Inhibition of estradiol binding to its receptor by the cupric ion

 Treatment of human recombinant estrogen receptor (hER) expressed in yeast with very low concentrations of the cupric ion decreased its ability to bind [³H]estradiol ([³H]E₂) and [¹²⁵I]tamoxifen aziridine (minimal Cu²⁺ concentration: 1 nM). This decrease was reflected in a loss of immunoreactivity for monoclonal antibodies raised against the hormone binding domain (HBD). An ER recombinant expressing solely the HBD confirmed that the ion operated at this level. Cysteines located within this domain contributed to the inhibitory action of the Cu²⁺ in view of a partial restoration of the [³H]E₂ binding activity with β-mercaptoethanol. Histidines were also implicated since the influence of Cu²⁺ on the [³H]E₂ binding parameters (Scatchard plot analysis) was maintained after oxidation of thiol groups by methyl methanethiosulfonate, and partly reversed by imidazole. Received: 10 December 1997 / Accepted: 26 June 1998     

Binding of calcium and zinc to the acetylcholine receptor purified from Torpedo Californica

Binding of [⁶⁵Zn⁺⁺] and [⁴⁵Ca⁺⁺] to the acetylcholine (ACh)-receptor, purified from the $\text{Torpedo}$ electric organ, was studied by equilibrium dialysis. Whereas [⁶⁵Zn⁺⁺] bound to 56 nmoles of sites per mg protein with a dissociation constant of 2.5 × 10⁻⁶M, no binding of [⁴⁵Ca⁺⁺] at concentrations up to 10⁻³M could be detected with this method. However, the binding of [acetyl-³H]choline to the receptor was blocked equally by very high Zn⁺⁺ or Ca⁺⁺ concentrations, and the K_i for this low affinity binding was 7 × 10⁻³M. The high affinity binding of [⁶⁵Zn⁺⁺] to the receptor was blocked best by Cd⁺⁺ then Co⁺⁺ and Mn⁺⁺, but least by Mg⁺⁺ and Ca⁺⁺. When the purified ACh-receptor itself was analyzed for the presence of cations by atomic absorption, it was discovered that 4.7% of its weight was due to bound Ca⁺⁺ that could not be removed even by extensive dialysis. When Ca⁺⁺-free solutions (containing 1 mM EDTA) were used during purification, 0.6% of the molecular weight of the receptor was still due to bound Ca⁺⁺. This was equivalent to 15 moles of Ca⁺⁺ for each mole of ACh bound at saturation. It is suggested that the source of this Ca⁺⁺ is endogenous, and that it is tightly bound to the ACh-receptor molecule.     

Oxidative stress disruption of receptor-mediated calcium signaling mechanisms

Background

Oxidative stress increases the cytosolic content of calcium in the cytoplasm through a combination of effects on calcium pumps, exchangers, channels and binding proteins. In this study, oxidative stress was produced by exposure to tert-butyl hydroperoxide (tBHP); cell viability was assessed using a dye reduction assay; receptor binding was characterized using [³H]N-methylscopolamine ([³H]MS); and cytosolic and luminal endoplasmic reticulum (ER) calcium concentrations ([Ca²⁺]_i and [Ca²⁺]_L, respectively) were measured by fluorescent imaging.

Results

Activation of M3 muscarinic receptors induced a biphasic increase in [Ca²⁺]_i: an initial, inositol trisphosphate (IP3)-mediated release of Ca²⁺ from endoplasmic reticulum (ER) stores followed by a sustained phase of Ca²⁺ entry (i.e., store-operated calcium entry; SOCE). Under non-cytotoxic conditions, tBHP increased resting [Ca²⁺]_i; a 90 minute exposure to tBHP (0.5-10 mM ) increased [Ca²⁺]_i from 26 to up to 127 nM and decreased [Ca²⁺]_L by 55%. The initial response to 10 μM carbamylcholine was depressed by tBHP in the absence, but not the presence, of extracellular calcium. SOCE, however, was depressed in both the presence and absence of extracellular calcium. Acute exposure to tBHP did not block calcium influx through open SOCE channels. Activation of SOCE following thapsigargin-induced depletion of ER calcium was depressed by tBHP exposure. In calcium-free media, tBHP depressed both SOCE and the extent of thapsigargin-induced release of Ca²⁺ from the ER. M3 receptor binding parameters (ligand affinity, guanine nucleotide sensitivity, allosteric modulation) were not affected by exposure to tBHP.

Conclusions

Oxidative stress induced by tBHP affected several aspects of M3 receptor signaling pathway in CHO cells, including resting [Ca²⁺]_i, [Ca²⁺]_L, IP3 receptor mediated release of calcium from the ER, and calcium entry through the SOCE. tBHP had little effect on M3 receptor binding or G protein coupling. Thus, oxidative stress affects multiple aspects of calcium homeostasis and calcium dependent signaling.     

3H-mepyramine binding to histamine H1-receptors in guinea pig lung: Characteristics and regulation by ions

The antogonist [³H]-mepyramine is used to label histamine H₁-receptors in guinea pig lung. Scatchard analysis reveals two classes of binding sites. Monovalent cations decrease steady-state binding (Na⁺ > Li⁺ > K⁺), while divalent cations (Mg⁺⁺, Ca⁺⁺, Mn⁺⁺, Ba⁺⁺) exhibit a biphasic curve, increasing binding at low concentrations and decreasing it at higher levels. Na⁺ decreases both affinity and number of binding sites. Dissociation curve shows two components, and Na⁺ accelerates the rate of dissociation of the slower component. GTP does not affect the binding of the antagonist ³H-Mepyramine.     

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin, 12-O-tetradecanoylphorbol-13-acetate and 17β-estradiol on estrogen receptor regulation in MCF-7 human breast cancer cells

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) exhibits remarkably potent antiestrogenic activity. To further elucidate the role of estrogen receptor (ER) regulation in this response, we examined the effects of exposure to TCDD in MCF-7 human breast cancer cells on ER mRNA levels by using an RNase protection assay, on ER accumulation by using an ER immunocytochemical essay (ER-ICA), and on ER function by competitive binding assays under conditions of saturating 17β-estradiol (E₂). Comparative studies were conducted with E₂ and 12-O-tetradecanoylphorbol-13-acetate (TPA), as both compounds are known to suppress ER expression. Our results indicate that 1 nM E₂ and 100 nM TPA both suppress ER mRNA levels as early as 4 h after exposure and to 33.6% and 16.5% of control levels, respectively, after 72 h. In contrast, no significant effect on ER mRNA levels was attributed to exposure to 10 nM TCDD. A greater than 50% reduction in positive staining was observed by ER-ICA after 72 h exposure to 1 nM E₂ and to 100 nM TPA, while only an 11% reduction in positive staining was observed with 10 nM TCDD. Specific binding of [³H]E₂ under saturating conditions (10 nM E₂) in whole cells was reduced by 50% in cultures exposed to 100 nM TPA, although no effect on binding was observed with exposure to 10 nM TCDD. In contrast, specific binding using subsaturating 1 nM [³H]E₂ was depressed by 49% in MCF-7 cells exposed to 10 nM TCDD for 72 h. This depression was inhibited by a 1-h treatment with 5 μM α-naphthoflavone, which inhibits TCDD-induced, P450-mediated, E₂ metabolism, and subsequent E₂ depletion. In conclusion, while TPA and E₂ effectively down-regulate ER expression, TCDD, under antiestrogenic conditions, has little if any effect on total ER levels in MCF-7 cells, and thus ER modulation is probably not necessary for the suppression of estrogenic activity in MCF-7 cells by TCDD. © 1996 Wiley-Liss, Inc.     

Gonadotropin and prostaglandins binding sites in nuclei of bovine corpora lutea

Highly purified nuclei isolated from bovine corpora lutea showed marked enrichment of NAD pyrophosphorylase, a marker for this organelle. Rough endoplasmic reticulum and lysosomal markers were undetectable, whereas plasma membrane and Golgi markers were detectable but not enriched in nuclei. These highly purified nuclei exhibited specific binding with ¹²⁵I-labeled human choriogonadotropin, [³H]prostaglandin E₁ and [³H]prostaglandin F_2α. However, these bindings were only 15.4% (human choriogonadotropin), 7.9% (prostaglandin E₁) and 8.9% (prostaglandin F_2α) of the plasma membrane binding observed under the same conditions. Washing of nuclei and plasma membranes twice with buffer containing 0.1% Triton X-100 resulted in gonadotropin and prostaglandin F_2α binding site and 5′-nucleotidase (EC 3.1.3.5) losses from nuclei that were different from those observed for plasma membranes. More importantly, the washed nuclei exhibited 44% (human choriogonadotropin), 21–26% (prostaglandins) of original specific binding despite virtual disappearance of 5′-nucleotidase activity. The nuclear membranes isolated from nuclei, specifically bound ¹²⁵I-labeled human choriogonadotropin and [³H]prostaglandin F_2α to the same extent or significantly more ([³H]prostaglandin E₁, P < 0.05) than nuclei themselves, despite the marked losses of chromatin. In summary, our data suggest that gonadotropin and prostaglandins bind to nuclei and that this binding was intrinsic and was primarily associated with the nuclear membrane.     

Beta-adrenergic receptor characteristics of postnatal rat myocardial cell preparations

Summary Primary mycolardial cell cultures and freshly isolated cardiac cells in suspension resprensent two isolated, whole cell models for investigating cellular transsarcolemmal⁴⁵Ca⁺⁺ exchange in response to a receptor-coupled stimulus. Studies were performed to characterize beta-adrenergic receptor binding, beta-adrenergic receptor mediated cellular calcium (⁴⁵Ca⁺⁺) exchange, and viability in purified primary myocardial cell cultures and freshly isolated cardiac cells in suspension obtained from 3-to 3-d-old Sprague-Dawley rats. In addition, beta-adrenergic receptor binding was characterized in whole-heart crude membrane preparations. All three preparations had saturable beta-adrenergic binding sites with the antagonist [¹²⁵I]iodopindolol ([¹²⁵I]IPIN). The suspensions had a significantly lower B _max (42±6 fmol/mg protein) than the membranes and cultures (77±8 and 95±10 fmol/mg protein, respectively). The K _D of the cultures (218±2.0 pM) was significantly higher than that for the suspensions (107 ±1.3 pM) and membranes (93±1.3 pM). Viability was significantly lower in the suspensions (57%) when compared to 94% viability in myocardial cell cultures after 3 h of incubation in Kreb's Henseleit buffer. Incubation of the cultures with 5.0×10⁻⁷ M isoproterenol resulted in a significant increase in⁴⁵Ca⁺⁺ exchange as early as 15 s. In contrast,⁴⁵Ca⁺⁺ exchange into the suspensions was not increased. Although both primary cell cultures and cardiac cells in suspension possess saturable beta-adrenergic receptors, only the monolayer cultures exhibited functional beta-adrenergic receptor-mediated⁴⁵Ca⁺⁺ exchange. Of the two intact cell models investigated, these data suggest that primary myocardial cell cultures are more suitable than cell suspensions for investigating beta-adrenergic receptor binding and functions in the postnatal rat heart. This research was supported by The University of Texas Research Institute, a grant from the Texas Advanced Research Technology Program awarded to S. W. Leslie and R. E. Wilcox, and contract 223-86-2109 from the Food and Drug Administration.     

R 24571: a new powerful inhibitor of red blood cell Ca++-transport ATPase and of calmodulin-regulated functions

Compound R 24571 (1-[bis(p-chlorophenyl)methyl]-3-[2,4-dichloro-β-(2,4-dichlorobenzyloxy)phenethyl]imidazoliniumchloride) is found to be a powerful inhibitor of red blood cell Ca⁺⁺-ATPase as well as Ca⁺⁺ transport into inside-out red blood cell vesicles with an IC₅₀-value of 0.5 and 2 μM, respectively. The inhibitory action of R 24571 is more specific on the calmodulin-dependent fraction of Ca⁺⁺-transport ATPase as compared to the basal Ca⁺⁺-transport ATPase (determined in the absence of calmodulin) and can be antagonized by increasing concentrations of calmodulin in an apparently competitive manner. With respect to other ATPases the action of R 24571 is relatively specific for red blood cell Ca⁺⁺-transport ATPase. Mg⁺⁺-ATPase requires a 40 times higher concentration for halfmaximal inhibition (IC₅₀ = 20 μM) whereas (Na⁺ + K⁺)-transport ATPase is only slightly affected in the investigated concentration range (≤20 μM).     

Receptors for gonadotropins and prostaglandins in lysosomes of bovine corpora lutea.

The total mitochondrial fraction of bovine corpus luteum specifically bound [³H]prostaglandin (PG) E₁, [³H] PGF_2α, and ¹²⁵I-labeled human lutropin (hLH) despite very little 5′-nucleotidase activity, a marker for plasma membranes. Since the total mitochondrial fraction isolated by conventional centrifugation techniques contains both mitochondria and lysosomes, it was subfractionated into mitochondria and lysosomes to ascertain the relative contribution of these fractions to the binding. Subfractionation resulted in an enrichment of cytochrome c oxidase (a marker for mitochondria) in mitochondria and of acid phosphatase (a marker for lysosomes) in lysosomes. The lysosomes exhibited little or no contamination with Golgi vesicles, rough endoplasmic reticulum, or peroxisomes as assessed by their appropriate marker enzymes. Subfractionation also re ulted in [³H] PGE₁, [³H] PGF_2α, and ¹²⁵I-labeled hLH binding enrichment with respect to homogenate in lysosomes but not in mitochondria. The lysosomal binding enrichment and recovery were, however, lower than in plasma membranes. The ratios of marker enzyme to binding, an index of organelle contamination, revealed that plasma membrane and lysosomal receptors were intrinsic to these organelles. Freezing and thawing had markedly increased lysosomal binding but had no effect on plasma membrane binding. Exposure to 0.05% Triton X-100 resulted in a greater loss of plasma membrane compared to lysosomal binding. In summary, the above results suggest that lysosomes, but not mitochondria, in addition to plasma membranes, intrinsically contain receptors for PGs and gonadotropins. Furthermore, lysosomes overall contain a greater number of PGs and gonadotropin receptors compared to plasma membranes and these receptors are associated with the membrane but not the contents of lysosomes.     

Differential effects of detergents and dimethylsulfoxide on membrane prostaglandin E1 and F2alpha receptors.

Pretreatment of membranes for 1 hr at 4° with up to 0.1% Triton X-100 (TX-100) and sodium desoxycholate (SDC), resulted in a greater loss of [³H] prostaglandin (PG)F₂α binding compared to E₁ binding. Lubrol WX (LWX) tended to cause a greater loss of [³H]PGF₂α than E₁ binding. However, the differential loss was not as marked as with TX-100 or SDC. Triton X-305 was relatively ineffective, but loss of [³H]PGE₁ binding was greater than for PGF₂α. Increasing concentrations of dimethylsulfoxide (DMSO) progressively inhibited PGF₂α binding without affecting PGE₁ binding. The detergent, but not DMSO, induced losses of membrane PG binding were due to solubilization of the receptors. Greater amounts of membrane protein and phospholipids were solubilized at detergent (TX-100 and SDC) concentrations that solubilized 100% of PGE₁ receptors compared to 100% solubilization of F₂α receptors. Neither the duration of preincubation nor the amount of membrane protein chosen were responsible for differential PGE₁ and F₂α receptor losses. These differential membrane PG receptor losses raise the possibility of differences in PGE₁ and F₂α receptors association with the membrane structure.     

High-affinity binding sites for prostaglandin E on human lymphocytes.

Binding sites on human lymphocytes for prostaglandins were examined by incubating cells with [³H]prostaglandin (PG) A₁, E₁, E₂, F_1α, and F_2α. Specific reversible binding for [³H]PGE₁ and E₂ was found with a K_d of ~2 × 10^?9M and a B max of ~200 binding sites per cell, assuming uniform distribution. We detected no specific binding of [³H]PGA₁, F_1α, or F_2α to lymphocytes. Also, the addition of 10- to 1000-fold greater amounts of unlabeled PGA, F_1α, or F_2α did not inhibit the binding of [³H]PGE. The time course of [³H]PGE binding appeared to be bimodal with one component complete within 5 min at 37 °C and another component of binding increasing over a 40-min incubation. We feel that the rapid component of binding may represent cell surface receptors for PGE while the slower component may represent a specific uptake mechanism for PGE into the cell. Glass adherent cells had fewer binding sites than nonadherent cells. Preincubation of the cells overnight resulted in a loss of binding sites.     

Binding of [3H]phencyclidine to membranes from housefly thoracic muscles

The binding of [³H]phencyclidine ([³H]PCP) to a preparation of housefly thoracic muscle membranes was studied. Specific [³H]PCP binding saturated with both time and at concentrations of the radiolabeled ligand greater than 20 nM. One binding site with a K_D of 10.7 nM and a B_max of 3.9 pmol/mg protein was observed. Specific [³H]PCP binding was also readily reversible with a half-time of dissociation (t_1/2) of 13.8 min and varied proportionately with tissue concentration. Of the drugs tested, specific [³H]PCP binding was inhibited by PCP analogs, antipsychotics, antidepressants, Ca²⁺ channel antagonists, and K⁺ channel blockers. [³H]PCP binding was unaffected by addition of carbamylcholine or L-glutamate in absence or presence of ATP, GTP, cAMP, or cGMP. Though the identity of the [³H]PCP binding protein in housefly muscle membranes is still unclear, it is more likely to be an ionic channel such as K⁺ or Ca²⁺ channels than a neurotransmitter receptor.     

Calcium alters the properties of [3H]diazepam binding sites in rat brain membranes

• 1.1. [³H]diazepam ([³H]DZ) was used as a ligand to study the effects of Ca²⁺ on benzodiazepine binding to rat brain membranes.
• 2.2. [³H]DZ bound at a single class of binding sites showing K_D and B_max values of 5.4 nM and 852 fmol/mg protein respectively. These values are consistent with previous reports.
• 3.3. Amongst the various divalent cations tested Mg²⁺, Fe²⁺, Cd²⁺, and Sr²⁺ had no significant effect on [³H]DZ binding. Mn²⁺, Ba²⁺, Co²⁺, Ni²⁺ and La²⁺ enhanced radioligand binding, whereas Ca²⁺, Zn²⁺ and Pb²⁺ inhibited [³H]DZ binding to brain freeze-thawed membranes.
• 4.4. The inhibition of [³H]DZ binding by Ca²⁺ was concentration-dependent. 50% inhibition occurred at a Ca²⁺ concentration of 5.6mM. The Hill coefficient for the inhibition was 1.03, displaying noncoperativity. The effect of Ca²⁺ on [³H]DZ binding could be prevented by La³⁺ but was not reversed by EGTA.
• 5.5. A kinetic analysis of Ca²⁺ inhibition of [³H]DZ binding indicates that Ca²⁺ inhibited competitively. Analysis of binding isotherms indicates that both K_D and B_max were altered at the [³H]DZ binding sites. The marked increase in K_D value in the presence of Ca²⁺ (5 mM) can be explained by a drastic increase in the dissociation rate constant.
• 6.6. It was suggested that Ca²⁺ may induce a conformational change in the diazepam binding sites on rat brain membranes. The unchanged Hill coefficient in the presence or absence of Ca²⁺ indicates that a single population of binding sites was labeled by [³H]DZ.
• 7.7. The calmodulin antagonists, trifluoperazine and W-7 were weak inhibitors of [³h]dz binding.

     

The Elusive Nature of Cerebellar Somatostatin Receptors: Studies in Rat,Monkey and Human Cerebellum

Abstract

The pharmacological profile and localization of somatostatin (SRIF) receptors were determined in rat, monkey and human cerebellum. In rat cerebellar cortex, low ss₁/sst₄, intermediate sst₂ and very high sst₃ receptor mRNA levels were found, sst₁ mRNA was also expressed in the deep cerebellar nuclei. [¹²⁵I]Tyr³-octreotide binding sites in cerebellar membranes correlated with recombinant sst₂, but not with sst₅ or sst₃ receptors and were found in the molecular layer of the cerebellum. [¹²⁵I]CGP 23996 (in Na⁺-buffer) binding in rat cerebellum correlated with sst₁ or sst₄, but not with sst₂, sst₃ or sst₅ receptor binding. Similar data were obtained in rhesus monkey cerebellum. mRNAs for all five receptors were found in the granule cell layer of the human cerebellum and/or in the dentate nucleus. [¹²⁵I]Tyr³-octreotide binding was strong in the molecular layer and correlated with that of recombinant sst₂ receptors, but not with sst₃ or sst₅ receptors. [¹²⁵I]CGP 23996 (in Mg⁺⁺-buffer) binding was heterogeneous (about 75%. to sst₂ and 25% to sst₁ and/or sst₄ receptors). The molecular and granular layers were equally and the dentate nucleus strongly labeled. Thus. SRIF receptors of the sst₂, sst₁ and/or sst₄ subtype are present in the rat, monkey and human cerebellum. In the latter two species, the sst₂ type appears to be predominant. Surprisingly, the high expression of sst₃ receptor mRNA is not supported by radioligand binding data in any of the species studied. The reason for this discrepancy remains to be elucidated.     

Effects of prostaglandin E1 on the metabolism in rat parathyroid gland in vitro

We investigated some effects of prostaglandin E₁ on the metabolism of rat parathyroid glands using a culture system containing basal Eagle's medium supplemented with 5–10% heat-inactivated rat serum. Rat parathyroid glands incorporate [³H]fucose and ¹⁴C-labeled amino acids into cellular glycoproteins and secrete some of these into the culture medium. Gel filtration chromatography separates these glycoproteins into three classes, the smallest of which (peak 3) is secreted with immunoreactive parathyroid hormone. In cultures of 48 h, prostaglandin E₁ (1 μg/ml) specifically inhibits the secretion of peak 3 and of parathyroid hormone but has no effect on the incorporation of [³H]-fucose, ¹⁴C-labeled amino acids, or [³H]uridine into parathyroid glands. Cytochalasin B inhibits the secretion of parathyroid hormone and the incroporation of isotopic fucose and amino acids. Cortisol stimulates incorporation of [³H]fucose and the secretion of parathyroid hormone even in the presence of inhibitory doses of prostaglandin E₁. It is concluded that, in organ culture, prostaglandin E₁ inhibits the secretion of parathyroid hormone and of a specific glycoprotein the function of which may be related to the secretion of the hormone.     

The binding of thyrotropin to liposomes containing gangliosides.

The relationship between uptake of Ca⁺⁺ and incorporation of sn-[¹⁴C]-glycerol-3-phosphate into phosphatidate, diglyceride, and triglyceride was evaluated in microsomes isolated from livers of normal fed male rats. Uptake of Ca⁺⁺ was dependent on concentration of Ca⁺⁺ (0.1 – 2.5 mM), and accompanied by a decrease in the rate of glycerolipid synthesis. The quantity of Ca⁺⁺ ion taken up at 20 μM CaCl₂ in the presence of ATP was equivalent to that observed with 2.5 mM CaCl₂ in the absence of ATP. The ATP dependent uptake of Ca⁺⁺, like the passive uptake at higher concentrations of Ca⁺⁺, was correlated with inhibition of incorporation of sn-glycerol-3-phosphate into phosphatidate. Accumulation of Ca⁺⁺ in hepatic microsomes, therefore, appears to result in a calcium-dependent decrease in biosynthesis of phosphatidate and other glycerolipids.     

Ribosome-free Terminals of Rough ER Allow Formation of STIM1 Puncta and Segregation of STIM1 from IP3 Receptors

Store-operated Ca²⁺ entry is a ubiquitous mechanism that prevents the depletion of endoplasmic reticulum (ER) calcium [1]. A reduction of ER calcium triggers translocation of STIM proteins, which serve as calcium sensors in the ER, to subplasmalemmal puncta where they interact with and activate Orai channels ([2], [3], [4], [5], [6], [7] and [8]; reviewed in [9]). In pancreatic acinar cells, inositol 1,4,5-trisphosphate (IP₃) receptors populate the apical part of the ER. Here, however, we observe that STIM1 translocates exclusively to the lateral and basal regions following ER Ca²⁺ loss. This finding is paradoxical because the basal and lateral regions of the acinar cells contain rough ER (RER); the size of the ribosomes that decorate RER is larger than the distance that can be spanned by a STIM-Orai complex [5] and [10], and STIM1 function should therefore not be possible. We resolve this paradox and characterize ribosome-free terminals of the RER that form junctions between the reticulum and the plasma membrane in the basal and lateral regions of the acinar cells. Our findings indicate that different ER compartments specialize in different calcium-handling functions (Ca²⁺ release and Ca²⁺ reloading) and that any potential interference between Ca²⁺ release and Ca²⁺ influx is minimized by the spatial separation of the two processes.     

Differential effects of detergents and dimethylsulfoxide on membrane prostaglandin E1 and F2α receptors

Pretreatment of membranes for 1 hr at 4° with up to 0.1% Triton X-100 (TX-100) and sodium desoxycholate (SDC), resulted in a greater loss of [³H] prostaglandin (PG)F₂α binding compared to E₁ binding. Lubrol WX (LWX) tended to cause a greater loss of [³H]PGF₂α than E₁ binding. However, the differential loss was not as marked as with TX-100 or SDC. Triton X-305 was relatively ineffective, but loss of [³H]PGE₁ binding was greater than for PGF₂α. Increasing concentrations of dimethylsulfoxide (DMSO) progressively inhibited PGF₂α binding without affecting PGE₁ binding. The detergent, but not DMSO, induced losses of membrane PG binding were due to solubilization of the receptors. Greater amounts of membrane protein and phospholipids were solubilized at detergent (TX-100 and SDC) concentrations that solubilized 100% of PGE₁ receptors compared to 100% solubilization of F₂α receptors. Neither the duration of preincubation nor the amount of membrane protein chosen were responsible for differential PGE₁ and F₂α receptor losses. These differential membrane PG receptor losses raise the possibility of differences in PGE₁ and F₂α receptors association with the membrane structure.     

Rapid action of estradiol in primate GnRH neurons: the role of estrogen receptor alpha and estrogen receptor beta

Estrogens play a pivotal role in the control of female reproductive function. Recent studies using primate GnRH neurons derived from embryonic nasal placode indicate that 17β-estradiol (E₂) causes a rapid stimulatory action. E₂ (1 nM) stimulates firing activity and intracellular calcium ([Ca²⁺]_i) oscillations of primate GnRH neurons within a few min. E₂ also stimulates GnRH release within 10 min. However, the classical estrogen receptors, ERα and ERβ, do not appear to play a role in E₂-induced [Ca²⁺]_i oscillations or GnRH release, as the estrogen receptor antagonist, ICI 182,780, failed to block these responses. Rather, this rapid E₂ action is, at least in part, mediated by a G-protein coupled receptor GPR30. In the present study we further investigate the role of ERα and ERβ in the rapid action of E₂ by knocking down cellular ERα and ERβ by transfection of GnRH neurons with specific siRNA for rhesus monkey ERα and ERβ. Results indicate that cellular knockdown of ERα and ERβ failed to block the E₂-induced changes in [Ca²⁺]_i oscillations. It is concluded that neither ERα nor ERβ is required for the rapid action of E₂ in primate GnRH neurons.     

Calcium transport and cellular distribution in quiescent and serum-stimulated primary cultures of bone cells and skin fibroblasts

Primary cultures of bone cells and skin fibroblasts were examined for their Ca⁺⁺ content, intracellular distribution and Ca⁺⁺ fluxes. Kinetic analysis of ⁴⁵Ca⁺⁺ efflux curves indicated the presence of three exchangeable Ca⁺⁺ compartments which turned over at different rates: a “very fast turnover” (S₁), a “fast turnover” (S₂), and a “slow turnover” Ca⁺⁺ pool (S₃). S₁ was taken to represent extracellular membrane-bound Ca⁺⁺, S₂ represented cytosolic Ca⁺⁺, and S₃ was taken to represent Ca⁺⁺ sequestered in some intracellular organelles, probably the mitochondria. Bone cells contained about twice the amount of Ca⁺⁺ as compared with cultured fibroblasts. Most of this extra Ca⁺⁺ was localized in the “slow turnover” intracellular Ca⁺⁺ pool (S₃). Serum activation caused the following changes in the amount, distribution, and fluxes of Ca⁺⁺: (1) In both types of cells serum caused an increase in the amount of Ca⁺⁺ in the “very fast turnover” Ca⁺⁺ pool, and an increase in the rate constant of ⁴⁵Ca⁺⁺ efflux from this pool, indicating a decrease in the strength of Ca⁺⁺ binding to ligands on cell membranes. (2) In fibroblasts, serum activation also caused a marked decrease in the content of Ca⁺⁺ in the “slow turnover” Ca⁺⁺ pool (S₃), an increase in the rates of Ca⁺⁺ efflux from the cells to the medium, and from S₃ to S₂, as well as a decrease in the rate of influx into S₃. (3) In bone cells the amount of Ca⁺⁺ in S₃ remained high in “serum activated” cells, the rate of efflux from S₃ to S₂ increased, and the rate of influx into S₃ also increased. The rate of efflux from the cells to the medium did not change. The results suggest specific properties of bone cells with regard to cell Ca⁺⁺ presumably connected with their differentiation. Following serum activation we investigated the time course of changes in the amount of exchangeable Ca⁺⁺ in bone cells and fibroblasts, in parallel with measurements of ³H-thymidine incorporation and cell numbers. Serum activation caused a rapid decrease in the content of cell Ca⁺⁺ which was followed by a biphasic increase lasting until cell division.