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P N Devreotes

Publications and source records attributed to P N Devreotes.

107 records · Page 6Linked to original sources
PubMed1984

Adenosine and its derivatives inhibit the cAMP signaling response in Dictyostelium discoideum.

In developmentally competent Dictyostelium discoideum amoebae, binding of cAMP to high-affinity surface receptors produces a rapid activation of adenylate cyclase which adapts within minutes. The result is a transient increase in intracellular cAMP which is rapidly secreted. Adenosine inhibited this cAMP signaling response with an apparent Ki of 300 microM. The apparent Ki's for 2'-O-methyladenosine and 2-chloroadenosine were approximately 30 and 100 microM, respectively. Inhibition by adenosine was rapid, reversible, and depended on the cAMP stimulus concentration. In addition, the adaptation of the cAMP signaling response was blocked by adenosine. As has been previously reported, adenosine inhibits cAMP binding to intact cells. Under the same developmental conditions as in the perfusion studies, we find the binding inhibition depends on both the cAMP and adenosine concentrations, with an apparent Ki of 100 microM. The apparent Ki's for 2'-O-methyl- and 2-chloroadenosine were approximately 8 and 35 microM, respectively. However, with cells developed for short times and with an axenic strain, inhibition was independent of cAMP concentration or cells showed mixed-type binding inhibition. The effect of adenosine on the cAMP signaling response is consistent with the reported effects of adenosine on other cAMP-mediated processes such as chemotaxis and the increase in intracellular cGMP, and may provide an explanation for the reported inhibition of center formation.

2-Chloroadenosine↗
PubMed1983

The effect of folic acid on cAMP-elicited cAMP production in Dictyostelium discoideum.

In Dictyostelium discoideum, folic acid serves as a chemoattractant, accelerates the appearance of several developmentally regulated enzymes, alters the phase of spontaneous light-scattering oscillations, and elicits increases in cGMP and cAMP. The effect of folic acid and pteridine analogs on rates of [3H]cAMP secretion, monitored by rapid perfusion, was studied. At low cell densities folic acid elicited only minimal [3H]cAMP secretion. At high cell densities, folic acid elicited greater increases in cellular cAMP and rates of [3H]cAMP secretion than did maximally effective doses of cAMP. Following pretreatment with 10(-8) M cAMP for 10 min or 10(-6) M cAMP for 2 min, no [3H]cAMP was secreted in response to folic acid. Conversely, pretreatment with folic acid did not impair subsequent responsiveness to 5 X 10(-9) M cAMP. Simultaneous addition of folic acid and cAMP increased the release of [3H]cAMP two- to threefold compared to control stimuli of the same concentration of cAMP. Pretreatment with a mixed stimulus attenuated the response to a subsequent increment in the cAMP concentration only if the higher cAMP stimulus did not include folic acid. The effects of folic acid are mimicked by the pteridine ring alone; analogs substituted at the 2- or 5-position are ineffective; non-reducible analogs are partially active. The observations presented here suggest significant differences in the mechanisms by which cAMP and folic acid stimulate cAMP production. Rather than there being separate receptors for each chemoattractant that interact in the same way with an intracellular pathway, it appears that folic acid and pteridine analogs potentiate the response to cAMP.

Adaptation, Physiological↗
PubMed1983

Quantitative analysis of cyclic AMP waves mediating aggregation in Dictyostelium discoideum.

We have previously reported the detection of cAMP waves within monolayers of aggregating Dictyostelium discoideum cells (K. J. Tomchik and P.N. Devreotes, 1981, Science 212, 443-446). The computer-assisted analysis presented here of the fluorographic images of the cAMP waves reveals (1) all the waves have a consistent width and height; (2) cAMP concentrations within centers of concentric aggregation territories oscillate periodically while at spiral centers the concentration builds up to a plateau value within 2 mm; (3) cells within the region of intersection of two oppositely directed cAMP waves are stimulated to produce more cAMP than those responding to a single wave; (4) cells start to move when the cAMP level begins to increase and cease movement when the peak cAMP concentration reaches the cell.

Computers↗
PubMed1983

Cyclic 3', 5'-AMP relay in Dictyostelium discoideum: adaptation is independent of activation of adenylate cyclase.

In Dictyostelium discoideum, binding of cAMP to high affinity surface receptors leads to a rapid activation of adenylate cyclase followed by subsequent adaptation within several minutes. The rate of secretion of [ 3H ]cAMP, which reflects the state of activation of the enzyme, was measured. Caffeine noncompetitively inhibited the response to cAMP. Inhibition was rapidly reversible and pretreatment of cells with caffeine for up to 22 min had little effect on the subsequent responsiveness to cAMP. However, cells pretreated with caffeine plus cAMP for greater than or equal to 8 min did not respond when caffeine was removed and the same concentration of cAMP was applied. The following observations indicate that both adaptation and deadaptation to cAMP occurred to the same extent and at the same rate whether or not cAMP synthesis was inhibited. First, when cells were pretreated with 10(-9)-10(-6) M cAMP in the presence or absence of caffeine and the stimulus was switched to a saturating dose of cAMP, the response to the increment was the same whether or not the initial response was blocked. Second, cells progressively lost responsiveness to 10(-6) M cAMP as pretreatment with 10(-6) M cAMP plus caffeine was extended from 0 to 8 min with the same time course as for those pretreated with 10(-6) M cAMP alone. Third, cells which were adapted in the presence of caffeine and cAMP deadapted within the same time period as controls when cAMP was removed. These observations demonstrate that while some part of the activation process is inhibited by caffeine the adaptation process is unaffected. Our conclusion is that adaptation does not depend on the activation of adenylate cyclase.

Adaptation, Physiological↗
PubMed1981

Adenosine 3',5'-monophosphate waves in Dictyostelium discoideum: a demonstration by isotope dilution--fluorography.

The distribution of adenosine 3',5'-monophosphate (cyclic AMP) in fields of aggregating amoebae of Dictyostelium discoideum was examined by a novel isotope dilution-fluorographic technique. Cellular cyclic AMP was visualized by its competition with exogenous 3H-labeled cyclic AMP for high-affinity binding sites on protein kinase immobilized on a Millipore filter used to blot the monolayer. The cyclic AMP was distributed in spiral or concentric circular wave patterns which centered on the foci of the aggregations. These patterns were correlated with those of cell shape change that propagate through the monolayers: cells in regions of high concentrations of cyclic AMP were elongated (presumably moving up a cyclic AMP gradient), whereas those in regions of low cyclic AMP concentrations were randomly directed. The highest cyclic AMP concentrations were about 10(-6)M. The widths of the regions of elevated cyclic AMP were about 0.3 to 1 millimeter which, assuming a wave velocity of 300 micrometers per minute, suggests that a cell signals for about 1 to 3 minutes. These observations support the hypothesis that the aggregation process in Dictyostelium is mediated by the periodic relay of cyclic AMP signals and suggest a simple scheme for the dynamics of the aggregation process.

Cell Aggregation↗
PubMed1980

Cyclic 3',5'-AMP relay in Dictyostelium discoideum III. The relationship of cAMP synthesis and secretion during the cAMP signaling response.

Refinement of a perfusion technique permitted the simultaneous measurement of cAMP-elicited [3H]cAMP secretion and intracellular [3H]cAMP levels in sensitive D. discoideum amoebae. These data were compared with measurements of the rate of [32P]cAMP synthesis by extracts of amoebae sonicated at different times during the cAMP signaling response. cAMP stimulation of intact cells led to a transient activation of adenylate cyclase, which was blocked if 10(-4) M NaN3 was added with the stimulus. During responses elicited by 10(-6) M cAMP, 10(-8) M cAMP, and an increment in cAMP from 10(-8) M to 10(-7) M, the rate of cAMP secretion was proportional to the intracellular cAMP concentration. Removal of a 10(-6) M cAMP stimulus 2 min after the initiation of the response led to a precipitous decline in intracellular cAMP. This decline was more rapid than could be accounted for by secretion alone, suggesting intracellular phosphodiesterase destruction of newly synthesized cAMP. Employing these data and a simple rate equation, estimates of the time-course of the transient activation of adenylate cyclase and the rate constants for cAMP secretion and intracellular phosphodiesterase activity were obtained. The calculated rate of cAMP synthesis rose for approximately 1 to 2 min, peaked, and declined to approach prestimulus levels after 3 to 4 min. This time-course agreed qualitatively with direct measurements of the time-course of activation, indicating that the activation of adenylate cyclase is a major in determining the time-course of the cAMP secretion response.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1980

Cyclic 3',5'-AMP relay in Dictyostelium discoideum IV. Recovery of the cAMP signaling response after adaptation to cAMP.

In dictyoselium discoideum, an increase in extracellular cAMP activates adenylate cyclase, leading to an increase in intracellular cAMP and the rate of cAMP secretion. Cells adapt to any constant cAMP stimulus after several minutes, but still respond to an increase in the concentration of the stimulus. We have now characterized the decay of adaptation (deadaptation) after the removal of cAMP stimuli. Levels of adaptation were established by the perfusion of [(3)H]adenosine-labeled amoebae with a defined cAMP stimulus. After a variable recovery period, the magnitude of the signaling response to a second stimulus was measured; its attenuation was taken as a measure of residual adaption to the first stimulus. The level of adaptation established by the first stimulus depended on both its magnitude and duration. Deadaptation began as soon as the first stimulus was removed. The magnitude of the response to the second stimulus increased with the recovery time in a first-order fashion, with a t(1/2)=3-4 min for stimuli of 10(-8) M to 10(-5) M cAMP. Responses to test stimuli, although reduced in magnitude, had an accelerated time-course when they closely followed a prior response that had not completely subsided. This effect is called priming; we believe it reveals a reversible, rate-limiting step that modulates the onset and termination of the signaling responses of amoebae that have not recently responded to a cAMP stimulus. We have suggested that the cAMP signaling response is controlled by two antagonistic cellular processes, excitation and adaptation. The data reported here imply that both the rate of rise in the adaptation process and the final level reached depend on the occupancy of cAMP surface receptors and that the decay of adaptation when external cAMP is removed proceeds with first-order kinetics.

Adaptation, Physiological↗
PubMed1980

Cyclic 3',5'-AMP relay in Dictyostelium discoideum V. Adaptation of the cAMP signaling response during cAMP stimulation.

In dictyostelium discoideum, extracellular cAMP activates adenylate cyclase, which leads to an increase in intracellular cAMP and the rate of cAMP secretion. The signaling response to a constant cAMP stimulus is terminated after several minutes by an adaptation mechanism. The time- course of adaptation stimuli of 10(-6) or 10(-7) M cAMP was assessed. We used a perfusion technique to deliver defined cAMP stimuli to [(3)H]adenosine-labeled amoebae and monitored their secretion of [(3)H]cAMP. Amoebae were pretreated with 10(-6) or 10(-7) M cAMP to periods of 0.33-12 minutes, and then immediately given test stimuli of 10(-8) M to 2.5 x 10(-7) M cAMP. The response to a given test stimulus was progressively attenuated and finally extinguished as the duration of the pretreatment stimulus increased. During concentration of the test stimulus. The responses to test stimuli of 10(-8), 5 x 10(-8), 10(-7), or 2.5 x 10(-7) M cAMP were extinguished after approximately 1, 2.25,2.5, and 10 min, respectively. 1.5 min of stimulation with 10(-7) M cAMP was necessary to extinguish the response of a test stimulus of 10(-8) M cAMP. Our data suggest that adaptation begins within 20 s of stimulation, rises rapidly for approximately 2.5 min, and reaches a plateau after approximately 10 min. The absolute rate of rise was faster during pretreatment with 10(-6) than with 10(-7) M cAMP. These results support a working hypothesis in which the occupancy of surface cAMP receptors leads to changes in two opposing cellular processes, excitation and adaptation, that control the activity of D. discoideum adenylate cyclase.

Adaptation, Physiological↗
PubMed1979

Cyclic 3',5' AMP relay in Dictyostelium discoideum. I. A technique to monitor responses to controlled stimuli.

A perfusion technique was developed to deliver [14C]adenosine 3',5'-cyclic monophosphate (cAMP) stimuli of well-defined magnitude and duration to tritium-labeled Dictyostelium discoideum amoebae and simultaneously monitor the elicited secretion of [3H]cAMP (i.e., the relay response). The tritiated compounds secreted in response to [14C]cAMP stimuli were highly enriched in [3H]cAMP and reflected an increase in intracellular cAMP accompanying stimulation rather than the release of a preexisting store or bulk cellular contents. The secretory response (per 10(6) cells) to 2-min stimuli increased during differentiation from about 0.2 pmol at 0.5 h to approximately 5 pmol of cAMP at 7 h. Without adequate perfusion, amoebae altered the level of cAMP in their environment in two ways: phosphodiesterases destroyed cAMP stimuli under some conditions so as to attenuate the relay response; under other circumstances, secreted cAMP magnified minimal exogenous stimuli into maximal responses. Amoebae, furthermore, would respond to their basal secretion of cAMP autocatalytically if its removal or destruction were interrupted. The perfusion system minimized these cell-induced modifications, allowing control of the level of the stimulus and response in quantitative studies.

Cyclic AMP↗
PubMed1979

Cyclic 3',5' AMP relay in Dictyostelium discoideum. II. Requirements for the initiation and termination of the response.

The secretion of 3H-cyclic adenosine 3',5'-monophosphate (cAMP) by prelabeled and suitably differentiated Dictyostelium discoideum amoebae was elicited in a perfusion apparatus by 10(-10) to 10(-5) M [14C]cAMP stimuli of defined magnitude and duration. Exogenous stimuli evoked an immediate increase in the rate of [3H]cAMP secretion which accelerated continuously to reach a peak of up to 100 times the unstimulated rate after 2--3 min of stimulation. Withdrawal of the stimulus at any time during the response led to a rapid decline to basal levels. Furthermore, a spontaneous decline in secretion rate was observed during prolonged cAMP stimulation, with a return to basal levels after 3--8 min of stimulation. After the initial secretory event, cells did not respond further to the continued presence of external [14C]cAMP unless (a) it was interrupted by a brief recovery period or (b) the level of the stimulus was increased sufficiently. Since the second increment could follow the first at any time, continuous secretion of [3H]cAMP could be sustained for up to 30 min by progressively increasing the stimulus between 10(-10) and 10(-5) M cAMP. The total magnitude of spontaneously terminated responses depended on the size of the increment in applied cAMP, larger stimuli evoking both a more rapid acceleration and a slower deceleration in [3H]cAMP secretion rate. The integrated response to a given increment in stimulus level was apparently independent of its "shape" - i.e., the duration, magnitude, and number of sub-steps in the increment. These data support two mechanistic inferences: that amoebae respond in proportion to relative increases in extracellular cAMP concentration, but adapt to the concentration of cAMP itself. The data further suggest that the initiation and termination of the response are mediated by cellular component(s) beyond cAMP-occupied receptors.

Adaptation, Physiological↗
PubMed1978

Newly synthesized acetylcholine receptors are located in the Golgi apparatus.

Chick skeletal muscle cells in tissue culture were fixed and treated with saponin to allow [125I]alpha-bungarotoxin access into the cells while preserving ultrastructure. The kinetics of binding of iodinated alpha-bungarotoxin to intracellular acetylcholine (ACh) receptors and to surface A Ch receptors were comparable. About half of the intracellular ACh receptors are newly synthesized and in the pathway leading to incorporation into the plasma membrane. Correlated electron microscope autoradiographic and kinetic studies of this receptor population suggest that a substantial fraction of the newly synthesized ACh receptors are located in the Golgi apparatus, where they reside for approx. 2 h.

Acetylcholine↗
PubMed1978

Metabolism of acetylcholine receptors in skeletal muscle.

The acetylcholine receptor in skeletal muscle is an integral plasma membrane glycoprotein. Its biosynthesis and incorporation into plasma membrane and its degradation are being studied with the use of biochemical, biophysical, and microscopic techniques. In this report, previously published data are combined with new information to yield a consistent and fairly detailed description ofthe mechanisms involved in receptor metabolism. It is proposed that the biosynthesis, transport, and incorporation of the receptor into plasma membranes involve a mechanism similar, or identical, to that used by the cell for production and secretion of secretory proteins. The receptor is degraded by a random-hit process, which involves internalization, transport to secondary lysosomes, and hydrolysis. Sites of regulation of receptor metabolism are discussed in the context of regulation of the number and distribution of receptors in plasma membranes, particularly with respect to the formation and stability of neuromuscular junctions.

Acetylcholine↗
PubMed1977

Kinetics of biosynthesis of acetylcholine receptor and subsequent incorporation into plasma membrane of cultured chick skeletal muscle.

20% of the acetylcholine receptors in cultured chick skeletal muscle remain unbound following long-term growth of muscle in medium containing a potent, essentially irreversible receptor-blocking agent, alpha-bungarotoxin. About half the receptors which are unavailable for interaction with extracellular alpha-bungarotoxin are newly synthesized molecules which presumably are being processed and transported to the plasma membrane. When the muscle cultures are switched to a medium containing 2H, 13C, 15N-amino acids, these receptors are rapidly labeled, the fraction of labeled molecules beginning to plateau at 3 hr. Few labeled receptors appear in the plasma membrane during the first 3 hr of labeling with 2H, 13C, 15N-amino acids. After 3.5 hr of labeling, virtually all the receptors being incorporated into the plasma membrane are labeled receptors. The kinetics of labeling of the "pool" and "surface" receptors with 2H, 13C, 15N-amino acids confirm the "precursor-product" type relationship of pool and surface acetylcholine receptors. In this study, receptors synthesized in medium containing 2H, 13C, 15N-amino acids were resolved from 1H, 12C, 14N-receptors by velocity sedimentation in sucrose-deuterium oxide and sucrose-H2O gradients, and their densities were estimated from sedimentation rates in shallow gradients of various average density. Estimated densities were 1.32 g/cm3 for 1H, 12C, 14N-receptors and 1.41 g/cm3 for 2H, 13C, 15N-receptors. This density difference corresponds to 80% substitution of normal aminoacyl residues by 2H, 13C, 15N-residues in the denser receptor.

Acetylcholine↗
PubMed1976

Synthesis of acetylcholine receptors by cultured chick myotubes and denervated mouse extensor digitorum longus muscles.

Mono-[125I]iodo- alpha - bungarotoxin - receptor complexes extracted from chick myotube cultures as well as from adult denervated extensor digitorum longus muscles of the mouse have been banded at their buoyant density in gradients of metrizamide-deuterium oxide. When cultures or denervated adult muscles are preincubated in media containing 2H- or 13C-substituted amino acids under conditions of active receptor accumulation, the mono-[125I]iodo-alpha-bungarotoxin-receptor complexes have an increased buoyant density and band in a position of higher density in the gradient relative to a marker of mono-[131I]iodo-alpha-bungarotoxin-receptor complexes extracted from cells preincubated in normal media. It is concluded that the accumulation of receptors on the surfaces of cultured chick myotubes and on the non-synaptic surfaces of extensor digitorum longus muscles following denervation are the result of de novo synthesis.

Animals↗
PubMed1975

Acetylcholine receptor turnover in membranes of developing muscle fibers.

[125I mono-iodo-alpha-bungarotoxin is used as a specific marker in a description of acetylcholine receptor metabolism. It is concluded that acetylcholine receptors in the surface membranes of chick and rat myotubes developing in cell cultures have a half-life of 22-24 h. Alpha-bungarotoxin (bound to a receptor which is removed from the membrane) is degraded to monoiodotyrosine which appears in the medium. Several observations are consistent with a model in which receptors or alpha-bungarotoxin-receptor complexes are internalized and then degraded: (a) the rate of appearance of iodotyrosine does not reach its maximal rate until 90 min after alpha-bungarotoxin is bound to the surface receptors; (b) 2,4-dinitrophenol, reduced temperature, and cell disruption all inhibit the degradation process. The degradation of surface receptors is not coupled to the process by which receptors are incorporated into the membrane. Evidence suggest that receptors are incorporated into the surface membrane from a presynthesized set of receptors containing about 10% as many alpha-bungarotoxin binding sites as does the surface. Additionally, a third set of acetylcholine receptors is described containing about 30% as amny binding sites as does the surface. These "hidden" recptors are not precursors yet are not readily accessible for binding of extracellular alpha-bungarotoxin. These findings are discussed in relation to both plasma membrane biosynthesis and control of chemosensitivity in developing and denervated skeletal muscle.

Animals↗