Krebs-Ringer Phosphate Medium Research Articles

Effect of in vitro ischemic or hypoxic treatment on mitochondrial electron transfer activity in rat brain slices assessed by gas–tissue autoradiography using [ 15O] molecular oxygen

We have investigated the effect of in vitro ischemic or hypoxic treatment on mitochondrial electron transport function in brain slices using gas–tissue autoradiography technique with [ 15O]O 2. Brain slices were preincubated in Krebs-Ringer phosphate medium bubbled with 100% O 2 for 30 min at 37°C. (1) Control culture was incubated in the same medium bubbled with 100% O 2 for 5–40 min at 37°C, then for another 30 min under the same conditions. (2) In vitro ischemia was induced by placing the culture in the medium deprived of glucose and bubbled with 100% N 2 for 5–40 min, then returning it to control conditions and culturing for another 30 min. (3) In vitro hypoxia was induced by placing the culture in the medium with glucose and bubbled with 100% N 2 for 5–40 min, then returning it to the control conditions for 30 min. After the three different treatments, the [ 15O]O 2 fixation by brain slices reflect to mitochondrial electron transport function was determined using gas–tissue autoradiography technique with [ 15O]O 2. The fixation of [ 15O]O 2 by striatum, cerebral cortex and hippocampus was reduced dependent upon the period of in vitro ischemic treatment. In contrast, the [ 15O]O 2 fixation by those brain regions was only slightly reduced by hypoxia treatment. The reduction in [ 15O]O 2 fixation induced by ischemic treatment was prevented by an antioxidant: glutathione, glutathione monoethyl ester or acetylsalicylic acid. The preventive effect of antioxidants on the mitochondrial damage induced by ischemia was more remarkable in the striatum than in the cerebral cortex and hippocampus. In the comparison of [ 15O]O 2 fixation between ischemia-treated young and senescent brain slices, reduction of 15O fixation by every brain region examined was more prominent in senescence than in the young. These results suggest that gas–tissue autoradiography using [ 15O]O 2 is useful to assess mitochondrial electron transport dysfunction induced by ischemia treatment in brain slices and that the oxidative stress participates in the mechanism of ischemia-induced dysfunction in mitochondria.

Effect of Mating Behavior on Luteinizing Hormone-Releasing Hormone Release in Female Rabbits as Monitored with Push-Pull Cannulae

To examine the effect of mating behavior on luteinizing hormone-releasing hormone (LHRH) release, intact New Zealand female rabbits were implanted with push-pull cannulae (PPC) aimed at the tuberal region of the hypothalamus and perfused with modified Krebs-Ringer phosphate medium at 11-13 microliters/min. In the mating experiments, does (n = 10) were initially perfused for a control period of 60-170 min followed by a mating period (100-160 min) which included the introduction of the male rabbit for an average time period of 30 min. Two groups of LHRH release patterns were observed: positive and negative responders. In the positive LHRH responders (n = 5), a clear rapid increase in LHRH release following mounting by the male occurred with a significant increase in the mean LHRH release (1.83 +/- 0.33 to 3.27 +/- 0.80 pg/10 min, p less than 0.040), in the mean LHRH amplitude (1.97 +/- 0.46 to 4.33 +/- 1.29 pg, p less than 0.022) and in the amplitude of the largest LHRH pulse (2.13 +/- 0.43 to 7.58 +/- 3.65 pg, p less than 0.022). In the negative LHRH responders (n = 5), no changes in LHRH release were detected although all rabbits ovulated, with some becoming pregnant. It appears from histological analysis that the difference between these two patterns of responses following mating are due to different cannula placements. In the positive responders, the tip of the PPC was localized in the tuberal region whereas in the negative responders, the placements were more dorsal and, in some cases, anterior.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies of the in vivo catabolism of exogenous dopamine as infused through a push-pull cannula implanted in the rat caudate nucleus

In this report, using a push-pull perfusion technique, we examined in vivo the effects of exogenous dopamine (DA) on the output of neurochemical substances from the caudate nucleus (CN) of freely behaving rats. Exogenous DA, at concentrations of 10 −5 M, 5 × 10 −5 M and 10 −4 M, in modified Krebs-Ringer phosphate medium (KRP) was infused directly into the CN for 15 min each. Exogenous DA at the doses tested elicited increases in 3,4-dihydroxyphenylacetic acid (DOPAC) output in a dose-dependent manner. In addition, the higher two doses of exogenous DA also induced increases in homovanillic acid (HVA) output from the rat CN. The increases in DOPAC output by 5 × 10 −5 M DA was partially blocked by the inclusion of 10 −3 M nomifensine in KRP. Interestingly, exogenous DA-induced increases in HVA output were little affected by the nomifensine treatment. However, the catabolism of exogenous DA was almost completely eliminated by a prior 6-hydroxydopamine lesion in the ipsilateral substantia nigra. Furthermore, infusions of exogenous DA did not change 5-hydroxyindoleacetic acid output from the CN. In conclusion, our results confirm in vivo that (a) DA catabolic pathway via DOPAC intermediate predominates over the alternative pathway via 3-methoxytyramine, (b) increases in extracellular DA will lead to increases in DOPAC and HVA levels in extracellular space and (c) the majority of the DA is oxidized by intradopaminergic monoamine oxidase.

Comparison of the effect of prolactin on dopamine release from the rat dorsal and ventral striatum and from the mediobasal hypothalamus superfused in vitro

In this paper, by means of a superfusion technique, we have examined the effect of ovine prolactin on dopaminergic neurons innervating the dorsal striatum, ventral striatum and the mediobasal hypothalamus of male rats. Fragments from dorsal striatum were superfused with ovine prolactin dissolved in normal medium (Krebs-Ringer phosphate) or medium containing tetrodotoxin (TTX, 10 −6 M). Ovine prolactin stimulated the in vitro release of dopamine from dorsal and ventral striatal fragments. In dorsal striatal fragments a linear dose-dependent dopamine release was observed only when fragments were superfused with Krebs-Ringer phosphate-TTX medium. In addition, [Leu 5]enkephalin (10 −6 and 10 −5 M) decreased the prolactin-induced in vitro dopamine release from dorsal striatal fragments superfused with Krebs-Ringer phosphate-TTX medium. Ovine prolactin (10 −9–10 −5 M) did not elicit changes in dopamine, 3,4-dihydroxyphenylacetic acid or 5-hydroxyindoleacetic acid outputs from mediobasal hypothalamic fragments superfused with Krebs-Ringer phosphate medium containing TTX. The possible regulatory mechanisms of ovine prolactin on dopaminergic neurons are discussed.

Effect of alkaline cations on cyclic 3′ 5′-AMP stimulated lipolysis in rat adipocytes

In Krebs-Ringer phosphate medium, cyclic AMP had little effect on production of free fatty acids by fat cells in vitro, whereas dibutyryl cyclic AMP or epinephrine stimulated the production of free fatty acids. On the other hand, although under non-physiological condition, cyclic AMP was found to stimulate the lipolysis in simple KC1-Tris medium. The stimulation level was similar to that elicited by dibutyryl cyclic AMP. Cyclic AMP also stimulated lipolysis in LiCl-Tris or RbCl-Tris medium, but not in NaCl-Tris medium. In KC1-Tris medium, addition of divalent alkaline cations (Mg 2+, Ca 2+ and Sr 2+, at 40 mM) completely inhibited the stimulation by cyclic AMP, but not those by dibutyryl cyclic AMP and epinephrine. No cyclic AMP-induced lipolysis was observed in homogenized or freeze-thawed cells.

In vitro release of LHRH from the bovine pituitary stalk-median eminence: Alterations during the postpartum period

Superfusion techniques were developed to investigate in vitro release of luteinizing hormone releasing hormone (LHRH) from the bovine pituitary stalk-median eminence (SME) obtained from suckled beef cows during the postpartum period. Mature beef cows were allotted at random to be slaughtered at either day 5 (n=5), 10 (n=6), 20 (n=6) or 30 (n=5) after calving. After isolation from the brain, the pituitary SME was divided mid-sagittally with one-half of the tissue placed in a vessel containing ice-cold superfusion medium and transported to the laboratory for superfusion. Krebs-Ringer phosphate solution supplemented with BSA, glucose and bacitracin served as the superfusion medium. A 10-min pulse of high K + Krebs-Ringer phosphate medium was passed through the superfusion chamber at two different times to provoke membrane depolarization causing release of LHRH. Treatment of SME tissue with high K + media increased LHRH release over basal pre-K + levels attesting to the viability of the preparation. The maximum K +-induced peak release of LHRH at the first K + pulse was greater on day 5 than day 20. These results indicate that: Dour superfusion system can be used to evaluate releasable pools of LHRH from the bovine pituitary SME, and 2) in vitro release of LHRH in response to K + depolarization is reduced by day 20 postpartum possibly indicating increases in in vivo release between day 5 and day 20.

Inhibition of the autoxidation of ascorbate and norepinephrine by extracts of [formula omitted] and [formula omitted

The autoxidation of ascorbate and of norepinephrine in Krebs Ringer phosphate medium, pH 7.4, was studied. The autoxidation of the two substances was determined spectrophotometrically at 265 and 480 nm respectively. The effect of dialyzed extracts (m.w. > 12,000) from Escherichia coli (aerobe), Megasphaera elsdenii , and Clostridium butyricum (obligate anaerobes) was examined and compared to similarly prepared extracts from rat serum and cerebral cortex. The assay medium contained cellular components diluted 10 3–10 6-fold. Up to 10 4-fold dilution there was a substantial reduction in the rate of both autoxidation reactions, but the preparations from M. elsdenii and C. butyricum were conspiciously less effective. After 5 min heat treatment at 100°C the anaerobic preparations produced <20% inhibition, while the activity of the other preparations remained unchanged at 75–95% inhibition. These and earlier experiments involving additional mammalian species (Mishra and Kovachich, Neurosci. Lett., 43: 103–108, 1983) and plants (Mishra and Kovachich, Life Sci., 34: 2207–2212, 1984) suggest that a high level of heat-stable antioxidant activity in one or both of these autoxidation tests (denatured plant extracts only inhibit ascorbate autoxidation) is a general characteristic of organisms that thrive in oxygen-rich atmosphere.

The effect of ascorbic acid on malonaldehyde formation, K+, Na+ and water content of brain slices

The ascorbate status of cortical brain slice preparation was correlated with malonaldehyde levels during the first hour of incubation in normal Krebs-Ringer phosphate medium. The ascorbate content of fresh cortical slice was 3.33 +/- 0.34 mumol/g. A portion of ascorbate was released from the tissue during incubation. The release of ascorbate was less extensive when 1 mM ascorbate was added to the medium, and in the presence of 3 mM ascorbate the initial tissue level of ascorbate was maintained during the experiment. Addition of ascorbate to the medium produced a biphasic effect on malonaldehyde formation: 1 mM ascorbate doubled, 3 mM ascorbate inhibited malonaldehyde production, as compared to control values. When in vivo cortical ascorbate level was increased by 25% through an intraperitoneal injection of 2 g/kg Na ascorbate, the relationship between tissue and medium ascorbate during incubation was unaltered, but malonaldehyde formation was delayed by approximately 30 min and the stimulatory effect of low concentration of ascorbate was not seen. During the lag period there was evidence of an ion and water pumping mechanism opposing the accumulation of water and the movement of K+ and Na+ along their concentration gradients.

Inhibition of ascorbate autoxidation by a rat brain cortical factor

The soluble fraction of rat brain cortex yielded an antioxidant factor that inhibits the autoxidation of ascorbic acid at physiological pH. The measurements were based on spectrophotometric detection of ascorbic acid at 265 nm in a physiological salt solution of the Krebs-Ringer type, pH 7.4, or in sodium phosphate buffer, pH 7.4. In these systems the autoxidation of ascorbate was linear with respect to time for at least 60 min. The potency of the antioxidant factor is indicated by the fact that half maximal inhibitory activity was obtained with an extract representing approximately 10,000-fold dilution of cell components. The inhibitory activity of the cortical tissue was solubilized during heat treatment. A considerable portion of the activity was released by brain slices into the medium during incubation in normal Krebs-Ringer phosphate medium.

On the mechanism of presynaptic autoreceptor-mediated inhibition of transmitter synthesis in dopaminergic nerve terminals

The effect of apomorphine (APO) upon dopamine (DA) synthesis and release from rat striatal slices was studied. The synthesis of DA was measured by incubating the slices in Krebs-Ringer phosphate (KRP) medium of variable ionic composition containing l-tyrosine[ 14C-U] as DA precursor. A superfusion system was used to study both spontaneous and K +-induced release of labeled DA from striatal slices. The addition of APO directly to the normal KRP medium markedly blocked the formation of [ 14C]DA from [ 14C]tyrosine with an ic 50 of 1.8 × 10 −7 M. Haloperidol (4 × 10 −7 M), a known DA antagonist, produced a shift to the right of the concentration-response curve for APO inhibition on DA synthesis, whereas the DA antagonist (+)butaclamol (4 × 10 −7 M) completely reversed the inhibition caused by APO (2 × 10 −7 M). DA uptake blockers, such as benztropine (2 × −6 M) or cocaine (1 × 10 −5 M), did not affect the ability of APO to inhibit DA synthesis. Furthermore, the α 2-adrenergic agonist clonidine produced only a mild inhibition and the β-adrenergic agonist isoproterenol produced no inhibition of [ 14C]DA formation. APO was able to inhibit DA formation both in the absence of calcium in the incubation medium or in the presence of high external calcium concentrations (4, 8 and 24 mM) which depress the rate of DA synthesis. Incubation conditions that cause an increase of free intraneuronal calcium concentrations, such as Na +-free medium, the presence of ouabain (1 × 10 −4 M), or K + depolarization, dramatically abolished or impaired the ability of APO to inhibit DA synthesis in striatal slices. It was not possible to demonstrate any change in spontaneous and K + (27 mM)-induced release of DA in the presence of APO concentrations that produced a marked inhibition of DA synthesis. The results reported in this work indicate that tissue slices can be used as a valuable experimental tool to study the inhibitory effect of APO on DA synthesis, and that this effect occurs through an interaction of APO with presynaptic DA autoreceptors located in striatal dopaminergic nerve terminals. The results obtained are not in keeping with the hypothesis that this autoreceptormediated inhibition of DA synthesis occurs through regulation of calcium influx into the DA nerve terminals. However, the possibility is raised that a sensitivity to high intraneuronal calcium concentration exists during the events that mediate APO-DA autoreceptor interaction and DA synthesis inhibition. It is further suggested that DA-synthesis-modulating autoreceptors do not participate in the modulation of DA release.

Metabolism of Babesia parasites in vitro. Glucose and energy-metabolism and survival of Babesia rodhaini in a basal medium with and without adenosine.

During in vitro incubation in Krebs Ringer phosphate (KRP) medium, rat erythrocytes about 20% parasitized with B. rodhaini used two to four times as much glucose as unparasitized erythrocytes. However, the AtP concentration and the ATP production of parasitized erythrocytes decreased. For example, before incubation the ATP concentration in parasitized erythrocytes was significantly higher thant unparasitized erythrocytes, but following 0.5 h incubation it was reduced to about the same as in unparasitized erythrocytes. Addition of 5 X 10(-3) M adenosine to the basal medium increased the ATP production and the adenylate energy charge of parasitized erythrocytes. Infectivity tests in susceptible mice showed that, despite these seemingly favourable changes in energy metabolism, adenosine had no detectable effect on the survival of B. rodhaini. Absence of infectivity showed the inability of KRP medium to support B. rodhaini for 22 h in vitro with or without adenosine. Adenosine also greatly decreased the glucose uptake by parasitized erythrocytes, apparently by competitive inhibition, since lactate production was unchanged.

Partial Inactivation of Na,K‐ATPase in Cortical Brain Slices Incubated in Normal Krebs‐Ringer Phosphate Medium at 1 and at 10 Atm Oxygen Pressures

Na,K-ATPase (ATP phosphohydrolase EC 3.6.1.3) activity was determined in homogenates of cortical brain slices after incubation in normal Krebs-Ringer phosphate medium at 1 atm oxygen pressure. After 10 min of incubation Na,K-ATPase activity was reduced by approximately 50%. Longer incubation did not cause further change in activity. The presence of 0.1 mM-MnCl2 in the medium offered significant protection, while an excursion to 10 atm oxygen pressure caused further inactivation. Measurements of malonaldehyde levels suggest that the inhibition of Na,K-ATPase is a result of lipid peroxidation. The evidence indicates that brain slices incubated under standard conditions suffer considerable oxidative damage.

The guinea pig neutrophil calcium-dependent lysosomal enzyme secretory process: Inhibition by nonsteroid anti-inflammatory agents

A selective release of lysosomal granule-associated β-glucuronidase and acid protease, but not cytoplasmic lactate dehydrogenase, occurs during contact between zymosan-treated serum and cytochalasin B-treated neutrophils. The calcium-dependent aspect of this secretory process is demonstrated by the absence of lysosomal enzyme discharge from neutrophils incubated with zymosan-treated serum in calcium-free Krebs-Ringer phosphate medium containing 7.5 mM glucose, pH 7.4, at 37°. The capacity of the divalent cation ionophore, A23187, to induce lysosomal enzyme release also implies a requirement of calcium for secretion to occur. Further, zymosan-treated serum and ionophore-induced release of β-glucuronidase was accompanied by a marked association of 45CaCl 2 with neutrophils. Indomethacin, diftalone, naproxen, ketoprofen and suprofen impeded A23187-stimulated extracellular extrusion of lysosomal enzymes from, and 45CaCl 2 association with, neutrophils. Acetylsalicylic acid and flazalone were inactive. The inhibitory effects of these nonsteroid anti-inflammatory agents could be reversed by increasing the extracellular calcium ion concentration. The specificity of calcium in reversing the effects of these agents was indicated by the observation that supplementing the incubation medium with magnesium would not attenuate the action of these nonsteroid agents. Therefore, the regulation of the selective secretion of lysosomal enzymes from neutrophils by nonsteroid anti-inflammatory agents may be related to their capacity to modulate the association of calcium with these cells.

Tyrosine hydroxylase regulation in rat striatal and olfactory tubercle slices—I: Activation induced by exposure to a calcium-free and high-magnesium medium

Slices from rat corpus striatum and olfactory tubercle were incubated for 15 min at 37° in Krebs-Ringer phosphate (KRP) medium or Ca 2+-free KRP medium both in the presence and absence of high Mg 2+ (12 mM). Tyrosine hydroxylase activity and kinetic parameters were determined in the 20,000 g supernatant fraction prepared from slices. The omission of Ca 2+ from the incubation medium resulted in a moderate increase in the activity of striatal tyrosine hydroxylase, assayed in the presence of subsaturating concentrations of tyrosine and pterin cofactor, and a significant increase in the activity of tyrosine hydroxylase isolated from olfactory tubercle slices. The presence of Mg 2+ (12 mM) in the Ca 2+-free KRP medium produced a further increase in the activity of the enzyme isolated both from striatal and olfactory tubercle slices. The per cent of stimulation of enzyme activity induced by incubating the slices in a Ca 2+-free, high-Mg 2+ KRP medium was maximal when assayed at pH 7.0. The Mg 2+-induced activation of tyrosine hydroxylase was antagonized by increasing the Ca 2+ concentration (3.75 to 15.0 mM) in the medium in which the slices were incubated. The direct addition of Mg 2+ (5–20 mM) to striatal and olfactory tubercle homogenates also resulted in an increase in the activity of tyrosine hydroxylase. The addition of high concentrations of Ca 2+ (10 mM) to the homogenates also resulted in an increase in enzyme activity but this effect was additive to that produced by Mg 2+ (10 mM). Incubation of striatal slices in a Ca 2+-free, high-Mg 2+ KRP medium produced changes in the kinetic properties of tyrosine hydroxylase. The apparent m of the enzyme for 6-methyl-5,6,7,8-tetrahydropterine HCl (6-MPH 4) was decreased significantly from 0.85 to 0.40 mM, with no significant change in the V max. However, the K i of the enzyme for dopamine (DA) was unchanged. Magnesium ions (12 mM) added to KRP-high K + (55 mM) medium blocked the activation of tyrosine hydroxylase induced by K +-depolarization of striatal slices. However, Mg 2+ (12 mM) addition to the incubation medium did not block but actually further increased the tyrosine hydroxylase activation that results after incubating striatal slices in a KRP medium enriched with dibutyryl cAMP (1 mM). Moreover, the stimulating effect on tyrosine hydroxylase observed when assays were conducted in the presence of Mg 2+, ATP and cAMP remained unchanged in homogenates prepared from slices previously incubated in a Ca 2+-free, high-Mg 2+ KRP medium. The results reported in this work clearly demonstrate that a kinetic activation of tyrosine hydroxylase in dopaminergic nerve terminals can occur under conditions of diminished extracellular Ca 2+ and blockade of transmitter release. Cyclic AMP does not seem to play a role in the tyrosine hydroxylase activation induced by incubation of slices in Ca 2+-free and high-Mg 2+ medium. The results support the hypothesis that the increase in DA biosynthesis observed in dopaminergic neurons after inhibition of impulse flow may result primarily as a consequence of a diminished entrance of Ca 2+ into the nerve terminal.

An energy requirement for the degradation of intravenously injected 125I-labeled albumin in mouse liver and kidney slices.

Liver and kidney slices prepared 30min after intravenous injections of formaldehyde-treated 125I-labelled bovine serum albumin into mice degrade approx. 25-40% of the protein to a trichloroacetic acid-soluble form during 60min incubation at 37 degrees C. The presence of bicarbonate in Krebs-Ringer phosphate medium inhibited intracellular proteolysis, and similar results were obtained at pH5 or pH7 in kidney or liver slices. Cellular integrity was required to obtain substantial rates of proteolysis. This intralysosomal intracellular degradation of an exogenous protein was partially inhibited by inhibitors of oxidative ATP formation, such as cyanide, azide, 2,4-dinitrophenol and absence of oxygen. Arsenite and iodoacetamide were also effective inhibitors, but the effects of fluoride were variable. These results suggest that an energy requirement exists for intralysosomal proteolysis in intact cells and are consistent with the hypothesis that energy may be required to maintain intralysosomal acidity.

Inhibitory effect of high oxygen pressure on potassiuminduced activation of pyruvate dehydrogenase and glucose metabolism in rat brain slices

The effects of high oxygen pressure on pyruvate dehydrogenase (pyruvate: lipoate oxidoreductase (decarboxylating and acceptor-acylating), EC 1.2.4.1) activity, tissue concentration of ATP, and CO 2 production from glucose were studied in rat brain cortical slices. The increase in pyruvate dehydrogenase activity and the lowering of cellular ATP, occurring during potassium-induced depolarization at 1 atm of oxygen, were reversed by increasing the oxygen pressure to 5 atm. When brain slices were incubated at 1 atm oxygen with [U- 14C]glucose, a high potassium medium approximately doubled the production of 14CO 2. Oxygen at 5 atm abolished this potassium-dependent increase in 14CO 2 production with no significant effect on glucose oxidation in normal Krebs-Ringer phosphate medium. Adding 4 atm helium to 1 atm oxygen did not interfere with the ability of potassium ions to activate pyruvate dehydrogenase, lower ATP, or increase glucose oxidation. The results show that toxic effects of hyperbaric oxygen, not manifest in “resting” tissue, may be revealed during stress such as potassium depolarization. The site of the toxic effects of oxygen is probably the cell membrane where excess oxygen appears to interfere with the action of the sodium pump, calcium transport or other processes stimulated by increased concentrations of extracellular potassium.

Phagocytic release of lysosomal enzymes from guinea pig neutrophils—regulation by corticosteroids, autonomic neurohormones and cyclic nucleotides

The effects of various pharmacologic agents on the capacity of guinea pig neutrophils to phagocytize serum-treated zymosan particles and release lysosomal enzymes were determined. Neutrophils (10 7) and zymosan were incubated in Krebs-Ringer phosphate (KRP) medium containing 7.5 mM glucose, pH 7.4, at 37° in the absence and presence of corticosteroids, cyclic nucleotides, and adrenomimetic and cholinomimetic agents. Methylprednisolone hemisuccinate, triamcinolone acetonide, dexamethasone acetate, paramethasone acetate and hydrocortisone hemisuccinate reduced particle uptake by and discharge of lysosomal enzymes from guinea pig neutrophils. Aldosterone hemisuccinate and deoxycorticosterone acetate were inactive. Adrenomimetic (e.g. epinephrine) agents inhibited particle uptake by and lysosomal enzyme secretion from neutrophils, and cholinomimetic (e.g. acetylcholine) agents accelerated lysosomal enzyme release but had no effect on phagocytosis. Cyclic 3',5'-adenosine monophosphate (cyclic AMP) and one of its analogs inhibited particle ingestion by and lysosomal enzyme release from neutrophils; and this inhibition was potentiated by theophylline. Cyclic 3',5'-guanosine monophosphate (cyclic GMP), in contrast to the actions of corticosteroids, adrenomimetic agents and cyclic AMP, accelerated lysosomal enzyme secretion but had no effect on particle uptake. Cyclic GMP did not affect release of cytoplasmic lactate dehydrogenase, thus indicating maintenance of cell viability during release of lysosomal enzymes. Cytochalasin B, an agent which blocked phagocytic uptake of zymosan, did not interfere with inhibition of lysosomal enzyme secretion by corticosteroids, adrenomimetic agents and cyclic AMP or acceleration of this event by cholinomimetic agents or cyclic GMP. These studies indicate that guinea pig neutrophils are capable of releasing lysosomal enzymes during phagocytosis of zymosan and that certain agents can modulate lysosomal enzyme secretion and/or phagocytosis.

Polyribosome Aggregation in Isolated Rat‐Liver Cells

Polyribosomes were prepared from isolated rat liver cells by homogenisation or Triton X‐100 lysis. Triton X‐100 lysis proved the more effective method of preparation of ribosomes and was therefore used to study polyribosome profiles obtained from isolated liver cells incubated in Krebs‐Ringer phosphate and Eagle's basal essential medium for various times. Polyribosome aggregation decreased throughout the three‐hour incubation period but cells in the basal essential medium maintained a more aggregated profile and higher rate of albumin synthesis than those in Krebs‐Ringer phosphate.

Quantitative studies of phagocytosis by polymorphonuclear leukocytes: use of emulsions to measure the initial rate of phagocytosis.

Polymorphonuclear leukocytes suspended in Krebs-Ringer phosphate medium ingest paraffin oil containing Oil Red O emulsified with a variety of substances. Spectrophotometric determination of Oil Red O in the cells after uningested particles have been removed by differential centrifugation provides a quantitative measure of phagocytosis. This system has been used to investigate the effects of several drugs and hormones on the initial rate of phagocytosis and to approach the question of how the surface of a particle influences its acceptability as a substrate for phagocytosis. The rate of uptake of paraffin oil emulsified with bovine albumin was constant for 6 min and was proportional to cell concentration when saturating concentrations of paraffin oil emulsion were used. At lower concentrations of substrate, the initial rate of phagocytosis was directly proportional to paraffin oil concentration. The increment in glucose oxidation associated with phagocytosis varied directly with the initial rate of particle uptake. The rate of ingestion of the albumin emulsion was not altered by serum (2-20%, v/v), glucose (5-20 mM), or omission of potassium from the medium. The rate of phagocytosis was decreased 65% if magnesium was omitted, and was essentially zero in the absence of divalent cations. The initial rate of uptake was inhibited by inhibitors of glycolysis, by N-ethylmaleimide (0.05-1 mM), colchicine (0.001-0.1 mM), theophylline (1 and 2 mM), dibutyryl cyclic AMP (1 mM), hydrocortisone (2.1 mM), and ethanol (85 mM). Inhibitors of oxidative phosphorylation and dexamethasone (0.01 mM) were without effect, while insulin (2 mU/ml) slightly stimulated the phagocytic rate. Paraffin oil emulsified with different agents was used to approach the question of how the surface of a particle influences its acceptability as a substrate for phagocytosis. Emulsions prepared with nonionic detergents, methylated proteins, and proteins with a weak net charge at pH 7.4 were poorly ingested. On the other hand emulsions prepared with agents of strong net positive or negative charge were rapidly taken up. The effect of divalent cations on the rate of phagocytosis varied with the nature of the emulsifier, but was not related in any simple, direct fashion to the net surface charge of the particles. However, it has not been conclusively established that charge was the only variable of the emulsion particles employed.

Effects of Lipolytic and Antilipolytic Agents on Cyclic 3',5'-Adenosine Monophosphate in Fat Cells

Abstract Isolated fat cells were incubated in Krebs-Ringer phosphate medium. Within 1 min after the addition of 1.1 µm epinephrine, the cyclic AMP content of the cells was elevated. It continued to rise, reaching a peak in 4 to 7 min, and fell thereafter, being only slightly above the control levels after 10 to 15 min. A maximal rate of glycerol production was established within 1 to 2 min, well before the peak in cyclic AMP concentration, and was maintained unchanged while the cyclic AMP level continued to rise and then fell. Both in magnitude and time course, the effect of adrenocorticotropin (ACTH), 0.2 unit per ml, on cyclic AMP levels and on lipolysis was exactly like that of epinephrine (or of both hormones together). Addition of more epinephrine or of ACTH at several times during 1 hour of incubation with 1.1 µm epinephrine did not change cyclic AMP levels. The effects of epinephrine on cyclic AMP levels and on glycerol production were unaltered by the presence in the medium of 4 mm glucose. When propranolol (or insulin) was added several times after epinephrine-simulated lipolysis had begun, the rate of glycerol production fell to zero within 1 to 2 min. In the same period, the cyclic AMP concentration fell to a basal level; i.e. the rate of decrease was much greater than that observed after 4 to 7 min of incubation with epinephrine alone. Furthermore, after incubation of cells for 20 min with epinephrine, the addition of 0.8 mm theophylline produced an 8-fold rise in cyclic AMP content measured 5 min later. This increment was, however, only a fraction of that observed during the first 5 min after addition of epinephrine in the presence of theophylline. Since propranolol inhibits the action of epinephrine but does not alter basal adenyl cyclase activity, and, since 0.8 mm theophylline alone did not alter cyclic AMP levels, it may be concluded that adenyl cyclase was still epinephrine-stimulated during the time when cyclic AMP levels were falling. Whether the rate of formation of cyclic AMP during this period was equal to that during the first minutes after addition of the hormone cannot be established. Attempts to show directly alterations in phosphodiesterase activity during incubation of cells with epinephrine have been unsuccessful. When the effect of epinephrine was terminated after 4 min by the addition of propranolol, introduction of ACTH at several times during the following 20 min caused an immediate resumption of glycerol production at a rate equal to that observed with the initial addition of epinephrine. Cyclic AMP levels, however, rose little if at all in 4 to 5 min after addition of ACTH. The ability of cells to elevate cyclic AMP levels a second time in response to hormonal stimulation was restored when cells that had been incubated for 20 min were washed and suspended in fresh medium. There appears to be something accumulated in the medium during incubation of cells with epinephrine that interferes with the effectiveness of the hormone in causing accumulation of cyclic AMP in fresh cells. Whether this material has its effect on the rate of formation or of degradation of cyclic AMP cannot be decided.