Pyridoxine induced puffing (II-48 C) and synthesis of a 40 KD protein in Drosophila hydei salivary glands

A specific heat shock puff (II-48 C) has been induced in isolated salivary glands of D. hydei by pyridoxine (vitamin B₆) at concentrations from 10^–7 to 10^–2 M. The puff begins to form within 5 min and continues to increase in size up to 2 h. Under conditions of puff induction the -amanitin sensitive synthesis of a 40 KD protein is induced.     

Rapid sequence divergence in a heat shock locus of Drosophila

In situ hybridization of cRNA transcribed from cloned D. melanogaster heat shock sequences to D. hydei chromosomes has shown that the D. hydei locus 2–32 A corresponds to the D. melanogaster locus 87 A/C and the D. hydei locus 2–36 A to the D. melanogaster locus 95 D, while the D. hydei locus 4–81 B corresponds to the D. melanogaster locus 63 BC. No hybridization to D. hydei chromosomes was found with cRNA transcribed from a clone containing the sequences encoded by the D. melanogaster locus 87 C. Neither D. melanogaster heat shock RNA nor D. virilis heat shock RNA hybridized significantly to the D. hydei heat shock locus 2–48 B. Furthermore, D. hydei heat shock RNA did not hybridize to the cytological homologs of locus 2–48 B found in D. repleta or in D. virilis. D, hydei heat shock. RNA did hybridize to the cytological homologs of locus 2–48 B in D. neohydei and D. eohydei, both of which belong to the hydei subgroup.     

Local protein accumulation during gene activation

Measurements of the integrated absorbancy of naphthol yellow S binding to protein (430 nm) and Feulgen-stained DNA (550 nm) of two puff regions in Drosophila hydei polytene chromosomes revealed a significant increase in the naphthol yellow S binding capacity during the first 5 min of puff induction. The ratio of integrated absorption values at 430 and 550 nm of two chromosome regions, 2-48 C and 4-81 B were determined relative to the ratio of absorption values at 430 and 550 nm of a reference band. These determinations were carried out in a non-puffed state and at 5, 10, 30, 60 and 120 min after onset of a temperature treatment inducing puffs in these regions. The quotient of the absorption ratio of the puff region and the ratio of the reference band provides a relative measure for naphthol yellow S binding to protein. The staining reaction was absent after pronase treatment.—The relative increase in naphthol yellow S binding was most obvious during the first 5 min after onset of puff induction. The binding of naphthol yellow S was increased by a factor 1.7 for puff 2-48 C, and a factor 1.9 for puff 4-81 B. The maximum value, indicating a relative increase by a factor 1.8 in puff 2-48 C and a factor 2.2 in puff 4-81 B was attained in both puffs at 30 min after onset of puff induction.—Among staining procedures performed on sulphydryl groups, free -amino acids and indole groups of tryptophane, only a positive result with the staining reaction on the indole groups was obtained for induced puffs.—Injection of tritiated sodium acetate, methionine-H³-methyl, ethionine-H³-ethyl, C¹⁴-sodium bicarbonate, a mixture of 15 H³-labelled L-amino acids and H³-tryptophane at various time intervals prior to puff induction failed to result in a specific incorporation of any of these radioactive substances into newly induced puffs.     

Cytochemical studies on RNP complexes produced by puff 2-48BC in Drosophila hydei

Differential staining of the core and RNP particles of RNP complexes in puff 2–48 BC in salivary gland chromosomes of Drosophila hydei was achieved with aqueous uranyl acetate (UA) at low pH, with UA in acetone, with phosphotungstic acid (PTA) in organic solvents, and with aqueous PTA at pH 5 and 6. A comparison of the results of UA and PTA staining under various conditions indicate that the proteins in the core region and in the RNP particles connected to it differ with respect to their amino-acid composition (arginine and lysine residues). — The staining mechanism of PTA and UA is discussed.     

The effect of changes in the respiratory metabolism upon genome activity in Drosophila

Inhibition of hydrogen transfer between NADH and Co Q by rotenone or amytal in salivary gland cells of Drosophila hydei maintained in vitro, results in the activation of a particular group of four loci in the polytene chromosomes (puff formation). The response of these loci to the same treatment is enhanced if Na-malonate is present in the incubation medium. — Three of the loci become active if the glands are kept in a medium supplied with antimycin A or 2-heptyl-4-hydroxyquinoline-N-oxide (H QNO), specific inhibitors of the electron transfer between cytochromes b and c. — It was established that a temperature treatment and DNP raise oxygen consumption of the cells to a certain level. Following the same treatments of glands supplied with Na-malate and Na-succinate the raise in oxygen consumption attains a significantly higher level. Under these conditions no response is observed at the genome level. — Whereas DNP, which uncouples oxidative phosphorylation and enhances the respiratory chain reactions, does induce the initiation of puff formation, oligomycin, which inhibits oxidative phosphorylation and suppresses the respiratory chain reactions, is ineffective in initiating puff formation at the specific loci. However, if oligomycin is supplied to the medium in combination with KCN which inhibits the cytochrome oxidase activity, three of the four loci become active. — The presence in the medium of substances which may act as hydrogen acceptors, e.g. menadione or methylene blue, can also result in activation of the chromosome loci. — These results are interpreted as indications for the existence of a regulatory mechanism between mitochondrial respiratory metabolism and the activity of a particular group of genome loci.     

The hormone ecdysone as effector of specific changes in the pattern of gene activities of Drosophila hydei

The hormone ecdysone induces a large number of changes in the puffing pattern of mid third instar larvae of Drosophila hydei. The pattern of changes occurring after experimental administration of the hormone are identical with those observed in normal development during a 6 hour period before puparium formation. After administration of the hormone a considerable number of puffs react with a change in activity within 15–20 min. During this period 3 puffs arise newly, 12 puffs show a strong increase in activity, 6 puffs show a less pronounced increase in activity and 12 puffs show a decrease in activity. At a period of 4–6 hours after administration of the hormone another 5 puffs arise newly. The effect of the hormone was identical in both in vivo and in vitro experiments. — Diameter measurements on several puffs reacting within 30 min with an increase in diameter showed that these puffs reacted simultaneously. Most of the puffs that showed a decrease in activity reacted with some delay. — A study of the effect of different hormone concentrations revealed that the kinetics of 4 puffs with respect to the relationship between concentration and puff size was identical over a range of concentrations from 33·10^–5 to 33CU/l. Three of these puffs showed a reaction with even lower concentrations. Maximum puff size is attained by all puffs at a concentration of 33·10^–4CU/l. Among the puffs studied no difference in their reaction threshold was found. — A study of the behavior of 5 puffs of the group reacting within 15–20 min and one of the group reacting after 4–6 hours in midintestine and Malpighian tubules revealed that these puffs showed the same reaction after injection of the hormone as observed in the salivary glands. — All puffs activated by administration of the hormone showed particularly strong uptake of tritiated uridine and accumulation of acidic protein. — It is concluded that the hormone ecdysone induces a pattern of changes in gene activity that is far more complex in Drosophila hydei than in Chironomus tentans.     

The heat shock protein hsp70 binds in vivo to subregions 2-48BC and 3-58D of the polytene chromosomes ofDrosophila hydei

Multiple interactions of members of the hsp70 family with cellular components have already been described. We present, however, the first evidence that upon heat shock treatment hsp70 molecules interact with specific chromosomal subdivisions of the polytene chromosomes ofDrosophila hydei. After a heat shock treatment of 20 min the protein binds to subdivision 3-58D₁ and to the heat shock inducible subdivisions 2-48B_3–6 and 2-48C_1–2. Hsp70 molecules were also observed in subdivision 3-58D₁ during recovery at 25°C but not in subdivisions 2-48B_3–6 and 2-48C_1–2. Our data suggest that this interaction is stress specific. DNase and RNase experiments suggest, moreover, that the hsp70 molecules bind to RNA from ribonucleoproteins (RNPs) in subdivisions 2-48B_3–6 and 2-48C_1–2 and to DNA in subdivision 3-58D₁. The DNA sequences in subdivision 3-58D₁ seem to have the potential to adopt the Z-DNA conformation.     

In situ hybridization of nuclear and cytoplasmic RNA to locus 2-48BC in Drosophila hydei

The maximum grain density over the heat-shock locus 2-48BC of Drosophila hydei polytene chromosomes obtained after in situ hybridization of nuclear RNA extracted from tissue culture cells labelled during incubation at 37° C is five times higher than that obtainable by using polysomal RNA isolated from the same cells. Furthermore, the addition of a large excess of unlabelled polysomal RNA reduced the amount of in situ hybridization of nuclear RNA by only 20% showing that nuclear 2-48BC RNA contains sequences not present in polysomal 2-48BC RNA. — The polysomal 2-48BC RNA is polyadenylated, as are the RNA sequences present in the polysomes complementary to the other two major heat shock loci 2-32A and 2-36A. Polyadenylated RNA, with an apparent size of 15S, complementary to locus 2-48BC is also found in the cytoplasm of D. hydei salivary glands.     

Optimization of a Propionibacterium acidipropionici continuous culture utilizing sucrose

To produce propionic acid and vitamin B₁₂ from sucrose, the strain Propionibacterium acidipropionici NRRL B3569 was selected by screening a number of Propionibacterium strains. The nutrient composition and the fermentation conditions for this strain were optimized in continuous culture. The investigations show that within a concentration range of 30–170 g l^–1 of sucrose in the fermentation medium, no significant substrate inhibition occurred. For the production of propionic acid and vitamin B₁₂, concentrations of 1.5 mg FeSO₄·7H₂O g^–1 dry biomass, 0.75 mg cobalt ions g^–1 dry biomass, 0.3 mg 5,6-dimethylbenzimidazole g^–1 dry biomass, and 12 g yeast extract 1^–1 were necessary additions to the sources of nitrogen, phosphate, and magnesium ions. The extra addition of up to 2.8 g betaine g^–1 dry biomass significantly increases the production of vitamin B₁₂. In the optimization of the pH value, temperature, and aeration, it was established that the conditions for propionic acid production and vitamin B₁₂ production are different. Whereas the optimal production of propionic acid took place under completely anaerobic conditions with a pH value of 6.5 and a temperature of 37°C, optimal vitamin B₁₂ production required a temperature of 40°C and aerobic conditions (0.5 vvm aeration at 100 rpm) with a pH value of 6.5.     

Photosynthetic activity of phytoplankton in a high altitude lake (Emerald Lake,Sierra Nevada,California)

Photosynthetic activity by phytoplankton was measured during the ice-free seasons of 1984, 1985 and 1987 using the ¹⁴C radioassay in high altitude Emerald Lake (California). Relative quantum yield (^B) and light-saturated chlorophyll-specific carbon uptake (P_m ^B) were calculated from the relationship of light and photosynthesis fitted to a hyperbolic tangent function. Temporal changes in P_m ^B showed no regular pattern. Seasonal patterns of ^B generally had peaks in the summer and autumn. Phytoplankton biomass (as measured by chlorophyll a) and light-saturated carbon uptake (P_m) had peaks in the summer and autumn which were associated with vertical mixing. Estimates of mean daily carbon production were similar among the three years: 57 mg C m^–2 2 d^–1 in 1984, 70 mg C m^–2 2 d^–1 in 1985 and 60 mg C m^–2 d^–1 in 1987. Primary productivity in Emerald Lake is low compared to other montane lakes of California and similar to high-altitude or high-latitude lakes in other regions.     

Local protein accumulation during gene activation

Relative values for the dry mass in puff-forming regions of Drosophila hydei salivary gland chromosomes were established with a Leitz double beam interference microscope. All measurements were made after RNA digestion.Optical path differences per unit area of the induced puffs 2-48C and 4-81B (temperature induced) and a cytoplasm-free background were recorded. In each of the nuclei used for these measurements, the same procedure was applied to two reference regions in the vicinity of the puff, region 2, 47A-48B and region 4, 81C-82C respectively. For comparison of the dry mass values of a puff region at various time intervals after the onset of puff induction, ratios of the optical path differences of the puff region over that of the reference region were calculated.These ratios were established at 5, 10, 30, 60 and 120 min after the onset of a temperature treatment and compared with the ratio in non-treated animals. From the data it can be concluded that the dry mass in the induced puffs increases gradually up to 30 min. At this time the dry mass ratio for puff 2-48C has reached a value of 150% and that of puff 4-81B, 210%. It is concluded that this increase in dry mass is due almost entirely to a local accumulation of non-histone protein.     

In vitro multiplication of Quercus leucotrichophora and Q. glauca: Important Himalayan oaks

Multiple shoots of Quercus leucotrichophora L. and Q. glauca Thunb. were induced from the intact embryos (decoated seeds) as well as from the cotyledonary nodes (with attached cotyledons but without radicle and primary shoot) of 3-weeks old in vitro grown seedlings on Woody Plant (WP; Lloyd and McCown, 1980) and Murashige and Skoog (MS; 1962) media supplemented with 6-benzyladenine (BA), either alone or in combination with gibberellic acid (GA₃)/ indole-3-butyric acid (IBA). BA (22.19 M) was effective for induction of multiple shoots and addition of GA₃ to the medium further enhanced the shoot number and shoot height but resulted in shoot thinness. High frequency shoot multiplication was achieved using cotyledonary nodes. Shoots were further multiplied from the original explant on WP medium supplemented with BA (22.19 M). Nearly 78% and 67% rooting was obtained in Q. leucotrichophora and Q. glauca microshoots (3–4 cm high), respectively on 1/2 strength WP medium supplemented with IBA (14.76 M). However, this was associated with basal callus formation. Treatment with IBA (25–100 M) for 24 or 48 h followed by transfer to PGR free 1/2 strength WP medium not only improved the rooting percentage but also avoided basal callus formation. IBA at 100 M for 24 h was most effective (90% and 100% rooting in Q. leucotrichophora and Q. glauca, respectively). In vitro rooted plants were hardened and established in garden soil.Growth performance of 6-month-old in vitro raised plants was compared with ex vitro plants (seedlings) of the same age. The photosynthesis and transpiration rates of eight months old in vitro and ex vitro raised plants of both species were measured under different light (0, 600, 900, 1200, 1500 and 2000 mol m^–2s^–1) and temperature (20, 25, 30, 35 and 40 °C). Light optimum for photosynthesis was around 2000 mol m^–2s^–1 in Q. leucotrichophora and around 1500 mol m^–2s^–1 in Q. glauca whereas optimum temperature for photosynthesis was 25 °C in Q. leucotrichophora and 30 °C in Q. glauca. The rate of transpiration at different temperatures (20–40 °C), in the two species, increased with increase in the light intensity up to the highest level, i.e., 2000 mol m^–2s^–1. Temperatures beyond 35 °C adversely affected the rate of transpiration in in vitro raised as well as ex vitro plants of both the species. In vitro raised and hardened plants of both the species were comparable to ex vitro plants in terms of gas and water vapour exchange characteristics, within the limits of this study.     

Growth,vitamin B12, and photopigment formations of Rhodopseudomonas sphaeroides S growing on propionate media under dark and light conditions

Summary The effects of light illumination and dissolved oxygen concentration (DO) on the growth characteristics of Rhodopseudomonas sphaeroides S and intracellular accumulations of vitamin B₁₂ and photopigments were studied in continuous cultures of aerobic-dark (S-1, DO>5 mg l^–1), aerobic-light (S-2, DO>5 mg l^–1, 4.5 klux), microaerobic-light (S-3, DO0,4.5 klux) and anaerobic-light (S-4, 4.5 klux) conditions using propionate media. Growth yields from propionic acid determined in S-3 and 4 were 1.5 to twofold greater than in S-1 and 2. Carbon dioxide evolution observed in S-3 and 4 was 0.05 to 0.1 times that in S-1 and 2. Overall carboxylase activity was maximal in S-4. Intracellular accumulations of bacteriochlorophyll and carotenoid were maximal in S-4, followed by S-3 and almost nil in S-1 and 2. The high growth yields observed in S-3 and 4 could be accounted for the high level of activity of carbon dioxide fixation and by the additional or effective utilization of light energy.Intracellular accumulation of vitamin B₁₂ in S-2, 3 and 4 was 1.5 to 1.8 times that in S-1. The maximum content of the vitamin was 74 g-B₁₂ g-cell^–1. The maximum productivity of vitamin B₁₂, g-B₁₂ l^–1 h^–1, was 6.6 in S-3 since specific growth rate, growth yield and the vitamin content of the cells were maximal in S-3.     

Modulation of allo-immune responsesin vivo andin vitro by sperm specific lactate dehydrogenase-C4

The role of sperm specific lactate dehydrogenase-C₄ (LDH-C₄) in allo-immune responses using mixed lymphocyte cultures (MLC) and cytotoxic T cell (CTL) generationin vitro and local graft versus host (LGVH) reaction and allograft enhancementin vivo has been ascertained. LDH was purified from testes (LDH-C₄) and kidney (LDH-B₄) of C57 Bl/Ks mice. MLC and CTL were performed using C57 Bl/Ks-anti A/J lymphocytes in presence of 10^–3-1 g LDH-B₄ or LDH-C₄ per culture. The MLC and CTL responses showed biphasic action depending on the dose of LDH-C₄. Early MLC culture gave significantly low stimulation index at 10^–2–10^–1 g LDH-C₄ as compared to non-treated control cultures. However, the MLC response in presence of LDH-C₄ was not different from the LDH-B₄ treated one which showed a similar biphasic trend. On the other hand,⁵¹Cr release from YAC-222 target cells was practically abolished by LDH-C₄ at 10^–3–1^–1 g, and this was strikingly different from LDH-B₄ or non-treated cultures. LGVH reactivity as performed by using C57 Bl/Ks lymphocytes along with LDH-C₄ in (C57 Bl/Ks x A/J) F₁ hybrids indicated a suppression of stimulation index in primary and secondary (i.e. preimmunized in presence of LDH-C₄ or LDH-B₄) LGVH. Allograft enhancement of Sa I (A/J) in C57 Bl/Ks mice in presence of LDH-C₄, was delayed slightly but significantly during primary or secondary transplantation reaction. The reaction of LDH-C₄ in the modulation of allo-immune responses was more specificin vivo thanin vitro since the B₄ isozyme did not modify LGVH and Sa1 allograft rejection. Resultsper se suggest that LDH-C₄ is immunosuppressive for cell mediated allo-immune responsesin vivo andin vitro.     

Effects of <Emphasis Type="Italic">in vitro</Emphasis>rooting environments and irradiance on growth and photosynthesis of strawberry plantlets during acclimatization

Strawberry (Fragaria ananassaDuch. cv. Fengxiang) plantlets were cultured under two in vitroenvironments for rooting, and then acclimatized under two ex vitroirradiance conditions. At the end of rooting stage plant height, fresh weight and specific leaf area of T1-plants grown under high sucrose concentration (3 sucrose), low photosynthetic photon flux density (30 mol m^–2 s^–1) and normal CO₂ concentration (350–400 l l^–1) were significantly higher than those of T2-plantlets grown under low sucrose concentration (0.5), high photosynthetic photon flux density (90 mol m^–2 s^–1) and elevated CO₂ concentration (700–800 l l^–1). But T2-plantlets had higher net photosynthetic rate (Pn), effective photochemical quantum yield of PSII (_PSII), effective photosynthetic electron transport rate (ETR), photochemical quenching (q_P) and ratio of chlorophyll fluorescence yield decrease (Rfd). After transfer, higher irradiance obviously promoted the growth of plantlets and was beneficial for the development of photosynthetic functions during acclimatization. T2-plantlets had higher fresh weight, leaf area, _PSII and ETR under higher ex vitroirradiance condition.     

Separation and quantification of the B6 vitamers in plasma and 4-pyridoxic acid in urine of adolescent girls by reversed-phase high-performance liquid chromatography

The vitamin B₆ status of seemingly healthy adolescent girls was determined using several accepted and proposed parameters in an effort to establish guidelines for status evaluation. High-performance liquid chromatography-derived plasma B₆ vitamers (pyridoxal phosphate, PLP; pyridoxine phosphate, PNP; pyridoxamine phosphate, PMP; pyridoxal, PL; pyridoxine, PN; and pyridoxamine, PM) and 4-pyridoxic acid (4-PA) concentrations and urinary 4-PA levels of 28 white adolescent females, 12–15 years, having radiomonitored plasma PLP concentrations and coenzyme stimulation of erythrocyte alanine aminotransferase activities indicative of adequate status were determined. Mean vitamin B₆ and protein intakes were 1.48 mg and 78.3 g. Ranges for plasma B₆ vitamer and 4-PA concentrations (nmol/1) were: PLP, 40.9–122.2; PNP, non-detectable (ND)—16.1; PMP, ND—8.1; PL, ND—15; PN, ND—21.9; PM, ND—17.8; and 4-PA, ND—55.7. PLP was the only vitamer found in plasma of all subjects. Urinary 4-PA concentrations ranged from 0.11 to 2.50 μmol/mmol of creatinine. B₆ vitamer values of these girls should be of use in the establishment of normal ranges for vitamin B₆ status parameters.     

Interaction between aflatoxin B1 and oxytetracycline in isolated rat hepatocytes

Isolated rat hepatocytes were used as an in vitro model to investigate A possible interaction between oxytetracycline (OXT) and aflatoxin B₁ (AFB₁). LDH leakage, RNA and protein synthesis and glycogen accumulation were measured in the presence of both drugs, either separately or in combination. The evolution of LDH leakage during the incubation was identical in untreated and treated cells. AFB₁ inhibited RNA and protein synthesis at a concentration of 10^–7 M and 10^–6 M, respectively, and higher, whereas OXT did not influence RNA synthesis but inhibited protein synthesis at the highest tested concentration, 10^–3 M. As far as glycogen is concerned, rats were injected with glucagon before sacrifice in order to obtain a constant synthesis rate in isolated hepatocytes. AFB₁ inhibited the accumulation of glycogen from 10^–6 M upward. This effect was never observed before 90 min of incubation. OXT had no effect on glycogen synthesis. In the presence of both drugs, no interaction was demonstrated as far as RNA and protein synthesis were concerned, but OXT opposed the inhibition induced by AFB₁ on glycogen accumulation. If the in vivo protection, provided by OXT against AFBI-induced toxicity, is due to a direct interference in the toxic mechanisms of the mycotoxin, these results show that OXT does not influence the AFB₁-inhibition of RNA and protein synthesis. The latter are early and sensitive parameters inhibited by AFB₁. On the contrary, taking into consideration the results on glycogen accumulation, it seems more interesting to investigate further this metabolism.Abbreviations AFB₁ Aflatoxin B₁ - OXT Oxytetracycline - DMEM Dulbecco's Modified Eagle's Medium - LDH Lactate Dehydrogenase - DMSO Dimethyl Sulfoxide - BSA Bovine Serum Albumin     

Radiation induced deletions in spermatids and spermatocytes ofDrosophila

Summary Males of the composition X^C2/B ^S Yy ⁺ were collected as prepupae, aged an additional 48 hours, then irradiated with 1000 r and brooded individually for eight days with three virginy w f females each day. Analysis suggests that brood days 1–3 represent the recovery of cells which were predominantly spermatids at the time of treatment, those from brood days 4–6 predominantly spermatocytes, and those from brood days 7–8 predominantly spermatogonia. The highest frequency of loss of individual Y markers and X0 males, as well as the smallest F₁ population size was found in broods 5 and 6, and especially in brood 6. Further analysis suggests that the vast majority of individual Y-marker losses recovered from diploid cells (spermatocytes, spermatogonia) arose independent of inter-Y arm exchange and Y-autosome translocations. In general, the data confirm the suggestive results of other investigators that deletions are recovered most readily from spermatocytes than from any other stage in spermatogenesis.     

Endothelin-1 binding to endothelin receptors in the rat anterior pituitary gland: possible formation of an ETA-ETB receptor heterodimer

1. Interaction in the recognition of endothelin-1 (ET-1), a typical bivalent ET receptor-ligand, between ET_A and ET_B receptors was investigated in the rat anterior pituitary gland, using our quantitative receptor autoradiographic method with tissue sections preserving the cell-membrane structure and ET receptor-related compounds.2. In saturation binding studies with increasing concentrations (0.77–200 pM) of ¹²⁵I-ET-1 (nonselective bivalent radioligand), ¹²⁵I-ET-1 binding to the rat anterior pituitary gland was saturable and single with a K _D of 71 pM and a B _max of 120 fmol mg^–1. When 1.0 M BQ-123 (ET_A antagonist) was added to the incubation buffer, binding parameters were 8.3 pM of K _D and 8.0 fmol mg^–1 of B _max, whereas 10 nM sarafotoxin S6c (ET_B agonist) exerted little change in these binding parameters (K _D, 72 pM; B _max, 110 fmol mg^–1).3. Competition binding studies with a fixed amount (3.8 pM) of ¹²⁵I-ET-1 revealed that when 1.0 M BQ-123 was present in the incubation buffer, ET_B receptor-related compounds such as sarafotoxin S6c, ET-3, IRL1620 (ET_B agonist), and BQ-788 (ET_B antagonist) competitively inhibited ¹²⁵I-ET-1 binding with K _is of 140, 18, 350 pM, and 14 nM, respectively, however, these compounds were not significant competitors for ¹²⁵I-ET-1 binding in the case of absence of BQ-123.4. In cold-ligand saturation studies with a fixed amount (390 pM) of ¹²⁵I-IRL 1620 (ET_B radioligand), IRL1620 bound to a single population of the ET_B receptor, and no change was observed in binding characteristics in the presence of 1.0 M BQ-123. ¹²⁵I-IRL1620 binding was competitively inhibited by ET-1 and ET-3 in the absence of BQ-123, with K _is of 20 and 29 pM, respectively, the affinities being much the same as those of 29 nM, in the presence of 1.0 M BQ-123.5. Two nonbivalent ET_A antagonists, BQ-123 and PD151242, were highly sensitive and full competitors for ¹²⁵I-ET-1 binding (5.0 pM), in the presence of 10 nM sarafotoxin S6c.6. Taken together with the present finding that mRNAs encoding the rat ET_A and the ET_B receptors are expressed in the anterior pituitary gland, we tentatively conclude that although there are ET_A and ET_B receptors with a functional binding capability for ET receptor-ligands, the ET_B receptor does not independently recognize ET-1 without the aid of the ET_A receptor. If this thesis is tenable, then ET-1 can bridge between the two receptors to form an ET_A–ET_B receptor heterodimer.