μ-opioid receptor-mediated inhibition of the release of radiolabelled noradrenaline and acetylcholine from rat amygdala slices

Presynaptic modulation by opioids of electrically-evoked neurotransmitter release from superfused rat amygdala slices prelabelled with [³H]noradrenaline (NA) and [¹⁴C]choline was examined. Both [³H]NA and [¹⁴C]acetylcholine release were strongly inhibited by morphine, the mixed δ/μ-receptor agonist [ -Ala², -Leu⁵]enkephalin (DADLE) and the highly selective μ-agonist [ -Ala², MePhe⁴, Gly-ol⁵]enkephalin (DAMGO), whereas the highly selective δ-agonist [ -Pen², -Pen⁵]enkephalin and the κ-agonist bremazocine were without effect. The inhibitory effects were potently antagonized by naloxone but not by the selective δ-receptor antagonist fentanylisothiocyanate. When the selective uptake inhibitor desipramine was used to prevent uptake of [³H]NA into noradrenergic nerve terminals, but sparing the uptake into dopaminergic nerve terminals, the electrically evoked release of tritium was strongly inhibited by bremazocine but not by DADLE or DAMGO.

The data indicate, that in the amygdala transmitter release from dopaminergic nerve fibres is inhibited only via activation of κ-receptors, whereas transmitter release from noradrenergic and cholinergic nerve fibers is subjected to inhibition by opioids via activation of μ-receptors only. Regional differences and similarities of modulation of neurotransmitter release by opioids in the rat brain are briefly discussed.     

μ Receptors and opioid cardiovascular effects in the NTS of rat

In order to assess the potential role of mu (μ) and delta (δ) opiate receptors in the central regulation of the cardiovascular and respiratory systems, the cardiovascular and respiratory effects of the relatively selective μ-opioid agonist D-Ala², MePhe⁴, Gly-ol⁵ enkephalin (DAGO) and relatively selective δ-agonist D-Ala²-D-Leu⁵ enkephalin (DADL) were compared following microinjection of these compounds into the nucleus tractus solitarius of pentobarbital-anesthetized rats. Both opioid agonists produced dose dependent increases in systolic and diastolic blood pressure as well as heart rate; but DAGO was nearly ten times more potent in eliciting these changes. Respiratory rate was increased by DADL and by lower doses of DAGO, but was depressed by higher doses of DAGO. Tidal volume was depressed by both peptides. These data support the concept that the cardiovascular pressor responses and tachycardia as well as the respiratory effects of opioids in the rat NTS are mediated by μ receptors.     

Regional interactions of opioid peptides at μ and δ sites in rat brain

Opiate binding sites in five brain regions were labeled with the μ and δ markers, ³H-morphine and ³H-[D-Ala²,D-leu⁵]enkephalin, respectively. The highest densities of both ³H-morphine and ³H-DADLE labeled sites are found in striatum and frontal cortex. Hypothalamus and midbrain contain predominantly ³H-morphine labeled sites. The selectivity of the opioid peptides [D-Ala²,D-leu⁵]enkephalin, β-endorphin and dynorphin(1–13) for the two opiate sites was investigated by comparing the potency of these unlabeled compounds against the μ and δ markers in different brain regions. This determination has the effect of controlling for the breakdown of peptides within each region. While the enkephalin analogue shows a preference for the δ binding site and β-endorphin is more nearly equipotent towards the two binding sites, dynorphin(1–13) shows a high affinity and selective preference for the μ binding site over the δ site. The potency of the opioid peptides in displacing the μ and δ markers varies from region to region according to the relative densities of the two opiate binding site populations.     

Effect of Chronic Administration of Morphine, U-50, 488H and [D-Pen, D-Pen]enkephalin on the Concentration of cGMP in Brain Regions and Spinal Cord of the Mouse

Bhargava, H. N. and Y. J. Cao. Effect of chronic administration of morphine, U-50,488H and [ -Pen², -Pen⁵]enkephalin on the concentration of cGMP in brain regions and spinal cord of the mouse. Peptides 18(10) 1629–1634, 1997.—The effects of chronic administration and subsequent withdrawal of μ-, κ- and δ-opioid receptor agonists on the levels of cyclic GMP in several brain regions and spinal cord of mice were determined in an attempt to further study the role of NO cascade in opioid actions. The agonists at μ-, κ- and δ-opioid receptor included morphine, U-50,488H and DPDPE, respectively. Tolerance to morphine was associated with highly significant increases in cGMP levels in corpus striatum (41%), cortex (36%), midbrain (73%) and cerebellum (51%) relative to controls. Abstinence caused increases in cGMP levels in corpus striatum (61%) and pons and medulla (45%). Tolerance to U-50,488H resulted in increases in cGMP levels in midbrain (52%) whereas abstinence from U-50,488H increased the cGMP levels in pons and medulla(76%). Tolerance to DPDPE was associated with increases in cGMP levels in hypothalamus (12%) and pons and medulla (33%) but decreases in cerebellum (66%) and spinal cord (58%). Abstinence from DPDPE produced increases in cGMP levels in pons and medulla (14%) but decreases in cerebellum (67%) and spinal cord (50%). Overall treatment with morphine and U-50,488H produced increases in cGMP levels in brain regions whereas DPDPE produced decreases in brain regions and spinal cord. Previous studies have shown that chronic administration of μ- and κ- opioid receptor agonists induce NO synthase (NOS) in certain brain regions and that the inhibitors of NO synthase attenuate tolerance to μ- and κ- but not to δ-opioid receptors agonists. Since activation of NO increases the production of cGMP, the present results demonstrating alterations of cGMP levels by μ-, κ- and δ-opioid receptor agonists are consistent with the behavioral results with NOS inhibitors on tolerance to μ-, κ- and δ-opioid receptor agonists.     

Selective labeling of κ2 opioid receptors in rat brain by [I]IOXY: Interaction of opioid peptides and other drugs with multiple κ2a binding sites

Recent studies from our laboratory resolved two subtypes of the κ₂ binding site, termed κ_2a and κ_2b, using guinea pig, rat, and human brain membranes depleted of μ and δ receptors by pretreatment with the site-directed acylating agents BIT (μ-selective) and FIT (δ-selective). 6β-Iodo-3,14-dihydroxy-17-cyclopropylmethyl-4,5-epoxymorphinan (IOXY), an opioid antagonist that has high affinity for κ₂ sites, was radioiodinated to maximum specific activity (2200 Ci/mmol) and purified by high pressure liquid chromotography and used to characterize multiple κ₂ binding sites. The results indicated that [¹²⁵I]IOXY, like [³H]bremazocine, selectively labels κ₂ binding sites in rat brain membranes pretreated with BIT and FIT. Using 100 nM [ -Ala²-MePhe⁴,Gly-ol⁵]enkephalin to block [¹²⁵I]IOXY binding to the κ_2b site, two subtypes of the κ_2a binding site were resolved, both in the absence and presence of 50 μM 5′-guanylyimidodiphosphate. Viewed collectively, these results provide further evidence for heterogeneity of the κ opioid receptor, which may provide new targets for drug design, synthesis, and therapeutics.     

Chronic intracerebroventricular infusion of the antiopioid peptide, Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2 (NPFF), downregulates mu opioid binding sites in rat brain

Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2 (NPFF), an endogenous mammalian antiopioid peptide, has been shown by other laboratories to attenuate the acute antinociceptive effects of morphine, the development of morphine tolerance, and naloxone-induced withdrawal in morphine-dependent rats. The present study determined the effect of chronic NPFF on mu opioid receptors and mRNA for the endogenous opioids dynorphin and enkephalin. Rats received ICV infusions of either saline or NPFF (5 μg/h) for 13 days via Alzet 2002 osmotic minipumps. Homogenate binding studies, which used whole brain membranes, demonstrated that NPFF decreased the B_max of mu binding sites (labeled by [³H][ -Ala²-MePhe⁴,Gly-ol⁵]enkephalin) from 262 ± 12 to 192 ± 12 fmolmg protein, and increased the K_d from 1.1 to 2.3 nM. Quantitative receptor autoradiography and in situ hybridization experiments were conducted with sections collected at the level of the striatum. The density of mu opioid binding sites labeled by [³H][ -Ala²-MePhe⁴,Gly-ol⁵]enkephalin was decreased in all brain areas measured except the corpus callosum, and there was no change in dynorphin mRNA or enkephalin mRNA in the caudate, the nucleus accumbens, or the ventral pallidum. Rats chronically administered ICV morphine sulfate (20 μg/h) for 14 days developed tolerance to morphine and a low degree of dependence, as measured by naloxone-precipitated withdrawal. Chronic administration of NPFF concurrently with morphine sulfate did not significantly alter naloxone-induced withdrawal signs or the development of morphine tolerance. Viewed collectively with previous findings that chronic ICV infusion of anti-NPFF IgG upregulates mu receptors, these data provide additional evidence that the density of CNS mu receptors is tonically regulated by NPFF in the extracellular fluid. The action of NPFF to decrease mu receptors is consistent with an antiopioid role for this peptide; however, the fact that NPFF (administered into the lateral ventricle) did not appreciably alter expression of morphine tolerance and dependence contrasts with previous findings and reinforces the view that this effect is most reliably seen after third ventricle administration.     

Passage of a δ-Opioid Receptor Selective Enkephalin, [d-Penicillamine2,5]Enkephalin, Across the Blood-Brain and the Blood-Cerebrospinal Fluid Barriers

Abstract: [ d -Penicillamine^2,5]enkephalin (DPDPE) is an enzymatically stable, δ-opioid receptor-selective peptide, which produces analgesia when given intracerebroventricularly. However, because only modest analgesic effects were seen after subcutaneous administration of DPDPE, it has been inferred that it does not cross the blood-brain barrier well. In this present study, a vascular brain perfusion technique in anesthetized rats was used to measure directly whether [³H]DPDPE could cross the blood-brain and/or the blood-CSF barriers. The results indicated that the brain uptake of [³H]DPDPE was significantly greater than that of [¹⁴C]sucrose, a vascular marker ( p < 0.01), and than that of [³H]DPDPE into the CSF ( p < 0.01). Furthermore, HPLC analysis confirmed the integrity of the ³H to DPDPE and demonstrated that intact [³H]DPDPE entered the brain. Although 1 m M leucine-enkephalin failed to inhibit uptake of [³H]DPDPE, unlabeled DPDPE (100 µ M ) caused a significant inhibition of the brain uptake ( p < 0.01) but not the CSF uptake of [³H]DPDPE. These data provide evidence that intact [³H]DPDPE enters the CNS of anesthetized rats by saturable and nonsaturable mechanisms. In addition, the saturable mechanism is likely to be found at the blood-brain barrier, with the blood-CSF barrier playing only a minor role in the brain uptake of this peptide.     

Probing the opioid receptor complex with (+)-trans-SUPERFIT. II. Evidence that mu ligands are noncompetitive inhibitors of the delta cx opioid peptide binding site.

Previous studies delineated two classes of δ binding sites; a δ binding site not associated with the opioid receptor complex, termed the δ_ncx site, and a δ site associated with the opioid receptor complex, termed the δ_cx site. The δ_ncx site has high affinity for [ -Pen², -Pen⁵]enkephalin, and is synonymous with what is now identified as the δ₁ binding site. Pretreatment of membranes with the δ-selective acylating agents FIT, or (+)-trans-SUPERFIT, deplete membranes of the δ_ncx binding site, which permits the selective labeling of the δ_cx binding site with [³H][ -Ala²,Leu⁵]enkephalin. The present study compared the properties of the δ_cx binding site present in brain membranes pretreated with (+)-trans-SUPERFIT with the properties of the δ_cx site present in untreated membranes. The major findings are: 1) pretreatment of membranes with (+)-trans-SUPERFIT decreased the IC₅₀ values of δ-preferring drugs, and increased the IC₅₀ values of μ-preferring drugs, for the δ_cx binding site; 2) the degree of δ selectivity was highly correlated with the magnitude of the (+)-trans-SUPERFIT-induced shift in the IC₅₀ values; 3) the ligand-selectivity patterns of the μ and δ_cx sites present in (+)-trans-SUPERFIT-pretreated membranes were poorly correlated; 4) whereas μ-preferring drugs were noncompetitive inhibitors of [³H][ -Ala²,Leu⁵]enkephalin binding to the δ_cx site, δ-preferring drugs were competitive inhibitors. Viewed collectively, these data support the hypothesis that the μ and δ_cx binding sites are distinct, provide additional evidence for δ receptor heterogeneity, and suggest that ( (+)-trans-SUPERFIT-pretreated membranes will provide a useful preparation for studying the δ_cx binding site.     

Differentiation of [H]phencyclidine and ( + )-[H]SKF-10,047 binding sites in rat cerebral cortex

The potency of a series of opioid and non-opioid psychotomimetic drugs to inhibit the specific binding of [³H]PCP and ( + )-[³H]SKF-10,047 to rat cerebral cortical membranes was examined. ( + )-PCMP, the 3-methylpiperidino analog of PCP, was a potent inhibitor of the specific binding of both ligands. All of the other 12 compounds examined, however, displayed a 3-277-fold selectivity for either [³H]PCP or (+)-[³H]SKF-10,047 binding. These results suggest that although these opioid and non-opioid psychotomimetics bind to both sites, most have significantly different affinities. The binding sites for [³H]PCP appear to be distinct from the ‘sigma’ binding sites labeled with (+)-[³H]SKF-10,047.

SKF-10,047 Sigma receptor Phencyclidine Phencyclidine receptor Psychotomimetic activity     

Endogenous heptapeptide Met-enkephalin-Gly-Tyr binds differentially to duplicate delta opioid receptors from zebrafish

Met-enkephalin-Gly-Tyr (MEGY) is an endogenous peptide that binds to opioid sites in zebrafish and in rat brain homogenates. The aim of this work is to characterize the binding profile of this opioid ligand on two duplicate delta receptors from zebrafish, ZFOR1 and ZFOR4. Our results show that, while ZFOR1 presents one single binding site for [³H]-MEGY (K_D = 4.0 ± 0.4 nM), the experimental data from ZFOR4 fit better to the two-site binding model (K_D1 = 0.8 ± 0.2 nM and K_D2 = 30.2 ± 10.2 nM). Two other MEGY synthetic analogues, (D-Ala²)-MEGY and (D-Ala², Val⁵)-MEGY were also prepared and tested, together with the original peptide MEGY and other opioid ligands, in competition binding assays. While these peptides presented K_i values on the nanomolar range when using [³H]-MEGY as radioligand, these parameters were two orders higher in competition binding assays with the antagonist [³H]-diprenorphine. Functional [³⁵S]GTPγS stimulation analysis has revealed that these two receptors can be activated by several opioid agonists. Our results prove that although the MEGY peptide acts as an agonist on ZFOR1 and ZFOR4, there are subtle pharmacological differences between these two delta opioid receptors from zebrafish.     

Toxicity and mutagenicity of X-rays and [125I]dUrd or [3H]TdR incorporated in the DNA of human lymphoblast cells

We measured the toxicity and mutagenicity induced in human diploid lymphoblasts by various radiation doses of X-rays and two internal emitters. [¹²⁵I]iododeoxyuridine ([¹²⁵I]dUrd) and [³H]thymidine ([³H]TdR), incorporated into cellular DNA. [¹²⁵I]dUrd was more effective than [³H]TdR at killing cells and producing mutations to 6-thioguanine resistance (6TG^R). No ouabain-resistant mutants were induced by any of these agents. Expressing dose as total disintegrations per cell (dpc), the D₀ for cell killing for [¹²⁵I]dUrd was 28 dpc and for [³H]TdR was 385 dpc. The D₀ for X-rays was 48 rad at 37°C. The slopes of the mutation curves were approximately 75 × 10⁻⁸ 6TG^R mutants per cell per disintegration for [¹²⁵I]dUrd and 2 × 10⁻⁸ for [³H]TdR. X-Rays induced 8 × 10⁻⁸ 6TG^R mutants per cell per rad. Normalizing for survival, [¹²⁵I]dUrd remained much more mutagenic at low doses (high survival levels) than the other two agents. Treatment of the cells at either 37°C or while frozen at −70°C yielded no difference in cytotoxicity or mutation for [¹²⁵I]dUrd or [³H]TdR, whereas X-rays were 6 times less effective in killing cells at −70°C.

Assuming that incorporation was random throughout the genome, the mutagenic efficiencies of the radionuclides could be calculated by dividing the mutation rate by the level of incorporation. If the effective target size of the 6TG^R locus is 1000–3000 base pairs, then the mutagenic efficiency of [¹²⁵I]dUrd is 1.0–3.0 and of [³H]TdR is 0.02–0.06 total genomic mutations per cell per disintegration. ¹²⁵I disintegrations are known to produce localized DNA double-strand breaks. If these breaks are potentially lethal lesions, they must be repaired, since the mean lethal dose (D₀) was 28 dpc. The observations that a single dpc has a high probability of producing a mutation (mutagenic efficiency 1.0–3.0) would suggest, however, that this repair is extremely error-prone. If the breaks need not be repaired to permit survival, then lethal lesions are a subset of or are completely different from mutagenic lesions.     

Synthesis, radiolabeling and receptor binding of [3H][(1S,2R)ACPC2]endomorphin-2

Previously, we have shown that substitution of Pro² for cis-2-aminocyclopentanecarboxylic acid, ACPC in endomorphin-2 results in an analogue with greatly augmented proteolytic stability, high μ-opioid receptor affinity and selectivity. We now report the synthesis and biochemical characterization of [³H][(1S,2R)ACPC²]endomorphin-2 with a specific activity of 1.41 TBq/mmol (38.17 Ci/mmol). Specific binding of [³H][(1S,2R)ACPC²]endomorphin-2 was saturable and of high affinity with an equilibrium dissociation constant, K_d = 1.80 ± 0.21 nM and receptor density, B_max = 345 ± 27 fmol × mg protein⁻¹ at 25 °C in rat brain membranes. Similar affinity values were obtained in kinetic and displacement assays. Both Na⁺ and Gpp(NH)p decreased the affinity proving the agonist character of the radioligand. [³H][(1S,2R)ACPC²]endomorphin-2 retained the μ-specificity of the parent peptide. The new radioligand will be a useful tool to map the topographical requirements of μ-opioid peptide binding due to its high affinity, selectivity and enzymatic stability.     

A differential interaction of putative μ-selective agonists with opiate binding sites in rat brain

The availability of tritium-labelled sufentanil ([³H]SUF) allowed for a further radioligand analysis of opiate binding sites in rat brain. A comparison of the binding characteristics of [³H]SUF and [³H]dihydromorphine ([³H]DHM) revealed a very similar potency in their mutual displacement by unlabelled analogues. Furthermore, a series of putative μ-opiate agonists displayed equal potencies in displacing either [³H]SUF and [³H]DHM, the only striking exception being the highly μ-selective opioid peptide morphiceptin which was 33 times less potent in inhibiting [³H]SUF as compared to [³H]DHM binding. Additional experiments revealed further pronounced differences in [³H]SUF and [³H]DHM binding characteristics: the total amount of binding sites for [³H]SUF was 4 times higher than that for [³H]DHM and the regional distribution within particular brain areas displayed considerable differences. Furthermore, the binding of [³H]SUF was differentially modulated by sodium and GTP as compared to [³H]DHM binding. These data suggest that in rat brain, [³H]SUF interacts both with μ-opiate sites recognizing [³H]DHM and another type of opiate site, which cannot be equated with any of the, as yet, described δ- or κ-binding sites, and rather, represents a subclass of μ-opiate receptor sites. These experiments, thus, support the notion of subclasses (isoreceptors) for different types of opiate receptors.     

Effect of hypoxia and glucose deprivation on ATP level, adenylate energy charge and [Ca]0-dependent and independent release of [H]dopamine in rat striatal slices

Release of [³H]dopamine ([³H]DA) from rat striatal slices kept under hypoxic or/and glucose-free conditions was measured using a microvolume perfusion method. The corresponding changes in nucleotide content were determined by reverse-phase high-performance liquid chromatography (RPHPLC). The resting release of [³H]DA was not affected by hypoxia, but under glucose-free conditions massive [Ca²⁺]₀-independent release of [³H]DA was observed. Hypoxia reduced the energy charge (E.C.) and the total purine content from 19.36 ± 4.15 to 6.98 ± 1.83 mol/mg protein. Glucose deprivation by itself, or in combination with hypoxia, markedly reduced the levels of adenosine 5′-triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP). The E.C. under glucose-free conditions was significantly reduced from 0.73 ± 0.04 to 0.44 ± 0.20. When the tissue was exposed to hypoxic and glucose-free conditions for 18 min the level of ATP was reduced to 3.15 ± 0.11 mol/mg protein. However, when the exposure time was 30 min the ATP level was further reduced to 1.11 ± 0.37 nmol/mg protein. The resting release was enhanced in a [Ca²⁺]₀-independent manner, but there was no release in response to stimulation, and tetrodotoxin did not affect the enhanced resting release, indicating that the release was not associated with axonal activity. Similarly, 50 μM ouabain, inhibitor of Na⁺/K⁺-activated ATPase, enhanced the release of [³H]DA at rest in a [Ca²⁺]₀-independent manner. It seems very likely that the reduced ATP level under glucose-free conditions leads to an inhibition of the activity of Na⁺/K⁺-ATPase that results in reversal of the uptake processes and in [Ca²⁺]₀-independent [³H]DA release from the axon terminals.     

Synthesis and preliminary evaluation of [3H]PSB-0413, a selective antagonist radioligand for platelet P2Y12 receptors

The selective antagonist radioligand [³H]2-propylthioadenosine-5′-adenylic acid (1,1-dichloro-1-phosphonomethyl-1-phosphonyl) anhydride ([³H]PSB-0413) was prepared by catalytic hydrogenation of its propargyl precursor with a high specific radioactivity of 74 Ci/mmol. In preliminary saturation binding studies, [³H]PSB-0413 showed high affinity for platelet P2Y₁₂ receptors with a K_D value of 4.57 nM. Human platelets had a high density of P2Y₁₂ receptors exhibiting a B_max value of 7.66 pmol/mg of protein.     

Synthesis and antitumor activity of a new class of pyrazolo[4,3-e]pyrrolo[1,2-a][1,4]diazepinone analogs of pyrrolo[1,4]benzodiazepines (PBDs)

A new class of Pyrrolo[1,4]benzodiazepines (PBDs) analogs featuring a pyrazolo[4,3-e]pyrrolo[1,2-a][1,4]diazepinone ring system has been designed and synthesized. In these compounds the A-benzene ring, characteristic of PBDs, has been replaced by a dimethylpyrazole ring, a modification suggested by modelling studies performed on the PBD base structure. Biological evaluation releaved appreciable antitumor activity for compounds 14 and 15 (8.84–22.4 μM) which encourages further investigation of the N⁶ and N⁷ alkyl pyrazole analogs.     

High affinity binding of [H]neurotensin to rat uterus

[³H]Neurotensin (NT) was found to bind specifically and with high affinity to crude membranes prepared from rat uterus. Scatchard analysis of saturation binding studies indicated that [³H]NT apparently binds to two sites (high affinity Kd 0.5 nM; low affinity Kd 9 nM) with the density of high affinity sites (41 fmoles/mg prot.) being about one-third that of the low affinity sites (100 fmoles/mg prot.). In competition studies, NT and various fragments inhibited [³H]NT binding with the following potencies (IC₅₀): NT 8–13 (0.4 nM), NT 1–13 (4 nM), NT 9–13 (130 nM), NT 1–11, NT 1–8 (>100 μM). Quantitatively similar results were obtained using brain tissue. These findings raise the possibility of a role for NT in uterine function.     

[D-Ala, Met]-Enkephalinamide: CNS-mediated inhibition of prostaglandin-stimulated intestinal fluid and ion transport in the rat

The opioid peptide [D-Ala², Met⁵]-enkephalinamide (DAMA), a non-selective opioid agonist, has previously been shown to inhibit cholera toxin-induced fluid accumulation in the rat and dog small intestine after its intracerebroventricular (ICV) administration. In the present study, we examined the time course of the antisecretory/proabsorptive effects of ICV DAMA on net fluid and ion transport across the rat jejunum in situ during intravenous prostaglandin E₁ (PGE) infusion. Net water and NaCl absorption were measured using a standard dilution marker technique in a 15–20 cm segment of proximal jejunum in urethaneanesthetized Sprague-Dawley rats. Infusion of PGE (5 μg/kg-min) over a 2 hr period produced a decrease in fluid and ion absorption that plateaued to a steady-state within 60 min. DAMA (1 and 3 μg/rat) administered by ICV bolus 60 min after the start of PGE infusion inhibited significantly PGE-induced decreases in water and chloride absorption relative to saline-treated controls. These dose-related peptide effects were expressed 15 min after DAMA treatment and were approximately 30 min in duration; they were antagonized by naloxone (1 mg/kg, IV) given at the time of DAMA injection. These results indicate that low concentrations of DAMA administered into the central nervous system rapidly and effectively inhibit changes in intestinal transport induced by a blood-borne secretagogue through an interaction with opiate receptors.     

Characterization of proctolin binding sites on locust oviduct membranes

The binding of [³H]proctolin to oviduct membranes has been analyzed to investigate the nature of proctolin binding sites in the oviduct. Proctolin binding was found to be time dependent, proportional to concentration of membrane protein, saturable, specific and reversible. Two apparent proctolin binding sites were recognized. The first had a K_d of 400 ± 82 nM and a B_max of 23.7 ± 6.7 pmol/mg protein. The second had a K_d of 2.4 ± 0.2 μM and a B_max of 96.3 ± 16.7 pmo/mg protein.

Binding was specific in thatcompetition experiments with a wide range of peptides showed that only Arg-Tyr-Leu-Pro-Ala was an effective competitor at μM concentrations. All other peptides examined weekly reduced proctolin binding at concentrations above 50 μM. Certain peptides were found to potentiate [³]pproctolin binding at low μM concentrations (1–10 μM) and to inhibit proctolin binding at higher concentrations. The significance of these findings is discussed.     

Phosphoinositide hydrolysis increase by angiotensin-(1--7) in neonatal rat brain

Angiotensin (Ang)-(1–7) is an endogenous peptide hormone of the renin–angiotensin system which exerts diverse biological actions, some of them counterregulate Ang II effects. In the present study potential effect of Ang-(1–7) on phosphoinositide (PI) turnover was evaluated in neonatal rat brain. Cerebral cortex prisms of seven-day-old rats were preloaded with [³H]myoinositol, incubated with additions during 30 min and later [³H]inositol-phosphates (IPs) accumulation quantified. It was observed that PI hydrolysis enhanced 30% to 60% in the presence of 0.01 nM to 100 nM Ang-(1–7). Neither 10 nM [D-Ala⁷]Ang-(1–7), an Ang-(1–7) specific antagonist, nor 10 nM losartan, an angiotensin II type 1 (AT₁) receptor antagonist, blocked the effect of 0.1 nM Ang-(1–7) on PI metabolism. The effect of 0.1 nM Ang-(1–7) on PI hydrolysis was not reduced but it was even significantly increased in the simultaneous presence of [D-Ala⁷]Ang-(1–7) or losartan. PI turnover enhancement achieved with 0.1 nM Ang-(1–7) decreased roughly 30% in the presence of 10 nM PD 123319, an angiotensin II type 2 (AT₂) receptor antagonist. The antagonists alone also enhanced PI turnover. Present findings showing an increase in PI turnover by Ang-(1–7) represent a novel action for this peptide and suggest that it exerts a function in this signaling system in neonatal rat brain, an effect involving, at least partially, angiotensin AT₂ receptors.