Membrane interaction of erythroid spectrin: surface-density-dependent high-affinity binding to phosphatidylethanolamine

Density-dependent spectrin binding to dimyristoylphosphatidylcholine/dimyristoylphosphatidylethanolamine (DMPC/DMPE) small uni-lamellar vesicles (SUVs) has been directly evaluated in this work from the increase in the extent of quenching of the tryptophan fluorescence of spectrin at two different temperatures, above and below the main phase transition temperatures (Tm). Results from the binding studies of spectrin to phospholipid SUVs indicated that the binding dissociation constant Kd, increased from 45 +/- 7 nM in pure DMPC SUVs to 219 +/- 20 nM in DMPC/DMPE (50:50) SUVs, both in the gel and liquid crystalline phase. However, in pure DMPE SUVs the Kd decreased drastically to 0.7 +/- 0.2 nM in the gel phase at 18 degrees C and to 2.6 +/- 0.7 nM in the fluid phase at 55 degrees C indicating a high affinity binding of spectrin for the bilayer-forming DMPE. The maximum extent of phospholipid-induced quenching and the number of spectrin molecules associated with one SUV particle, evaluated in the present work, led to a model in DMPC/DMPE bilayer membranes indicating the PE-binding site of spectrin to localize at one of the terminal domains of the dimeric spectrin. A direct evidence of the localization of the PE-binding site at one of the terminal ends of the spectrin dimer also came from electron microscopic observation in fluid membranes made of bovine brain PE.     

Effect of spectrin on structure properties of lipid bilayers formed from mixtures of phospholipids. Fluorescence and microcalorimetric studies.

Effect of spectrin from human erythrocytes on structure properties of lipid bilayers formed from a mixture of phosphatidylethanolamine/phosphatidylserine (PE/PS) and/or phosphatidylethanolamine/phosphatidylcholine (PE/PC) was studied with the use of fluorescence and microcalorimetric methods. Spectrin did not affect the order parameter of lipids in PE/PS vesicles. However, spectrin binding to liposomes did influence temperature, half-width and enthalpy of phase transitions in mixtures of dimyristoylphosphatidylethanolamine (DMPE) and dimyristoylphosphatidylcholine (DMPC), and this effect was dependent on DMPE to DMPC weight ratio. A change in miscibility of the components in the presence of spectrin was observed and it might be due to spectrin-PE interactions.     

Apparent “negative cooperativity” kinetics in the absence of a nonlinear Scatchard plot of thyrotropin-receptor interaction in a human thyroid adenoma

A human thyroid adenoma (benign nodule) was identified which exhibited a linear Scatchard plot of ¹²⁵I-TSH binding, characteristic of a single class of binding site with high affinity (K_d = 0.5±0.1 nM) and low binding capacity (0.8±0.2 pmol/mg protein). In contrast, Scatchard analysis of binding to adjacent normal thyroid was nonlinear, suggesting the presence of high and low-affinity binding sites with K_d's of 0.4±0.2 and of 27.9±11.0 nM and capacities of 0.7±0.3 and 1.8±1.0 pmol/mg protein, respectively. Dissociation of bound ¹²⁵I-TSH from membranes of both adenoma and normal tissue revealed identical enhancement of dissociation in the presence of excess native hormone, thought to be evidence for the “negative cooperativity” model of hormone-receptor interaction. Furthermore, adenylate cyclase from both tissues was equally responsive to TSH. Thus, a thyroid adenoma which contains TSH-responsive adenylate cyclase still exhibited enhanced dissociation by native hormone, even though Scatchard analysis yielded a single, non-cooperative class of binding sites. This suggests that enhanced dissociation of bound hormone does not provide a demonstration of negatively-cooperative site-site interaction. Furthermore, nonlinear Scatchard plots, typical of TSH binding in normal thyroid, represent two classes of binding sites, of which the high affinity type is responsible for stimulation of adenylate cyclase.     

Studies on protein kinase activity and the binding of adenosine 3'5-monophosphate by membranes of hereditary spherocytosis erythrocytes.

Evidence has been presented recently of a deficiency of an endogenous membrane-associated protein kinase in erythrocytes of patients with hereditary spherocytosis (HS). We have measured endogenous protein kinase activity in erythrocyte membranes of 4 HS subjects using different membrane isolation and reaction conditions and find that the phosphorylation of the spectrin component (mean ± S.E. 17.1 ± 1.2 pmoles/10 mins per mg protein) is not significantly different to that of 4 normal controls (mean ± S.E. 20.7 ± 1.1 pmoles/10 mins per mg protein). Phosphorylation of exogenous proteins such as casein and protamine is also not deficient in HS erythrocyte membranes. Adenosine 3′5-monophosphate (cyclic AMP) binding to normal and HS erythrocyte membranes was also studied using a Millipore filtration assay. The affinity of cyclic AMP for erythrocyte membranes as determined by Hill plots of binding data from 4 HS subjects (K_D mean ± S.E. = 2.2 ± 0.2 nM) was not significantly different to 4 normal controls (K_D mean ± S.E. = 2.8 ± 0.6 nM). The rate of dissociation of bound cyclic AMP from HS membranes was also similar to control membranes. We thus cannot confirm the prediction by others that an abnormality of cyclic AMP interaction with the erythrocyte membrane underlies HS.     

Binding of synthetic peptide TPLVTLFK to nonopioid beta‐endorphin receptor on rat brain membranes

The synthetic peptide TPLVTLFK corresponding to the sequence 12–19 of β‐endorphin (referred to as octarphin) was found to bind to high‐affinity naloxone‐insensitive binding sites on membranes isolated from the rat brain cortex (K_d = 2.6 ± 0.2 nM ). The binding specificity study revealed that these binding sites were insensitive not only to naloxone but also to α‐endorphin, γ‐endorphin, [Met⁵]enkephalin, and [Leu⁵]enkephalin, as well. The [³H]octarphin specific binding with brain membranes was inhibited by unlabeled β‐endorphin (K_i = 2.4 ± 0.2 nM ) and a selective agonist of nonopioid β‐endorphin receptor decapeptide immunorphin SLTCLVKGFY (K_i = 2.9 ± 0.2 nM ). At the same time, unlabeled octarphin completely (by 100%) inhibited the specific binding of [³H]immunorphin with membranes (K_i = 2.8 ± 0.2 nM ). Thus, octarphin binds with a high affinity and specificity to nonopioid receptor of β‐endorphin on rat brain cortex membranes. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Allosteric effects of atropine and carbachol on the activity of α<Subscript>2</Subscript>-adrenoceptors in rat brain cortex membranes

Activation and inhibition of muscarinic cholinoceptors by atropine and carbachol are shown to exert allosteric effects on the binding of specific nonselective α₂-adrenoceptor antagonist [³H]RX821002 in rat brain cortex membranes. The ligand-receptor interaction for α₂-adrenoceptors corresponded to the model suggesting the presence of one homogeneous pool of receptors with two specific binding sites. The parameters of the [³H]RX821002 binding were as follows: [³H]RX821002 -K _d = 1.94 ± 0.08 nM, B _max = 13.4 ± 1.8 fmol/mg protein, n = 2. The inhibition of muscarinic cholinoceptors by atropine induced an increase of affinity (K _d = 1.36 ± 0.12 nM) and a decrease of the α2-adrenoceptor density (B _max = 10.18 ± 0.48 fmol/mg protein). The muscarinic cholinoceptor agonist carbachol induced an increase of the affinity (K _d = 1.56 ± 0.05 nM) and quantity of binding sites (B _max = 16.61 ± 0.29 fmol/mg protein). As a result, under the influence of atropine and carbachol, the efficiency of binding (E = B _max/2K _d) increased from 3.50 ± 0.40 to 5.60 ± 0.79 and 6.86 ± 0.20 fmol/mg protein/nM, respectively. The data suggest that α₂-adrenoceptors exist in rat brain cortex as homodimers.     

Phase transitions as a function of osmotic pressure in Saccharomyces cerevisiae whole cells, membrane extracts and phospholipid mixtures

Fourier Transform Infrared spectroscopy (FTIR) was used to determine the phase transition temperature of whole Saccharomyces cerevisiae W303-1 A cells as a function of Aw in binary water-glycerol media. A phase transition occurred at 12 °C in water, at 16.5 °C at Aw=0.75, and at 19.5 °C at Aw=0.65. The temperature ranges over which transition occurred increased with decreasing Aw. A total lipid extract of the plasma membranes isolated from S. cerevisiae cells was also studied, with a phase transition temperature determined at 20 °C in pure water and at 27 °C in binary water-glycerol solutions for both Aw levels tested. The pure phospholipids dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE) and three binary mixtures of these phospholipids (percentage molar mixtures of DMPC/DMPE of 90.5/9.5, 74.8/25.2, and 39.7/60.3) were studied. For DMPC, there was no influence of Aw on the phase transition temperature (always 23 °C). On the other hand, the phase transition temperature of DMPE increased with decreasing Aw for the three aqueous solutions tested (glycerol, sorbitol and sucrose), from 48 °C in water, to 64 °C for a solution at Aw=0.67. For the DMPC/DMPE mixtures, transitions were found intermediate between those of the two phospholipids, and a cooperative state was observed between species at the gel and at the fluid phases.     

Effects of GM1 on brain spectrin-aminophospholipid interactions

Spectrin, a major component of the membrane skeletal meshwork of metazoan cells, is implicated to associate with membrane domains and is known to act as a scaffold for stabilization and activation of different signalling modules. We have studied the effect of GM1 (monosialotetrahexosyl ganglioside), a well-known model ganglioside and a signalling moiety, on the interaction of non-erythroid brain spectrin with both saturated and unsaturated aminophospholipids by spectroscopic methods. We observe that GM1 modulates brain spectrin-aminophospholipid interaction to the greatest degree whereas its effect on erythroid spectrin is not as pronounced. Fluorescence quenching studies show that brain spectrin interacts with DMPC/DMPE-based vesicles with a 10-fold increased affinity in presence of very low amounts of 2% and 5% GM1, and the extent of quenching decreases progressively in presence of increasing amounts of GM1. Interaction of brain spectrin with unsaturated membrane systems of DOPC/DOPE weakens in presence GM1. Increase in the mean lifetime of the Trp residues of brain spectrin in presence of GM1 indicates change in the microenvironment of spectrin, without affecting the secondary structure of the protein significantly. Studies on pressure – area isotherm of Langmuir-Blodgett monolayer and Brewster's angle microscopy show that GM1 has an expanding effect on the aminophospholipid monolayers, and ordered regions in DMPC/DMPE mixed monolayers are formed and are stabilized at higher pressure. GM1-induced fluidization of the phospholipid membranes and probable physical contact between bulky sugar head group of GM1 and spectrin, may explain the modulatory role of GM1 on aminophospholipid interactions with nonerythroid brain spectrin.     

Phase transitions as a function of osmotic pressure in Saccharomyces cerevisiae whole cells, membrane extracts and phospholipid mixtures

Fourier Transform Infrared spectroscopy (FTIR) was used to determine the phase transition temperature of whole Saccharomyces cerevisiae W303-1 A cells as a function of Aw in binary water-glycerol media. A phase transition occurred at 12 degrees C in water, at 16.5 degrees C at Aw=0.75, and at 19.5 degrees C at Aw=0.65. The temperature ranges over which transition occurred increased with decreasing Aw. A total lipid extract of the plasma membranes isolated from S. cerevisiae cells was also studied, with a phase transition temperature determined at 20 degrees C in pure water and at 27 degrees C in binary water-glycerol solutions for both Aw levels tested. The pure phospholipids dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE) and three binary mixtures of these phospholipids (percentage molar mixtures of DMPC/DMPE of 90.5/9.5, 74.8/25.2, and 39.7/60.3) were studied. For DMPC, there was no influence of Aw on the phase transition temperature (always 23 degrees C). On the other hand, the phase transition temperature of DMPE increased with decreasing Aw for the three aqueous solutions tested (glycerol, sorbitol and sucrose), from 48 degrees C in water, to 64 degrees C for a solution at Aw=0.67. For the DMPC/DMPE mixtures, transitions were found intermediate between those of the two phospholipids, and a cooperative state was observed between species at the gel and at the fluid phases.     

Interaction of the synthetic immunomodulatory dipeptide bestim with murine macrophages and thymocytes

The tritium-labeled dipeptide bestim (γ-D-Glu-L-Trp) with a specific activity of 45 Ci/mmol was obtained by high-temperature solid-state catalytic isotope exchange. It was found that [³H]bestim binds with a high affinity to murine peritoneal macrophages (K _d 2.1 ± 0.1 nM) and thymocytes (K _d 3.1 ± 0.2 nM), as well as with plasma membranes isolated from these cells (K _d 18.6 ± 0.2 and 16.7 ± 0.3 nM, respectively). The specific binding of [³H]bestim to macrophages and thymocytes was inhibited by the unlabeled dipeptide thymogen (L-Glu-L-Trp) (K _i 0.9 ± 0.1 and 1.1 ± 0.1 nM, respectively). After treatment with trypsin, macrophages and thymocytes lost the ability to bind [³H]bestim. Bestim in the concentration range of 10^?10 to 10^?6 M reduced the adenylate cyclase activity in the membranes of murine macrophages and thymocytes.     

The LKEKK synthetic peptide as a ligand of rat intestinal epithelial cell membranes

A tritium-labeled synthetic LKEKK pentapeptide corresponding to the sequences 16–20 of human thymosin-α₁ and 131–135 of human interferon-α₂ was obtained with a specific activity of 28 Ci/mmol. [³H]LKEKK was found to bind with high affinity (K _d 3.7 ± 0.3 nM) to the membranes isolated from epithelial cells of rat small intestinal mucosa. The trypsin treatment of the membranes did not affect the binding, thus supporting the nonprotein nature of the peptide receptor. The binding of the labeled peptide was inhibited by unlabeled thymosin-α₁, interferon-α₂, and cholera toxin B subunit (K _i 4.2 ± 0.4, 3.5 ± 0.3, and 4.7 ± 0.3 nM respectively). The pentapeptide did not affect the adenylate cyclase activity within the concentration range of 1–1000 nM.     

Synthetic peptide KKRR corresponding to the human ACTH fragment 15–18 is an antagonist of the ACTH receptor

Tritium-labeled synthetic fragments of human adrenocorticotropic hormone (ACTH) [³H]ACTH (11–24) and [³H]ACTH (15–18) with a specific activity of 22 and 26 Ci/mmol, respectively, were obtained. It was found that [³H]ACTH-(11–24) binds to membranes of the rat adrenal cortex with high affinity and high specificity (K _d 1.8 ± 0.1 nM). Twenty nine fragments of ACTH (11–24) were synthesized, and their ability to inhibit the specific binding of [³H]ACTH (11–24) to adrenocortical membranes was investigated. The shortest active peptide was found to be an ACTH fragment (15–18) (KKRR) (K _i 2.3 ± 0.2 nM), whose [³H] labeled derivative binds to rat adrenocortical membranes (K _d 2.1 ± 0.1 nM) with a high affinity. The specific binding of [³H]ACTH-(15–18) was inhibited by 100% by unlabeled ACTH (11–24) (K _i 2.0 ± 0.1 nM). ACTH (15–18) in the concentration range of 1–1000 nM did not affect the adenylate cyclase activity of adrenocortical membranes and, therefore, is an antagonist of the ACTH receptor.     

Serotoninergic agonists and antagonists are the modulators of α1-Adrenoceptor activity in rat brain cortical membranes

The effects of activation and inhibition of serotonin receptors by serotonin (5-HT) and mianserin on the specific nonselective α₁-antagonist [³H]prazosine binding in rat cerebral cortex membranes was studied. It was shown that the ligand-receptor interaction of α₁-adrenoceptors corresponded to the model suggesting the presence of one pool of receptors and the binding of two ligand molecules to the receptor. The parameters of [³H]prazosine binding to α₁-adrenoceptors were as follows: K _d =1.85 ± 0.16 nM, B _max = 31.1 ± 0.3 fmol/mg protein, n = 2. In case of activation of 5HT-receptors by serotonin, the character of ligand binding was different: two pools of receptors were detected with the parameters K _d1 = 0.61 ± 0.04, K _d2 = 3.82 ± 0.15 nM, B _m1 = 6.6 ± 0.7, B _m2 = 25.6 ± 0.4 fmol/mg protein, n = 2. The sensitivity of the high-affinity pool increased threefold and the sensitivity of the low-affinity pool decreased twofold as compared to the control. The value of maximal reaction (B _max) did not change. In the case of inhibition of 5HT-receptors by mianserin, radioactive ligand is bound to α₁-adrenoceptors according to the same model as in the control conditions. The affinity of α₁-adrenoceptors to [³H]prazosine decreases twofold and the concentration increases (K _d = 3.97 ± 0.12 nM, B _max = 40.0 ± 0.5 fmol/mg protein). The data suggest that α₁-adrenoceptors in rat cerebral cortex exist as a dimer. The modulatory effects of serotonin and mianserin on the specific binding of [³H]prazosine to α₁-adrenoceptors was detected, manifesting itself as changes in the binding parameters and in the general character of ligand-receptor interactions.     

Binding of chemotactic peptide to the outer surface and to whole human spermatozoa with different affinity states

Binding of N-formyl-methionyl-L-leucyl-[³H]phenylalanine (fML[³H]Ph) to human ejaculated spermatozoa and to its isolated plasma membrane was studied. Our data confirm the presence of specific receptors for f-MLPh in the human spermatozoa and suggest that whole spermatozoa receptors exist in two affinity states, one high-affinity, low-capacity specific receptor (K_d = 12.3 ± 0.5 nM, n = 22,285 ± 65,008 binding sites per sperm cell) and a second one (K_d = 700 ± 47 nM) that is not saturable, indicating a low-affinity, high-capacity nonspecific site. In contrast, sperm membrane showed only one class of binding site (K_d = 6.4 ± 0.12 nM), which was statistically different from that of the high-affinity binding site of intact spermatozoa. To explain this difference we discuss the possibility that first, the two binding affinities represent two interconvertible states of a single receptor population, which, depending on the metabolic activity of spermatozoa, may change its physicochemical properties; or second, they reflect two different processes, binding and/or transport into the spermatozoa.     

Transbilayer and interbilayer phospholipid exchange in dimyristoylphosphatidylcholine/dimyristoylphosphatidylethanolamine large unilamellar vesicles

The rates of spontaneous interbilayer and transbilayer exchange of [3H]dimyristoylphosphatidylcholine ([3H]DMPC) were examined in DMPC and DMPC/dimyristoylphosphatidylethanolamine (DMPE) large unilamellar vesicles in the liquid-crystalline-, gel-, and mixed-phase states. DMPC desorption rates from either gel or liquid-crystalline phases containing DMPE are very similar to the corresponding rates from pure DMPC gel or liquid-crystalline phases. This is not the case for DMPC desorption from distearoylphosphatidylcholine (DSPC)-containing gel phases, where the desorption rates are significantly faster than from a pure DMPC gel phase [Wimley, W. C., & Thompson, T. E. (1990) Biochemistry 29, 1296-1303]. We proposed that the DMPC/DSPC behavior results from packing defects in gel phases composed of both DMPC and DSPC molecules because of the four-carbon difference in the acyl chain lengths of the two species. The present results strongly support this hypothesis because no such anomalous behavior is observed in DMPC/DMPE, which is similar to DMPC/DSPC in phase behavior but does not have the chain length difference. The inclusion of 10-30 mol % DMPE in DMPC bilayers was also found to have a significant effect on the rate of transbilayer movement (flip-flop) of [3H]DMPC in the liquid-crystalline phase. Between 10 and 30 mol % DMPE, flip-flop of DMPC is slowed by at least 10-fold relative to flip-flop in DMPC bilayers, and the entropy and enthalpy of flip-flop activation are both substantially decreased.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthetic peptide TPLVTLFK (octarphin) reduces the corticosterone production by rat adrenal cortex through nonopioid β‐endorphin receptor

The synthetic peptide octarphin (TPLVTLFK) corresponding to the sequence 12–19 of β‐endorphin, a selective agonist of nonopioid β‐endorphin receptor, was labeled with tritium to a specific activity of 29 Ci/mmol. [³H]Octarphin was found to bind to high‐affinity naloxone‐insensitive binding sites on membranes isolated from rat adrenal cortex (K_d = 35.7 ± 2.3 nM, B_max = 41.0 ± 3.6 pmol/mg protein). The binding specificity study revealed that these binding sites were insensitive not only to naloxone but to α‐endorphin, γ‐endorphin, [Met⁵]enkephalin, and [Leu⁵]enkephalin as well. At the same time, the [³H]octarphin‐specific binding with adrenal cortex membranes was inhibited by unlabeled β‐endorphin (K_i = 32.9 ± 3.8 nM). Octarphin at concentrations of 10^?9–10^?6 M was found to inhibit the adenylate cyclase activity in adrenocortical membranes, whereas intranasal injection of octarphin at doses of 5 and 20 µg/rat was found to reduce the secretion of corticosterone from the adrenals to the bloodstream. Thus, octarphin decreases the adrenal cortex functional activity through the high affinity binding to nonopioid receptor of β‐endorphin. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Interaction of the synthetic peptide octarphin with rat adrenal cortex membranes

The synthetic peptide octarphin (TPLVTLFK, fragment 12?C19 of ??-endorphin), a selective agonist of the nonopioid ??-endorphin receptor, was labeled with tritium yielding specific activity of 28 Ci/mmol. The binding of [³H]octarphin to rat adrenal cortex membranes was studied under normal conditions as well as after cold and heat shocks. It was found that under normal conditions [³H]octarphin specifically binds to the membranes with high affinity: K _d1 = 36.3 ± 2.5 nM, B_max1 = 41.0 ± 3.8 pmol/mg protein. The specific binding of [³H]octarphin to the membranes was inhibited by unlabeled ??-endorphin (K _i = 33.9 ± 3.6 nM) and the agonist of the non-opioid receptor decapeptide immunorphin (K _i = 36.8 ± 3.3 nM). Unlabeled naloxone, [Leu⁵]- and [Met⁵]enkephalins, ??- and ??-endorphins, and corticotropin were inactive (K _i > 1 ??M). Both cold and heat shocks decreased the binding affinity: K _d2 = 55.6 ± 4.2 nM and K _d3 = 122.7 ± 5.6 nM, respectively. In both cases, the maximal binding capacity of the receptor did not change. Thus, even a short-term thermal shock significantly affects the sensitivity of the non-opioid ??-endorphin receptor of adrenal cortex membranes.     

The role of electrostatic interactions in the membrane binding of melittin

The binding of melittin and the C-terminally truncated analogue of melittin (21Q) to a range of phospholipid bilayers was studied using surface plasmon resonance (SPR). The phospholipid model membranes included zwitterionic dimyristylphosphatidylcholine (DMPC) and dimyristylphosphatidylethanolamine (DMPE), together with mixtures DMPC/dimyristylphosphatidylglycerol (DMPG), DMPC/DMPG/cholesterol and DMPE/DMPG. Melittin bound rapidly to all membrane mixtures, whereas 21Q, which has a reduced charge, bound much more slowly on the DMPC and DMPC/DMPG mixtures reflecting the role of the initial electrostatic interaction. The loss of the cationic residues also significantly decreased the binding of 21Q with DMPC/DMPG/Cholesterol, DMPE and DMPE/DMPG. The role of electrostatics was also highlighted with NaCl in the buffer, which affected the way melittin bound to the different membranes, causing a more uniform, concentration dependant increase in response. The biosensor results were correlated with the conformation of the peptides determined by circular dichroism analysis, which indicated that high α-helicity was associated with high binding affinity. Overall, the results demonstrate that the positively charged residues at the C-terminus of melittin play an essential role in membrane binding, that modulation of peptide charge influences selectivity of binding to different phospholipids and that manipulation of the cationic regions of antimicrobial peptides can be used to modulate membrane selectivity.     

Characteristics of an Adenylate Cyclase Coupled β-Adrenergic Receptor in a Smooth Muscle Tumor Cell Line

Abstract

We have shown that binding of ³H-dihydroalprenolol ([³H] DHA) to DDT₁ MF-2 cells and cell membranes was of high affinity, saturable, stereoselective and reversible. The [³H]DHA dissociation constants were 0.63 ± 0.15 nM (n=6) and 0.83 ± 0.04 nM (n=5) for intact cells and cell membranes, respectively, with a binding site concentration for cells of 27,300 ± 5,200 sites/ cell (n=6) and for membranes 468 ± 24 fmoles/mg protein (n=5). The order of agonist competition for the [³H]-DHA binding site of DDT₁ cell membranes was isoproterenol (K_i = 0.20 ± 0.07 μM) > epinephrine (K_i = 0.4 ± 0.2 μM) > norepinephrine (K_i = 66.5 ± 5.15 μM) consistent with a β₂-selective receptor interaction. Zinterol, a β₂-selective antagonist, (K_i = 0.05 ± 0.01 μM) was 18x more effective than metoprolol, a β₁-selective antagonist (K_i = 0.9 ± 0.1 μM), in competing for the DHA binding site. A nonlinear iterative curve fitting analysis of zinterol and metoprolol binding isotherms indicated that (p>0.05) DDT₁ cells possess a pure population of β₂-adrenergic receptors. Finally, we have shown that DDT₁ MF-2 cell β₂-adrenergic receptor is functionally coupled to adenylate cyclase via a G/F protein complex as demonstrated in part by a guanine nucleotide requirement for isoproterenol stimulation of adenylate cyclase activity. In addition, guanine nucleotide mediated a reduction in the affinities of isoproterenol and epinephrine for the [³H]DHA binding site.     

Potassium Depletion and Regulation of Angiotensin II Receptors in Vascular Smooth Muscle Cells

Abstract

Mesenteric artery smooth muscle cells were grown in culture media containing high, normal, or low concentrations of potassium to study the effects on angiotensin II (Ang II) receptor regulation. Cell growth was similar among cells grown in the different culture media. Cells grown in high potassium media (K=5.8 mEq/L) had an equilibrium dissociation constant, K_d, of 1.59 ± 0.2 nM, whereas those grown in normal potassium media (K=4.1 mEq/L) had a K_d of 1.79 ± 0.2 nM and those grown in a low potassium media (K=2.9 mEq/L) had a K_d of 1.19 ± 0.12 nM (not significantly different, NS). Binding capacity of smooth muscle cells grown in high potassium media was 81 ± 16.7 fmol/mg prot, 95.1 ± 12.4 fmol/mg prot in those grown in normal potassium media and those grown in low potassium media 86.4 ± 24.1 fmol/mg prot (NS). Binding of radiolabelled Ang II was reduced by approximately 70% in cells exposed to unlabelled Ang II for 30 or 60 minutes. However, this effect of exposure to Ang II to reduce subsequent binding of Ang II was identical in cells grown in high and low potassium medium. Therefore, we were unable to identify a direct effect of low potassium to induce changes in Ang II receptor binding affinity or binding capacity. Previously observed changes in these Ang II binding parameters in potassium-depleted rats was probably a consequence of other factors which were simultaneously altered by potassium deficiency.