The CCK-2 Receptor is Located on the ECL Cell,But Not on the Parietal Cell

Background: The interrelationship between histamine and gastrin in the physiological regulation of gastric acid secretion is still a matter of dispute. CCK-2 receptors are located on enterochromaffin-like (ECL) cells in corpus mucosa and gastrin stimulates acid production by releasing histamine from the ECL cells, which in turn stimulates the parietal cells. Whether parietal cells also possess gastrin receptors of physiological significance is unclear. The aim of the present study was to localize the CCK-2 receptor cellularly and concomitantly demonstrate a gastrin receptor response (histamine release). Methods: Fluorescein labelled cholecystokinin-8 (Fluo-CCK-8) was added to the arterial infusion to totally isolated, vascularly perfused rat stomachs to a final concentration of 130 pmol L -1 for 1 min, either alone or along with 520 nmol -1 CCK-8 after 10-min pre-perfusion with CCK-8. Immediately after the FluoCCK-8 had reached the oxyntic mucosa, biopsies were taken and the binding sites were localized by double immunohistochemistry combined with the tyramide signal amplification (TSA) technique. Venous histamine was measured before and during stimulation. Results: Fluo-CCK-8 (130 pM) evoked histamine release, and binding sites were found in the basal part of corpus mucosa, co-localized with histidine decarbocylase (HDC) immunoreactive ECL cells. No binding of Fluo-CCK was found in the midglandular region of corpus, dominated by parietal cells. Binding of Fluo-CCK-8 was abolished by concomitant perfusion with excess CCK-8. Conclusion: Fluo-CCK-8 given to isolated rat stomachs in a physiological concentration binds to CCK-2 receptors on ECL cells and causes histamine release, whereas no binding of Fluo-CCK-8 to parietal cells was found.     

The gene for the human immune interferon receptor is located on chromosome 6.

When 32P-labeled human recombinant immune interferon gamma (Hu-[32P]IFN-gamma) is crosslinked to human cells with disuccinimidyl suberate, a complex with a molecular size of approximately equal to 117,000 Da was identified by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The formation of this complex is inhibited when the binding is performed in the presence of excess unlabeled Hu-IFN-gamma. The specific formation of the 117,000-Da complex is not observed in mouse L cells or Chinese hamster ovary cells. This complex shows all of the criteria that identify it as the Hu-IFN-gamma receptor or its binding subunit. The same complex can be formed following binding and covalent crosslinking of Hu-[32P]IFN-gamma to some hamster-human or mouse-human somatic cell hybrids. The presence of human chromosome 6 in the hybrids is necessary and sufficient for the formation of this complex. More specifically, the long arm of chromosome 6 seems sufficient. Therefore, we have localized the gene for the Hu-IFN-gamma receptor (or its binding subunit) to the long arm of human chromosome 6. The presence of this chromosome in the somatic cell hybrids is not adequate, however, to confer antiviral resistance to the hybrids in the presence of Hu-IFN-gamma.     

An epitope of the transferrin receptor is exposed on the cell surface of high-grade but not low-grade human lymphomas

In attempting to identify antigens that are differentially expressed on tumor cells following transformation from follicular small cleaved cell lymphoma (FSC) to immunoblastic lymphoma (IL), we identified a unique epitope of the transferrin receptor (TfR). The epitope is available for binding in aggressive lymphomas but not in indolent lymphomas or normal cells. An immunoglobulin G2a (IgG2a) antibody that binds this epitope, Trump, was produced by screening on tumor cells from a patient who initially had a low-grade lymphoma which subsequently converted to a high-grade lymphoma. Immunoprecipitation and comodulation studies show that Trump binds to the TfR, but blocking studies and immunostaining reveal that the TfR epitope seen by Trump is distinct from the OKT9 and anti-TfR binding sites. The ability of Trump to discriminate a separate population of more highly malignant cells suggests that the expression of the Trump epitope is determined by the state of activation or degree of malignancy of the cell. In addition, it may be possible to use the Trump antibody diagnostically or therapeutically in the management of lymphomas.     

Notch receptor cleavage depends on but is not directly executed by presenilins

Notch receptors undergo three distinct proteolytic cleavages during maturation and activation. The third cleavage occurs within the plasma membrane and results in the release and translocation of the intracellular domain into the nucleus to execute Notch signaling. This so-called gamma-secretase cleavage is under the control of presenilins, but it is not known whether presenilins themselves carry out the cleavage or whether they act by means of yet-unidentified gamma-secretase(s). In this article, we show that Notch intracellular cleavage in intact cells completely depends on presenilins. In contrast, partial purification of the Notch cleavage activity reveals an activity, which is present only in protein extracts from presenilin-containing cells, and which does not comigrate with presenilin. This finding provides evidence for the existence of a specific Notch-processing activity, which is physically distinct from presenilins. We conclude from these experiments that presenilins are critically required for Notch intracellular cleavage but are not themselves directly mediating the cleavage.     

The muscarinic receptor gene expressed in rabbit parietal cells is the m3 subtype.

To investigate the nature of the muscarinic receptors present on parietal cell membranes, binding studies and polymerase chain reaction (PCR) amplification of parietal cell messenger (m) RNA were undertaken. Displacement of N-[3H]methylscopolamine by various muscarinic antagonists showed displacement with a single affinity. The apparent dissociation constant values were as follows: atropine (nonselective), 1.95 +/- 0.28 nmol/L; pirenzepine (M1), 169 +/- 24 nmol/L; AF-DX 116 (M2), 1542 +/- 33 nmol/L; and hexahydrosiladifenidol (M3), 29 +/- 3.4 nmol/L. These data confirmed the existence of only an M3 receptor linked to acid secretion as defined pharmacologically. PCR amplification of parietal cell mRNA with primers designed for detection of all known muscarinic receptor subtypes showed that only m3 fragments were produced from parietal cell mRNA, whereas m1 and m2 products could be detected in brain or cardiac mRNA. The m3 nature of the PCR product was confirmed by Southern blotting with 32P-labeled human m3 complementary DNA. Hence the two carbachol affinities and the separable cellular responses following muscarinic activation are caused by separate coupling pathways of the M3 receptor.     

Pentagastrin gastroprotection against acid is related to H2 receptor activation but not acid secretion

Background—Pentagastrin enhancesgastric mucosal defence mechanisms against acid and protects thegastric mucosa from experimental injury.
Aims—To investigate whether thisgastroprotection is mediated by histamine receptors or occurs as asecondary effect of acid secretion stimulation.
Methods—The effects ofomeprazole (100 µmol/kg), ranitidine (20 mg/kg), and pyrilamine (10 mg/kg) on pentagastrin (80 µg/kg/h) induced gastroprotection againstacidified aspirin injury were examined in a luminal pH controlledmodel. The effects of these compounds on pentagastrin enhancedgastroprotective mechanisms were investigated using intravitalmicroscopy, in which intracellular pH of gastric surface cells(pH_i), mucus gel thickness, gastric mucosal blood flow, andacid output were measured simultaneously.
Results—Pentagastrin protected ratgastric mucosa from acidified aspirin injury. This gastroprotection wasabolished by ranitidine, but not omeprazole or pyrilamine. Pentagastrininduced a hyperaemic response to luminal acid challenge, increasedmucus gel thickness, and elevated pH_i during acidchallenge. Ranitidine reversed these enhanced defence mechanisms,whereas omeprazole and pyrilamine preserved these effects.
Conclusions—These dataindicate that pentagastrin associated gastroprotection and enhanceddefence mechanisms against acid result mainly from activation ofhistamine H₂ receptors, and not as an effect of thestimulation of acid secretion.

Keywords:gastric injury; gastric defence mechanisms; omeprazole; pyrilamine; ranitidine; intracellular pH

     

The unliganded glucocorticoid receptor is localized in the nucleus, not in the cytoplasm.

The glucocorticoid receptor (GR) is often described to be localized in the cytoplasm in the absence of hormone and to translocate to the nucleus upon binding of the hormone. This apparently different behavior of the GR compared to that of other members of the steroid receptor superfamily is unexpected because similarities in the molecular structures of steroid hormone receptors would predict similarities in their working mechanisms. The absence of the unliganded GR from the nuclear compartment may be due to an artefactual redistribution, occurring during the immunocytochemical procedure. We systematically studied the effects of various fixation and permeabilization procedures on the distribution of the GR in hepatoma cells that were incubated in steroid-free or steroid-containing medium. Immunofluorescent labeling of the GR in formaldehyde-fixed and detergent-permeabilized cells resulted in the almost complete absence of immunoreactivity of cells when the receptor was in its unliganded form, whereas the liganded receptor was detected mainly in the nucleus. On the other hand, labeling of cryosectioned glutaraldehyde/formaldehyde-fixed cells demonstrated that receptor antigenicity is present in the nucleus in the absence as well as the presence of steroid. We conclude that the results obtained with the cryosectioning procedure reflect the receptor distribution in the living cell, since glutaraldehyde fixation of the cells prevented the washout of loosely bound receptor. The predominantly nuclear location of the unliganded GR in hepatoma cells and the absence of changes in distribution after steroid stimulation imply that the nuclear translocation model is not true for the GR.     

The epidermal growth factor receptor (EGF-R) is present on the basolateral, but not the apical, surface of enterocytes in the human gastrointestinal tract.

BACKGROUND: While it is clear that luminal epidermal growth factor (EGF) stimulates repair of the damaged bowel, its significance in maintaining normal gut growth remains uncertain. If EGF is important in maintaining normal gut growth, the EGF receptor (EGF-R) should be present on the apical (luminal) surface in addition to the basolateral surface. AIMS/SUBJECTS/METHODS: This study examined the distribution of the EGF-R in the epithelium throughout the human gastro-intestinal tract using immunohistochemistry, electron microscopy, and western blotting of brush border preparations. RESULTS: Immunostaining of the oesophagus showed circumferential EGF-R positivity in the cells of the basal portions of the stratified squamous epithelium but surface cells were EGF-R negative. In the normal stomach, small intestine, and colon, immunostaining localised the receptor to the basolateral surface with the apical membranes being consistently negative. EGF-R positivity within the small intestine appeared to be almost entirely restricted to the proliferative (crypt) region. Western blotting demonstrated a 170 kDa protein in whole tissue homogenates but not in the brush border vesicle preparations. CONCLUSIONS: As the EGF-R is located only on the basolateral surfaces in the normal adult gastrointestinal tract, the major role of luminal EGF is probably to stimulate repair rather than to maintain normal gut growth.     

Loss of Tie2 receptor compromises embryonic stem cell-derived endothelial but not hematopoietic cell survival

Tie2 is a receptor-type tyrosine kinase expressed on hematopoietic stem cells and endothelial cells. We used cultured embryonic stem (ES) cells to determine the function of Tie2 during early vascular development and hematopoiesis. Upon differentiation, the ES cell-derived Tie2+ Flk1+ fraction was enriched for hematopoietic and endothelial progenitor cells. To investigate lymphatic differentiation, we used a monoclonal antibody against LYVE-1 and found that LYVE-1+ cells derived from Tie2+ Flk1+ cells possessed various characteristics of lymphatic endothelial cells. To determine whether Tie2 played a role in this process, we analyzed differentiation of Tie2-/- ES cells. Although the initial numbers of LYVE-1+ and PECAM-1+ cells derived from Tie2-/- cells did not vary significantly, the number of both decreased dramatically upon extended culturing. Such decreases were rescued by treatment with a caspase inhibitor, suggesting that reductions were due to apoptosis as a consequence of a lack of Tie2 signaling. Interestingly, Tie2-/- ES cells did not show measurable defects in development of the hematopoietic system, suggesting that Tie2 is not essential for hematopoietic cell development.     

The apoptotic-cell receptor CR3, but not alphavbeta5, is a regulator of human dendritic-cell immunostimulatory function

Dendritic cells (DCs) that capture apoptotic cells (ACs) in the steady state mediate peripheral tolerance to self-antigens. ACs are recognized by an array of receptors on DCs, the redundancy of which is not completely defined. We made use of an AC surrogate system to address the individual roles of the alphavbeta5 and complement receptors (CRs) in the phagocytosis and induction of immunity. CR3 and CR4, while substantially less efficient than alphavbeta5 in internalizing ACs, initiate signals that render DCs tolerogenic. Responding T cells show impaired proliferation and IFNgamma production and subsequently die by apoptosis. While tolerogenic DCs are not induced via alphavbeta5, coligation of CR3 and alphavbeta5 maintains the DC's tolerogenic profile. This immunomodulatory role, however, is countered by a significant inflammatory stimulus such as bacterial infection. Overall, our data suggest that under steady-state conditions, signaling via CRs predominates to render DCs tolerogenic.     

The P2Y1 receptor, necessary but not sufficient to support full ADP-induced platelet aggregation, is not the target of the drug clopidogrel

Recently we showed that the P2Y₁ receptor coupled to calcium mobilization is necessary to initiate ADP-induced human platelet aggregation. Since the thienopyridine compound clopidogrel specifically inhibits ADP-induced platelet aggregation, it was of interest to determine whether the P2Y₁ receptor was the target of this drug. Therefore we studied the effects of clopidogrel and of the two specific P2Y₁ antagonists A2P5P and A3P5P on ADP-induced platelet events in rats. Although clopidogrel treatment (50 mg/kg) greatly reduced platelet aggregation in response to ADP as compared to untreated platelets, some residual aggregation was still detectable. In contrast, A2P5P and A3P5P totally abolished ADP-induced shape change and aggregation in platelets from both control and clopidogrel-treated rats. A2P5P and A3P5P (100 μM ) totally inhibited the [Ca²⁺]_i rise induced by ADP (0.1 μM ) in control and clopidogrel-treated platelets, whereas clopidogrel treatment had no effect. Conversely, the inhibition of adenylyl cyclase induced by ADP (5 μM ) was completely blocked by clopidogrel but not modified by A2P5P or A3P5P (100 μM ). A3P5P (1 m M ) reduced the number of [³³P]2MeSADP binding sites on control rat platelets from 907 ± 50 to 611 ± 25 per platelet. After clopidogrel treatment, binding of [³³P]2MeSADP decreased to 505 ± 68 sites per platelet and further decreased to 55 ± 12 sites in the presence of A3P5P (1 m M ). In summary, these results demonstrate that the platelet P2Y₁ receptor responsible for the initiation of aggregation in response to ADP is not the target of clopidogrel. Platelets may express another, as yet unidentified, P2Y receptor, specifically coupled to the inhibition of adenylyl cyclase and necessary to induce full platelet aggregation, which could be the target of this drug.     

Cointegrate formation by Tn5, but not transposition, is dependent on recA.

We have studied the effect of the recA-dependent homologous recombination system of Escherichia coli on both Tn5-mediated cointegrate formation and Tn5 transposition. We demonstrate here that, whereas transposition of Tn5 is independent of the recA gene product (as has been shown by other workers), Tn5-mediated cointegrate formation is strongly dependent on recA. The structures of both the simple transposition products and the cointegrates formed in a recA- background seem to be the same as those produced in a recA+ background. These results provide strong evidence that Tn5 does not transpose via an obligate cointegrate intermediate and suggest that the recA effect on cointegrate formation is exerted during the process of transposition.     

Promotion of murine hepatocarcinogenesis by testosterone is androgen receptor-dependent but not cell autonomous.

Tfm (testicular feminization) mutant mice lack functional androgen receptors. By studying liver tumor development in Tfm mice, we have shown that the greater susceptibility of male mice relative to female mice for liver tumor induction by N,N-diethylnitrosamine is androgen receptor-dependent. C57BL/6J normal and Tfm mutant mice were injected at 12 days of age with N,N-diethylnitrosamine (0.2 mumol/g, i.p.), and liver tumors were enumerated in 50-week-old animals. Normal males averaged 20 liver tumors per animal; Tfm males, 0.7; normal females, 0.6; and Tfm/+ heterozygous females, 1.5. The androgen receptor gene and the Tfm mutation are X chromosome linked. Because of random X chromosome inactivation, hepatocytes from Tfm/+ heterozygous female mice are mosaic with respect to the expression of mutant or wild-type receptors. To determine if testosterone acts directly as a liver tumor promoter, through the androgen receptor in preneoplastic hepatocytes, or by an indirect mechanism, we chronically treated these mosaic female mice with testosterone and measured the androgen receptor content of the resulting tumors. B6C3F1 Tfm/+ mosaic and +/+ wild-type female mice were injected i.p. at 12 days of age with N,N-diethylnitrosamine (0.1 mumol/g) and ovariectomized at 8 weeks of age. Half of the mice of each group subsequently received biweekly s.c. injections of testosterone (0.15 mg per mouse) for 30 weeks. Tumor multiplicity was the same for wild-type and Tfm/+ mosaic females treated with testosterone (31-32 tumors per animal at 38 weeks of age) and was increased relative to females not treated with testosterone (13-17 tumors per animal at 50 weeks of age). Testosterone treatment did not significantly increase the percentage of androgen receptor-positive tumors in Tfm/+ mosaic females: 58% of the tumors from Tfm/+ mosaic females treated with testosterone were receptor positive compared to 48% in Tfm/+ females not treated with testosterone and 92% in wild-type females treated with testosterone. Finally, the number of androgen receptors in the majority of liver tumors examined was greatly decreased relative to the surrounding normal liver tissue. We conclude that liver tumor promotion by testosterone requires a functional androgen receptor in the intact animal. However, this promotion is not cell autonomous; that is, the response of the preneoplastic hepatocyte is not dependent on the expression of functional receptor in the target cell.     

Estrogen receptor beta, but not estrogen receptor alpha, is present in the vascular endothelium of the human and nonhuman primate endometrium

Estrogen action is dependent upon the presence of specific ligand-activated receptors in target tissues. The aim of the present experiments was to compare the spatial and temporal pattern of expression of estrogen receptor beta (ERbeta) with that of ERalpha in full thickness endometrial samples (from the superficial to the basal zone) obtained from both women and rhesus macaques. Immunohistochemical localization with specific antibodies revealed that ERalpha and ERbeta were both expressed in nuclei of the glands and stroma. Consistent with previous studies, expression of ERalpha declined in the glands and stroma of the functionalis during the secretory phase. The luminal epithelium also displayed positive immunoreactivity for ERbeta. Expression of ERbeta declined in glandular cell nuclei, but not stroma, within the functionalis during the late secretory phase. Levels of expression of ERalpha and ERbeta in all cellular compartments remained unchanged in the basalis. Both receptor subtypes were detected on Western blots using proteins extracted from uterine samples obtained throughout the menstrual cycle. There was a striking contrast between the pattern of expression of ERalpha and ERbeta in the vascular endothelium and the perivascular cells surrounding endometrial blood vessels; only ERbeta was present in the endothelial cell population, although both forms of ER were expressed in perivascular cells. We conclude that estrogen action(s) within the vascular endothelium in the endometrium may be mediated via direct binding to the ERbeta isoform and that these cells could therefore be a target for agonists or antagonists that selectively target the beta form of the ER.