Prostaglandin E1 specific binding in rhesus myometrium

Myometrial low speed supernatant prepared from non-pregnant rhesus uteri was incubated with ³H-Prostaglandin (PG)E₁ with or without addition of unlabelled prostaglandins. The uptake of ³H-PGE₁ was inhibited in a dose dependent fashion by PGE₂>PGE₁>PGA₁>PGF_2α=PGA₁>PGB₁=PGB₂≥PGD₂. PGE₁ metabolites inhibited ³H-PGE₁ binding in the following order: 13,14-dihydro-PGE₁>13,14-dihydro-15-keto-PGE₁=15-keto-PGE₁. The specific binding of ³H-PGE₁ and ³H-PGF_2α was similarly affected by the temperature and time of incubation. Equilibrium binding constants determined using rhesus uteri obtained during the luteal phase of the menstrual cycle indicate the presence of high affinity PGE₁ binding sites with an average (n=3) apparent dissociation constant of 2.2 × 10⁻⁹M and a lower affinity PGE₁ binding site with a K_d ≅ 1 × 10⁻⁸M. No high affinity — low capacity ³H-PGF_2α sites could be demonstrated.Relative uterine stimulating potencies of some natural prostaglandins and prostaglandin analogs tested after acute intravenous administration in mid-pregnant rhesus monkeys corresponded with the PGE₁ binding inhibition of the respective compound. The uterine stimulating potencies of the prostaglandin analogs tested were: (15S)-15-methyl-PGE₂=16,16-dimethyl-PGE₂>17-phenyl-18,19,20-trinor-PGE₂>16 phenoxy-17,18,19,20-tetranor-PGF_2α=PGE₂=PGE₁=(15S)-15-methyl-PGF_2α>PGF_2α.     

Comparisons of affinity constants of PGEs-receptor interaction with concentrations of PGEs needed for half maximal stimulation of adenylate cyclase

The specific binding of ³H-prostaglandin E₁ (³H-PGE₁) to bovine corpus luteum cell membranes was not affected by cholesterol or various progestins at concentrations of up to 9.0 × 10⁻⁶M. At concentrations above 2.5 × 10⁻⁶M; estrone, 17β-estradiol (but not 17α-estradiol or 17β-estradiol glucuronide), estriol, equilin, D-equilenin, 17-ethynyl estradiol, diethylstilbestrol, cortisol, corticosterone, deoxycorticosterone and aldosterone inhibited specific binding of ³H-PGE₁. On the other hand, testosterone and dihydrotestosterone (DHT) (but not androstenedione) significantly enhanced ³H-PGE₁ binding. These findings permitted the following correlations between steroid structure and modulation of ³H-PGE₁ binding: steroids with a free phenolic ring and a 17β-hydroxyl or 17-keto group or C-21 steroids with a C-20 ketone and a C-21 hydroxy group decrease, whereas C-19 steroids with a C-17 hydroxy group enhance specific binding of ³H-PGE₁. PGE receptors are heterogeneous with respect to affinity for ³H-PGE₁. The steroids that decreased ³H-PGE₁ binding caused a lowering to a complete loss of low affinity PGE receptors. Steroids that increased ³H-PGE₁ binding caused appearance of new low affinity PGE receptors. Association rate constants for ³H-PGE₁ binding were decreased by 17β-estradiol (61%) and increased by DHT (59%).     

Receptors for prostaglandins and gonadotropins in the cell membranes of bovine corpus luteum

The cell membranes exhibited specific binding to ³H-prostaglandin E₁ (³H-PGE₁) and ¹²⁵I-human chorionic gonadotropin (¹²⁵I-HCG). Unlabeled PGE₁,PGE₂ (1.4 × 10^?7M), PGF_1α and PGF_2α (1.4 × 10^?5M) decreased ³H-PGE₁ binding by more than 80%. The binding of ¹²⁵I-HCG was completely inhibited by 5 × 10^?8M unlabeled HCG. However, the unlabeled PGE₁ (1.15 × 10^?6M) and HCG (8.4 × 10^?7M) had no effect on ¹²⁵I-HCG and ³H-PGE₁ binding respectively. A PG antagonist, 7-oxa-13-prostynoic acid, inhibited only ³H-PGE₁ binding but not ¹²⁵I-HCG binding. These results suggest the presence of specific receptors for PGE₁ and HCG in the cell membranes and that the binding occurs either at two different sites on the same receptor or that each binds to a “different” receptor molecule.     

The presence of discrete receptors for prostaglandin F2 alpha in the cell membranes of bovine corpora lutea.

The cell membranes isolated from bovine corpora lutea bound ³H-prostaglandin (PG) F₂α with high affinity and specificity. The specific binding of ³H-PGF₂α was detectable at 10^?10M added ³H-PGF₂α and reached saturation at 10^?7M to 10^?6M. Unlabeled PGF₂α, as low as 10^?9M, inhibited the binding of ³H-PGF₂α with complete inhibition occurring at 10^?6M. The Scatchard analysis of equilibrium binding data revealed that the PGF₂α receptors are heterogeneous: Kd₁?5.1 × 10^?9M, n?289 fmoles/mg protein; Kd₂?1.8 × 10^?8M, n?780 fmoles/mg protein. The relative affinities of various other PGs for binding to PGF₂α receptors were (PGF₂α?100%): PGF₁α?17.5; PGE₁?0.8; PGE₂?22.4; PGA₁?0.007; PGB₁?0.01. The specificity and affinity of ³H-PGF₂α binding is consistent with the possibility that this receptor interaction may reflect an initial event in the action of PGF₂α as a luteolytic agent.     

Histamine alters prostaglandin output from diestrous rat uteri. Involvement of H2-receptors and 9-keto-reductase

The effects of exogenous histamine (H) on prostaglandin (PG) generation and release in uteri isolated from diestrous rats and the influences of H₂-receptors blockers (cimetidine and mitiamide) on the output of uterine PGs, were explored. Moreover, the action of H on the uterine 9-keto-reductase, was also studied. Histamine (10⁻⁴M) failed to alter the basal output of PGE₁ but reduced significantly the generation and release of PGE₂ and augmented the output of PGF_2α. On the other hand, cimetidine (10⁻⁵M) enhanced the basal release of PGE₂ but had no action on the outputs of PGs E₁ or F_2α. The enhancing effect of H on the production and release of PGF_2α was abolished in the presence of cimetidine. Also, the antagonist reversed the influence of H on the output of PGE₂. Metiamide, another H₂-receptor antagonist, did not alter the basal control generation and release of uterine PGs, but antagonized the augmenting influence of H on PGF_2α uterine output, as much as cimetidine did, and prevented the depressive action of H on the release of PGE₂ from uteri. Histamine (10⁻⁴M) significantly stimulated uterine formation of cyclic-adenosine monophosphate, an action which was antagonized by the presence of cimetidine (10⁻⁵M), a blocker of H₂ receptors. Also, histamine (10⁻⁵M) and dibutyril-cyclic-adenosine monophosphate (DB-cAMP) at 10⁻³M, enhanced significantly the formation ³H-PGF_2α from ³H-PGE₂. Results presented herein demonstrate that H is able to diminish the generation of PGE₂ in uteri from rats at diestrus augmenting the synthesis of PGF_2α, apparently via the activation of H₂-receptors, enhancing adenylate-cyclase. These effects appear to increase uterine 9-keto-reductase activity which transforms PGE₂ into PGF_2α. Relationships between the foregoing results and those evoked by estradiol, are also discussed.     

Modulation of human myometrial PGE2 receptor by GTP characterization of receptor subtype

We studied PGE₂ specific binding sites in human myometrial microsomes prepared from uterine specimens obtained by hysterectomy (women between 38 and 55 years of age). Competition experiments showed that the potency order for various prostaglandins (PGs) was : PGE₂ ≥ PGE₁ PGF_2α > Iloprost ≥ Carbacyclin ZK 110841 (PGD₂ analogue). These relative affinities indicated that the receptor was of the EP type.In kinetic experiments GTP, GppNHp and GTPγS increased the rate of PGE₂ binding (steady state was reached more rapidly in the presence of nucleotides) but maximal specific binding was not significantly different. Complete dissociation could not be obtained, even in the presence of GTP. Only 50% of maximal binding was readily dissociable. The dissociation rate was 4.56.10⁻⁴ sec⁻¹ (half time of about 660 sec) and in the presence of GTP analogues it was slightly increased (k−1 = 7.16 10⁻⁴ sec⁻¹ half time 420 sec.). Scatchard analysis of saturation curves showed an increase in ligand receptor affinity in the presence of GTP or nucleotide analogues: the Kd shifted from 9.66 ± 2.8.10⁻⁹ M to 4.96 ± 1.25.10⁻⁹M, but the number of binding sites did not change significantly (310 ± 37 to 350 ± 17 fmol/mgP). The effect of GTP was observed at a concentration of 5.10⁻⁴M. GppNHp and GTPγS were effective at 1.10⁻⁵M. Pretreatment of myometrial membranes with pertussis or cholera toxins had no effect on PGE₂ binding to membrane sites. Our conclusion is that GTP induced conversion of a population of low affinity sites into a population of higher affinity sites. This effect of guanine nucleotides was described in adipocytes and kidney medulla.Competition studies with PGE₂ analogues (sulprostone, 17-phenyl-ω-trinor PGE₂, M&B 28,767, misoprostol, butaprost) showed that this receptor mediates a contractile response and is probably an EP₃ subtype.     

Prostaglandin specific binding in hamster myometrial low speed supernatant

A charcoal adsorption method was developed to measure specific prostaglandin binding in low speed supernates of hamster myometrial homogenates. This method was used to characterize and quantitate PGE₁-specific binding. The equilibrium binding constants and the concentration of specific PGE₁ binding sites were determined during the hamster estrous cycle. The apparent association constant for 12 different preparations was 1.16 ± 0.08 × 10⁹M⁻¹. The concentration of PGE₁ specific binding sites was significantly higher on Days 2 and 3 of the estrous cycle than it was on Days 1 or 4. The competition for PGE₁ binding sites by PGE₂, PGF_2α, PGA₁ and various PGE₁ metabolites and derivatives was measured in hamster myometrial homogenates. Relative affinities of the natural prostaglandins for the PGE₁ binding sites, calculated by parallel line assay, were: PGE₂>PGE₁>PGA₁>PGF_2α. For PGE₁ metabolites the relative affinities were: PGE₁>13,14-dihydro-PGE₁>13,14-dihydro-15-keto-PGE₁>15-keto-PGE₁. For the analogs and derivatives the compounds tested ranked as: 16,16-dimethyl-PGE₁≥PGE₁>PGE₁ methyl ester>17-phenyl-18,19,20-trinor-PGE₁>15(S)15-methyl-PGE₁ methyl ester. Arachidonic acid, bis-homo-γ-linolenic acid and 7-oxa-13 prostynoic acid had relative affinities ≥0.1 compared to PGE₁=100. Indomethacin had a relative affinity of 0.4 compared to PGE₁.     

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.     

Prostaglandin E2: Midterm abortion in rats using Silastic-PVP tubes

The results of the present study establish that 1.5 mg PGE₂ (lyophilized sodium salt) incorporated in one cm long open-ended Silastic-polyvinylpyrrolidone (PVP) tube when inserted into 10 day pregnant rats induced abortion within 70–72 hours in all the treated rats. A combined treatment of PGE₂ and 17β-estradiol failed to increase the abortion inducing effect of a Silastic-PVP-PGE₂ tube. It is observed that PGE₂ is about 4 times less potent than PGF_2α in inducing midterm abortion in rats. It is suggested that either PGE₂ exerts luteolytic effect after being converted to PGF_2α, although how it occurs is not clear; or PGE₂ causes expulsion of the fetuses by its uterine stimulating property. 17β- estradiol increases the uterine synthesis of PGF_2α as described earlier but seems not affecting the production of PGE₂ by the uterus. The release rate of ³H-PGE₂ from Silastic-PVP tube and is also described.     

Biphasic effect of sodium fluoride and guanyl nucleotides on binding to prostaglandin E2 receptors in rat epididymal adipocyte membranes

Both NaCl and NaF promoted PGE₂ binding to epididymal adipocyte membranes by apparent increase in the binding affinity. In order to distinguish between the effect of fluoride and the ‘salt effect’ of sodium on PGE₂ binding, the effects of Mg²⁺ and guanyl nucleotides on PGE₂ binding in the presence of NaCl or NaF were compared. Mg²⁺ decreased PGE₂ binding; high NaF concentration abolished this inhibition, while increased NaCl concentratipns did not affect the Mg²⁺ inhibition. In the presence of Mg²⁺ the effects of NaCl and NaF were additive. The enhancement of PGE₂ binding by fluoride, unlike sodium, was dependent on the presence of Mg²⁺. Induction of the membranes with GDPβS, Gpp(NH)p, GTP or GTPγS increased PGE, binding. Gradual increase in NaF concentrations in the presence of guanyl nucleotides resulted in stimulation of PGE₂ binding at low NaF concentrations and inhibition of PGE₂ binding at higjh NaF concentrations. No changes in the stimulatory action of NaCl on PGE₂ binding were observed in the simulatenous presence of NaCl and guanyl nucleotides. A biphasic effect on PGE₂ binding was observed with a wide concentration range of guanyl nucleotides. Treatment of the isolated membranes with cholera or pertussis toxins stimulated the adenylyl cyclase activity of the membranes, but failed to influence PGE₂ binding. The implications of these findings are discussed.     

Prostaglandin (PG) analysis in urine of humans and rats by different radioimmunoassays: Effect on PG-excretion by PG-synthetase inhibitors, laparotomy and furosemide

Thw radioimmunological (RIA) determination of prostaglandin (PG) E₂ and of PGF_2α in urine humans and rats is described in detail. After extraction and chromatography PGE₂ was determined by using a PGE specific antibody or by using either PGB or PGF_2α specific antibodies after the respective conversion procedures. The three different RIA procedures were compared to each other. PGF_2α was determined by a specific antibody to PGF_2α. Basal excretion of PGE₂ and of PGF_2α in healthy women on free diet was 9.3 ng/hour ± 0.96 and 18.3 ng/hour ± 2.5 respectively. Furosemide increased the excretion of PGE₂ and of PGF_2α in humans significantly, while PG-excretion rates decreased on indomethacin. In rat urine PGE₂ and PGE_2α increased markedly from 46.2 pg/min ± 9.3 and 27 ± 3.4 to 253.8 ± 43.3 and 108 ± 12.6 pg/min (per one kidney) in the anesthetized-laparotomized animal. This increase was abolished after giving two different PG synthetase inhibitors.     

Effect of in vivo prostaglandin treatment on H-PGF2α uptake in hamster corpora lutea

Pregnant hamsters were administered (SC) prostaglandin or vehicle on the morning of the 4th day of pregnancy. Serum progesterone was significantly depressed (p<.01) at 0.5, 2, and 6 hours after treatment with 100 μg PGF_2α. Serum progesterone levels were unchanged 2 hours and 6 hours after treatment with 100 μg PGF_2β and 2 hours after treatment with 1 mg PGF_2β. Progesterone levels were depressed to less than 1 ng/ml 6 hours after treatment with 1 mg PGF_2β. The specific uptake of ³H-PGF_2α in whole hamster corpora lutea was significantly depressed 2 hours and 6 hours following 100 μg PGF_2α treatment. A 15% depression in specific uptake occurred 0.5 hour post-treatment. Treatment with 100 μg PGF_2β resulted in no change. Administration of 1 mg PGF_2β resulted in depressed ³H-PGF_2α uptake at both 2 and 6 hours post-treatment.Prostacyclin (PGI₂) treatment resulted in no change in either ³H-PGF_2α specific uptake or serum progesterone 2 hours after 100 μg treatment SC. These parameters were both reduced approximately 30% 6 hours post-treatment. Treatment with 6-keto-PGF_1α resulted in a complete lack of measurable ³H-PGF_2α uptake and serum progesterone levels less than 1 ng/ml at both 2 and 6 hours after treatment with 1 mg SC.     

The use of stable prostaglandins to investigate prostacyclin (PGI2)-binding sites and PGI2-sensitive adenylate cyclase in human platelet membranes

Prostacyclin, (PGI₂) is a potent but unstable inhibitor of platelet aggregation, probably acting through stimulation of adenylate cyclase.A stable analogue of prostacyclin with antiaggregatory properties, 5,6-dihydro-PGI₂ (6β-PGI), and PGE₁ can compete for the binding sites labelled by ³H-PGI₂ in human platelet membranes (the affinity being PGI₂ > PGE₁ > 6β -PGI₁). Both 6β-PGI₁ and PGE₁, as well as PGI₂, bind to two classes of binding sites. 6β -PGI₁ and PGE₁ activate adenylate cyclase to the same extent as PGI₂,with a rank order of potency which parallels that observed in binding experiments. The stimulation of this enzyme is brought about by interaction of each these prostanoids with two different classes of components. The comparison of binding and adenylate cyclase data suggests that the sites to which PGI₂, 6β -PGI₁ and PGE₁ bind might be coupled to the activation of adenylate cyclase. Since 6β-PGI₁ seems to act through the same molecular mechanisms as PGI₂, because of its stability it is an useful tool to investigate the mode of action of prostacyclin in platelets.     

Steroid hormone modulation of 3H-prostaglandin E1 binding to bovine corpus luteum cell membranes

The specific binding of ³H-prostaglandin E₁ (³H-PGE₁) to bovine corpus luteum cell membranes was not affected by cholesterol or various progestins at concentrations of up to 9.0 × 10⁻⁶M. At concentrations above 2.5 × 10⁻⁶M; estrone, 17β-estradiol (but not 17α-estradiol or 17β-estradiol glucuronide), estriol, equilin, D-equilenin, 17-ethynyl estradiol, diethylstilbestrol, cortisol, corticosterone, deoxycorticosterone and aldosterone inhibited specific binding of ³H-PGE₁. On the other hand, testosterone and dihydrotestosterone (DHT) (but not androstenedione) significantly enhanced ³H-PGE₁ binding. These findings permitted the following correlations between steroid structure and modulation of ³H-PGE₁ binding: steroids with a free phenolic ring and a 17β-hydroxyl or 17-keto group or C-21 steroids with a C-20 ketone and a C-21 hydroxy group decrease, whereas C-19 steroids with a C-17 hydroxy group enhance specific binding of ³H-PGE₁. PGE receptors are heterogeneous with respect to affinity for ³H-PGE₁. The steroids that decreased ³H-PGE₁ binding caused a lowering to a complete loss of low affinity PGE receptors. Steroids that increased ³H-PGE₁ binding caused appearance of new low affinity PGE receptors. Association rate constants for ³H-PGE₁ binding were decreased by 17β-estradiol (61%) and increased by DHT (59%).     

Midterm abortion in hamsters induced by Silastic-PVP-PGE2 tubes

Intrauterine insertion of a 0.5 cm long Silastic-PVP tube containing 750 μg PGE₂ (lyophilized sodium salt) caused midterm abortion in hamsters within 48 hours. An earlier study using a similar Silastic-PVP tube delivery system showed that 200 μg of PGF_2α (Tham) was sufficient to induce abortion in 100% of pregnant hamsters (18). Prostaglandin E₂ is, therefore, about 3.5–4 times less potent than PGF_2α as an abortifacient in the hamster. The release of ³H-PGE₂ from Silastic-PVP tube and is also described. It is suggested that an increase in LH release might be one of the factors leading to luteolysis; and that either PGE₂ exerts a direct luteolytic effect or this effect is manifested after its being converted to PGF_2α.     

Metabolism of arachidonic acid in vitro by ovine conceptuses recovered during early pregnancy

Metabolism of [³H] arachidonic acid ([³H] AA) and synthesis of prostaglandins were examined with ovine conceptuses and endometrial slices collected on various days after mating. Tissues were incubated for 24 hr with or without 5 μCi of [³H] AA and with 200 μg radioinert AA. In experiment 1, results of chromatography indicated that conceptuses collected on days 14 and 16 after mating metabolized [³H] AA to PGE₂, PGF_2α, PGFM, 6-keto-PGF_1α, and to unidentified compounds in three chromatographic regions. One of these regions (region 1) contained triglycerides. Endometrial slices metabolized only small amounts of the [³H] AA to prostaglandins. In experiment 2, results of radioimmunoassays indicated that day 14 conceptuses released somewhat similar amounts (ng/mg tissue) of PGF_2α (32.1 ± 17.9), PGFM (8.4 ± 6.2), PGE₂ (12.3 ± 7.5) and 6-keto-PGF_1α (41.4± 4.8), whereas day 16 conceptuses released more (P<.05) PGF_2α (9.0 ± 4.1) and 6-keto-PGF_1α (15.9 ± 2.7) than PGE₂ (0.9 ± 0.2) or PGFM (0.5 ± 0.08). Day 14 and 16 endometrial slices released (ng/mg tissue) more (P<.05) PGFM (3.0 ± 0.2) and 6-keto-PGF_1α (4.0 ± 0.4) than PGF_2α (0.5 ± 0.08) or PGE₂ (0.05 ± 0.02). In experiment 3, conceptuses were recovered on days 16, 20 and 24 of pregnancy and incubated with [³H] AA to determine the effects of indomethacin on [³H] AA metabolism. In general, indomethacin (Id; 4 × 10⁻⁴ M) reduced (P<.05) the percentage of total dpm recovered as prostaglandins, but Id increased the release of chromatographic region I. Experiment 4 was conducted with day 16, 20 and 24 conceptuses to evaluate the time course of metabolism of [³H] AA, and the appearance of region I and of prostaglandins. In general, the percentage of total dpm in region I increased as the percentage of dpm as [³H] AA decreased. The percentage of dpm as prostaglandins increased as the percentage of dpm in region I decreased. Prostaglandins, probably essential for embroynal survival and development, were synthesized in vitro by ovine conceptuses.     

Subcellular distribution of prostaglandin and gonadotrop in receptors in bovine corpora lutea.

The subcellular distribution of prostaglandin (PG) E₁, F₂α and gonadotropin receptors in bovine corpora lutea was critically examined by preparing various subcellular fractions, assaying for various marker enzymes to assess the purity and examining ³H-PGE₁, ³H-PGF₂α and ¹²⁵I-human lutropin (hLH) specific binding. The marker enzyme data suggested that subcellular fractions were relatively pure with little or no cross contamination. The binding of ³H-PGs and ¹²⁵I-hLH was markedly enriched in plasma membranes with respect to homogenate. The other subcellular fractions also exhibited binding despite very little or no detectable 5′-nucleotidase activity. If 5′-nucleotidase was assumed to lack sensitivity and reliability to detect minor contamination with plasma membranes and ³H-PGs or ¹²⁵I-hLH binding were used as sensitive plasma membrane markers, it was still difficult to explain binding in other fractions based on plasma membrane contamination. Therefore, these results lead to the inevitable conclusion that plasma membranes were primary (or one of the primary) but not exclusive sites for PGE₁, PGF₂α and gonadotropin receptors.     

Characterization, size estimation and solubilization of α-macroglobulin complex receptors in liver membranes

Receptors for α₂-macroglobulin-proteinase complexes have been characterized in rat and human liver membranes. The affinity for binding of ¹²⁵I-labelled α₂-macroglobulin · trypsin to rat liver membranes was markedly pH-dependent in the physiological range with maximum binding at pH 7.8–9.0. The half-time for association was about 5 min at 37°C in contrast to about 5 h at 4°C. The half-saturation constant was about 100 pM at 4°C and 1 nM at 37°C (pH 7.8). The binding capacity was approx. 300 pmol per g protein for rat liver membranes and about 100 pmol per g for human membranes. Radiation inactivation studies showed a target size of 466 ± 71 kDa (S.D., n = 7) for α₂-macroglobulin · trypsin binding activity. Affinity cross-linking to rat and human membranes of ¹²⁵I-labelled rat α₁-inhibitor-3 · chymotrypsin, a 210 kDa analogue which binds to the α₂-macroglobulin receptors in hepatocytes (Gliemann, J. and Sottrup-Jensen, L. (1987) FEBS Lett. 221, 55–60), followed by SDS-polyacrylamide gel electrophoresis, revealed radioactivity in a band not distinguishable from that of cross-linked α₂-macroglobulin (720 kDa). This radioactivity was absent when membranes with bound ¹²⁵I-α₁-inhibitor-3 complex were treated with EDTA before cross-linking and when incubation and cross-linking were carried out in the presence of a saturating concentration of unlabelled complex. The saturable binding activity was maintained when membranes were solubilized in the detergent 3-[(3-cholamidopropyl)dimethylammonio]profane sulfonate (CHAPS) and the size of the receptor as estimated by cross-linking experiments was shown to be similar to that determined in the membranes. It is concluded that liver membranes contain high concentrations of an approx. 400–500 kDa α₂-macroglobulin receptor soluble in CHAPS. The soluble preparation should provide a suitable material for purification and further characterization of the receptor.     

The effect of adrenergic stimulation on urinary prostaglandin E2 and 6 keto PGF1α in man

To evaluate the details of the adrenergic stimulation of urinary prostaglandins in man, ten normal volunteers were given various agonists and antagonists. The effect of 4 hour IV infusions of norepinephrine (NE), NE + phentolamine (PHT), NE + phenoxybenzamine (PHB), NE + prazosin (PZ), isoproterenol (ISO), and PHT alone on urinary PGE₂ and PGI₂ (6 keto PGF_1α) were determined. PGE₂ and 6 keto PGF_1α were measured by radioimmunoassay from 4 hour urine samples. NE stimulated both PGE₂ (196±40 to 370±84 ng/4 hrs/g creatinine and 6 keto PGF_1α(184±30 to 326±36), both p<0.01. In contrast, ISO had no effect on either PGE₂ or 6 keto PGF_1α excretion. Alpha blockade with PHT. PHB, or PZ inhibited the NE induced systemic pressor effect. However, the effect of the alpha blockers on the NE induced stimulation of PGE₂ and 6 keto PGF_1α varied. PHT did not alter the NE stimulated PGE₂ or 6 keto PGF_1α release (370±84 vs. 381±80) PGE₂ and (326±50 vs. 315±40) 6 keto PGF_1α, both p>0.2). PHT alone stimulated only 6 keto PGF_1α. PHB and the specific α₁ antagonist PZ similarly eliminated the NE induced prostaglandin release. These results suggest that adrenergically mediated urinary prostaglandin release in man is via an alpha receptor with α₁ characteristics.     

Characterization of adrenergic receptors and related transduction pathways in the liver of the rainbow trout

Epinephrine (EPI) is thought to act by stimulating adenylyl cyclase (ACase) and cAMP production through β-adrenoceptors in the liver of more primitive vertebrates. Recent observations, however, point to an involvement of α₁-adrenoceptors in EPI action, at least in some fish species. The role of the α₁- and β-adrenergic transduction pathways has been investigated in rainbow trout (Oncorhynchus mykiss) hepatic tissue. Radioligand-binding assays with the β-adrenergic antagonist ³H-CGP-12177 using hepatic membranes purified on a discontinuous sucrose gradient confirmed the presence of β-adrenoceptors (K_d0.36 nM, B_max 8.61 fmol · mg⁻¹ protein). We provide the first demonstration of α₁-adrenoceptors in these same membranes; analysis of binding data with the α₁-adrenergic antagonist ³H-prazosin demonstrated a single class of binding sites with a K_dof 15.4 nM and a B_max of 75.2 fmol · mg⁻¹ protein. There is a straight correlation between β-adrenoceptor occupancy, ACase activation and cAMP production. On the contrary, the role of inositol 1,4,5-trisphosphate (IP₃) has to be elucidated; in fact, despite the presence of specific microsomal binding sites for IP₃ (K_d 6.03 nM, B_max 90.2 fmol · mg⁻¹ protein), its cytosolic concentration was not modulated by EPI. On the other hand, we have previously shown in American eel and bullhead hepatocytes that α₁-adrenergic agonists are able to increase intracellular concentrations of IP₃ and Ca²⁺ and to activate glycogenolysis. These data suggest a marked variation in the liver of different fish both in terms of α₁-binding sites affinity and of α₁-adrenoceptor/IP₃/Ca²⁺ transduction systems.