Biomedical subjects

P Needleman

Publications and source records attributed to P Needleman.

At least 73 records · Page 4Linked to original sources

PubMed1988

Isolation and characterization of the complementary DNA for sheep seminal vesicle prostaglandin endoperoxide synthase (cyclooxygenase).

An oligonucleotide probe was used to isolate a clone encoding prostaglandin endoperoxide synthetase (cyclooxygenase, EC 1.14.99.1) from a sheep seminal vesicle cDNA library. The protein predicted from nucleic acid sequence contains 599 amino acids including a 23-amino acid signal sequence. Thus, the mature cyclooxygenase deduced from the cDNA compares favorably in molecular size to the 70-kDa protein determined by gel electrophoresis. A putative transmembrane region and potential carbohydrate addition sites for N-linked sugars can be inferred from the amino acid sequence. Significantly, sequence similarities exist between cyclooxygenase, myeloperoxidase, and several other heme-containing proteins. The putative glycosylation sites, transmembrane domain, and sequence similarities with functionally related enzymes have been incorporated into a model for the topology of cyclooxygenase in the endoplasmic reticulum.

Amino Acid Sequence↗

PubMed1988

Regulation of fibroblast cyclooxygenase synthesis by interleukin-1.

We have prepared polyclonal antiserum against sheep seminal vesicle prostaglandin H synthase (also termed cyclooxygenase) which cross-reacted with human cyclooxygenase, thereby enabling us to directly determine the synthetic rate of cyclooxygenase protein and its modulation by the monokine interleukin-1 (IL-1). Cultured human dermal fibroblast cells were labeled with [35S]methionine, and the membrane-bound cyclooxygenase was solubilized and immunoprecipitated 35S-labeled fibroblast cyclooxygenase migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular size of approximately 73,000 daltons, similar to that of native sheep cyclooxygenase and of cyclooxygenase covalently labeled by [3H]aspirin, i.e. [3H]acetylcyclooxygenase. Additional validation of the immunoprecipitated 35S-labeled cyclooxygenase band indicated that it was specifically displaced by unlabeled sheep cyclooxygenase. N-terminal amino acid radiosequence analysis of [3H]proline-labeled cyclooxygenase revealed [3H]proline residues in positions 3, 6, and 8, consistent with the previously reported N-terminal sequence of sheep cyclooxygenase. Endoglycosidase H treatment of 35S-labeled fibroblast cyclooxygenase caused a decline in apparent molecular size (due to removal of mannose residues) which was similar to that seen with the native sheep cyclooxygenase. [35S]Methionine pulse-chase experiments indicated a half-life of 1 h for fibroblast cyclooxygenase. The monokine interleukin-1 stimulated fibroblast cyclooxygenase synthesis in a time- and dose-dependent fashion; as little as 0.03 unit/ml of IL-1 produced significant stimulation of 35S-labeled cyclooxygenase synthesis. Maximum stimulation was 3-10-fold after preincubation of the cells with 0.3 unit/ml of IL-1 for 12-16 h. IL-1 treatment of cells yielded parallel dose-response curves for stimulation of prostaglandin E2 formation, increased cellular cyclooxygenase activity, and increased synthetic rate of newly formed cyclooxygenase, suggesting that the IL-1 effect is mediated mainly, if not solely, via induction of cyclooxygenase synthesis.

Animals↗

PubMed1988

Expression of the gene for preproatriopeptin in the central nervous system of the rat.

Atriopeptin (AP) is a peptide hormone synthesized and secreted by the atria of the heart that participates in the regulation of fluid and electrolyte balance. AP-like materials have been detected immunologically in neurons in the central nervous system of the rat. In this study, we have used a solution hybridization-nuclease protection assay to determine whether the brain of the rat contains RNA coding preproatriopeptin, the atrial biosynthetic precursor of AP, and to study the regional distribution of preproatriopeptin mRNA in the brain. We have found that the brain contains mRNA identical to the atrial messenger RNA for preproatriopeptin. AP mRNA is differentially distributed in the brain; the highest concentration was found in the hypothalamus, followed by the cortex and septum, hippocampus, midbrain, spinal cord, olfactory bulb, striatum, and pons and medulla. Very low levels were found in the cerebellum, while no detectable AP mRNA was observed in retina, anterior pituitary, or rat liver. The presence of AP mRNA in the brain demonstrates that neurons have the capacity to synthesize preproatriopeptin identical to that produced in the heart. Bioactive peptides produced from this precursor may be endogenous central neuromodulators as well as a circulating hormone.

Animals↗

PubMed1988

Manipulation of stretch-induced atriopeptin prohormone release and processing in the perfused rat heart.

Atriopeptin (AP) is stored as the prohormone AP-126 [atrial natriuretic factor-(1-26)] in atrial granules. Cultured atrial myocytes synthesize and release only prohormone into the medium. HPLC analysis of the coronary venous effluent of media from perfused rat hearts subjected to right atrial stretch indicated the presence of the C-terminal mature hormone AP-28 [atrial natriuretic factor-(99-126)] and little or no prohormone. Absence of calcium from the perfusion medium increased total AP release and surprisingly blocked the proteolytic cleavage of the prohormone. Similarly, addition of the proteolytic inhibitor aprotinin to the perfusion medium suppressed the processing of the endogenous AP-126 released by atrial stretch. Aprotinin would be restricted to the extracellular space, which is therefore implicated as the site of prohormone processing. This suggestion was validated by the demonstration that the perfused rat heart could readily cleave exogenous prohormone to mature hormone, a process blocked by aprotinin. Hypothetically, the stimulus-release-processing event initiated by atrial stretch may require the concerted action of the synthetic cell (i.e., atrial myocyte) and a processing cell or site (e.g., the adjacent atrial mesenchymal cell) for the production of the mature AP-28, which is the circulating molecular form of this endocrine system.

Animals↗

PubMed1988

Paradoxical relationship between atriopeptin plasma levels and diuresis-natriuresis induced by acute volume expansion.

Surgical removal of one or both atrial appendages was employed in rats to reduce the intrinsic stores of atriopeptin (AP). In conscious rats (with intact baroreceptor reflexes), bilateral or unilateral atrial appendectomy suppressed the diuresis and natriuresis produced by acute volume expansion. Surprisingly, volume expansion (with 4% bovine serum albumin in saline at 1.5 ml/kg per min for 15 min) did not result in an increase in plasma AP immunoreactivity (APir) in control or atrial-appendectomized conscious rats. Previous studies demonstrated that acute volume expansion in anesthetized animals caused increased plasma APir. Indeed, we found that volume expansion causes comparable diuresis-natriuresis in conscious and chloral hydrate-anesthetized rats, but only the latter group exhibits an increase in plasma APir. Brattleboro rats, which are deficient in vasopressin, exhibit the same response as Long-Evans controls in that acute volume expansion in conscious animals produces a pronounced diuresis and natriuresis but no APir release, but when these same animals are anesthetized, there is a simultaneous induction of diuresis-natriuresis and APir release by volume expansion. Plasma AP does not increase in conscious rats despite a large volume load, 30-40% of the total blood volume given in 15 min, and the natriuresis-diuresis appears to also be independent of vasopressin. On the other hand, the diuresis induced by acute volume expansion in anesthetized rats seems dependent on the elevated APir, since rats made autoimmune to AP (which are nonresponsive to exogenous AP infusions) exhibit a diuresis in conscious but not anesthetized rats. We therefore conclude that the participation of AP in volume homeostasis is more likely in pathophysiological states and that another mechanism or possibly another atrial factor mediates the diuresis-natriuresis induced by volume expansion in conscious rats.

Anesthesia↗

PubMed1988

Gadolinium-DTPA liposomes as a potential MRI contrast agent. Work in progress.

To evaluate the use of liposomes containing Gadolinium-DTPA (Gd-DTPA) as potential intravascular contrast agents, we synthesized and tested Gd-DTPA liposomes. A freeze-thaw extrusion process was used to synthesize neutral unilamellar vesicles. Using this technique, we prepared 0.4 micron vesicles with encapsulation efficiency as high as 39% for Gd-DTPA. In vitro dialysis showed that essentially 100% of the Gd-DTPA was retained with the liposomes after 72 hours of dialysis. MR imaging of in vitro samples showed concentration-dependent increase in signal intensity with Gd-DTPA liposomes. Imaging of rats after intravenous injection of Gd-DTPA liposomes showed sustained intravascular contrast enhancement of vascular structures and liver greater than free Gd-DTPA. There was no evidence of acute toxicity in rats during the imaging experiments or on follow-up of two months. Paramagnetic liposomes may be useful to enhance the vasculature, liver, and spleen.

Animals↗

PubMed1988

Reversible induction of right ventricular atriopeptin synthesis in hypertrophy due to hypoxia.

Right ventricular hypertrophy produced in rats exposed to 10% oxygen for 3 weeks resulted in a ninefold increase in atriopeptin immunoreactivity (APir) and a 160-fold increase in atriopeptin messenger RNA (AP mRNA) in the right ventricular myocardium. A small but significant increase in left ventricular APir and AP mRNA was also present, probably representing the interventricular septum. Right atrial APir was decreased by 50%, but left atrial APir was not different from normoxic controls. Purification of ventricular tissue extracts by high-performance liquid chromatography revealed primarily the high molecular weight prohormone. The development of right ventricular hypertrophy and right ventricular APir content followed a similar time course, each evident at 7 days of hypoxia and reaching a plateau at 14 days. Hypoxia followed by normoxia caused right ventricular APir to fall to control levels within 3 days, despite persistent right ventricular hypertrophy. This data demonstrates that hypoxia can reversibly induce extra-atrial expression of atriopeptin synthesis in the cardiac ventricle.

Animals↗

PubMed1988

Is atriopeptin a physiological or pathophysiological substance? Studies in the autoimmune rat.

Atriopeptin (AP), a natriuretic-diuretic and vasodilatory peptide, is synthesized and secreted from mammalian atria. The definitive role of this peptide on cardiovascular physiology and pathophysiology has yet to be determined. We developed a population of autoimmune rats sensitized against their own AP to evaluate the consequences of prolonged AP deficiency on physiological and pathophysiological processes. Natriuresis in response to acute intravenous volume expansion was inhibited in the autoimmune rat, however, natriuresis produced by chronic oral salt loading was not suppressed in these animals. Plasma AP increased threefold in the spontaneously hypertensive rat when evaluated as a function of blood pressure. Immunization of these rats had no effect on the rate of development, magnitude of their developing hypertension, or their daily sodium excretion when compared with nonimmunized controls. Mineralocorticoid escape occurred during desoxycorticosterone acetate administration to rats. The ability of rats to escape from the sodium-retaining effects of this steroid was not affected by prior immunization against AP. These results suggest that AP is an important natriuretic substance in response to acute intravascular volume loading. However, atriopeptin does not appear to be involved in the natriuretic response to chronic intravascular volume loading, blood pressure regulation, or mineralocorticoid escape.

Animals↗

PubMed1988

Essential fatty acid deficiency reduces the inflammatory cell invasion in rabbit hydronephrosis resulting in suppression of the exaggerated eicosanoid production.

The rabbit hydronephrotic kidney (HNK) is a model of renal inflammation characterized by a marked increase in arachidonic acid metabolism which is temporally associated with an inflammatory cell influx into the injured tissue. The HNK exhibits an exaggerated elaboration of eicosanoids ex vivo in response to inflammatory agonists (bradykinin and the chemotactic peptide, n-formyl-methionyl-leucyl-phenylalanine). Essential fatty acid (EFA) deficiency [i.e., deprivation of (n-6) fatty acids] attenuated markedly the ex vivo elaboration of eicosanoids and prevented the enhancement of the microsomal cyclooxygenase and thromboxane synthase activity associated with 3 days of ureter occlusion. In contrast, postobstructive release prevented the ex vivo elaboration of eicosanoids by the HNK. When the HNK was assessed morphologically by electron microscopy, both EFA deficiency and postobstructive release markedly reduced the population of interstitial macrophages normally seen in the HNK. Apparently, EFA deficiency blocked the influx of macrophages whereas postobstructive release resulted in the efflux of macrophages from the HNK. Because EFA deficiency has been shown to inhibit the synthesis of leukotriene B4, a potential chemotaxin, it was hypothesized that EFA deficiency might prevent the influx of macrophages due to an inhibition of leukotriene B4 synthesis. Indeed, EFA deficiency suppressed the synthesis of this eicosanoid in blood whereas prostaglandin E2 and thromboxane A2 production were unaffected. In summary, this study demonstrates that EFA deficiency prevents the influx of macrophages into the HNK and prevents the enhanced arachidonate metabolism which normally occurs after ureter obstruction. A potential role for leukotriene B4 as a chemotactic agent in this model of renal inflammation also is suggested.

Animals↗

PubMed1987

Metabolic and functional alterations in macrophages induced by essential fatty acid deficiency.

Essential fatty acid (EFA) deficiency has been shown to protect against the glomerulonephritis in a murine model of systemic lupus erythematosus. Since macrophages are an important cellular constituent of the inflammatory lesion, the effects of EFA deficiency on the eicosanoid metabolism and function of these cells were determined. EFA-deficient macrophages exhibited a depletion of phospholipid arachidonate and an accumulation of 20:3(n-9); phosphatidylinositol was the phospholipid most affected. When these macrophages were stimulated with unopsonized zymosan, they produced markedly less leukotriene C4 and B4 than control macrophages. EFA-deficient macrophages also synthesized leukotriene C3 from endogenous 20:3(n-9). No leukotriene B3 was detected. In contrast to the effects on leukotriene production, prostaglandin and thromboxane production were only minimally affected by EFA deficiency. When challenged with zymosan, EFA-deficient macrophages released less arachidonate relative to control macrophages and released half again as much 20:3(n-9) as arachidonate. Release of arachidonate from phosphatidylcholine in the EFA-deficient cells was highly selective for arachidonate; however, release of arachidonate from phosphatidylinositol was depressed relative to control and was not selective. Incubation of macrophages with exogenous arachidonate and 20:3(n-9) established that 20:3(n-9) decreased leukotriene C4 and B4 synthesis from arachidonate but did not affect prostaglandin production. To determine the functional effects of the deficiency state, receptor-mediated pinocytosis and phagocytosis were also examined in EFA-deficient cells. EFA-deficient macrophages exhibited a marked reduction in receptor-mediated pinocytosis. Phagocytosis, however, was unaffected by the deficiency state. These effects on macrophage eicosanoid metabolism and function may comprise a significant component of the anti-inflammatory effect of EFA deficiency.

Animals↗

PubMed1987

Cellular confluence determines injury-induced prostaglandin E2 synthesis by human keratinocyte cultures.

When keratinocyte cultures become confluent, their prostaglandin E2 synthesis is suppressed. To determine whether the injury response is characterized by increased prostaglandin E2 synthesis, an in vitro injury model was developed. When confluent keratinocyte cultures were focally lethally irradiated using ultraviolet light B, a dose-dependent increase in prostaglandin E2 synthesis was induced by the injury. After irradiation, confluent cultures' prostaglandin E2 synthesis increased for 2 days to 8-fold more than controls, then decreased to control values by day 6. Increased prostaglandin E2 synthesis was first detected 8 h after injury. Focal irradiation of non-confluent cultures (killing isolated colonies) caused no change in prostaglandin E2 synthesis, indicating that culture continuity must be disrupted before synthesis increases. In addition, partial irradiations of petri dishes demonstrated that enhanced metabolism was confined to cells adjacent to the injury site and was not mediated by a soluble factor. When confluent and injured cultures were incubated with [14C]arachidonic acid, and the products formed analyzed by thin layer chromatography, 10-fold more prostaglandin E2 microgram protein was seen in irradiated cultures relative to confluent controls. The products formed by each group were the same, and no consistent increases in metabolites other than prostaglandin E2 were observed. The increased synthesis of prostaglandin E2 by injured cultures was apparently due to an increase in cyclooxygenase activity as determined by kinetic experiments. These data indicate that the pattern of metabolism of arachidonic acid seen in non-confluent cultures is similar to that seen in injury, and that cell-cell contact modulates enhanced prostaglandin E2 synthesis.

Cells, Cultured↗

PubMed1987

Development, validation and application of an enzyme immunoassay (EIA) of atriopeptin.

A rapid, convenient, and sensitive enzyme immunoassay (EIA) for atriopeptin (AP) has been developed. The tracer-ligand for the assay is the 24-amino acid peptide, AP24, which has been covalently coupled to the tetrameric form of acetylcholinesterase (AChE) (EC 3.1.1.7). Tracer, unknown, and primary antibody are incubated in a 96-well microtiter plate precoated with secondary antibody. After washing, a colorimetric reaction is used to measure acetylcholinesterase activity. A direct linear correlation was obtained when comparing the conventional radioimmunoassay and the EIA by using the same primary antibody to assay: plasma samples (rat or human), HPLC column fractions, or atrial extracts. Besides being technically much less demanding and not requiring the use of the radioisotopes, the EIA is more sensitive than the radioimmunoassay and thereby lends itself to a "flash" same-day assay of samples.

Acetylcholinesterase↗

PubMed1987

Long-acting angiotensin II inhibitors containing hexafluorovaline in position 8.

An improved synthesis of hexafluorovaline (Hfv) derivatives, i.e., DL-Hfv-OBzl and Boc-DL-Hfv, is described. Incorporation of hexafluorovaline into angiotensin resulted in [Sar1,Hfv8]AII and [Sar1,D-Hfv8]AII. At the nanogram/milliliter dose range, the L congener was 20-100 times more active as either angiotensin agonist or angiotensin antagonist than its D diastereomer on isolated tissue preparations. At the microgram dose range, both [Sar1,Hfv8]AII and [Sar1,D-Hfv8]AII were significantly more effective than [Sar1,Leu8]AII as angiotensin II inhibitors, producing prolonged blockade of the pressor response toward angiotensin II for over 1 h.

Angiotensin II↗

PubMed1987

Ventricular atriopeptin. Unmasking of messenger RNA and peptide synthesis by hypertrophy or dexamethasone.

Left ventricular hypertrophy or treatment with dexamethasone caused a 2.5-fold to threefold increase in both immunoreactive atriopeptin (AP) and AP messenger RNA (mRNA), primarily in left ventricular tissue. The combined treatments increased immunoreactive AP and AP mRNA more than either treatment alone. In the animals in which cardiac hypertrophy had been produced by abdominal aortic constriction, there was a decrease in atrial levels of AP and an increase in plasma levels of immunoreactive AP. The increase in left ventricular immunoreactive AP was confirmed by immunohistochemical staining of tissue from hypertrophied and/or dexamethasone-treated rats. The mRNA accumulated in the left ventricle was identical to atrial AP mRNA, as judged by transcriptional start site and by size on Northern blots. Because the mass of ventricular tissue is substantially greater than that of atrial tissue, the induced mRNA levels may represent a total abundance approaching one third of the total AP mRNA in the atria. High performance liquid chromatographic purification of ventricular extracts primarily demonstrated the presence of the high molecular precursor and small amounts of C-terminal peptide AP. Induction of ventricular AP (mRNA and peptide) may represent regression of the tissue to an earlier developmental form. These data provide a unique example of regulation of AP biosynthesis in nonatrial tissue.

Animals↗

PubMed1987

Heparin interferes with the biological effectiveness of atriopeptin.

The chromatographic mobility of atriopeptin-28 or of the prohormone is markedly altered by preincubation of the peptides with heparin before separation on reverse-phase high performance liquid chromatography. Protamine prevented the heparin effect and reestablished the original migration pattern of the atrial peptides. The addition of heparin to either rat or human plasma samples did not interfere with the atriopeptin immunoreactivity. The influence of heparin on the biological activity of the atriopeptin-28 in anesthetized rats was also investigated. Infusion of heparin (30 U/min) significantly reduced the dose-dependent fall of blood pressure produced by atriopeptin-28, but did not interfere with the hypotensive effect of nitroglycerin. Similarly, infusion of heparin in volume-expanded rats markedly decreased the diuresis produced by atriopeptin-28 without altering the urine volume excreted in response to furosemide. These data suggest that the highly charged molecule heparin can modify the physical and biological properties of atriopeptins, perhaps by binding to the numerous arginine residues (i.e., 5 arginine residues in atriopeptin-28) in the atriopeptin molecules.

Animals↗

PubMed1987

Effect of endogenously produced leukotrienes and thromboxane on renal vascular resistance in rabbit hydronephrosis.

Ureteral obstruction in rabbits is characterized by mononuclear cell invasion of the renal cortex and proliferative fibrosis that is associated with exaggerated prostaglandin synthesis in response to vasoactive and inflammatory cell agonists. In this investigation, we studied the effects of the chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (fMLP) and bradykinin (BK) on eicosanoid synthesis and renal vascular resistance in the ex vivo perfused hydronephrotic kidney (HNK). Administration of fMLP resulted in the dose-dependent synthesis of leukotrienes, thromboxane A2 (TXA2), prostaglandin E2 (PGE2), and prostacyclin (PGI2). Peptidoleukotriene synthesis was monitored by specific radioimmunoassay and by guinea pig ileum bioassay and it was then validated by inhibition of the ileal contractile activity with the peptidoleukotriene receptor antagonist FPL-55712. The leukotrienes produced were identified as LTB4, LTC4, LTD4, and LTE4 by comigration with authentic standards on reverse phase high-performance liquid chromatography (RP-HPLC) and by ultraviolet spectroscopy. BK administration stimulated the synthesis of TXA2, PGE2, and PGI2 but not the synthesis of leukotrienes, in contrast to the results with fMLP, suggesting the involvement of different cell types. Administration of fMLP to the HNK also resulted in a renal vasoconstriction that was partially inhibited by FPL-55712 and that was completely inhibited by the thromboxane synthase inhibitor OKY-1581. Consistent with this result, exogenous administration of LTC4 resulted in the synthesis of TXA2 and in a renal vasoconstriction that was inhibited by either FPL-55712 or OKY-1581.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1987

Exaggerated atrial arachidonate metabolism in rabbit left ventricular myocardial infarction.

Isolated perfused rabbit hearts that have previously been subjected to in vivo left ventricular myocardial infarction respond to N-formylmethionyl-leucyl-phenylalanine (fMLP) or bradykinin (BK) administration with the synthesis of large quantities of eicosanoids. To anatomically localize these synthetic responses we studied the effects of fMLP and BK on eicosanoid synthesis in isolated atria and isolated perfused ventricles from normal and infarcted (4 d in vivo) rabbit hearts. These studies revealed that enhanced agonist-stimulated eicosanoid synthesis occurs largely in the right atria of infarcted hearts, a site distant from the zone of injury. Studies of exogenous arachidonate metabolism in microsomes prepared from various regions of the heart showed that while prostaglandin synthetic capacity is preferentially localized to the right atrium, right atria from normal and infarcted hearts have similar thromboxane and PGE2 synthetic capacity. These results demonstrate that enhanced agonist-stimulated eicosanoid synthesis following rabbit left ventricular myocardial infarction occurs largely in the right atrium, and that this effect is independent of the activity of prostaglandin synthetic enzymes.

4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyraz↗

PubMed1987

Indomethacin in vivo increases the sensitivity to Listeria infection in mice. A possible role for macrophage thromboxane A2 synthesis.

This paper demonstrates that in the presence of indomethacin, a cyclooxygenase inhibitor, 100% of the mice died when infected with live Listeria, whereas none of the animals died in the absence of the drug. The death of the animals correlated with the numbers of bacteria found extraperitoneally in the spleen and not with the Ia expression of the peritoneal macrophages. Increases in the spleen bacterial numbers between mice treated with either indomethacin or a specific thromboxane synthase inhibitor, OKY1581, and those not receiving either drug, were found as early as 2-4 h after infection. The differences in the initial increased bacterial spleen counts in the presence of indomethacin were reversed by administration of a stable thromboxane A2 analog or another potent vasoconstrictor, phenylephrine. Because thromboxane A2 does not regulate macrophage or T cell functions directly (Tripp, C.S., A. Wyche, E.R. Unanue, and P. Needleman, 1986, J. Immunol., In press; and Ceuppens, J.S., S. Vertessen, H. Deckmyn, and J. Vermylen, 1985, Cell Immunol., 90:458-463), but is probably generated at the site of an infection (Tripp, C.S., K.M. Leahy, and P. Needleman, 1985, J. Clin. Invest., 76:898-901), these data suggest an important role for the vasoconstrictive properties of thromboxane A2 in the regulation of immunity to Listeria infection.

Animals↗