Kemal Payza

Analog of neuropeptide FF attenuates morphine abstinence syndrome

The octapeptide FLFQPQRFamide (neuropeptide FF or F8Fa) may play a role in opiate dependence and subsequent abstinence syndrome. Previously, NPFF precipitated opiate abstinence syndrome, while IgG from NPFF antiserum attenuated subsequent naloxone-precipitated abstinence signs in dependent rats. The peptide desamino YFLFQPQRamide (daY8Ra) was synthesized as a possible NPFF antagonist. At a dose of 600 ng ICV, daY8Ra significantly attenuated (p less than 0.001) the number of abstinence-like signs subsequently induced by 10 micrograms NPFF ICV, suggesting that daY8Ra does have antagonist activity against NPFF. Pretreatment of morphine-dependent rats with the same dose of daY8Ra also significantly attenuated (p less than 0.001) the abstinence signs subsequently precipitated by 10 micrograms naloxone ICV. Pretreatment with 600 ng of NPFF itself, or of NPFF modified at the N-terminal only (daY9Fa), failed to attenuate subsequent naloxone-precipitated abstinence, suggesting that the C-terminal modification is critical for NPFF antagonist activity. It should be noted, however, that higher doses of daY8Ra (2 micrograms or more) can precipitate some abstinence signs in a manner similar to NPFF.

Analog of neuropeptide FF attenuates morphine tolerance

Previous studies suggest that neuropeptide FF (NPFF) plays a role in opiate dependence and subsequent abstinence syndrome. Endogenous NPFF also appears to play a role in opiate tolerance since third ventricle injection of IgG from NPFF antiserum selectively restores morphine sensitivity in morphine-tolerant rats. The NPFF analog, desamino YFLFQPQRamide (daY8Ra) has previously antagonized behavioral effects of NPFF and has attenuated morphine dependence. The present study assessed whether daY8Ra could similarly attenuate morphine tolerance. Third ventricle (i.c.v.) injection of daY8Ra restored the analgesic response to i.c.v. morphine in morphine-tolerant rats (radiant heat tail flick test). Saline injection failed to produce this effect. In opiate-naive rats, however, the same treatment with daY8Ra did not affect the analgesic response to i.c.v. morphine. Thus, daY8Ra appears to selectively restore morphine sensitivity in opiate-tolerant animals. These results further support the hypothesis that endogenous NPFF contributes to opiate tolerance.

Modulation of Neuropeptide FF Receptors by Guanine Nucleotides and Cations in Membranes of Rat Brain and Spinal Cord

Abstract: Using a radioligand binding assay, we examined ionic modulation and G protein coupling of neuropeptide FF(NPFF) receptors in membranes of rat brain and spinal cord. We found that NaCl (but not KCl or LiCl) and MgCl2 increased specific 125I‐YLFQPQRFamide (125I‐Y8Fa) binding to NPFF receptors in both tissues in a dose‐dependent manner, with optimal conditions being 60 mM NaCl and 1 mM MgCl2. Guanine nucleotides dose‐dependently inhibited specific 125I‐Y8Fa binding to rat brain and spinal cord membranes with maximal effects of 64 ± 6 and 71 ± 2%, respectively. The order of potency was nonhydrolyzable GTP analogues > GTP GDP > GMP, ATP. The guanine nucleotide inhibition was observed in the absence and presence of NaCl and MgCl2. The mechanism of inhibition in spinal cord membranes appeared to be a reduction in the number of NPFF receptors; in one experiment, control KD and Bmax values were 0.068 nM and 7.2 fmol/mg of protein, respectively, and with 0.1 μM guanylylimidodiphosphate the respective values were 0.081 nM and 4.9 fmol/mg, a 32% reduction in receptor number. Similar results were obtained with guanosine 5′‐0‐(3‐thiotriphosphate). Our data suggest that 125I‐Y8Fa binding sites in rat CNS are G protein‐coupled NPFF receptors regulated by GTP and cations.

Subcutaneous injection of an analog of neuropeptide FF prevents naloxone-precipitated morphine abstinence syndrome.

There is evidence that neuropeptide FF (NPFF) has antiopiate activity and may play a role in opiate dependence and subsequent abstinence syndrome. A fragment of NPFF was modified at the C-terminal in an effort to convert it to an NPFF antagonist. It was also dansylated at the N-terminal in an effort to render it more lipophilic and increase its penetration of the blood-brain barrier. Third ventricle administration of the resulting compound, dansyl-PQRamide (0.75 microgram and 1 microgram), dose-dependently antagonized the quasi-morphine abstinence activity of NPFF (10 micrograms) in opiate-naive rats. Subcutaneous injection of dansyl-PQRamide (13 mg/kg) in chronically morphine-infused rats attenuated opiate dependence as indicated by prevention of naloxone-precipitated abstinence syndrome. Dansyl-PQRamide displaced radiolabelled ligand from NPFF receptors in a concentration-dependent manner with a Ki of 13 microM, and had a half-life over 300 times longer than NPFF under aminopeptidase digestion.

Enhanced antiopiate activity in peptidomimetics of FMRFamide containing Z-2,3-methanomethionine

FMRFa is a molluscan peptide that has shown antiopiate activity in a number of mammalian test systems. The current study determined the antiopiate potency of FMRFa and two conformationally constrained peptidomimetics of FMRFa containing stereoisomers of Z-2,3-methanomethionine. Morphine abstinence signs were observed after varying doses (0.25-25.0 micrograms) of these substances were injected into the third ventricle of morphine-dependent rats. Although both peptidomimetics were far more potent than FMRFa itself, they bound with lower affinity than FMRFa to rat spinal cord receptors for the mammalian FMRFa-like peptide, NPFF.

Enhanced antiopiate activity and enzyme resistance in peptidomimetics of FMRFamide containing (E)-2,3-methanomethionine.

FMRFamide is a molluscan peptide that has shown antiopiate activity in a number of mammalian test systems. The current study determined the antiopiate potency of FMRFamide and two conformationally constrained peptidomimetics of FMRFamide containing stereoisomers of (E)-2,3-methanomethionine. Morphine abstinence signs were observed after varying doses (0.25-25.0 microgram) of these substances were injected into the third ventricle of morphine-dependent rats. Both peptidomimetics were far more potent than FMRFamide itself. In addition, although both peptidomimetics bound with lower affinity than FMRFamide to rat spinal cord receptors for NPFF (the mammalian FMRFamide-like peptide), they were far more resistant than FMRFamide to enzymatic degradation by leucine aminopeptidase.

Receptor inactivation by dye-neuropeptide conjugates: 1. The synthesis of Cys-containing dye-neuropeptide conjugates

In an attempt to attenuate specifically identified receptors through photolysis, a four-step synthesis is of a useful tethered derivative of Azure-B (Az) was developed After characterization, this derivative was covalently attached to CFMRFamide, CFMRF, and CLRFamide (i.e., three different neuropeptide analogues of the putative neurotransmitter FMRFamide. This resulted in the formation of three dye-neuropeptide conjugates: Az-CFMRFamide, Az-CFMRF, and Az-CLRFamide.

Secretion from rat neurohypophysial nerve terminals (neurosecretosomes) rapidly inactivates despite continued elevation of intracellular Ca2

Cytoplasmic calcium concentration was measured in neurosecretory nerve terminals (neurosecretosomes) isolated from rat neurohypophyses by fura-2 fluorescence measurements and digital video microscopy. Hormone release and cytoplasmic calcium concentration were measured during depolarizations induced by elevated extracellular potassium concentration. During prolonged depolarizations with 55 mM [K+]o, the cytoplasmic calcium concentration remained elevated as long as depolarization persisted, while secretion inactivated after the initial sharp rise. The amplitude and duration of the increase in [Ca2+]i was dependent on the degree of depolarization such that upon low levels of depolarizations (12.5 mM or 25 mM [K+]o), the calcium responses were smaller and relatively transient, and with higher levels of depolarization (55 mM [K+]o) the responses were sustained and were higher in amplitude. Responses to low levels of depolarization were less sensitive to the dihydropyridine calcium channel blocker, nimodipine, while the increase in [Ca2+]i induced by 55 mM [K+]o became transient, and was significantly smaller. These observations suggest that these peptidergic nerve terminals possess at least two different types of voltage-gated calcium channels. Removal of extracellular sodium resulted in a significant increase in [Ca2+]i and secretion in the absence of depolarizing stimulus, suggesting that sodium-calcium exchange mechanism is operative in these nerve terminals. Although the [Ca2+]i increase was of similar magnitude to the depolarization-induced changes, the resultant secretion was 10-fold lower, but the rate of inactivation of secretion, however, was comparable.

Enhanced antiopiate activity and enzyme resistance in a peptidomimetic of FMRFamide containing E-2,3-methanomethionine and E-2,3-methanophenylalanine

FMRFamide is a molluscan peptide that has shown antiopiate activity in a number of mammalian test systems. Peptidomimetics of FMRFamide substituted with conformationally constrained stereoisomers of Z-2,3-methanomethionine or E-2,3-methanomethionine precipitated abstinence syndrome far more potently than FMRFamide itself. The current study determined the effect on antiopiate potency of an additional rigid substitution. A peptidomimetic containing a stereoisomer of E-2,3-methanomethionine was compared with a peptidomimetic additionally substituted at the C-terminal with E-2,3-methanophenylalanine. Morphine abstinence signs were observed after varying doses (0.125-25.0 micrograms) of these two peptidomimetics were injected into the third ventricle of morphine-dependent rats. The peptidomimetic containing both rigid substitutions was far more potent than the peptidomimetic of FMRFamide containing methanomethionine alone. The increased potency appears to be related to enzyme resistance rather than receptor affinity.

Sodium inhibits hormone release and stimulates calcium efflux from isolated nerve endings of the rat neurohypophysis

Summary1.We studied the effects of extracellular sodium on the secretion of vasopressin (VP) and oxytocin (OT) and the efflux of45Ca from isolated, perfused nerve endings of the rat neurohypophysis (neurosecretosomes).2.Upon removal of sodium from the perfusing medium, basal release of VP and OT increased by 3.95 ± 0.23- and 3.71 ± 0.22-fold, respectively, followed by a decline to about double the levels in normal (150 mM) sodium (P ⩽ 0.1).3.Compared to neurosecretosomes perfused in normal (150 mM) sodium, omission of sodium from the medium augmented ionomycin-induced VP and OT secretion by 66 ± 5- and 20 ± 3-fold, respectively, and A23187-induced secretion was increased 1.3 ± 0.4- and 1.3 ± 0.1-fold (P ⩽ 0.01 for both ionophores).4.The inhibition of ionomycin-induced secretion by sodium was concentration dependent (P ⩽ 0.01 for sodium ⩾ 5 mM); the IC50 was about 10 mM sodium for both hormones, and the Hill slope was close to -1.5.The rate of45Ca efflux from neurosecretosomes showed 2.7 ± 0.1-fold stimulation upon increasing sodium from 4.5 to 150 mM (P ⩽ 0.01).6.Our results suggest that sodium inhibits basal and stimulated secretion at the nerve terminal, possibly by reducing intraterminal calcium through sodium/calcium exchange.