Calcitonin gene-related peptide-like immunoreactivities in pheochromocytomas

Calcitonin gene-related peptide (CGRP) is reported to exist in high concentrations in plasma and tumor tissues of medullary thyroid carcinomas. CGRP-like immunoreactivity (LI) in tumor tissues of pheochromocytomas was investigated by radioimmunoassay. CGRP-LI in 9 pheochromocytomas ranged from 0.50 to 1240 ng/g wet tissue. Sephadex G-50 column chromatography revealed that most of CGRP-LI in tumor extracts was eluted in an identical position to synthetic human CGRP. Reverse-phase high pressure liquid chromatography revealed that CGRP-LI in tumor extracts was eluted in an identical position to synthetic human CGRP and in a more hydrophobic position. These results indicate that high concentrations of CGRP-LI also exist in tumor tissues of pheochromocytomas.     

Detection of neuropeptide Y-like immunoreactivity and messenger RNA in rat platelets: the effects of vinblastine, reserpine, and dexamethasone on NPY expression in blood cells.

Rat plasma contains high basal levels (220 pmol/liter) of neuropeptide Y (NPY)-like immunoreactivity (LI) compared to pig (30 pmol/liter) and man (25 pmol/liter). The platelet-enriched fraction (PEF), obtained from rat blood contained 10,061 pmol/g NPY-LI. However, in human and pig blood, the PEF contained very low levels of NPY-LI. Gradient centrifugation of rat blood showed the highest concentration of NPY-LI (10.8 +/- 0.4 pmol/g) in the platelet fraction. The mononuclear cell fraction contained 1.64 +/- 0.16 pmol/g, whereas only 0.56 +/- 0.06 pmol/g of NPY-LI was found in the red blood cell/polymorphonuclear cell fraction. Characterization of NPY-LI in rat plasma and platelets by high-pressure liquid chromatography showed one predominating peak which coeluted with synthetic NPY (1-36) as well as three minor peaks, one of which coeluted with oxidized NPY. Analysis of NPY messenger RNA (mRNA) in bone marrow of the rat revealed a 0.79-kb-long NPY mRNA. This size is intermediate to the 0.82-kb NPY mRNA in brain and the 0.76-kb NPY mRNA in spleen. The highest level of NPY mRNA in rat blood was found in the mononuclear cell fraction but NPY mRNA was also detected in the platelet fraction. No NPY mRNA was detected in bone marrow or blood from pig and rabbit or from human blood or bone marrow. Forty-eight hours after treatment of rats with vinblastine the content of NPY mRNA and NPY-LI in rat blood was decreased, while the level of NPY-LI in bone marrow was markedly enhanced. Reserpine treatment caused an increase in NPY mRNA content in bone marrow and spleen. After administration of dexamethasone the level of NPY mRNA increased in both spleen and peripheral blood cells with increased NPY-LI content in the spleen. It is concluded that in addition to megakaryocytes in spleen and bone marrow, platelets and possibly also lymphocytes/monocytes in peripheral blood of the rat contain NPY mRNA and peptide. The expression of NPY mRNA in bone marrow, spleen, and blood is influenced by vinblastine, reserpine, and dexamethasone.     

Analysis of rat uterine estrogen receptors by high-pressure liquid chromatography methods

Cytosolic (ERc) and nuclear (ERn) estrogen receptors prepared from rat uteri were characterized by size-exclusion and ion-exchange HPLC. The oligomeric ERc eluted as a single, sharp peak near the exclusion volume of the gel column; ERn eluted as a broad peak. When salt-extracted ERn was partially purified sequentially by Sephadex G-200, DEAE-cellulose chromatography and polyacrylamide gel electrophoresis, the partially purified receptor moieties were not distinguishable by the sucrose gradient method, but showed characteristic retention times in the size-exclusion HPLC column. Further distinction in net surface charges was observed between ERc and ERn moieties by ion-exchange high-pressure liquid chromatography (HPLC). Molybdate-stabilized ERc was eluted as sharp peak at 0.27 M salt gradient. In contrast, fresh extracts of ERn emerged as a broad peak in the region of 0.1-0.2 M salt gradient. In the absence of molybdate, ERc dissociated into several 4-5 S molecules, which were well resolved in the DEAE column. This report, therefore, demonstrates the usefulness of size-exclusion and ion-exchange HPLC for steroid receptor analysis.     

Human brain natriuretic peptide-like immunoreactivity in human brain.

The presence of immunoreactive human brain natriuretic peptide in the human brain was studied with a specific radioimmunoassay for human brain natriuretic peptide-32. This assay showed no significant cross-reaction with human alpha atrial natriuretic peptide, porcine brain natriuretic peptide or rat brain natriuretic peptide. Immunoreactive human brain natriuretic peptide was found in all 5 regions of human brain examined (cerebral cortex, thalamus, cerebellum, pons and hypothalamus) (0.6-6.7 pmol/g wet weight, n = 3). These values were comparable to the concentrations of immunoreactive alpha atrial natriuretic peptide in human brain (0.5-10.1 pmol/g wet weight). However, Sephadex G-50 column chromatography showed that the immunoreactive human brain natriuretic peptide in the human brain eluted earlier than synthetic human brain natriuretic peptide-32. These findings suggest that human brain natriuretic peptide is present in the human brain mainly as larger molecular weight forms.     

Studies on the tissue distribution and stability of "big" CRF (corticotropin-releasing factor).

Extracts of various bovine or rat neural tissues made with 0.1 N HCl, 2N acetic acid or distilled water were fractionated on Sephadex G-100 column with 0.2 N acetic acid as the eluant. A distinct peak of “big” CRF which elutes in the void volume of Sephadex G-100 was observed only with hypothalamic median eminence and hypophyseal stalk. Human serum and extracts of cerebral cortex, neurohypophysis and an ACTH-producing lung tumor, had CRF activity which eluted from Sephadex G-100 with diffuse patterns without a distinct peak. “Big” CRF is stable during storage at ?20 C in water or at 4 C in acid, but progressively disappeared when stored at ?20 C in acid.     

Simultaneous microdetermination of homovanillic acid and 5-hydroxyindoleacetic acid in brain tissue using Sephadex G-10 and QAE-Sephadex A-25

Homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) in animal brains were simultaneously purified by two steps of column chromatography on Sephadex G-10 and QAE-Sephadex A-25. Perchloric acid extracts of brain tissue were directly passed through a column of Sephadex G-10. The gel retained both HVA and 5-HIAA, thereby separating them from Cl0^?₄ which interferes with subsequent purification process and from endogenous substances which give blank fluorescence. HVA was loosely adsorbed on the gel and was easily desorbed with dilute acetic acid. This effluent was successively passed onto a column of QAE-Sephadex A-25 placed beneath the G-10 column and the adsorbed HVA was eluted with 0.1 M Na₂HPO₄. The 5-HIAA remaining on the Sephadex G-10 without being desorbed by acetic acid was eluted with dilute ammonia. The recovery of both acid metabolites by this column procedure was more than 90%. Thus, it is possible to determine the levels of HVA and 5-HIAA in single brains of small rodents.     

Neuropeptide Y-like immunoreactivity in adrenaline cells of adrenal medulla and in tumors and plasma of pheochromocytoma patients

The occurrence of neuropeptide Y (NPY)-like immunoreactivity (LI) in the adrenal gland of several species as well as in tumor tissue and plasma from pheochromocytoma patients was investigated. NPY-LI was present in chromaffin cells of the adrenaline type in all species investigated except in the pig, as demonstrated by a colocalization of NPY-LI and the adrenaline-synthetizing enzyme phenylethanolamine N-methyltransferase (PNMT). NPY-LI in the adrenaline cells of the cat was clearly separated from the neurotensin-LI in the noradrenaline dopamine-beta-hydroxylase-positive, PNMT-negative cells. NPY-LI seems to co-exist with enkephalin-like material in the chromaffin cells. In addition, NPY-LI was present in nerves both within the adrenal cortex and medulla. The highest levels of NPY-LI were found in mouse and cat, while only a very low amount of NPY-LI was present in the pig adrenal. Characterization of the adrenal NPY-LI by reversed-phase high-performance liquid chromatography revealed that the main peak was similar to porcine NPY. In addition, two minor peaks of NPY-LI were present. High levels of NPY-LI were found in plasma and tumors from the pheochromocytoma patients. During manipulation of the tumors upon surgical removal, there was a marked increase in plasma NPY-LI in parallel with the raise in catecholamines and in blood pressure. At least two forms of NPY-LI were present in plasma and tumor extracts from pheochromocytoma patients with the main peak corresponding to porcine NPY. Since NPY exerts vasoconstrictor effects, it may be postulated that NPY contributes to the adrenal cardiovascular response and to the hypertension seen in pheochromocytoma patients.     

Corticotropin releasing factor (CRF)-like immunoreactivity in the hypothalamus and posterior pituitary of the goat, bovine, rat, monkey and human

Goat hypothalamic extract prepared by HCl extraction and chromatographed on a Sephadex G-50 column showed two immunoreactive CRF peaks. Most of the immunoreactivity coeluted with synthetic ovine CRF, and a small peak eluted near the void volume. Bovine, monkey, rat and human hypothalamic extracts prepared by acid-acetone or acid-methanol extraction showed three immunoreactive peaks. Most of the immunoreactivity coeluted with ovine CRF, and other smaller peaks eluted near the void volume and slightly before arginine vasopressin. Goat hypothalamic extract showed the highest cross-reactivity with anti-ovine CRF serum, followed by bovine hypothalamic extract. Less cross-reactivity was found in human, rat and monkey hypothalamic extracts. CRF immunoreactivity in goat hypothalamic extract coeluted with ovine CRF on reversed phase high performance liquid chromatography (HPLC) and main CRF immunoreactivity in human and rat hypothalamic extracts eluted slightly later than ovine CRF. These results suggest that there is a heterogeneity among the CRF molecules in these species and that goat CRF may be more similar to that of sheep CRF and the amino acid sequence or molecular weight of other animals CRF may be different from that of sheep CRF. The monkey posterior pituitary and rat neurointermediate lobe showed similar elution patterns of CRF immunoreactivity to their hypothalamic extracts on Sephadex gel filtration and HPLC. These results indicate that the posterior pituitary contains a similar CRF to hypothalamic CRF.     

Neuropeptide Y and sympathetic vascular control in man

A parallel increase in systemic plasma levels of neuropeptide Y (NPY)-like immunoreactivity (LI) and noradrenaline (NA) was found during thoracotomy and surgery involving cardiopulmonary bypass in man. Thus, plasma levels of NPY-LI increased from 29 +/- 4 pmol/l before anaesthesia to 59 +/- 10 after thoracotomy and to 87 +/- 8 pmol/l upon cardiopulmonary bypass. The corresponding NA levels increased from 1.3 +/- 0.1 nmol/l before anaesthesia to 3.0 +/- 0.6 and 4.2 +/- 5 nmol/l after thoracotomy and cardiopulmonary bypass, respectively. A significant correlation was found between plasma levels of NPY-LI and NA during the operation but not between NPY-LI and adrenaline. The NPY-LI in human plasma was found to be similar to synthetic porcine NPY on reversed phase high performance liquid chromatography. Human submandibular arteries contained high levels of NPY-LI (24 +/- 3 pmol/g). In in vitro experiments on isolated human submandibular arteries, NPY in low concentrations (1000 pmol/l) was found to potentiate the contractile effects of NA or transmural nerve stimulation and to exert vasoconstrictor activity per se in higher concentrations. The calcium-entry antagonist nifedipine abolished both the NPY-induced contractions and the enhancement of NA-evoked contractions. NPY depressed the nerve stimulation-evoked 3H-NA release from human submandibular arteries via a prejunctional mechanism which was resistant to nifedipine. NPY contracted human mesenteric veins and renal arteries, but not mesenteric arteries. In conclusion, NPY seems to be co-released with NA upon sympathetic activation in man. Furthermore, NPY exerts both pre- and postjunctional effects on sympathetic control of human blood vessels.     

Pro-enkephalin peptides possessing Met-enkephalin-Arg6-Gly7-Leu8-immunoreactivity in rat CSF, striatum and adrenal gland

Cerebrospinal fluid (CSF) taken from rats implanted with chronic cisternal cannulae and extracts prepared from rat adrenal gland and striatum were subjected to Sephadex G-50 chromatography and HPLC. Fractions were monitored using specific radioimmunoassays (RIA) for the pentapeptide methionine enkephalin (Met-Enk) and methionine enkephalin-Arg6-Gly7-Leu8 (Met-EnkRGL). In rat CSF, striatum and adrenal gland, three Met-EnkRGL-immunoreactive (IR) peaks of Mrs 8000, 5000 and 1000 daltons were detected. The same peaks were also found to possess Met-Enk-immunoreactivity after enzyme digestion of Sephadex G-50 fractions with trypsin and carboxypeptidase B (CPB), suggesting their derivation from proenkephalin. HPLC of the 8K and 5K peaks on a column of Ultrapore RPSC showed them to elute discretely with similar retention times, indicative of hydrophobic peptides of large molecular weight. Their similar hydrophobicities yet significant separation during gel filtration would suggest that the 8K and 5K peptides are structurally closely related yet different with respect to their molecular weights. HPLC of the small molecular weight material from rat striatum and adrenal gland revealed the presence of Met-EnkRGL and Met-EnkRGL sulphoxide in both tissues. In rat striatum Met-Enk and its sulphoxide were also detected. The oxidised pentapeptide was found to be present in rat CSF, together with two unidentified small molecular weight Met-Enk-IR peaks detected without prior enzyme digestion of fractions. The small molecular weight Met-EnkRGL-IR material in rat CSF was found to be comprised of two unknown peptides which were less hydrophobic than Met-EnkRGL and its sulphoxide derivative.     

An amino-terminal fragment of cholecystokinin-58 is present in the gut: evidence for a similar processing site of procholecystokinin in canine gut and brain

Radioimmunoassays using antibodies specific for the carboxyl terminus of cholecystokinin (CCK) and the midportion of CCK-58 (raised against synthetic canine CCK-33-(1-27] revealed the existence of a CCK fragment in canine gut and brain extracts which lacks the biologically active carboxyl terminal immunoreactivity. This material eluted on Sephadex G-50 gel permeation chromatography in the region of CCK-58, on high-pressure liquid chromatography (HPLC) after CCK-39 and before CCK-58, and on cation-exchange FPLC it eluted after CCK-58. The immunoreactive pattern, the ratio of absorbance at 280-220 nm and the chromatographic elution positions suggest that this large CCK-like molecule represents an amino-terminal fragment of CCK-58. This fragment is present in canine gut and brain. Therefore, a similar processing site of procholecystokinin is suggested in both tissues.     

Characterization of parathyroid hormone-related protein in the human term placenta.

To characterize parathyroid hormone-related protein (PTHrP) in the human placenta, we measured PTHrP-like immunoreactivity (PRP-LI) in the term placenta and studied the elution profiles of placental tissue extracts on Sephadex G-75 chromatography with a specific RIA. We also examined the gene expression of PTHrP mRNA by Northern blot analysis and the localization of PRP-LI in the placenta by immunohistochemistry. The amount of PRP-LI in placental extracts (n = 7) was 20.9 +/- 2.2 pg/g wet tissue (mean +/- SE). Dilution curves of placental tissue ran parallel to those of synthetic PTHrP (1-34) standards. Sephadex G-75 gel chromatography demonstrated two major PRP-LI peaks; the first peak was eluted around the molecular size between 10 kilodaltons (Kda) and 20 Kda and the other around 5 Kda. Northern blot analysis of PTHrP mRNA extracted from placental tissues showed a major hybridization signal around 18S. PTHrP immunohistochemistry showed PRP-LI staining in the cytoplasm of syncytiotrophoblasts and stroma cells (Hofbauer cells) in the term placenta. These results suggest that syncytiotrophoblasts and stroma cells in the term placenta synthesize PTHrP in two major molecular forms, 10 Kda-20 Kda and around 5 Kda.     

Androphilic proteins in cytosols of human benign prostatic hypertrophy.

To examine the properties of androphilic proteins in human benign prostatic hypertrophy, the binding capacity and affinity of the proteins were determined after acetone-treatment, ammonium sulfate precipitation and chromatographies of DEAE and Sephadex G-200. Androphilic proteins in the extract of acetone-dried cytosol from the hypertrophic human prostate was precipitated at 30-50% saturation of ammonium sulfate. The binding of this fraction to dihydrotestosterone and testosterone was high affinity, but the binidng to estradiol-17 beta was the one of non-specific. Androphilic proteins in the 30-50% fraction were eluted from DEAE-cellulose column by buffer containing 0.05 M KCL. On Sephadex G-200 chromatography of 30-50% fraction, the androphilic proteins were observed in three peaks; one was eluted in the void volume and other two were eluted at the sites of IgG and albumin. The amount and ratio of proteins eluted in the void volume and the site of IgG from Sephadex G-200 column were variable in individual tissue samples. The chromatographic behavior of the 30-50% fraction in Sephadex G-200 was not changed significantly by introducing 0.4 M KCl in the system. Polyacrylamide gel electrophoresis was applied for further separation of the proteins.     

Tetrodotoxin-sensitive protein in the extracts from excitable tissues

The sodium permeability of liposomes preincubated with the soluble fraction of brain and heart muscle homogenates was increased veratrine. The veratrine increment was decreased by tetrodotoxin. The effect was specific for the extracts from excitable tissues. Bovine serum and soluble fraction of liver homogenate induced neither veratrine- nor tetrodotoxin-sensitivity of the liposomes. Treatment of the excitable tissue extracts by pronase and heat denaturation caused their complete inactivation. Tetrodotoxin-sensitive factor could be fractionated by ammonium sulfate precipitation and by DEAE-Servacel chromatography. On a column of Sephadex G-200 it was eluted with the void volume. It is suggested that the tetrodotoxin-sensitive factor is a protein which could be a soluble precursor of the voltage-dependent sodium channels.     

Multiple forms of ACTH biological activity in the pars intermedia of the rat adenohypophysis.

Acid extracts of a) acutely dispersed rat pars intermedia (PI) cells, b) media after incubation of PI cells, c) whole nervosa-intermedia, and d) whole pars distalis, were chromatographed on Sephadex G-50 Fine in 1% acetic acid. Three peaks of ACTH biological activity were resolved in all four extracts. Peak I eluted in the void volume of the column, peak III co-eluted with synthetic ACTH^1–39, and peak II eluted in an intermediate position. The predominant ACTH activity derived from the PI tissue was peak I, amounting to over 70% of the total ACTH activity present in that lobe. The positions of PI peaks I and II remained unaltered after rechromatography as well as after treatment with and chromatography in 8 M urea. However, peak I of PI ACTH was further resolved into two separate peaks by chromatography on Sephadex G-100 SF. Thus pars intermedia ACTH activity appears to be composed of four separate entities, with the predominant forms being larger than ACTH^1–39.     

The structure and function of acid proteases. VII. Distribution and some properties of acid proteases in monkey tissues.

1. The distribution of acid protease activity in various tissues of Japanese monkey (Macaca fuscata fuscata) was investigated with hemoglobin as a substrate at pH 3.0. The activity per protein weight in crude extracts was highest in spleen and lung, and decreased in the order: spleen, lung greater than kidney, testis greater than brain greater than liver, placenta greater than thyroid gland, muscle. The activity in crude muscle extract was about one-tenth those of spleen and lung. The activity per wet tissue weight was in roughly the same order except for a lower activity per wet weight of brain. 2. Upon chromatography of each crude extract on a Sephadex G-100 column, one major activity peak was eluted at a position corresponding to a molecular weight of about 41,000. This enzyme activity is attributed to cathepsin D [EC 3.4.23.5]. In addition, a minor activity peak was eluted in the case of spleen, lung and kidney at the break-through position, corresponding to a molecular weight of more than 100,000. This activity peak is presumably due to cathepsin E. These acid protease activities were, in most cases, strongly inhibited by pepstatin, an acid protease-specific peptide inhibitor. 3. The distribution of acid protease activity was investigated in the brain of crab-eating monkey (Macaca fascicularis). The activity was fairly evenly distributed among several regions of the brain, and its distribution was similar to those of other acid hydrolases, especially N-acetyl-beta-D-glucosaminidase [EC 3.2.1.30] and acid phosphatase [EC 3.1.3.2], which are marker enzymes of lysosomes.     

Purification and chemical characterization of human hexosaminidases A and B.

N-Acetyl-beta-hexosaminidases A and B were purified to homogeneity from human placenta. In the initial step of purification, the enzymes were adsorbed on concanavalin A-Sepharose 4B and eluted from the column with alpha-methyl D-mannosides. Subsequent purification steps included DEAE-cellulose column chromatography, QAE-Sephadex [diethyl-(2-hydroxypropyl)aminoethyl-Sephadex] column chromatography, Sephadex G-200 gel filtration and preparative disc polyacrylamide-gel electrophoresis, followed by another QAE-Sephadex chromatography for the hexosaminidase A preparation, and DEAE-cellulose column chromatography, calcium phosphate gel chromatography, Sephadex G-200 gel filtration, QAE-Sephadex chromatography and CM-cellulose chromatography for the hexosaminidase B preparation. The purified preparations, particularly hexosaminidase A, had significantly higher specific enzyme activities than previously reported. The preparations moved on polyacrylamide-gel electrophoresis as single protein bands, which also stained for enzyme activity. Sedimentation-equilibrium centrifugation indicated homogenous dispersion of the enzymes, and the molecular weight was estimated as about 110000 for both enzymes. Complete amino acid and carbohydrate compositions of the two isoenzymes were determined, and, in contrast with previous suggestions, no sialic acid was found in the enzymes.     

NH2-terminal big gastrin immunoreactivity in human urine

The concentrations and molecular forms of urinary and plasma gastrin from normal subjects were studied by radioimmunoassays using two region-specific antisera. Urinary concentration of NH2-terminal big gastrin (G-34) immunoreactivity was several hundred times as great as that of COOH-terminal gastrin immunoreactivity. Fractionation of urine extract showed a broad giant peak of NH2-terminal G-34 immunoreactivity (gastrin fragments "U") eluting in a later position than G-34(1-17) by Sephadex G-50 column chromatography. HPLC revealed that urinary NH2-terminal G-34 immunoreactivity was composed of four fragments including G-34(1-8), G-34(1-9), and G-34(1-10). Sephadex G-50 column chromatography of plasma extract revealed two or three peaks of NH2-terminal G-34 immunoreactivity, and a major peak eluted in the same position as urinary gastrin fragments "U". These results and data on renal clearances suggest that most of all gastrin fragments "U" in plasma are excreted in urine without renal reabsorption, whereas almost all of plasma COOH-terminal gastrin peptides including G-34 and little gastrin (G-17) are removed and metabolized in the kidney.     

Presence of immunoreactive dynorphin in human cerebrospinal fluid

Immunoreactive dynorphin (I-dynorphin) was measured by radioimmunoassay in human cerebrospinal fluid (CSF). I-dynorphin concentration in CSF was 30 +/- 2 pg/ml. Sephadex G-25 column chromatography showed the main peak eluted at the position of dynorphin-(1-17). HPLC elution profile of this major peak from gel filtration showed a large peak corresponding to the position of dynorphin-(1-17) and small peaks corresponding to the positions of dynorphin-(1-13), dynorphin-(1-12) and other unknown peptides.