Transport and metabolism of glucose by dissociated brain cells: Effects of trypsin

A study was carried out to determine the effect of trypsin on glucose transport into brain cells. Two suspensions of dissociated cells were prepared from the two brain hemispheres of adult rats—one using only mechanical means to dissociate the cells and one using trypsin. The use of trypsin for preparation of dissociated brain cells caused a marked reduction in the rate of transport of [1,2-³H]-2-deoxy-d-glucose compared to uptakes of this glucose analog by cells prepared without trypsin. Responses of the two cell preparations to inhibitors of glucose transport (cytochalasin B and phloretin) were similar. Rates of oxidation of [6-¹⁴C]glucose to¹⁴CO₂ by trypsin-treated cells were nearly double those in cells prepared without trypsin. Electron microscopic examination of the two preparations revealed much less preservation of structural integrity if trypsin was used to prepare the cells. The findings suggest that trypsin alters cell structure and affects receptor-regulated events in brain cells.     

Metabolic activities in human skin fibroblasts preloaded with labeled GM2-ganglioside

Confluent cultures of human skin fibroblasts were maintained for 10 days with sphingosine labeled [3H]GM2. Labeled medium was then replaced with normal medium and the cells maintained for 42 days with weekly medium changes. Cells were harvested at regular intervals and cells, medium, and trypsin digest supernatant analyzed for [3H]GM2 and its metabolic products. The ganglioside can be membrane associated and removed by trypsin, or membrane incorporated and trypsin insensitive. The membrane incorporated material is apparently transported to the lysosomes slowly by membrane flow, where 80% of the cellular GM2 can be metabolized by day 42. [3H]GM2 as well as its metabolic products in control cells is continuously released into the medium, during which it can also become associated with the cell surface membrane. There is no detectable metabolism of the [3H]GM2 in GM2 gangliosidosis cell lines over the extended post-labeling period, indicating that there is no residual enzyme activity in these cells. Undegraded GM2 is continuously released into the medium and remains associated with the cell surface membrane as well.     

Migration and keratinization of cells in wool follicles

Migration of cells in wool follicles of an adult Merino sheep was studied autoradiographically in skin samples taken at intervals after an intravenous injection of [3H]thymidine. Fibre and inner root sheath cells incorporated [3H]thymidine in a cone-shape region of the follicle bulb. Labelled inner sheath cells migrated out of the bulb ahead of contemporaneous cells in the fibre and remained in advance, although to a progressively lesser extent, until the inner sheath cells sloughed into the follicle lumen. Outer root sheath cells incorporated [3H]thymidine along the length of the follicle. Cells in the proximal half of the outer sheath migrated inwards and distally and sloughed into the follicle lumen before contemporaneous inner sheath cells. Other cells in the distal half of the outer sheath migrated past the level where cells from the proximal population were shed and also sloughed into the lumen. In the most distal part of the outer sheath, which formed the epidermis-like lining of the follicle canal, little migration of cells was observed during 8 days of observation. The specific activity of tritium in fibres plucked from the same sheep at intervals after the intravenous injection of [3H]thymidine was determined by scintillation counting and assessed in terms of cell migration and hardening of the fibres. The time which the specific activity of solvent-degreased fibres reached a maximum was found to give an estimate of the time for cells in the fibre to migrate to the upper limit of the keratogenous zone. When the plucked fibres were extracted with 8 M urea the times of the maximum specific activities of the urea-dispersible and urea-insoluble material provided respectively estimates of the times at which hardening of the fibres began and ended. The effects of different planes of nutrition were examined in two other Merino sheep by radioassay of fibres plucked after intravenous injections of [3H]thymidine given after equilibration period of at least 2 months on each level of feeding. A high plane of nutrition the rate of cell migration and hastened the onset of hardening of the fibres, but prolonged the hardening process. The prolongation of the hardening process was confirmed by the specific activities of fibres plucked after intravenous injections of [35S]cystine.     

Leukotriene C4 metabolism by hepatoma cells deficient in the uptake of cysteinyl leukotrienes

Uptake and metabolism of the cysteinyl leukotrienes C4 and E4 (LTC4 and LTE4) were studied in AS-30D hepatoma cell suspensions and compared with rat hepatocytes. The hepatoma cells were deficient in the uptake of [3H]LTC4 and [3H]LTE4 but took up, in control experiments, L-[14C]glutamine and [14C]adenosine in a time-dependent manner. By contrast, isolated hepatocyte suspensions incubated under the same conditions took up [3H]LTC4 and [3H]LTE4 as well as L-[14C]glutamine and [14C]adenosine. The hepatoma cells deficient in the uptake of cysteinyl leukotrienes metabolized extracellular [3H]LTC4 to [3H]LTD4 and to [3H]LTE4. Addition of acivicin, an inhibitor of gamma-glutamyltransferase, largely prevented metabolism of [3H]LTC4 by the hepatoma cells. Sonication of the cells did not enhance the formation of [3H]LTD4 and [3H]LTE4 from [3H]LTC4. We conclude that ectoenzymes of AS-30D hepatoma cells catalyze the conversion of LTC4 to LTE4 via LTD4. As compared to hepatocytes, these neoplastic cells have lost the uptake system for cysteinyl leukotrienes and may serve in studies on leukotriene metabolism by cell-surface enzymes.     

Uptake and metabolism of female sex steroids by isolated small neurons and other cell fractions from the rat medial basal hypothalamus

Rat medial basal hypothalami (MBH) and sections of cerebral cortex (CC) were dissociated with trypsin to prepare single cells and subcellular fractions. They were then separated into four fractions on a discontinuous sucrose gradient. The small neurons in Fraction D were highly purified. Fraction A had synaptosomes, myelin and other cell particulates. Fraction B had glial cells, neurons and a few synaptosomes. Fraction C had large neurons and red blood cells. All four fractions contained LHRH, but most (62.5%) of this hormone was present in Fraction A. Dissociated cell suspensions were incubated with [³H]-steroids, with and without a 100-fold excess of unlabeled steroids, then separated on sucrose gradients. In most fractions the total uptake and specific uptake of [³H]-progesterone, [³H]-5α-pregnane-3,20-dione (5α-dihydroprogesterone) and [³H]-l7β-estradiol were greater for the dissociated cells from the MBH than the CC. The dissociated cells and cell particulates in all four fractions from the MBH and CC metabolized progesterone, 5α-dihydroprogesterone and l7β-estradiol.These results indicate that hypothalamic neurons contain small amounts of LHRH and retain the ability to take up and metabolize progesterone, 5α-dihydroprogesterone and 17β-estradiol.     

Selective loss of S-phase cells when making cell suspensions from lymphoid tissue.

The relative yield of S-phase cells when making cell suspensions from lymph nodes was determined by two different methods: by estimating the proportions of S-phase cells in sections and smears from lymph nodes undergoing a local graft-versus-host reaction, and by measuring the [³H]thymidine activity relative to DNA content in intact tissue and cell suspensions from normal lymph nodes. Both methods showed a large selective loss of S-phase cells in the process of making cell suspensions. The cell types preparing for division in the GVH nodes were then determined by light microscopic autoradiography combined with electron microscopy of neighboring ultrathin sections. The majority of dividing cells were lymphoid; some of these showed advanced signs of cell death.     

Pulsatile secretion of bioactive luteinizing hormone in adult male rhesus macaques: acute and chronic effects of orchidectomy

Glycosaminoglycans (GAGs) were purified from bovine follicular fluid, and their effectiveness to compete for heparin-binding sites in granulosa cells was evaluated. The GAGs dermatan sulfate (DS) and heparan sulfate (HS) were purified by anion-exchange high-performance liquid chromatography. Approximately 5 micrograms of protein from suspensions of bovine granulosa cells were incubated with 101 pmoles of [3H]heparin and 0.01-5.0 mg/ml of HS or DS for 2 h at 37 degrees C in 40 mM tris(hydroxymethyl)aminomethane (Tris), pH 7.35. Heparan sulfate obtained from small and medium follicles displaced [3H]heparin in a dose-dependent manner from 0.1 to 5 mg/ml, but HS from large follicles did not displace [3H]heparin. The DS obtained from small, medium, and large follicles displaced [3H]heparin in a dose-dependent manner, and the potency of the DS to displace [3H]heparin increased as the size of the follicles from which the DS was purified increased. Those results were independent of the maturational state of the granulosa cells. In a separate experiment, heparin (17.1% sulfate) was N-desulfated (11.8%), and the desulfated heparin did not displace [3H]heparin. It was concluded that the effectiveness of follicular HS and DS to compete for heparin-binding sites on granulosa cells was dependent on the maturation of the follicle from which the fluid was obtained rather than on the source of granulosa cells. The binding interaction of the GAGs relies, to some extent, on the presence and positions of sulfate moieties.     

DNA synthesis in Langerhans' cells of mouse ear epithelium revealed by tritiated thymidine autoradiography and histochemical staining for non-specific esterase and beta-glucuronidase activity

The proportion of Langerhans' cells in DNA synthesis in normal mouse skin was assessed by combining tritiated thymidine [3H]TdR autoradiography with enzyme histochemistry. After injection of [3H]TdR, ear skin was treated in two ways. Epithelial sheet preparations were stained for the presence of non-specific esterase and cytospin preparations of epithelial cell suspensions were stained for beta-glucuronidase activity. The labelling index (+/- SE mean) for cytospins, 40 min after injecting [3H]TdR, was 1.6 +/- 0.15%, doubling to 3-4% from 7-17 days after injection. The sheet preparations showed the proportion of label attributable to paired Langerhans' cells rising from 18% at 40 min after injection, to approximately 45%, on days 1-4 after injection. These results suggest that the proliferation of Langerhans' cells in normal mouse skin might be higher than was previously thought to be the case.     

Comparative analysis of different approaches to investigate cell kinetics

The potential of different methods to investigate proliferative activity of cell populations was analysed for non-Hodgkin's lymphomas. Cells in S phase and all cycling cells were determined on cell suspensions obtained from fresh lymph node material by [3H]-thymidine autoradiography [( 3H]TdR LI), a monoclonal antibody to bromodeoxyuridine (BrdU LI), and the monoclonal antibody Ki67. A good correlation was observed between the values of [3H]TdR LI and BrdU LI (rs = 0.90; P less than 0.01), [3H]TdR LI and S phase (rs = 0.62; P less than 0.01) and [3H]TdR LI and Ki67 (rs = 0.64; P less than 0.01) in individual lymphomas. Using the median values obtained from the different approaches as cut-off points to define slowly and rapidly proliferating tumours, the best agreement was observed between [3H]TdR LI and BrdU LI (91%) and poorer agreements, even though statistically significant, were observed between [3H]TdR LI and S phase (73%) or Ki67 (76%). In conclusion, the kinetic information derived from different approaches was more or less concordant and newly proposed approaches should be directly and carefully verified for their prognostic relevance before using them as alternatives to conventional methods.     

Steroid metabolism by avian ovarian cells during follicular maturation

The profiles of steroid hormones produced by ovarian cells from the domestic hen were examined. Theca cells from the immature, small white follicles (SWFT), the third largest (T3), and largest (T1) preovulatory follicles, and the ruptured, postovulatory follicle (POFT) were incubated for 3 h at 37 degrees with [3H] progesterone (Prog) or [3H] pregnenolone (Preg). Granulosa cells from the largest preovulatory follicle were incubated with [3H] Preg or were coincubated with theca cells and [3H] Preg. The production of specific steroid metabolites was determined on the basis of coelution of radioactivity with known standard compounds, using an isocratic high-pressure liquid chromatography (HPLC) technique. Granulosa cells converted 93% of [3H] Preg substrate to Prog. More Prog was utilized by T3 cells than by T1 and SWFT cells, either when [3H] Prog was the substrate or when coincubated with granulosa cells and [3H] Preg. The major metabolites of Prog were androstenedione, 17-hydroxyprogesterone, and an unidentified compound with an elution time of 53 min. The POFT cells metabolized [3H] Prog to the same extent as T3 cells did, but their profile of steroidogenesis favored production of the unidentified 53 min metabolite. SWFT cells utilized the least amount of [3H] Preg substrate. The results point to marked changes in enzyme activities in theca cells during maturation and following ovulation.     

Phosphatidylcholine metabolism in endothelial cells: evidence for phospholipase A and a novel Ca2+-independent phospholipase C

The metabolism of phosphatidylcholine (PC) was investigated in sonicated suspensions of bovine pulmonary artery endothelial cells and in subcellular fractions using two PC substrates: 1-oleoyl-2-[3H]oleoyl-sn-glycero-3-phosphocholine and 1,2-dipalmitoyl-sn-glycero-3-phospho[14C]choline. When these substrates were incubated with the whole cell sonicate at pH 7.5, all of the metabolized 3H label was recovered in [3H]oleic acid (95%) and [3H]diacylglycerol (5%). All of the 14C label was identified in [14C]lysoPC (92%) and [14C]phosphocholine (8%). These data indicated that PC was metabolized via phospholipase(s) A and phospholipase C. Substantial diacylglycerol lipase activity was identified in the cell sonicate. Production of similar proportions of diacylglycerol and phosphocholine and the low relative activity of phospholipase C compared to phospholipase A indicated that the phospholipase C-diacylglycerol lipase pathway contributed little to fatty acid release from the sn-2 position of PC. Neither phospholipase A nor phospholipase C required Ca2+. The pH profiles and subcellular fractionation experiments indicated the presence of multiple forms of phospholipase A, but phospholipase C activity displayed a single pH optimum at 7.5 and was located exclusively in the particulate fraction. The two enzyme activities demonstrated differential sensitivities to inhibition by p-bromophenacylbromide, phenylmethanesulfonyl fluoride and quinacrine. Each of these agents inhibited phospholipase A, whereas phospholipase C was inhibited only by p-bromophenacylbromide. The unique characteristics observed for phospholipase C activity towards PC indicated the existence of a novel enzyme that may play an important role in lipid metabolism in endothelial cells.     

Stimulation of the biosynthesis of membrane glycoproteins from Zajdela ascites hepatoma cells by Robinia lectin.

Membrane glycoprotein biosynthesis of ascites hepatoma cells is followed by [14C]glucosamine and [3H]leucine incorporation into cells in culture. The rate of incorporation is strongly increased by the addition of Robinia lectin in culture medium. Labeled glycoproteins are released from lectin stimulated and non-stimulated cells by trypsin digestion. Studies of labeled trypsinates on sodium dodecyl sulfate gel electrophoresis and Sephadex G-200 filtration exhibit two fractions both labeled with [14C]glucosamine and [3H]leucine and having different molecular weights, one over 200000 and the other about 2000. Identical results are obtained when external membrane glycoproteins are solubilized by sodium deoxycholate. Comparison of surface glycoproteins isolated by trypsinization from control cells labeled with [3H]-glucosamine and from lectin stimulated cells labeled with [14C]glucosamine displays no significant qualitative differences between glycoprotein fractions released from both cell groups.     

Activation of ornithine decarboxylase in monolayer cells treated with trypsin

When monolayer Chinese hamster cells are treated with trypsin for short periods of time, ornithine decarboxylase (ODCase) activity increases two- to fourfold. This increase can be blocked by aprotinin, a protease inhibitor, and is not observed when cultures are dislodged from substrate mechanically prior to contact with exogenous trypsin. The trypsin-induced increase in ornithine decarboxylase activity is not due to degradation of enzyme or inhibitor molecules or to new enzyme synthesis. Immunoprecipitable protein, radiolabeled with [3H]alpha-difluoromethylornithine in vitro, is the same molecular weight in cells harvested with or without trypsin. Protein-bound levels of this specific enzyme-activated irreversible inhibitor of ornithine decarboxylase are unchanged by trypsin treatments that increase enzyme activity. Trypsin treatment of rat embryonic fibroblasts, transformed by a temperature-sensitive mutant of Rous sarcoma virus, increases ODCase activity in cells growing at the nonpermissive, but not at the permissive, temperature for the transformed phenotype. These results suggest that ornithine decarboxylase can be activated by exogenous trypsin treatment in a manner that is dependent on cell adhesion properties, which are modified in transformed cells.     

Leishmania donovani: cell-surface heparin receptors of promastigotes are recruited from an internal pool after trypsinization

With the use of [3H]heparin, we recently demonstrated that Leishmania donovani promastigotes express a cell-surface receptor that is specific for the glycosaminoglycan heparin (Mukhopadhyay et al. 1989, The Biochemical Journal, 264, 517-525.). Treatment of the parasite with trypsin abolishes 75-90% of this [3H]heparin-binding activity. When trypsinized promastigotes were resuspended in fresh culture medium in the absence and presence of cycloheximide (10 micrograms/ml), approximately 25-30% of the original heparin-binding capacity was restored within 1 hr, indicating that recruitment of receptors from an internal pool occurred without de novo protein synthesis. Scatchard analysis of the regenerated receptor revealed that the number of regenerated binding sites per cell was 2.3 x 10(5); these sites have a binding affinity of 6.7 x 10(-7) M. Like the native heparin receptors on the surface of freshly isolated cells, the receptors recruited after trypsinization are also highly specific for heparin, as a 25-fold excess of four other glycosaminoglycans displaced less than 10% of bound [3H]heparin from the trypsinized cells. The structural requirements of the ligand heparin, namely the number of monosaccharide units and degree of sulfation, were compared for both the native and regenerated receptor: for both receptors, oversulfated polysaccharide heparin fragments of at least six to eight sugar residues were most efficient at displacing [3H]heparin. The concentrations of oligosaccharide fragments required to displace 50% of [3H]heparin were 0.32 and 0.035 microM for the hexa- and octasaccharides, respectively. Colloidal gold-labeled heparin was bound to promastigotes and visualized by electron microscopy. This analysis revealed that the heparin bound almost exclusively to the flagella of control cells (not subjected to trypsin) and those which had regenerated receptor after trypsinization. The physiological significance of this heparin-binding activity on the surface of promastigotes is discussed.     

Neuroblastoma × Glioma Hybrid Cells Synthesize Enkephalin-Like Opioid Peptides

Partially purified extracts from neuroblastoma X glioma hybrid cells 108CC15 inhibit, like opioids, the prostaglandin E1-evoked formation of cyclic AMP in a dose-dependent manner in the same hybrid cells. The inhibition is prevented by the opioid antagonist naloxone. In addition, the same extract competes with [3H]naloxone and [3H]Leu-enkephalin for binding to opioid receptors of hybrid cell membranes and to a specific antiserum, respectively. The opioid activity in the extracts is destroyed by carboxypeptidase A and leucine aminopeptidase, but not by trypsin. Further purification of the extracts by HPLC, TLC, or high-voltage paper electrophoresis reveals in each case two active fractions which behave like Met- and Leu-enkephalin. The Met-enkephalin-like, but not the Leu-enkephalin-like, fraction is inactivated by treatment with BrCN. Dimethylaminonaphtylsulfonyl (dansyl) derivatives of Met- and Leu-enkephalin correspond to [3H]dansyl derivatives of Met-like substances from hybrid cells. Three to four times as much Met-enkephalin-like as Leu-enkephalin-like material is present in the extract. The overall concentration of opioid peptides in the hybrid cells varies between 0.03 and 1.0 pmol Leu-enkephalin equivalents per mg protein. The amount of opioids in the hybrid cells is strongly dependent on the cell density. The findings suggest that neuroblastoma X glioma hybrid cells contain opioid peptides that are very similar, if not identical, to Met- and Leu-enkephalin. Opioid activity can also be detected in other neuronal cell lines and even in glioma cells.     

In vivo uptake and metabolism of [3H]ecdysone in the vitellogenic follicles of Sarcophaga bullata (Diptera) and its localisation by autoradiography

Within 4 h after injection of [³H]ecdysone, almost all tritiated material has disappeared from the haemolymph, indicating that the uptake by the tissues is very fast. After only 15 min, 19% of the label was found in the ecdysterone fraction and 4% in the highly polar products (HPP) fraction. The uptake of [³H]ecdysone by the ovary (mid-vitellogenic) is almost complete within 1 h after injection. The pattern of [³H]ecdysteroids in the ovaries follows a well ordered sequence: firstly, [³H]ecdysone is the major component of the [³ H]ecdysteroids but it disappears within 2 h, next a peak value of [³H]ecdysterone was found at 1 h, whereafter this also disappeared, and from 2 h on, there was a considerable increase in HPP. The HPP consisted of 3 fractions (A, B and C). Glusulase treatment revealed that apparently only fraction B consisted of glucuronide and/or sulphate-conjugates of ecdysteroids. Autoradiographic experiments confirmed that the uptake of [³H]ecdysone was a very rapid process. In ovaries fixed 1 h after injection, the silver grains were abundant in the ooplasm but were also found in the follicle cell cytoplasm and in trophocytes. In follicles examined 16 h after injection, only a few silver grains were observed in the trophocytes and follicle cells. However, the cytoplasm of the oocyte was labelled. The border cells also accumulated label.

The major results indicate that all cell types of the follicle seem to be able to absorb ecdysone from the haemolymph and that there seems to be a rather selective uptake of ecdysone. In the ooplasm, ecdysone is converted to highly polar conjugates.     

Gossypol inhibits aromatase activity in cultured porcine granulosa cells

The present study investigated whether gossypol inhibited aromatase activity in cultured porcine granulosa cells. Aromatase activity was assayed by measuring (3)H-H(2)O released from [1beta-(3)H]-androstenedione. First, immature porcine granulosa cells were cultured with various doses of follicle stimulating hormone (FSH, 1 to 1000 ng/ml) for 1 to 5 d to determine optimal culture conditions for aromatase activity assay. Second, porcine granulosa cells were cultured with or without FSH in the presence or absence of gossypol. Gossypol, at 4 muM, significantly inhibited FSH-induced aromatase activity while showing no effect on basal aromatase activity. Gossypol did not inhibit cell proliferation during cell culture. These results suggest that gossypol inhibits aromatase activity by interfering with FSH induction of aromatase in cultured porcine granulosa cells.     

Direct flow cytometric quantification of alkaline phosphatase activity in rat bone marrow stromal cells.

Cell preparations in cytochemistry are conventionally analyzed with transmitted light after fixation and reaction with agents such as azo-coupling dyes. With cell suspensions stained with fluorescent cytochemical dyes, cells can also be analyzed and sorted by flow cytometry. We have exploited the intense red fluorescence of Fast Red Violet LB generated in cytochemical reactions to perform flow cytometric analyses of alkaline phosphatase (AP) expression in rat bone marrow stromal cells. By modifying staining protocols of single-cell suspensions, we demonstrate that in comparison to staining with Fast Red TR, the method is specific, can distinguish among various levels of enzyme expression within the whole population, and permits enzyme kinetic studies of heterogeneous cell populations. The method was applied to study the effect of the glucocorticoid dexamethasone (Dx) on cell proliferation and AP expression. In low AP-expressing cells, Dx treatment at 10(-8) M increased the [3H]-thymidine labeling index from 3.85% to 5.24% (p less than 0.01). In contrast, high AP-expressing cells were unlabeled by [3H]-thymidine. The staining and analytical methods reported here facilitate the detection, isolation, and quantification of subpopulations of bone marrow stromal cells that express alkaline phosphatase activity. These experiments demonstrate the value of flow cytometry as an adjunct to conventional cytochemical methods.     

Identification of a domain in cytochrome C that displaces [3H]TCP binding from rat brain membrane receptors: synthesis of beta-neuroprotectin

1. A stereospecific radioreceptor binding assay for the phencyclidine analogue, [3H]TCP, was utilized to screen for inhibition of binding in extracts of rat brain. 2. Extracts were prepared from rat cerebral cortex and hippocampus by methods employing aqueous acid. The extracts were fractionated by reversed phase-HPLC (RP-HPLC) and tested for activity in the radioreceptor assay. Three zones of activity were detected. The middle zone was further purified by high performance-size exclusion chromatography (HP-SEC). 3. Size exclusion chromatography revealed a single zone of activity corresponding to mol. wts of ca 12,000-31,000 daltons. A fraction from this zone was digested with trypsin, and the resulting enzyme fragments, isolated by a combination of HP-SEC and RR-HPLC, were identified as fragments of rat cytochrome C. 4. Horse cytochrome C was digested with trypsin and the fragments were similarly purified on the basis of the [3H]TCP binding displacement assay. The fragments were sequenced and found to be trypsin cleavage products of a single largely invariant domain of the cytochrome C molecule: Lys-Lys-Lys-Asp-Glu-Arg-Ala-Asp-Leu-Ile-Ala-Tyr-Leu-Lys-Lys. 5. beta-neuroprotectin (D)-Ala-Asp-Leu-Ile-Ala-Tyr-Leu-NH2, inhibits [3H]TCP binding and provides protection against NMDA mediated neuronal cell death at low concentrations.     

The development of responsiveness to juvenile hormone in the follicle cells of Rhodnius prolixus

During the previtellogenic phase of the development of the follicle in Rhodnius, the JH-sensitive Na/K ATPase in the membranes of the follicle cells displays display a rapid increase in activity, so that the level in vitellogenic follicle cells is about six times that in early previtellogenic cells. The sharp increase in activity during the development of previtellogenic cells is abolished in an allatectomized female. Topical application of JH I early in development restores the level in previtellogenic cells and produces a nearly normal level in vitellogenic cells. Treatment with JH I later in the cycle fails to yield an increase in the activity of the JH-sensitive Na/K ATPase in the previtellogenic cell and leads to only partial restoration of the activity in vitellogenic follicles. These effects of allatectomy and early and late hormone replacement are duplicated by effects on the maximal binding capacity for [³H]JH I of the membranes of follicle cells. These results are interpreted in the light of “activation”, the JH-mediated process by which follicles acquire the competence to respond to JH by becoming patent.