On the histochemistry of epimerases

Summary A histochemical method for hydroxyproline epimerase together with its mechanism of enzymatic action is described. The epimerase is studied in different tissues and organs. The data resulting from the investigation reveal a particular epimerase activity in connective tissue and skeletal muscular tissue.     

The quantitative histochemistry of enzymes of the pentose phosphate pathway in the central nervous system of the rat

Abstract— Methods are presented for the measurement of the non-oxidative enzymes of the pentose phosphate pathway in freeze-dried samples of tissue weighing 2 μg or less. The activities of transketolase (EC 2.2.1.1), transaldolase (EC 2.2.1.2), ribosephosphate isomerase (EC 5.3.1.6), and ribulosephosphate epimerase (EC 5.1.3.1), together with glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (EC 1.1.1.44) have been measured in seven specific regions in the central nervous system of the rat. Michaelis constants and temperature coefficients of these enzymes were obtained on homogenates of whole rat brain. The entire enzymic complement of the pentose phosphate pathway was detected in each of the regions examined. The activities of the non-oxidative enzymes and 6-phosphogluconate dehydrogenase did not vary greatly among the different regions examined, whereas the activity of glucose-6-phosphate dehydrogenase varied in close correspondence with the lipid content of the various structures. The cellular, granular layer of the cerebellum was exceptional, since it exhibited at least three times more transaldolase activity than that observed in other structures, an observation suggesting an association of transaldolase with nerve cell bodies.     

An immunohistochemical study of the pancreatic endocrine cells of the nude mouse, Balb/c-nu/nu

The regional distribution and frequency of the pancreatic endocrine cells in the nude mouse, Balb/c-nu/nu were studied by immunohistochemical (peroxidase anti-peroxidase; PAP) methods using specific antisera against insulin, glucagon, somatostatin and human pancreatic polypeptide (hPP). The pancreas of the mouse was divided into two lobes, the splenic and duodenal lobes, and each lobe was subdivided into three regions, the pancreatic islets (central and peripheral regions), the exocrine region and the pancreatic duct region (consisting of duct epithelium and surrounding connective tissue--sub-epithelial connective tissue). In the pancreatic islets, most of insulin-immunoreactive (IR) cells were located in the central region, and glucagon-, somatostatin and hPP-IR cells were located in the peripheral region regardless of the lobe. In the splenic part, glucagon-IR cells were also located in the central regions, and more numerous somatostatin-IR cells were detected in the central regions compared to those of the duodenal part. hPP-IR cells were restricted to the peripheral regions in both lobes but more numerous cells were detected in the duodenal portion as compared to those of the splenic portion. In the exocrine parenchyma of the splenic lobe, only insulin-, glucagon- and somatostatin-IR cells were detected.. Here, the insulin- and glucagon-IR cells formed cell clusters, while somatostatin-IR cells were present as solitary cells. In the exocrine region of the duodenal portion, only insulin-, somatostatin- and hPP-IR cells were observed, with the same distributional pattern as that found in the splenic lobe. However, clusters of cells consisting only of hPP-IR cells were distributed in the pancreas parenchyma as small islets. In the pancreatic duct region, only solitary hPP-IR cells were demonstrated in the sub-epithelial connective tissue regions of the splenic portion. In conclusion, some strain-dependent characteristic distributional patterns of pancreatic endocrine cells, especially of the hPP-IR cells, were found in the nude mouse. In addition, somewhat different distributional patterns were found between the two pancreatic lobes.     

Biosynthesis of lysine in plants: the putative role of meso-diaminopimelate dehydrogenase

Extracts from Chlamydomonas, corn, soybean and tobacco were tested for enzymes of the lysine biosynthetic pathway. Dihydrodipicolinic acid (DHD) synthase, DHD reductase, diaminopimelate (DAP) epimerase and DAP decarboxylase were present in all. However, in contrast to the report of Wenko et al., meso-DAP dehydrogenase could not be detected in extracts prepared from soybean. Moreover, it was not found in Chlamydomonas, corn and tobacco as well. In order to set an upper limit to the amount of meso-DAP dehydrogenase that might be present, reconstruction experiments were performed with soybean and corn extracts in which the conversion of dihydrodipicolinate to lysine was made dependent on the addition of limited amounts of the meso-DAP dehydrogenase purified from Bacillus sphaericus. The presence of DAP epimerase and the absence of meso-DAP dehydrogenase indicates that the meso-DAP dehydrogenase abbreviated pathway for lysine synthesis is not operative in plants.     

Enzymatic Evidence for a Complete Oxidative Pentose Phosphate Pathway in Chloroplasts and an Incomplete Pathway in the Cytosol of Spinach Leaves

The intracellular localization of transaldolase, transketolase, ribose-5-phosphate isomerase, and ribulose-5-phosphate epimerase was reexamined in spinach (Spinacia oleracea L.) leaves. We found highly predominant if not exclusive localization of these enzyme activities in chloroplasts isolated by isopyknic centrifugation in sucrose gradients. Glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, glucose phosphate isomerase, and triose phosphate isomerase activity was present in the chloroplast fraction but showed additional activity in the cytosol (supernatant) fraction attributable to the cytosol-specific isoforms known to exist for these enzymes. Anion-exchange chromatography of proteins of crude extracts on diethylaminoethyl-Fractogel revealed only a single enzyme each for transaldolase, transketolase, ribose-5-phosphate isomerase, and ribulose-5-phosphate epimerase. The data indicate that chloroplasts of spinach leaf cells possess the complete complement of enzymes of the oxidative pentose phosphate path-way (OPPP), whereas the cytosol contains only the first two reactions, contrary to the widely held view that plants generally possess a cytosolic OPPP capable of cyclic function. The chloroplast enzymes transketolase, ribose-5-phosphate isomerase, and ribulose-5-phosphate epimerase appear to be amphibolic for the Calvin cycle and OPPP.     

Reticular meshwork of the spleen in rats studied by electron microscopy

The reticular meshwork of the rat spleen, which consists of both fibrous and cellular reticula, was investigated by transmission electron microscopy. The fibrous reticulum of the splenic pulp is composed of reticular fibers and basement membranes of the sinuses. These reticular fibers and basement membranes are continuous with each other. The reticular fibers are enfolded by reticular cells and are composed of two basic elements: 1) peripheral basal laminae of the reticular cells, and 2) central connective tissue spaces in which microfibrils, collagenous fibrils, elastic fibers, and unmyelinated adrenergic nerve fibers are present. The basement membranes of the sinuses are sandwiched between reticular cells and sinus endothelial cells and are composed of lamina-densalike material, microfibrils, collagenous fibrils, and elastic fibers. The presence of these connective tissue fibrous components indicates that there are connective tissue spaces in these basement membranes. The basement membrane is divided into three parts: the basal lamina of the reticular cell, the connective tissue space, and the basal lamina of the sinus endothelial cell. When the connective tissue space is very small or absent, the two basal laminae may fuse to form a single, thick basement membrane of the splenic sinus wall. The fibrous reticulum having these structures is responsible for support (collagenous fibrils) and rebounding (elastic fibers). The cells of the cellular reticulum--reticular cells and their cytoplasmic processes, which possess abundant contractile microfilaments, dense bodies, hemidesmosomes, basal laminae, and a well-developed, rough-surfaced endoplasmic reticulum, and Golgi complexes, which are characteristic of both fibroblasts and smooth muscle cells--are considered to be myofibroblasts. They may play roles in splenic contraction and in fibrogenesis of the fibrous reticulum. The contractile ability may be influenced by the unmyelinated adrenergic nerve fibers that pass through the reticular fibers. The three-dimensional reticular meshwork of the spleen consists of sustentacular fibrous reticulum and contractile myofibroblastic cellular reticulum. This meshwork not only supports the organ but also contributes to a contractile mechanism in circulation regulation, in collaboration with major contractile elements in the capsulo-trabecular system.     

Analogs of diaminopimelic acid as inhibitors of meso-diaminopimelate dehydrogenase and LL-diaminopimelate epimerase

Analogs 1-8 of diaminopimelic acid (DAP) were synthesized and tested for inhibition of purified meso-DAP D-dehydrogenase from Bacillus sphaericus and of LL-DAP epimerase from Escherichia coli. The dehydrogenase was assayed by monitoring NADPH formation spectrophotometrically at 340 nm. N-Hydroxy DAP 4, N-amino DAP 5, and 4-methylene DAP 6 are substrates of the dehydrogenase with relative rates exceeding those of the meso isomers of the thia analogs 1ab, 2ab, and 3ab. DAP epimerase was assayed by coupling the epimerization of LL-DAP to DL-DAP (Km = 0.26 mM) with the dehydrogenase-catalyzed oxidation of DL-DAP by NADP. Lanthionine isomers 1ab and 1c were stronger inhibitors of the epimerase (Ki = 0.18 mM, Ki' = 0.67 mM, and Ki = 0.42 mM, respectively) than the corresponding meso-sulfoxide 2ab or the meso-sulfone 3ab. Other isomers of 2 and 3, as well as compounds 7 and 8, showed no epimerase inhibition. N-Hydroxy DAP 4 was the most potent competitive inhibitor (Ki = 0.0056 mM) of the epimerase, whereas N-amino DAP 5 is weaker (Ki = 2.9 mM) and 4-methylene DAP 6 is a noncompetitive inhibitor (Ki' = 0.95 mM). Although none of the analogs tested showed time-dependent inactivation of either enzyme, compounds 4, 5, 6, and 7 display substantial antibacterial activities. Possible mechanisms of epimerase inhibition and significance of the DAP pathway as a target for antibiotics are discussed.     

The pentose phosphate pathway of glucose metabolism. Hormonal and dietary control of the oxidative and non-oxidative reactions of the cycle in liver

1. Measurements were made of the non-oxidative reactions of the pentose phosphate cycle in liver (transketolase, transaldolase, ribulose 5-phosphate epimerase and ribose 5-phosphate isomerase activities) in a variety of hormonal and nutritional conditions. In addition, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities were measured for comparison with the oxidative reactions of the cycle; hexokinase, glucokinase and phosphoglucose isomerase activities were also included. Starvation for 2 days caused significant lowering of activity of all the enzymes of the pentose phosphate cycle based on activity in the whole liver. Re-feeding with a high-carbohydrate diet restored all the enzyme activities to the range of the control values with the exception of that of glucose 6-phosphate dehydrogenase, which showed the well-known ;overshoot' effect. Re-feeding with a high-fat diet also restored the activities of all the enzymes of the pentose phosphate cycle and of hexokinase; glucokinase activity alone remained unchanged. Expressed as units/g. of liver or units/mg. of protein hexokinase, glucose 6-phosphate dehydrogenase, transketolase and pentose phosphate isomerase activities were unchanged by starvation; both 6-phosphogluconate dehydrogenase and ribulose 5-phosphate epimerase activities decreased faster than the liver weight or protein content. 2. Alloxan-diabetes resulted in a decrease of approx. 30-40% in the activities of 6-phosphogluconate dehydrogenase, ribose 5-phosphate isomerase, ribulose 5-phosphate epimerase and transketolase; in contrast with this glucose 6-phosphate dehydrogenase, transaldolase and phosphoglucose isomerase activities were unchanged. Treatment of alloxan-diabetic rats with protamine-zinc-insulin for 3 days caused a very marked increase to above normal levels of activity in all the enzymes of the pentose phosphate pathway except ribulose 5-phosphate epimerase, which was restored to the control value. Hexokinase activity was also raised by this treatment. After 7 days treatment of alloxan-diabetic rats with protamine-zinc-insulin the enzyme activities returned towards the control values. 3. In adrenalectomized rats the two most important changes were the rise in hexokinase activity and the fall in transketolase activity; in addition, ribulose 5-phosphate epimerase activity was also decreased. These effects were reversed by cortisone treatment. In addition, in cortisone-treated adrenalectomized rats glucokinase activity was significantly lower than the control value. 4. In thyroidectomized rats both ribose 5-phosphate isomerase and transketolase activities were decreased; in contrast with this transaldolase activity did not change significantly. Hypophysectomy caused a 50% fall in transketolase activity that was partially reversed by treatment with thyroxine and almost fully reversed by treatment with growth hormone for 8 days. 5. The results are discussed in relation to the hormonal control of the non-oxidative reactions of the pentose phosphate cycle, the marked changes in transketolase activity being particularly outstanding.     

Regulation of resynthesis of the CO2-acceptor in photosynthesis. Feedback inhibition of transketolase

The presence of ribulose-5-phosphate epimerase (EC 5.1.3.1, epimerase) in samples of ribose-5-phosphate isomerase (EC 5.3.1.6, isomerase) obtained from spinach ( Spinacea aleracea L. cv. Bloomsdale Long Standing) was determined using (i) a sampling procedure which measured the quantity of xylulose-5-phosphate formed in the reaction mixture and (ii) a coupled enzyme assay in which the rate of oxidation of NADH was measured after establishing steady-state concentrations of xylulose-5-phosphate, dihydroxacetonephosphate and glyceraldehyde-3-phosphate by the action of epimerase, transketolase (EC 2.2.1.1), triosephosphate isomerase (EC 5.3.1.1) and glycerol-3-phosphate dehydrogenase (EC 1.1.1.8). In preparations where the ratio of isomerase to epimerase activities was less than 100, both assay procedures yielded valid indications of epimerase activity. The steady-state assay system was found, however, to seriously underestimate epimerase activity in enzyme preparations which were enriched in isomerase. Cross plots of epimerase activity determined by the sampling and steady-state procedures demonstrated that an inhibitor of the coupling enzyme mixture was formed in the presence of high relative concentrations of the isomerase. The inhibited coupling enzyme mixture was fully active with glycer-aldehyde-3-phosphate. Inhibition of the coupling enzyme mixture was attributed to transketolase. Feedback inhibition of transketolase is proposed to be of physiological significance in the photosynthesis cycle, operating to restrict resynthesis of CO₂-acceptor under conditions where high steady-state concentrations of the intermediates of the photosynthesis cycle are maintained.     

Identification and Characterization of Bifunctional Proline Racemase/Hydroxyproline Epimerase from Archaea: Discrimination of Substrates and Molecular Evolution

Proline racemase (ProR) is a member of the pyridoxal 5’-phosphate-independent racemase family, and is involved in the Stickland reaction (fermentation) in certain clostridia as well as the mechanisms underlying the escape of parasites from host immunity in eukaryotic Trypanosoma. Hydroxyproline epimerase (HypE), which is in the same protein family as ProR, catalyzes the first step of the trans-4-hydroxy-L-proline metabolism of bacteria. Their substrate specificities were previously considered to be very strict, in spite of similarities in their structures and catalytic mechanisms, and no racemase/epimerase from the ProR superfamily has been found in archaea. We here characterized the ProR-like protein (OCC_00372) from the hyperthermophilic archaeon, Thermococcus litoralis (TlProR). This protein could reversibly catalyze not only the racemization of proline, but also the epimerization of 4-hydroxyproline and 3-hydroxyproline with similar kinetic constants. Among the four (putative) ligand binding sites, one amino acid substitution was detected between TlProR (tryptophan at the position of 241) and natural ProR (phenylalanine). The W241F mutant showed a significant preference for proline over hydroxyproline, suggesting that this (hydrophobic and bulky) tryptophan residue played an importance role in the recognition of hydroxyproline (more hydrophilic and bulky than proline), and substrate specificity for hydroxyproline was evolutionarily acquired separately between natural HypE and ProR.　A phylogenetic analysis indicated that such unique broad substrate specificity was derived from an ancestral enzyme of this superfamily.     

Comparative analysis of the structure and thermal stability of sea urchin peristome and rat tail tendon collagen

We have purified collagen from two distinct sources; the vertebrate, rat tail tendon and an invertebrate, sea urchin adult tissue, the peristome. The collagenous nature of the purification products was confirmed by amino acid compositional analysis. Both preparations had high contents of glycine and proline residues and hydroxyproline was also present. The total pyrrolidine (proline+hydroxyproline) content decreased from 17.9 mole% in rat tail collagen to 12.9 mole% in peristome collagen. Distinctly different circular dichroic spectra were measured for these collagens. Analyses of spectra, measured as a function of temperature, revealed distinct thermal denaturation profiles. The melting temperature for rat tail collagen was 38.5 degrees C, while the corresponding value for peristome collagen was significantly lower at 27 degrees C. A similar thermal denaturation profile was obtained for rat tail collagen in digestion experiments using a 41-kDa gelatinase activity, isolated from sea urchin eggs. These results identify structural differences between a typical, vertebrate type I fibrillar collagen and an echinoderm collagen which serves as a constituent of a mutable connective tissue. These differences may relate to the functional roles played by collagen in these distinctly different tissues.     

Peroxisomal beta-oxidation system of Candida tropicalis. Purification of a multifunctional protein possessing enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase and 3-hydroxyacyl-CoA epimerase activities

A multifunctional protein from oleate-grown cells of Candida tropicalis has been purified and partially characterized. A simple two-step purification has been developed involving ion-exchange chromatography followed by dye-ligand chromatography on blue Sepharose CL-6B. Homogeneous enzyme with a subunit Mr of 102 000 is obtained in 60% yield. The native relative molecular mass, determined by three different methods, yielded values which suggest that the enzyme is dimeric. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of the purified protein revealed a single polypeptide band and reverse-phase high-performance liquid chromatography indicated a single component suggesting that this protein may consist either of two identical or very similar subunits. Three beta-oxidation activities, enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase and 3-hydroxyacyl-CoA epimerase, co-purified with this protein. The ratio of the three beta-oxidation enzyme activities remained constant during purification and was unchanged by additional chromatographic methods (adsorption and affinity chromatography), thus indicating the multifunctional nature of this protein. Enzymatic staining of the purified protein for 3-hydroxyacyl-CoA dehydrogenase and epimerase, following electrophoresis in a polyacrylamide density gradient, further supported the multifunctionality of this protein. After isopycnic centrifugation of a particulate fraction from oleate-grown cells in a linear sucrose gradient the activities of all individual beta-oxidation enzymes cosedimented with catalase and with the glyoxylate bypass enzymes. This result demonstrated the peroxisomal localization of the multifunctional enzyme. The relationship of this multifunctional protein to the two bifunctional beta-oxidation enzymes isolated from peroxisomes of rat liver and from glyoxysomes of cucumber seeds is discussed.     

The pentose phosphate pathway in the endoplasmic reticulum

Approximately the same levels of six of the seven enzymes catalyzing reactions of the pentose phosphate pathway are in the cisternae of washed microsomes from rat heart, spleen, lung, and brain. Renal and hepatic microsomes also have detectable levels of these enzymes except ribulose-5-phosphate epimerase and ribose-5-phosphate isomerase. Their location in the cisternae is indicated by their latencies, i.e. requirement for disruption of the membrane for activity. In addition, transketolase, transaldolase, and glucose-6-phosphatase, a known cisternal enzyme, are inactivated by chymotrypsin and subtilisin only in disrupted hepatic microsomes under conditions in which NADPH-cytochrome c reductase, an enzyme on the external surface, is inactivated equally in intact and disrupted microsomes. The failure to detect the epimerase and isomerase in hepatic microsomes is due to inhibition of their assays by ketopentose-5-phosphatase. Xylulose 5-phosphate is hydrolyzed faster than ribulose 5-phosphate. A mild heat treatment destroys hepatic xylulose-5-phosphatase and glucose-6-phosphatase without affecting acid phosphatase. These results plus the established wide distribution of glucose dehydrogenase, the microsomal glucose-6-phosphate dehydrogenase, and its localization to the lumen of the endoplasmic reticulum suggest that most mammalian cells have two sets of enzymes of the pentose phosphate pathway: one is cytoplasmic and the other is in the endoplasmic reticulum. The activity of the microsomal pentose phosphate pathway is estimated to be about 1.5% that of the cytoplasmic pathway.     

Presence of bound substrate in lactate dehydrogenase from carp liver

While attempting to purify UDP-galactose 4-epimerase from carp liver extract at pH 8.0, it was observed that the preparation even after dialysis could reduce NAD to NADH, interfering epimerase assay. The NAD reduction activity and the epimerase were co-eluted in a series of chromatographic steps. Mass spectrometric analysis of semi-purified fraction revealed that carp liver lactate dehydrogenase (LDH) contained bound lactate which was converted to pyruvate in the presence of NAD. The enzyme-bound lactate and the association with epimerase stabilized LDH from trypsin digestion and thermal inactivation at 45 degrees C by factors of 2.7 and 4.2 respectively, as compared to substrate-free LDH. LDH and epimerase do not belong to any one pathway, but are the rate-limiting enzymes of two different pathways of carbohydrate metabolism. Typically, strongly associated enzymes work in combination, such as two enzymes of the same metabolic pathway. In that background, co-purification of LDH and epimerase as reloaded in this study was an unusual phenomenon.     

Evidence that the fadB gene of the fadAB operon of Escherichia coli encodes 3-hydroxyacyl-coenzyme A (CoA) epimerase, delta 3-cis-delta 2-trans-enoyl-CoA isomerase, and enoyl-CoA hydratase in addition to 3-hydroxyacyl-CoA dehydrogenase.

Genetic complementation of a mutant defective in fatty acid oxidation (fadAB) with plasmids containing DNA inserts from the fadAB region of the Escherichia coli genome was studied. The mutant containing the hybrid plasmid with a 5.2-kilobase (kb) PstI-SalI fragment was found to overproduce 3-hydroxyacyl-coenzyme A (CoA) epimerase and delta 3-cis-delta 2-trans-enoyl-CoA isomerase as well as three other beta-oxidation enzymes by 16- to 18-fold compared with the wild-type parental strain LE392. The purification of a fully functional multienzyme complex of fatty acid oxidation from the transformant ultimately established that the 5.2-kb DNA fragment contained an entire fadAB operon. Since immunotitration of cell extracts with antibodies against the fatty acid oxidation complex proved that all 3-hydroxyacyl-CoA epimerase and delta 3-cis-delta 2-trans-enoyl-CoA isomerase activities were associated with the complex, no genetic loci other than the fadAB operon encoded these two enzymes. Moreover, the binding of antibodies caused parallel inhibition of four component enzymes, whereas 3-ketoacyl-CoA thiolase activity was slightly increased. These findings support the suggestion that the epimerase and isomerase as well as enoyl-CoA hydratase and L-3-hydroxyacyl-CoA dehydrogenase are located on the same polypeptide. The results of this study, together with published data (S.-Y. Yang and H. Schulz, J. Biol. Chem. 258:9780-9785, 1983), lead to the conclusion that 3-hydroxyacyl-CoA epimerase, delta 3-cis-delta 2-trans-enoyl-CoA isomerase, and enoyl-CoA hydratase in addition to 3-hydroxyacyl-CoA dehydrogenase are encoded by the fadB gene.     

Biochemical parameters of the metabolism of the intercellular substance of connective tissue, correlating with morphometric signs of long bone lesions in an open aseptic fracture]

The aim of the research was experimental study of dynamics and correlative dependences between biochemical connective tissue matrix metabolism indices (blood serum collagenase, cathepsin B, elastase, antielastase activity, hydroxyproline fractions and glycosaminoglycans concentration) and tissue damage morphometric indices after long bone aseptic osteotomy of rat in terms 3 h-60 days. The most strong and significant correlation was found between cathepsin B, elastase activity indices and dimensions of bone marrow ischemic damage focuses and summarised periosteal regenerates volume in bone fragments.     

A new robust kinetic assay for DAP epimerase activity

DAP epimerase is the penultimate enzyme in the lysine biosynthesis pathway. The most versatile assay for DAP epimerase catalytic activity employs a coupled DAP epimerase–DAP dehydrogenase enzyme system with a commercial mixture of DAP isomers as substrate. DAP dehydrogenase converts meso-DAP to THDP with concomitant reduction of NADP⁺ to NADPH. We show that at high concentrations, accumulation of NADPH results in inhibition of DAPDH, resulting in spurious kinetic data. A new assay has been developed employing DAP decarboxylase that allows the reliable characterisation of DAP epimerase enzyme kinetics.     

Heroin-induced alterations in leukocyte numbers and apoptosis in the rat spleen

The present study assessed the effects of acute heroin treatment on the cellularity of the rat spleen and the rate of splenocyte death by necrosis or apoptosis. The results showed that 1 h after a single injection of heroin, the total number of leukocytes in the spleen was decreased in a dose-dependent manner. Prior injection of naltrexone completely blocked heroin's effect, and the heroin-induced decrease in splenic leukocytes was not associated with a heroin-induced increase in circulating leukocytes. A 1-h exposure to heroin did not increase levels of lactate dehydrogenase, a cytosolic enzyme, in supernatants of splenic mononuclear cells cultured for 45 min or 24 h, suggesting that heroin does not increase necrotic death in the spleen. In contrast, a 1-h heroin treatment did increase the percentage of Annexin V(+) cells in 0- and 24-h cultures of splenic mononuclear cells, indicating that heroin increases apoptotic death in the spleen. A 3-h exposure to heroin also produced a significant increase in apoptosis in the spleen. DNA fragmentation, a marker of cells in late stages of apoptosis, could not be detected in fresh splenocytes, but was evident in 24-h cultures of splenic mononuclear cells from saline- and heroin-treated rats. These results demonstrate that a single administration of heroin produces a decrease in the number of splenic leukocytes and an increase in the apoptotic death of splenic mononuclear cells.     

Pro-enkephalin opioid peptides are abundant in porcine and bovine splenic nerves,but absent from nerves of rat,mouse, hamster,and guinea-pig spleen

The opioidergic innervation of the mammalian spleen and possible species differences were investigated. Light-microscopic immunohistochemistry revealed that splenic nerves of bovine and porcine spleen, but not of rat, mouse, hamster and guinea-pig spleen contained proenkephalin-derived opioidergic innervation. Immunoreactivity to both prodynorphin and pro-opiomelanocortin was absent from splenic nerves. In bovine and porcine spleen, fibers immunoreactive for met-enkephalin, met-enkephalin-Arg-Phe, met-enkephalin-Arg-Gly-Leu, leu-enkephalin and peptide F formed perivascular plexus, traveled in trabecular connective tissue, and extended into the capsule. Spatial relationships with immune cells were apparent in the white and red pulp, excluding lymphoid follicles. Colocalization of enkephalin immunoreactivity with immunoreactivities for tyrosin hydroxylase, dopamin--hydroxylase, and neuropeptide Y, but not for substance P or calcitonin gene-related peptide were found. Our results provide evidence that opioid expression in splenic innervation is strongly species-dependent and exclusively proenkephalin-derived. Colocalization with marker enzymes of noradrenergic neurons indicates a mainly postganglionic sympathetic origin of proenkephalinergic splenic innervation. Opioidergic perivascular nerves probably control the splenic blood flow. A close interrelationship of opioidergic fibers with immune cells provides the anatomical basis for direct effects of neurally released opioids on splenic immune functions.     

An atypical population of NK cells that spontaneously secrete IFN-gamma and IL-4 is present in the intraepithelial lymphoid compartment of the rat

The intestinal lymphoid compartment of the rat is large and diverse, but the phenotype and functions of its constituent cell populations are not fully characterized. Using new methodology for the isolation and purification of rat intestinal intraepithelial lymphocytes (IELs), we previously identified a population of alphabeta- and gammadelta-TCR- NKR-P1A+ NK cells. These cells were almost completely restricted to the CD4-CD8- IEL population, and unlike peripheral NK cells in the rat, they were CD2-. We now report that rat intraepithelial NK (IENK) and peripheral NK cells are similar in morphology, in their ability to lyse NK-sensitive targets, and in their ability to suppress a one-way mixed lymphocyte culture. In contrast, however, intraepithelial and splenic NK cells differ markedly in two respects. First, IENK cells express high levels of ADP-ribosyltransferase 2 (a marker of regulatory T cells in the rat) and CD25, whereas peripheral NK cells do not. Second, unlike splenic NK cells, a substantial fraction of IENK cells appear to spontaneously secrete IL-4 and/or IFN-gamma. We conclude that the rat IEL compartment harbors a large population of NKR-P1A+CD3- cells that function as NK cells but display an activated phenotype and unusual cytokine profile that clearly distinguish them from splenic NK cells. Their phenotypic and functional characteristics suggest that these distinctive IENK cells may participate in the regulation of mucosal immunity.