Characteristics of stably expressed human dopamine D1a and D1b receptors: atypical behavior of the dopamine D1b receptor.

Human dopamine D1a and D1b receptors were stably expressed in Baby Hamster Kidney (BHK) or Chinese Hamster Ovary (CHO) cells. [3H]SCH23390 saturation experiments indicated the presence of only a single binding site in the D1a expressing cell line with a Kd of 0.5 nM. In D1b expressing cell lines, two binding sites were observed with Kd values of 0.5 and 5 nM in CHO cells and 0.05 and 1.6 nM in BHK cells, respectively. Neither of the receptors affected Ca2+ metabolism whereas they both were coupled in a stimulatory fashion to adenylyl cyclase. The pharmacological profile of both the D1a and D1b receptors as assessed from inhibition of specific [3H]SCH 23390 binding was classical D1-like. Thus, benzazepine derivatives as well as the atypical neuroleptics, clozapine and fluperlapine, exhibited high affinity whereas D2 selective compounds like sulpiride and spiperone had low affinity for these receptors. Besides SCH 23390, only NNC 112, fluphenazine and bulbocapnine were able to discriminate between the two states of the D1b receptor. In case of the D1a receptor, the Ki values obtained in binding experiments were very similar to Ki values obtained from inhibition of dopamine stimulated adenylyl cyclase. In the D1b expressing cell line, the Ki values obtained from inhibition of the dopamine stimulated adenylyl cyclase indicated a significantly better correlation with the state of the D1b receptor showing high affinity for antagonists. In agreement with observations from binding experiments, dopamine was around 20 fold more potent in stimulating adenylyl cyclase via the D1b receptor as compared to the D1a receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

The Horseshoe Crab Tachypleus tridentatus has Two Kinds of Hemocytes: Granulocytes and Plasmatocytes.

For the first time, the fine structure of the hemocytes from the horseshoe crab Tachypleus tridentatus is investigated by transmission electron microscopy and light microscopy serial sectioning. Two morphologically distinct, ellipsoidal, and mononucleate hemocytes--granulocytes (amebocytes) and plasmatocytes--are revealed. Granulocytes constitute about 97% of the hemocytes. They have a marginal band of microtubules, a heterochromatic nucleus, distended but poorly developed RER, few free ribosomes, few mitochondria, and many large secretory granules. The majority of these granules have a uniform content and are mature. Structured granules located in the proximity of Golgi complexes may be immature transitional stages leading to the mature uniform granules. Upon stimulation with endotoxin from gram negative bacteria, the mature granules become transitory structured before exocytosis. In contrast, the immature granules are not exocytosed. Plasmatocytes constitute about 3% of the hemocytes. They differ from granulocytes by having an euchromatic nucleus, a well-developed RER of flattened or tubular cisternae, many free ribosomes, many mitochondria, but only few, if any, large secretory granules. Apparently, plasmatocytes are not affected by endotoxin. The relationship and possible functions of granulocytes and plasmatocytes are discussed and compared with those of the horseshoe crab, Limulus polyphemus.

Pronuclear fusion failure: an alternate conjugational pathway in Tetrahymena thermophila, induced by vinblastine.

Vinblastine is shown to induce pronuclear fusion failure in conjugating Tetrahymena thermophila. In this alternate conjugational pathway gametic pronuclei are exchanged between conjugants but do not fuse. Each pronucleus undergoes one mitotic division to produce a new macro- and micronucleus. Genetic consequences of pronuclear fusion failure include the following: (1) the progeny are whole genome homozygotes with nuclei derived from single meiotic products, and (2) half of the progeny are heterokaryons with micro- and macronuclei of different genetic origins. These facts make this process extremely useful in strain construction and mutant isolation. The induction of pronuclear fusion failure by vinblastine suggests that microtubules play an essential role in pronuclear fusion.

Polypeptides during early conjugation in Tetrahymena thermophila.

As Tetrahymena thermophila cells differentiate from their vegetative life cycle to sexual reproduction, their polypeptide pattern undergoes a series of changes. These changes have been traced in extracellular, cellular, and subcellular compartments. The first alteration is induced by the nutritional shift-down and results in stimulation of at least one ciliary polypeptide and affects a series of polypeptides from other compartments. The second alteration is induced by mixing starved cells of complementary mating types and this stimulates the synthesis of nine ciliary polypeptides before pairs have formed and eight afterwards. At least five of these early and one of the late conjugation-related ciliary polypeptides are removed by low concentrations of EDTA, indicating that they are located on the external side of the plasma membrane. No differences were observed between polypeptides excreted during starvation and after mixing of complementary mating types. At Tris concentrations restrictive for conjugation, cilia lack the conjugation-related polypeptides. Some of these are instead found among the excreted polypeptides. Using O'Farrell gels and silver staining on isogenic cells of all possible mating types, we have been unable to correlate changes in polypeptide patterns to specific mating types.

Utilization of iron complexes in an animal cell.

The ciliate protozoan Tetrahymena thermophila was grown in synthetic nutrient medium in the absence of the iron chelator citrate. Utilization and toxicity of various iron compounds or complexes in iron-starved cells were assessed from the number of cell doublings obtained within a standard time. The compounds tested included complexes formed between ortho-phosphates and two forms of ferric hydroxides, native and cationized ferritin, and tris-acetylacetonato Fe(III). The ferric hydroxo ortho-phosphate particles are toxic and can be removed from the medium by Millipore filtration. Uptake of ferritin and tris-acetylacetonato-Fe(III) is independent of food vacuole formation and seems to occur by micropinocytosis and by plasma membrane translocation, respectively.

Tetrahymena. Adaptation to high iron.

Fe-starved ciliates Tetrahymena thermophila cease to multiply at Fe(III) concentrations above 10 microM in a synthetic nutrient medium lacking a good iron chelator such as citrate. If, however, the Fe(III) concentration is gradually increased over a series of subcultivations the cells will tolerate up to 300 microM Fe(III). Our experiments rule out the possibility of genetic selection of Fe-tolerant clones and suggest a physiological type of adaptation.

Stage-specific changes in protein synthesis during conjugation in Tetrahymena thermophila.

Conjugation in the free-living ciliate Tetrahymena thermophila is an inducible developmental system which results in a synchronized reorganization of the genetic material in both mates of a pair. The cytological events were followed by Feulgen stainings of simultaneously mating cells and protein synthesis was revealed using [35S]methionine pulse labelling and two-dimensional gel electrophoresis. At least 33 proteins, including 24 conjugation-specific proteins, with apparent molecular weights (Mr) between 61 and 200 X 10(3) are stimulated during conjugation. Two slightly acidic proteins (Mr 89 and 73 X 10(3), respectively) are stimulated shortly after mixing of mating-competent cells and mainly before tight pairs are formed. Ten proteins are stimulated during meiosis, and two of these (Mr 90 and 78 X 10(3), respectively) are particularly interesting, since they are highly stimulated and more basic (pI values around 8.5) than most other proteins detected. Twelve proteins are stimulated essentially between pairing and early macronuclear development, three are stimulated from shortly before zygote formation and during the post-zygotic divisions, and six are stimulated during late conjugation, at various parts of macronuclear development. The functions of the conjugation-stimulated proteins are discussed.

Mutants of TETRAHYMENA THERMOPHILA with Temperature-Sensitive Food Vacuole Formation. I. Isolation and Genetic Characterization.

Germ-line mutants have been isolated in Tetrahymena thermophila that have recessive, temperature-sensitive defects in phagocytosis. Nitrosoguanidine-mutagenized cells were induced to undergo cytogamy, and clones were isolated that were unable to form food vacuoles after two days of growth at 39 degrees . Most of the mutants belong to a single complementation group, designated vacA. They have defects in oral development-not in phagocytosis per se-that are undetectable under light microscopy. One fertile mutant, phenotypically indistinguishable from the vacA group, has its vac mutation(s) restricted to the macronucleus, and it is a heterokaryon for two other markers. This clone probably resulted from a failure of the two gametic nuclei to fuse after normal exchange. Two additional mutants were studied, but their sterility prevented a full genetic analysis. One of these clones has a rudimentary oral apparatus and defective contractile vacuole pores; both defects may be determined by the same mutation. The other clone has a structurally normal oral apparatus and may be defective in phagocytosis per se.-The induction and characterization of germ-line mutations that affect oral development open the way for the genetic dissection of the morphogenesis of a complex eukaryotic organelle, and make available additional useful mutants for the study of nutrition and transmembrane active transport.