Thermodynamic mixing of molecular states of the epidermal growth factor receptor modulates macroscopic ligand binding affinity.

The epidermal growth factor receptor (EGFr), when expressed on the cell surface, has long been known to display two distinct affinities for epidermal growth factor (EGF) binding. In addition, the treatment of cells expressing the EGFr with phorbol esters has been shown to cause a loss of the high-affinity binding capacity of the receptor. In the present study, point mutations that alter acidic or phosphorylation sites have been made in an intracellular domain near Tyr-992 (residues 988-992) of the EGFr. Equilibrium (125)I-EGF binding studies demonstrate that the conversion of Tyr-992 into glutamate induces a 4-fold decrease in the EGFr apparent low-affinity dissociation constant, whereas the mutation of two acidic residues, Asp-988 and Glu-991, or the conversion of Tyr-992 into phenylalanine does not alter EGFr affinity. Phorbol ester treatment of EGFr-expressing Chinese hamster ovary cells results in a loss of high-affinity binding and an increase in the apparent low-affinity dissociation constant of the receptor, similar to the effect of a truncation mutant in which the C-terminal 190 residues are deleted. These results are examined in the context of a new model for regulation of the affinity of the EGFr for EGF in which a cytosolic particle stabilizes the high-affinity conformation of the EGFr and a rapid equilibrium exists between EGFr high-affinity and low-affinity conformations. This model demonstrates that the macroscopic affinities of the EGFr can differ from the affinities of individual EGFr molecules and provides a theoretical framework whereby the measured affinities of the EGFr are modulated by intracellular interactions.

Epidermal growth factor receptor internalization rate is regulated by negative charges near the SH2 binding site Tyr992.

This study examines the effects of mutations at and in the vicinity of tyrosine 992 of the epidermal growth factor receptor (EGFr) on epidermal growth factor- (EGF-) stimulated internalization of the receptor. Two regions of the EGFr adjacent to this domain have been defined previously as internalization domains. The present work shows that the mutation of negatively charged amino acid residues near Tyr992 to their uncharged analogues increases the rate of EGF receptor internalization. In addition, the conversion of Tyr992, which is an EGFr ligand-induced autophosphorylation site, to phenylalanine also increases the rate of receptor internalization. However, the mutation of Tyr992 to a glutamate residue does not alter the receptor internalization rate. In addition, the truncation of the EGFr at glutamate 996 reduces the internalization rate by half. This result confirms previous reports that residues immediately C-terminal to Glu996 are necessary to allow the normal rate of ligand-induced receptor endocytosis. The data suggest that negative charge in the vicinity of Tyr992, and potentially the phosphorylation of the EGFr at Tyr992, reduces the rate of ligand-induced receptor endocytosis. This reduction in internalization rate increases the lifetime of the activated EGFr in the plasma membrane by about 70%, thus suggesting that phosphorylation of Tyr992 acts to increase the signaling capacity of the EGF receptor even as it directly acts as an SH2 binding site.

Time course of the initial [Ca2+]i response to extracellular ATP in smooth muscle depends on [Ca2+]e and ATP concentration.

In response to extracellular application of 50 microM ATP, all individual porcine aortic smooth muscle cells respond with rapid rises from basal [Ca2+]i to peak [Ca2+]i within 5 s. The time from stimulus to the peak of the [Ca2+]i response increases with decreasing concentration of ATP. At ATP concentrations of 0.5 microM and below, the time to the [Ca2+]i peak varies more significantly from cell to cell than at higher concentrations, and each cell shows complicated initiation and decay kinetics. For any individual cell, the lag phase before a response decreases with increasing concentration of ATP. An increase in lag time with decreasing ATP concentration is also observed in the absence of extracellular Ca2+, but the lag phase is more pronounced, especially at concentrations of ATP below 0.5 microM. Whole-cell patch-clamp electrophysiology shows that in porcine aortic smooth muscle cells, ATP stimulates an inward current carried mainly by Cl- ion efflux with a time course similar to the [Ca2+]i changes and no detectable current from an ATP-gated cation channel. A simple signal cascade initiation kinetics model, starting with nucleotide receptor activation leading to IP3-mediated Ca2+ release from IP3-sensitive internal stores, fits the data and suggests that the kinetics of the Ca2+ response are dominated by upstream signal cascade components.

Kinetics of extracellular ATP hydrolysis by microvascular endothelial cells from rat heart.

We have characterized the ectonucleotidases that catalyse the reaction sequence ATP-->ADP-->AMP-->adenosine on microvascular endothelial cells cultured from the rat heart. Computer simulation and data fitting of progress of reaction curves showed that depletion of substrate at the cell surface dominates the regulation of the rate of hydrolysis of ATP when it is presented to the cells. Preferential delivery of AMP by ADPase to 5'-nucleotidase makes a significant contribution to the regulation of adenosine production from ATP or ADP. By contrast, we found no evidence for the preferential delivery of ADP from ATPase to ADPase. Feed-forward inhibition of AMP hydrolysis by ADP and/or ATP also modulated the rate of adenosine production. The properties of the ectonucleotidases on rat heart microvascular cells are such that adenosine is produced at a steady rate over a wide range of ATP concentrations.

Role of phosphodiesterase isoenzymes in regulating intracellular cyclic AMP in adenosine-stimulated smooth muscle cells.

Three phosphodiesterase (PDE) isoenzymes were separated by Mono Q h.p.l.c. column chromatography from the soluble fraction of a homogenate of pig aortic smooth muscle cells. The first peak of PDE activity was stimulated by calmodulin in the presence of calcium. The second broad peak contained at least two activities, which were sensitive to inhibition by CI-930 or rolipram respectively. The distribution of total cellular enzyme activity in different subcellular fractions was also determined. The majority (78%) of the total activity was present in the cytosolic fraction, 18% of activity was in a membrane-bound form and 4% of activity was associated with the cytoskeleton. Rolipram-sensitive PDE was present predominantly in the cytosolic fraction, whereas cyclic GMP-inhibited, CI-930-sensitive PDE was evenly distributed between the cytosolic and particulate fractions. All of the calmodulin-dependent PDE activity was found in the soluble fraction. CI-930 and rolipram enhanced, by 2-fold and 3-4-fold respectively, the adenosine-stimulated rise in cellular cyclic AMP level. The increase in cyclic AMP levels due to CI-930 or rolipram was dose-dependent. Removal of adenosine once cyclic AMP had risen resulted in a rapid fall in cyclic AMP levels even in the presence of rolipram and CI-930. M&B 22,948, the calmodulin-dependent PDE inhibitor, caused less than a 25% increase of the adenosine-stimulated cyclic AMP levels by itself, but it contributed substantially to controlling the cyclic AMP levels after the removal of adenosine when used together with CI-930 and rolipram. These phenomena suggested that all three PDE isoenzymes participated in modulating cellular cyclic AMP levels after adenosine stimulation, and that differential importance of the individual isoenzymes depends on cellular cyclic AMP levels.

Effect of 5'-deoxy-5'-isobutylthioadenosine on formation and release of adenosine from neonatal and adult rat ventricular myocytes.

1. Studies in rat polymorphonuclear leucocytes have suggested that 5'-deoxy-5'-isobutylthioadenosine (IBTA), an inhibitor of the IMP-selective cytosolic 5'-nucleotidase, may be used to test its role in adenosine formation in intact cells. We investigated adenosine formation in neonatal and adult rat cardiomyocytes. 2. 2-Deoxyglucose (30 mM) with oligomycin (2 micrograms/ml) induced a 90-100% fall in ATP concentration in 10 min in neonatal and 60 min in adult heart cells. Adenosine accumulation was substantially increased, accounting for 13% of the fall in ATP concentration in neonatal cells and 56% in adult cells. 3. Anti-(rat liver ecto-5'-nucleotidase) serum did not inhibit adenosine accumulation. Furthermore, dipyridamole (10 microM), a nucleoside-transport blocker, inhibited by 80% the appearance of the newly formed adenosine in the medium, showing that adenosine is produced intracellularly by both adult and neonatal-rat myocytes in response to inhibition of oxidative metabolism. 4. IBTA (3 mM) inhibited by 80% the appearance of adenosine in the medium, but did not inhibit total adenosine accumulation by neonatal-rat myocytes and only modestly inhibited total adenosine accumulation by adult myocytes. 5. IBTA, like dipyridamole, inhibited incorporation of extracellular adenosine (10 microM) into neonatal and adult ventricular myocyte nucleotides by 60-70%. Transport of IBTA (100 microM) into the cells did not appear to be inhibited by dipyridamole (30 microM). 6. We conclude that IBTA acted primarily to inhibit adenosine release from myocytes. The small effect on adenosine formation rates implies that the IMP-selective cytosolic 5'-nucleotidase plays a minor role in this tissue.

Independent modes of propagation of calcium waves in smooth muscle cells.

The purinergic agonist adenosine triphosphate (ATP) stimulates an initial transient followed by subsequent oscillations in cytosolic calcium ion concentration ([Ca2+]i) in individual porcine aortic smooth muscle cells. Using microinjection of fura-2 covalently coupled to dextran, we have analyzed in detail the spatial and temporal features of the oscillations. We have observed both cytoplasmic calcium waves and gradients within single cells. Single cells can contain multiple loci of initiation of oscillations. Independent oscillations in a single cell can have independent frequencies and these oscillations can propagate without interference across the same region of the cell, suggesting that they arise either from separately regulated stores of Ca2+ or a single Ca2+ store operated by two separate release mechanisms. The shape of the wave front and the manner of the waye's decay can vary from one oscillation to the next. Ca2+ signaling in individual arterial smooth muscle cells thus displays complex spatial and temporal organization.

Gastric and salivary mucins inhibit angiotensin-converting enzyme. Inhibition is partly due to oligosaccharides.

Pig gastric mucin, a highly glycosylated glycoprotein, inhibits angiotensin-converting enzyme (ACE) with an IC50 of 2 mM-neutral hexose content. Pig submaxillary mucin at 2.3 mM inhibits by 73%. To determine whether the oligosaccharide moieties of the mucins contribute to this inhibition, oligosaccharides were prepared from each mucin by reductive beta-elimination and their effects on enzyme activity determined. Total oligosaccharides from gastric mucin inhibited enzyme activity with an IC50 of 0.3 mM based on the neutral hexose content of the oligosaccharide solution. Fractions isolated from gastric mucin by chromatography on DEAE-cellulose and Bio-Gel P-2 inhibited ACE with IC50 values ranging from 2 to 16 mM-oligosaccharide. Larger oligosaccharides inhibited with lower IC50 values than did smaller oligosaccharides. Fractions of average molecular mass 1100 and 740 Da prepared from submaxillary mucin inhibited with IC50 values of 40 and 80 mM-oligosaccharide respectively. Monosaccharides commonly present in serum and membrane glycoproteins were also tested for their effect on ACE. Galactose, N-acetylglucosamine, N-acetylgalactosamine and glucosamine were inhibitory. N-Acetylneuraminic acid stimulated the activity of ACE. Fucose, ethylene glycol and sucrose had no effect on the activity of the enzyme. The influences of different buffers, ion concentrations, pH and substrate structure on the effect of carbohydrate on enzyme activity were also evaluated. The extent of inhibition by the monosaccharide galactose was strongly influenced by buffer ion and substrate concentration. The effects of the oligosaccharide moieties and intact mucins were less sensitive to assay conditions.

Regulation of extracellular adenosine production by ectonucleotidases of adult rat ventricular myocytes.

We have investigated the kinetic properties of the extracellular reaction sequence ATP----ADP----AMP----adenosine catalyzed by ectonucleotidases at the surface of adult rat cardiac myocytes. Analysis of progress of reaction curves indicates that depletion of substrate at cell surfaces dominates the regulation of the rate of hydrolysis of ATP or of ADP when it is the initial substrate. Preferential delivery of intermediate products to be substrates at cell surfaces makes a significant contribution to the regulation of adenosine production from ATP or ADP. Preferential delivery has more impact on the delivery of ADP from adenosinetriphosphatase (ATPase) to adenosinediphosphatase (ADPase) than on delivery of AMP from ADPase to 5'-nucleotidase. At high initial ATP concentrations, feed-forward inhibition of AMP hydrolysis also modulates the rate of adenosine production. Taken together, the properties of the ectonucleotidases on the myocyte provide a milieu at the cell surface that tends to be poor in nucleotides, especially ATP and ADP (P2 purinoceptor agonists), and rich in adenosine (a P1 purinoceptor agonist) during periods of supply of extracellular nucleotides.

Independent pathways regulate the cytosolic [Ca2+] initial transient and subsequent oscillations in individual cultured arterial smooth muscle cells responding to extracellular ATP.

Stimulation with extracellular ATP causes a rapid initial transient rise followed by asynchronous periodic oscillations in cytosolic calcium ion activity ([Ca2+]i) in individual aortic smooth muscle cells in either HEPES-buffered or HCO3(-)-buffered saline. The dose at which one-half of the cells display an initial rise in cytosolic calcium is 0.11 microM ATP in the presence of external Ca2+ and 0.88 microM ATP in the absence of external Ca2+; the corresponding value for oscillations in the presence of external Ca2+ is 2.6 microM ATP. While the initial transient displays rapid desensitization, the oscillations persist for greater than 30 min in the continuous presence of ATP. The presence of the agonist ATP is also absolutely required for the maintenance of the oscillations, presumably to provide continuous activation of P2 purinoceptors. The average frequency of oscillation is approximately 0.9 min-1. The frequency depends only slightly on the concentration of ATP, and oscillations do not collapse into a prolonged elevated [Ca2+]i at high concentrations of ATP. Both Ca2+ influx and release from internal stores participate in the initial transient. Oscillations are not produced in the absence of external Ca2+ but are initiated upon the addition of external Ca2+ in the continued presence of ATP. Oscillations in progress are abolished by the removal of extracellular Ca2+ with one additional peak occurring after the Ca2+ removal. These data suggest that extracellular Ca2+ influx is required for the maintenance of the posttransient oscillations, presumably to provide the Ca2+ necessary for refilling intracellular Ca2+ pools that are the source of the oscillating [Ca2+]i. The Ca2+ influx is not regulated by voltage-gated Ca2+ channels. The data in this report are consistent with the view that the initial transient has contributions from two receptor-mediated pathways, and the oscillations are controlled either by a mechanism separate from the ones that control the initial transient or by steps whose control diverges before the point of desensitization.

Modulation of response to adenosine in vascular smooth muscle cells cultured in defined medium.

Cultured pig aortic smooth muscle cells maintain a viable, quiescent state in a chemically defined medium that contains 10(-6) M insulin, 5 micrograms/ml transferrin, and 0.2 mM ascorbate. DNA synthesis and DNA content were determined by measuring tritiated thymidine incorporation and DNA-binding to the fluorescent probe 4',6-diamidino-2-phenylindole, respectively. The majority of the population of cells in defined medium cultures were diploid. Tritiated thymidine uptake in cells in defined medium was one-tenth that observed in cells in fetal bovine serum-containing medium. The study of cellular cyclic AMP level in response to extracellular adenosine stimulation in dividing cells and quiescent cells showed that cells in defined medium had a lower extent of response to adenosine compared to cells cultured in serum-containing medium. Both the cell growth index and the response to adenosine of cells cultured in defined medium were reversible after replacing the medium with 10% fetal bovine serum-containing medium, which suggests that the cells in defined medium were healthy and were capable of modulating cellular metabolism depending on culture conditions.

Cyclic AMP efflux is regulated by occupancy of the adenosine receptor in pig aortic smooth muscle cells.

Cultured pig aortic smooth muscle cells respond to extracellular adenosine by activating adenylate cyclase and by initiating the efflux of cAMP. In the presence of extracellular adenosine, efflux is first order with respect to intracellular cAMP concentration up to at least 125 pmol/10(6) cells. The apparent first-order rate constant for the efflux of cAMP increases in a dose-dependent manner in response to extracellular adenosine or 5-N-ethylcarboxamide adenosine. The EC50 for adenosine for promoting cAMP efflux is 12 microM. For cells stimulated with 5-N-ethylcarboxamide adenosine, the EC50 is 5 microM. When extracellular adenosine is removed, efflux stops abruptly. Cellular cAMP content falls but is still in a range that supports cAMP efflux when agonist is present. Efflux is not affected by H8 (N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride), an inhibitor of cAMP-dependent protein kinase. These data suggest that in pig aortic smooth muscle cells, the efficiency of cAMP efflux is regulated by A2 receptor occupancy.

Charge-coupled device imaging of rapid calcium transients in cultured arterial smooth muscle cells.

Transient changes in the concentration of intracellular free calcium are associated with the transduction of primary signals and the subsequent employment of Ca2+ as a second messenger in a multitude of cell types. These transients, typically monitored with the calcium-sensitive fluorescent dye Fura-2, are known to occur with a time course in the order of seconds. In order to accurately monitor such rapid changes in intracellular free calcium concentration in both single cells and simultaneously in several cells in a single field, we have developed a digital fluorescence imaging system based on a charge-coupled device (CCD) camera. We report here on the detailed kinetics of calcium increases in cultured arterial swine smooth muscle cells in response to the agonist ATP.

The hydrolysis of extracellular adenine nucleotides by arterial smooth muscle cells. Regulation of adenosine production at the cell surface.

The extracellular reaction sequence ATP----ADP----AMP----adenosine participates in regulating the time course of cellular response during crisis or signaling events, such as thrombus formation or neurotransmission. We have investigated the whole time course of hydrolysis of ATP to adenosine by recirculating adenine nucleotide substrates over smooth muscle cells attached to polystyrene beads. Kinetic parameters were estimated for each reaction by fitting observed time courses to models of the pathway. In spite of the inhibition of 5'-nucleotidase by ADP, adenosine was produced very rapidly by smooth muscle cells. Comparisons of the apparent Km values of ADPase and 5'-nucleotidase (determined from experiments in which each substrate was used as the initial substrate with Km values observed when each substrate was supplied from the upstream reaction) suggest that the local concentrations of substrate supplied from the preceding reactions are very much higher than those in the bulk phase. This enhancement of efficiency overcomes the effect of the feed-forward inhibition to give rise to very rapid adenosine production from ADP or ATP. These observations are in marked contrast to our previous findings with endothelial cells (Gordon, E. L., Pearson, J. D., and Slakey, L. L. (1986) J. Biol. Chem. 261, 15496-15504), on which feed-forward inhibition causes a profound lag in adenosine production from adenine nucleotides and on which there are no apparent surface effects on substrate delivery.

Adenosine receptors activate adenylate cyclase and enhance secretion from bovine adrenal chromaffin cells in the presence of forskolin.

Cells of the adrenal medulla release not only catecholamines but also high concentrations of neuropeptides and nucleotides. Chromaffin cells, like many neuronal cells, have a diversity of receptors: adrenergic receptors, peptide receptors, histamine receptors, and dopamine receptors. We recently reported that these cells have nucleotide receptors that can mediate inhibition of the secretory response. The present studies show that adenosine, in the presence of enabling concentrations of forskolin, can potently enhance response to nicotinic stimulation. Neither adenosine nor forskolin alone produces a significant effect. A marked rise in intracellular cyclic AMP (cAMP) concentration is associated with the enhancement of secretion caused by forskolin plus adenosine. A phosphodiesterase inhibitor, Ro 20-1724, used together with forskolin produces significant increases in both cellular cAMP content and catecholamine secretion. However, the adenosine agonist 5'-N-ethylcarboxyadenosine elevates cellular cAMP content in the presence of forskolin without having any positive effect on secretion. This finding suggests that the rise in cAMP level may not be the sole cause of the increase in secretion by adenosine.

Time course of release into the medium of newly synthesized proteins by cultured aortic endothelial cells. Role of serum in preventing proteolytic degradation.

Endothelial cells from pig aortas were labeled with 35S-methionine, and the soluble proteins that were released into the culture medium were examined by SDS-PAGE. Proteins were collected during labeling, and during the intervals 0 to 3, 3 to 6, 6 to 12, and 12 to 24 hours of chase after a 1-hour labeling period, and during the intervals 0 to 12, 12 to 24, and 24 to 48 hours of chase after a 24-hour labeling period. Release of radiolabeled soluble protein from cells into the medium continued over the longest time periods examined. If the cells were labeled for 1 hour, characteristic patterns of proteins appeared in the medium during the labeling period, early (0- to 3-hour postlabeling) and late (6- to 24-hour postlabeling). The time course of release of fibronectin (Fn) and of angiotensin-converting enzyme (ACE) was determined by using immunoprecipitation. ACE appeared only after 6 hours of chase. Fn appeared throughout the chase period. The effects of serum and of the protease inhibitors alpha-2 macroglobulin, pepstatin, and leupeptin on protein release were examined. In the absence of serum, endothelial cell culture medium contained substantial protease activity capable of completely degrading most of the released proteins; a major effect of serum was to protect newly released protein from degradation.

The hydrolysis of extracellular adenine nucleotides by cultured endothelial cells from pig aorta. Feed-forward inhibition of adenosine production at the cell surface.

The time course of the extracellular reaction sequence ATP----ADP----AMP----adenosine has been examined during recirculation of substrate solutions over cultured pig aortic endothelial cells attached to polystyrene beads. This permits the study of reactions at volume to cell surface ratios approaching those of small blood vessels. When endothelial cells were presented with an initial bolus of ATP, high concentrations of the intermediates ADP and AMP developed before significant conversion of AMP to adenosine occurred. Further, the higher the initial ATP concentration, the slower the conversion of AMP to adenosine. Kinetic constants for each reaction were estimated by fitting simulated reaction curves to observed time courses. Apparent Km values estimated in this way agreed well with those reported for initial velocity measurements (ATPase = 300 microM; ADPase = 240 microM; and 5'-nucleotidase = 26 microM). The ratio of maximum velocities was ATPase:ADPase:AMPase = 6:1.5:1, with absolute values varying among cell batches. The data could only be fitted if the model incorporated inhibition of 5'-nucleotidase by ATP or ADP, and satisfactory fitting was achieved with a Ki value for ADP of 5 microM. These kinetic properties maximize the time separation of the intermediate pools. In vivo, at sites of platelet degranulation, they would create a time gap proportional to the size of the initial release between release of ADP (a proaggregatory milieu) and the appearance of adenosine (an anti-aggregatory milieu).

Hydrolysis of diadenosine 5',5''-P',P''-triphosphate (Ap3A) by porcine aortic endothelial cells.

Diadenosine triphosphate is present in platelet-dense granules and released quantitatively on platelet aggregation. We have found that intact porcine aortic endothelial cells can efficiently hydrolyze extracellular diadenosine triphosphate. The products of diadenosine triphosphate hydrolysis are adenosine monophosphate and adenosine diphosphate. Adenosine diphosphate is a potent stimulus of platelet aggregation. Since platelet-dense granules contain high concentrations of adenosine triphosphate and adenosine diphosphate, we examined endothelial cell hydrolysis of a mixture of diadenosine triphosphate and adenosine triphosphate. We find that the presence of adenosine triphosphate severely inhibits the hydrolysis of diadenosine triphosphate. Thus, although endothelial cells can rapidly clear extracellular diadenosine triphosphate, during platelet aggregation the hydrolysis of diadenosine triphosphate may be slow due to the presence of high concentrations of other adenine nucleotides. This phenomenon may be important physiologically if, as current evidence implies, diadenosine triphosphate is involved in the maintenance of hemostasis.