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

1983 ◽  
Vol 97 (1) ◽  
pp. 173-177 ◽  
Author(s):  
A Theibert ◽  
P N Devreotes
Keyword(s):  
Adenylate Cyclase ◽  
Dictyostelium Discoideum ◽  
Time Course ◽  
Initial Response ◽  
Activation Process ◽  
Surface Receptors ◽  
High Affinity ◽  
Camp Synthesis ◽  
Time Period ◽  
Rapid Activation

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.

scholarly journals cAMP induces a rapid and reversible modification of the chemotactic receptor in Dictyostelium discoideum.

1985 ◽  
Vol 100 (3) ◽  
pp. 715-720 ◽  
Author(s):  
C Klein ◽  
J Lubs-Haukeness ◽  
S Simons
Keyword(s):  
Dictyostelium Discoideum ◽  
Concentration Dependence ◽  
Time Course ◽  
Camp Synthesis ◽  
Sds Page ◽  
Reversible Modification ◽  
Camp Receptor ◽  
Camp Stimulation ◽  
Stimulation Of

Stimulation, within 1 min after cAMP stimulation, of aggregation-competent Dictyostelium discoideum amebae was found to cause a rapid (within 1 min) modification of the cell's surface cAMP receptor. The modified receptor migrated on SDS PAGE as a 47,000-mol-wt protein, as opposed to a 45,000-mol-wt protein labeled on unstimulated cells. The length of time this modified receptor could be detected depended upon the strength of the cAMP stimulus: 3-4 min after treatment with 10(-7) M cAMP, cells no longer possessed the 47,000-mol-wt form of the cAMP receptor. Instead, the 45,000-mol-wt form was present. Stimulation of cells with 10(-5) M cAMP, however, resulted in the persistent (over 15 min) expression of the modified receptor. The time course, concentration dependence, and specificity of stimulus for this cAMP-induced shift in the cAMP receptor were found to parallel the cAMP-stimulated phosphorylation of a 47,000-mol-wt protein. In addition, both phenomena were shown to occur in the absence of endogenous cAMP synthesis. The possibility that the cAMP receptor is phosphorylated in response to cAMP stimulation, and the role of this event in cell desensitization, are discussed.

Effects of a novel hypothalamic peptide, pituitary adenylate cyclase-activating polypeptide, on pituitary hormone release in rats

1992 ◽  
Vol 134 (1) ◽  
pp. 33-41 ◽  
Author(s):  
G. R. Hart ◽  
H. Gowing ◽  
J. M. Burrin
Keyword(s):  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Time Course ◽  
Pituitary Hormone ◽  
Pituitary Cells ◽  
Hormone Release ◽  
Α Subunit ◽  
Time Period ◽  
Pituitary Adenylate Cyclase ◽  
Pretreatment Time

ABSTRACT We have demonstrated that the novel hypothalamic peptide pituitary adenylate cyclase-activating poly-peptide (PACAP-38; 0·1–100 nmol/l) caused an increase in the release of GH, ACTH, LH and α-subunit and accumulation of intracellular cyclic AMP from dispersed rat anterior pituitary cells in static culture for 24 h. There were no significant effects on TSH or prolactin release over the same time-period. PACAP-38 (10 nmol/l) increased the release of GH by 1·3-fold (P<0·05), ACTH by 1·9-fold (P<0·05), LH by 3·5-fold (P<0·001) and α-subunit by 2·0-fold (P< 0·005) and the accumulation of intracellular cyclic AMP by >2-fold (P<0·001) after 24 h. However, the time-course for the effect of PACAP-38 (1 mmol/l) on hormone release and intracellular cyclic AMP levels showed a temporal dissociation. The effect of PACAP-38 on GH and ACTH levels did not reach significance until 24 h whereas the effect of PACAP-38 on LH and α-subunit release reached significance after 4 h implying a different mechanism of action for their release. To investigate the PACAP-induced secretion of LH and α-subunit further, we examined the effects of PACAP after down-regulation of protein kinase C (PKC). PACAP-38 at a dose maximal for the stimulation of LH and α-subunit release (10 nmol/l) added together with the PKC activator, 12-0-tetradecanoyl-phorbol-13-acetate (TPA; 0·1 μmol/l) had no greater effect on LH and α-subunit release than TPA alone over a 4 h incubation period. Increasing the pretreatment time with TPA (0–5 h) at a dose (0·1 μmol/l) known to deplete PKC activity substantially, reduced the ability of PACAP-38 to stimulate LH and α-subunit release and intracellular cyclic AMP levels significantly. We conclude that the stimulatory actions of PACAP on LH and α-subunit relies in part on PKC activity. Journal of Endocrinology (1992) 134, 33–41

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

1980 ◽  
Vol 86 (2) ◽  
pp. 537-544 ◽  
Author(s):  
M C Dinauer ◽  
S A MacKay ◽  
P N Devreotes
Keyword(s):  
Adenylate Cyclase ◽  
Time Course ◽  
Camp Signaling ◽  
Intracellular Camp ◽  
Intact Cells ◽  
Camp Synthesis ◽  
Transient Activation ◽  
Direct Measurements ◽  
Precipitous Decline ◽  
Relationship Of

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.

scholarly journals Dictyostelium discoideum mutant synag 7 with altered G-protein-adenylate cyclase interaction

1988 ◽  
Vol 91 (2) ◽  
pp. 287-294
Author(s):  
B.E. Snaar-Jagalska ◽  
P.J. Van Haastert
Keyword(s):  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
G Protein ◽  
Dictyostelium Discoideum ◽  
High Speed ◽  
Wild Type ◽  
High Affinity ◽  
Gtp Binding

Previous results have shown that Dictyostelium discoideum mutant synag 7 is defective in the regulation of adenylate cyclase by receptor agonists in vivo and by GTP gamma S in vitro; the guanine nucleotide activation of adenylate cyclase is restored by the high-speed supernatant from wild-type cells. Here we report that in synag 7 membranes: (1) cyclic AMP receptors had normal levels and were regulated by guanine nucleotides as in wild-type; (2) GTP binding and high-affinity GTPase were reduced but still stimulated by cyclic AMP; (3) the supernatant from wild-type cells restored GTP binding to membranes of this mutant, and partly restored high-affinity GTPase activity; (4) the supernatant of synag 7 was ineffective in these reconstitutions and did not influence GTP binding and GTPase activities in mutant or wild-type membranes. These results suggest that the defect in mutant synag 7 is located between G-protein and adenylate cyclase, and not between receptor and G-protein. A factor in the supernatant is absent in synag 7 and appears to be essential for normal GTP binding, GTPase and activation of adenylate cyclase. This soluble heat-labile factor may represent a new molecule required for receptor- and G-protein-mediated activation of adenylate cyclase.

scholarly journals Cyclic 3', 5'-AMP relay dictyostelium discoideum. V. Adaptation of the cAMP signaling response during cAMP stimulation

1980 ◽  
Vol 86 (2) ◽  
pp. 554-561 ◽  
Author(s):  
M Dinauer ◽  
TL Steck ◽  
P Devreotes
Keyword(s):  
Adenylate Cyclase ◽  
Test Stimulus ◽  
Dictyostelium Discoideum ◽  
Time Course ◽  
Intracellular Camp ◽  
Perfusion Technique ◽  
Adaptation Mechanism ◽  
Cellular Processes ◽  
Extracellular Camp ◽  
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.

The Binding of Calcium Ions to Bovine Factor X by Rate Dialysis

1978 ◽  
Vol 40 (02) ◽  
pp. 350-357
Author(s):  
Robert H Yue ◽  
Menard M Gertler
Keyword(s):  
Molecular Weight ◽  
Dissociation Constant ◽  
Calcium Ions ◽  
Activation Process ◽  
Factor X ◽  
The Real ◽  
High Affinity ◽  
Binding Data ◽  
Sequential Mechanism ◽  
Bovine Factor

SummaryThe binding of Ca+2 to bovine factor X (molecular weight of 74,000) (Yue und Gertler 1977) was studied by the technique of rate dialysis and with the use of 45Ca+2. The binding data are consistent with a model of sequential mechanism. One mole of Ca+2 binds to the glycoprotein with a dissociation constant of 5.2 × 10-5 M and an additional 39 ± 4 moles of Ca+2 bind to this zymogen with a dissociation constant of 3.7 × 10-3M. The binding of the high affinity Ca+2 causes a functionally significant change in the zymogen, and (calcium) (factor X) complex is the real substrate in the activation process by the protease in Russell’s viper venom.

UPTAKE OF OXYTOCIN IN TISSUES AFTER INTRAVENOUS ADMINISTRATION OF TRITIATED OXYTOCIN IN RATS

1969 ◽  
Vol 61 (3) ◽  
pp. 432-440 ◽  
Author(s):  
Ingvar Sjöholm ◽  
Gunnar Rydén
Keyword(s):  
Skeletal Muscle ◽  
Distribution Pattern ◽  
Intravenous Injection ◽  
Intravenous Administration ◽  
Time Course ◽  
Tritiated Water ◽  
Preferential Distribution ◽  
Time Period ◽  
Initial Uptake

ABSTRACT The distribution of oxytocin in the kidneys, liver, uterus and skeletal muscle of the rat was followed during 10 min after intravenous injection of tritium labelled oxytocin. Oxytocin was found to be taken up and degraded mainly in the kidneys and the liver. After 150 seconds no intact oxytocin could be detected in these organs. The time course of the distribution of the radioactivity in the liver and the skeletal muscle showed no noteworthy characteristics, whereas a different course was found in the kidneys and in the uterus. In the kidneys, the radioactivity increased continuously from 60 to 200 seconds after the injection, indicating an accumulation of oxytocin or its metabolites in the kidneys. In the uterus a high initial uptake was observed, followed by a decrease of the radioactivity from 60 to 100 seconds after the injection. This distribution pattern was specific to oxytocin, since the uptake of tritiated tyrosine and tritiated water was almost constant during the same time period. These findings may indicate a preferential distribution of oxytocin to the uterus.

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