Autoradiographic localization of tritiated ouabain-sensitive sodium pump sites in ion transporting epithelia

The freeze-dry autoradiographic method devised originally by Stirling (J Cell Biol 53:704, 1972) to localize Na+ pump sites with (3H)ouabain is reviewed. Biochemical, physiological, and autoradiographic data are discussed which establish that ouabain binding to intact tissue conforms to rigid criteria for high Na+ pump specificity. Among these are that glycoside binding exhibits saturation kinetics, ligand dependence, and close correlation with degrees of inhibition of Na+-K+-ATPase and Na+ transport. Moreover, localization of Na+ pump sites by this technique shows a cell and membrane specificity which mirrors that obtained by cytochemical and immunocytochemical methods. In addition to resolving cell-specific patterns of localization in heterogeneous tissues, the demonstration of Na+-K+-ATPase by these techniques indicates that Na+ pumps are distributed uniformly along plasmalemmal surfaces and are restricted to the basolateral interface in reabsorptive and secretory epithelia despite the opposing polarity of net transepithelial electrolyte transport.     

Gangliosides of sea urchin embryos. Their localization and participation in early development

The influence of antibodies to gangliosides of sea urchin Strongylocentrotus intermedius eggs on early embryos of this species was studied. gamma-Globulins were isolated from rabbit anti-ganglioside serum by micropreparative electrophoresis. These gamma-globulins produced anomalies in the development of embryos permeabilized in Triton X-100. The anomalies were not observed when anti-ganglioside gamma-globulins were added to the incubation medium together with gangliosides or when the permeabilized embryos were incubated with gamma-globulins of normal rabbit serum. Pretreatment of S. intermedius embryos with serotonin, tryptamine or some other indole derivatives led to the disappearance of ganglioside determinants from the cell surface and sharply increased immunofluorescence within the cell. Such pretreatment of embryos increased the amount of cell-associated gangliosides more than threefold as compared to untreated embryos. Serotonin was shown to bind specifically to sea urchin gangliosides immobilized on octyl-Sepharose. These observations suggest that cell-surface gangliosides, after binding drugs, are internalized and that serotonin and its antagonists inhibit the transport of newly synthesized gangliosides to the cell-surface membrane.     

Presence of serotonin in early chick embryos

With biochemical analysis and with autoradiography based on injection of 5-[3H]hydroxytryptophan, it was possible to demonstrate the presence of serotonin (5-hydroxytryptamine) in early chick embryos as early as the pre-streak stage. The biochemical analysis which covered the early developmental period (0.5-6 days of incubation) revealed an elevated concentration of serotonin at gastrulation; from then it stayed at a lower and fairly even level. Autoradiographs of embryos at the pre-streak stage, the primitive streak stage, the head fold stage and the 4-6 somites stage indicated the presence of serotonin in intracellular yolk granules and in cell nuclei. Moreover, the amine appeared associated with microfilaments and microtubules, particularly in developing neural cells. Notably the elevated concentration of serotonin at gastrulation, but also the intracellular distribution of the amine during early organogenesis, indicates a prominent role for it in cell-shape changes and morphogenesis in the early chick embryo.     

Autoradiographic localization of angiotensin II receptors in the rat brainstem

The microscopic localization of angiotensin II receptors in the rat brainstem has been accomplished utilizing in vitro receptor autoradiographic techniques. These receptors are highly localized to discrete nuclear regions of the brainstem. Significant densities of autoradiographic grains, indicating the presence of specifically bound [125I]-angiotensin II, were observed in regions of the film corresponding to the nucleus of the solitary tract, dorsal motor nucleus of the vagus nerve, nucleus intercalatus, nucleus commissuralis, substantia gelatinosa of the trigeminal nerve and the inferior olivary nucleus. Previous immunohistochemical studies have indicated the presence of immunoreactive angiotensin II in several of these regions, especially those concerned with central cardiovascular regulatory mechanisms. These results provide additional evidence in support of the hypothesized role of angiotensin II as a central neuromodulator in the regulation of systemic blood pressure.     

Autoradiographic localization of angiotensin II receptors in developing rat cerebellum and brainstem

The role of Angiotensin II (Ang II) as a growth promoting or modulating factor has recently become a field of intensive research. A central issue in developmental neurobiology is the understanding of mechanisms governing the formation of spatially ordered connections. In this study, we show the localization of Ang II receptor subtypes by autoradiography in 2-week-old rat hindbrains confronting these data with membrane binding assays. Competition studies done on membrane preparations evidence no major changes on the relative affinities for both receptor subtypes between 2-week-old and adult rat tissues. By autoradiography, we found that all the areas (1-10) of the 2-week-old cerebellum showed both receptor subtypes present in complementary adjacent layers. Areas expressing a high level of AT2 receptors follow: inferior colicullus (IC), dorso tegmental nucleus, central (DTgC), subcoeruleus, alpha, sensory root of the trigeminal nerve, principal sensory root trigeminal nucleus (Pr5, Pr5VL) supragenual nucleus, genu facial nerve, facial nucleus, cerebellar peduncles, vestibular and lateral nuclei. Spinal trigeminal, (oral) and Raphe nuclei express AT1 receptor subtype. The high level of Ang II AT2 receptors present in the cerebellar peduncles might have a meaning on the establishment of the olivo-cerebellar connection. The high expression of Ang II AT2 receptors on 2-week-old rat hindbrains, a critical age on development, as well as its disappearance in the adult, strongly suggests a probable role of these receptors in cell migration and neuronal synaptogenesis.     

SAR development of a selective 5-HT1D antagonist/serotonin reuptake inhibitor lead using rapid parallel synthesis

Incorporation of an SRI (serotonin reuptake inhibitor) pharmacophore into a selective 5-HT(1D) agonist has led to the discovery of a molecule having both 5-HT(1D) antagonist and SRI activity. RPS methodology was used to develop the SAR and identify potential approaches to reduce unwanted adrenergic alpha 1 and dopamine D(2) cross-reactivities.     

Immunocytochemical localization of serotonin in embryos, larvae and adults of the lancelet, Branchiostoma floridae

Serotonin (5-hydroxytryptamine) is a biogenic amine distributed throughout the metazoans and has an old evolutionary history. It is involved as a developmental signal in the early morphogenesis of both invertebrates and vertebrates, whereas in adults it acts mainly as a neurotransmitter and gastrointestinal hormone. In vertebrates, serotonin regulates the morphogenesis of the central nervous system and the specification of serotonergic as well as dopaminergic neurons. The present study uses, as an experimental model, an invertebrate chordate, the lancelet Branchiostoma floridae, characterized by its remarkable homologies with vertebrates that allows the 'bauplan' of the probable ancestor of vertebrates to be outlined. In particular, the involvement of serotonin as a developmental signal in embryos and larvae, as well as a neurotransmitter and gastrointestinal hormone in adult specimens of Branchiostoma floridae, gives further support to a common origin of cephalocordates and vertebrates.     

Transplacental delivery of the Wnt antagonist Frzb1 inhibits development of caudal paraxial mesoderm and skeletal myogenesis in mouse embryos.

Axial structures (neural tube/notochord) and surface ectoderm activate myogenesis in the mouse embryo; their action can be reproduced, at least in part, by several molecules such as Sonic hedgehog and Wnts. Recently, soluble Wnt antagonists have been identified. Among those examined only Frzb1 was found to be expressed in the presomitic mesoderm and newly formed somites and thus its possible role in regulating myogenesis was investigated in detail. When presomitic mesoderm or newly formed somites were cultured with axial structures and surface ectoderm on a feeder layer of C3H10T1/2 cells expressing Frzb1, myogenesis was abolished or severely reduced in presomitic mesoderm and the three most recently formed somites. In contrast, no effect was observed on more mature somites. Inhibition of myogenesis did not appear to be associated with increased cell death since the final number of cells in the explants grown in the presence of Frzb1 was only slightly reduced in comparison with controls. In order to examine the possible function of Frzb1 in vivo, we developed a method based on the overexpression of the soluble antagonist by transient transfection of WOP cells with a Frzb1 expression vector and injection of transfected cells into the placenta of pregnant females before the onset of maternofoetal circulation. Frzb1, secreted by WOP cells, accumulated in the embryo and caused a marked reduction in size of caudal structures. Myogenesis was strongly reduced and, in the most severe cases, abolished. This was not due to a generalized toxic effect since only several genes downstream of the Wnt signaling pathway such as En1, Noggin and Myf5 were downregulated; in contrast, Pax3 and Mox1 expression levels were not affected even in embryos exhibiting the most severe phenotypes. Taken together, these results suggest that Wnt signals may act by regulating both myogenic commitment and expansion of committed cells in the mouse mesoderm.     

Autoradiographic studies on the distribution of labeled maternal RNA in early mouse embryos

Maternal RNA of mouse eggs and embryos was labeled by exposure of growing ovarian oocytes to 3H-uridine in vivo 8 to 16 days before ovulation and fertilization. Labeled embryos from the 1-cell stage to the blastocyst stage were collected, fixed, and autoradiographs of plastic sections prepared. The observed grain density was similar in the pronuclei and in the cytoplasm of 1-cell embryos. Knowing the volumes of nucleus and cytoplasm, it was determined that 3% of the maternal RNA was found in the pronuclei. It is suggested that some of this nuclear RNA may be stable small nuclear RNAs (e.g. U1 RNA) retained from the germinal vesicle stage through meiotic maturation. During the 2-cell stage and beyond, maternal RNA is degraded and labeled precursor is reincorporated into nuclear RNA, making it difficult to accurately quantitate the amount of nuclear maternal RNA. It is known that about one third of the total maternal RNA is lost between the 8-cell and blastocyst stages. It was found that cytoplasmic grain densities in inner and outer cells of the morula and blastocyst were not significantly different. Thus, the loss of maternal RNA does not proceed more rapidly in the differentiating trophoblast than in the inner cell mass.     

Autoradiographic localization of vasoactive intestinal polypeptide receptors in the rat mesenteric vascular tree

By the use of combined in vitro radioreceptor binding and autoradiographic techniques, we analyzed the pharmacological properties and the anatomical localization of the vasoactive intestinal polypeptide (VIP) receptor in rat superior mesenteric artery and in medium and small mesenteric artery branches. 125I-VIP was bound by sections of rat superior mesenteric artery in a manner consistent with the labeling of specific VIP receptors, with Kd and Bmax values of 0.23 nM and 0.71 pmol/mg protein respectively. Inhibition of 125I-VIP binding with VIP and related peptides gives the following rank order of potency: VIP greater than peptide histidine methionine greater than secretin. Light microscope autoradiography reveals specific VIP binding sites within the medial layer of superior mesenteric artery and its branches. Medium and small sized vessels are richer in 125I-VIP binding sites than the larger ones.     

Identification of genes required for cytoplasmic localization in early C. elegans embryos

We have isolated and analyzed eight strict maternal effect mutations identifying four genes, par-1, par-2, par-3, and par-4, required for cytoplasmic localization in early embryos of the nematode C. elegans. Mutations in these genes lead to defects in cleavage patterns, timing of cleavages, and localization of germ line-specific P granules. Four mutations in par-1 and par-4 are fully expressed maternal effect lethal mutations; all embryos from mothers homozygous for these mutations arrest as amorphous masses of differentiated cells but are specifically lacking intestinal cells. Four mutations in par-2, par-3, and par-4 are incompletely expressed maternal effect lethal mutations and are also grandchildless; some embryos from homozygous mothers survive and grow to become infertile adults due to absence of functional germ cells. We propose that all of these defects result from the failure of a maternally encoded system for intracellular localization in early embryos.     

Autoradiographic and functional development of gastric cholecystokinin receptors in the rat

To determine the functional role for and the pharmacological specificity of developing gastrointestinal CCK receptors, in vitro pyloric contractility and autoradiographic CCK receptor binding were examined in pups aged 1–20 days. CCK contracted the gastroduodenal junction at all ages, while nonsulfated CCK-8 (d-CCK) was less potent. Autoradiographic studies revealed CCK binding localized to the gastroduodenal junction throughout development. MK-329, a specific type A CCK receptor antagonist, completely displaced ¹²⁵I CCK-8 binding at all ages, while d-CCK displaced binding at ages at which d-CCK elicited gastroduodenal contractility. The results demonstrate a physiological role for and pharmacological specificity of neonatal gastroduodenal CCK receptors.     

The early development of the lymphatic system in mouse embryos.

The early development of the lymphatic system was studied in embryos of an inbred strain of the laboratory mouse. During the first stage of its development the system is represented by a more or less regular series of small and blind-ending outgrowths of the major embryonic veins which develop in a cranio-caudalward direction from the jugular to the pelvic region. As a result of differences in growth rates of adjacent anatomical structures this series of early lymphatic primordia becomes subdivided into 4 singular primordia and 12 groups of primordia. After the constituents of each group of early primordia have fused, 16 isolated lymphatic plexuses (sacs) are formed of which 14 are in bilaterally symmetric and 2 are in a median line position: i.e. bilaterally: (1) the jugulo-axillary lymph sac situated lateral to the anterior cardinal vein and dorsal to the primitive ulnar vein and its major branch, the external mammary vein, (2) the paratracheal lymph plexus situated medial to the anterior cardinal vein, (3) the internal thoracic lymph plexus situated lateral to the thoracic part of the posterior cardinal vein, (4) the thoracic ducts situated medial to the thoracic part of the posterior cardinal vein, (5) the lumbar lymph plexus situated dorso-lateral to the abdominal part of the posterior cardinal vein, (6) the subcardinal lymph plexus and (7) the iliac lymph plexus situated ventro-lateral to the abdominal part of the posterior cardinal vein; and in the median line: (8) the subtracheal lymph plexus situated at the confluence of the pulmonary veins and (9) the mesenteric lymph plexus situated near the confluence of the splenic and the superior mesenteric veins. Except for some openings at the jugulo-subclavian confluence all connections with the veins disappear. From the primordia extensions grow out centrifugally. They invade the surrounding tissues and, in part, fuse with similar sprouts of adjacent primordia. In this way a continuous system of lymph truncs is formed that opens into the venous system at the jugulo-subclavian confluence.     

PAN GU: a protein kinase that inhibits S phase and promotes mitosis in early Drosophila development

Following completion of meiosis, DNA replication must be repressed until fertilization. In Drosophila, this replication block requires the products of the pan gu (png), plutonium (plu) and giant nuclei (gnu) genes. These genes also ensure that S phase oscillates with mitosis in the early division cycles of the embryo. We have identified the png gene and shown that it encodes a Ser/Thr protein kinase expressed only in ovaries and early embryos, and that the predicted extent of kinase activity in png mutants inversely correlates with the severity of the mutant phenotypes. The PLU and PNG proteins form a complex that has PNG-dependent kinase activity, and this activity is necessary for normal levels of mitotic cyclins. Our results reveal a novel protein kinase complex that controls S phase at the onset of development apparently by stabilizing mitotic cyclins.     

Autoradiographic visualization of alpha 1-adrenergic receptors in cervix of early pregnant rat

alpha 1-Adrenergic receptors were identified, characterized, and localized in rat cervix on Day 6 of pregnancy by autoradiography. Autoradiographic study was performed in slide-mounted rat cervix sections using [3H]-prazosin ([3H]-PRAZ) as ligand. Binding was time dependent and specific. Pharmacological study indicated that specific [3H]-PRAZ binding was inhibited with high affinity by prazosin and phenylephrine and low affinity by yohimbine and clonidine. In cervix, the alpha 1-adrenergic receptors were localized mainly to the inner circular layer of the myometrium. Binding to the outer longitudinal layer of myometrium was moderate, and binding was absent in the endometrium. The regional distribution of alpha 1-adrenergic receptors strongly suggests that the circular layer of myometrium may function as an important modulator of contractile response of the cervix, probably involved in the retention of blastocysts at the utero-cervical end of the horn.     

Ultrastructural localization of dopaminergic receptors in the rat corpus striatum by radioautography using tritiated domperidone

An attempt to localize dopaminergic receptors in the rat neostriatum by high-resolution radioautography was realized using intracerebral injections of the new ligand (antagonist) 3H-domperidone. In tissue regions located far from the injection site, the weaker diffuse radioautographic reaction permitted us to observe the existence of clusters of silver grains over some cerebral structures. The specificity of this type of labelling was tested using intraperitoneal injections of large amounts of haloperidol in order to block the studied receptors. Thus, we observed specific labelling over some nerve terminals as well as a low number of synaptic contact of the asymmetric type (however some synapses of the symmetric type were also labelled). These result agree with our previous work (1), and confirm the existence of dopaminergic synapses in the rat caudate-putamen.     

Human platelet alpha 2-adrenergic receptors: labeling with 3H-yohimbine, a selective antagonist ligand

³H-Yohimbine, a potent and selective pharmacological antagonist of α₂-adrenergic receptors, labeled human platelet membrane α₂-receptors with high affinity. Binding was rapid and reversible at 25°C. Both saturation and kinetic experiments indicated a single order of binding sites, with an equilibrium K_D value of 1.0–1.5 nM. Low Mg²⁺ concentrations increased the K_D for ³H-yohimbine without altering the B_max. The ³H-yohimbine site exhibited α₂-receptor specificity: (?)-norepinephrine and (?)-isoproterenol were 4.8 and 330 times less potent than (?)-epinephrine; (?)-catecholamines were 17–35 times more potent than corresponding (+)-catecholamines; the selective α₁-antagonist prazosin was 340 times less potent than yohimbine. Catecholamine agonists exhibited shallow curves in inhibiting ³H-yohimbine binding, with pseudo-Hill coefficients (n_H) of less than 1.0, whereas the n_H of antagonists was 1.0. No specific binding of ³H-prazosin to platelet membranes was observed, indicating the absence of α₁-receptors. ³H-Yohimbine labeled fewer platelet sites than did ³H-dihydroergocryptine under identical conditions (80 vs 130 receptors/ cell), and may be a more specific and useful antagonist probe of platelet α₂-receptors than ³H-dihydroergocryptine.