Krebs-Ringer Phosphate Buffer Research Articles

Quantification of Prostaglandin E2 Concentration in Interstitial Fluid from the Hypothalamic Region of Free-moving Mice

Prostaglandin E2 (PGE2) is a well-established chemical mediator for the generation of the fever at the hypothalamus of the brain. PGE2 mediates fever generation via PGE receptor 3 (i.e., EP3) on neurons in the preoptic area. The role of PGE2 has been analyzed by measuring PGE2 concentration in cerebrospinal fluid (Ccsf); however, local PGE2 concentration at the hypothalamus may not necessarily be consistent with Ccsf. In this protocol, we introduce our method to measure directly the alteration in PGE2 concentration in interstitial fluid in the hypothalamus (Cisf) of awake (free-moving) mice using a microdialysis technique. Male mice (c57BL/6J) were anesthetized and fixed in the stereotaxic instrument, and a microdialysis probe was inserted into the hypothalamus through a guide cannula. On the fifth postoperative day, Cisf was monitored in free-moving mice that were intraperitoneally (i.p.) injected with lipopolysaccharide (LPS). PGE2 and other eicosanoids recovered in Krebs-Ringer phosphate buffer and defused through a microdialysis probe were extracted into ethyl acetate/formic acid and then quantified with LC-MS/MS. Our method is useful to understand the role of key regulators of prostaglandin concentration such as those of transporters, which have been unappreciated in inflammation-based brain diseases.

Distribution of 7-hydroxymethotrexate in human blood

We have examined the in-vitro distribution of 7-hydroxymethotrexate (7-OH-MTX), a cytotoxic metabolite of methotrexate (MTX), in human blood, and its protein binding in serum. The distribution of 7-OH-MTX (10(-6) M) in fresh samples of whole blood was studied at 37 degrees C and pH 7.51 +/- 0.05 (mean +/- s.d.), and its protein binding was assessed by equilibrium dialysis of serum against Krebs Ringer phosphate buffer at 37 degrees C and pH 7.41 +/- 0.07 (mean +/- s.d.). 7-OH-MTX had a mean cell/plasma concentration ratio of 0.03 (range 0-0.27, n = 18). It was extensively bound in human serum, with a bound fraction of 90.4 +/- 3.3% (mean +/- s.d.) in healthy volunteers (n = 11), and significantly lower, 82.3 +/- 4.0% (mean +/- s.d.), in hypoalbuminaemic surgical patients (n = 7). The binding of 7-OH-MTX was correlated with serum albumin (HSA) concentrations (r = 0.72, P less than 0.0007, n = 18). Blood distribution data support the contention that 7-OH-MTX has a small volume of distribution, and HSA appears to be mainly responsible for the high degree of its protein binding in serum.

Infection decreases fatty acid oxidation and nuclear hormone receptors in the diaphragm

Respiratory failure is a major cause of mortality during septic shock and is due in part to decreased ventilatory muscle contraction. Ventilatory muscles have high energy demands; fatty acid (FA) oxidation is an important source of ATP. FA oxidation is regulated by nuclear hormone receptors; studies have shown that the expression of these receptors is decreased in liver, heart, and kidney during sepsis. Here, we demonstrate that lipopolysaccharide (LPS) decreases FA oxidation and the expression of lipoprotein lipase (LPL), FA transport protein 1 (FATP-1), CD36, carnitine palmitoyltransferase beta, medium chain acyl-CoA dehydrogenase (MCAD), and acyl-CoA synthetase, key proteins required for FA uptake and oxidation, in the diaphragm. LPS also decreased mRNA levels of PPARalpha and beta/delta, RXRalpha, beta, and gamma, thyroid hormone receptor alpha and beta, and estrogen related receptor alpha (ERRalpha) and their coactivators PGC-1alpha, PGC-1beta, SRC1, SRC2, Lipin 1, and CBP. Zymosan resulted in similar changes in the diaphragm. Finally, in PPARalpha deficient mice, baseline CPT-1beta and FATP-1 levels were markedly decreased and were not further reduced by LPS suggesting that a decrease in the PPARalpha signaling pathway plays an important role in inducing some of these changes. The decrease in FA oxidation in the diaphragm may be detrimental, leading to decreased diaphragm contraction and an increased risk of respiratory failure during sepsis.

Growth Hormone Inhibition of Glucose Uptake in Adipocytes Occurs without Affecting GLUT4 Translocation through an Insulin Receptor Substrate-2-Phosphatidylinositol 3-Kinase-dependent Pathway

Growth hormone (GH) pretreatment of 3T3-L1 adipocytes resulted in a concentration- and time-dependent inhibition of insulin-stimulated glucose uptake. Surprisingly, this occurred without significant effect on insulin-stimulated glucose transporter (GLUT) 4 translocation or fusion with the plasma membrane. In parallel, the inhibitory actions of chronic GH pretreatment also impaired insulin-dependent activation of phosphatidylinositol (PI) 3-kinase bound to insulin receptor substrate (IRS)-2 but not to IRS-1. In addition, insulin-stimulated Akt phosphorylation was inhibited by GH pretreatment. In contrast, overexpression of IRS-2 or expression of a constitutively active Akt mutant prevented the GH-induced insulin resistance of glucose uptake. Moreover, small interfering RNA-mediated IRS-2 knockdown also inhibited insulin-stimulated Akt activation and glucose uptake without affecting GLUT4 translocation and plasma membrane fusion. Together, these data support a model in which chronic GH stimulation inhibits insulin-dependent activation of phosphatidylinositol 3-kinase through a specific interaction of phosphatidylinositol 3-kinase bound to IRS-2. This inhibition leads to suppression of Akt activation coupled to glucose transport activity but not translocation or plasma membrane fusion of GLUT4.

Hyperosmolarity Induced Cystine Transport and Cystine-cysteine Cycle between Erythrocytes and the Plasma

The objective of the present study was to determine if a cystine-cysteine cycle operates between the erythrocytes and the plasma. In the present study we incubated the erythrocytes in krebs ringer phosphate buffer with different osmolarity containing different amounts of cystine. Our results show that erythrocytes do not uptake cystine from the environment when the osmolarity of the buffer is 310 mOsmol/l. Erythrocytes also do not operate a cystinecysteine cycle in this isoosmolar buffer. However, when exposed to hyperosmolar buffer in the ranges that occur in th ek idne ym edulla which is in between 1200–1400 mOsmol/l erythrocytes start to uptake cystine from the environment and induce a cystine-cysteine cycle. The cystine uptake and cystine-cysteine cycle were characterized by measurement of changes in the free -SH concentrations in erythrocytes and in the buffer. Following incubation of erythrocytes in 1 mM cystine containing 1250 and 1300 mOsmol/l buffer, the free -SH concentrations in the buffer reached to 0.102 ± 0.002 and 0.241 ± 0.013µmol/ml erythrocyte respectively. Our results demonstrate that erythrocytes display a cystine-cysteine cycle in hyperosmolar environment which is prevailed mainly in the kidney medulla. Our results also display that this process is biologically active and energy dependent. The observed cystine-cysteine cycle is inhibited when the erythrocytes are incubated at lower temperatures and in the absence of glucose. Our results suggest that erythrocytes uptake cystine, intracellulary reduce it to cysteine and release it back to the environment whe ne xposed to hyperosmolar conditions. Erythrocytes may have a role in the regulation of plasma cystine and cysteine concentrations and may contribute to the regulation of plasma redox status.

Cytosolic Aspartate Aminotransferase, a New Partner in Adipocyte Glyceroneogenesis and an Atypical Target of Thiazolidinedione

We show that cytosolic aspartate aminotransferase (cAspAT) is involved in adipocyte glyceroneogenesis, a regulated pathway that controls fatty acid homeostasis by promoting glycerol 3-phosphate formation for fatty acid re-esterification during fasting. cAspAT activity, as well as the incorporation of [(14)C]aspartate into the neutral lipid fraction of 3T3-F442A adipocytes was stimulated by the thiazolidinedione (TZD) rosiglitazone. Conversely, the ratio of fatty acid to glycerol released into the medium decreased. Regulation of cAspAT gene expression was specific to differentiated adipocytes and did not require any peroxisome proliferator-activated receptor gamma (PPARgamma)/retinoid X receptor-alpha direct binding. Nevertheless, PPARgamma is indirectly necessary for both cAspAT basal expression and TZD responsiveness because they are, respectively, diminished and abolished by ectopic overexpression of a dominant negative PPARgamma. The cAspAT TZD-responsive site was restricted to a single AGGACA hexanucleotide located at -381 to -376 bp whose mutation impaired the specific RORalpha binding. RORalpha ectopic expression activated the cAspAT gene transcription in absence of rosiglitazone, and its protein amount in nuclear extracts is 1.8-fold increased by rosiglitazone treatment of adipocytes. Finally, the amounts of RORalpha and cAspAT mRNAs were similarly increased by TZD treatment of human adipose tissue explants, confirming coordinated regulation. Our data identify cAspAT as a new member of glyceroneogenesis, transcriptionally regulated by TZD via the control of RORalpha expression by PPARgamma in adipocytes.

Perilipin regulates the thermogenic actions of norepinephrine in brown adipose tissue

In response to cold, norepinephrine (NE)-induced triacylglycerol hydrolysis (lipolysis) in adipocytes of brown adipose tissue (BAT) provides fatty acid substrates to mitochondria for heat generation (adaptive thermogenesis). NE-induced lipolysis is mediated by protein kinase A (PKA)-dependent phosphorylation of perilipin, a lipid droplet-associated protein that is the major regulator of lipolysis. We investigated the role of perilipin PKA phosphorylation in BAT NE-stimulated thermogenesis using a novel mouse model in which a mutant form of perilipin, lacking all six PKA phosphorylation sites, is expressed in adipocytes of perilipin knockout (Peri KO) mice. Here, we show that despite a normal mitochondrial respiratory capacity, NE-induced lipolysis is abrogated in the interscapular brown adipose tissue (IBAT) of these mice. This lipolytic constraint is accompanied by a dramatic blunting ( approximately 70%) of the in vivo thermal response to NE. Thus, in the presence of perilipin, PKA-mediated perilipin phosphorylation is essential for NE-dependent lipolysis and full adaptive thermogenesis in BAT. In IBAT of Peri KO mice, increased basal lipolysis attributable to the absence of perilipin is sufficient to support a rapid NE-stimulated temperature increase ( approximately 3.0 degrees C) comparable to that in wild-type mice. This observation suggests that one or more NE-dependent mechanism downstream of perilipin phosphorylation is required to initiate and/or sustain the IBAT thermal response.

Energy Depletion Inhibits Phosphatidylinositol 3-Kinase/Akt Signaling and Induces Apoptosis via AMP-activated Protein Kinase-dependent Phosphorylation of IRS-1 at Ser-794

Energy depletion activates AMP-activated protein kinase (AMPK) and inhibits cell growth via TSC2-dependent suppression of mTORC1 signaling. Long term energy depletion also induces apoptosis by mechanisms that are not well understood to date. Here we show that AMPK, activated by energy depletion, inhibited cell survival by binding to and phosphorylating IRS-1 at Ser-794. Phosphorylation of IRS-1 at this site inhibited phosphatidylinositol 3-kinase/Akt signaling, suppressed the mitochondrial membrane potential, and promoted apoptosis. Of the treatments promoting energy depletion, glucose deprivation, hypoxia, and inhibition of ATP synthesis in the mitochondria stimulated phosphorylation of IRS-1 at Ser-794 via an LKB1/AMPK-dependent manner, whereas oxidative stress and 2-deoxyglucose stimulated phosphorylation at this site via a Ca2+/calmodulin-dependent protein kinase kinase beta/AMPK axis. These data define a novel pathway that cooperates with other adaptive mechanisms to formulate the cellular response to energy depletion.

A role of lipin in human obesity and insulin resistance: relation to adipocyte glucose transport and GLUT4 expression

The mouse lipin gene, Lpin1, is important for adipose tissue development and is a candidate gene for insulin resistance. Here, we investigate the adipose tissue expression levels of the human LPIN1 gene in relation to various clinical variables as well as adipocyte function. LPIN1 gene expression was induced at an early step in human preadipocyte differentiation in parallel with peroxisome proliferator-activated receptor gamma. Lipin mRNA levels were higher in fat cells than in adipose tissue segments but showed no difference between subcutaneous and omental depots. Moreover, LPIN1 expression levels were reduced in obesity, improved following weight reduction in obese subjects, and were downregulated in women with the metabolic syndrome. With respect to adipocyte function, adipose LPIN1 gene expression was strongly associated with both basal and insulin-mediated subcutaneous adipocyte glucose transport as well as mRNA levels of glucose transporter 4 (GLUT4). We show that body fat accumulation is a major regulator of human adipose LPIN1 expression and suggest a role of LPIN1 in human preadipocyte as well as mature adipocyte function.

The cholinergic influence on the mesenteric ganglion affects the liberation of ovarian steroids and nitric oxide in oestrus day rats: characterization of an ex vivo system

The axons that constitute the ovarian nervous plexus originate mostly in the principal neurons of the superior mesenteric ganglion (SMG) that is part of the sympathetic ganglionic chain and exhibits cholinergic receptors. In order to observe the effect of acetylcholine, the main neurotransmitter in the ganglionic transmission, the purpose of the present work was: first, to standardize an integrated ex vivo superior mesenteric ganglion-ovarian nervous plexus-ovary (SMG-ONP-O) system in oestrus day rats; secondly, to determine if the ganglionic cholinergic stimulus modifies the release of nitric oxide and steroids in the ovary compartment in the absence of humoral factors; and thirdly, to investigate if there are differences in the responses between the left and right ovaries caused by the neural stimulus. The ex vivo experimental left and right systems were developed and standardized. The systems were incubated in Krebs-Ringer phosphate buffer in a Dubnoff metabolic shaker. The progesterone release was determined to standardize the incubation times, obtaining different responses between the left and right systems, which shows that both systems have their own autonomic tone. Non-specific stimulation with KCl in the ganglion compartment provoked different responses in terms of release of progesterone and oestradiol. Progesterone decreased in the left and right systems. However, oestradiol diminished at short times and increased at 60 and 120 min in the left ovary, whereas it increases at 30 and 60 min in the right ovary. These different responses show the sensitivity and viability of both systems. When acetylcholine was used in the ganglion compartment, the release of nitric oxide, progesterone, androstenedione and oestradiol was evaluated. The liberation of nitrite increased at 15, 30 and 60 min in the left system and decreased in the right system at 120 min. Progesterone showed a decrease in its release at 15, 30 and 120 min and androstenedione at 15 min in the left ovary compartment. In the right ovary, only progesterone decreased in relation to the control at 120 min while androstenedione did not show significant changes. Oestradiol showed an increase in the left ovary compartment at all the studied times, while in the right ovary it did not show any changes. These results indicate that the neural stimulus from the superior mesenteric ganglion through the ovarian nervous plexus is one of the factors modulating the secretory activity of the ovarian steroids and nitric oxide. The system is viable and also shows a different sensitivity of the left ovary in relation to the right one at least in this cycle stage, characterized by marked irrigation and profound structural changes in the ovary.

Exopolysaccharides from Burkholderia cenocepacia Inhibit Neutrophil Chemotaxis and Scavenge Reactive Oxygen Species

Bacteria belonging to the Burkholderia cepacia complex are important opportunistic pathogens in compromised hosts, particularly patients with cystic fibrosis or chronic granulomatous disease. Isolates of B. cepacia complex may produce large amounts of exopolysaccharides (EPS) that endow the bacteria with a mucoid phenotype and appear to facilitate bacterial persistence during infection. We showed that EPS from a clinical B. cenocepacia isolate interfered with the function of human neutrophils in vitro; it inhibited chemotaxis and production of reactive oxygen species (ROS), both essential components of innate neutrophil-mediated host defenses. These inhibitory effects were not due to cytotoxicity or interference with intracellular calcium signaling. EPS also inhibited enzymatic generation of ROS in cell-free systems, indicating that it scavenges these bactericidal products. B. cenocepacia EPS is structurally distinct from Pseudomonas aeruginosa alginate, yet they share the capacity to scavenge ROS and inhibit chemotaxis. These properties could explain why the two bacterial species resist clearance from the infected cystic fibrosis lung.

Dual Roles of P2 Purinergic Receptors in Insulin-stimulated Leptin Production and Lipolysis in Differentiated Rat White Adipocytes

ATP is co-localized with norepinephrine at the sympathetic nerve terminals and may be released simultaneously upon neuronal stimulation, which results in activation of purinergic receptors. To examine whether leptin synthesis and lipolysis are influenced by P2 purinergic receptor activation, the effects of ATP and other nucleotides on leptin secretion and glycerol release have been investigated in differentiated rat white adipocytes. Firstly, insulin-induced leptin secretion was inhibited by nucleotide treatment with the following efficacy order: 3'-O-(4-benzoyl)benzoyl ATP (BzATP) > ATP >> UTP. Secondly, treatment of adipocytes with ATP increased both intracellular Ca(2+) concentration and cAMP content. Intracellular calcium concentration was increased by ATP and UTP, but not BzATP, an effect attributed to phospholipase C-coupled P2Y(2). On the other hand, cAMP was generated by treatment with BzATP and ATPgammaS, but not UTP, indicating functional expression of adenylyl cyclase-coupled P2Y(11) receptors in white adipocytes. Thirdly, lipolysis was significantly activated by BzATP and ATP, which correlated with the characteristics of the P2Y(11) subtype. Taken together, the data presented here suggest that white adipocytes express at least two different types of P2Y receptors and that activation of P2Y(11) receptor might be involved in inhibition of leptin production and stimulation of lipolysis, suggesting that purinergic transmission can play an important role in white adipocyte physiology.

INTERLEUKIN 8 DIMERIZATION AS A MECHANISM FOR REGULATION OF NEUTROPHIL ADHERENCE-DEPENDENT OXIDANT PRODUCTION

Interleukin 8 (IL-8), a member of the CXC subfamily of chemoattractant cytokines, induces a range of functional responses in human neutrophils via its interactions with two high-affinity cell-surface receptors, CXCR1 and CXCR2. Like other CXC chemokines, IL-8 forms homodimers at physiologic concentrations. Monomers and dimers bind to CXC receptors with high affinity and induce various functions. Binding to glycosaminoglycans decreases the dimerization constant, enhancing surface-bound dimer formation. However, a specific role for IL-8 dimerization has not been identified. We explored the hypothesis that certain neutrophil responses to IL-8 were induced primarily by the IL-8 dimers. To this end, two dimerization-deficient IL-8 mutant proteins, M3 and M4, were used in various functional assays. In contrast to native IL-8, these proteins existed primarily as monomers at micromolar concentrations. The mutants retained high-affinity binding to both CXC receptors and potently induced neutrophil calcium flux, chemotaxis, and elastase release. In contrast to native IL-8, neither mutant inhibited tumor necrosis factor alpha-induced oxidant production. Additionally, M4 was less effective than native IL-8 at desensitizing neutrophil migration. These data suggest that although IL-8 dimers or monomers are sufficient for several neutrophil functions, dimers may participate in suppression of specific surface-dependent neutrophil responses.

Iron-mediated H2O2 Production as a Mechanism for Cell Type-specific Inhibition of Tumor Necrosis Factor α-Induced but Not Interleukin-1β-induced IκB Kinase Complex/Nuclear Factor-κB Activation

Coordinated and specific regulation of tumor necrosis factor (TNF) and interleukin (IL)-1 signaling pathways and how and whether they are modified by different agents are key events for proper immune responses. The IkappaB kinase complex (IKK)/NF-kappaB and JNK/AP-1 pathways are central mediators of TNF and IL-1 during inflammatory responses. Here we show that l-mimosine, a toxic non-protein amino acid that has been shown to reduce serum TNFalpha levels and affect inflammatory responses, specifically inhibits TNF-induced IKK but not JNK in a cell type-specific manner. l-Mimosine did not affect IKK and NF-kappaB activation by IL-1beta. l-Mimosine caused cell cycle arrest at G(1)-S phase, but inhibition of IKK was found to be independent of cell cycle arrest. Treatment of cells with l-mimosine resulted in production of H(2)O(2). Addition of FeSO(4) restored IKK activation by TNFalpha as did ectopic expression of catalase or pretreatment of cells with N-aceltyl-l-cysteine, indicating a role for intracellular H(2)O(2) as a mediator of inhibition. Cleavage and degradation of TNF pathway components TNFR1, RIP, and Hsp90 were observed in l-mimosine and H(2)O(2) treated cells indicating a putative mechanism for selective inhibition of TNF but not IL-1beta-induced IKK activation.

Suppression of the accumulation of triosephosphates and increased formation of methylglyoxal in human red blood cells during hyperglycaemia by thiamine in vitro.

The accumulation of triosephosphates and the increased formation of the potent glycating agent methylglyoxal in intracellular hyperglycaemia are implicated in the development of diabetic complications. A strategy to counter this is to stimulate the anaerobic pentosephosphate pathway of glycolysis by maximizing transketolase activity by thiamine supplementation, with the consequent consumption of glyceraldehyde-3-phosphate and increased formation of ribose-5-phosphate. To assess the effect of thiamine supplementation on the accumulation of triosephosphates and methylglyoxal formation in cellular hyperglycaemia, we incubated human red blood cell suspensions (50% v/v) in short-term culture with 5 mM glucose and 50 mM glucose in Krebs-Ringer phosphate buffer at 37 degrees C as models of cellular metabolism under normoglycaemic and hyperglycaemic conditions. In hyperglycaemia, there is a characteristic increase in the concentration of the triosephosphate pool of glycolytic intermediates and a consequent increase in the concentration and metabolic flux of the formation of methylglyoxal. The addition of thiamine (50-500 microM) increased the activity of transketolase, decreased the concentration of the triosephosphate pool, decreased the concentration and metabolic flux of the formation of methylglyoxal, and increased the concentration of total sedoheptulose-7-phosphate and ribose-5-phosphate. Biochemical changes implicated in the development of diabetic complications were thereby prevented. This provides a biochemical basis for high dose thiamine therapy for the prevention of diabetic complications.

Gas-tissue autoradiography using [ [formula omitted]] molecular oxygen for visualization of mitochondrial oxygen fixation in brain slices

We have developed a novel in vitro autoradiographic method of visualizing oxygen fixation using [ 15 O ]O 2. Brain slices (400 μm) are preincubated in Krebs–Ringer phosphate buffer and exposed to [ 15 O ]O 2 with carrier O 2 for 10 min on slide glass or on a Millicell-CM membrane in a chamber. After the 15 O 2 exposure, the brain slices are placed in contact with an imaging plate. The autoradiograms are analyzed with MacBas to obtain images of the distribution of radioactivity. The [ 15 O ]O 2 fixation was more extensive in the gray matter (striatum, thalamus and cerebral cortex) than in the white matter. The fixation of [ 15 O ]O 2 in brain slices decreased dose dependently with NaCN concentration. These results indicate that [ 15 O ]O 2 is converted into [ 15 O ]H 2O in the brain tissue according to the activity of mitochondrial electron transfer and remains in the tissue. This method provides information regarding the basic oxygen consumption of brain slices of rat and mouse under conditions of sufficient O 2 delivery, which reflects mitochondrial electron transport. Themes: Cellular and Molecular Biology Topics: Staining, Tracing, and imaging techniques

Visualization of mitochondrial oxygen fixation in brain slices by gas–tissue autoradiography

We have developed a novel autoradiographic method of visualizing oxygen fixation with sufficient delivery of [ 15 O ]O 2/O 2. Brain slices (400 μm) were preincubated in Krebs–Ringer phosphate buffer and exposed to [ 15 O ]O 2 in a chamber. Fixation of [ 15 O ]O 2 correlated with the polarographically measured oxygen consumption among tissue slices from various organs ( r=0.84). The fixation of [ 15 O ]O 2 by brain slices was significantly reduced (7.2% of the control) by heat-treatment or dose dependently by NaCN (18.2% of the control on 50 mM NaCN pretreatment). The 15 O radioactivity in the brain slices prepared from rotenone injected rats was also reduced compared to the control (56.8% of the control side). In an autoradiographic study, 15 O radioactivity showed a heterogeneous distribution both in coronal and sagittal sections. Autoradiography of young and senescent rat brain sections showed reduction of oxygen uptake with aging in the cerebrum, the senescent being 77.4% of the young. This method provides information regarding basic oxygen consumption of tissue slices under condition of sufficient O 2 delivery, which reflects mitochondrial electron transport.

Small Peptide of Soybean Hydrolysate Is Absorbed More Rapidly than Large Peptide in the Perfused Rat Small Intestine.

The effects of peptide chain length on the absorption of soybean hydrolysates by the rat small intestine were investigated by the perfusion. Two types of soybean hydrolysates, a small peptide (SP; average peptide chain length: 3.2) and a large peptide (LP; average peptide chain length: 5.2), were prepared for this experiment. Krebs-Ringer phosphate buffer (pH 7.0) containing 0.5% (w/v) of either of the two types of soybean hydrolysates was perfused through a 15-cm length of rat jejunum for 70min. After the perfusion, samples were collected; and the total amount of amino acid was then measured by an amino acid analyzer (Hitachi L-8500) after hydrolysis. The absorption rate of each amino acid was subsequently determined. The absorption rate for glycine, alanine, isoleucine, leucine histidine, arginine, phenylalanine, and proline in SP was significantly greater than that for those in LP. The total amino acid absorption of SP was significantly greater than that of LP. There were no significant differences in the net absorption of water in the buffer solutions of SP and LP. We thus concluded that SP is more greatly absorbed than LP in the intestinal perfusion model of the rat small intestine.

Relationship between motility and oxygen consumption of sperm from the cauda epididymides of the rat.

The oxygen consumption of rat sperm was low (2.7 microL O2 10(8) sperm(-1) h(-1)) in caudal epididymal semen (CES) when stimulation of motility was avoided. The addition of 1 microL of Krebs Ringer phosphate buffer (KRP) to 40 microL of CES (CES:KRP = 40:1) did not activate motility, but stimulated oxygen consumption 2-fold. Inclusion of 1-5 mM glucose, acetate, pyruvate or lactate in the KRP further stimulated respiration rate (up to 4.3-fold) without activating motility, but respiration was reduced when 2-deoxyglucose replaced energy substrates. Inclusion of dibutyryl cAMP (1 mM) activated sperm motility in all samples and stimulated oxygen consumption 2.9-fold. Dilution of CES at the ratio of CES:KRP = 40:1000 also activated sperm motility and stimulated respiration rate 2.9-fold. The combined effect of dibutyryl cAMP and glucose in stimulating respiration was greater than their individual effects. However, the response to cAMP or substrates was not altered by incubation in KRP containing either 0 or 0.5 mM Ca2+. It was concluded that the motility and metabolism of rat epididymal sperm are suppressed in vivo. Respiration can be stimulated by a small (1.025-fold) dilution and further stimulated by the inclusion of energy substrate, without activating motility. However, a larger dilution or inclusion of cAMP activated motility and simultaneously stimulated metabolism, with exogenous substrate being required to stimulate respiration to the maximum rate. This suggests that prior to activation, the rate of oxygen consumption and sperm motility are not coupled.

Difference and Similarity of Serotonin and Pteridines to Act on Lipid Metabolism

Summary Incorporation of fatty acids into phospholipids has been investigated using samples of rat liver homogenate, Krebs-Ringer phosphate buffer (pH=7.4) containing 0.3% albumin, fatty acid mixture and glycerol. The addition of neopterin, serotonin (0.2 and 2.0 nmoljg wet weight) without iproniasid and 5- hydroxyindoleacetic acid (20.0 nmoljg wet weight) induced an increase of saturated and a decrease of unsaturated, especially arachidonic acid, incorporation into phospholipids. These changes were accompanied with elevated cholesterol content in samples. The addition of 5,6,7,8-tetrahydrobiopterin (5 and 30 pmolj g wet weight) and serotonin (0.2 and 2.0 nmoljg wet weight) together with iproniasid (10 nmoljg wet weight) to incubation medium for phospholipid biosynthesis in vitro induced a decrease of saturated and an increase of unsaturated, especially arachidonic acid, incorporation into phospholipids. These changes were accompanied with decreased cholesterol content in samples. The influence of serotonin without iproniasid and 5-hydroxyindoleacetic acid was similar to that of neopterin, kynurenine and noradrenaline observed earlier, while the influence of serotonin together with iproniasid was similar to that of 5,6,7,8- tetrahydrobiopterin and its precursors found earlier. Our results allow to suggest that in the studied concentrations serotonin increases, while 5-hydroxyindoleacetic acid decreases membrane fluidity.