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Biomedical subjects

P Needleman

Publications and source records attributed to P Needleman.

At least 163 records · Page 9Linked to original sources
PubMed1983

Cell culture of renal epithelium derived from rabbit microdissected cortical collecting tubules.

Cortical collecting tubules were dissected from rabbit kidney and cultured in a hormonally defined serum-free medium. Morphologic studies of the cultured cells derived from the collecting tubule indicated that the cells maintained their epithelial nature. These studies also revealed the presence of two distinct cell types that closely resemble the principal and intercalated cell types of the cortical collecting tubule. Several biochemical characteristics of the cultured cells were found to be similar to previously reported values for the cortical collecting tubule. The cells retain hormonal responsiveness to antidiuretic hormone (ADH), as demonstrated by a 12-fold increase in cAMP in response to ADH. Cultured cortical collecting tubule cells produce prostaglandins, with prostaglandin E2 as the predominant cyclooxygenase product. This study presents the first morphologic and biochemical characterization of cortical collecting tubule epithelial cells grown in culture.

Animals↗
PubMed1983

Metabolic and cellular alterations underlying the exaggerated renal prostaglandin and thromboxane synthesis in ureter obstruction in rabbits. Inflammatory response involving fibroblasts and mononuclear cells.

Unilateral ureter obstruction in rabbits produced profound changes in endogenous and exogenous renal arachidonic acid metabolism. Isolated perfused hydronephrotic kidneys (removed after 3 or 10 d of ureter obstruction) responded to bradykinin stimulation with a markedly enhanced release of prostaglandin E2 and thromboxane A2. Reversal (3 or 10 d) of the ureter obstruction resulted in a reduction in the vasoactive peptide-induced release of prostaglandin E2 and thromboxane A2 from the perfused hydronephrotic kidney. However, postobstruction reversal of prostaglandin production by the agonist-stimulated perfused kidney was not reflected in the cortical microsomal cyclooxygenase activity, which is greatly enhanced during ureter obstruction and does not decrease after removal of the obstruction. Histological analysis of the renal cortex in rabbits with ureteral obstruction revealed a proliferation of fibroblast-like cells and the presence of mononuclear cells; removal of the obstruction did not result in a disappearance of cortical fibroblasts but did result in a decrease of monocytes. The critical involvement of mononuclear cells in the exaggerated arachidonate metabolism that occurs during hydronephrosis was exhibited by the demonstration that: (a) only the perfused hydronephrotic rabbit kidney responded to administration of endotoxin with a sustained release of prostaglandin E2 and thromboxane A2; (b) the contralateral rabbit kidney, which is devoid of mononuclear cells, did not respond to endotoxin; and (c) the hydronephrotic cat kidney, which exhibits a fibroblast proliferation with a low number of mononuclear cells, did not respond to endotoxin. Thus, proliferation of fibroblast-like cells and the presence of mononuclear cells appear to be involved in the exaggerated prostaglandin and thromboxane production underlying hydronephrosis. The increase in microsomal cyclooxygenase activity is apparently most closely correlated with the increased fibroblastic activation and cellularity, whereas mononuclear cells (possibly via monokines) seem to be critical for the markedly enhanced prostaglandin and thromboxane release induced by endotoxin and bradykinin.

Animals↗
PubMed1983

Presence of inflammatory cells associated with exaggerated arachidonic acid metabolism in renal injury.

Unilateral ureter obstruction induces an exaggerated prostaglandin release from isolated perfused rabbit kidneys in response to vasoactive peptides. Perfused hydronephrotic kidneys also exhibit the release of thromboxane A2 which is not detected with normal or contralateral kidneys. Reversal of the ureteral obstruction causes a decreased production of PGs and TxA2 in response to bradykinin. Morphological examination of the HNK demonstrates an enlarged interstitial space containing a fibroblast-like cell and the presence of mononuclear cells. Administration of endotoxin to the perfused HNK elicits the release of PGE2 and TxB2 consistent with the ability of endotoxin to stimulate arachidonic acid metabolism in cultured macrophage. Rabbit CLK and the cat HNK, which are deficient in macrophages, exhibit minimal PGE2 and no detectable TxA2 release after endotoxin stimulation. Cells cultured from the rabbit HNK cortex contain fibroblast-like cells and phagocytic cells which respond to BK with a profound PG production. Conditioned media from mononuclear cells have been shown by others to stimulate PGE2 production from fibroblasts. Other models of renal disease (renal venous constriction and glycerol-induced tubular necrosis) exhibit exaggerated PG and TxA2 release and facilitated cortical microsomal AA metabolism. These data suggest that proliferation of fibroblast-like cells and the presence of mononuclear cells may be involved in this exaggerated PG and TxA2 production underlying renal injury.

Animals↗
PubMed1983

Endotoxin induces chronic prostaglandin and thromboxane synthesis from ureter-obstructed kidneys: role of inflammatory cells.

A bolus injection of a very low dose (100 ng) of endotoxin into an isolated perfused hydronephrotic (3 day unilateral ureter obstructed) kidney resulted in the appearance of substances in the venous effluent, which caused a biphasic chronic contraction of intestinal and vascular smooth muscle bioassay strips. Indomethacin pretreatment blocked the effect. The contralateral (unobstructed) rabbit kidney, postobstructed hydronephrotic rabbit kidney (i.e., release of ureter obstruction for 3-10 days) and the hydronephrotic cat kidney did not respond to endotoxin. Histological examination demonstrated that the hydronephrotic rabbit kidney contains mononuclear cells, whereas the unobstructed contralateral rabbit kidney as well as the postobstructed rabbit hydronephrotic kidney and the hydronephrotic cat kidney have considerably fewer mononuclear cells. The responsiveness of the hydronephrotic rabbit kidney to endotoxin is dependent on ex vivo perfusion time and is suppressed by inhibition of protein synthesis with cycloheximide. These data suggest that ureter obstruction in the rabbit stimulates the infiltration of macrophages which are sensitive to endotoxin and which participate in the exaggerated prostaglandin production.

Animals↗
PubMed1982

Prostaglandin endoperoxide E2 isomerase is dissociated from prostaglandin endoperoxide synthetase in the renal cortex.

The rate of arachidonic acid metabolism by prostaglandin (PG) endoperoxide synthetase by the rabbit renal cortex (approximately 200 pmol/mg of protein/min) is very slow compared to medulla (approximately 2000 pmol/mg/min). However, by using PGH2 as a substrate and limiting reaction times, we were able to directly measure the endoperoxide-dependent PGE2 isomerase and found that both the cortex and medulla possess high levels of this activity (approximately pmol/mg/min). The PG endoperoxide E2 isomerase is dependent on reduced glutathione, but not cysteine, and is inactivated by p-hydroxymercuribenzoate or boiling. Thus, the renal medulla appears to exhibit an efficient coupling of cyclooxygenase and PG endoperoxide E2 isomerase, whereas the cortex has a vast excess of endoperoxide-dependent enzyme.

Animals↗
PubMed1982

Evidence for two distinct forms of fatty acid cyclooxygenase in brain.

The enzymatic metabolism of [14C]arachidonic acid (AA) was studied with microsomes prepared from rabbit medulla. Prostaglandin E2 (PGE2) levels, measured either by radiochemistry or radioimmunoassay, rose rapidly and abruptly plateaued within 5 min, while prostaglandin F2 alpha (PGF2 alpha) levels continued to rise for 30 min. The rapid termination of PGE2 biosynthesis was not the result of limited cofactor, substrate, or product feedback inhibition, nor was it due to PGE2-9-ketoreductase activity. Inhibition of the PGH2 leads to PGE2 isomerase by arachidonic acid or its metabolites could not explain the abrupt half in PGE2 biosynthesis. Proof for two separate cyclooxygenases comes from our observation that a preincubation of the brain microsomes with unlabeled AA eliminated PGE2 biosynthesis while PGF2 alpha production continued. Further evidence to suggest two cyclooxygenases in brain is derived from the observation that indomethacin inhibited PGE2 production at concentrations that did not affect PGF2 alpha biosynthesis. These results suggest that one fatty acid cyclooxygenase is closely associated with PGH2 leads to PGE2 isomerase and readily undergoes autodestruction and the second cyclooxygenase is associated with a PGH2 leads to PGF2 alpha reductase and is somewhat resistant to arachidonate-induced destruction and to nonsteroidal antiinflammatory agents.

Animals↗
PubMed1982

Contribution of thromboxane to renal resistance changes in the isolated perfused hydronephrotic rabbit kidney.

Thromboxane A2 is not produced in normal rabbit kidneys, but its synthesis is induced in numerous renal pathological states. The presence of this potent vasoconstrictor could readily compromise renal hemodynamics. We found that the thromboxane synthetase inhibitor, OKY-1581 (sodium-3-[4,3-pyridylmethyl)phenyl]-2-methylacrylate) is effective in the perfused hydronephrotic kidney in selectively inhibiting thromboxane production without altering prostaglandin E2 or prostacyclin release. The vasoactive peptides bradykinin and angiotensin cause the hydronephrotic kidney to produce thromboxane A2, which results in a profound renal vasoconstriction which is reversed by pretreatment with OKY-1581. Thus, OKY-1581 provides a powerful tool which can be used to assess the participation of thromboxane in pathophysiological states and to ascertain the therapeutic potential of thromboxane synthetase inhibitors in numerous disease states.

Animals↗
PubMed1982

The arachidonic acid metabolic capacity of canine myocardium is increased during healing of acute myocardial infarction.

The relative capacity for metabolizing [14C]arachidonic acid into biologically active products was studied in microsomes prepared from both normal and infarcted regions of myocardium at three different times after circumflex coronary artery occlusion in the dog. At 3 days after infarction, when polymorphonuclear leukocytes were the predominant invading cell, the ability of infarcted left ventricle microsomes to produce arachidonic acid metabolites was greater than that of microsomes from normal areas of the same hearts. At 3 weeks after infarction, when macrophages were the predominant infiltrating cell and there was a proliferation of blood vessels and fibroblasts, there continued to be significant increases in the production of both prostacyclin (measured as 6-keto PGF1 alpha) and thromboxane (measured as TxB2). This enhanced production was still seen at 3 months after infarction at a time when histological examination of the tissue showed that it was still healing with both blood vessels and fibroblasts present. The production of 6-keto PGF1 alpha was 31.7 +/- 4 picomoles per milligram protein per hour (pmol/mg per hr) in noninfarcted regions of left ventricle, whereas the production was significantly increased to 71.7 +/- 15 at 3 days, 64.1 +/- 10 at 3 weeks, and 67.2 +/- 15 even at 3 months after infarction. The thromboxane synthetase activity rose significantly from 30.1 +/- 5 pmol mg per hr in noninfarcted regions to 73.7 +/- 18 at 3 days, 71.2 +/- 5 at 3 weeks, and 92.4 +/- 40 at 3 months. The enhanced ability to metabolize arachidonic acid may result from the inflammatory cell invasion or fibroblast activation which accompany healing of acute infarcts.

Animals↗
PubMed1982

Selective inhibitors of platelet arachidonic acid metabolism: aggregation independent of lipoxygenase.

The use of acetylenic acid analogs differing in chain length or position of the triple bonds permitted the systemic study of structure activity relationships for the arachidonate metabolizing enzymes (i.e, cyclo-oxygenase and lipoxygenase) in platelets and the relationship of these enzymes to aggregation. We were able to demonstrate analogs that were differentially selective in altering platelet arachidonic acid metabolism. We found analogs that preferentially: (a) inhibited cyclo-oxygenase only, (b) inhibited the 12-lipoxygenase only, (c) inhibited the cyclo-oxygenase and lipoxygenase, and (d) inhibited neither enzyme in platelets. There was a direct correlation between the rank order of potency of the acetylenic analogs to inhibit platelet cyclo-oxygenase and to suppress aggregation. Certain structural features of the triynoic acetylenic analogs were critical in influencing platelet function; thus the presence of a triple bond at position 14 as well as the lack of triple bond at position 5 resulted in analogs which inhibited both cyclo-oxygenase and platelet aggregation. On the other hand, analogs that inhibited only platelet 12-lipoxygenase were very weak inhibitors of platelet aggregation. These inhibitors provide potentially powerful tools for dissociating the two arachidonate metabolic pathways. If other tissues are are readily manipulated as platelets, the analogs may be especially useful for gaining insight into the contribution of lipoxygenase products to biological function.

Arachidonate Lipoxygenases↗
PubMed1981

Peptide-induced prostaglandin biosynthesis in the renal-vein-constricted kidney.

The ipsilateral kidney was removed from a rabbit 48h after unilateral partial renal-vein-constriction and was perfused with Krebs-Henseleit media at 37 degrees C. Hourly administration of a fixed dose of bradykinin to the renal-vein-constricted kidney demonstrated a marked time-dependent increase in the release of bioassayable prostaglandin E(2) and thromboxane A(2) into the venous effluent as compared with the response of the contralateral control kidney. The renal-vein-constricted kidney produced up to 60 times more prostaglandin E(2) in response to bradykinin after 6h of perfusion as compared with the contralateral kidney; thromboxane A(2) was not demonstratable in the contralateral kidney. Inhibition of protein synthesis de novo in the perfused renal-vein-constricted kidney with cycloheximide lessened the hormone-stimulated increase in prostaglandin E(2) by 94% and in thromboxane A(2) by 90% at 6h of perfusion. Covalent acetylation of the renal cyclo-oxygenase by prior oral administration of aspirin to the rabbit inhibited initial bradykinin-stimulated prostaglandin E(2) biosynthesis 71% at 1h of perfusion. However, there was total recovery from aspirin in the renal-vein-constricted kidney by 2h of perfusion after bradykinin stimulation. Total cyclo-oxygenase activity as measured by [(14)C]arachidonate metabolism to labelled prostaglandins by renal cortical and renal medullary microsomal fractions prepared from 6h-perfused kidneys demonstrated that renal-vein-constricted kidney-cortical cyclo-oxygenase activity was significantly greater than the contralateral-kidney-cortical conversion, whereas medullary arachidonate metabolism was comparable in both the renal-vein-constricted kidney and contralateral kidney. These data suggest that perfusion of a renal-vein-constricted kidney initiates a time-dependent induction of synthesis of prostaglandin-producing enzymes, which appear to be primarily localized in the renal cortex. The presence of the synthetic capacity to generate very potent vasodilator and vasoconstrictor prostaglandins in the renal cortex suggests that these substances could mediate or modulate changes in renal vascular resistance in pathological states.

Animals↗
PubMed1981

A novel prostaglandin 11-keto reductase found in rabbit liver.

The prostaglandin 11-keto reductase of rabbits, which catalyzes the conversion of prostaglandin D2 to prostaglandin F2 alpha, is only found in the liver. This enzyme, which is primarily localized in the soluble fraction, requires NADPH for activity; its Km value for prostaglandin D2 is approx. 200 muM. [14C]Prostaglandin D2 administered to rabbits via the portal vein escapes from the liver unchanged, suggesting that exogenous prostaglandin D2 may be inaccessible to the enzyme. The possible role of this enzyme in the metabolism of endogenous hepatic prostaglandin D2 is suggested by the demonstration that the liver synthesizes prostaglandin D2 from arachidonic acid.

Animals↗
PubMed1981

Localization of exaggerated prostaglandin synthesis associated with renal damage.

Regional localization of the exaggerated prostaglandin E2 (PGE2) synthesis caused by hydronephrosis was studied in unilateral ureteral ligated rabbits. The renal distribution of PGE2 production was compared in the hydronephrotic and contralateral kidneys. Basal and bradykinin-stimulated PGE2 synthesis were increased in cortical and medullary slices of the hydronephrotic kidneys. Contralateral (control) cortical slices produced very low levels of PGE2 and were insensitive to stimulation by bradykinin (BK). The hydronephrotic cortex produced 10 times more PGE2 than the contralateral cortex and responded to BK stimulation with increased PGE2 synthesis. Cortical slices from the hydronephrotic kidney exhibited a time-dependent increase in PGE2 release, presumably as a result of new protein synthesis. The division of the hydronephrotic cortex into outer and inner regions revealed that the inner cortex produced more PGE2 than the outer cortex. A similar division of the hydronephrotic medulla showed that the inner medulla produced slightly greater amounts of PGE2 than the outer medulla. The present study demonstrates that hydronephrosis causes increases in prostaglandin synthesis throughout the kidney. We suggest from these results and other studies that a possible explanation for this finding is the involvement of the collecting duct system in this response. The gradient of PGE2 production detected in the cortex may have a very significant role in the control of renal hemodynamics and could provide an explanation for the large decrease in blood flow to the inner cortex caused by indomethacin treatment.

Animals↗
PubMed1981

Ductus arteriosus closure may result from suppression of prostacyclin synthetase by an intrinsic hydroperoxy fatty acid.

Fetal sheep ductus arteriosus readily synthesizes prostacyclin from exogenous prostaglandin endoperoxides, and in the presence of high oxygen tension, this synthesis is markedly suppressed. Fetal aorta and pulmonary artery also synthesize prostacyclin; however, this synthesis is much less suppressed by high oxygen tension. We propose that ductal closure may be regulated by the oxygen dependent synthesis of hydroperoxy fatty acid which would block the production of the vasodilatory prostacyclin and expose the direct contractile properties of intrinsic prostaglandin endoperoxide. This mechanism would result in ductal closure at birth.

6-Ketoprostaglandin F1 alpha↗
PubMed1981

Stimulation of renal prostaglandin synthesis by the pressor activity of vasopressin.

Renal prostaglandin E2 (PGE2) synthesis is enhanced by exogenous vasopressin (AVP). To determine whether the pressor or the antidiuretic activities of AVP are related to its effect on PGE2 synthesis, AVP and the nonpressor analog, 1-deamino-8-D-arginine vasopressin (dD'AVP), were administered to the isolated perfused rabbit kidney, PGE2 was measured in the renal venous effluent by RIA and validated by bioassay. AVP in doses of 20, 200, and 2,000 ng progressively increased perfusion pressore by 12.1 +/- 2.4, 62.0 +/- 7.9, and 74.3 +/- 13.5 mm Hg, respectively, and increased PGE2 by 28 +/- 7, 80 +/- 3, and 129 +/- 57 ng/50 ml effluent, respectively. Bradykinin and angiotensin II induced a similar dose response on PGE2 release. However, dD'AVP in doses up to 20,000 ng minimally enhanced PGE2 release (20 +/- 16 ng/50 ml) and did not alter perfusion pressure. Simultaneous administration of AVP (200 ng) with the specific AVP pressor antagonist, d-cyclo-O-methyl-tyrosine-AVP, in a 1:10 (agonist to antagonist) weight ratio blunted the increase in perfusion pressure by 59 +/- 9% and blunted the increase in PGE2 release by 59 +/- 10% (P < 0.05). In a 1:100 weight ratio, the pressure antagonist reduced the AVP pressure response by 86 +/- 6% and reduced PGE2 by 84 +/- 7% (P < 0.01). These data suggest that the AVP stimulation of renal PGE2 synthesis is related primarily to its pressor and not to its antidiuretic activity in this in vitro kidney model. (Endocrinology 108: 495, 1981)

Angiotensin II↗