Prostaglandin responses in isolated perfused rat liver: Ca2+ and K+ fluxes, hemodynamic and metabolic effects

Addition of prostaglandin F2 alpha and prostaglandin E2 to isolated perfused rat liver led to a dose-dependent, transient net Ca2+ release, which was completed within 3 min. Withdrawal of the prostaglandins resulted in a Ca2+ re-uptake over a period of about 10 min. Simultaneously, these prostaglandins induced an increase of portal pressure, stimulated hepatic glucose output and 14CO2 production from [1-14C]glutamate and led to K+ movements across the hepatocyte plasma membrane similar to those observed with other Ca2+-mobilizing agents. With prostaglandin F2 alpha there was a close correlation between the net Ca2+ release and the maximal rate of initial net K+ uptake by the liver (linear regression coefficient r = 0.902; n = 20). Prostaglandin F2 alpha was more effective than prostaglandin E2 or D2. Because prostaglandins are known to be produced by hepatic non-parenchymal cells during stimulation by phagocytosis or by addition of extracellular ATP or UTP, these data suggest an interaction between non-parenchymal and parenchymal liver cells and point to a modulating role of prostaglandins in hepatic metabolism and microcirculation, which is mediated by Ca2+-mobilizing mechanisms.     

Calcium ion fluxes induced by the action of alpha-adrenergic agonists in perfused rat liver.

Phenylephrine (2.0 microM) induces an alpha 1-receptor-mediated net efflux of Ca2+ from livers of fed rats perfused with medium containing physiological concentrations (1.3 mM) of Ca2+. The onset of efflux (7.1 +/- 0.5 s; n = 16) immediately precedes a stimulation of mitochondrial respiration and glycogenolysis. Maximal rates of efflux are observed between 35 s and 45 s after alpha-agonist administration; thereafter the rate decreases, to be no longer detectable after 3 min. Within seconds of terminating phenylephrine infusion, a net transient uptake of Ca2+ by the liver is observed. Similar effects were observed with vasopressin (1 m-unit/ml) and angiotensin (6 nM). Reducing the perfusate [Ca2+] from 1.3 mM to 10 microM had little effect on alpha-agonist-induced Ca2+ efflux, but abolished the subsequent Ca2+ re-uptake, and hence led to a net loss of 80-120 nmol of Ca2+/g of liver from the tissue. The administration at 5 min intervals of short pulses (90 s) of phenylephrine under these conditions resulted in diminishing amounts of Ca2+ efflux being detected, and these could be correlated with decreased rates of alpha-agonist-induced mitochondrial respiration and glucose output. An examination of the Ca2+ pool mobilized by alpha-adrenergic agonists revealed that a loss of Ca2+ from mitochondria and from a fraction enriched in microsomes accounts for all the Ca2+ efflux detected. It is proposed that the alpha-adrenergic agonists, vasopressin and angiotensin mobilize Ca2+ from the same readily depleted intracellular pool consisting predominantly of mitochondria and the endoplasmic reticulum, and that the hormone-induced enhanced rate of mitochondrial respiration and glycogenolysis is directly dependent on this mobilization.     

Possible involvement of cyclooxygenase products in the actions of platelet-activating factor and of lipoxygenase products in the vascular effects of epinephrine in perfused rat liver

Platelet-activating factor (PAF) stimulates glycogenolysis and induces vasoconstriction in perfused rat liver. The effect of PAF was rapid but transient and it was blocked by indomethacin and bromophenacyl bromide which suggests a role of cyclooxygenase metabolites in its action. The homologous desensitization of glycogenolysis produced by PAF and the sensitivity of its actions to inhibitors of cyclooxygenase and phospholipase A2 markedly differentiate the mechanism of action of this agent with that of alpha 1-adrenergic agents, vasopressin or angiotensin II. No effect of PAF in isolated hepatocytes was observed which suggest that cells other than hepatocytes could be involved in its action in perfused liver. In addition nordihydroguaiaretic acid and bromophenacyl bromide abolished the vascular effect (but not the glycogenolysis) produced by epinephrine which suggest a role for lipoxygenase products in this effect.     

Inhibition of the glycogenolytic effects of alpha-adrenergic stimulation and glucagon by cobalt ions in perfused rat liver

Cobalt ions (2 mM) inhibited the glycogenolysis induced by phenylephrine and glucagon in perfused rat liver. Cobalt ions also inhibited 45Ca++ efflux from prelabelled livers induced by phenylephrine and glucagon. In addition, they inhibited the rise in tissue levels of cyclic AMP caused by glucagon, but did not inhibit the stimulation of 45Ca++ efflux or glycogenolysis by cyclic AMP or dibutyryl cyclic AMP. The specific binding of glucagon and alpha-agonist to hepatocytes was not inhibited by cobalt ions. These data suggest that cobalt ions, presumably through their high affinity for calcium binding sites on membranes inhibit the stimulation of glycogenolysis by phenylephrine and glucagon in distinct ways; one by inhibiting calcium mobilization and the other by inhibiting cyclic AMP production. Therefore, it is conceivable that membrane-bound calcium plays an important role in stimulating Ca++ mobilization by phenylephrine, and cyclic AMP production by glucagon.     

Stimulation by alpha-adrenergic agonists of Ca2+ fluxes, mitochondrial oxidation and gluconeogenesis in perfused rat liver.

Glucose output from perfused livers of 48 h-starved rats was stimulated by phenylephrine (2 microM) when lactate, pyruvate, alanine, glycerol, sorbitol, dihydroxyacetone or fructose were used as gluconeogenic precursors. Phenylephrine-induced increases in glucose output were immediately preceded by a transient efflux of Ca2+ and a sustained increase in oxygen uptake. Phenylephrine decreased the perfusate [lactate]/[pyruvate] ratio when sorbitol or glycerol was present, but increased the ratio when alanine, dihydroxyacetone or fructose was present. Phenylephrine induced a rapid increase in the perfusate [beta-hydroxybutyrate]/[acetoacetate] ratio and increased total ketone-body output by 40-50% with all substrates. The oxidation of [1-14C]octanoate or 2-oxo[1-14C]glutarate to 14CO2 was increased by up to 200% by phenylephrine. All responses to phenylephrine infusion were diminished after depletion of the hepatic alpha-agonist-sensitive pool of Ca2+ and returned toward maximal responses after Ca2+ re-addition. Phenylephrine-induced increases in glucose output from lactate, sorbitol and glycerol were inhibited by the transaminase inhibitor amino-oxyacetate by 95%, 75% and 66% respectively. Data presented suggest that the mobilization of an intracellular pool of Ca2+ is involved in the activation of gluconeogenesis by alpha-adrenergic agonists in perfused rat liver. alpha-Adrenergic activation of gluconeogenesis is apparently accompanied by increases in fatty acid oxidation and tricarboxylic acid-cycle flux. An enhanced transfer of reducing equivalents from the cytoplasmic to the mitochondrial compartment may also be involved in the stimulation of glucose output from the relatively reduced substrates glycerol and sorbitol and may arise principally from an increased flux through the malate-aspartate shuttle.     

Accumulation of Ca2+ induced by cytotoxic levels of menadione in the isolated, perfused rat liver

Previous studies have indicated that the presence of cytotoxic levels of menadione (2-methyl-1,4-naphthoquinone) causes rapid changes in intracellular thiol and Ca2+ homeostasis in isolated rat hepatocytes. The present investigation was undertaken to examine these effects in the intact liver. Rat livers were therefore perfused with Krebs-Henseleit buffer containing 1.3 mM Ca2+ using a single-pass mode, and the perfusate Ca2+ level was monitored with an on-line Ca2+-selective electrode. Infusion of menadione elicited an increased O2 uptake by the liver, followed by a dose-dependent decrease in the perfusate level of Ca2+. Hepatic accumulation of Ca2+ was accompanied by stimulation of cytosolic phosphorylase a activity. Cessation of menadione infusion resulted in gradual recovery of perfusate Ca2+ to base levels. Ca2+ uptake was not accompanied by decreases in reduced pyridine nucleotide or ATP levels in the liver as evidenced by measurements either during maximal Ca2+ uptake or after recovery. However, Ca2+ uptake was correlated with decreased glutathione and increased glutathione disulfide levels in the liver, both of which reversed during recovery from Ca2+ uptake. Moreover, depletion of hepatic glutathione by pretreatment with diethylmaleate resulted in increased Ca2+ uptake during menadione infusion. The amount of protein-bound mixed disulfides showed a particularly striking relationship to Ca2+ uptake, reaching a maximal level during Ca2+ uptake and reversing toward normal value during recovery from Ca2+ accumulation. The present findings suggest that menadione-induced Ca2+ uptake is due to plasma membrane dysfunction as a result of loss of protein thiol groups critical for maintaining the plasma membrane Ca2+ extrusion mechanism. Our model offers a particularly useful opportunity to study mechanisms underlying toxic disturbances in Ca2+ homeostasis in the intact liver, since Ca2+ fluxes can be monitored under conditions in which cellular control mechanisms are not obliterated by excessive toxicity.     

Assessment of the role of Ca2+ mobilization from intracellular pool(s), using dantrolene, in the glycogenolytic action of alpha-adrenergic stimulation in perfused rat liver

To identify the role of Ca2+ mobilization from intracellular pool(s) in the action of alpha-adrenergic agonist, the effects of dantrolene on phenylephrine-induced glycogenolysis were investigated in perfused rat liver. Dantrolene (5 X 10(-5) M) inhibited both glycogenolysis and 45Ca efflux induced by 5 X 10(-7) M phenylephrine. The inhibition by dantrolene was observed in the presence and absence of perfusate calcium. In contrast, dantrolene did not inhibit glycogenolysis induced by glucagon. To confirm the specificity of dantrolene action on calcium release in liver, experiments were also carried out using isolated hepatocytes. Dantrolene did not affect phenylephrine-induced production of inositol 1,4,5-trisphosphate. The compound did inhibit a rise in cytoplasmic Ca2+ concentration induced by phenylephrine both in the presence and absence of extracellular Ca2+. Thus, these results suggest that calcium release from an intracellular pool is essential for the initiation of alpha-adrenergic stimulation of glycogenolysis in the perfused rat liver.     

Synergistic stimulation of Ca2+ uptake by glucagon and Ca2+-mobilizing hormones in the perfused rat liver. A role for mitochondria in long-term Ca2+ homoeostasis.

A perfused liver system incorporating a Ca2+-sensitive electrode was used to study the long-term effects of glucagon and cyclic AMP on the mobilization of Ca2+ induced by phenylephrine, vasopressin and angiotensin. At 1.3 mM extracellular Ca2+ the co-administration of glucagon (10 nM) or cyclic AMP (0.2 mM) and a Ca2+-mobilizing hormone led to a synergistic potentiation of Ca2+ uptake by the liver, to a degree which was dependent on the order of hormone administration. A maximum net amount of Ca2+ influx, corresponding to approx. 3800 nmol/g of liver (the maximum rate of influx was 400 nmol/min per g of liver), was induced when cyclic AMP or glucagon was administered about 4 min before vasopressin and angiotensin. These changes are over an order of magnitude greater than those induced by Ca2+-mobilizing hormones alone [Altin & Bygrave (1985) Biochem. J. 232, 911-917]. For a maximal response the influx of Ca2+ was transient and was essentially complete after about 20 min. Removal of the hormones was followed by a gradual efflux of Ca2+ from the liver over a period of 30-50 min; thereafter, a similar response could be obtained by a second administration of hormones. Dose-response measurements indicate that the potentiation of Ca2+ influx by glucagon occurs even at low (physiological) concentrations of the hormone. By comparison with phenylephrine, the stimulation of Ca2+ influx by vasopressin and angiotensin is more sensitive to low concentrations of glucagon and cyclic AMP, and can be correlated with a 20-50-fold increase in the calcium content of mitochondria. The reversible uptake of such large quantities of Ca2+ implicates the mitochondria in long-term cellular Ca2+ regulation.     

Evidence that platelet and skeletal sarcoplasmic reticulum Ca2+-ATPase are structurally distinct

Proteolytic digestion and indirect immunostaining were used to compare the platelet and sarcoplasmic reticulum Ca2+-ATPase proteins. When the platelet and sarcoplasmic reticulum Ca2+-ATPase proteins were digested in the native state with trypsin, the platelet Ca2+-ATPase, which had an apparent undigested molecular mass of 103 kDa, yielded 78-kDa and 25-kDa fragments. Calcium transport activity depended on the integrity of the 103-kDa protein, while the digested protein had residual ATPase activity. Tryptic digestion of the sarcoplasmic reticulum pump protein, which also had an undigested molecular mass of 103 kDa, yielded products with apparent molecular masses of 55 kDa, 36 kDa, and 26 kDa. Distinct patterns were also observed when the platelet and sarcoplasmic reticulum calcium pump proteins were digested with chymotrypsin and Staphylococcus aureus protease in the presence of sodium dodecyl sulfate. Chymotrypsin digestion of the platelet protein resulted in the appearance of products with apparent molecular masses of 70 kDa, 39 kDa, and 31 kDa, while a similar digestion of the sarcoplasmic reticulum calcium pump protein yielded 54-kDa, 52.5-kDa, 46-kDa, 41-kDa, and 36-kDa fragments. Exposure of the sarcoplasmic reticulum and platelet Ca2+-ATPase proteins to S. aureus protease also yielded dissimilar fragmentation patterns. These results indicate that the Ca2+-ATPases from platelets and sarcoplasmic reticulum are distinct proteins.     

Insulin action on adipocytes. Evidence that the anti-lipolytic and lipogenic effects of insulin are mediated by the same receptor.

1. The dose-response relationships of insulin stimulation of lipogenesis and inhibition of lipolysis were studied simultaneously by using rat adipocytes to determine whether these different effects of insulin are mediated through the same or different sets of receptors. 2. The sensitivity (defined as the concentration of insulin required to produce a half-maximal effect) of the stimulated lipogenic response to insulin was not significantly different from the sensitivity of the anti-lipolytic response to insulin. The addition of different adrenaline and glucose concentrations did not alter the half-maximal concentration of insulin required to inhibit lipolysis. 3. The specificities of the lipogenic and antilipolytic responses were studied by using insulin analogues. The sensitivities of the lipogenic and anti-lipolytic responses were the same for five chemically modified insulins and hagfish insulin, which have potencies compared with bovine insulin of between 3 and 90%. 4. Starving rats for 48h significantly increased the sensitivities of both the antilipolytic and lipogenic responses to insulin, but the changes in the sensitivities of both lipogenesis and anti-lipolysis returned to that of fed rats. 5. We conclude that insulin stimulates lipogenesis and inhibits lipolysis over the same concentration range. These observations provide powerful evidence that the different effects of insulin are mediated through the same set of receptors.     

Mitochondrial and extramitochondrial Ca2+ pools in the perfused rat liver. Mitochondria are not the origin of calcium mobilized by vasopressin

A nondisruptive technique developed by Bellomo et al. (Bellomo, G., Jewell, S. A., Thor, H., and Orrenius, S. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 6842-6846) has been used to examine the distribution of calcium ions between mitochondrial and extramitochondrial compartments in the perfused rat liver. The amount of calcium released by the uncoupler 2,4-dinitrophenol from the mitochondrial compartment was 19 +/- 2 nmol X g-1, wet weight, which is equivalent to a total calcium concentration of 3.5 X 10(-4) M in the mitochondria and is by several orders of magnitude smaller than the concentration thought to be present in these organelles. The amount of calcium released from the liver in the presence of the divalent cation ionophore A 23187 was 96 +/- 7 nmol X g-1, wet weight, which is of the same order of magnitude as the amount released by the calcium-dependent hormone vasopressin (97 +/- 11 nmol X g-1, wet weight). Experiments with different sequential combinations of hormone with uncoupler or ionophore reveal that in the perfused liver, in contrast to isolated hepatocytes or isolated mitochondria, the amount of calcium attributable to the mitochondria is too small to account for the calcium released during hormonal stimulation. Consequently extramitochondrial calcium stores are the main source of cellular calcium mobilized under this condition. In addition these findings imply that in the liver several mitochondrial enzymes, e.g. alpha-oxoglutarate dehydrogenase, can be effectively regulated by calcium and that the role of mitochondria in buffering the cytosolic free calcium in vivo has to be reconsidered.     

Ca2+-dependent activation of the malate-aspartate shuttle by norepinephrine and vasopressin in perfused rat liver

The role of Ca2+ in stimulation of the malate-aspartate shuttle by norepinephrine and vasopressin was studied in perfused rat liver. Shuttle capacity was indexed by measuring the changes in both the rate of production of glucose from sorbitol and the ratio of lactate to pyruvate during the oxidation of ethanol. (T. Sugano et al. (1986) Amer. J. Physiol. 251, E385-E392). Asparagine (0.5 mM), but not alanine (0.5 mM) decreased the ethanol-induced responses. Norepinephrine and vasopressin had no effect on the ethanol-induced responses when the liver was perfused with sorbitol or glycerol. In the presence of 0.25 mM alanine, norepinephrine, vasopressin, and A23187 decreased the ethanol-induced responses that occurred with the increase of flux of Ca2+. In liver perfused with Ca2+-free medium, asparagine also decreased the ethanol-induced responses, but norepinephrine and vasopressin had no effect. Aminooxyacetate inhibited the effects of norepinephrine, A23187, and asparagine. Regardless of the presence or absence of perfusate Ca2+, the combination of glucagon and alanine had no effect on the ethanol-induced responses. Norepinephrine caused a decrease in levels of alpha-ketoglutarate, aspartate, and glutamate in hepatocytes incubated with Ca2+. The present data suggest that the redistribution of cellular Ca2+ may activate the efflux of aspartate from mitochondria in rat liver, resulting in an increase in the capacity of the malate-aspartate shuttle.     

The influx of Ca2+ induced by the administration of glucagon and Ca2+-mobilizing agents to the perfused rat liver could involve at least two separate pathways.

The Ca2+-mobilizing actions of ADP, ATP and epidermal growth factor (EGF) and their interaction with glucagon were studied in a perfused liver system incorporating a Ca2+-selective electrode. ADP (1-100 microM), ATP (1-100 microM) and EGF (10-50 nM) all induced a net efflux, followed by a net uptake of Ca2+ in the intact liver. The co-administration of glucagon (or of cyclic AMP) with these agents resulted in a synergistic potentiation of the Ca2+ uptake response in a way which resembles the synergism observed when glucagon is administered with phenylephrine, vasopressin or angiotensin [Altin & Bygrave (1986) Biochem J. 238, 653-661]. The inability of diltiazem, verapamil and nifedipine to inhibit the Ca2+-influx response suggests that the stimulation of Ca2+ influx does not occur through voltage-sensitive Ca2+ channels. By contrast, the synergistic effects of glucagon in the stimulation of Ca2+ influx are inhibited by 10 mM-neomycin, and a lowering of the extracellular pH to 6.8. Simultaneous measurements of perfusate Ca2+ and pH changes suggest that the Ca2+ influx response is not mediated by a Ca2+/H+ exchange. The inability of neomycin and low extracellular pH to inhibit the refilling of the hormone-sensitive pool of Ca2+, after the administration of Ca2+-mobilizing agents alone, provides evidence for the existence in liver of at least two Ca2+-influx pathways, or mechanisms for regulating Ca2+ influx.     

Evidence that isoproterenol-induced Ca2(+)-mobilization in rat parotid acinar cells is not mediated by activation of beta-adrenoceptors

The effects of isoproterenol (ISO), a beta-adrenoceptor agonist, on cytosolic free Ca2+ ([Ca2+]i) in rat parotid acinar cells were examined using the fluorescent Ca2(+)-indicator fura-2. At concentrations up to 1 mM, ISO caused a rapid increase in [Ca2+]i in a dose-dependent manner, while addition of 1 microM ISO, which evokes the maximum amylase secretion, had only a slight effect on [Ca2+]i. There was no such increase in [Ca2+]i with the addition (2 mM) of 8-bromo-cyclic AMP, a permeant cyclic AMP analogue. The alpha-adrenoceptor antagonist phentolamine blocked the ISO-induced [Ca2+]i increase better than the beta-adrenoceptor antagonist, propranol, and the muscarinic receptor antagonist, atropine. The IC50 value (the concentration which reduces the ISO-induced increase in [Ca2+]i by 50%) of phentolamine was estimated to be 7.6 nM, for propranolol 13.2 microM and for atropine 3.5 microM. The difference in potency between the three antagonists was similar to the difference in blocking the [Ca2+]i increase induced by phenylephrine, an alpha-adrenoceptor agonist. These results suggest that the Ca2(+)-mobilization in response to high concentrations of ISO results from an activation of alpha-adrenoceptors rather than beta-adrenoceptors.     

Phosphate and calcium uptake by mitochondria and by perfused rat liver induced by the synergistic action of glucagon and vasopressin.

Co-administration of glucagon and vasopressin to rat liver perfused with buffer containing 1.3 mM-Ca2+ induces a 4-fold increase in Pi in the subsequently isolated mitochondria (from approx. 9 to approx. 40 nmol/mg of mitochondrial protein). This increase is not attributable to PPi hydrolysis, and is not observed if the perfusate Ca2+ is lowered from 1.3 mM to 50 microM. The increase in mitochondrial Pi closely parallels that of mitochondrial Ca2+; when the increase in Pi and Ca2+ accumulation is maximal, the molar ratio is close to that in Ca3(PO4)2. Measurement of changes in the perfusate Pi revealed that, whereas administration of glucagon or vasopressin alone brought about a rapid decline in perfusate Pi, the largest decrease (reflecting net retention of Pi by the liver) was observed when the hormone was co-administered in the presence of 1.3 mM-Ca2+. The synergistic action of glucagon plus vasopressin was nullified by lowering the perfusate Ca2+ to 50 microM. The data provide evidence that, whereas glucagon may be able to alter Pi fluxes directly in intact liver, any alterations induced by vasopressin are indirect and result only from its action of mobilizing Ca2+.     

Nerve growth factor action is mediated by cyclic AMP- and Ca+2/phospholipid-dependent protein kinases

Nerve growth factor (NGF) mediates the phosphorylation of tyrosine hydroxylase in PC12 cells on two distinct peptide fragments, separable by two-dimensional tryptic phosphopeptide mapping (phosphopeptides T1 and T3). Phorbol diester derivatives capable of activating Ca+2/phospholipid-dependent protein kinase (C-kinase) cause a specific phosphorylation of peptide T3 in a dose-dependent, saturable manner. Derivatives of the endogenous C-kinase activator diacylglycerol, also cause the phosphorylation of tyrosine hydroxylase on peptide T3. The C-kinase inhibitors chlorpromazine and trifluoperazine inhibit the phorbol diester stimulated phosphorylation of site T3 in a dose-dependent manner. These agents inhibit the phosphorylation of T3 in response to NGF, but have no effect on NGF's ability to cause T1 phosphorylation. In a PC12 mutant deficient in cAMP-dependent protein kinase activity, NGF mediates the phosphorylation of tyrosine hydroxylase on peptide T3 but not on T1. We conclude that NGF mediates the activation of both the cAMP-dependent protein kinase and the C-kinase to phosphorylate substrate proteins. These kinases can act independently to phosphorylate tyrosine hydroxylase, each at a different site, and each of which results in the enzyme activation. A molecular framework is thus provided for events underlying NGF action.     

Ca2+ transport by rat liver plasma membranes: the transporter and the previously reported Ca2+-ATPase are different enzymes

A rat liver plasma membrane fraction showed an ATP-dependent uptake of Ca2+ which was released by the ionophore A23187. This activity represents a plasma membrane component and is not due to microsomal contamination. The Ca2+ transport displayed several properties which were different from those of the high-affinity Ca2+-ATPase previously observed in these membranes (Lotersztajn et al. (1981) J. Biol. Chem. 256, 11209-11215; Birch-Machin, M.A. and Dawson, A.P. (1986) Biochim. Biophys. Acta 855, 277-285). These observations have shown that Ca2+-ATPase does not require added Mg2+ whereas we have demonstrated that, in the same membrane preparation, Ca2+ uptake required millimolar concentrations of added Mg2+. The Ca2+-ATPase has a broad specificity for the nucleotides ATP, GTP, UTP and ITP while Ca2+ uptake remains specific for ATP. Ca2+ uptake also displayed different affinities for free Ca2+ and MgATP compared to Ca2+-ATPase activity, with apparent Km values of 0.25 microM Ca2+, 0.15 mM MgATP and 1.0 microM Ca2+, 4 microM MgATP respectively. The apparent maximum rate of Ca2+ uptake was about 150-fold less than Ca2+-ATPase activity. These features suggest that the high-affinity Ca2+-ATPase is not the enzymic expression of the ATP-dependent Ca2+ transport mechanism.     

The effects of platelet-activating factor on flow rate and eicosanoid release in the isolated perfused rat gastric vascular bed

In the isolated rat stomach perfused via the vasculature in situ under constant pressure bolus injections of platelet-activating factor (PAF, 3, 16, or 50 ng) induced dose-dependent, long-lasting reductions of flow rates and simultaneously significant increases in the release of cysteinyl-leukotrienes (cys-LT), thromboxane (TX) B₂ and 6-keto-prostaglandin (PG) F_1α. Reversed phase high pressure liquid chromatography demonstrated the release of a mixture of comparable amounts of LTC₄, LTD₄ and LTE₄ by PAF. Inhibition of cys-LT sythesis by the lipoxygenase inhibitors nordihydroguaiaretic acid (NDGA) and L-651, 896 did not significantly affect PAF-induced flow reduction indicating that endogenous cys-LT are of minor importance for the PAF effect on gastric vascular flow. This conclusion is supported by the fact that the cys-LT receptor antagonist FPL 55712 in a concentration (1 × 10⁻⁶ M) that completely antagonized gastric flow reduction by exogenous LTC₄ (1 × 10⁻⁷ M) had no effect on the PAF-induced reduction of flow. The cyclooxygenase inhibitor indomethacin aggravated the PAF-induced flow reduction suggesting that the endogenous vasodilator PGI₂ might act as a functional PAF antagonist in the rat gastric vascular bed. In contrast to FPL 55712 the PAF antagonist BN 52021 significantly and concentration-dependently antagonized the PAF effect on gastric vascular flow. The results demonstrate that PAF and LTC₄ induce flow reductions in the rat gastric vascular bed by activating different receptors and that endogenous eicosanoids released by PAF do not contribute significantly to the PAF effect on gastric vascular flow.     

The effects of platelet-activating factor on flow rate and eicosanoid release in the isolated perfused rat gastric vascular bed

In the isolated rat stomach perfused via the vasculature in situ under constant pressure bolus injections of platelet-activating factor (PAF, 3, 16, or 50 ng) induced dose-dependent, long-lasting reductions of flow rates and simultaneously significant increases in the release of cysteinyl-leukotrienes (cys-LT), thromboxane (TX) B2 and 6-keto-prostaglandin (PG) F1 alpha. Reversed phase high pressure liquid chromatography demonstrated the release of a mixture of comparable amounts of LTC4, LTD4 and LTE4 by PAF. Inhibition of cys-LT synthesis by the lipoxygenase inhibitors nordihydroguaiaretic acid (NDGA) and L-651,896 did not significantly affect PAF-induced flow reduction indicating that endogenous cys-LT are of minor importance for the PAF effect on gastric vascular flow. This conclusion is supported by the fact that the cys-LT receptor antagonist FPL 55712 in a concentration (1 x 10(-6) M) that completely antagonized gastric flow reduction by exogenous LTC4 (1 x 10(-7) M) had no effect on the PAF-induced reduction of flow. The cyclooxygenase inhibitor indomethacin aggravated the PAF-induced flow reduction suggesting that the endogenous vasodilator PGI2 might act as a functional PAF antagonist in the rat gastric vascular bed. In contrast to FPL 55712 the PAF antagonist BN 52021 significantly and concentration-dependently antagonized the PAF effect on gastric vascular flow. The results demonstrate that PAF and LTC4 induce flow reductions in the rat gastric vascular bed by activating different receptors and that endogenous eicosanoids released by PAF do not contribute significantly to the PAF effect on gastric vascular flow.     

Insulin-like action of catecholamines and Ca2+ to stimulate glucose transport and GLUT4 translocation in perfused rat heart

The uptake of 2-deoxyglucose by perfused rat hearts was compared to the distribution of the insulin-regulatable glucose transporter (GLUT4) in membrane preparations from the same hearts. The hearts were treated with the alpha-adrenergic combination of epinephrine + propranolol, the beta-adrenergic agonist isoproterenol, high (8 mM) Ca2+ concentrations, insulin and the alpha adrenergic combination or insulin alone. Epinephrine (1 microM) + propranolol (10 microM), isoproterenol (10 microM), high Ca2+, insulin (1 microM) + epinephrine (1 microM) + propranolol (10 microM) and insulin (1 microM) each led to an increase in 2-deoxyglucose uptake and a shift in the recovery of the GLUT4 from a high-speed pellet membrane fraction (putatively intracellular) to a low-speed pellet membrane fraction (putatively sarcolemmal). There were significant correlations (r = -0.673, P less than 0.001) between the stimulation of 2-deoxyglucose uptake and the loss of GLUT4 from the intracellular membrane fraction, or the increase in the sarcolemmal fraction. The data provide evidence that the GLUT4 is translocated by agents that stimulate glucose transport in heart, and therefore this mechanism is not restricted to insulin.