5-trisphosphate for its receptor.

Luminal Ca2+ has been proposed to regulate the sensitivity of intracellular Ca2+ stores to InsP3 and perhaps thereby to contribute to the mechanisms responsible for regenerative intracellular Ca2+ signals. Since Ca2+ release from intracellular stores is accompanied by K+ influx into the stores, InsP3-stimulated Ca2+ mobilization can be inhibited by substantially reducing the [K+] of incubation media. By measuring [3H]InsP3 binding to permeabilized hepatocytes in K(+)-deficient media, thereby preventing InsP3-stimulated Ca2+ mobilization, we have examined the effects of Ca2+ within the intracellular stores on equilibrium binding of InsP3 to its receptor. Our results demonstrate a small, but significant (P < 0.05, n = 8-9), effect of luminal Ca2+ on InsP3 binding; the concentration of InsP3 causing half-maximal displacement of [3H]InsP3 (1.25 nM) was 3.5 nM for empty stores and 2.1 nM for stores containing Ca2+. Our results suggest that at least part of the stimulatory effect of luminal Ca2+ on InsP3-stimulated Ca2+ mobilization may result from an effect of luminal Ca2+ on InsP3 binding to its receptor.

Animals↗
PubMed1993

Effects of Ca2+ chelators on purified inositol 1,4,5-trisphosphate (InsP3) receptors and InsP3-stimulated Ca2+ mobilization.

Changes in medium free [Ca2+] ([Ca2+]m) (< or = 1 microM) did not affect binding of [3H]InsP3 to inositol 1,4,5-trisphosphate (InsP3) receptors purified from cerebellum, but the chelators normally used to buffer [Ca2+]m inhibited InsP3 binding. 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) (1 mM) fully and reversibly inhibited specific [3H]InsP3 (0.6 nM) binding; the half-maximal effect (IC50) occurred with 0.34 +/- 0.03 mM BAPTA. Similar effects were observed with fura-2 (IC50 = 0.12 +/- 0.03 mM) and EDTA (IC50 = 8.7 +/- 1.9 mM). The order of potency of these Ca2+ chelators in inhibiting InsP3 binding was the reverse of their relative affinities for Ca2+; Ca2+ chelation is not, therefore, the cause of the inhibition. When [Ca2+]m was fixed (250-280 nM), InsP3-stimulated 45Ca2+ mobilization from permeabilized hepatocytes was competitively inhibited by increasing concentrations of BAPTA (KD = 1.8 mM). The effects of BAPTA were not a consequence of chelation of polyvalent cations, because TPEN (0.1 mM), which chelates heavy metal ions, did not affect InsP3 binding and pretreatment of all media with a polymetal sponge (DTPA-polyacrylamide) did not affect the sensitivity of InsP3 binding to BAPTA. We conclude that BAPTA and related Ca2+ chelators, in their Ca2+ -free forms, are competitive antagonists of InsP3 binding to its receptor.

Animals↗
PubMed1993

Caffeine-stimulated Ca2+ release from the intracellular stores of hepatocytes is not mediated by ryanodine receptors.

Caffeine has been much used to examine the possibility that ryanodine receptors similar to those found in skeletal and cardiac muscle may be more widely distributed and perhaps contribute to regenerative Ca2+ signals in electrically inexcitable cells. In permeabilized hepatocytes loaded with 45Ca2+, caffeine (> or = 5 mM) decreased the 45Ca2+ content of the intracellular stores by up to 60%; the effect was substantially reversible and it was not mimicked by the closely related methylxanthine theophylline (20 mM). Ryanodine (5 microM) stimulated a far smaller Ca2+ mobilization (7 +/- 1%). Procaine (1 mM), Ruthenium Red (10 microM) and ryanodine (5 microM) did not affect the Ca2+ release evoked by InsP3 (3 microM) or caffeine (30 mM). We conclude that caffeine can specifically cause Ca2+ release from the intracellular stores of hepatocytes, but the effect is unlikely to be mediated by ryanodine receptors.

Animals↗
PubMed1993

Spontaneous Ca2+ spiking in a vascular smooth muscle cell line is independent of the release of intracellular Ca2+ stores.

Monolayers of fura-2-loaded A7r5 cells, a cell line derived from rat embryonic aorta, generated spontaneous Ca2+ spikes that were synchronized within the cell population. These Ca2+ spikes were abolished by removal of extracellular Ca2+ or addition of nimodipine (50 nM), and their frequency was increased by depolarization with high K+ or by treatment with BAYK 8644 (1 microM), indicating that Ca2+ entry through L-type Ca2+ channels is required for Ca2+ spiking. Several lines of evidence indicate that mobilization of intracellular Ca2+ stores is not necessary for this Ca2+ spiking. 1) Ryanodine (0.1-50 microM) neither stimulated Ca2+ mobilization nor affected Ca2+ spiking; 2) the complex effects of caffeine were mimicked by theophylline, 8-bromo-cyclic adenosine 3':5'-monophosphate (8-bromo-cAMP), and forskolin, suggesting that the caffeine effects may be mediated by cAMP and not by ryanodine receptors; 3) prolonged incubation with thapsigargin (50 nM), which depletes intracellular Ca2+ stores, did not affect the frequency of Ca2+ spiking; 4) Ba2+ or Sr2+ could substitute for Ca2+ in the spike-generating mechanism even when intracellular stores were depleted of Ca2+. Under conditions where the sarcoplasmic reticulum (SR) contained Ca2+, Ba2+ spikes did not cause Ca2+ mobilization. The mechanisms involved in generating spontaneous Ca2+ spiking in A7r5 cells are therefore likely to reside in the sarcolemma and to operate independently of SR Ca2+ uptake and release.

Animals↗
PubMed1993

Measuring and predicting the performances of practicing physicians: an overview of two decades of research at the University of Utah.

This paper presents the major findings of research carried out by a team of investigators at the University of Utah School of Medicine in the 1960s and 1970s (1) to better understand the multidimensional and complex aspects of practicing physicians' performances, and (2) to investigate relationships between assessments of students in medical school and physicians' performances. The major research began in the 1960s, and data were assembled from a variety of sources to identify factors of physicians' performances. The original study sampled 507 practicing physicians, classified into four highly diverse and representative groups. The team also investigated to what degree these physicians' medical school performances (measured by grades) were predictors of their practice performances. Factor analysis of the data on the physicians' performances resulted in the identification of 25 to 29 factors within each of the four groups. A representative profile of the performances of each physician studied showed how well the physician performed on each of the factors appropriate to his or her specialty and background. Correlation analysis demonstrated that these physicians' performances in medical school, as measured by grade-point averages (GPAs), were almost completely independent of their later performances in their practices, a disturbing finding and one that the team maintained cannot be dismissed by arguments of "restriction of range." In the teams' later studies, 61 measures of physicians' performances were correlated to medical school GPAs in the two basic science years and the two clinical science years. No association beyond chance was observed, and nonsignificant relationships held up regardless of the physicians' specialties or years of experience.(ABSTRACT TRUNCATED AT 250 WORDS)

Achievement↗