Defective prodynorphin processing in mice lacking prohormone convertase PC2

Prodynorphin, a multifunctional precursor of several important opioid peptides, is expressed widely in the CNS. It is processed at specific single and paired basic sites to generate various biologically active products. Among the prohormone convertases (PCs), PC1 and PC2 are expressed widely in neuroendocrine tissues and have been proposed to be the major convertases involved in the biosynthesis of hormonal and neural peptides. In this study we have examined the physiological involvement of PC2 in the generation of dynorphin (Dyn) peptides in mice lacking active PC2 as a result of gene disruption. Enzymological and immunological assays were used to confirm the absence of active PC2 in these mice. The processing profiles of Dyn peptides extracted from brains of these mice reveal a complete lack of Dyn A-8 and a substantial reduction in the levels of Dyn A-17 and Dyn B-13. Thus, PC2 appears to be involved in monobasic processing, leading to the generation of Dyn A-8, Dyn A-17, and Dyn B-13 from prodynorphin under physiological conditions. Brains of heterozygous mice exhibit only half the PC2 activity of wild-type mice; however, the levels of Dyn peptides in these mice are similar to those of wild-type mice, suggesting that a 50% reduction in PC2 activity is not sufficient to significantly reduce prodynorphin processing. The disruption of the PC2 gene does not lead to compensatory up-regulation in the levels of other convertases with similar substrate specificity because we find no significant changes in the levels of PC1, PC5/PC6, or furin in these mice as compared with wild-type mice. Taken together, these results support a critical role for PC2 in the generation of Dyn peptides.     

Opioid and other peptides as inhibitors of leumorphin (dynorphin B-29) converting activity

A thiolprotease from rat brain membranes was shown to convert synthetic dynorphin B-29 (Dyn B-29, "leumorphin") to the tridecapeptide dynorphin B (Dyn B, "rimorphin"). This represents a "single-arginine cleavage" between threonine-13 and arginine-14 of the substrate. The dynorphin converting activity displayed typical Michaelis-Menten kinetics with an apparent Km for the substrate of 0.58 microM. Surprisingly, a synthetic peptide, Dyn B-29-(9-22), which contains the cleavage site, did not inhibit the activity. Dyn A inhibited the activity competitively with an apparent Ki of 3.7 microM. The converting activity was also inhibited by Dyn A-(6-17) but not by Dyn A-(8-17), suggesting a role of Arg6-Arg7 in the inhibition of converting activity. Bovine adrenal medulla Peptide E inhibited the converting activity substantially whereas metorphamide did not, suggesting the importance of COOH-terminal residues in recognition. Beta-Endorphin was an effective inhibitor of converting activity, and [alpha-N-acetyl]beta-endorphin was not, indicating a crucial role of the free NH2-terminus in recognition by the enzyme. ACTH inhibited the activity competitively with an apparent Ki of 39 nM. The converting activity was also inhibited substantially by ACTH-(1-13) but not by alpha-MSH, again indicating a requirement of the free NH2-terminus for recognition. The above results suggest that the converting enzyme recognizes peptides of the three known opioid gene families.     

A-18-famide and F-8-famide, endogenous mammalian equivalents of the molluscan neuropeptide FMRFamide (Phe-Met-Arg-Phe-NH2), inhibit colonic bead expulsion time in mice

Morphine and the two endogenous mammalian FMRFamide (Phe-Met-Arg-Phe-NH2)-related peptides known as morphine-modulating neuropeptides, F-8-Famide (Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2) and A-18-Famide (Ala-Gly-Glu-Gly-Leu-Ser-Ser-Pro-Phe-Trp-Ser-Leu-Ala-Ala-Pro-Gln-Arg-Phe -NH2), were administered intracerebroventricularly (ICV) to mice and the effect of each on colonic bead expulsion time was measured. Each of the three compounds delayed expulsion of a 3 mm glass bead placed in the distal colon. A-18-Famide was more potent than F-8-Famide [ED 50 = 2.3 micrograms (1.2 nmole) and 13.9 micrograms (13.0 nmole), respectively]. A-18-Famide: 1) did not block morphine-induced delay of bead expulsion time, and 2) was blocked by simultaneous administration (ICV) of 1.0 microgram of the competitive opiate antagonist naloxone. These data demonstrate apparent opioid modulatory or agonist-like, rather than antagonist-like, properties of A-18-Famide and F-8-Famide.     

Inhibition of thimet oligopeptidase by siRNA alters specific intracellular peptides and potentiates isoproterenol signal transduction

Mammalian cells have a large number of intracellular peptides that are generated by extralysosomal proteases. In this study, the enzymatic activity of thimet oligopeptidase (EP24.15) was inhibited in human embryonic kidney (HEK) 293 cells using a specific siRNA sequence. The semi-quantitative intracellular peptidome analyses of siRNA-transfected HEK293 cells shows that the levels of specific intracellular peptides are either increased or decreased upon EP24.15 inhibition. Decreased expression of EP24.15 was sufficient to potentiate luciferase gene reporter activation by isoproterenol (1-10 μM). The protein kinase A inhibitor KT5720 (1 μM) reduced the positive effect of the EP24.15 siRNA on isoproterenol signaling. Thus, EP24.15 inhibition by siRNA modulates the levels of specific intracellular peptides and isoproterenol signal transduction.     

Dynorphin peptides differentially regulate the human kappa opioid receptor

Dynorphins, endogenous peptides for the kappa opioid receptor, play important roles in many physiological and pathological functions. Here, we examined how prolonged treatment with three major prodynorphin peptides, dynorphin A (1-17) (Dyn A), dynorphin B (1-13) (Dyn B) and alpha-neoendorphin (alpha-Neo), regulated the human kappa opioid receptor (hKOR) stably expressed in Chinese hamster ovary (CHO) cells. Results from receptor binding and [(35)S]GTPgammaS binding assays showed that these peptides were potent full agonists of the hKOR with comparable receptor reserve and intrinsic efficacy to stimulate G proteins. A 4-h incubation with alpha-Neo at a concentration of approximately 600xEC(50) value (from [(35)S]GTPgammaS binding) resulted in receptor down-regulation to a much lower extent than the incubation with Dyn A and Dyn B at comparable concentrations ( approximately 10% vs. approximately 65%). Extending incubation period and increasing concentrations did not significantly affect the difference. The plateau level of alpha-Neo-mediated receptor internalization (30 min) was significantly less than those of Dyn A and Dyn B. Omission of the serum from the incubation medium or addition of peptidase inhibitors into the serum-containing medium enhanced alpha-Neo-, but not Dyn A- or Dyn B-, mediated receptor down-regulation and internalization; however, the degrees of alpha-Neo-induced adaptations were still significantly less than those of Dyn A and Dyn B. Thus, these endogenous peptides differentially regulate KOR after activating the receptor with similar receptor occupancy and intrinsic efficacy. Both stability in the presence of serum and intrinsic capacity to promote receptor adaptation play roles in the observed discrepancy among the dynorphin peptides.     

Dynorphin A analogs containing a conformationally constrained phenylalanine derivative in position 4: reversal of preferred stereochemistry for opioid receptor affinity and discrimination of kappa vs. delta receptors

Analogs of the opioid peptide [D-Ala8]dynorphin A-(1-11)NH2 containing optically pure (R)- and (S)-2-aminotetralin-2-carboxylic acid (Atc) in position 4 were synthesized and evaluated for opioid receptor affinity. These peptides are the first reported dynorphin A analogs containing a conformationally constrained amino acid in place of the important aromatic residue Phe4. By incorporating resolved Atc isomers, the opioid receptor affinity and the stereochemistry of the constrained residue could be unambiguously correlated. Both Dyn A analogs containing Atc in position 4 retained nanomolar affinity for kappa and mu opioid receptors. Unexpectedly the peptide containing (R)-Atc, corresponding to a conformationally constrained D-Phe analog, displaying higher affinity for both kappa and mu receptors than the peptide containing (S)-Atc. In contrast [D-Phe4,D-Ala8]Dyn A-(1-11)NH2 exhibited significantly lower affinity for kappa and mu receptors than the parent peptide, as expected. Conformational restriction of the Phe4 sidechain or incorporation of D-Phe in position 4 had the largest effect on delta receptor affinity, yielding compounds with negligible affinity for these receptors. Thus, there appear to be distinctly different structural requirements for this residue for kappa vs. delta receptors, and it is possible to completely distinguish between these two receptors by changing a single residue in Dyn A.     

Site and mechanism of action of dynorphin A-(1-13) and N-methyl-D-aspartate on ACTH release in fetal sheep

Dynorphin A (Dyn A) stimulates the release of ACTH in fetal sheep, a response that involves N-methyl-D-aspartate (NMDA) receptors but not the secretogogues corticotropin-releasing hormone or arginine vasopressin. We now find that neither Dyn A-(1-13) (0.5 mg/kg, i.v.) nor NMDA (4 mg/kg, i.v.) elicits ACTH release in postnatal lambs. This led us to hypothesize that Dyn A-(1-13) and NMDA might act to release placental ACTH. However, the ability of Dyn A-(1-13), NMDA, and the kappa-opioid receptor agonist U-50488H (1 mg/kg, i.v.) to release ACTH was lost after either fetal hypophysectomy (n = 4) or hypothalamo-pituitary disconnection (n = 4). These results indicate that neither the placenta nor the fetal pituitary is the site of action for these agonists and suggest a hypothalamic or suprahypothalamic site of action. Furthermore, the release of ACTH by Dyn A-(1-13) and NMDA was abolished after pretreatment with indomethacin, suggesting that they might cause the release of a prostanoid, possibly from the placenta, that subsequently acts at the hypothalamus or serves as a permissive factor in the action of Dyn A-(1-13) and NMDA at the hypothalamus.     

Complex correlations between the morphology, electrophysiology and peptide immunohistochemistry of guinea-pig enteric neurones.

Neuroanatomical, electrophysiological and immunohistochemical techniques were used to describe correlations between soma morphology and electrophysiological properties in two groups of guinea-pig enteric neurones posing particular challenges. Lucifer Yellow-staining of 542 myenteric plexus neurones of duodenum revealed a great diversity of neuronal morphology. The distribution was: Dogiel Type I 27%, Dogiel Type II 54%, Stach Type IV 9%; 10% were unclassified. Correlations were sought in 59 of these cells between morphology and electrophysiological properties but no particular association was recognised. Dynorphin A(1-8)-like immunoreactivity (Dyn A(1-8)-IR) was found in up to 90% of identified submucous neurones of guinea-pig ileum. Of 62 S-neurones, 41 showed 'weak' and 19 had 'intense' Dyn A (1-8)-IR. There was no evidence of Dyn A(1-8)-IR in 2 S-neurones, nor in 8/8 AH-neurones. As for 11/16 vasoactive intestinal peptide- (VIP-) IR neurones, there was a strong correlation between the presence of 'weak' Dyn A(1-8)-IR and the occurrence of inhibitory (IPSPs) and slow excitatory synaptic potentials (EPSPs) (13/16 cells tested), which were never observed in neurones with 'intense' Dyn A(1-8)-IR (16/16) or neuropeptide Y (NPY)-IR (8/8). Similarly, 7/7 neurones with 'weak' Dyn A(1-8)-IR, but not those (7/7) with 'intense' Dyn A(1-8)-IR, hyperpolarised or showed a conductance change to noradrenaline. It was concluded that dynorphin A(1-8)-like-IR was contained in two populations of submucous neurone that are anatomically, immunohistochemically, electrophysiologically and pharmacologically distinct and closely related to those containing VIP and NPY. Furthermore, as in the myenteric plexus throughout the small intestine, opioid peptides are not expressed in Dogiel Type II cells.     

VI. Measurement of proenkephalin A-derived peptides in biological tissues by high pressure liquid chromatography coupled with amperometric electrochemical detection

We describe an analytic method for the separation and quantitation of a number of proenkephalin A-derived peptides using high pressure liquid chromatography coupled with amperometric electrochemical detection (HPLC-AECD). Initially, we coupled our HPLC separation system with AECD in series with a UV detector for additional confirmation of peak specificity. AECD provided a 10⁶ - fold increase in sensitivity over UV detection for these peptides. In addition to Met-enkephalin (ME), ME-Arg, ME-Arg-Phe, ME-Arg-Gly-Leu, Leu-enkephalin (LE) and LE-Arg (Dyn 1–6), we separated and detected the sulfoxides of ME and its extended peptides. Subsequently, we used minor modifications of the isocratic mobile phase to separate and detect enkephalin-related peptides with greater sensitivity and shorter chromatographic run times; each of these mobile phases was used to separate and detect two to three peptides. We have applied this HPLC-AECD methodology to quantitate ME, ME-Arg-Phe, ME-Arg-Gly-Leu and LE in pheochromocytoma tumors.     

Dynorphin A processing enzyme: tissue distribution, isolation, and characterization

Limited proteolysis of the dynorphin precursor (prodynorphin) at dibasic and monobasic processing sites results in the generation of bioactive dynorphins. In the brain and neurointermediate lobe of the pituitary, prodynorphin is processed to produce alpha and beta neo endorphins, dynorphins (Dyn) A-17 and Dyn A-8, Dyn B-13, and leucine-enkephalin. The formation of Dyn A-8 from Dyn A-17 requires a monobasic cleavage between Ile and Arg. We have identified an enzymatic activity capable of processing at this monobasic site in the rat brain and neurointermediate lobe of the bovine pituitary; this enzyme is designated "dynorphin A-17 processing enzyme." In the rat brain and neurointermediate lobe, a majority of the Dyn A processing enzyme activity is membrane-associated and can be released by treatment with 1% Triton X-100. This enzyme has been purified to apparent homogeneity from the membrane extract of the neurointermediate lobe using preparative iso-electrofocussing in a granulated gel pH 3.5 to 10, FPLC using anion exchange chromatography, and non-denaturing electrophoresis. The Dyn A processing enzyme exhibits a pI of about 5.8 and a molecular mass of about 65 kDa under reducing conditions. The Dyn A processing enzyme is a metalloprotease and has a neutral pH optimum. It exhibits substantial sensitivity to metal chelating agents and thiol agents suggesting that this enzyme is a thiol-sensitive metalloprotease. Specific inhibitors of other metallopeptidases such as enkephalinase [EC 3.4.24.11], the enkephalin generating neutral endopeptidase [EC 3.4.24.15], or NRD convertase do not inhibit the Dyn A processing enzyme activity. In contrast, specific inhibitors of angiotensin converting enzyme inhibit the activity. The purified enzyme is able to process a number of neuropeptides at both monobasic and dibasic sites. These characteristics are consistent with a role for the Dyn A processing enzyme in the processing of Dyn A-17 and other neuropeptides in the brain.     

Non-opioid nociceptive activity of human dynorphin mutants that cause neurodegenerative disorder spinocerebellar ataxia type 23

We previously identified four missense mutations in the prodynorphin gene that cause human neurodegenerative disorder spinocerebellar ataxia type 23 (SCA23). Three mutations substitute Leu(5), Arg(6), and Arg(9) to Ser (L5S), Trp (R6W) and Cys (R9C) in dynorphin A(1-17) (Dyn A), a peptide with both opioid activities and non-opioid neurodegenerative actions. It has been reported that Dyn A administered intrathecally (i.t.) in femtomolar doses into mice produces nociceptive behaviors consisting of hindlimb scratching along with biting and licking of the hindpaw and tail (SBL responses) through a non-opioid mechanism. We here evaluated the potential of the three mutant peptides to produce similar behaviors. Compared to the wild type (WT)-peptide, the relative potency of Dyn A R6W, L5S and R9C peptides for SBL responses was 50-, 33- and 2-fold higher, and Dyn A R6W and L5S induced the SBL responses at a 10-30-fold lower doses. Dyn A R6W was the most potent peptide. The SBL responses induced by Dyn A R6W were dose dependently inhibited by morphine (i.p.; 0.1-1 mg/kg) or MK-801, an NMDA ion channel blocker (i.t. co-administration; 5-7.5 nmol). CP-99,994, a tachykinin NK1 receptor antagonist (i.t. co-administration; 2 nmol) and naloxone (i.p.; 5 mg/kg) failed to block effects of Dyn A R6W. Thus, similarly to Dyn A WT, the SBL responses induced by Dyn A R6W may involve the NMDA receptor but are not mediated through the opioid and tachykinin NK1 receptors. Enhanced non-opioid excitatory activities of Dyn A mutants may underlie in part development of SCA23.     

Synaptosomal Membrane-Bound Form of Endopeptidase-24.15 Generates Leu-Enkephalin from Dynorphin 1-8, α- and β-Neoendorphin, and Met-Enkephalin from Met-Enkephalin-Arg6-Gly7-Leu

Brain contains a membrane-bound form of endopeptidase-24.15, a metalloendopeptidase predominantly associated with the soluble protein fraction of brain homogenates. Subcellular fractionation of the enzyme in rat brain showed that 20-25% of the total activity is associated with membrane fractions including synaptosomes. Solubilization of the enzyme from synaptosomal membranes required the use of detergents or treatment with trypsin. The specific activity of the enzyme in synaptosomal membranes measured with tertiary-butoxycarbonyl-Phe-Ala-Ala-Phe-p-aminobenzoate as substrate was higher than that of endopeptidase-24.11 ("enkephalinase"), a membrane-bound zinc-metalloendopeptidase believed to function in brain neuropeptide metabolism. Purified synaptosomal membranes converted efficiently dynorphin1-8, alpha- and beta-neoendorphin into leucine enkephalin and methionine-enkephalin-Arg6-Gly7-Leu8 into methionine enkephalin in the presence of captopril, bestatin, and N-[1-(R,S)-carboxy-2-phenylethyl]-Phe-p-aminobenzoate, inhibitors of angiotensin converting enzyme (EC 3.4.15.1), aminopeptidase (EC 3.4.11.2), and membrane-bound metalloendopeptidase (EC 3.4.24.11), respectively. The conversion of enkephalin-containing peptides into enkephalins was virtually completely inhibited by N-[1-(R,S)-carboxy-2-phenylethyl]-Ala-Ala-Phe-p-aminobenzoate, a specific active-site-directed inhibitor of endopeptidase-24.15, indicating that this enzyme was responsible for the observed interconversions. The data indicate that synaptosomal membranes contain enzymes that can potentially generate and degrade both leucine- and methionine-enkephalin.     

Synthesis and opioid activity of 2-substituted dynorphin A-(1-13) amide analogues.

A series of 2-substituted dynorphin A-(1-13) amide (Dyn A-(1-13)NH2) analogues was prepared by solid phase peptide synthesis and evaluated for opioid receptor affinities in radioligand binding assays and for opioid activity in the guinea pig ileum (GPI) assay. Amino acid substitution at the 2 position produced marked differences in both opioid receptor affinities and potency in the GPI assay; Ki values for the analogues in the radioligand binding assays and IC50 values in the GPI assay varied over three to four orders of magnitude. The parent peptide, Dyn A-(1-13)NH2, exhibited the greatest affinity and selectivity for kappa receptors and was the most potent peptide examined in the GPI assay. The most important determinant of opioid receptor selectivity and opioid potency for the synthetic analogues was the stereochemistry of the amino acid at the 2 position. Except for [D-Lys2]Dyn A-(1-13)NH2 in the kappa receptor binding assay, the analogues containing a D-amino acid at position 2 were much more potent in all of the assays than their corresponding isomers containing an L-amino acid at this position. The L-amino acid-substituted analogues generally retained some selectivity for kappa opioid receptors. The more potent derivatives with a D-amino acid in position 2, however, preferentially interacted with mu opioid receptors. Introduction of a positively charged amino acid into the 2 position generally decreased opioid receptor affinities and potency in the GPI assay.     

Big dynorphin as a putative endogenous ligand for the kappa-opioid receptor

The diversity of peptide ligands for a particular receptor may provide a greater dynamic range of functional responses, while maintaining selectivity in receptor activation. Dynorphin A (Dyn A), and dynorphin B (Dyn B) are endogenous opioid peptides that activate the kappa-opioid receptor (KOR). Here, we characterized interactions of big dynorphin (Big Dyn), a 32-amino acid prodynorphin-derived peptide consisting of Dyn A and Dyn B, with human KOR, mu- (hMOR) and delta- (hDOR) opioid receptors and opioid receptor-like receptor 1 (hORL1) expressed in cells transfected with respective cDNA. Big Dyn and Dyn A demonstrated roughly similar affinity for binding to hKOR that was higher than that of Dyn B. Dyn A was more selective for hKOR over hMOR, hDOR and hORL1 than Big Dyn, while Dyn B demonstrated low selectivity. In contrast, Big Dyn activated G proteins through KOR with much greater potency, efficacy and selectivity than other dynorphins. There was no correlation between the rank order of the potency for the KOR-mediated activation of G proteins and the binding affinity of dynorphins for KOR. The rank of the selectivity for the activation of G proteins through hKOR and of the binding to this receptor also differed. Immunoreactive Big Dyn was detected using the combination of radioimmunoassay (RIA) and HPLC in the human nucleus accumbens, caudate nucleus, hippocampus and cerebrospinal fluid (CSF) with the ratio of Big Dyn and Dyn B being approximately 1:3. The presence in the brain implies that Big Dyn, along with other dynorphins, is processed from prodynorphin and secreted from neurons. Collectively, the high potency and efficacy and the relative abundance suggest that Big Dyn may play a role in the KOR-mediated activation of G proteins.     

Metabolism of dynorphin A1–13 in human CSF

In-vitro incubation of human cerebrospinal fluid (CSF) obtained from patients ranging from 22–78 years with 10 μM of dynorphin A1–13 (Dyn A1–13) resulted in several cleavage products. Dyn A1–12 and A2–13 were identified as the major CSF metabolites by matrix-assisted laser desorption mass spectrometry (LD-MS). Further metabolites were Dyn A1–6, A2–12 and A4–12. LD-MS further suggested the formation of Dyn A1–8, A1–7, A1–10, A7–10, A3–12, A7–12, A3–13, A7–13 and A8–13. The metabolic half-life of Dyn A1–13 at 37°C was approximately 2.5 h (range 1.75–8.5 h), compared to less than one minute in plasma. The half-life of Dyn A1–13 decreased markedly with age or age-associated processes (n=20, r²=0.498). Noncompartmental kinetic analysis in the absence or presence of enzyme inhibitors (leucinethiol 10 μM, captopril 100 μM and GEMSA 20 μM) suggested that Dyn A1–13 is mainly metabolized by carboxypeptidase to A1–12 (51%) and by aminopeptidases to A2–13 (35%). The generation of A1–6 (13%) was only detected under enzyme inhibition. The extent of conversion into the main metabolites did not follow an age-associated trend, thus over-all enzyme levels but no specific enzymatic systems are elevated with age.     

Habenular modulation of dynorphinergic systems in rat ventral mesencephalon

Bilateral electrolytic lesion of the striatonigral pathways (which convey massive afferents to the substantia nigra) caused a marked lowering of alpha-neo-endorphin (alpha-Neo) and dynorphin A(1-8) [Dyn A(1-8)] levels in the substantia nigra without affecting the alpha-Neo content in the ventral tegmental area. Moreover, unilateral infusion of the axon sparing neurotoxin ibotenate into the striatum, but not into the substantia nigra, decrease these two opioid peptides in the substantia nigra on the side ipsilateral to the lesion, failing to modify the alpha-Neo levels in the ventral tegmental area. Bilateral electrolytic lesion of the habenula augmented alpha-Neo content in the substantia nigra and ventral tegmental area at 8-30 days postlesion without affecting the nigral Dyn A(1-8). These results add further support to the view that alpha-Neo- and Dyn A(1-8)-containing neurons projecting to the substantia nigra originate in the striatum and descend through striatonigral pathways. The present data provide evidence that the habenula may participate in the regulation of the activity of alpha-Neo-immunoreactive neurons in the substantia nigra and ventral tegmental area.     

PVN lesions prevent the endothelin 1-induced increase in arterial pressure and vasopressin

Endothelin (ET) acts within the central nervous system to increase arterial pressure and arginine vasopressin (AVP) secretion. This study assessed the role of the paraventricular nuclei (PVN) in these actions. Intracerebroventricular ET-1 (10 pmol) or the ET(A) antagonist BQ-123 (40 nmol) was administered in conscious intact or sinoaortic-denervated (SAD) Long-Evans rats with sham or bilateral electrolytic lesions of the magnocellular region of the PVN. Baseline values did not differ among groups, and artificial cerebrospinal fluid (CSF) induced no significant changes. In sham-lesioned rats, ET-1 increased mean arterial pressure (MAP) 15.9 +/- 1.3 mmHg in intact and 22.3 +/- 2.7 mmHg in SAD (P < 0.001 ET-1 vs. CSF) rats. PVN lesions abolished the rise in MAP: -0.1 +/- 2.8 mmHg in intact and 0.0 +/- 2.9 mmHg in SAD. AVP increased in only in the sham-lesioned SAD group 8.6 +/- 3.5 pg/ml (P < 0.001 ET-1 vs. CSF). BQ-123 blocked the responses. Thus the integrity of the PVN is required for intracerebroventricularly administered ET-1 to exert pressor and AVP secretory effects.     

Effect of vasopressin administration and deficiency upon 3H-AVP binding sites in the CNS and periphery during development.

Arginine8-vasopressin (AVP, 40 micrograms/100 g b.wt., SC) was administered to male Long-Evans (LE) pups from day 1 to 7 of life and the pups were sacrificed on day 8 or 60. 3H-AVP binding was performed on membranes prepared from the liver, kidney, and septum. No significant changes were observed in the kidney or septum of animals 8 or 60 days old. However, the chronic AVP treatment did result in a significant increase in the density of 3H-AVP binding sites in the liver when compared to control day 8 pups (control 44 +/- 2 vs. AVP 56 +/- 3 fmol/mg protein), with no change in affinity. This effect was maintained into adulthood, as the day 60 AVP-treated LE rats also showed a significant increase in liver 3H-AVP binding sites compared to control (control 186 +/- 9 vs. AVP 239 +/- 14 fmol/mg protein), with no change in affinity. A comparison of 3H-AVP binding sites in 8-day-old LE, heterozygous Brattleboro (HET-BB), and homozygous Brattleboro rats (HOM-BB) was performed to assess the effect of complete (HOM-BB) and partial (HET-BB) VP deficiency on binding sites in the CNS and periphery. The liver again was the only tissue in which a change in 3H-AVP binding characteristics was noted. The HOM-BB rat (Bmax 144 +/- 6 fmol/mg protein) displayed a significant increase in AVP binding sites from the LE rat (Bmax 100 +/- 7 fmol/mg protein), while the 3H-AVP binding sites in the HET-BB rat liver (Bmax 69.8 +/- 9 fmol/mg protein) were significantly lower than LE rats. Thus hepatic AVP receptors appear most sensitive to the presence or absence of vasopressin during the early postnatal period.     

Dynorphins and leu-enkephalin in brain nuclei and pituitary of WKY and SHR rats

The distribution of dynorphin 1–13 (Dyn-1–13, Dyn-(1–8) and Leu⁵-enkephalin (LE) immunoreactivities (ir) were determined in discrete brain nuclei of normotensive (WKY) and hypertensive (SHR) rats. The concentration of ir-Dyn-(1–13) and ir-Dyn-(1–8) varied markedly among the various nuclei studies with a predominance of ir-Dyn-(1–13) over ir-Dyn-(1–8) in all the nuclei of both WKY and SHR rats. Ir-LE also showed large variations in different sites and no consistent relationships were found between the distribution of ir-Dyn-(1–8), Dyn-(1–13) and LE. SHR rats had lower levels of ir-Dyn-(1–13), Dyn-(1–8) and LE in the suprachiasmatic nucleus compared with WKY rats. In addition, SHR rats had lower levels of ir-Dyn-(1–8)- in the paraventricular and central amygdala, and higher ir-Dyn-(1–13) levels in the substantia nigra. The level of ir-Dyn-(1–13) in the neurointermediate lobe (NIL) of SHR rats was decreased substantially compared with that of WKY rats. The localization of these opioid peptides suggests that dynorphin-like peptides may serve a variety of hypothalamic and extrahypothalamic functions which might differ between SHR and WKY rats.     

Peripheral opioidergic regulation of the tracheobronchial mucociliary transport system.

We hypothesized that, in the airway mucosa, opioids are inhibitory neural modulators that cause an increase in net water absorption in the airway mucosa (as in the gut). Changes in bidirectional water fluxes across ovine tracheal mucosa in response to basolateral application of the opioid peptides beta-endorphin, dynorphin A-(1-8), and [d-Ala(2), d-Leu(5)]-enkephalin (DADLE) were measured. beta-Endorphin and dynorphin A-(1-8) decreased luminal-to-basolateral water fluxes, and dynorphin A-(1-8) and DADLE increased basolateral-to-luminal water flux. These responses were electroneutral. In seven beagle dogs, administration of aerosolized beta-endorphin (1 mg) to the tracheobronchial airways decreased the clearance of radiotagged particles from the bronchi in 1 h from 34.7 to 22.0% (P < 0.001). Naloxone abrogated the beta-endorphin-induced changes in vitro and in vivo. Contrary to our hypothesis, the opioid-induced changes in water fluxes would all lead to a predictable increase in airway surface fluid. The beta-endorphin-induced increases in airway fluid together with reduced bronchial mucociliary clearance may produce procongestive responses when opioids are administered as antitussives.