Ecdysteroid titer and oocyte growth in the northern house mosquito, Culex pipiens L

In an anautogenous strain of the northern house mosquito, Culex pipiens, the ovaries reached the resting stage (follicle length = 90 microns) three days after adult emergence. Follicle length increased from 90 to 550 microns between 0 and 60 hr after a blood meal. Total ecdysteroids reached a peak at 400 fmol/insect at 36 hr after a blood meal then declined rapidly. The ratio of 20-hydroxyecdysone to ecdysone increased in conjunction with the total ecdysteroid level. Oocyte growth beyond the resting stage and initiation of vitellogenesis was dependent on a head factor which was released within 4-8 min of the start of the blood meal.     

Insulin stimulates fluid-phase endocytosis and exocytosis in 3T3-L1 adipocytes

Fluid phase endocytosis by monolayers of 3T3-L1 adipocytes has been followed by measuring [14C]sucrose uptake, a well characterized pinocytic marker. Insulin, at a maximal stimulatory concentration, increased the pinocytic rate by 2-fold within 5 min of its addition; this activation persisted for at least 2 h. The dose-response curve for the enhancement of fluid-phase endocytosis by insulin was identical with that for the stimulation of hexose transport, as measured by the uptake of 2-deoxyglucose. The concentration of insulin eliciting half-maximal effects was 6 nM. These results suggest that activation of endocytosis and hexose uptake by insulin are triggered by the same signalling event. Insulin-activated pinocytosis was not dependent upon the increased metabolism of D-glucose that occurs in response to the hormone, since the stimulation of fluid-phase endocytosis occurred in the absence of 5 nM glucose. Fluid-phase exocytosis was examined by loading cells with [14C]sucrose for various times and then measuring tracer efflux. The rate of sucrose release was biphasic; a portion of the internalized sucrose was rapidly released from the cell (t1/2 approximately 5 min), whereas the remainder was released slowly (t1/2 approximately to 5 h). These results are consistent with a sequential two-compartment model in which the [14C] sucrose first enters a compartment from which about 70% of the sucrose is rapidly released back into the medium and the remaining 30% is transferred to a second compartment. Therefore, the true rate of endocytosis is much greater than the observed accumulation rates, except after short uptake times. Insulin increases the rate of sucrose efflux from both compartments as well as the rate of transfer from the first compartment to the second compartment by about 2-fold. Furthermore, insulin increased the apparent size of the first and second compartments by 1.6- and 3-fold, respectively. The lysosomotropic agent chloroquine (200 muM) had only a small effect on fluid movements in these cells. The rapid and prolonged stimulation of fluid-phase endocytosis and exocytosis by insulin are hitherto unrecognized effects of this hormone.     

Initiation and termination of host-seeking inhibition in Aedes aegypti during oöcyte maturation

The inhibition of host-seeking behaviour that accompanies vitellogenesis in the mosquito, Aedes aegypti, was examined by the removal and implantation of ovaries. Mosquitoes ovariectomized before a blood meal and between 1 and 6 hr after a blood meal responded to a host at 48 hr after a blood meal. However, when ovariectomy was delayed until 10 hr after the meal or later, most mosquitoes did not respond to the host. When a partial ovary was present for only the first 12 hr after a meal, there was no host-seeking inhibition at 48 hr, and only 58% of females with one complete ovary present during this time interval responded. Howver, these same amounts of ovarian tissue inhibited host-seeking when they remained for 48 hr after a meal. Vitellogenic ovaries from donors blood-fed 8–24 hr before, implanted into sugar-fed recipients, did not affect the host-seeking behaviour of these recipients. Ovaries removed and reimplanted before the blood meal inhibited host-seeking at 72 hr after the blood meal only in the absence of oviposition from intact ovaries. It is concluded that 2 humoral factors are involved in the promotion of host-seeking inhibition: the first factor is produced by the ovaries, and after reaching a critical threshold in the haemolymph, stimulates the release of a second factor that acts directly to inhibit mosquito behaviour. An ovary which retains 2 or fewer eggs after oviposition terminates the inhibition via nervous pathways. The role of 20-hydroxyecdysone in the behavioural inhibition is discussed.     

Effect of LH on the release of cyclic AMP by the rabbit ovary perfused in vivo and in vitro.

After perfusion of 10 rabbit ovaries in vitro with a modified Krebs bicarbonate buffer containing dextran and glucose, the concentration of cAMP in the perfusion medium was significantly increased 2-5 min after stimulation with 10 mug LH/ml medium and was higher at 15 and 30 min. Intravenous injection of 100 mug LH/rabbit caused a significant increase of cAMP concentrations in the ovarian venous blood from 8 ovaries 10 min after the injection and the cAMP concentrations were higher after 15 and 30 min. The ovarian blood flow was not changed after the LH injection. It is concluded that perfusion techniques can be useful in analysis of the mechanisms and physiological significance of release of cAMP from the ovary after hormonal stimulation.     

Stimulation of 3-O-methylglucose transport by anaerobiosis in rat thymocytes.

Transport of 3-O-methylglucose by rat thymocytes occurs by facilitated diffusion and follows a biphasic time course. The half-times of the two phases of uptake are 0.8 min and 20 to 30 min; the rapid phase contributes 10 to 20% of the total 3-O-methylglucose taken up at equilibrium. Cells incubated under anaerobic conditions for 1 hour undergo a 3- to 4-fold increase in the initial rate of 3-O-methylglucose uptake. The relative contribution of the rapid phase of uptake increases nearly 4-fold in anaerobically incubated cells, although the half-time of the rapid phase remains the same. Anaerobiosis also reduces the half-time of the slow phase of uptake by a factor of three. In the absence of exogenous glucose, anaerobiosis reduces cellular ATP by 97% after 1 hour at 37 degrees. However, full stimulation of transport activity does not occur in cells with such low levels of ATP. When anaerobically incubated cells are re-exposed to oxygen, ATP synthesis proceeds and transport activity increases by 100% within 5 to 10 min. Adding 1 mM 2,4-dinitrophenol at the time the anaerobic cells are reexposed to oxygen completely blocks the subsequent ATP synthesis and the associated increase in transport activity. Cells incubated aerobically in the presence of 1 mM 2,4-dinitrophenol show a 90% reduction in ATP levels and a 2-fold increase in the rate of 3-O-methylglucose uptake. An additional 70% increase in transport activity is observed when the cells are washed free of uncoupler and incubated an additional 10 min. The results suggest that transport activity is stimulated when cellular ATP levels decline but that the stimulation process requires some minimal level of ATP for full expression.     

Exo-endocytosis in isolated peptidergic nerve terminals occurs in the sub-second range

Exo- and endocytotic processes induced by depolarization of isolated neurosecretory nerve terminals show a close temporal correlation, which suggests a short time of integration of the neurosecretory granule membrane with the plasma membrane. In order to determine minimal time requirements for exocytosis-coupled endocytosis to occur, we have analyzed by electron microscopy uptake of horserdish peroxidase (HRP) as a fluid phase marker at the onset of depolarization. We have applied rapid mixing and sampling (quenched flow) to assess events in subsecond time peroids after stimulation. A significant number of labelled endocytotic vacuoles was observed during the first second of depolarization. This number then further increased by a factor of about 2 (within 5 s) and 4 (within 50s). Thus, as for exocytosis, the rate of endocytosis decreased considerably during prolonged stimulation. These data indicate i) that a substantial proportion of secretory granules undergoes exocytosis very shortly after stimulation, and ii) that, following exocytosis, the minimal time required for consecutive membrane retrieval is in the sub-second range.     

Effect of wortmannin and phorbol ester on Paramecium fluid-phase uptake in the presence of transferrin

The kinetics of the uptake of the fluid phase marker Lucifer Yellow (LY), and its alteration by wortmannin, an inhibitor of phosphatidylinositol-3 kinase (PI-3K), and the PKC modulators: GF 109203 X, an inhibitor, and phorbol ester, an activator was studied in eukaryotic model Paramecium aurelia. Spectrophotometric quantification of LY accumulation was performed in the presence or absence of transferrin, a marker of receptor-mediated endocytosis. Internalization of LY showed a curvilinear kinetics: the high initial rate of LY uptake (575 ng LY/mg protein/hr) decreased almost 5-fold within 15 min, reaching plateau at 126 ng/mg protein/hr. Transferrin induced a small increase (7.5%) in the fluid phase uptake rate (after 5 min) followed by a small decrease at longer incubation times. Lucifer Yellow and transferrin (visualized by streptavidin-FITC) were localized in Paramecium by 3-D reconstruction by confocal microscopy. LY showed a scattered, diffuse fluorescence typical of fluid phase uptake whereas transferrin accumulated in membrane-surrounded endosomes. Wortmannin did not affect LY accumulation but decreased it when transferrin was present in the incubation medium. This suggests an effect on the transferrin uptake pathway, presumably on the stage of internalization in "mixing" endosomes to which transferrin and LY were targeted. Phorbol ester diminished LY accumulation by 22% and this effect persisted up to 25 min of incubation. PKC inhibitor did not affect LY uptake. However, in the presence of transferrin, the LY uptake increased within the first 15 minutes followed by a rapid 20% decrease in comparison to the control. Such an effect of PKC modulators suggests that PMA action on fluid phase uptake is not directly mediated by PKC.     

The effects of blood loss on the performance of physical exercise

Seven male subjects were studied before and up to 53 days after the loss of 11 of blood. The resting hematocrit fell from 44.0 to 38.7% and returned to control level after 3 weeks. Maximal oxygen uptake decreased from 4.00 1/min to 3.54 1/min and returned to the initial level within 2 weeks. Submaximal oxygen uptake, pulmonary ventilation, maximal heart rate and blood lactate were not found to change significantly. Submaximal heart rate was increased from 125 beats . min-1 to about 135 beats . min-1 and remained elevated for 3 weeks, whereas blood lactate was increased only in the first week. Maximal work time decreased from 5.1 min to 3.8 min and remained low for the first 2 weeks, but rose thereafter above the starting level. Comparison with a control study suggested that there is some training effect, which, when allowed for, indicates that maximal work time returns to starting values at the same time as does the maximal oxygen uptake. It is concluded that the drop in Hct, maximal oxygen uptake and work capacity, found after the loss of 11 of blood, are related to each other both in magnitude and duration.     

Effects of cyclic AMP and dibutyryl cyclic AMP on amino acid transport in the isolated rat ovary.

Prepubertal rat ovaries were incubated in medium containing the non-utilizable amino acids alpha-aminoisobutyric acid (AIB-14C) or 1-aminocyclo-pentane-carboxylic acid (cycloleucine-14C). The rate of uptake of the two amino acids was studied in the isolated ovaries after different incubation periods. Addition of 5mM cyclic AMP (cAMP) caused a slight stimulation of the AIB-transport but in higher concentrations (10-25 mM) an inhibition was noted. With dibutyrl cyclic AMP (dbcAMP) a dose-dependent increase was seen with 0.5-5 mM concentrations with no further effect of higher concentrations. Time course studies were performed with both AIB and cycloleucine in presence of 10 mM dbcAMP and increased uptake values were noted at each time studied (30-240 min). The phosphodiesterase inhibitor aminophyline in lower concentrations did not influence AIB-transport but 5-10 mM caused increased uptake values in the ovaries. The stimulatory action of dbcAMP on amino acid transport was augmented by a low concentration of aminophylline (0.5 mM). Experiments were in addition carried out in the presence of puromycin and under these circumstances it was still possible to enhance amino acid transport by addition of dbcAMP. The results are discussed in relation to earlier reported effects of gonadotropins on ovarian amino acid transport.     

Insulin-like peptides and the target of rapamycin pathway coordinately regulate blood digestion and egg maturation in the mosquito Aedes aegypti

Background

Mosquitoes are insects that vector many serious pathogens to humans and other vertebrates. Most mosquitoes must feed on the blood of a vertebrate host to produce eggs. In turn, multiple cycles of blood feeding promote frequent contacts with hosts and make mosquitoes ideal disease vectors. Both hormonal and nutritional factors are involved in regulating egg development in the mosquito, Aedes aegypti. However, the processes that regulate digestion of the blood meal remain unclear.

Methodology/Principal Findings

Here we report that insulin peptide 3 (ILP3) directly stimulated late phase trypsin-like gene expression in blood fed females. In vivo knockdown of the mosquito insulin receptor (MIR) by RNA interference (RNAi) delayed but did not fully inhibit trypsin-like gene expression in the midgut, ecdysteroid (ECD) production by ovaries, and vitellogenin (Vg) expression by the fat body. In contrast, in vivo treatment with double-stranded MIR RNA and rapamycin completely blocked egg production. In vitro experiments showed that amino acids did not simulate late phase trypsin-like gene expression in the midgut or ECD production by the ovaries. However, amino acids did enhance ILP3-mediated stimulation of trypsin-like gene expression and ECD production.

Conclusions/Significance

Overall, our results indicate that ILPs from the brain synchronize blood meal digestion and amino acid availability with ovarian ECD production to maximize Vg expression by the fat body. The activation of digestion by ILPs may also underlie the growth promoting effects of insulin and TOR signaling in other species.     

Ecdysteroids regulate yolk protein uptake by Drosophila melanogaster oocytes

Juvenile hormones (JHs) are thought to drive the regulation of yolk protein uptake by ovaries in Drosophila melanogaster. However, the level of JH production in a mutant stock (ap(56f)) is depressed yet the flies are normally vitellogenic. The production of ecdysteroids by these ap(56f) ovaries in vitro is elevated above that of wild-type ovaries. The incubation of wild-type ovaries in the presence of 0.1mM JHB(3) increased ecdysteroid biosynthesis only during the first 18h following eclosion. Female Drosophila melanogaster undergo a pre-vitellogenic reproductive diapause when exposed to low temperature (11 degrees C) and a short-day photoperiod (L12:D12). The rate of ecdysteroid synthesis by the ovaries, but not JH production, increased within 12h of a temperature upshift to 25 degrees C from a basal level of 20+/-1pg/10 pair of ovaries/5h to a sustained level of 150+/-20pg/10 pair/5h. Vitellogenic oocytes were noted in all females within 12h of this temperature upshift. Diapause was also terminated by the injection of 1&mgr;g of 20-hydroxyecdysone into the abdomens of diapausing females as determined by an increase in ovary size, and the appearance of vitellogenic oocytes as compared to controls. These results are consistent with a revised model for the regulation of yolk protein uptake by ovaries in which ecdysteroids, and not JHs, play the prominent role.     

Quantitative changes in polyphosphoinositides 1,2-diacylglycerol and inositol 1,4,5-trisphosphate by platelet-derived growth factor and prostaglandin F2 alpha

We developed a novel method to quantify trace amounts of phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) using antibodies against PIP and PIP2. With this method, polyphosphoinositides can be measured in the range from 20 to 500 pmol. We applied the method to quantify changes in PIP and PIP2 levels in Balb/c/3T3 cells stimulated by platelet-derived growth factor (PDGF) and prostaglandin F2 alpha (PGF2 alpha), growth factors that stimulate the hydrolysis of PIP and PIP2. PIP2 content decreased rapidly to about 60% of control within 1 min while PIP content decreased gradually but significantly to 60% (PDGF) or 70% (PGF2 alpha) of control. Simultaneously we measured the mass levels of inositol 1,4,5-trisphosphate and 1,2-diacylglycerol (DG). Inositol 1,4,5-trisphosphate levels rapidly increased and reached a maximum at 30 s after PDGF or PGF2 alpha stimulation and then decreased to the control level within 2 min. On the other hand, DG formation showed biphasic changes. In the first phase, DG rapidly accumulated and reached a maximum at 30 s after PDGF or PGF2 alpha stimulation and then quickly decreased. In the second phase, DG accumulated gradually, but very markedly, 2 min after PDGF or PGF2 alpha stimulation. Considering the changes in PIP2, DG in the first phase seems to be derived mainly from PIP2 while most of the DG in the second phase derived from other lipids.     

Differentiation-Dependent Decline of DNA Synthetic Activities in Naegleria gruberi

SYNOPSIS. DNA of Naegleria gruberi strain NEG, grown in axenic culture, forms a band at a density of 1.6912 in CsCl gradient and has a GC content of 31.8%. Incorporation of [³H]thymidine into DNA is much reduced in differentiating Naegleria immediately after the stimulation to transforms, primarily because of the reduction in thymidine uptake by differentiating cells. In addition, there is a marked decrease in the rate of incorporation of [³H]thymidine and [³H]uracil into DNA at from 45 to 60 min after the stimulation for differentiation. This decrease in the rate of precursor incorporation into DNA appears to be due to the differentiation-dependent cessation of nuclear DNA synthesis. The differentiated phenotype (the flagellate) emerges at ∼ 70 min after the stimulation, and over 90% of the population differentiates within the next 30 min. Synthesis of mitochondrial DNA is detectable until 190 min after the stimulation. Since the S phase of Naegleria lasts ∼ 180 min, some cells in the population must cease synthesizing nuclear DNA in the middle of the S phase.     

The effects of decapitation and the influence of size and sex on diuresis in newly emerged mosquitoes

ABSTRACT. Diuresis in the newly emerged adult mosquito occurs in two phases, a short initial peak immediately after eclosion and a longer peak in the middle of day 1. The present work lends support to the view that the second phase of diuresis is controlled by a hormone which is either released from the head, or from the thorax under the control of the head, 3–4 h after eclosion. The influence of the head ceases to be effective some 9 h later. The rate of water loss is normally higher in unfed females than in unfed males. Whereas the higher rate of diuresis in females during the two peaks is a function of size, the higher rate in unfed females during the second and third day following eclosion is mostly a result of ovarian activity during this period.     

Effects of adenosine diphosphate on Ca2+ fluxes and Ca2+ accumulation of sarcoplasmic reticulum

Ca2+ transport by sarcoplasmic reticulum vesicles was examined by incubating sarcoplasmic reticulum vesicles (0.15 mg/ml) at 37 degrees C in, either normal medium that contained 0.15 M sucrose, 0.1 M KCl, 60 microM CaCl2, 2.5 mM ATP and 30 mM Tes at pH 6.8, or a modified medium for elimination of ADP formed from ATP hydrolysis by including, in addition, 3.6 mM phosphocreatine and 33 U/ml of creatine phosphokinase. In normal medium, Ca2+ uptake of sarcoplasmic reticulum vesicles reached a plateau of about 100 nmol/mg. In modified medium, after this phase of Ca2+ uptake, a second phase of Ca2+ accumulation was initiated and reached a plateau of about 300 nmol/mg. The second phase of Ca2+ accumulation was accompanied by phosphate uptake and could be inhibited by ADP. Since, under these experimental conditions, there was no significant difference of the rates of ATP hydrolysis in normal medium and modified medium, extra Ca2+ uptake in modified medium but not in normal medium could not be explained by different phosphate accumulation in the two media. Unidirectional Ca2+ influx of sarcoplasmic reticulum near steady state of Ca2+ uptake was measured by pulse labeling with 45Ca2+. The Ca2+ efflux rate was then determined by subtracting the net uptake from the influx rate. At the first plateau of Ca2+ uptake in normal medium, Ca2+ influx was balanced by Ca2+ efflux with an exchange rate of 240 nmol/mg per min. This exchange rate was maintained relatively constant at the plateau phase. In modified medium, the Ca2+ exchange rate at the first plateau of Ca2+ uptake was about half of that in normal medium. When the second phase of Ca2+ uptake was initiated, both the influx and efflux rates started to increase and reached a similar exchange rate as observed in normal medium. Also, during the second phase of Ca2+ uptake, the difference between the influx and efflux rates continued to increase until the second plateau phase was approached. In conditions where the formation of ADP and inorganic phosphate was minimized by using a low concentration of sarcoplasmic (7.5 micrograms/ml) and/or using acetyl phosphate instead of ATP, the second phase of Ca2+ uptake was also observed. These data suggest that the Ca2+ load attained by sarcoplasmic reticulum vesicles during active transport is modulated by ADP accumulated from ATP hydrolysis. ADP probably exerts its effect by facilitating Ca2+ efflux, which subsequently stimulates Ca2+ exchange.     

Evidence for hormonal control of diuresis after a blood meal in the mosquito Aedes aegypti

In previous studies we have presented evidence for the role of peptides, isolated from heads of the mosquito Aedes aegypti, in stimulating fluid secretion by isolated Malpighian tubules. In the present study we conducted experiments to investigate whether these peptides are involved in hormone-mediated diuresis after a blood meal. In vivo experiments showed that the head was required to maintain diuresis after the blood meal. Whereas feeding on blood triggered a prompt diuresis in the intact mosquito, subsequent decapitation caused a gradual, not an abrupt, decline in urine excretion rate. Hemolymph collected from mosquitoes fed blood significantly stimulated fluid secretion in vitro by isolated Malpighian tubules, whereas hemolymph from unfed or blood-fed decapitated mosquitoes did not. These results indicate that a diuretic factor was released into the hemolymph after a blood meal. This factor was not present in the hemolymph of decapitated females. We identified the head as a source of diuretic factors. Peptides isolated from a head extract by high-performance liquid chromatography, when injected into the hemocoel of blood-fed decapitated mosquitoes, triggered diuresis in vivo and also stimulated fluid secretion in isolated Malpighian tubules. These studies support the hypothesis that the head is a storage site for diuretic peptides that may be released after a blood meal to control diuresis.     

1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholines: influence on aggregation and [3H]serotonin release of human thrombocytes

1-O-Alkyl-2-O-acetyl-sn-glycero-3-phosphocholines (platelet activating factor, PAF) aggregate human thrombocytes in a concentration dependent fashion. After a short lag-phase, maximum aggregation is reached within 2 min. PAF releases serotonin from human thrombocytes within 1 min. Indomethacin and creatine phosphate (CP)/creatine phosphokinase (CPK) are able to inhibit the second phase of the aggregation by PAF, while xylocain reduces both the first and second phase of aggregation of human thrombocytes. Hirudine neither influences the first nor the second phase of aggregation by PAF.     

Transmission dynamics of lymphatic filariasis: density-dependence in the uptake of Wuchereria bancrofti microfilariae by vector mosquitoes

Gaining a better understanding of parasite infection dynamics in the vector mosquito (Diptera: Culicidae) population is central to improving knowledge regarding the transmission, persistence and hence control of lymphatic filariasis. Here, we use data on mosquito feeding experiments collated from the published literature to examine the available evidence regarding the functional form of the first component of this parasite-vector relationship for Wuchereria bancrofti (Filarioidea: Onchocercidae) causing Bancroftian filariasis, i.e. the rate of microfilariae (mf) uptake from the blood of infected humans by the feeding mosquito vector. Using a simple logarithmic regression model for describing the observed relationships between the mean numbers of mf ingested per mosquito and parasite load in humans in each study, and a linear mixed-effects meta-analytical framework for synthesizing the observed regressions across studies, we show here for the first time clear evidence for the existence of density-dependence in this process for all the three major filariasis transmitting mosquito vectors. An important finding of this study is that this regulation of mf uptake also varies significantly between the vector genera, being weakest in Culex, comparatively stronger in Aedes and most severe and occurring at significantly lower human mf loads in Anopheles mosquitoes. The analysis of the corresponding mf uptake prevalence data has further highlighted how density-dependence in mf uptake may influence the observed distributions of mf in vector populations. These results show that whereas strong regulation of mf uptake, especially when it leads to saturation in uptake at low human parasite intensities, can lead to static distributions of mf per mosquito with host parasite intensity, a weaker regulation of mf ingestion can give rise to changes in both mean mf loads and in the frequency distribution of parasites/mosquito with increasing human parasite intensity. These findings highlight the importance of considering local vector infection dynamics when attempting to predict the impacts of community-based filariasis control. They also emphasize the value of developing and applying robust meta-analytic methods for estimating functional relationships regarding parasitic infection from population ecological data.     

Epidermal growth factor stimulates fluid phase endocytosis in human fibroblasts through a signal generated at the cell surface

We have investigated the stimulation of fluid phase endocytosis by epidermal growth factor (EGF) in normal human fibroblasts using ¹²⁵I-labeled polyvinylpyrrolidone (¹²⁵I-PVP) as a fluid phase marker. We found that EGF initially induced a thereefold increase in the rate of ¹²⁵I-PVP uptake. This initial burst of fluid uptake terminated within 10 min. Thereafter, the rate of fluie uptake in EGF-treated cells was approximately 40% higher than in control cells. To identify the cellular site of EGF action in stimulating fluid phase endocytosis, we examined the kinetics of the induction of this response as well as the kinetics of cell surface binding and internalization of ¹²⁵I-EGF. Although there was no detectable lag between binding of EGF to the cell surface and its internalization, the kinetics of the two processes were quite different. Significantly, the kinetics of induction of ¹²⁵I-PVP uptake matched the kinetics of binding of ¹²⁵I-EGF to its cell surface receptors, indicating that the signal for the increase in fluid phase endocytosis is generated at the cell surface. To determine if EGF-stimulated fluid phase endocytosis was related to EGF-stimulated endocytosis of its own receptor, we compared the EGF dose dependency and time course of the two processes. Although the stimulated endocytosis of the EGF receptor was not saturable with respect to the concentration of EGF used, the stimulation of fluid phase endocytosis was half maximal at an EGF concentration of 1 ng/ml and saturated at a concentration of 5 ng/ml. Also, the stimulation of fluid phase endocytosis was sevenfold greater initially after adding EGF than after a 30-min continuous incubation with the hormone, whereas the enhanced clearance of the EGF receptor did not change during this time period. We conclude that the EGF-stimulated increase in fluid phase endocytosis is not directly coupled to EGF-stimulated endocytosis of its own receptor but instead to a separate signal generated at the cell surface.