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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.

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.

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)

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.

Activation of cell-surface receptors often evokes changes in Ca2+ fluxes leading to an increase in cytosolic Ca2+, a generally accepted mediator of many cell responses. The molecular mechanisms by which surface agonists elicit these changes in Ca2+ flux have remained elusive. An increase in the polyamines putrescine, spermidine and spermine, and their rate-regulating, synthetic enzyme ornithine decarboxylase (ODC), is one of the earliest events that occur during cell growth, replication and differentiation. However, the precise physiological roles of the polyamines remain enigmatic. Recently, we found that testosterone induces an early (less than 60s), Ca2+- and receptor-dependent stimulation of endocytosis, hexose transport and amino acid transport in mouse kidney cortex involving the proximal tubules. This response is associated with increased Ca2+ fluxes and a mobilization of intracellular calcium, and is thought to represent a direct, receptor-mediated action of testosterone on the surface membrane. Polyamine synthesis was previously found to be essential for the long-term effects of testosterone on mouse kidney. We now report that testosterone evokes a rapid (less than 30 s), transient increase in ODC activity and a sustained increase in polyamines in kidney cortex. This polyamine synthesis is obligatory for stimulation of membrane transport functions and Ca2+ fluxes. These findings form the basis for a new theory of information flow in stimulus-response coupling in which the polyamines serve as messengers to generate a Ca2+ signal by increasing Ca2+ influx and mobilizing intracellular calcium via a cation-exchange reaction.

We recently found that the beta-adrenergic agonist 1-isoproterenol evokes a rapid (less than 5 min) Ca2+- and receptor-dependent stimulation of endocytosis, hexose transport, and amino acid transport in mouse renal cortex involving proximal tubule cells. This response is associated with increased Ca2+ fluxes and a mobilization of mitochondrial calcium, suggesting that stimulus-response (stimulus-"transport") coupling is mediated by cytosolic Ca2+. We show here that 1 microM isoproterenol evokes a rapid (less than 60 sec) transient increase in the activity of ornithine decarboxylase followed by an early (less than 2 min) sustained increase in putrescine, spermidine, and spermine concentrations in mouse kidney cortex slices in vitro. Small doses of isoproterenol (down to 24 nmol/kg) elicited a rapid (less than 2 min) increase in polyamines in vivo. The ornithine decarboxylase inhibitor alpha-difluoromethylornithine (5 mM) suppressed the testosterone-induced increase in polyamine levels and rates of endocytosis, hexose transport, and amino acid transport, measured by horseradish peroxidase, [14C]aminoisobutyric acid, and deoxy[3H]glucose uptake. alpha-Difluoromethylornithine also blocked the isoproterenol-induced increase in 45Ca influx and efflux and 45Ca redistribution; 0.5 mM putrescine nullified alpha-difluoromethylornithine inhibition and restored the increment in polyamines, 45Ca fluxes, endocytosis, hexose transport, and amino acid transport. These data implicate polyamine synthesis in isoproterenol stimulation of Ca2+ fluxes and membrane transport processes and support a model for signal transduction and stimulus-response coupling in which ornithine decarboxylase activation and polyamine synthesis play a pivotal role in regulating Ca2+ fluxes. In this model the polyamines generate local Ca2+ signals by stimulating Ca2+ influx or mobilizing intracellular calcium (or both) through a cation exchange reaction.

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.

Testosterone was previously shown to induce an early (less than 1 min) receptor-dependent stimulation of endocytosis, hexose and amino acid transport in mouse kidney cortex (Koenig, H., Goldstone, A. and Lu, C.Y. (1982) Biochem. Biophys. Res. Commun. 104, 165-172). Testosterone (10(-8) M) has now been found to stimulate rapidly (less than 30 s) the influx and efflux of 45Ca2+ in cortex slices. Testosterone also decreased mitochondrial 45Ca and augmented soluble 45Ca, indicating a mobilization of intracellular calcium. Incubation of cortex slices in calcium-free medium without or with 2.5 mM EGTA decreased basal endocytosis, hexose and amino acid transport and blocked the hormonal response. 100 microM verapamil blocked the hormonal response without affecting basal transport. The calcium ionophore A23187 rapidly stimulated endocytosis, hexose and amino acid transport. These data indicate that androgenic stimulation of membrane transport functions involves an increased influx of extracellular calcium and a mobilization of intracellular calcium. Increased cytosolic Ca2+ is probably the regulatory signal for these transport processes.

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.