Development of C5a receptor antagonists. Differential loss of functional responses.

C5a is a 74-amino acid glycoprotein generated on activation of the C system. The responses evoked by C5a, both in vitro and in vivo, and its association with inflammatory diseases, suggest that a receptor antagonist would be of considerable therapeutic importance. However, efforts at generating antagonists have so far been unsuccessful. Structure/activity studies of the C terminus of C5a have generated peptide analogues with nanomolar affinities, but all of these retain strong agonist properties. We now report hexapeptides of the form NMePhe-Lys-Pro-dCha-X-dArg in which increasing aromaticity at position 5 leads to a progressive loss of agonism with little change in binding affinity. The different responses induced by C5a are lost in the order: degranulation before Ca(2+)-flux before chemotaxis. We also describe the first full antagonist of C5a, because the peptide in which x = Trp is not only devoid of all agonist properties, but it inhibits C5a induced degranulation and C5a stimulated G protein activation.

The amino terminus of the human C5a receptor is required for high affinity C5a binding and for receptor activation by C5a but not C5a analogs.

The binding domain of the human C5a receptor consists of two distinct and physically separable subsites. One of these sites binds the C-terminal 8 amino acids of C5a and is as yet undefined, while the second site lies in the N terminus of the receptor and interacts with the core of C5a. Two deletion mutants were prepared to probe the importance of this second site. Removal of residues 2-22 decreased the binding affinity for C5a by 600-fold, while extending the deletion through residue 30 caused a further 75-fold decrease. Thus, the N terminus is responsible for at least 45% of the total energy for the binding of C5a. The five aspartic acids present in the deleted segments appear to be critical residues, as their conversion to alanines accounts for most of the affinity lost in the two truncations. Despite its importance for binding, the N terminus is not necessary for signal transduction, as a C-terminal peptide analog of C5a was able to stimulate G protein activation and to generate a Ca2+ flux through a receptor lacking residues 2-22. However, intact C5a was a very poor activator of this truncated receptor. These results imply that interaction between the N terminus of the receptor and C5a produces a conformational change in C5a that allows it's C terminus to properly interact with and activate the receptor.

Modulatory effect of endopeptidase inhibitors on bradykinin-induced contraction of rat uterus.

Bradykinin (BK) affects a variety of smooth muscle types, including uterus. These effects are generally short-lived due to metabolism by a variety of enzymes including angiotensin converting enzyme (ACE), endopeptidase 24.11 (EP-24.11) and endopeptidase 24.15 (EP-24.15). The uterotonic action of BK and the limitation of that action by peptidases were examined using isolated rat uterus. BK contracted the estrus, diestrus and day 22 pregnant rat uterus. N-[1(R,S)-carboxy-3-phenylpropyl]-Phe-p-aminobenzoate (10(-7) M), a specific inhibitor of EP-24.11, and N-[1(R,S)-carboxy-3-phenylpropyl]-Ala-Ala-Phe-p-aminobenzoate (10(-6) M), a specific inhibitor of EP-24.15, enhanced BK-induced contraction in the estrus and pregnant uterus. Enalaprilat (6 x 10(-8) M), an inhibitor of ACE, also enhanced BK-induced contraction. The enzyme inhibitors alone did not contract the uterus. Bradykinin B2 receptor antagonism blocked the effects of the inhibitors. ACE is present in the rat uterus, but there are no reports of EP-24.11 or EP-24.15. Here we report that EP-24.11 and EP-24.15 activities are present in the estrus and pregnant rat uterus. Partially purified uterine homogenates metabolized specific model substrates for EP-24.11 and EP-24.15. The enzyme activities were inhibited by N-[1(R,S)-carboxy-3-phenylpropyl]-Phe-p-aminobenzoate and N-[1(R,S)-carboxy-3-phenylpropyl]-Ala-Ala-Phe-p-aminobenzoate, respectively, and increased 5- to 8-fold at term pregnancy as compared to estrus.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of beta-endorphin secretion by corticotropin-releasing factor in the intact rat testis.

CRF stimulates beta-endorphin (beta EP) secretion from adult rat Leydig cells in vitro. To evaluate the relevance of this action of CRF in a physiological context, we studied the effect of CRF on beta EP secretion in the testis in whole animals. In the pubertal rat, intratesticular infusion of CRF resulted in a 3-fold stimulation of immunoreactive beta EP (ir beta EP) levels in testicular interstitial fluid (TIF) compared with levels in contralateral saline-infused testes. Coadministration of the antagonist CRF-(12-41) resulted in TIF ir beta EP concentrations that were reduced compared to levels in paired saline controls. Infusion of the competitive antagonist CRF-(9-41) alone slightly stimulated ir beta EP concentrations in TIF. In adult animals, all of the peptides tested were without effect. These results suggest a developmental regulation of the action of CRF on beta EP levels in the pubertal rat testis. Our studies in conjunction with documented results demonstrate that in the testis, beta EP is actively secreted. These results imply that testicular ir beta EP is derived from a POMC mRNA that encodes a signal peptide containing preprohormone similar or identical to pituitary POMC. Previous studies of rodent testicular mRNA have found only 5'-truncated forms of POMC message. The current study provides direct evidence for a low abundance POMC transcript that could encode the preprohormone. Therefore, the major components of a pituitary-like CRF/POMC stimulus/secretion system appear to be present in the rat testis, including the full-length POMC mRNA and release of POMC-derived beta EP that is stimulated by CRF.

Inhibition of endopeptidase-24.15 decreases blood pressure in normotensive rats.

The potent vasodilatory peptide bradykinin is cleaved at the Phe5-Ser6 bond in vitro by the metalloenzyme endopeptidase-24.15 (E.C.3.4.24.15). We now report that intravenous infusion of N-[1-(R,S)-carboxy-3-phenylpropyl]-Ala-Ala-Phe-p-aminobenzoate, a specific active site-directed inhibitor of endopeptidase-24.15, produces an immediate drop in mean arterial pressure of as much as 50 mm Hg in pentobarbital-anesthetized, normotensive rats. Arterial pressure recovers within 5 minutes. The B2 bradykinin antagonist [Arg0,Hyp3,Thi5,8,D-Phe7]-bradykinin attenuates the decrease in mean arterial pressure resulting from treatment with the inhibitor. The endopeptidase-24.15 inhibitor potentiates the hypotensive effect of intravenous bradykinin infusion, increasing the maximal effect of the peptide by 47% and increasing the potency by almost 10-fold, while the response to intra-arterial bradykinin is less affected by the inhibitor. These results support a role for endopeptidase-24.15 in the inactivation of endogenous and exogenous bradykinin and suggest a direct involvement of the enzyme in the control of blood pressure.

Endopeptidase-24.15 in rat hypothalamic/pituitary/gonadal axis.

Endopeptidase-24.15 (E.C. 3.4.24.15; EP-24.15) cleaves several substrates found in the hypothalamic/pituitary/gonadal axis, including gonadotropin-releasing hormone (GnRH) and the opioid peptides of the dynorphin family. We have examined the activity of EP-24.15 in these tissues as a function of maturation, of the estrous cycle, and in response to ovariectomy and estrogen replacement. A developmental regulation of EP-24.15-specific activity is apparent in anterior pituitary, in hypothalamus, and in the gonads. EP-24.15 is increased in the preoptic area and is decreased in the anterior pituitary in both male and female rats prior to puberty. The specific activity of EP-24.15 was increased following ovariectomy in the anterior pituitary and within medial and lateral preoptic nuclei. Testicular specific activity of EP-24.15 increased with age in a linear fashion, while ovarian EP-24.15 activity increased immediately prior to puberty, but returned to prepubertal levels by 65 days of age. The relevance of EP-24.15 to the metabolism of specific peptides is discussed.

Antinociceptive properties of inhibitors of endopeptidase 24.15.

Endopeptidase 24.15, a metalloendopeptidase active in brain, rapidly converts prodynorphin-derived peptides into leu-enkephalin. Inhibitors of this enzyme slow the degradation of these peptides in vivo and in vitro. The present study evaluated two inhibitors of endopeptidase 24.15, N-[1-(RS)-carboxy-3-phenyl-propyl]-Ala-Ala-Phe-p-aminobenzoate (cFP-AAF-pAB), and N-[1-(RS)-carboxy-3-phenylpropyl]-Ala-D-Ala-Phe-p-aminobenzoate (cFP-A(D)AF-pAB), for antinociception on the tail-flick and jump tests in rats following intracerebroventricular administration relative to an inhibitor of endopeptidase 24.11, N-(1-(RS)-carboxy-3-phenylpropyl]-Phe-p-aminobenzoate (cFP-F-pAB). cFP-AAF-pAB, cFP-A(D)AF-pAB and cFP-F-pAB produced equipotent dose-dependent (25-250 nmol) and time-dependent (5-7 h) antinociception with larger effects on the jump (49-51% increase) relative to the tail-flick (28-41% increase) test. Naloxone (1 mg/kg, SC) significantly reduced antinociception elicited by all inhibitors on the jump test. Motor performance failed to be affected by inhibitor administration. The gradual appearance of antinociception and its naloxone sensitivity suggest that these effects are mediated through inhibition of opioid peptide degradation.

Distribution of endopeptidase-24.15 in rat brain nuclei using a novel fluorogenic substrate: comparison with endopeptidase-24.11.

A novel fluorogenic substrate for the neutral metalloendopeptidase-24.15 (E.C.3.4.24.15; EP-24.15) was synthesized which allowed continuous assay of the enzyme. The substrate, Glutaryl-Phe-Ala-Ala-Phe-4-methoxynaphthylamide (G-FAAF-4MN) is cleaved at the Phe-Ala bond by EP-24.15 (Km = 0.026 mM). The product, AAF-4MN is subsequently hydrolyzed to its constituent amino acids and the potent fluorophore 4MN by aminopeptidase M. This method has allowed the measurement of the specific activity EP-24.15 within microdissected nuclei of rat brain. The enzyme was found to have a relatively broad distribution within brain nuclei, and the activity ranged from 15-80 nmol 4MN/mg prot/h in all areas examined. The activity of EP-24.15 was relatively high in the medial and lateral pre-optic nuclei, where potential substrates include the dynorphin-like peptides and LHRH. The activity of EP-24.15 was compared with that of endopeptidase-24.11 (E.C.3.4.24.11, 'enkephalinase', EP-24.11), another peptide-cleaving metalloenzyme. EP-24.11 appeared to have a much more narrow distribution, with very high specific activity in basal ganglia as well as in the supraoptic and suprachiasmatic nuclei.

An inhibitor of endopeptidase-24.15 blocks the degradation of intraventricularly administered dynorphins.

Conversion of the octapeptide dynorphin (Dyn) A-(1-8) to Leu5-enkephalin (LE) by endopeptidase EC 3.4.24.15 (EP-24.15) in vivo was examined using the technique of ventriculocisternal perfusion. Peptides were administered intracerebroventricularly in the presence or absence of the EP-24.15 inhibitor N-[1-(R,S)-carboxy-3-phenylpropyl]-Ala-Ala-Phe-p-aminobenzoate (cFPAAF-pAB) via cannulae placed into the lateral ventricle of urethane-anesthetized rats. The concentration of Dyn-like peptides and LE within the CSF was monitored by radioimmunoassay in samples of CSF taken from a second cannula placed in the cisterna magna. In the absence of inhibitor, less than 5% of the Dyn A-(1-8) administered was recovered in CSF. Immunoreactive LE, which is normally not found in CSF, increased rapidly in content following Dyn A-(1-8) infusion, an observation suggesting that the larger peptide is converted to LE. When the inhibitor cFPAAF-pAB was coadministered with Dyn A-(1-8), the concentration of immunoreactive Dyn A-(1-8) after 5 min was 40 times higher than that found in the absence of inhibitor. The angiotensin converting enzyme inhibitor captopril reduced the degradation of Dyn A-(1-8) to a much lesser degree. The inhibitor of EP-24.15 also afforded some protection of other Dyn-like peptides. No EP-24.15 activity was found in rat CSF, whereas high activity was found in the choroid plexus. Taken together, these data clearly indicate that an ectoenzyme form of EP-24.15 rapidly converts intracerebroventricularly administered Dyn-like peptides to LE.

Inhibition of endopeptidase 24.15 slows the in vivo degradation of luteinizing hormone-releasing hormone.

Endopeptidase (EP) 24.15 cleaves the Tyr5-Gly6 bond of luteinizing hormone-releasing hormone (LHRH) (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2), and is the primary LHRH degrading enzyme in pituitary and hypothalamic membrane preparations. Potent and specific inhibitors were used to identify the enzymes involved in the in vivo degradation of LHRH. After i.c.v. administration of LHRH, only about 1% of the peptide was recovered from brain after 1 hr. Concurrent administration of LHRH and N-[1-(RS)-carboxy-3-phenylpropyl]-Ala-Ala-Phe-p-aminobenzoate (cFP-AAF-pAB), a specific inhibitor of EP 24.15, led to a more than 10-fold increase in LHRH recovery. Administration of N-[1-(RS)-carboxy-3-phenylpropyl]-Phe-pAB (cFP-F-pAB) or captopril, inhibitors of "enkephalinase" (EP 24.11) and angiotensin converting enzyme, respectively, did not significantly increase LHRH recovery. Intravenous administration of LHRH and either cFP-F-pAB or cFP-AAF-pAB but not captopril, led to an increase in the half-life of LHRH from 10 min to 15 and 20 min, respectively. Concurrent administration of both inhibitors resulted in a dramatic 8-fold increase in the half-life of LHRH, similar to values reported for "superactive" analogs of LHRH which are rendered resistant to enzymatic degradation by introduction of a D-amino acid in position 6. Concentrations of plasma LHRH 65 to 80 min after administration of inhibitors were 100- to 200-fold higher than those in controls. The potentiating effect of cFP-F-pAB resulted from inhibition of the in vivo degradation of cFP-AAF-pAB by EP 24.11.(ABSTRACT TRUNCATED AT 250 WORDS)

Endopeptidase-24.15 is the primary enzyme that degrades luteinizing hormone releasing hormone both in vitro and in vivo.

The concentration of luteinizing hormone releasing hormone (LHRH) (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2), which reaches the anterior pituitary via the hypothalamo-hypophyseal portal system, appears to be controlled in part by the rate of LHRH degradation within the hypothalamus and/or pituitary. Specific, active site-directed endopeptidase inhibitors synthesized in our laboratory were used to identify the enzyme(s) involved in LHRH degradation by hypothalamic and pituitary membrane preparations, and by an intact anterior pituitary tumor cell line (AtT20). Incubation of LHRH with pituitary and hypothalamic membrane preparations led to the formation of pGlu-His-Trp (LHRH1-3) as the main reaction product. Under the same conditions, addition to the incubation mixtures of captopril, an inhibitor of the angiotensin converting enzyme, led to accumulation of pGlu-His-Trp-Ser-Tyr (LHRH1-5) and, to a lesser extent, pGlu-His-Trp-Ser-Tyr (LHRH1-6). The degradation of LHRH and the formation of the N-terminal tri- and pentapeptides was blocked by N-[1-(R,S)-carboxy-3-phenylpropyl]-Ala-Ala-Phe-p-aminobenzoate (cFP-AAF-pAB), a specific, active site directed inhibitor of endopeptidase-24.15. Some inhibition of LHRH degradation and formation of the N-terminal hexapeptide was also obtained in the presence of N-[1-carboxy-2-phenylethyl]-Phe-p-aminobenzoate (cFE-F-pAB), an inhibitor of endopeptidase-24.11. Similar results were obtained with AtT20 cell membranes and with intact AtT20 cells in monolayer culture. Following cleavage by endopeptidases the C-terminal part of LHRH was rapidly degraded by aminopeptidases. Superactive analogs of LHRH in which Gly6 was replaced by a D-amino acid are resistant to degradation by both endopeptidase-24.11 and -24.15. In vivo, when LHRH was injected directly into the third ventricle of rats, the presence of cFP-AAF-pAB inhibited LHRH degradation. It is concluded that LHRH degradation is primarily initiated by the membrane-bound form of endopeptidase-24.15 to yield pGlu-His-Trp-Ser-Tyr and to a lesser extent by endopeptidase-24.11 to yield pGlu-His-Trp-Ser-Tyr-Gly.

Substrate-related potent inhibitors of brain metalloendopeptidase.

Rat brain metalloendopeptidase (EC 3.4.24.15) generates Leu- and Met-enkephalin from several larger opioid peptides and is capable of degrading a number of neuropeptides. Substrate-related N-(1-carboxy-3-phenylpropyl) peptide derivatives were synthesized and tested for enzyme inhibition. The best of these derivatives, N-[1(RS)-carboxy-3-phenylpropyl]-Ala-Ala-Tyr-p-aminobenzoate, inhibited the enzyme in a competitive manner with a Ki of 16 nM. The data indicate that the carboxyl group of the N-(1-carboxy-3-phenylpropyl) moiety coordinates with the active site zinc atom and that the remaining part of the inhibitor is necessary for interaction with the substrate recognition site of the enzyme. Replacement of the 1-carboxy-3-phenylpropyl group by a carboxymethyl group decreased the inhibitory potency by more than 3 orders of magnitude, emphasizing the importance of the hydrophobic phenyl group for inhibitor binding to a hydrophobic pocket at the S1 subsite. Replacement of the Tyr residue by an Ala residue decreased the inhibitory potency by more than 20-fold. Changes in the structure of the residue interacting with the S1' subsite could cause a more than 60-fold change in inhibition. The inhibitors were either ineffective or only weakly inhibitory against membrane-bound metalloendopeptidase ("enkephalinase", EC 3.4.24.11), an enzyme highly active in rabbit kidney but also present in brain. The data indicate the presence of an extended binding site in the enzyme with residues interacting with S1, S1', and S3' subsites largely determining inhibitor binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Response of rat pituitary anterior lobe prodynorphin products to changes in gonadal steroid environment.

The total content of rat pituitary anterior lobe (AL) immunoreactive (ir) dynorphin A (ir-Dyn A) and ir-dynorphin B (Dyn B) increased in male rats between 15 and 58 days of age, but there was little alteration in the concentration of ir-Dyn A or B expressed relative to protein content. Adult rats (90 days of age) had lower concentrations of these peptide immunoreactivities in the AL. Castration of 58-day-old male rats produced a testosterone-reversible loss of ir-Dyn A and B by 50-60% 3 days after surgery. Thereafter, the levels of these peptides gradually increased to 2.5 times the levels found in control animals at 1 month after castration. These effects of castration on AL dynorphin were not seen in 15-day-old rats and were much less marked in adults. Similar changes were seen in the levels of other prodynorphin products, alpha- and beta-neo-endorphin (ir-alpha-nEnd and ir-beta-nEnd), and ir-[Leu5]enkephalin (ir-LE). Administration of testosterone (100 micrograms/100 g BW) to castrated rats for 2 days largely prevented the drop in the levels of AL ir-Dyn A and B. Ovariectomy produced an increase in the levels of ir-Dyn A, Dyn B, alpha-nEnd, beta-nEnd, and LE 2 weeks after surgery, but, in contrast to castration, no significant decrease was seen 3 days after ovariectomy. These changes in AL content of dynorphin-related peptides after castration or ovariectomy directly reflect those previously reported for AL content of LH. The mechanisms regulating storage (and perhaps secretion) of AL peptides derived from prodynorphin may be similar to those regulating storage and secretion of LH and FSH in rat AL. AL ir-LE could potentially arise from proenkephalin A or prodynorphin (proenkephalin B). Ir-LE levels in AL were approximately 10 times higher than the levels of ir-[Met5]-enkephalinyl-Arg-Gly-Leu (ME-RGL) in male rat AL, and changes in ir-LE content after castration were very similar to those observed in other prodynorphin-derived peptides, but different from the effects of castration on ir-ME-RGL. It is possible that prodynorphin is a major source of AL ir-LE.

Hemorrhagic shock and the central vasopressin and opioid peptide system of rats.

The effect of hemorrhagic shock (40% of blood vol) on the distribution of immunoreactive dynorphin A (Dyn A-IR), [Arg8]vasopressin (AVP-IR), and [Leu5]enkephalin (LE-IR) in the pituitary and brain nuclei was studied in the conscious rat. At 24 h after hemorrhage, the neurointermediate lobe (NIL) showed a reduction in Dyn A-IR (52%) and AVP-IR (32%) and an increase in LE-IR (72%); at this time, the anterior lobe also showed decreased Dyn A-IR (50%) and increased LE-IR (210%). Dyn A-IR, but not LE-IR, was also significantly depleted in some forebrain nuclei in all experimental groups as compared with intact controls, whereas Dyn A-IR in the hypothalamic ventromedial nucleus was elevated only in the sham-control rats. AVP-IR was elevated in the supraoptic nucleus and median eminence (200 and 31%, respectively) 2 and 24 h after bleeding, although plasma AVP returned to normal levels. These data indicate that stress and hypovolemic hypotension produce site and time-dependent change in distribution of dynorphins, AVP, and LE in the central nervous system.

Increased dynorphin immunoreactivity in spinal cord after traumatic injury.

Opiate antagonists, at high doses, have been shown to improve physiological variables and outcome after experimental spinal injury. Dynorphin appears to be unique amongst opioids in producing hindlimb paralysis after intrathecal injection. Taken together, these findings suggest a possible pathophysiological role for endogenous opioids, particularly dynorphin, in spinal injury. In the present studies we examined the relationship between changes in dynorphin immunoreactivity (Dyn-ir) in rat spinal cord after traumatic injury and the subsequent motor dysfunction. Trauma was associated with significantly increased Dyn-ir at the injury site, but not distant from the lesion. Dyn-ir was found elevated as early as 2 h and as late as 2 weeks after trauma, and was significantly correlated with the degree of injury. These data are consistent with the hypothesis that dynorphin systems may be involved in the secondary injury that follows spinal trauma.

Endogenous opioid immunoreactivity in rat spinal cord following traumatic injury.

It has been postulated that endogenous opioids play a pathophysiological role in spinal cord injury, based on the therapeutic effects of the opiate receptor antagonist naloxone in certain experimental models. The high doses of naloxone required to exert a therapeutic action suggest that naloxone's effects may be mediated by non-mu opiate receptors, such as the kappa receptor. This notion is supported by recent pharmacological studies demonstrating that an opiate antagonist more active at kappa sites is effective and far more potent than naloxone in improving outcome after spinal cord injury. Moreover, dynorphin--postulated to be the endogenous ligand for the kappa receptor--is unique among opioids in producing hindlimb paralysis following intrathecal administration in the rat. In the present studies we have examined changes in endogenous opioid immunoreactivity following traumatic spinal cord injury in the rat. Dynorphin A was found to increase progressively with graded injury; changes were restricted to the injury segment and adjacent areas and were time dependent. Dynorphin A-(1-8) showed no marked changes. Methionine and leucine enkephalin were either unaltered or reduced at the injury site; changes were not well localized and were not clearly related to the injury variables. These findings provide further support for a potential pathophysiological role of prodynorphin-derived peptides in spinal cord injury.

Effects of traumatic injury on dynorphin immunoreactivity in spinal cord.

Traumatic spinal cord injury in rats resulted in a significant eleation of dynorphin A immunoreactivity in spinal cord tissue at the level of, and below, the site of injury. [Leu5]enkephalin levels in the same tissue samples were not significantly altered following severe injury. Dynorphin A immunoreactivity was found in the fraction relatively enriched in synaptosomes after subcellular fractionation of spinal cord tissue. The dynorphin A content of this fraction was not significantly changed following injury, suggesting that dynorphin containing nerve terminals and axons are not severely damaged as a result of the injury.

Subcellular distribution of opioid peptides in rat hypothalamus and pituitary.

Homogenates of rat anterior lobe (AL) and neurointermediate lobe (NIL) pituitary and rat hypothalamus were subjected to subcellular fractionation and density gradient centrifugation. The subcellular distribution of immunoreactive dynorophin A (ir-Dyn A) in NIL was found to be similar to that of ir-arginine vasopressin (ir-AVP). ir-Dyn A migrated as a discrete band on sucrose density gradients, which corresponded in sedimentation rate to that of ir-AVP, suggesting that these two peptides are stored within organelles of similar size and density. Two other products of prodynorphin, ir-alpha-neoendorphin (ir-alpha-nEND) and ir-Dyn A-(1-8) also comigrated with ir-AVP. ir-[Leu5]-enkephalin (ir-LE), which may be a product of prodynorphin or proenkephalin, was also found to migrate in this region of the gradient. When a homogenate of rat hypothalamus was prepared using a method that has been developed for synaptosome isolation, ir-Dyn A was found to comigrate with Na+/K+-activated adenosine triphosphatase (Na/K-ATPase), a synaptosomal marker enzyme. Using a more concentrated homogenate ir-Dyn A was found to migrate to a less dense region where peptide-containing synaptic vesicles have previously been localized. When a synaptosomal preparation was lysed in hypotonic solution a shift was seen in the migration rate of ir-Dyn A to this region of the gradient (containing putative synaptic vesicles). Thus the bulk of hypothalamic dynorphin appears to be present within synaptosome-like structures which, upon lysis, release a less dense, smaller subcellular organelle corresponding in sedimentation characteristics to other types of peptide-containing synaptic vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)

Dynorphin-A-(1-8) is contained within vasopressin neurosecretory vesicles in rat pituitary.

Dynorphin-A-(1-8), an opioid peptide widely distributed in the rat central nervous system, is present in vasopressin-containing neurosecretory cells terminating in the neural lobe of the pituitary. Electron microscopic immunocytochemistry reveals that dynorphin-A-(1-8) is contained within the same neurosecretory vesicles as vasopressin and vasopressin-associated neurophysin in the neural lobe of the rat. The results indicate that dynorphin may be released in the pituitary concomitantly with vasopressin during the antidiuretic response.