Neuropeptide Y 3-36 is an endogenous ligand selective for Y2 receptors.

Neuropeptide Y (NPY 1-36) binds to Y1 and Y2 receptors with similar affinity. No endogenous molecular form of NPY with selectivity for Y1 or Y2 receptors has been described so far. We report the presence of an endogenous fragment of NPY in porcine brain, NPY 3-36, which lacks the amino-terminal dipeptide Tyr-Pro of NPY 1-36. NPY 3-36 accounts for 35% of NPY-like immunoreactivity in porcine brain. We have compared binding of NPY 3-36 and NPY 1-36 in model systems of Y1-like (SK-N-MC cells) and Y2-like receptors (CHP234 cells). NPY 3-36 and NPY 1-36 had similarly high affinity for Y2-like receptors on CHP234 cells, but NPY 3-36 had a 1000-fold lower affinity than NPY 1-36 for Y1-like receptors on SK-N-MC cells. Thus amino-terminal cleavage of NPY 1-36 generating NPY 3-36 converts an unselective Y1/Y2 receptor ligand into a highly Y2 selective ligand. This may be a means of fine tuning NPY biological actions.

Regulation of NPY/NPY Y1 receptor/G protein system in rat brain cortex.

Neuropeptide Y (NPY) content, NPY receptors, and alpha-subunits of the G proteins Go and Gi were determined in cerebral cortex of male normotensive Wistar-Kyoto and spontaneously hypertensive rats at 3-28 wk of age and of adult female rats. NPY lacked major effects on adenylate cyclase or inositol phosphate formation. NPY content was similar in all normotensive groups but lower in spontaneously hypertensive rats at all ages. 125I-NPY labeled a homogeneous population of Y1-like receptors. The Y1 NPY receptor number gradually increased with age with similar values in both strains but was significantly smaller in female than in male rats. The Y1 NPY receptor affinity was similar in all male groups but greater in female rats. The abundance of immunodetectable Go alpha and Gi alpha and of pertussis toxin substrates was less at 3 wk than in older rats but similar in both sexes and strains. We conclude that rat cerebral cortex contains Y1-like receptors; sex, age, and blood pressure differentially regulate NPY content, Y1 NPY receptors, and Go alpha and Gi alpha.

[Pro34]peptide YY is a Y1-selective agonist at peptide YY/neuropeptide Y receptors.

We have investigated binding and functional effects of a new peptide YY analogue, [Pro34]peptide YY, at Y1 and Y2-like subtypes of receptors for peptide YY and neuropeptide Y. In binding studies [Pro34]peptide YY had a similarly high affinity as peptide YY to human Y1-like receptors in SK-N-MC cells, a human neuroblastoma cell line of presumed neurogenic origin, and HEL cells, a human cell line derived from a patient with Hodgkin's disease. In functional studies [Pro34]peptide YY stimulated Ca2+ elevations in both Y1-like receptor cell lines with similar potency and efficacy as peptide YY. In contrast to peptide YY [Pro34]peptide YY was 1000-fold less potent in binding to Y2-like receptors in porcine splenic membranes and lacked agonistic effects in another Y2-like receptor-mediated model system, i.e. inhibition of [3H]serotonin release from rat cerebral cortical slices. Thus, [Pro34]peptide YY is a highly Y1-selective full agonist of peptide YY/neuropeptide Y receptors. [Pro34]peptide YY could be useful for studying the importance of Y receptor subtypes in mediating peptide YY physiological actions.

Elevated urinary excretion of endothelin-like immunoreactivity in children with renal disease is related to urine flow rate.

Endothelin 1-21 belongs to a family of locally produced regulatory peptides with potent vasoconstrictor activity and profound renal effects. To study the biological significance of endothelin in children with renal diseases, we measured urinary endothelin-like immunoreactivity (ETir) excretion in children and adolescents (60 normal controls and 57 patients with renal disease). ETir excretion was constant during childhood and adolescence (4-18 years). Compared to these normal controls elevated urinary excretions of ETir were found in children with chronic renal failure, following renal transplantation and with idiopathic hypercalciuria (all groups p < 0.001). However, ETir excretion was unchanged in children with idiopathic steroid sensitive nephrotic syndrome, with stable chronic glomerulonephritis and in 4 patients with hemolytic uremic syndrome. Urinary ETir concentrations were similar in controls and in various patient groups. ETir excretion correlated positively with urine flow rate in normal controls (r = 0.71) and in all patients studied (r = 0.91). Fractional excretion of ETir correlated negatively with glomerular filtration rate. Eight healthy volunteers (23-27 years old, 4 female, 4 male) were studied before and after oral water load (20 ml/kg) to investigated the effect of ETir excretion on urine flow rate. Urine flow rose tenfold in response to water load and urine concentration of ETir fell only by factor 3 and urinary ETir excretion rose fivefold. These results indicate that urinary ETir excretion is related to and depends at least in part on urine flow rate. ETir excretion may so reflect a role of ETir in renal disease, especially in the diuretic state.

Serial measurements of neuropeptide Y in plasma for monitoring neuroblastoma in children.

Neuropeptide Y (NPY) was studied as a marker for neuroblastoma in 12 children. All but one patient with neuroblastoma had elevated plasma NPY concentrations at diagnosis. During treatment NPY values returned to normal in 9 of 12 children. All three children without normalization of plasma NPY values died; two of them had a relapse and the third died of toxic effects. Plasma NPY appears to be a sensitive marker of neuroblastoma.

Clinical settings and vasopressin function in hyponatraemic children.

Hyponatraemia is one of the most common electrolyte abnormalities in hospitalised children. In a prospective study we tested whether hyponatraemia is associated with sustained release of the antidiuretic hormone arginine vasopressin (AVP). Out of 27 children with persistent hyponatremia (serum sodium < 130 mmol/l), 25 had measurable plasma concentrations of AVP [median and quartiles 5.0 pg/ml (1.5-8.3)]. Volume contraction as consequence of sodium loss caused hyponatraemia in 16 patients. Hyponatraemia in the presence of extracellular volume expansion and reduced effective arterial blood volume occurred in 5 patients. Only 3 patients had normovolaemic hyponatraemia (so-called syndrome of inappropriate antidiuretic hormone secretion) and 3 suffered from chronic renal failure. It is concluded that plasma AVP concentration is measurable in most children with hyponatraemia. Non-osmotic stimulation of AVP release and lack of suppression of this hormone is an important pathogenetic mechanism of hyponatraemia in children.

Is PP56 (D-myo-inositol-1,2,6-trisphosphate) an antagonist at neuropeptide Y receptors?

PP56 (D-myo-inositol-1,2,6,-trisphosphate) has been reported to specifically inhibit neuropeptide Y-mediated effects in vasculature, heart and brain; because of its reversible but non-competitive antagonism interaction with neuropeptide Y receptor signalling or allosteric modulation of neuropeptide Y binding have been postulated. These possibilities were tested in the present study. PP56 did not affect [125I]neuropeptide Y binding to HEL- or SK-N-MC-cells or to porcine splenic membranes. PP56 did not inhibit neuropeptide Y-stimulated Ca2+ increases in HEL- or SK-N-MC-cells or in cultured porcine aortic vascular smooth muscle cells but if anything slightly enhanced it. PP56 did not antagonize the neuropeptide Y-mediated inhibition of forskolin-stimulated cAMP accumulation in HEL-cells. We conclude that previously reported antagonistic effects of PP56 occur distal to the neuropeptide Y receptor or its second messenger systems Ca2+ and cAMP or may be restricted to neuropeptide Y receptors in certain model systems.

Novel generation of hormone receptor specificity by amino terminal processing of peptide YY.

The physiological significance of multiple Y receptors has not been determined since until recently only one form of endogenous agonists was known, namely PYY1-36 and NPY1-36. Recently, a new molecular form of PYY was characterized as des(Tyr-Pro)PYY (PYY3-36 or PYY-II). Its ability to interact at various Y receptors was not characterized. Analytical chromatography of fresh canine colon extracts shows two peaks of immunoreactivity eluting in the positions of PYY-II and PYY1-36 (PYY). PYY-II was about 40% of the total PYY immunoreactivity indicating that it is one of the major forms of PYY expressing its biological activity. It is shown that PYY-II will not displace label from the Y1 receptors found on a human neuroblastoma cell line. It is further shown that PYY-II is as potent as PYY for the inhibition of pancreatic secretion, which must occur through Y2 receptors. The enzymatic removal of Tyr-Pro from PYY to form PYY-II must therefore regulate the relative expression of a non-selective agonist (PYY) to a highly selective Y2 agonist (PYY-II). Amino terminal processing of PYY represents a novel type of regulation of peptide hormone specificity. It has important biological implications for PYY and potential relevance for other peptide hormone receptor systems.

NPY and carbachol raise Ca2+ in SK-N-MC cells by three different mechanisms. Evidence for inositol phosphate-independent Ca2+ mobilization by NPY.

We have compared the mechanism of NPY- and carbachol-stimulated Ca2+ increases in SK-N-MC cells. NPY stimulated Ca2+ mobilization via a pertussis toxin-sensitive mechanism. Carbachol stimulated Ca2+ mobilization and influx via pertussis toxin-insensitive and -sensitive mechanisms, respectively. Carbachol but not NPY stimulated inositol phosphate accumulation by a pertussis toxin-insensitive mechanism. We conclude that carbachol promotes Ca2+ influx via a pertussis toxin-sensitive G protein and Ca2+ mobilization via a pertussis toxin-insensitive G-protein coupling to inositol phosphate generation; NPY stimulates Ca2+ mobilization via a pertussis toxin-sensitive G protein without apparent involvement of inositol phosphates.

NPY-stimulated Ca2+ mobilization in SK-N-MC cells is enhanced after isoproterenol treatment.

Neuropeptide Y (NPY) receptors in the SK-N-MC human neuroblastoma cell line couple to mobilization of intracellular Ca2+ and inhibition of adenylylcyclase. Pretreatment of SK-N-MC cells with isoproterenol enhanced the NPY-stimulated Ca2+ mobilization, mainly by increasing the maximal response to NPY. The enhancement was time-(maximal after 24 h) and concentration-dependent (maximal at 10 microM isoproterenol), blocked by the beta-adrenergic antagonist propranolol, and mimicked by forskolin. Concomitant treatment with cycloheximide prevented the enhancing effect of isoproterenol, suggesting the involvement of protein synthesis. Isoproterenol treatment did not alter the number or affinity of 125I-labeled NPY binding sites, the amount of pertussis toxin substrates, or NPY-mediated inhibition of cAMP accumulation. Similarly, isoproterenol treatment had no effect on basal intracellular Ca2+ and on Ca2+ increases elicited by carbachol, caffeine, or ionomycin. We conclude that isoproterenol treatment can sensitize NPY receptor responsiveness in a way that is specific for Ca2+ mobilization mechanisms used by this hormone.

Neuropeptide Y (NPY) receptors in HEL cells: comparison of binding and functional parameters for full and partial agonists and a non-peptide antagonist.

1. We have compared the binding and Ca2+ mobilizing properties of various full agonists, partial agonists and a non-peptide antagonist at the neuropeptide Y (NPY) receptor of human erythroleukemia (HEL) cells. 2. [125I]-NPY binding to intact HEL cells was rapid, saturable, of high affinity and with a specificity typical for the Y1-like subtype: NPY, peptide YY (PYY) and [Pro34]-NPY competed for [125I]-NPY binding with high affinity whereas NPY13-36 and NPY18-36 had only low affinity. 3. NPY, PYY and [Pro34]-NPY potently increased intracellular Ca2+ in HEL cells and had equal efficacy. NPY13-36, vasoactive intestinal peptide (VIP) and pancreatic polypeptide (PP) increased intracellular Ca2+ only poorly. 4. Whereas VIP and PP did not significantly affect NPY-stimulated Ca2+ mobilization, NPY13-36 inhibited NPY-stimulated Ca2+ increases and shifted the NPY concentration-response curve to the right without altering its maximal effect. 5. The agonist (pEC50) potencies of the various peptides corresponded well with the affinities of these compounds in the binding assay (pKi), whereas the antagonist potencies (pKb) of the peptide partial agonists and the pA2 value of the non-peptide NPY antagonist (He 90481), calculated from functional data, were lower than the respective affinities determined in the binding studies. 6. A plot of the fractional Ca2+ response vs the fractional receptor occupancy did not reveal any non-linear receptor-effector coupling for NPY or [Pro34]-NPY; a small receptor reserve might exist for PYY. 7. We conclude that the binding and functional properties of HEL cell NPY receptors are very similar. NPY, PYY and [Pro34]NPY are full agonists at these receptors, whereas NPY13-36 is a partial agonist.

G-protein coupling and signalling of Y1-like neuropeptide Y receptors in SK-N-MC cells.

We have studied [125I]neuropeptide Y-binding sites and neuropeptide Y-mediated second messenger responses in human SK-N-MC neuroblastoma cells with special reference to the role of G-proteins. Neuropeptide Y stimulated two second messenger responses in SK-N-MC cells, inhibition of cAMP accumulation and mobilization of Ca2+ from intracellular stores. Both effects were completely abolished by pretreatment with pertussis toxin. Binding of [125I]neuropeptide Y to intact cells or SK-N-MC cell membranes was rapid, reversible, characterized by high affinity and low capacity, and had pharmacological characteristics of a homogeneous population of Y1-like neuropeptide Y receptors. In permeabilized cells, [125I] neuropeptide Y binding was inhibited by GTP gamma S in a concentration-dependent manner. Saturation experiments in the absence and presence of GTP gamma S demonstrated a reduction in the number of high-affinity [125I]neuropeptide Y-binding sites without a decrease in affinity of the remaining sites. Pretreatment of intact cells with pertussis toxin completely abolished the inhibition of [125I]neuropeptide Y binding by GTP gamma S. Moreover, pertussis toxin treatment reduced the number of high-affinity [125I]neuropeptide Y binding sites. We conclude that the agonist ligand [125I]neuropeptide Y identifies functional neuropeptide Y receptors in SK-N-MC cells; however, the number of specific [125I]neuropeptide Y-binding sites may not necessarily reflect the number of neuropeptide Y receptors, because the former is affected by the functional state of cellular G-proteins.

Medium scale production of L-myo-inositol 1-phosphate.

L-myo-Inositol-1-phosphate synthetase was purified from baker's yeast, grown in a fermenter in an inositol-deficient medium and analyzed using a new HPLC assay for inositol. This enzyme was used in a procedure, developed from methods partially described in the literature, for the medium scale production and purification of L-myo-inositol 1-phosphate. The identity and purity of the product were confirmed by 1H and 31P NMR spectroscopy.

Enzyme assay of L-myo-inositol 1-phosphate synthetase based on high-performance liquid chromatography of benzoylated inositol.

A procedure for the determination of inositol by reversed-phase HPLC is described which is based on a precolumn benzoylation and detection at 230 nm. This procedure was used to assay the activity of L-myo-inositol 1-phosphate synthetase (EC 5.5.1.4) after treatment of the enzymatic product by a phosphatase.

The acidic peptide-specific type II protein kinases from the nucleus and the cytosol of porcine liver are distinct.

The second messenger-independent acidic peptide-specific protein kinase II (casein kinase II) from the cytosol of porcine liver has been purified to apparent homogeneity by using DEAE-cellulose, hydroxyl apatite, and phosphocellulose chromatography. The native enzyme has an apparent Mr of 150,000. After sodium dodecyl sulfate-gel electrophoresis a band of Mr = 39,000 and a slightly diffuse band of Mr = 27,000 were found indicating an alpha 2 beta 2 structure of this protein kinase. A thorough comparison with the corresponding enzyme from the nucleus was performed. The two enzymes differ in the subunit composition, as the nuclear enzyme is composed of subunits with a Mr of 95,000; they further differ in the heparin sensitivity and binding to blue dextran-Sepharose. Distinct differences in their nucleotide binding sites were found upon mapping with ATP analogs, although both enzymes utilize ATP as well as GTP. On the other hand, both enzymes phosphorylate identical sites in the casein variants beta A2 and alpha S1B at comparable rates. These results demonstrate for the first time the existence of distinct nucleus and cytoplasm specific type II "casein kinases".

Regulation in tobacco callus of enzyme activities of the nicotine pathway : I. The route ornithine to methylpyrroline.

Nicotine synthesis was stimulated by reduction of the medium auxin concentration (induction medium) in callus tissue originating from Nicotiana tabacum cv. Samsun. The enzyme activities of the route ornithine to methylpyrroline, which are those of ornithine decarboxylase, putrescine methyltransferase and methylputrescine oxidase, were determined during callus growth in the induction medium and as a control under non-nicotine-stimulating conditions (growth medium). The enzymes were assayed by high-performance liquid chromatography. Whereas the activities of ornithine decarboxylase were very similar under nicotine-stimulating and non-stimulating conditions, those of putrescine methyltransferase and methyl-putrescine oxidase increased strongly in the induction medium. In addition, the pools of putrescine and methylputrescine were determined throughout the callus growth cycle. Both sets of data strongly confirm the supposition that putrescine methyl-transferase is the enzyme under stringent control for nicotine biosynthesis, whereas the subsequent methylputrescine oxidase is co-regulated, although less stringently.

Regulation in tobacco callus of enzyme activities of the nicotine pathway : II. The pyridine-nucleotide cycle.

In tobacco callus, the induction of nicotine synthesis, which stimulates enzyme activities of the ornithine-methylpyrroline route (see the preceding paper), also leads to marked changes in the enzyme activities of the pyridine-nucleotide cycle. This cycle provides the metabolite (probably nicotinic acid) for condensation with methylpyrroline to produce nicotine. The activities of eight enzymes of the pyridine-nucleotide cycle and of quinolinic-acid phosphoribosyltransferase, the anaplerotic enzyme, were determined by high-performance liquid chromatography assays. The distinct changes of their activities upon induction of nicotine synthesis lead to the following conclusions: i) nicotinic acid is the relevant metabolite which is provided by the pyridine-nucleotide cycle and consumed for nicotine synthesis. ii) The enhancement of the nicotinic-acid pool arises in two ways, by synthesis of NAD and degradation via nicotinamide mononucleotide and by a direct route from nicotinic-acid mononucleotide (NaMN) which is degraded by a glycohydrolase with a rather high K m value. Such a K m value prevents the complete depletion of the NaMN pool.

The pyridine-nucleotide cycle in tobacco : Enzyme activities for the recycling of NAD.

In order to elucidate the NAD-recycling pathway the following enzyme activities have been characterized in different tobacco tissues and in tomato root: NAD pyrophosphatase, nicotinamide mononucleotide (NMN)/nicotinic acid mononucleotide (NaMN) glycohydrolases, nicotinamidase and nicotinic acid phosphoribosyltransferase. The investigations were performed with protein extracts purified by gel filtration and enzymatic activities were determined by high-performance liquid chromatography methods. The kinetic parameters of the different enzymes from tobacco root and their specificity are reported. The data are in favor of the so-called pyridine-nucleotide cycle VI (NAD→NMN→nicotinamide→nicotinic acid→NaMN→nicotinic acid adenine dinucleotide→NAD). In the nicotine-producing tobacco root a further direct route leading from NaMN to nicotinic acid is proposed. These data are reconciled with the assumption that it is nicotinic acid which is provided by the pyridine-nucleotide cycle for the synthesis of nicotine.