Biomedical subjects

J J Trout

Publications and source records attributed to J J Trout.

At least 19 recordsLinked to original sources

PubMed1995

Polyamines and NMDA receptors modulate pericapillary astrocyte swelling following cerebral cryo-injury in the rat.

Four hours following cryo-injury rat cerebral pericapillary astrocytes from the perilesional area were markedly swollen occupying 17% of the pericapillary space as compared to 11% in sham operated controls. Ornithine decarboxylase activity and polyamine levels were increased over sham controls. The astrocytic swelling, the percentage of the pericapillary space occupied by astrocytic processes, and polyamine levels were reduced to near control levels by the following: (1) alpha-difluoromethylornithine; (2) Ifenprodil; and (3) MK-801. alpha-Difluoromethylornithine is a specific inhibitor of ornithine decarboxylase, Ifenprodil is an inhibitor of the polyamine binding site on the n-methyl-d-aspartate receptor, and MK-801 is an antagonist to n-methyl-d-aspartate binding to the n-methyl-d-aspartate receptor. Addition of putrescine, the product of ornithine decarboxylase activity, reversed the effect of alpha-difluoromethylornithine and restored the pericapillary swelling. Putrescine did not affect the MK-801-induced reduction in pericapillary astrocytic swelling. Therefore, polyamines and the n-methyl-d-aspartate receptor modulate excitotoxic responses to cryo-injury in pericapillary cerebral astrocytes.

Animals↗

PubMed1994

Polyamines mediate coronary transcapillary macromolecular transport in the calcium paradox.

This study addresses the role of polyamines and their rate limiting enzyme, ornithine decarboxylase in regulation of macromolecular transport of two macromolecules, fluorescein and horseradish peroxidase, across coronary capillaries. Rat hearts were isolated and retrogradely perfused through the aorta (Langendorff method), stabilized by 10 min perfusion with Krebs-Henseleit medium containing Ca2+, followed by 5 min perfusion with Krebs-Henseleit medium without Ca2+ and an additional 30 s to 2 min with Krebs-Henseleit medium containing 1 mg/ml horseradish peroxidase. alpha-Difluoromethylornithine, the only known function of which is inhibition of ornithine decarboxylase, and putrescine were added as needed. Perfusion with Krebs-Henseleit medium without Ca2+ caused a two-fold increase in fluorescein transport but a decrease in horseradish peroxidase transport. Reperfusion with Krebs-Henseleit medium caused a four-fold increase in fluorescein transport and a ten-fold increase in horseradish peroxidase positive intraendothelial cell vesicles over control values. alpha-Difluoromethylornithine inhibited these increases and putrescine negated the alpha-difluoromethylornithine effect. Other morphological measures of horseradish peroxidase transport and associated membrane activities including modulation of endothelial cells luminal and abluminal pits were effected in the same manner. Transport of macromolecules through coronary capillaries, over the short term studied, appears to be regulated by the ornithine decarboxylase/polyamine pathway.

Animals↗

PubMed1994

Ligation-induced acute pancreatitis in rats and opossums: a comparative morphologic study of the early phase.

Bile and pancreatic duct ligation (BPDL) in rats does not induce severe acute pancreatitis but only mild inflammation, which is self-limiting and eventually leads to pancreatic atrophy. However, BPDL in opossums induces severe acute necrotizing pancreatitis which uniformly leads to death within 14 days. We compared pancreatic morphologic changes after 24 hr of BPDL in rats and opossums. Pancreatitis histology score and acinar cell ultrastructural changes were evaluated. In both species, BPDL was associated with significant increases in histology score compared to sham controls (5.0 +/- 0.3 vs 1.5 +/- 0.3 in rats, 5.3 +/- 0.4 vs 1.1 +/- 0.1 in opossums; mean +/- SEM, ANOVA, P < 0.05). However, there was no significant difference in histology score between rats and opossums following BPDL; histologic changes, such as white blood cell infiltration, acinar cell vacuolation, and focal acinar cell necrosis, were similar. Acinar cell ultrastructural changes after BPDL in both species included dilated endoplasmic reticulum and autophagic vacuole formation. These findings indicate that the early morphologic changes after BPDL in rats are quite similar to those seen early in the course of BPDL-induced acute necrotizing pancreatitis in opossums. As the rat is a more economical and convenient model to study than the opossum, this study supports the use of the rat model to conduct pilot studies of early events in the development of BPDL-induced acute pancreatitis. This study also suggests the potential for investigating mechanisms that may be present in the rat which protect against progressive and fatal acute necrotizing pancreatitis as observed in opossums after longer periods of BPDL.

Acute Disease↗

PubMed1993

N-methyl-D-aspartate receptor excitotoxicity involves activation of polyamine synthesis: protection by alpha-difluoromethylornithine.

We investigated the role of polyamines and their regulatory enzyme ornithine decarboxylase in N-methyl-D-aspartate-induced excitotoxicity in embryonic chick retina. N-Methyl-D-aspartate (200 microM) produced an early increase in ornithine decarboxylase activity, putrescine concentration, and Ca2+ entry, leading to selective neuronal death by 30 min. This response was attenuated by the ornithine decarboxylase inhibitor alpha-difluoromethylornithine and the N-methyl-D-aspartate receptor antagonist 5-aminophosphonovaleric acid. Exogenous putrescine increased intracellular putrescine and spermine levels and reversed neuroprotection by alpha-difluoromethylornithine, but not by 5-aminophosphonovaleric acid. N-Methyl-D-aspartate-receptor stimulation of putrescine/polyamine synthesis mediates abnormal Ca2+ entry and acute excitotoxic neuronal death. Postreceptor inhibition of the ornithine decarboxylase/polyamine cascade by alpha-difluoromethylornithine may provide neuroprotection against N-methyl-D-aspartate-induced excitotoxicity.

Animals↗

PubMed1992

Capillary NMDA receptors regulate blood-brain barrier function and breakdown.

Polyamines and their regulatory synthetic enzyme ornithine decarboxylase (ODC) have been implicated in blood-brain barrier (BBB) breakdown following cryogenic injury. ODC activation and BBB breakdown are prevented by MK-801, indicating involvement of NMDA receptors. Studies in isolated rat cerebral capillaries supports the presence of NMDA receptors linked to ODC. NMDA (1-50 microM) stimulated capillary uptake of horseradish peroxidase, 2-deoxy-[14C]glucose, and 45 Ca in a receptor-, concentration-, polyamine- and Ca(2+)-dependent manner. We suggest that NMDA receptors may couple capillary transport of nutrients to glutamate-mediated neuronal excitation, and when overestimated disrupt normal BBB function.

Animals↗

PubMed1990

Brain polyamines are controlled by N-methyl-D-aspartate receptors during ischemia and recirculation.

We studied the time course and molecular mechanisms of changes in brain polyamines and their rate-regulatory synthetic enzyme ornithine decarboxylase during reversible forebrain ischemia and recirculation in the gerbil. Bilateral carotid occlusion induced an acute (less than 2 minutes), transient increase in ornithine decarboxylase activity and putrescine level. After 15 minutes of ischemia, recirculation evoked an immediate (less than 1 minute) increase in ornithine decarboxylase activity and putrescine concentration that progressed over a 15-minute period. A small rise in spermidine and spermine also was observed. A secondary increase in ornithine decarboxylase activity and the levels of putrescine and spermidine commenced after 6 hours of recirculation. Pretreatment with a-difluoromethylornithine, a specific suicide inhibitor of ornithine decarboxylase, or MK-801, a noncompetitive N-methyl-D-aspartate receptor antagonist, abolished all early and delayed increases in ornithine decarboxylase activity and polyamine levels. This is the first demonstration that both ischemia and postischemic recirculation evoke rapid, transient increases in the activity of ornithine decarboxylase and the levels of polyamines, most notably the ornithine decarboxylase product, putrescine. Our results indicate that N-methyl-D-aspartate receptor activation (by an ischemically induced elevation of extracellular glutamate) is responsible for initiating the early and the delayed stimulation of ornithine decarboxylase activity. Ornithine decarboxylase activation causes the rapid rise in the levels of putrescine and higher polyamines observed in the acute response to ischemia and the acute and delayed response to postischemic recirculation. These polyamine changes may be involved in the pathophysiology of Ca2+ entry and neuronal death after brain ischemia.

Animals↗

PubMed1989

Polyamines and Ca2+ mediate hyperosmolal opening of the blood-brain barrier: in vitro studies in isolated rat cerebral capillaries.

We recently presented evidence that the reversible opening of the blood-brain barrier (BBB) by the infusion of 1.6 M mannitol into the rat internal carotid artery is mediated by a rapid stimulation of ornithine decarboxylase (ODC) activity and putrescine synthesis in cerebral capillaries. We have now investigated this hypothesis further, using isolated rat cerebral capillaries as an in vitro model of the BBB. The ODC activity of cerebral capillary preparations was enriched up to 15-fold over that of the cerebral homogenate. Hyperosmolal mannitol in physiological buffer evoked a rapid (less than 15 s), concentration- and time-dependent increase in capillary ODC activity and an accumulation of putrescine and spermidine which was blocked by the specific ODC inhibitor, alpha-difluoromethylornithine (DFMO, 10 mM). Mannitol (1 M), as well as 2 M urea, evoked a two- to fivefold increase in the temperature-sensitive influx of 45Ca2+ and uptake of horseradish peroxidase (HRP) and 2-deoxy-D-[1-3H]glucose (DG), but not alpha-[1-14C]aminoisobutyrate, during a 2-min incubation. DFMO (10 mM) abolished 1 M mannitol-mediated stimulation of 45Ca2+ influx and uptake of HRP and DG, whereas 1 mM putrescine replenished capillary polyamines and reversed the DFMO effects. Mannitol (1 M)-induced stimulation of ODC activity and membrane transport processes was Ca2+-dependent and verapamil- and nisoldipine-sensitive. Phorbol myristate acetate (PMA, 10 nM), a protein kinase C activator, also evoked a two- to threefold stimulation of 45Ca2+ transport and HRP and DG uptake. This PMA effect was abolished by DFMO, suggesting involvement of rapid, ODC-controlled polyamine synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Aminoisobutyric Acids↗

PubMed1989

Polyamines mediate androgenic stimulation of calcium fluxes and membrane transport in rat heart myocytes.

The androgenic steroid hormone testosterone induced an early (less than 30-60 seconds) stimulation of endocytosis, hexose transport, and amino acid transport, monitored by the temperature-sensitive uptake of horseradish peroxidase, 2-deoxyglucose, and alpha-aminoisobutyrate, respectively, in rat ventricle cubes and acutely isolated ventricular myocytes. This stimulation was time- and concentration-dependent and was maximal at 10(-9) to 10(-8) M testosterone, consistent with androgen-receptor mediation. EGTA (2.5 mM), La3+ (1 mM), and verapamil (100 microM) ablated the hormonal response. The calcium ionophore A23187 (10 microM) induced an acute stimulation of endocytosis, amino acid transport, and hexose transport which was not further increased by testosterone (10(-8) M), suggesting a common effector pathway. Testosterone (10(-8) M) also evoked a rapid (less than 30 seconds) stimulation of 45Ca influx and efflux. Testosterone (10(-8) M) induced a rapid (less than 5 seconds) transient increase in ornithine decarboxylase (ODC) activity peaking (twofold to threefold) at 60 seconds, and an early (15 seconds) transient accumulation of polyamines peaking at 60 seconds in isolated myocytes. The specific, irreversible ODC inhibitor alpha-difluoromethylornithine (DFMO, 5-10 mM) blocked the testosterone-evoked increase in ODC activity and polyamine levels and the stimulation of Ca2+ fluxes, endocytosis, hexose transport, and amino acid transport. Putrescine (0.5-1 mM), the ODC product, reversed DFMO inhibition and restored the increase in polyamines, 45Ca fluxes, and Ca2+-dependent membrane transport processes. These results demonstrate that rapid, transient ODC-regulated polyamine synthesis is essential for androgenic stimulation of Ca2+ fluxes and membrane transport processes in ventricular myocytes. These findings support a model for signal transduction in which newly synthesized polyamines serve as intracellular messengers to regulate transmembrane Ca2+ movements, Ca2+-dependent membrane transport functions, and other Ca2+- and polyamine-sensitive processes in cardiac myocytes.

Amino Acids↗

PubMed1987

Polyamines mediate uncontrolled calcium entry and cell damage in rat heart in the calcium paradox.

Brief perfusion of heart with calcium-free medium renders myocardial cells calcium-sensitive so that readmission of calcium results in uncontrolled Ca2+ entry and acute massive cell injury (calcium paradox). We investigated the hypothesis that polyamines may be involved in the mediation of abnormal Ca2+ influx and cell damage in the calcium paradox. The isolated perfused rat heart was used for these studies. Calcium-free perfusion promptly (less than 5 min) decreased the levels of polyamines and the activity of their rate-regulating synthetic enzyme, ornithine decarboxylase (ODC), and calcium reperfusion abruptly (less than 15-180 s) increased these components. alpha-Difluoromethylornithine (DFMO), a specific suicide inhibitor of ODC, suppressed the calcium reperfusion-induced increase in polyamines and the concomitant increase in myocardial cellular 45Ca influx, loss of contractility, release of cytosolic enzymes, myoglobin, and protein, and structural lesions. Putrescine, the product of ODC activity, nullified DFMO inhibition and restored the calcium reperfusion-induced increment in polyamines and the full expression of the calcium paradox. Putrescine itself enhanced the reperfusion-evoked release of myoglobin and protein in the absence of DFMO. Hypothermia blocked the changes in heart ODC and polyamines induced by calcium-free perfusion and calcium reperfusion and prevented the calcium paradox. These results indicate that rapid Ca2+-directed changes in ODC activity and polyamine levels are essential for triggering excessive transsarcolemmal transport of Ca2+ and explosive myocardial cell injury in the calcium paradox.

Animals↗

PubMed1986

Blood-brain barrier breakdown by cold injury. Polyamine signals mediate acute stimulation of endocytosis, vesicular transport, and microvillus formation in rat cerebral capillaries.

Polyamines have been previously implicated in the mediation of blood-brain barrier breakdown induced by cryogenic injury (H Koenig, AD Goldstone, CY Lu, Biochem Biophys Res Commun 116:1039, 1983). We studied acute (less than 5 minute) changes in capillary ultrastructure, microvascular permeability, and the levels of polyamines and their rate regulating synthetic enzyme ornithine decarboxylase (ODC) in rat cerebral cortex after focal cold injury. Microvascular permeability was measured by relative transport of intravenously administered fluorescein. Capillary ultrastructure was studied by quantitative stereology and morphometry after intravenous administration of horseradish peroxidase. Focal cold injury induced a 2.5-, 3.8-, 1.7-, and 1.4-fold increase in the levels of ODC, putrescine, spermidine and spermine, and a 46-fold increase in fluorescein uptake in perilesional cortex. Few capillaries in control cortex contained endocytic pits or horseradish peroxidase-positive vesicles, whereas most capillaries near lesions showed these structures. Cryoinjury induced a 5-fold increase in the relative volume of microvilli and horseradish peroxidase vesicles, a 2.3-fold increase in area of luminal endocytic pits, and a 6.3-fold increase in area of abluminal exocytic pits. The ODC inhibitor alpha-difluoromethylornithine blocked the cryoinjury-induced changes in ODC, polyamines, fluorescein uptake, and capillary ultrastructure. Putrescine negated the effect of alpha-difluoromethylornithine or capillary ultrastructure, and was previously shown to nullify the alpha-difluoromethylornithine effects on polyamines and fluorescein permeability (cited above). These data link rapid changes in ODC and polyamines to blood-brain barrier breakdown, and suggest that the abnormal permeability is associated with an acute, polyamine-mediated stimulation of microvillus formation, endocytosis, and vesicular transport in capillary endothelium.

Animals↗

PubMed1984

Cytochemistry of myocardial structures related to degenerative processes in spontaneously hypertensive and normotensive rats.

Using electron microscopy and cytochemical techniques we investigated structures which are associated with long-term hypertension and ageing in the myocardial cell of the rat. Lysosomes, demonstrated by acid phosphatase and aryl sulfatase activities, were found mainly in the perinuclear region in young rats. With age these organelles appeared with increasing frequency in other regions of the cell. Spontaneously hypertensive rats (SHR) showed an earlier apparent migration of lysosomes than did normotensive rats (WKY). Our observations indicate that lysosomes were closely associated with autophagic vacuoles, membrane swirls , translucent mitochondria, myelin figures and other structures linked with degenerative events. In the oldest SHR lysosomes, autolysosomes (autophagic vacuoles with lysosomal activity), and degenerative structures were observed in various regions of the myocardial cell. Peroxisomes, as demonstrated by catalase activity, did not seem to be affected by hypertension or age. A number of dense osmophilic structures did not react for any of the enzymes studied; these included myelin figures, mitochondrial inclusions and diffuse dense bodies. Our observations implicate both ageing and hypertension in the enhancement of lysosomes and their end products.

Acid Phosphatase↗

PubMed1983

Beta-adrenergic stimulation evokes a rapid, Ca2+-dependent stimulation of endocytosis, hexose and amino acid transport associated with increased Ca2+ fluxes in mouse kidney cortex.

The beta-adrenergic agonist 1-isoproterenol evokes an acute (less than 5 min) stimulation of endocytosis, hexose transport and amino acid transport, measured by the temperature-sensitive uptake of HRP, 3H-DG and 14C-AIB, in mouse kidney cortex slices. This stimulation is concentration dependent and is maximal at 10(-8)-10(-7) M isoproterenol. Peroxidase cytochemistry showed that the hormonal increase in HRP uptake is confined to proximal tubules. The rapid membrane response is abolished in a calcium-free medium and by the beta-adrenergic antagonist propranolol, indicating Ca2+- and beta-adrenoreceptor-dependence. Isoproterenol (1 microM) rapidly (less than 30 sec) stimulates the influx and efflux of 45Ca in cortex slices. Isoproterenol also decreased mitochondrial 45Ca and increased soluble 45Ca. These results indicate that beta-adrenergic stimulation of membrane transport functions involves an increased influx of extracellular calcium and a mobilization of intracellular (mitochondrial) calcium. An increase in cytosolic Ca2+ concentration appears to be the regulatory signal for these membrane transport processes.

Amino Acids↗

PubMed1982

Ultrastructure of the human intervertebral disc: II. Cells of the nucleus pulposus.

The cells of the intervertebral disc exist in a unique environment; not only are discs subject to large mechanical loads, they are the largest avascular structures in the body. To describe the ultrastructure and age changes in cells from human nucleus pulposus, we studied these cells in individuals ranging in age from the 26th week of fetal life to 91 years. Viable chondrocyte-like cells existed in specimens from all ages. The presence of Golgi cisternae and well-developed endoplasmic reticulum in these cells suggests that they are capable of producing and maintaining the extracellular matrix. Necrotic cells were also present in all samples, and many cells which appeared viable when examined by light microscopy proved to be necrotic when examined by electron microscopy. The percentage of necrotic cells increased with age from 2% or less in fetal specimens to over 50% in adults. In addition, with age, a distinct pericellular matrix or "nest," consisting of collagen fibrils, fine filaments, dense particles, and banded structures, formed around most cells with no apparent preference for viable or necrotic cells. Nest formation and increasing density of the cell nests may reflect accumulation of cell products.

Adolescent↗

PubMed1982

Fiber splitting in tonic and phasic skeletal muscles following denervation, chloroquine and triton WR-1339 treatments.

Fiber splitting was studied in the tonic, anterior (ALD) and phasic, posterior (PLD) latissimus dorsi muscles using three experimental models, denervation, chloroquine administration and Triton WR-1339 administration. Fiber splitting was observed in the PLD in all three models, but in the ALD only after chloroquine administration. A basement membrane was always present in the split fibers, and acid phosphatase was localized between the split fibers in the PLD. However, no evidence of lysosomal involvement in the fiber splitting process was observed.

Acid Phosphatase↗

PubMed1982

Ultrastructure of the human intervertebral disc. I. Changes in notochordal cells with age.

We examined nucleus pulposus notochordal cells of individuals ranging in age from the eighth week of fetal life to 32 years of age. With increasing age, notochordal cell structure changed, as did the cell-to-cell relationships and the cell-to-chordal relationships. All notochordal cells contained normal organelles, including well-developed endoplasmic reticulum, but, in addition, fetal notochordal cells demonstrated an unusual relationship between rough endoplasmic reticulum and mitochondria: elements of the rough endoplasmic reticulum encircled almost every mitochondrion. Fetal notochordal cells contained large amounts of glycogen, while older cells had much smaller glycogen deposits. Cytoplasmic filaments were observed in cells of all ages. The cells formed tightly packed clusters in the fetus with little, if any, extracellular matrix between individual cells. Cells separated from each other with age and by the twenty-first week of fetal life, only slender strands of cytoplasm connected them. Previous light microscopic studies described notochordal cells as 'physaliphorus' cells since they appeared to contain large cytoplasmic vacuoles. However, electron microscopy showed that these apparent vacuoles consist of extracellular matrix surrounded by cells or cell processes. The structure of notochordal cells and their persistence in the nucleus pulposus after fetal life suggest that they may have a significant role in the formation and maintenance of the nucleus pulposus.

Adolescent↗

PubMed1981

Further studies on a unique T-tubular acid phosphatase in avian skeletal muscle.

With the unique observation, using conventional cytochemistry, of acid phosphatase reaction production in the T-tubules of the posterior latissimus dorsi muscle of the chicken, the possibility of endocytosis of lysosomal enzymes by muscle cells came into question. After testing the substrate specificity of this T-tubular phosphatase, it was clear that the enzyme hydrolysed glucose 6-phosphate and beta-glycerophosphate at pH 5.0 but not cytidine-5'-monophosphate which was hydrolysed by dense bodies and autophagic vacuoles. The cytochemical evidence points to a unique phosphatase present on muscle cell membranes which apparently does not belong to the vacuolar apparatus of skeletal muscle and is not 5'-nucleotidase.

Acid Phosphatase↗

PubMed1981

Degeneration and regeneration in denervated tonic and phasic skeletal muscle: morphology and acid phosphatase cytochemistry.

Following denervation, ultrastructural alterations were observed in the tonic, anterior (ALD) and phasic posterior (PLD) latissimus dorsi muscles of the chicken. In the ALD muscle these changes were characteristic of both degeneration and regeneration, while in the PLD muscle, the changes were characteristic only of degeneration. Acid phosphatase positive structures, which included dense bodies in the ALD and PLD as well as T-tubules in the PLD, were observed intact with no evidence of release of enzyme into the sarcoplasm. No evidence of an increase in the number of autophagic vacuoles was found. The morphological evidence presented in this communication does not support the hypothesis that lysosomes are involved in denervation atrophy through autophagy of muscle cell constituents.

Acid Phosphatase↗

PubMed1981

Skeletal muscle myopathy caused by triton WR-1339.

After administration (1 g/kg every other day for a total of five injections) of Triton WR-1339, the tonic, anterior (ALD) and phasic, posterior (PLD) latissimus dorsi muscles of the chicken underwent distinct pathological modifications. Some of the morphological alterations in the muscles paralleled those seen after administration of chloroquine, increased autophagic vacuole formation in the ALD muscle and swelling of the sarcoplasmic reticulum in the PLD muscle, but other changes were unique to Triton WR-1339. These included loss of myofilaments and whole myofibrils, indentation of the sarcolemma as well as increased numbers of ribosomes in the ALD muscle and swelling of the T-tubular system in the PLD muscle. These results are compared with other lysosome mediated pathologies, as well as with other myopathies.

Animals↗