Ryanodine and dihydropyridine binding patterns and ryanodine receptor mRNA levels in myopathic hamster heart

1994 ◽  
Vol 267 (3) ◽  
pp. H1205-H1213 ◽  
Author(s):  
W. G. Lachnit ◽  
M. Phillips ◽  
K. J. Gayman ◽  
I. N. Pessah
Keyword(s):  
Binding Sites ◽  
Receptor Activation ◽  
Mrna Levels ◽  
Functional Changes ◽  
Cardiomyopathic Hamster ◽  
Receptor Mrna ◽  
Significant Difference ◽  
Voltage Dependent ◽  
Hamster Heart ◽  
Ca2 Channels

We have determined the densities of sarcolemmal voltage-dependent Ca2+ channels (VDCC) and Ca(2+)-induced Ca2+ release channels (CICR) of sarcoplasmic reticulum (SR) in the cardiomyopathic hamster heart using [3H]PN-200 and [3H]ryanodine, respectively. Partially purified cardiac membrane preparations from myopathic animals exhibit a twofold higher capacity to bind both [3H]PN-200 and [3H]ryanodine. Crude particulate membrane fractions from normal and cardiomyopathic animals reveal no significant difference in receptor densities for [3H]PN-200, whereas densities for [3H]ryanodine binding sites and mRNA levels are significantly (P < 0.05) diminished in cardiomyopathic animals. Inhibition of [3H]ryanodine binding by either Ca2+ or Mg2+ (in mM) as well as temperature dependence for receptor activation for [3H]ryanodine (Q10) is not significantly different, whereas membranes isolated from cardiomyopathic hearts are 1.4-fold and threefold more sensitive to activation by doxorubicin and Ca2+ (in microM), respectively. Vesicles isolated from myopathic hearts are more sensitive to inhibition of Ca2+ uptake by doxorubicin. The higher densities of binding sites for [3H]PN-200 and [3H]ryanodine observed in partially purified membrane fractions from cardiomyopathic hearts are more likely the result of altered patterns with which T-tubule and CICR channels fractionate in preparations from cardiomyopathic hamster heart rather than transcriptional upregulation and may be a consequence of the deficiency in a dystrophin-associated glycoprotein recently identified. Downregulation and functional changes in CICR channels may alter SR Ca2+ transport and contribute to the progression of cardiomyopathy in the hamster.

Increased mechanical extraction of T-tubule/junctional SR from cardiomyopathic hamster heart

1993 ◽  
Vol 264 (5) ◽  
pp. H1447-H1453 ◽  
Author(s):  
Y. Tawada-Iwata ◽  
T. Imagawa ◽  
A. Yoshida ◽  
M. Takahashi ◽  
H. Nakamura ◽  
...  
Keyword(s):  
Binding Sites ◽  
Binding Assay ◽  
Radioligand Binding ◽  
Immunoblot Assay ◽  
Mechanical Disruption ◽  
Significant Difference ◽  
Number Of Binding Sites ◽  
Hamster Heart ◽  
Mechanical Extraction ◽  
Normal Controls

We determined the contents of L-type calcium channels (LCC) and other membrane proteins in ventricular homogenates and microsomes prepared from hearts of 30- to 70-day-old Syrian cardiomyopathic (Bio 14.6) and normal hamsters. Quantitative immunoblot assay revealed that myopathic microsomes, as compared with normal controls, were enriched about twofold with the alpha 1-subunit of LCC, the ryanodine receptor calsequestrin, and Na(+)-K(+)-adenosinetriphosphatase (ATPase), whereas the contents of these proteins in ventricular homogenates were not different. In contrast, Na(+)-H+ antiporter and sarcoplasmic reticulum (SR) Ca(2+)-ATPase showed no difference in their contents in both homogenates and microsomes. Radioligand binding assay further showed no significant difference in the number of binding sites for [3H]prazosin, [125I]iodocyanopindolol, and [3H]saxitoxin between myopathic and normal microsomes. These result suggest that whereas membrane densities of LCC and the other proteins examined are not increased in myopathic cardiomyocytes, T-tubule/junctional SR membranes are more easily extracted from them by mechanical disruption. This, together with 1.5-fold higher yield of microsomal fractions from myopathic heart muscle, shows that abnormality exists in the mechanical property of cell membrane in the myopathic heart.

scholarly journals Block of N-type Calcium Channels in Chick Sensory Neurons by External Sodium

1997 ◽  
Vol 109 (6) ◽  
pp. 693-702 ◽  
Author(s):  
Luis Polo-Parada ◽  
Stephen J. Korn
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Dependent Manner ◽  
Full Time ◽  
Channel Conductance ◽  
Concentration Dependent Manner ◽  
Voltage Dependent ◽  
Ion Pore ◽  
Channel Currents ◽  
Ca2 Channels

L-type Ca2+ channels select for Ca2+ over sodium Na+ by an affinity-based mechanism. The prevailing model of Ca2+ channel permeation describes a multi-ion pore that requires pore occupancy by at least two Ca2+ ions to generate a Ca2+ current. At [Ca2+] &lt; 1 μM, Ca2+ channels conduct Na+. Due to the high affinity of the intrapore binding sites for Ca2+ relative to Na+, addition of μM concentrations of Ca2+ block Na+ conductance through the channel. There is little information, however, about the potential for interaction between Na+ and Ca2+ for the second binding site in a Ca2+ channel already occupied by one Ca2+. The two simplest possibilities, (a) that Na+ and Ca2+ compete for the second binding site or (b) that full time occupancy by one Ca2+ excludes Na+ from the pore altogether, would imply considerably different mechanisms of channel permeation. We are studying permeation mechanisms in N-type Ca2+ channels. Similar to L-type Ca2+ channels, N-type channels conduct Na+ well in the absence of external Ca2+. Addition of 10 μM Ca2+ inhibited Na+ conductance by 95%, and addition of 1 mM Mg2+ inhibited Na+ conductance by 80%. At divalent ion concentrations of 2 mM, 120 mM Na+ blocked both Ca2+ and Ba2+ currents. With 2 mM Ba2+, the IC50 for block of Ba2+ currents by Na+ was 119 mM. External Li+ also blocked Ba2+ currents in a concentration-dependent manner, with an IC50 of 97 mM. Na+ block of Ba2+ currents was dependent on [Ba2+]; increasing [Ba2+] progressively reduced block with an IC50 of 2 mM. External Na+ had no effect on voltage-dependent activation or inactivation of the channel. These data suggest that at physiological concentrations, Na+ and Ca2+ compete for occupancy in a pore already occupied by a single Ca2+. Occupancy of the pore by Na+ reduced Ca2+ channel conductance, such that in physiological solutions, Ca2+ channel currents are between 50 and 70% of maximal.

scholarly journals Agonist-induced phasic and tonic responses in smooth muscle are mediated by InsP3

2002 ◽  
Vol 115 (10) ◽  
pp. 2207-2218 ◽  
Author(s):  
John G. McCarron ◽  
John W. Craig ◽  
Karen N. Bradley ◽  
Thomas C. Muir
Keyword(s):  
Smooth Muscle ◽  
Muscle Contraction ◽  
Channel Blocker ◽  
Single Cells ◽  
Smooth Muscle Contraction ◽  
Receptor Activation ◽  
Tonic Component ◽  
Voltage Dependent ◽  
Ca2 Channels ◽  
Insp3 Receptor

Many cellular functions are regulated by agonist-induced InsP3-evoked Ca2+ release from the internal store. In non-excitable cells, predominantly, the initial Ca2+release from the store by InsP3 is followed by a more sustained elevation in [Ca2+]i via store-operated Ca2+ channels as a consequence of depletion of the store. Here, in smooth muscle, we report that the initial transient increase in Ca2+, from the internal store, is followed by a sustained response also as a consequence of depletion of the store (by InsP3), but, influx occurs via voltage-dependent Ca2+ channels. Contractions were measured in pieces of whole distal colon and membrane currents and [Ca2+]i in single colonic myocytes. Carbachol evoked phasic and tonic contractions; only the latter were abolished in Ca2+-free solution. The tonic component was blocked by the voltage-dependent Ca2+ channel blocker nimodipine but not by the store-operated channel blocker SKF 96365. InsP3 receptor inhibition, with 2-APB, attenuated both the phasic and tonic components. InsP3 may regulate tonic contractions via sarcolemma Ca2+ entry. In single cells,depolarisation (to ∼-20 mV) elevated [Ca2+]i and activated spontaneous transient outward currents (STOCs). CCh suppressed STOCs, as did caffeine and InsP3. InsP3 receptor blockade by 2-APB or heparin prevented CCh suppression of STOCs; protein kinase inhibition by H-7 or PKC19-36did not. InsP3 suppressed STOCs by depleting a Ca2+ store accessed separately by the ryanodine receptor (RyR). Thus depletion of the store by RyR activators abolished the InsP3-evoked Ca2+ transient. RyR inhibition (by tetracaine) reduced only STOCs but not the InsP3transient. InsP3 contributes to both phasic and tonic contractions. In the former, muscarinic receptor-evoked InsP3 releases Ca2+ from an internal store accessed by both InsP3 and RyR. Depletion of this store by InsP3 alone suppresses STOCs, depolarises the sarcolemma and permits entry of Ca2+ to generate the tonic component. Therefore, by lowering the internal store Ca2+ content,InsP3 may generate a sustained smooth muscle contraction. These results provide a mechanism to account for phasic and tonic smooth muscle contraction following receptor activation.

Up-regulation of insulin-like growth factor-I (IGF-I) receptor gene expression in patients with reduced serum IGF-I levels

1993 ◽  
Vol 10 (2) ◽  
pp. 115-120 ◽  
Author(s):  
R Eshet ◽  
H Werner ◽  
B Klinger ◽  
A Silbergeld ◽  
Z Laron ◽  
...  
Keyword(s):  
Gene Expression ◽  
Binding Sites ◽  
Dissociation Constants ◽  
Receptor Gene ◽  
Control Group ◽  
Mrna Levels ◽  
Receptor Mrna ◽  
Igf I ◽  
Low Levels ◽  
Receptor Gene Expression

ABSTRACT We have analysed the expression of the IGF-I receptor gene in lymphocytes of patients with low levels of circulating IGF-I (four patients with isolated GH deficiency (IGHD) and one Laron-type dwarf (LTD)) in comparison with a control group exhibiting normal serum IGF-I levels and endocrine profiles. 125I-Labelled IGF-I binding assays were performed on erythrocytes to determine the number of IGF-I binding sites per cell and their dissociation constants. Erythrocytes from patients with IGHD or LTD contained significantly (P=0·002) more receptors per cell (10·9±3·1 binding sites/cell), with a reduced affinity (Kd = 0·49±0·05 nm), than erythrocytes from controls (2·0±0·4 sites/cell; Kd = 0·14 nm). The levels of IGF-I receptor mRNA in circulating lymphocytes were determined by an RNA template-specific reverse transcription/polymerase chain reaction method. There was a statistically significant increase in IGF-I receptor mRNA levels in lymphocytes from patients with LTD or IGHD when compared with controls (3108·1±775·9 vs 576·0±465·7 arbitrary units, P=0·006). The increased level of IGF-I binding due to increased IGF-I receptor gene expression may represent a compensatory up-regulation process activated in response to the low levels of IGF-I in the circulation of patients with LTD or IGHD.

Sodium Status Affects Gc-B Natriuretic Peptide Receptor mRNA Levels, but Not Gc-A Or C Receptor Mrna Levels, in the Sheep Kidney

1994 ◽  
Vol 86 (5) ◽  
pp. 517-522 ◽  
Author(s):  
Margaret B. Fraenkel ◽  
G. Peter Aldred ◽  
John G. McDougall
Keyword(s):  
Natriuretic Peptide ◽  
Transcriptional Control ◽  
Renal Cortex ◽  
Renal Medulla ◽  
Mrna Levels ◽  
Binding Studies ◽  
Renal Vasculature ◽  
Receptor Mrna ◽  
Natural Ligand ◽  
Significant Difference

1. In humans and experimental animals the natriuresis and diuresis resulting from infusion of atrial natriuretic peptide varies with the sodium status of the subject. Tissue binding studies have suggested that this may be related to changes in the renal receptors for the hormone. 2. In order to establish whether these changes are under transcriptional control, we examined the levels of mRNA for the three natriuretic peptide receptors [GC-A, GC-B and clearance (C) receptors] in renal cortex and medulla from six sodium-loaded, six sodium-depleted and four control sheep. cDNA probes specific to each receptor were generated using the polymerase chain reaction. 3. GC-B receptor mRNA levels were increased approximately two-fold in the renal cortex of sodium-depleted animals, whereas there was no influence on GC-B receptor mRNA levels in the renal medulla. There was no significant difference in mRNA levels for the GC-A and C receptors. 4. At present the role of the GC-B receptor and its natural ligand C-type natriuretic peptide in the control of renal function is unknown. The present experiments imply some intrarenal function for the GC-B receptor and its natural ligand, although the site of any such function, e.g. renal vasculature or tubules, remains unclear. In addition, we have shown that if GC-A and C receptor levels in the sheep are modulated by sodium, the regulation occurs beyond the level of gene transcription.

scholarly journals Regulation of transferrin receptor expression in myeloid leukemia cells

Blood ◽  
1987 ◽  
Vol 70 (3) ◽  
pp. 852-859
Author(s):  
R Taetle ◽  
S Ralph ◽  
S Smedsrud ◽  
I Trowbridge
Keyword(s):  
Steady State ◽  
Binding Sites ◽  
Myeloid Leukemia ◽  
Receptor Gene ◽  
Receptor Expression ◽  
Leukemia Cells ◽  
Mrna Levels ◽  
Serum Free ◽  
Receptor Mrna ◽  
Free Media

Surface transferrin (Tf) receptors are displayed by cultured human hematopoietic cells and provide Fe required for cell growth. Cell cycle status, cell density in culture, exposure to Fe, and differentiation alter Tf receptor display by myeloid leukemia cells. To investigate mechanisms controlling Tf receptor expression, rates of receptor synthesis and steady-state mRNA levels were measured in HL60 promyelocytic cells grown in serum and serum-free media or after differentiation in response to dimethylsulfoxide (DMSO). Although surface binding sites were unchanged during the first three days in culture with serum or in serum-free media containing Tf, by the third day, rates of receptor biosynthesis and steady-state mRNA levels declined, consistent with cell density-dependent, receptor regulation. Cells grown with soluble Fe instead of Tf showed reduced Tf binding sites, rates of receptor synthesis, and Tf receptor mRNA. When cells grown with Fe were subcultured, Tf receptor mRNA levels increased within 15 minutes and peaked by 24 hours. This was followed by a decline in receptor and gene expression so that by three days cells grown in the presence of Fe expressed approximately four times fewer receptors and had half the rates of Tf receptor synthesis and mRNA levels of cells grown in serum or Tf. Cells treated with DMSO showed a rapid decline in surface receptors, receptor synthesis, and steady- state mRNA levels. Modulation of Tf receptor expression was not due to redistribution between the cell surface and an internal receptor pool. In each instance, concurrent assessment of N-ras transcripts showed that changes in Tf receptor mRNA levels were not due to generalized alterations in protein synthesis. Exposure of cells grown in Fe or treated with DMSO to cycloheximide did not alter Tf receptor mRNA levels, thereby suggesting that receptor expression was not regulated by posttranscriptional processes dependent on protein synthesis. Actinomycin D inhibition of Tf receptor mRNA was compatible with a transcript half-life of approximately 2.2 hours. Nuclear transcription studies showed reduced rates of Tf receptor transcription after culture with Fe or exposure to DMSO. The present studies show complex patterns of Tf receptor gene regulation in cultured myeloid leukemia cells and demonstrate that transcriptional regulation is a major mechanism controlling Tf receptor gene expression in response to Fe and differentiation.

scholarly journals Regulation of transferrin receptor expression in myeloid leukemia cells

Blood ◽  
1987 ◽  
Vol 70 (3) ◽  
pp. 852-859 ◽  
Author(s):  
R Taetle ◽  
S Ralph ◽  
S Smedsrud ◽  
I Trowbridge
Keyword(s):  
Steady State ◽  
Binding Sites ◽  
Myeloid Leukemia ◽  
Receptor Gene ◽  
Receptor Expression ◽  
Leukemia Cells ◽  
Mrna Levels ◽  
Serum Free ◽  
Receptor Mrna ◽  
Free Media

Abstract Surface transferrin (Tf) receptors are displayed by cultured human hematopoietic cells and provide Fe required for cell growth. Cell cycle status, cell density in culture, exposure to Fe, and differentiation alter Tf receptor display by myeloid leukemia cells. To investigate mechanisms controlling Tf receptor expression, rates of receptor synthesis and steady-state mRNA levels were measured in HL60 promyelocytic cells grown in serum and serum-free media or after differentiation in response to dimethylsulfoxide (DMSO). Although surface binding sites were unchanged during the first three days in culture with serum or in serum-free media containing Tf, by the third day, rates of receptor biosynthesis and steady-state mRNA levels declined, consistent with cell density-dependent, receptor regulation. Cells grown with soluble Fe instead of Tf showed reduced Tf binding sites, rates of receptor synthesis, and Tf receptor mRNA. When cells grown with Fe were subcultured, Tf receptor mRNA levels increased within 15 minutes and peaked by 24 hours. This was followed by a decline in receptor and gene expression so that by three days cells grown in the presence of Fe expressed approximately four times fewer receptors and had half the rates of Tf receptor synthesis and mRNA levels of cells grown in serum or Tf. Cells treated with DMSO showed a rapid decline in surface receptors, receptor synthesis, and steady- state mRNA levels. Modulation of Tf receptor expression was not due to redistribution between the cell surface and an internal receptor pool. In each instance, concurrent assessment of N-ras transcripts showed that changes in Tf receptor mRNA levels were not due to generalized alterations in protein synthesis. Exposure of cells grown in Fe or treated with DMSO to cycloheximide did not alter Tf receptor mRNA levels, thereby suggesting that receptor expression was not regulated by posttranscriptional processes dependent on protein synthesis. Actinomycin D inhibition of Tf receptor mRNA was compatible with a transcript half-life of approximately 2.2 hours. Nuclear transcription studies showed reduced rates of Tf receptor transcription after culture with Fe or exposure to DMSO. The present studies show complex patterns of Tf receptor gene regulation in cultured myeloid leukemia cells and demonstrate that transcriptional regulation is a major mechanism controlling Tf receptor gene expression in response to Fe and differentiation.

Regulation of 5-HT2A receptor mRNA levels and binding sites in rat frontal cortex by the agonist DOI and the antagonist mianserin

2000 ◽  
Vol 39 (11) ◽  
pp. 1996-2005 ◽  
Author(s):  
A Anji ◽  
M Kumari ◽  
N.R Sullivan Hanley ◽  
G.L Bryan ◽  
J.G Hensler
Keyword(s):  
Frontal Cortex ◽  
Binding Sites ◽  
Mrna Levels ◽  
Receptor Mrna ◽  
Rat Frontal Cortex

scholarly journals Effect of thrombin, the thrombin receptor activation peptide, and other mitogens on vascular smooth muscle cell urokinase receptor mRNA levels

Blood ◽  
1994 ◽  
Vol 84 (11) ◽  
pp. 3700-3708 ◽  
Author(s):  
U Reuning ◽  
SP Little ◽  
EP Dixon ◽  
NU Bang
Keyword(s):  
Smooth Muscle ◽  
Growth Factor ◽  
Cell Surface ◽  
Time Course ◽  
Transcriptional Activity ◽  
Receptor Activation ◽  
Thrombin Receptor ◽  
Mrna Levels ◽  
Mrna Stabilization ◽  
Receptor Mrna

Bovine vascular smooth muscle cells (SMC) express the urokinase-type plasminogen activator receptor (u-PAR) claimed to be important in cell invasion. Receptor numbers and affinity are regulated by thrombin and several other mitogens involved in SMC proliferation. We investigated the effects of these mitogens on u-PAR mRNA levels. On continuous thrombin stimulation the u-PAR message in SMC was 10 +/- 2.3-fold elevated reaching a maximum between 6 and 9 hours and declining to control values within 48 hours. Thrombin present for 30 minutes on the cell surface produced similar effects. Stimulation with the thrombin receptor activation peptide S-F-L-L-R-N representing the NH2-terminus of the tethered ligand also increased u-PAR mRNA levels with an identical time course. D-Phe-Pro-Arg-chloromethyl ketone (PPACK) active site blocked thrombin and the catalytically inactive thrombin mutant S205A did not affect u-PAR mRNA levels. Thrombin stimulation also resulted in a 2 +/- 0.2-fold transient increase in thrombin receptor mRNA preceding the rise in u-PAR message. Transforming growth factor beta 1 (TGF beta 1) and platelet-derived growth factor (PDGF) showed similar time courses for the elevation of u-PAR mRNA levels with a maximal 5.5 +/- 0.9 and 12 +/- 2.5-fold increase, respectively. Basic fibroblast growth factor (bFGF) and phorbol myristate acetate (PMA) showed a more prolonged effect increasing u-PAR mRNA levels 8 +/- 2.0- fold and 12.3 +/- 2.5-fold, respectively, within 6 hours but remaining 5 to 10-fold elevated at 48 hours. In order to decide if the u-PAR mRNA increase was due to message stabilization or a consequence of transcriptional activation we used the RNA polymerase II inhibitor 5,6- dichloro-1-beta-D-ribofuranosyl benzimidazole (DRB) during the stimulation experiments. u-PAR mRNA levels on TGF beta 1 stimulation of SMC decayed after the addition of DRB indicating that enhancement of transcriptional activity was involved in the induction. In contrast, the time course of u-PAR mRNA elevation on thrombin, bFGF, and PMA stimulation was not significantly altered in the presence of DRB suggesting that in these latter cases u-PAR mRNA message accumulation was at least in part due to mRNA stabilization. Increased transcriptional activity, mRNA stabilization and expression of u-PAR protein on the SMC surface in response to growth factors may facilitate enhanced cell surface protease activity, cell migration, and development of atheromatous lesions.

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