Pre-and post-natal ontogeny of neutral endopeptidase 24-11 (‘enkephalinase’) studied by in vitro autoradiography in the rat

Neutral endopeptidase (NEP, enkephalinase, CALLA) which is present in various neural and non-neural tissues, is able to cleave a variety of regulatory peptides. The distribution of NEP has been studied during rat pre-and post-natal development by autoradiography after in vitro binding of the tritiated inhibitor [³H]HACBO-Gly to whole-body and organ sections. In the central nervous system (CNS), where the presence of NEP has been related to the termination of the action of enkephalins, the external layer of the olfactory bulbs is the only structure prominently labeled before birth. Other CNS structures rich in NEP in the adult, such as the nigrostriatal tract, are progressively labeled after birth. Outside the CNS, the progressive appearance of NEP in the kidney, the lungs and the salivary glands suggests its concomitant involvement in adult physiological functions, including fluid balance control, possibly by cleaving the atrial natriuretic peptide (ANP) and other peptides. On the other hand, transient or enhanced expression of NEP is observed during the development of several organs such as the sensory organs, the heart and the major blood vessels, the intestine, the bones and the genital tubercle. In addition to the still incompletely known physiological functions of the enzyme, the developmental pattern of its expression in several tissues strongly suggests a modulatory role for NEP in the ontogeny of a large number of organs.     

Nuclear localization of aldosterone in rat brain cells assessed by autoradiography.

Autoradiographic studies with 3H aldosterone demonstrate nuclear concentration of hormone in neurons of the hippocampus, septum, allocortical regions and brain stem reticular formation and motor nuclei of cranial nerves and in the meninges. The results suggest that mineralocorticoids have wide ranging effects on different parts of the central nervous system.     

Enzymatic phosphorylation of proteins of rat liver chromatin by (γ-32P) ATP in vitro

Zusammenfassung Isoliertes Rattenleberchromatin baut die endständige Phosphat-Gruppe von (-³²P) ATP in seine Proteine in Form von Phosphatester von Serin und Threonin ein. Der Einbau ist Mg⁺⁺ und Mn⁺⁺ abhängig und steigt mit Erhöhung des pH des Inkubationsmediums an. Phosphat und Pyrophosphat stimulieren die Phosphorylierung, zyklisches AMP hat keinen Effekt. Die sauren Proteine werden viel stärker als die Histone phosphoryliert. Das eingebaute Phosphat wird sehr schnell freigesetzt, ein Prozess, der von der Anwesenheit von dephosphorylierenden Enzymen abhängt.     

Induction of apoptosis and CD10/neutral endopeptidase expression by jaspamide in HL-60 line cells

Jaspamide (jasplakinolide) is a natural peptide isolated from marine sponges of Jaspis species and has fungicidal and growth-inhibiting activities. We characterized the jasplakinolide-induced loss of viability by programmed cell death in the HL-60 human promyelocytic leukemia cell line and found that this process was accompanied by neutral endopeptidase (NEP)/CD10 expression on the surface of the apoptotic cells. HL-60 cells do not normally express detectable amounts of NEP/CD10 on their surface or intracytoplasmically, but upon jaspamide treatment, CD10 was synthesized de novo, its expression being inhibited by cycloheximide pretreatment. Once synthesized, NEP/CD10 interfered with the jasplakinolide signal delivered to HL-60 cells. Inhibition of NEP/CD10 by the NEP inhibitor phosphoramidon or by an anti-CD10 monoclonal antibody significantly increased apoptosis induction. The appearance of CD10 on the cell surface was blocked by preincubation of the cells with the monocytic/macrophage-differentiating agents vitamin D3 and phorbol 12-myristate 13-acetate, but not by the granulocytic differentiating agents retinoic acid or dimethyl sulfoxide. Moreover, in the promonocytic U937 and mature monocytic THP-1 cell lines, jaspamide induced apoptosis but not CD10 expression. In HL-60 cells, CD10 expression was partially but not totally blocked by the broad-spectrum caspase inhibitor benzyloxacarbonyl-Val-Ala-Asp-fluoromethylketone, indicating a connection between apoptosis induction and CD10 synthesis. Our findings suggest that the CD10 expression is related to the programmed cell death induction by jaspamide, and also with the process of granulocytic differentiation in HL-60 cells. Received 22 April 2002; received after revision 8 June 2002; accepted 10 June 2002     

Pre- and postnatal lead exposure affects the serotonergic system in the immature rat brain.

The effect of pre- and postnatal lead exposure on the development of the serotonergic system in striatum and brain stem was investigated. Serotonin and its metabolite 5-HIAA where determined by HPLC-EC. A significant decrease of 5-HT was detected in the brain stem at postnatal day 28. At both days 6 and 28 postnatal, 5-HIAA was reduced in striatum and brain stem. The results provide support to the hypothesis that developing 5-HT neurons are sensitive to relatively low levels of lead exposure.     

The responsiveness to (1-24) ACTH of Mongolian gerbil adrenals superfused in vitro as a function of basal secretion

Wide variations have been found in the responsiveness to (1-24)ACTH of Mongolian gerbil adrenal glands superfused in vitro. These variations were not correlated to corticosteroid plasma levels or to adrenal weights. In contrast, (1-24)ACTH-stimulated secretion greatly depended on basal in vitro secretion (quarters: r = 0.97, p less than 0.01; slices: r = 0.90, p less than 0.001) indicating that the fine adjustment of basal corticosteroidogenesis forms an important part of the regulatory mechanisms modulating (1-24)ACTH-stimulated corticosteroid secretion in vitro.     

Pre- and post-partum plasma amine oxidase differences in the rhesus monkey (Macaca mulatta)

Summary Plasma amine oxidase activity increased from 23.4 nmol/ml/h during pregnancy to 49.5 nmol/ml/h during an extended post partum period in 10 rhesus monkeys. Comparison with non-pregnant control monkeys sampled at similar times indicated that the significant differences were in the extended post partum period.We thank Dr.C. H. Conaway and Dr.J. D. Loy of the Caribbean Primate Research Center andJ. G. Vandenbergh andW. Post of the North Carolina Department of Mental Health for the use of their facilities and animals. We also acknowledge the technical assistance ofC. H. Donnelly andMs. Suzanne Baulu. This work was supported by a grant from Dr.H. C. Robinson, Jr., through Yale University Medical School and by NIH Contract No. 71-2003.     

Occurrence and biosynthesis of 11 (R)-hydroxy-eicosatetraenoic acid (11-R-HETE) in the Caribbean soft coralPlexaurella dichotoma

Gorgonian soft corals from the Caribbean Sea are known to contain prostaglandin-like compounds as well as other products of arachidonic acid lipoxygenation, and the formation of the latter has been suggested to represent the first step in prostaglandin biosynthesis. Here we present evidence for the presence of 11-R-hydroxy-5Z, 8Z, 12E, 14Z-eicosatetraenoic acid (11-R-HETE), as well as of the enzyme responsible for its biosynthesis, in the Caribbean gorgonianPlexaurella dichotoma. Lipid extracts fromP. dichotoma were purified by conventional SiO₂ column chromatography followed by reversed phase high-pressure liquid chromatography (HPLC). These yielded a component having chromatographic and spectroscopic properties identical to synthetic 11-HETE. Electron impact mass spectrometric analysis of the acetoxy-, methyl ester derivative of the compound confirmed its identity as 11-HETE, while chiral phase HPLC of the methyl ester derivative showed that the stereochemistry of the alcoholic carbon atom wasR. Enzymatically active homogenates fromP. dichotoma were able to convert both unlabelled and [³H] arachidonic acid into 11-HETE. In vitro biosynthesis of the latter metabolite was also observed with homogenates of the Mediterranean gorgonianParamuricea clavata, another non-prostaglandin-containing soft coral, thus suggesting that 11-R-HETE production is not necessarily accompanied by prostaglandin formation in gorgonian corals.     

Antagonism by haloperidol of the suppression of exploratory locomotor activity induced by the local application of (-)3-(3-hydroxyphenyl)-N-n-propylpiperidine into the nucleus accumbens of the rat

The injection of (-)3-PPP into the nucleus accumbens, 10 microgram/side, produced a suppression of exploratory locomotor activity without affecting treadmill locomotion. Furthermore, the suppression of exploratory locomotor activity produced by (-)3-PPP was antagonized by the administration of haloperidol, 25-50 micrograms/kg i.p.     

The localization of [3H]-desipramine in central nerve terminals studied with electron microscope autoradiography and subcellular fractionation.

The cellular and subcellular distribution of [3H]-desipramine (DMI) in rat brain was studied by electron microscope (EM) autoradiography and by subcellular fractionation. A considerable proportion of label was found to be bound to the membranes of presynaptic nerve terminals, as well as to sites inside those terminals.     

Production of insulin-like growth factor I (IGF-I) and IGF-binding proteins by rat intestinal stromal cells in vitro

To determine if intestinal stromal cells secrete diffusible factors such as insulin-like growth factors (IGFs) capable of regulating epithelial cell growth in vitro, stromal cells were isolated by enzymatic digestion of rat intestine. Incorporation of [³H]thymidine into DNA and [¹⁴C]leucine into protein of IEC-6 cells, a model intestinal epithelial cell line, was significantly increased (two- to threefold) when the IEC-6 cells were co-cultured with stromal cells, relative to IEC-6 cells grown alone. Medium conditioned by stromal cells stimulated DNA synthesis of IEC-6 cells in a dose-dependent manner. Analysis of the conditioned medium revealed that intestinal stromal cells secreted IGF-I, but little IGF-II, in addition to an M _r 32,000 IGF-binding protein (IGFBP-2) and an IGFBP having M _r∼ 24,000. We conclude that rat intestinal stromal cells secrete one or more diffusible factors, which may include IGF-I and IGFBPs, capable of stimulating proliferation of IEC-6 cells in vitro. Received 25 August 1997; received after revision 7 November 1997; accepted 20 November 1997     

Hashish VI1: Conversion of (-)-Δ 1(6)-Tetrahydrocannabinol to (-)-Δ 1(7)-Tetrahydrocannabinol. Stability of (-)-Δ 1 and (-)-Δ 1(6)-Tetrahydrocannabinols

Zusammenfassung Die Synthese von (—)-¹⁽⁷⁾-Tetrahydrocannabinol (THC) auf (—)-¹⁽⁶⁾ wird beschrieben. Das Hauptprodukt der oxidativen Zerstörung von (—)-¹-THC ist Cannabinol. Beschleunigte Versuche der Luftoxidation von (—)-¹-THC und (—)-¹⁽⁶⁾-THC haben gezeigt, dass das letztere beständiger ist. Dieser Unterschied kann mit der Reaktivität des C₃-benzylisch-allylischen Wasserstoffes in (—)-¹-THC erklärt werden.     

Electrolyte transport in the seminiferous tubules of the rat studied by the stopped-flow microperfusion technique

Zusammenfassung Mit Hilfe der «stopped-flow»-Mikroperfusionstechnik wurden am Samenkanälchen Elektrolyttransportprozesse untersucht. Es wurde gefunden, dass die Kanälchen ein kaliumreiches Primärsekret bilden. Dieses unterscheidet sich in seiner Zusammensetzung von dem Sekret, das man gewöhnlich unter ungestörten Fliessbedingungen findet.

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This project was supported by the Rural Bank of Australia. One of us (R.D.H.) thanks the National Health and Medical Research Council of Australia for the award of a B.Sc. (med.) studentship for 1970.     

D(-)Lactic acid—a physiological isomer in the rat

Summary D(-)Lactate is produced in significant amounts together with L(+)lactate in the stomach of normal experimental rats. It is absorbed into the blood and constitutes a physiological isomer in this animal species.     

Cleavage of p-nitroanilides of N-acylated tri- and tetrapeptides by alanine endopeptidase from the brush border membranes of rat enterocytes

The activity of the alanine endopeptidase from the intestinal brush border was studied using chromogenic substrates of the general formula Sc-Ala2-X-pNA. Sc-Y-Z-Ala-pNA and W-Ala3-pNA respectively. Substrates with C-terminal Leu or Nle are hydrolyzed more readily than Ala-analogues. At least one Ala-residue in one of the positions adjacent to the C-terminus is necessary for the enzyme activity. An Na-substituent has no effect on the activity.     

Study on the distribution of methyl-mercury (203Hg) by whole body autoradiography of macaque monkeys (Macaca irus)]

The distribution of methyl-mercury labelled with mercury-203 has been studied in Monkeys by whole body autoradiography technique. The mercury is rapidly absorbed. It is localized in all the body and mainly in well defined areas of the central nervous system.     

Aminergic innervation of the hypothalamo-hypophyseal system in Gambusia sp. (Teleost, Poecilidae) studied by two fluorescence techniques]

Using fluorescence techniques (Falck-Hillarp, de La Torre and Surgeon) 2 paired groups of perikarya were observed in the hypothalamus of Gambusia, one in the nucleus recessus lateralis (NRL) and the other in the nucleus recessus posterioris (NRP). No fluorescent neuron could be detected in the nucleus recessus preopticus (NRPO) nor in the nucleus lateralis tuberis (NLT). Fluorescent aminergic fibers demonstrated in the rostral neurohypophysis and in the neurointermediate lobe might originate from the NRL. These two techniques are complementary, but the last one, more rapidly performed, appears promising.