Tissue Wet Wt Research Articles

Concurrent quantification of tissue metabolism and blood flow via 2H/31P NMR in vivo. III. Alterations of muscle blood flow and metabolism during sepsis.

In the conclusion of this series of reports, the application of 31P/2H NMR to investigate the pathophysiology of sepsis in rat hindlimb muscle is demonstrated. Sepsis decreased muscle [PCr] by 18%, 18 +/- 4 SD vs 22 +/- 4 SD mmol/kg tissue wet wt (P = 0.01) in control rats but [ATP] was unchanged, 6 mmol/kg tissue wet wt (P = 0.2). The derived free cytosolic [ADP] in the two groups was similar, [ADP]septic = 0.023 +/- 0.004 SD and [ADP]control = 0.021 +/- 0.003 SD mmol/kg tissue wet wt, and not statistically different (P = 0.14). Likewise [Pi] in the septic and control groups was not statistically different, [Pi]septic = 1.1 +/- 0.5 SD and [Pi]control = 1.2 +/- 0.4 SD mmol/kg tissue wet wt (P = 0.2). Septic rats presented the symptom of respiratory alkalosis evidenced by elevated blood pH. Sepsis decreased muscle blood flow by 33%, P = 0.003, but examination of individual subjects did not demonstrate a correlation with the reduction in [PCr]. Thus, a metabolic energy deficit caused by cellular ischemia/hypoxia is not a likely cause of cellular abnormality in rat hindlimb muscle during sepsis.

Concurrent quantification of tissue metabolism and blood flow via 2H/31P NMR in vivo. I. Assessment of absolute metabolite quantification.

In a series of three papers, we demonstrate and validate an approach for concurrent absolute quantification in situ of blood flow and energy metabolism with a modification of the NMR method for absolute concentration determination put forth by Thulborn and Ackerman [J. Magn. Reson. 55, 357 (1983)] and later expanded upon by Tofts and Wray. In this first paper of the series, we briefly review the theoretical basis for the concentration measurement and present, for the first time, a successful paired validation of metabolite quantification via 31P surface-coil NMR through corroborative in vitro enzymatic assays. The paired radiolabeled microsphere validation of blood flow measurement via 2H surface-coil NMR employing D2O as a freely diffusible tracer and the concurrent determination of blood flow and energy metabolism in a septic rat model are presented in the accompanying second and third paper to complete the series. In this article a classical RF tank circuit is employed to describe the effect of conductive sample loading on the NMR receiver by considering its apparent series resistance. It is shown in an easily visualized generalizable manner that the effect of sample loading on the observed NMR signal intensity can be accounted for quantitatively by monitoring changes in 90 degrees pulse width at constant power at a fixed reference point, i.e., Ssample = Sphantom (PW90phantom/PW90sample). In a series of paired experiments the absolute concentrations of high energy phosphates obtained from resting rat leg muscle (n = 4) in situ (NMR) and in vitro (enzymatic) were determined as follows: [PCr]NMR = 17.2 +/- 0.8 SD, [PCr]enzymatic = 17.3 +/- 2 SD, [ATP]NMR = 5.1 +/- 0.8 SD, [ATP]enzymatic = 5.0 +/- 0.2 SD mmol/kg tissue wet wt. Results of these two independent methods of concentration determination were not statistically different (P = 0.94 and P = 0.74 respectively) and serve to rigorously validate the Thulborn approach for absolute quantification of phosphorous metabolites in situ via NMR. Furthermore, these results strongly suggest that ATP and PCr in resting rat leg muscle under normal physiologic conditions are 100% NMR visible. The free cytosolic [ADP]NMR was estimated from the creatine kinase reaction equilibrium expression to be 0.022 +/- 0.003 SD mmol/kg tissue wet wt.

Metabolic and cardiodynamic responses of isolated turtle hearts to ischemia and reperfusion.

We used 31P and 1H nuclear magnetic resonance spectroscopy to measure intracellular pH, high energy phosphates, and lactate levels in hearts of turtles (Chrysemys picta bellii) subjected to 1.5 h of global ischemia followed by reperfusion. We simultaneously monitored maximum ventricular developed pressure (Pmax), maximal rate of pressure development (dP/dtmax), rate-pressure product (RPP), cardiac output, and heart rate and also measured lactate efflux from the hearts during reperfusion. Our goal was to test the hypothesis that turtle hearts would prove tolerant of prolonged global ischemia at 20 degrees C and would recover completely on reperfusion without any indication of ischemia-or reperfusion-related injury. The 1.5 h of ischemia resulted in decreases in phosphocreatine and ATP to 31.4 +/- 2.8 and 87.3 +/- 6.3% of control, respectively, while Pi rose to 236.6 +/- 26.3%. Intracellular pH decreased during this period from 7.38 +/- 0.02 to 6.87 +/- 0.04. Most of these changes occurred during the first 30 min. Tissue lactate rose during 1.5 h of ischemia from approximately 1.5 to 22.3 mumol/g wet tissue wt. However, the rate of lactate production was much higher during the first 21 min of ischemia (0.41 mumol.g-1.min-1) than during the remaining 70 min (0.10 mumol.g-1.min-1). With the onset of ischemia, Pmax, dP/dtmax, RPP, and heart rate all decreased dramatically with roughly the same time course as the changes in high-energy phosphates and intracellular pH. On reperfusion, turtle hearts rapidly restored high-energy phosphates, intracellular pH, lactate, and cardiodynamics to control levels, usually within 15-30 min, with no evidence of reperfusion injury.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of the oxytocin gene in rat placenta.

The placenta is an endocrinologically active organ and expresses an important number of polypeptide hormone genes. Although oxytocin (OT)-like immunoreactivity has been detected in placental extracts by RIA, the precise nature and origin of placental OT has remained unclear. In the present study, we examined OT gene expression in rat placental tissue at various stages of gestation using northern blot analysis, polymerase chain reaction, in situ hybridization, HPLC, and immunocytochemistry. Northern blot analysis of RNA extracted from rat placenta revealed a single type of OT gene transcript (0.66 kilobases) which differed in size from hypothalamic OT transcripts (0.75 kilobases). Deadenylation of placental and hypothalamic messenger RNA (mRNA) showed that this size difference was due to differences in poly(A) tail lengths. Polymerase chain reaction amplification of placental and hypothalamic complementary DNAs using four different exon-specific primers provided no evidence for the existence of any additional structural differences between hypothalamic and placental OT-gene transcripts. Quantitative evaluation of northern blots showed that OT mRNA abundance per microgram of total RNA was stage specific and declined by a factor of 6 from day 14 to day 21 of gestation. In contrast to the marked variation of mRNA abundance, the OT peptide content, as measured by RIA, underwent no significant change during the time period studied and varied between 0.37-0.51 ng/g wet tissue wt. Characterization of placental OT immunoreactivity by HPLC and gel filtration identified two peaks of immunoreactivity: one peak (70% of immunoreactivity) corresponded to synthetic OT; whereas the other peak (Mr 11,000, 30% of immunoreactivity) represented a noncovalent association between OT and another molecule, consistent with the formation of a neurophysin/OT complex. By in situ hybridization and immunocytochemistry, we localized OT mRNA and OT immunoreactivity to cells of the trophoblastic epithelium covering the septa of the labyrinth as well as to cytotrophoblastic elements and giant cells of the maternally derived basal zone of the placenta. Placental OT may act locally, may interact with uterine OT receptors, or may play a role in fetal development.

Cerebellar glutamine synthetase in children after hypoxia or ischemia.

Glutamate has been implicated in the pathophysiology of acute hypoxic-ischemic encephalopathy. Glutamine synthetase is an enzyme found in astrocytes that converts glutamate to its nontoxic analogue, glutamine. The present study tests the hypothesis that brain glutamine synthetase activity increases in response to acute hypoxic-ischemic insults and not in response to chronic hypoxia-ischemia or non-hypoxic-ischemic neurological disease. Frozen sections of cerebellum from children who died with acute or chronic hypoxic-ischemic insults or chronic non-hypoxic-ischemic neurological disease were spectrophotometrically assayed for glutamine synthetase activity by an observer who was blinded to the clinical group assignment of each specimen. Enzyme activity was elevated in specimens from children with acute hypoxic-ischemic insults (mean 6.5; range 5.4-7.2 units/g wet tissue wt) as compared with those from patients with chronic hypoxia-ischemia (mean 2.8; range 0.7-10.2 units/g wet tissue wt) or with non-hypoxic-ischemic neurological disease (mean 2.6; range 1.3-3.9 units/g wet tissue wt). This difference was not due to differences in the degree of histological astrocytosis or edema among the specimens. Statistical analysis by the Kruskal-Wallis one-way analysis of variance by ranks test indicates that the three data groups do not come from one population (p less than 0.05). These results support the notion that glutamine synthetase activity increases in response to acute hypoxic-ischemic nervous system injury in children and that other compensatory mechanisms prevail in the case of chronic hypoxic-ischemic insults.

A comparison of water loss and gill areas in two supralittoral amphipods from New Zealand

Total gill area and gill distribution were measured for the sandhopper Talorchestia quoyana (Milne-Edwards) and the beach flea Transorchestia chiliensis (Milne-Edwards). For both species the gill structure and proportional area contributed by individual gills was similar. Gill 6 (G6) was the largest, providing 36% of the gill area in Tal. quoyana and 30% in Tr. chiliensis. The gill area/total dry weight relationships were similar, Y = 1.3 X0.79 for Tal. quoyana and 1.4 X0.78 for Tr. chiliensis. Small, medium and large amphipods survived >24 h in aerial conditions close to 100% RH at 15 °C. Rates of water loss in desiccating conditions increased with decreasing RH. Lethal water loss exceeded 30% weight loss for both species. Rate of water loss, (R) mg water loss. mg wet wt tissue. h−1 exposed to 75% RH for Tr. chiliensis was 0.21, resulting in total mortality within 2 h. Medium Tal. quoyana were the most resistant group surviving 4 h exposure to 75% RH with R = 0.08. Differences in desiccation tolerances of the two amphipods are not explained by body water content, gill area relationships or the larger maximal size of T. quoyana. Results were combined with those from other talitrids to examine the relationship between gill area, water content, desiccation habitat and oxygen consumption in aerial and aquatic conditions. There were no consistent relationship between gill area, O2 uptake and desiccation resistance. Amphipods show compensatory respiratory adaptation with individuals from all habitats, showing similar rates of oxygen uptake, either in air or in water, whichever was their most usual respiratory medium. Q10 values close to 2.0 were found in all ecomorphological groups. Sandhoppers, including Tal. quoyana, are best able to survive terrestrial conditions associated with a low humidity environment. It is concluded that the water loss characteristics of Tr. chiliensis limit its distribution on sand beaches to areas of high relative humidities.

Enzymatic determination of total CO 2 in freeze-clamped animal tissues and plasma

An enzymatic method for measuring total carbon dioxide content in freeze-clamped animal tissues is described. Total carbon dioxide content [TCO 2] was defined as the sum of the dissolved CO 2, the bicarbonate concentration, and the carbonate concentration. Tissue was extracted in 80% methanol, 20 m m 2-amino-2-methyl-1-propanol, pH 9.5 at 25°C and homogenized in a 1.5-ml Sardstat screw-top test tube containing 0.5-mm glass beads and a minibead beater. Total CO 2 was determined as bicarbonate/carbonate by monitoring the oxidation of NADH at 340 nm using the coupled assay of phosphoenolpyruvate carboxylase (EC 4.1.1.31) and malate dehydrogenase (EC 1.1.1.37). In the coupled assay system, 1 μmol of bicarbonate/carbonate consumed is equivalent to the oxidation of 1 μmol NADH at 340 nm. The assay medium comprised 50 m m 2-amino-2-methyl-1-propanol, pH 9.0 at 25°C, 5 m m phosphoenolpyruvate (PEP), 0.25 m m NADH, 5 m m MgCl 2, 5 m m mercaptoethanol, 0.02% bovine serum albumin, 10 m m oxamate, PEP carboxylase (0.5 units/ml), and malate dehydrogenase (0.5 units/ml). The total CO 2 content measured in freeze-clamped rat heart, liver, brain, and skeletal muscle was 20.53 ± 0.64, 17.34 ± 0.67, 17.00 ± 0.48, 16.06 ± 0.53 μmol/g wet wt tissue, respectively ( n = 5). The total CO 2 in the crusher muscle of the lobster was found to be 5.0 ± 0.33 μmol/g wet wt. Total CO 2 was also enzymatically measured in arterial plasma from four chronically cannulated male wistar rats and was 24.65 ± 1.81 μmol/ml plasma. It was concluded that the enzymatic method of total CO 2 determination can be used to measure bicarbonate in amounts of tissue ranging in weight from 50 to 100 mg or 50 to 100 μl of plasma. By substituting the bicarbonate concentration into the Henderson-Hasselbalch equation, and knowing the venous plasma pCO 2, the method can be used to calculate the intracellular pH in a variety of tissues.

Fatty acid content and composition of five fish species from the Persian Gulf

Abstract 1. 1. The fatty acid compositions of muscle total, polar and neutral lipids in five commonly eaten fish species in Kuwait were determined. 2. 2. The total amount of fatty acids varied from 0.3 to 1.9% of tissue wet wt. 3. 3. Fatty acid compositions and their total amounts were very similar in polar lipids of all species studied. The amount of neutral lipids varied more. 4. 4. n-3 Fatty acids, mainly docosahexaenoic and eicosapentaenoic acids, were the dominant unsaturated fatty acids (15.4–43.4%), but n- 6 fatty acids, mainly arachidonic, linoleic and docosapentaenoic acids, were also well represented (7.3–21.8%).

Normal and Adenomatous Human Pituitaries Secrete Thyrotropin-Releasing HormoneIn vitro: Modulation by Dopamine, Haloperidol, and Somatostatin

TRH is present in human normal pituitaries and in pituitary adenomas. In this study we demonstrated that the same tissues can release TRH in vitro. Fragments from seven normal pituitaries (10-15 mg/syringe) and dispersed cells from eight prolactinomas, four GH-secreting and two nonsecreting adenomas (1-3 x 10(6) cells/syringe) were perifused using a Krebs-Ringer culture medium. After 1 h of equilibration the perifusion medium was collected every 2 min (1 mL/fraction) for 3 h. TRH, PRL, and GH were measured by RIA under basal conditions and in the presence of 10(-10) to 10(-6) mol/L dopamine (DA), alone or concomitant with haloperidol, or in the presence of 10(-10) or 10(-6) mol/L somatostatin. Both normal pituitary fragments and pituitary adenomatous cells (from all types of adenomas studied) spontaneously released TRH in vitro. TRH was detected in the perifusion medium either immediately after the end of the equilibration period or 30-60 min later. The molecular identity of TRH was assessed by high pressure liquid chromatography. There was no difference in the profile and the rate of TRH secretion between normal and tumoral tissues, and no correlation was found between the level of TRH release and that of PRL or GH secretion. DA stimulated TRH release from normal pituitaries and from PRL- and GH-secreting adenomas at doses as low as 10(-10) mol/L. A concomitant decrease in PRL and GH release was observed from adenomatous cells and in one case of normal tissue. Haloperidol (10(-7) mol/L) antagonized the effect of 10(-8) mol/L DA on both TRH and PRL secretion in normal pituitary and in prolactinomas. DA had no effect on TRH release from two nonsecreting tumors. The amounts of TRH released during 1 h of perifusion were 60-1640 pg/2 mg wet wt tissue in normal pituitaries and 54-2174 pg/10(6) cells in adenomas; these values were very high compared to those precedently reported within the tissues. These results indicate that pituitary cells can release TRH in vitro and suggest that TRH might be synthesized in situ. We suggest that TRH could act on pituitary hormone secretion and/or cell proliferation via a paracrine and/or an autocrine mechanism.

Characterization of the Substance P Receptor in Guinea Pig Lung Tissues

The biologic activity of substance P has been demonstrated to be limited both in in vivo and in vitro by a membrane-bound protease, neutral endopeptidase (EC 3.4.24.11). The interaction of substance P with its receptor on guinea pig lung tissues was studied in the presence of an inhibitor of neutral endopeptidase under conditions that protect the peptide from degradation. Uptake of 0.1 nM [125I]-BH-substance P in lung membrane preparations was rapid at 4 degrees C, reaching equilibrium in 30 to 40 min, and binding was stable for at least 30 min thereafter. Binding was reversible and saturable. Scatchard analyses of saturation binding data are consistent with a single class of receptor molecules in both lung parenchymal and airway membranes, with a Kd of 2 to 3 nM and a receptor density of 4,000 to 5,000 fmol/g wet wt of tissue. In competitive binding experiments, neurokinin A and substance P methyl ester were equipotent and required approximately 100-fold higher concentrations to effect equivalent displacement than unlabeled substance P. Eledoisin also competed for [125I]-BH-substance P binding, but was less effective than the other analogs. The spasmogenically inactive derivative, substance P 1-9, did not compete for substance P binding at concentrations as high as 1 microM. Binding of [125I]-BH-substance P was rapidly and completely reversed by addition of 0.1 mM GTP, suggesting that association with a GTP binding protein is required for high affinity binding of substance P to its receptor in lung. The substance P receptor molecule was further characterized by covalently crosslinking [125I]-BH-substance P to membrane preparations followed by SDS-PAGE of the solubilized material.(ABSTRACT TRUNCATED AT 250 WORDS)

Post-translational processing of oxytocin-neurophysin prohormone in the ovine corpus luteum: activity of peptidyl glycine α-amidating mono-oxygenase and concentrations of its cofactor, ascorbic acid

Peptidyl glycine alpha-amidating mono-oxygenase (PGA), the terminal enzyme in the pathway of oxytocin synthesis, was measured in extracts of ovine corpora lutea throughout the oestrous cycle. Activity of PGA was low early in the cycle but increased between days 2 and 10 (from 2.3 to 9.0 pmol/mg protein per h) and remained high until day 15. Thereafter, activity declined rapidly at structural luteolysis and was low in corpora albicantia collected 18 and 20 days after ovulation (1.28 and 1.07 pmol/mg protein per h respectively). Luteal concentrations of ascorbic acid, a cofactor for PGA, were high (4.7 mumol/g wet wt tissue) by day 4 after oestrus; concentrations fell rapidly after day 15 (to 2.1 mumol/g on day 16). Concentrations of ascorbic acid were also high in the pituitary gland and in the adrenal medulla and cortex. Concentrations of oxytocin in luteal tissue, which were low (0.3 nmol/g wet wt) on day 2 after oestrus, were highest (2.73 nmol/g) on day 6 and declined thereafter (0.56 nmol/g on day 10, 0.08 nmol/g on day 15 and not detectable on days 18 and 20). Concentrations of oxytocin, progesterone, PGA and protein were measured in subcellular fractions obtained after density gradient centrifugation of extracts of corpora lutea collected on days 6, 7 and 12 of the oestrous cycle, and on day 7 from an anaesthetized ewe before and after treatment with the prostaglandin F2 alpha analogue, cloprostenol. PGA colocalized with particle-associated oxytocin in fractions of density 1.049-1.054 g/ml. Exogenous [3H]oxytocin and [3H]progesterone and endogenous progesterone localized in fractions of density 1.035 g/ml. Oxytocin and PGA were depleted from fractions of density 1.049-1.054 g/ml following cloprostenol treatment in vivo. Fractionation of extracts of ovine corpora lutea by high-performance liquid chromatography (HPLC) followed by radioimmunoassay and radioreceptor assay for oxytocin demonstrated the presence of at least two cross-reacting substances with elution characteristics distinct from oxytocin. Concentrations of these peptides increased as the cycle progressed. These compounds differed from the putative C-terminally extended post-translational processing intermediates, oxytocinyl-glycine, oxytocinyl-glycine-lysine and oxytocinyl-glycine-lysine-arginine, as indicated by their elution positions on HPLC and the specificities of the assays used to detect them, and no conclusions could be drawn on which post-translational processing step was rate-limiting in oxytocin synthesis. These data are consistent with the suggestion that post-translational processing of oxytocin-neurophysin prohormone takes place in secretory granules in luteal cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Epidermal Growth Factor Precursor in Mouse Lactating Mammary Gland Alveolar Cells

Previous studies have demonstrated that high levels of epidermal growth factor (EGF) occur in human and rodent milk and that oral administration of this polypeptide stimulates rodent gastrointestinal development. It is not known whether EGF in milk originates from cells of the lactating mammary gland or is sequestered from an extramammary source. In the present study, prepro-EGF mRNA (approximately 4.7 kilobases) was detected in the CD-1 mouse mammary gland throughout the period of lactation; by comparison, negligible levels of this EGF transcript were found in the gland during pregnancy. Low levels of EGF immunoreactivity (4-5 ng/g wet wt tissue) were extracted from lactating (day 18) mammary glands with dilute acetic acid. Immunolocalization was evident with antisera to either EGF or two other regions of the EGF precursor in essentially all alveolar cells of the lactating gland. The most prominent staining with antiserum to EGF was observed along the luminal borders of cells; this pattern of cellular staining required proteolytic pretreatment of tissue sections. Western blot analyses of cell membranes isolated from the day 16 lactating mammary gland revealed an EGF-immunoreactive band at about 145K, which was equivalent in size to the EGF precursor found in mouse kidney cell membranes. Despite these findings, labeling of lactating mammary gland mince with L-[35S]methionine and cysteine for up to 4 h did not reveal any specific bands in immunoprecipitates. These cumulative findings suggest that the precursor form of EGF occurs in alveolar cells of lactating mammary gland and that this protein is translocated to the cell membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Production of GAWK (chromogranin-B 420-493)-like immunoreactivity by endocrine tumors and its possible diagnostic value.

GAWK (chromogranin-B 420-493) is a 74 amino acid peptide recently isolated from human pituitaries. Using two different antibodies (directed against GAWK [1-17] and [20-38] fragments) GAWK-LI was measured in tumors from 194 patients and in the plasma of 434 patients by RIA. The highest tissue concentrations of GAWK-LI were found in pheochromocytoma (GAWK [1-17]-LI, 18,173 +/- 3,915; GAWK [20-38]-LI, 17,852 +/- 2,763 [mean +/- SEM] pmol/g wet wt tissue; n = 9), which were at least ten times higher than any other tumors producing GAWK-LI. High concentrations of GAWK-LI were also found in other types of endocrine tumors including carcinoid, medullary carcinoma of thyroid, pancreatic, and ACTH-producing lung tumors. On the other hand, low concentrations of GAWK-LI were found in nonendocrine tumors. Plasma concentrations of GAWK-LI were found to be elevated in patients with endocrine tumor, but more so in those with pancreatic tumors than with pheochromocytomas. Plasma concentrations returned to normal after successful tumor removal. Chromatographic profiles of GAWK-LI in extracts of pheochromocytomas and normal adrenals showed high molecular weight peaks that were absent in the extracts of other endocrine tumors and normal pancreas, suggesting differential tissue-specific processing. Thus GAWK-LI is produced by a variety of endocrine tumors and may serve as a plasma tumor marker, especially in patients with pancreatic endocrine tumors.

Regional distribution of chromogranin B 420–493-like immunoreactivity in the pituitary gland and central nervous system of man, guinea-pig and rat

The distribution of chromogranin B 420–493-like immunoreactivity was investigated in the pituitary gland and central nervous system of man, guinea-pig and rat, by using two different antibodies. In man, highest chromogranin B 420–493-like immunoreactivity concentrations were found in the pituitary gland, chromogranin B 420–493 (1–17)-like immunoreactivity74.3 ± 8.5 (mean ± S.E.M.pmol/g wet wt tissue) and chromogranin B 420–493 (20–38)-like immunoreactivity2017.0 ± 142.3, followed by the hypothalamus and substantia nigra. In the spinal cord, the highest concentration was found in the sacral dorsal area. Chromogranin B 420–493-like immunoreactivity was detected in human cerebrospinal fluid, chromogranin B 420–493 (1–17)-like immunoreactivity376.7 ± 77.9 and chromogranin B 420–493 (20–38)-like immunoreactivity1174.7 ± 259.3pmol/l cerebrospinal fluid. Chromogranin B 420–493-like immunoreactivity was also found in high concentrations in the guinea-pig and rat pituitary glands, but very low chromogranin B 420–493-like immunoreactivity levels were present in different parts of the brain of both species. Gel permeation chromatography and fast protein liquid chromatography analysis of eight different regions of human CNS showed multiple chromogranin B 420–493-like immunoreactivity peaks. The profile of pituitary extracts was different from those of other parts of the CNS and the cerebrospinal fluid. The profiles of guinea-pig and rat pituitary on gel chromatography and fast protein liquid chromatography were different again from those of human CNS. Examination of subcellular fractionation of whole rat brain showed highest concentrations of chromogranin B 420–493-like immunoreactivity in the synaptosome fractions. Chromogranin B 420–493-like immunoreactivity release was stimulated from rat pituitary cells by high potassium ion concentrations. These findings show that (1) chromogranin B 420–493-like immunoreactivity is widely distributed throughout the pituitary gland and CNS of three mammalian species, with the highest concentration in the pituitary gland, and it is also present in human cerebrospinal fluid, and (2) the processing of chromgranin B in the pituitary gland may be different from that in the other area of the CNS, so that it is possible chromogranin B or its fragments may play a neuroeffector role in the mammalian CNS.

Extraction and measurement of myocardial nucleotides, nucleosides, and purine bases by high-performance liquid chromatography

Nucleotides, nucleosides, and pruine bases were extracted from human endomyocardial biopsies, freeze-clamped rat hearts, and porcine coronary sinus plasma. Perchloric acid extracts were neutralized with Freon-trioctylamine and analyzed at 250 nm by reverse-phase ion-pairing high-performance liquid chromatography. To achieve the sensitivity necessary for analyzing small (1–3 mg wet wt) tissue samples, a small-bore, 2.1-mm-internal-diameter, C18, 5-μm reverse-phase column and a flow rate of 0.2 ml/min were used. All of the myocardial nucleotides and AMP degradation products were resolved in a total separation time of 27 min with 30 m m KH 2PO 4, 7.5 m m tetrabutylammonium phosphate buffers, and binary pH and acetonitrile gradients.

Organ distribution of aldehyde dehydrogenase activity in the rainbow trout ( Salmo gairdneri Richardson)

1. 1. Aldehyde dehydrogenase activity was measured in gills, muscle, brain, intestine, kidney, heart and liver of rainbow trout, using 3,4-dihydroxyphenylacetaldehyde (the biogenic aldehyde derived from dopamine) as the substrate. 2. 2. Aldehyde dehydrogenase activity was found to be present in all of the organs studied. 3. 3. The highest activity was found in the liver (276 nmol/min · g wet wt of tissue). 4. 4. A remarkably high activity was found in the heart (117 nmol/min · g). 5. 5. The gills showed the lowest activity (1.9 nmol/min · g).

Determination of β-phenylethylamine in catfish ( Parasilurus asotus) tissues by gas chromatography/mass spectrometry

1. 1. β-Phenylethylamine (PEA) was detected and quantitated in tissues of the catfish, Parasilurus asotus, by very specific and sensitive gas chromatography/mass spectrometry. 2. 2. The selected ion monitoring was made with a strong quasi-molecular ion of the pentafluoropropionic derivative of PEA in the positive chemical ionization mode. 3. 3. PEA was found in all tissues tested ranging from 2.8 to 38.2 ng/g wet wt tissue. It was highest in the spinal cord, followed by the skin, brain and intestine.

Evidence for increased aortic plasma membrane calcium transport caused by experimental atherosclerosis in rabbits.

Several lines of evidence, including the reported ability of calcium channel blockers to prevent atherogenesis in cholesterol-fed rabbits, suggest that calcium mediates one or more of the pathologic changes in atherosclerosis. Moreover, it has long been known that calcium accumulates in atherosclerotic blood vessels. To test the hypothesis that a substantial fraction of this accumulated calcium is intracellular and to identify possible causes of this accumulation, calcium fluxes and contents were determined in aortic segments from cholesterol-fed rabbits and age-matched controls. A new method, based on 45Ca efflux experiments and computer-assisted kinetic analysis, was used to measure intracellular and extracellular calcium contents (nmol calcium/g wet wt tissue) and fluxes. Total intracellular calcium increased from 269 +/- 11.6 to 1,300 +/- 352 nmol/g in cholesterol-fed animals compared with controls (p less than 0.01). This change was sufficient to account for the observed increase in total tissue calcium from 4,190 +/- 211 to 5,240 +/- 477 nmol/g (p less than 0.05). Thus, the fraction of tissue calcium that is intracellular increased significantly from 0.065 +/- 0.006 to 0.223 +/- 0.048 (p less than 0.01) in experimental atherosclerosis. In addition, the data were quantitatively consistent with the hypothesis that these changes are brought about by a 4.8-fold increase in the plasma membrane calcium permeability of aortic smooth muscle cells. These results provide evidence that increased intracellular calcium is a possible mediator of cholesterol-induced atherogenesis.

The level of natural antioxidant glutathione and histidine-containing dipeptides in skeletal muscles of developing chick embryos

1. 1. The levels of glutathione and histidine-containing dipeptides in skeletal muscles change in different ways during ontogenesis. 2. 2. The glutathione content in skeletal muscles increases between the 9th and 18th days of embryongenesis—from 0.5 to 2.0 μmol/g of tissue wet wt and then drops to zero in 3-week-old chickens. 3. 3. The level of histidine-containing dipeptides increases throughout the observation period beginning with their appearance on the 14th day in leg muscles and on the 15th day in breast muscles of chicken embryos up to the 21st postnatal day. 4. 4. There is a negative correlation between the antioxidative systems of glutathione and histidine-containing dipeptides in muscle tissue, i.e. dipeptide-rich tissues contain little or no glutathione and vice versa.

Quantification of naloxone binding sites in brains from suckled beef cows during postpartum anestrus and resumption of estrous cycles.

Thirty beef cows, approximately 3 yr of age, were randomly assigned to be slaughtered on d 7, 14, 28, 42 or 56 postpartum. Each cow suckled one calf until slaughter. Data from cows slaughtered on d 42 and 56 were pooled and further classified as anestrous or cyclic based on the presence of a corpus luteum and elevated serum concentrations of progesterone at slaughter. Specific binding of [3H]naloxone (3H-NAL) to homogenates of tissue from hypothalamus (HYP), preoptic area (POA) and basal forebrain (BF) was assessed using multiple-point Scatchard analyses. Nonspecific binding was estimated in the presence of 10(-6) M naloxone. Separation of bound from free 3H-NAL was achieved by centrifugation at 20,000 X g. Concentration (fmol/mg original tissue wet wt) of 3H-NAL binding sites in POA tissue was higher (P less than .05) on d 28 postpartum in anestrous cows than in cyclic cows on d 42 + 56 postpartum (2.58 +/- .32 vs 1.58 +/- .10). When all anestrous cows were compared with cyclic cows, concentrations of 3H-NAL binding sites in POA tissues and in BF tissue were higher (P less than .05) in anestrous cows (anestrous POA, 2.12 +/- .17, cyclic POA, 1.58 +/- .10; anestrous BF, 2.94 +/- .41, cyclic BF, 2.19 +/- .16). Compared across brain regions for all cows, the concentration of specific binding sites for 3H-NAL was greater (P less than .01) in BF (2.5 +/- .2) than in POA (1.9 +/- .1) and greater (P less than .01) in POA than in HYP (1.5 +/- .1).(ABSTRACT TRUNCATED AT 250 WORDS)