Binding of <Superscript>3</Superscript>H-WIN-35,428 and <Superscript>125</Superscript>I-RTI-121 to Human Platelet Membranes

The dopamine transporter (DAT) is a protein regulating dopamine concentration in the synaptic cleft through the re-uptake mechanism. The DAT is the main target of psychostimulants and seems to play a pivotal role in neuronal degeneration and different neuropsychiatric disorders involving the dopamine system. Exhaustive research, however, regarding the presence of this protein in human platelets is still inconclusive, although it is thought that it might provide a peripheral tool to serve as a mean of exploring the same structure present in the brain. Therefore, we assessed some binding assays in platelets derived from healthy human subjects by means of ³H-WIN 35,428, a compound which is considered a selective ligand for the labelling of this protein, and by means of ¹²⁵I-RTI-121, another compound with high specificity for DAT. The results showed that the binding of ³H-WIN-35,428 was too low to enable the detection of any structure; the binding of ¹²⁵I-RTI-121, on the other hand, revealed the presence of two binding sites with pharmacological profiles similar to that of the serotonin transporter (SERT). In conclusions, therefore, platelets would not seem to be a useful model for exploring the DAT, given the prevalence therein of the SERT and the difficulty of labelling the DAT with the currently available ligands.     

Rat mesencephalic neuronal cells cultured for different periods as a model of dopamine transporter ontogenesis

Ventral mesencephalic neurons contained only low-affinity and sodium-independent binding sites of [³H]WIN 35,428 (marker of dopamine transporter) during the first 10d in primary cultures. These sites were present in cytosol, and they are not very probably related to dopamine transporter. After 12 d in culture, membrane-bound, high-affinity, and sodium-dependent [³H]WIN 35,428 binding sites were detected. In membranes prepared from cells 14 d in culture, cocaine displaced [³H]WIN 35,428 binding with similar potency to that in striatal membranes of adult rat brain. The high-affinity [³H]WIN 35,428 binding sites in mesencephalic neuronal cell cultures are very probably related to dopamine transporter. The development of high-affinity [³H]WIN 35,428 binding sites in neurons cultured for different time periods could be a useful model of dopamine transporter ontogenesis.     

Distribution of [3H]GR65630 Binding in Human Brain Postmortem

We investigated the distribution of serotonin (5-HT) receptors of type 3 (5-HT₃) in human brain areas, by means of the the specific binding of [³H]GR65630. The brains were obtained during autoptic sessions from 6 subjects. Human brain membranes and the binding of [³H]GR65630 were carried out according to standardized methods. The highest density (Bmax ± 6 SD, fmol/mg protein) of [³H]GR65630 binding sites was found in area postrema (13.1 ± 9.7), followed at a statistically lower level, by nucleus tractus solitarius (6.7 ± 3.4), nervus vagus (5.5 ± 2.1), striatum (4.8 ± 2.4) with a progressive decrease in amygdala, olivar nuclei, hippocampus, olfactory bulbus and prefrontal cortex, and then by the other cortical areas and the cerebellum, where no binding was detected. These observations extend previous findings on the distribution of 5-HT₃ receptors and confirm interspecies variations that might explain the heterogeneous properties of 5-HT₃ receptors in different animals.     

No Orcadian Rhythms of Serotoninergic,Alpha-, Beta-Adrenergic and Imipramine Binding Sites in Rat Brain Regions

B_max values of the specific binding of [³H]-WB 4101, [³H]-dihydroalprenolol, [³H]-spiperone and [³H]-imipramine to various rat brain regions were determined at 4 hr intervals over 24 hr under circadian conditions. No significant circadian rhythm of binding sites number was found for any receptor investigated in cerebral cortex, hypothalamus or brain stem. Some methodological issues are discussed.     

Characterization of quisqualate recognition sites in rat brain tissue usingDl-[3H]α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and a filtration assay

The binding of [³H]AMPA (Dl--amino-3-hydroxy-5-methylisoxazole-4-propionic acid), a ligand for the putative quisqualate excitatory amino acid receptor subtype, was evaluated using centrifugation and filtration receptor binding techniques in rat brain crude synaptosomal membrane preparations. Maximal specific binding of [³H]AMPA occurred in Triton X-100 treated membranes in the presence of the chaotropic agent potassium thiocyanate (KSCN). The effects of KSCN on binding were reversible and optimal at 100 mM. Supernatant obtained from detergent-treated membranes inhibited specific [³H]AMPA and [³H]kainic acid binding, suggesting the presence of an inhibitory agent which was tentatively identified as glutamate. Using centrifugation, saturation analysis revealed two distinct binding sites in both the absence and presence of KSCN. The chaotrope was most effective in increasing binding at the low affinity binding site, enhancing the affinity (K _d) without a concommitant change in the total number of binding sites. Using filtration, a single binding site was detected in Triton-treated membranes. Like the data obtained by centrifugation, KSCN enhanced the affinity of the receptor (K _d value=10 nM) without altering the number of binding sites (B _max=1.2 pmol/mg protein). The rank order of potency of various glutamate analogs in the [³H]AMPA binding assay was quisqualate > AMPA > l-glutamate > kainate > d-glutamate, consistent with the labeling of a quisqualate-type excitatory amino acid receptor subtype.l-glutamic acid diethylester, and 2-amino-7-phosphonoheptanoic acid (AP₇) were inactive. The present technique provides a rapid, reliable assay for the evaluation of quisqualate-type excitatory amino acid agonists and/or antagonists that may be used to discover more potent and selective agents.     

TRP expression pattern and the functional importance of TRPC3 in primary human T-cells

TRP proteins form ion channels which are activated following receptor stimulation. In T-cell lines, expression data of TRP proteins have been published. However, almost no data about TRP expression is available in primary human T-cells. Using RT-PCR and quantitative RT-PCR, we compare the expression of TRP mRNA in 1) human peripheral blood lymphocytes, which are a mix of mostly mono-nuclear blood lymphocytes but contain other leucocytes, 2) a pure human CD4⁺ T-helper cell population in the resting (= naïve) and activated (= effector) state, and 3) two commonly used CD4⁺ Jurkat T-cell lines, E6-1 and parental. To mimic physiological cell stimulation, we analyzed TRP expression in primary human cells in a quantitative way over several days following formation of an immunological synapse through stimulation with antibody-coated beads. The TRP expression profile of primary human T-cells was significantly different from Jurkat T-cells. Among the TRP mRNAs of the TRPC, TRPM, and TRPV family, we found consistent expression of TRPC1, TRPC3, TRPV1, TRPM2, and TRPM7 in primary human CD4⁺ T-cells of all analyzed blood donors. Among these, TRPC3 and TRPM2 were strongly up-regulated following stimulation, but with different kinetics. We found that TRPC3 modulates Ca²⁺-dependent proliferation of primary CD4⁺ T-cells indicating that TRPC3 may be involved in Ca²⁺ homeostasis in T-cells besides the well-established STIM and ORAI proteins which are responsible for store-operated Ca²⁺ entry.     

Comparison of [3H]WIN 35,428 Binding, a Marker for Dopamine Transporter, in Embryonic Mesencephalic Neuronal Cultures with Striatal Membranes of Adult Rats

Abstract: In contrast to striatal membranes of adult rats, where high- ( K _D1= 34 n M ) and low- ( K _D2= 48,400 n M ) affinity binding sites for [³H]WIN 35,428 are present, in primary cultures of ventral mesencephalon neurons (CVMNs) only low-affinity binding sites were found ( K _D= 336,000 n M ). The binding of [³H]WIN 35,428 in CVMNs prepared from rat embryos was reversible, saturable, and located in cytosol. Although dopamine (DA) uptake blockers inhibited [³H]DA uptake at nanomolar concentrations in CVMNs, the displacement of [³H]WIN 35,428 binding in CVMNs by DA uptake inhibitors required 100-8,000 times higher concentrations than were needed to displace [³H]WIN 35,428 binding in striatal membranes. Piperazine derivatives, e.g., GBR-12909, GBR-12935, and rimcazole, inhibited [³H]WIN 35,428 binding in CVMNs more effectively than did cocaine, WIN 35,428, mazindol, nomifensine, or benztropin. A positive correlation ( r = 0.779; p < 0.001) was found between drug affinities for the striatal membrane sites labeled by [³H]WIN 35,428 and their abilities to inhibit DA uptake in CVMNs, whereas no correlation existed between the IC₅₀ values of drugs that inhibited [³H]WIN 35,428 binding and [³H]DA uptake in CVMNs. The cytosolic [³H]WIN 35,428 binding sites may be a piperazine acceptor and may not be involved in the regulation of the DA transporter.     

Latch and trigger role for R445 in DAT transport explains molecular basis of DTDS

A recent study reports on five different mutations as sources of dopamine transporter (DAT) deficiency syndrome (DTDS). One of these mutations, R445C, is believed to be located on the intracellular side of DAT distal to the primary (S1) or secondary (S2) sites to which substrate binding is understood to occur. Thus, the molecular mechanism by which the R445C mutation results in DAT transport deficiency has eluded explanation. However, the recently reported X-ray structures of the endogenous amine transporters for dDAT and hSERT revealed the presence of a putative salt bridge between R445 and E428 suggesting a possible mechanism. To evaluate whether the R445C effect is a result of a salt bridge interaction, the mutants R445E, E428R, and the double mutant E428R/R445E were generated. The single mutants R445E and E428R displayed loss of binding and transport properties of the substrate [³H]DA and inhibitor [³H]CFT at the cell surface while the double mutant E428R/R445E, although nonfunctional, restored [³H]DA and [³H]CFT binding affinity to that of WT. Structure based analyses of these results led to a model wherein R445 plays a dual role in normal DAT function. R445 acts as a component of a latch in its formation of a salt bridge with E428 which holds the primary substrate binding site (S1) in place and helps enforce the inward closed protein state. When this salt bridge is broken, R445 acts as a trigger which disrupts a local polar network and leads to the release of the N-terminus from its position inducing the inward closed state to one allowing the inward open state. In this manner, both the loss of binding and transport properties of the R445C variant are explained.     

Effects of Valeriana Officinalis Extracts on [3H]Flunitrazepam Binding,Synaptosomal [3H]GABA Uptake,and Hippocampal [3H]GABA Release

Extracts of Valeriana officinalis have been used in folkloric medicine for its sedative, hypnotic, tranquilizer and anticonvulsant effects, and may interact with -aminobutyric acid (GABA) and/or benzodiazepine sites. At low concentrations, valerian extracts enhance [³H]flunitrazepam binding (EC₅₀ 4.13 × 10^–10 mg/ml). However, this increased [³H]flunitrazepam binding is replaced by an inhibition at higher concentrations (IC₅₀ of 4.82 × 10^–1 mg/ml). These results are consistent with the presence of at least two different biological activities interacting with [³H]flunitrazepam binding sites. Valerian extracts also potentiate K⁺ or veratridine-stimulated release of radioactivity from hippocampal slices preloaded with [³H]GABA. Finally, inhibition of synaptosomal [³H]GABA uptake by valerian extracts also displays a biphasic interaction with guvacine. The results confirm that valerian extracts have effects on GABA_A receptors, but can also interact at other presynaptic components of GABAergic neurons.     

Isolation and characterization of monoclonal antibodies to (Na + K)-ATPase

Four stable hybridoma cell lines secreting antibodies specific to the membrane (Na⁺ + K⁺)-dependent ATPase isolated from lamb kidney medulla have been produced by fusing mouse myeloma cells with spleen cells from immunized mice. These cell lines produce IgG γ₁ heavy chain and κ light chain antibodies which are directed against the catalytic or α-subunit of the (Na⁺ + K⁺)-ATPase enzyme. Binding studies, using antibodies that were produced by growing hybridomas in vivo and purified by affinity column chromatography, suggest a somewhat higher affinity of these antibodies for the isolated α-subunit than for the ‘native’ holoenzyme. In addition, these monoclonal antibodies show no reactivity with either the glycoprotein (β) subunit of the lamb enzyme nor the (Na⁺ + K⁺)-ATPase from rat kidney, an ouabain-insensitive organ. Cotitration binding experiments have shown that the antibodies from two cell lines originally isolated independently from the same culture plate well population of fused cells bind to the same determinant site and are probably the same antibody. Cotitration and competition binding studies with two other antibodies have revealed two additional distinct antibody binding sites which appear to have little overlap with the first site. One of the three different antibodies isolated caused a partial inhibition of the (Na⁺ + K⁺)-ATPase activity. This antibody appears to be directed against a specific functionally important site of the α-subunit and is a competitive inhibitor of ATP binding. Under optimum conditions of ATPase activity, this inhibitory effect is not altered by the presence of the other two antibodies.     

Errata

1. (1) The significance of the specific (ouabain-sensitive) ⁸⁶Rb⁺ or ⁴²K⁺ uptake by cardiac muscle preparations which are not ‘sodium-loaded’ was studied.

2. (2) In left atrial preparations of guinea-pig heart, resting ⁸⁶Rb⁺ uptake was relatively low. It was markedly increased by electrical stimulation. This stimulated uptake was further enhanced by isoproterenol and inhibited by verapamil.

3. (3) In rat atria, the resting ⁸⁶Rb⁺ uptake was somewhat higher than in guinea-pig atria, and the increase in uptake caused by electrical stimulation was smaller. In guinea-pig right ventricular papillary muscle, the resting uptake was highest among those tissues studied, and the response to electrical stimulation was smallest. In the latter tissue, verapamil produced only a minimal inhibition of the specific ⁸⁶Rb⁺ uptake.

4. (4) The effect of the frequency of electrical stimulation on ⁸⁶Rb⁺ uptake paralleled its influence on the force of contraction, suggesting the involvement of intracellular sodium in both events.

5. (5) In both left atrial and right papillary muscle preparations of guinea-pig heart, specific ⁴²K⁺ uptake observed with 5.8 mM K⁺ was relatively high, and was increased only slightly by electrical stimulation. This electrical stimulation, however, increased ouabain-induced inhibition of ⁴²K⁺ uptake, suggesting that the stimulation increases the amount of Na⁺ available to the sodium pump.

6. (6) When the K⁺ concentration was 1 mM, the resting ⁴²K⁺ uptake was low, and could be enhanced by electrical stimulation.

Modeling of the interaction of Na+ and K+ with the binding of dopamine and [3H]WIN 35,428 to the human dopamine transporter

Although much is known about the effects of Na+, K+, and Cl- on the functional activity of the neuronal dopamine transporter, little information is available on their role in the initial event in dopamine uptake, i.e., the recognition step. This was addressed here by studying the inhibition by dopamine of the binding of [3H]WIN 35,428 [2beta-carbomethoxy-3beta-(4-fluorophenyl)[3H]tropane], a phenyltropane analogue of cocaine, to the cloned human dopamine transporter expressed in HEK-293 cells. The decrease in the affinity of dopamine (or WIN 35,428) binding affinity with increasing [K+] could be fitted to a competitive model involving an inhibitory cation site (1) overlapping with the dopamine (or WIN 35,428) domain. The K+ IC50 for inhibiting dopamine or WIN 35,428 binding increased linearly with [Na+], indicating a K(D,Na+) of 30-44 mM and a K(D,K+) of 13-16 mM for this cation site. A second Na+ site (2), distal from the WIN 35,428 domain but linked by positive allosterism, was indicated by model fitting of the WIN 35,428 binding affinities as a function of [Na+]. No strong evidence for this second site was obtained for dopamine binding in the absence or presence of low (20 mM) Cl- and could not be acquired for high [Cl-] because of the lack of a suitable substitute ion for Na+. The K(D) but not Bmax of [3H]WIN 35,428 binding increased as a function of the [K+]/[Na+] ratio regardless of total [Cl-] or ion tonicity. A similar plot was obtained for the Ki of dopamine binding, with Cl- at > or = 140 mM decreasing the Ki. At 290 mM Cl- and 300 mM Na+ the potency of K+ in inhibiting dopamine binding was enhanced as compared with the absence of Cl- in contrast to the lack of effect of Cl- up to 140 mM (Na up to 150 mM). The results indicate that Cl- at its extracellular level enhances dopamine binding through a mechanism not involving site 1. The observed correspondence between the WIN 35,428 and dopamine domains in their inclusion of the inhibitory cation site explains why many of the previously reported interrelated effects of Na+ and K+ on the binding site of radiolabeled blockers to the dopamine transporter are applicable to dopamine uptake in which dopamine recognition is the first step.     

Specificity of ligand binding to transport sites: Ca2+ binding to the Ca2+ transport ATPase and its dependence on H+ and Mg2+

Ligand binding to transport sites constitutes the initial step in the catalytic cycle of transport ATPases. Here, we consider the well characterized Ca²⁺ ATPase of sarcoplasmic reticulum (SERCA) and describe a series of Ca²⁺ binding isotherms obtained by equilibrium measurements in the presence of various H⁺ and Mg²⁺ concentrations. We subject the isotherms to statistical mechanics analysis, using a model based on a minimal number of mechanistic steps. The analysis allows satisfactory fits and yields information on occupancy of the specific Ca²⁺ sites under various conditions. It also provides a fundamental method for analysis of binding specificity to transport sites under equilibrium conditions that lead to tightly coupled catalytic activation.     

1-Methyl-4-phenylpyridinium-induced down-regulation of dopamine transporter function correlates with a reduction in dopamine transporter cell surface expression

The mechanisms whereby 1-methyl-4-phenylpyridinium (MPP(+)) mediates cell death and Parkinsonism are still unclear. We have shown that dopamine transporter (DAT) is required for MPP(+)-mediated cytotoxicity in HEK-293 cells stably transfected with human DAT. Furthermore, MPP(+) produced a concentration- and time-dependent reduction in the uptake of [3H]dopamine. We observed a significant decrease in [3H]WIN 35428 binding in the intact cells with MPP(+). The saturation analysis of the [3H]WIN 35428 binding obtained from total membrane fractions revealed a decrease in the transporter density (B(max)) with an increase in the dissociation equilibrium constant (K(d)) after MPP(+) treatment. Furthermore, biotinylation assays confirmed that MPP(+) reduced both plasma membrane and intracellular DAT immunoreactivity. Taken together, these findings suggest that the reduction in cell surface DAT protein expression in response to MPP(+) may be a contributory factor in the down-regulation of DAT function while enhanced lysosomal degradation of DAT may signal events leading to cellular toxicity.     

The Role of GlycineB Binding Site and Glycine Transporter (GlyT1) in the Regulation of [3H]GABA and [3H]Glycine Release in the Rat Brain

The effect of N-methyl-D-aspartic acid (NMDA), a selective glutamate receptor agonist, on the release of previously incorporated [³H]-aminobutyric acid(GABA) was examined in superfused striatal slices of the rat. NMDA (0.01 to 1.0 mM) increased [³H]GABA overflow with an EC₅₀ value of 0.09 mM. The [³H]GABA releasing effect of NMDA was an external Ca²⁺-dependent process and the GABA uptake inhibitor nipecotic acid (0.1 mM) potentiated this effect. These findings support the view that NMDA evokes GABA release from vesicular pool in striatal GABAergic neurons. Addition of glycine (1 mM), a cotransmitter for NMDA receptor, did not influence the NMDA-induced [³H]GABA overflow. Kynurenic acid (1 mM), an antagonist of glycine_B site, decreased the [³H]GABA-releasing effect of NMDA and this reduction was suspended by addition of 1 mM glycine. Neither glycine nor kynurenic acid exerted effects on resting [³H]GABA outflow. These data suggest that glycine_B binding site at NMDA receptor may be saturated by glycine released from neighboring cells. Glycyldodecylamide (GDA) and N-dodecylsarcosine, inhibitors of glycineT1 transporter, inhibited the uptake of [³H]glycine (IC₅₀ 33 and 16 M) in synaptosomes prepared from rat hippocampus. When hippocampal slices were loaded with [³H]glycine, resting efflux was detected whereas electrical stimulation failed to evoke [³H]glycine overflow. Neither GDA (0.1 mM) nor N-dodecylsarcosine (0.3 mM) influenced [³H]glycine efflux. Using Krebs-bicarbonate buffer with reduced Na⁺ for superfusion of hippocampal slices produced an increased [³H]glycine outflow and electrical stimulation further enhanced this release. These experiments speak for glial and neuronal [³H]glycine release in hippocampus with a dominant role of the former one. GDA, however, did not influence resting or stimulated [³H]glycine efflux even when buffer with low Na⁺ concentration was applied.     

Imipramine-Induced Changes in 5-HT2 Receptor Sites and Inositoltrisphosphate Levels in Rat Brain

The effect of chronic administration of Imipramine on [³H]Spiperone binding to 5-HT₂ sites and inositoltrisphosphate (IP₃) levels in rat cerebral cortex was studied. Our data shows that treatment with imipramine (5 mg/kg body weight, intraperitoneally) for 30 days significantly down regulates 5-HT₂ receptors sites (262 ± 29 fmol/mg protein) in cerebral cortex (38%), compared to control rats (425 ± 60 fmol/mg protein., P < 0.001). However there was no significant change in the affinity of [³H]-Spiperone binding (kd) to 5-HT₂ sites in cerebral cortex after exposure to imipramine (Kd = 0.84 ± 0.11 nM). It is also observed that imipramine treatment significantly reduces 5-HT stimulated [³H]IP₃ formation in cerebral cortex (6,411 ± 708 dpm/mg protein), compared to the saline treated rats (12,238 ± 1,544 dpm/mg protein; P < 0.001), with concomitant decrease in Pdtlns-4–5-P₂. This study suggests that the therapeutic action of imipramine in brain might be by reducing hypersensitivity of 5-HT₂ receptors by down regulation, which leads to reduced levels of inositolphospholipids. This inturn reduces the levels of IP₃. In conclusion, imipramine acts at presynaptic site by blocking the reuptake of serotonin and at post synaptic site it downregulates 5-HT₂ sites with decreased IP₃ levels after chronic exposure.     

Cations Affect [3H]Mazindol and [3H]WIN 35,428 Binding to the Human Dopamine Transporter in a Similar Fashion

Abstract: The present study addresses the possibility that there are different cocaine-related and mazindol-related binding domains on the dopamine transporter (DAT) that show differential sensitivity to cations. The effects of Zn²⁺, Mg²⁺, Hg²⁺, Li⁺, K⁺, and Na⁺ were assessed on the binding of [³H]mazindol and [³H]WIN 35,428 to the human (h) DAT expressed in C6 glioma cells under identical conditions for intact cell and membrane assays. The latter were performed at both 0 and 21°C. Zn²⁺ (30–100 µ M ) stimulated binding of both radioligands to membranes, with a relatively smaller effect for [³H]mazindol; Mg²⁺ (0.1–100 µ M ) had no effect; Hg²⁺ at ∼3 µ M stimulated binding to membranes, with a relatively smaller effect for [³H]mazindol than [³H]WIN 35,428 at 0°C, and at 30–100 µ M inhibited both intact cell and membrane binding; Li⁺ and K⁺ substitution (30–100 m M ) inhibited binding to membranes more severely than to intact cells; and Na⁺ substitution was strongly stimulatory. With only a few exceptions, the patterns of ion effects were remarkably similar for both radioligands at both 0 and 21°C, suggesting the involvement of common binding domains on the hDAT impacted similarly by cations. Therefore, if there are different binding domains for WIN 35,428 and mazindol, these are not affected differentially by the cations studied in the present experiments, except for the stimulatory effect of Zn²⁺ at 0 and 21°C and Hg²⁺ at 0°C.     

Ion channels in the immune system as targets for immunosuppression

The discovery of a diverse and unique subset of ion channels in T lymphocytes has led to a rapidly growing body of knowledge about their functional roles in the immune system. Potent and specific blockers have provided molecules tools to probe channel structure—function relations and to elucidate the involvement of K⁺, Ca²⁺, and Cl⁻ channels in T-cell activation and cell volume regulation. Recent advances in analyzing Kv1.3 channel structure—function relationships have defined binding sites for channel blockers, which have now been shown to be effective in suppressing T-cell function in vivo. Ion channels may provide excellent pharmaceutical targets for modulating immune system function.     

Estradiol receptor binding to the epithelium of uterine lumen and glands: region- and time-related changes during preimplantation and periimplantation periods studied by autoradiography

The presence and changes of estradiol nuclear binding and related functions in uterine luminal and glandular epithelium were studied before and after blastocyst implantation using receptor autoradiography with ³H-estradiol-17 in association with ³H-thymidine incorporation and immunocytochemical binding of antibody to estrogen receptor ER-. ³H-estradiol nuclear binding is present but variable during days 1.5–7.5 of pregnancy. Sites of strong nuclear binding of ³H-estradiol exhibit strong immunocytochemical staining with ER- antibody. Qualitative and quantitative evaluation of autoradiograms reveal that there is a general increase of nuclear ³H-estradiol binding during the first 3 days after fertilization in both luminal and glandular epithelium. The binding of estradiol is stronger in glandular epithelium from day 2.5 to day 7.5, paralleled by a rise in ³H-thymidine incorporation on day 2.5. By comparison, in the epithelium of the uterine lumen ³H-estradiol nuclear binding is low, but relatively high in epithelial cells at lateral branching of the lumen where the increase in ³H-estradiol binding corresponds to an increased labeling index with ³H-thymidine. A highly differentiated binding of ³H-estradiol to luminal and glandular epithelium was demonstrated with region- and time-specific changes of related effects on cell proliferation, differentiation, and secretion, probably involving involution and remodeling. The strong ³H-estradiol binding to glandular epithelium suggests that estradiol exerts pronounced effects on glandular activities in the periimplantation period.