Harold Koenig

Polyamines appear to be second messengers in mediating Ca2+ fluxes and neurotransmitter release in potassium-depolarized synaptosomes

High potassium (50 mM) depolarization induces a rapid (less than 15 sec) increase in the levels of the polyamines putrescine, spermidine and spermine and their rate-regulating synthetic enzyme ornithine decarboxylase in synaptosomes from rat cerebral cortex. The ornithine decarboxylase inhibitor alpha-difluoromethylornithine blocked the K+-stimulated increase in enzyme activity and polyamines and also suppressed the increase in 45Ca2+ influx and efflux and the Ca2+-dependent release of GABA and norepinephrine. Added putrescine attenuated or negated the effects of alpha-difluoromethylornithine. These results suggest that enhanced polyamine synthesis is required for potassium depolarized stimulation of synaptic function.

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.

Blood brain barrier breakdown in brain edema following cold injury is mediated by microvascular polyamines

A focal freeze injury to rat cerebral cortex induces an early (less than 5 min) increase in brain ornithine decarboxylase activity and an accumulation of polyamines involving cerebral microvessels. This polyamine synthesis correlates with the abnormal increase in microvascular permeability, monitored by uptake of Evans Blue and sod. fluorescein. The ornithine decarboxylase inhibitor alpha-difluoromethylornithine suppressed the injury-induced increment in spermidine and spermine and microvascular permeability. Putrescine nullified alpha-difluoromethylornithine inhibition and restored microvessel spermidine and spermine and the pathological increase in microvascular permeability. These results indicate that polyamine synthesis is obligatory for blood-brain barrier breakdown. alpha-Difluoromethylornithine may be useful in the treatment of vasogenic brain edema.

Blood–Brain Barrier Breakdown in Cold-Injured Brain Is Linked to a Biphasic Stimulation of Ornithine Decarboxylase Activity and Polyamine Synthesis: Both Are Coordinately Inhibited by Verapamil, Dexamethasone, and Aspirin

Abstract: An early increase in ornithine decarboxylase (ODC) activity and polyamine levels in rat cerebral capillaries was previously implicated in the mediation of blood–brain barrier (BBB) breakdown in cold‐injured brain. A time course study in rat cerebrum indicated that cold injury evokes a biphasic increase in ODC activity and polyamine levels in perilesional cortex. ODC activity rose sharply (fourfold) within 1 min, remained elevated for 5 min, and then returned to the basal level by 10 min. A transient rise in polyamine concentration followed in the rank order of putrescine > spermidine > spermine. A secondary rise in ODC activity commenced in perilesional tissue at 2–6 h and peaked (8.8‐fold) at 48 h. Major increases in the content of putrescine (330%), spermidine (103%), and spermine (50%) developed at 48–72 h. α‐Difluoromethylornithine (DFMO), a specific irreversible inhibitor of ODC, suppressed the evoked increase in ODC activity and abolished the associated increase in content of polyamines, findings indicating that the accumulation of polyamines in cryoinjured brain reflects enhanced synthesis resulting from an ODC‐mediated increase in putrescine content. Cycloheximide and actinomycin D were without effect on the early increase in ODC activity but inhibited the delayed increase in ODC activity, an observation suggesting that the initial increase in activity reflects an activation of a cryptic ODC via a posttranslational process, whereas the delayed increase in activity results from ODC synthesis mainly under transcriptional control. Because membrane phospholipid degradation, release of diacylglycerol and free arachidonic acid, and prostaglandin formation are early events in cold‐injured brain, we assessed the effects of verapamil (a calcium channel blocker), dexamethasone (which inhibits arachidonic acid release), and aspirin (a cyclooxygenase inhibitor). These agents resembled DFMO in that they inhibited the early (2‐min) and delayed (24‐h) increase in ODC activity and polyamine concentrations and concurrently attenuated BBB breakdown in the perilesional cortex, as monitored by fluorescein transport. Exogenous putrescine nullified the protective effect of verapamil, dexamethasone, and aspirin on BBB breakdown following cryogenic injury. These results implicate Ca2+ influx via calcium channels, phospholipid hydrolysis, and prostaglandin synthesis in cryogenically induced stimulation of ODC activity and further strengthen the evidence linking polyamines to BBB breakdown. Changes in ODC‐regulated polyamine synthesis in brain cells may play an important role in other aspects of the pathophysiology of cerebral injury.

Isolation and characterization of acidic lipoprotein in renal and hepatic lysosomes

Abstract Purified lysosomal fractions from rat liver and kidney were sonicated with Triton X-100 (.2%), and soluble material subjected to flotation ultracentrifugation in KBr. The soluble protein, 85% of the total, was separated into a low density lipoprotein (LDLP, d in situ .

Polyamines and Ca2+ Mediate Hyperosmolal Opening of the Blood‐Brain Barrier: In Vitro Studies in Isolated Rat Cerebral Capillaries

Abstract 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 (<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, α‐ difluoromethylornithine (DFMO, 10 m M). 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 α‐[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. The effects of 10 nM PMA and 1 M mannitol were additive, suggesting that hyperosmolal stimulation of ODC‐activated polyamine synthesis does not involve protein kinase C. These data support the hypothesis that ODC‐activated polyamine synthesis and Ca2+ influx (via Ca2+ channels) play a key role in mediating the effects of hyperosmolality on BBB permeability.

Polyamines mediate the reversible opening of the blood-brain barrier by the intracarotid infusion of hyperosmolal mannitol

The blood-brain barrier (BBB) can be opened transiently by infusing a hyperosmolal solution of a non-electrolyte into the internal carotid artery. We investigated the hypothesis that capillary polyamines and their rate-regulating synthetic enzyme, ornithine decarboxylase (ODC), may be involved in mediating BBB breakdown in this model, as they are in BBB breakdown by focal cold injury. The intracarotid infusion of 1.6 M mannitol induced a prompt (less than 2 min) increase in ODC activity and the levels of polyamines in the ipsilateral hemisphere. Isolated cerebral capillary preparations and neural elements showed similar increases in ODC activity. The rank order of increase at 2 min, ODC (170%) greater than putrescine (90%) greater than spermidine (15%) greater than spermine (7%), was consistent with an activation of the ODC-regulated pathway of polyamine synthesis. The specific ODC inhibitor alpha-difluoromethylornithine (DFMO) blocked the 1.6 M mannitol-induced increase in ODC activity and the accumulation of polyamines, and concurrently prevented BBB breakdown, monitored by transport of intravenously administered Evans blue and alpha-[3H]aminoisobutyrate into cerebral tissue. Exogenous putrescine, the product of ODC activity, replenished brain polyamines and negated DFMO protection allowing BBB breakdown by 1.6 M mannitol. These experiments support the hypothesis that BBB breakdown induced by the intracarotid infusion of hyperosmolal mannitol is mediated by rapid, ODC-regulated synthesis of microvascular polyamines. In addition, increases in ODC-controlled polyamine synthesis in nerve cells may play a significant role in the pathophysiology of the reversible neuronal dysfunction, e.g. diazepam-sensitive seizure-like activity, enhanced glucose utilization, evoked by the intracarotid infusion of hyperosmolal mannitol.

Testosterone induces a rapid stimulation of endocytosis, amino acid and hexose transport in mouse kidney cortex

Abstract Testosterone induced a rapid ( tfm Y mice did not respond to testosterone. The rapid increase in endocytosis, amino acid and hexose transport may represent a direct, receptor-mediated response of the surface membrane of target cells to testosterone.

Polyamines are intracellular messengers in the β-adrenergic regulation of Ca2+ fluxes, [Ca2+]i and membrane transport in rat heart myocytes

The beta-adrenergic agonist 1-isoproterenol (0.1 microM) evokes an acute (less than 5-10 sec) transient increase in the activity of ornithine decarboxylase (ODC), and the levels of polyamines (putrescine, spermidine, spermine) in acutely isolated rat ventricular myocytes. Isoproterenol rapidly (less than 15 sec) increases 45Ca influx and efflux, decreases [Ca2+]i, and stimulates Ca2+-dependent membrane transport (endocytosis, hexose transport, amino acid transport). The beta-adrenergic antagonist propranolol blocks isoproterenol-induced membrane transport. The ODC inhibitor alpha-difluoromethylornithine (DFMO, 5-10 mM) blocks the isoproterenol-evoked increase in ODC activity and polyamine levels and the changes in 45Ca fluxes, [Ca2+]i and membrane transport. Putrescine (0.5-1 mM) replenishes cellular polyamines and reverses the DFMO effect. These data exclude an increase in [Ca2+]i in stimulus-transport coupling, and support the hypothesis that polyamines are messengers in beta-adrenoceptor-mediated regulation of transmembrane Ca2+ fluxes, [Ca2+]i, and Ca2+-dependent membrane transport.

Synthesis and turnover of lysosomal glycoproteins. Relation to the molecular heterogeneity of the lysosomal enzymes.

Many, if not all, of the lysosomal acid hydrolases exist in several molecular forms. For example, using polyacrylamide gel electrophoresis [l] and isoelectric focusing [2,3] we found two to five distinct forms of seven different hydrolases in rat kidney lysosomes. That this molecular heterogeneity may have biological importance is strongly suggested by the recent discovery that the acidic (A) form of a lysosomal hydrolase is lacking in two inherited lipid storage diseases, namely aryl sulfatase A in metachromatic leukodystrophy [4,5] and hexosaminidase A in TaySachs disease [6,7] . Apparently the corresponding basic (B) form of these hydrolases which is present in normal or elevated amounts in these diseases is incapable of hydrolyzing the accumulated sphingolipid, cerebroside sulfate in metachromatic leukodystrophy [4,5] and GM, ganglioside (and its asialo derivative) in Tay-Sachs disease [6,7] . These observations imply that the different molecular forms may display divergent substrate specificities and hydrolytic activities in vivo. We have shown that the various acid hydrolases in purified lysosomal fractions from rat kidney and liver are glycoproteins (GPs) [ 1,8,9] . A study of the effects of bacterial neuraminidase on the electrophoretic mobility [8] , and pIs [3] of a number of lysosomal hydrolases indicated that N-acetylneuraminic acid (NANA) residues are largely responsible for the solubility and electronegative charge of these enzymes. On the basis of the solubility and electrophoretic mobility of five acid hydrolases in rat kidney subcellular fractions [9, lo] , and