Methionine and leucine enkephalin in rat neurohypophysis: different responses to osmotic stimuli and T2 toxin.

Specific radioimmunoassays were used to measure the effects of hypertonic saline (salt loading), water deprivation, and trichothecene mycotoxin (T2 toxin) on the content of methionine enkephalin (ME), leucine enkephalin (LE), alpha-neoendorphin, dynorphin A, dynorphin B, vasopressin, and oxytocin in the rat posterior pituitary. Concentrations of vasopressin and oxytocin decreased in response to both osmotic stimuli and treatment with T2 toxin, but the decrease was greater with osmotic stimulations. Similarly, concentrations of LE and dynorphin-related peptides declined after salt loading and water deprivation; LE concentrations also decreased after treatment with T2 toxin. The concentration of ME decreased after water deprivation, did not change after salt loading, and increased after T2 toxin treatment. The differentiating effects of these stimuli on the content of immunoreactive LE and ME are consistent with the hypothesis that LE and ME may be localized in separate populations of nerve endings with different roles in the posterior pituitary.

Combinatorial chemistry--applications of light-directed chemical synthesis.

Combinatorial methods in biology and chemistry are proving to be powerful methods for generating molecular diversity. One approach, light-directed chemical synthesis, combines semiconductor-based photolithography technologies with solid-phase organic chemistry to synthesize large arrays of molecules with potential biological activity. This novel technology has the potential to provide libraries of both natural and synthetic molecules that might be screened rapidly for biological activity.

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.

On the origin of Leu-enkephalin and Met-enkephalin in the rat neurohypophysis.

The posterior lobe of the pituitary contains large amounts of Leu- and Met-enkephalin (LE and ME, respectively). A marked depletion of ME (81.9%) and LE (94.5%) in the posterior pituitary occurred after transection of the pituitary stalk. This indicates that most, if not all, of the enkephalins are in processes of central neurons. In the present study, I attempted to determine the source(s) of the LE- and ME-containing fibers in the posterior pituitary by examining the effects of hypothalamic lesions or fiber transections on the LE and ME levels. Lesions of the hypothalamic paraventricular nuclei caused ME and LE levels in the posterior pituitary to decrease significantly (55.6% and 27.6%, respectively). Deafferentation of the medial basal hypothalamus (creating islands of tissue containing the ventromedial and arcuate nuclei) resulted in a marked reduction in LE (94.1%) and ME (54.7%). Treating neonatal rats with monosodium glutamate resulted in a selective destruction of arcuate nucleus neurons, but did not affect LE and ME concentrations in the posterior pituitary. Thus, about half of the ME in the posterior pituitary seems to be provided by neurons in the vicinity of the paraventricular and ventromedial nuclei, whereas only about one quarter of the LE in the posterior pituitary is in processes of the paraventricular nucleus neurons. The remainder of the LE is contributed to the posterior pituitary by neurons outside the medial basal hypothalamus, probably by the supraoptic nucleus neurons. These findings are consistent with the hypothesis that LE and ME may be localized in separate populations of nerve endings in the neurohypophysis and may have different roles.

Stimulation by leumorphin of prolactin secretion from the pituitary in rats.

The effect of leumorphin (LM), one of big leu-enkephalins derived from preproenkephalin B, on PRL secretion was studied in the rat in vivo and in vitro. Intracerebroventricular injection of synthetic porcine LM (0.06-6 nmol/rat) caused a dose-related increase in plasma PRL levels in urethane-anesthetized male rats and in conscious freely moving rats. Intravenous injection of LM (3 nmol/100 g BW) also raised plasma PRL levels in these animals. The plasma PRL response to intracerebroventricular LM (0.6 nmol/rat) was blunted by naloxone (125 micrograms/100 g BW, iv). The stimulating effect of LM on PRL release was the most potent among the peptides derived from preproenkephalin B. In in vitro studies, PRL release from superfused anterior pituitary cells was stimulated in a dose-related manner by LM (10(-9)-10(-6) M), and the effect was blunted by naloxone (10(-5) M). These results suggest that LM has a potent stimulating effect on PRL secretion from the pituitary in the rat by acting, at least in part, directly at the pituitary through an opiate receptor.

Effect of hypertonic saline infusion on the level of immunoreactive dynorphin in extracted human plasma.

Dynorphin-A and its related peptides are derived from prodynorphin, one of the three known endogenous opioid precursors. The prodynorphin gene is expressed in the vasopressinergic magnocellular neurons of the hypothalamus, while its peptide products are present in the vasopressin (AVP) neurosecretory vesicles of the neurohypophysis. The concentration of immunoreactive (IR) dynorphin is orders of magnitude higher in the neurohypophysis than in any other tissue, suggesting that perhaps the prodynorphin-derived peptides are secreted from the hypothalamic-neurohypophyseal unit into the general circulation. Experiments in rats have shown that osmotic stimuli increase both AVP and prodynorphin in the hypothalamus. To determine whether human hypothalamic prodynorphin is also under osmotic regulation, we measured plasma IR-dynorphin, plasma IR-AVP, and serum sodium immediately before and during the infusion of normal or hypertonic saline in normal human volunteers. Because of the unusual susceptibility of the prodynorphin-derived peptides to cleavage by endopeptidases, we also developed an appropriate plasma dynorphin extraction technique. We found that the IR-dynorphin present in human plasma was composed of 6K- and 4K-sized peptides and that no larger than 6K or smaller than 4K dynorphins were present. The infusion of normal saline did not have any significant effect on plasma IR-dynorphin, while 3% hypertonic saline increased its plasma levels. Thus, the mean IR-dynorphin level in the plasma of the volunteers infused with normal saline was 40.3 +/- 6.4 fmol/mL (mean +/- SE; n = 6) at zero time; after 30 min of infusion, plasma IR-dynorphin was 36.0 +/- 6.3, after 60 min it was 29.9 +/- 5, after 90 min it was 36.0 +/- 4.7, after 120 min it was 36.8 +/- 3.2, and after 150 min it was 36.0 +/- 6.1. The plasma IR-dynorphin level in the volunteers infused with hypertonic saline was 31.7 +/- 3.5 fmol/mL (mean +/- SE; n = 10) at zero time. After 30 min of infusion it increased to 37.4 +/- 3.8, after 60 min to 46.4 +/- 7.7, after 90 min to 56.2 +/- 9.1, after 120 min to 53.6 +/- 8.7, and after 150 min to 99.0 +/- 14.2. The increase in plasma IR-dynorphin with time was significant (P less than 0.0001) and correlated positively with serum sodium and plasma AVP. The physiological role of the prodynorphin-derived peptides of the hypothalamic-neurohypophyseal unit is not yet known.(ABSTRACT TRUNCATED AT 400 WORDS)

Distribution of opiate receptor subtypes and enkephalin and dynorphin immunoreactivity in the hippocampus of squirrel, guinea pig, rat, and hamster.

The distribution of enkephalin and dynorphin immunoreactivity in the hippocampus of four rodent species (gray squirrel, guinea pig, rat, and hamster) is compared with the pattern of opiate receptor subtypes (mu, delta, and kappa). The distribution of opioid peptides is fairly consistent in the anterior hippocampus of these four species. Intense immunoreactivity for dynorphin and enkephalin is found in the hilus of the dentate gyrus and in the mossy fiber system. Occasional immunoreactive processes are seen in the dentate molecular layer and scattered throughout the CA1 and CA3 fields. In the rat and hamster, an additional plexus of enkephalinergic fibers straddles both sides of the hippocampal fissure. Cells immunoreactive for both opioid peptides are located in and just superficial to the dentate granule cell layer. Opiate receptors are variably distributed in these rodent species. In the squirrel, guinea pig, and hamster, mu and kappa binding is dense in the stratum lucidum of CA3 and the molecular layer of the dentate gyrus. In the rat, dense mu and kappa binding is localized within and adjacent to the pyramidal and granule cell layers. Delta receptor patterns show additional species differences. In the rat, the delta distribution is similar to the mu and kappa patterns. In the other species, the delta binding pattern is generally the inverse of the mu/kappa pattern: most areas of the hippocampus are enriched in delta sites, whereas the stratum lucidum and the pyramidal cell layer are receptor-sparse. Thus, the stratum lucidum--site of dense terminations of mossy fibers containing opioid peptides--is characterized by selectively sparse delta receptors in four species and by selectively dense kappa receptors in three species. The three receptor subtypes, taken either individually or together and compared to the peptides, are more variably and more widely distributed throughout the hippocampus and fail to show a correspondence with opioid-peptide-containing terminals. The mismatches suggest that receptor locations and densities are organized without relation to the sites of relevant transmitter release.

Prodynorphin peptide distribution in the forebrain of the Syrian hamster and rat: a comparative study with antisera against dynorphin A, dynorphin B, and the C-terminus of the prodynorphin precursor molecule.

The neuroanatomical distribution of the prodynorphin precursor molecule in the forebrain of the male Syrian hamster (Mesocricetus auratus) has been studied with a novel antiserum directed against the C-terminus of the leumorphin [dynorphin B (1-29)] peptide product. C-peptide staining in sections from colchicine-treated hamsters is compared to staining in sections from untreated animals. In addition, the pattern of C-peptide immunostaining in hamster brain is compared to that in the rat brain. Finally, the C-peptide immunolabeling patterns in hamsters and rats are compared to those obtained with antisera to dynorphin A (1-17) and dynorphin B (1-13). Areas of heaviest prodynorphin immunoreactivity in the hamster include the hippocampal formation, lateral septum, bed nucleus of the stria terminalis, medial preoptic area, medial and central amygdaloid nuclei, ventral pallidum, substantia nigra, and numerous hypothalamic nuclei. Although this C-peptide staining pattern is similar to dynorphin staining reported previously in the rat, several species differences are apparent. Whereas moderate dentate gyrus granule cell staining and no CA4 cell staining have been reported in the rat hippocampal formation, intense immunostaining in the dentate gyrus and CA4 cell labeling are observed in the hamster. In addition, the medial preoptic area, bed nucleus of the stria terminalis, and medial nucleus of the amygdala stain lightly for prodynorphin-containing fibers and cells in the rat, compared to heavy cell and fiber staining in the hamster in all three of these regions. In the rat there is no differential staining between tissues processed with the C-peptide, dynorphin A, and dynorphin B antisera, but numerous areas of the hamster brain show striking differences. In most hamster brain areas containing prodynorphin peptides, the C-peptide antiserum immunolabels more cells and fibers than the dynorphin B antiserum, which in turn labels more cells and fibers than dynorphin A antiserum. However, exceptions to this hierarchy of staining intensity are found in the lateral hypothalamus, substantia nigra, arcuate nucleus, and habenula. The differences in staining patterns between rat and hamster are greatest when C-peptide antiserum is used; apparent species differences are present, though less pronounced, in dynorphin B- and dynorphin A-immunostained material.

Immunoreactive pro-enkephalin and prodynorphin products are differentially distributed within the nucleus of the solitary tract of the rat.

In this study we examined the distribution of two different endogenous opioid peptides in the nucleus of the solitary tract of the rat medulla. As a marker for immunoreactive enkephalin, we used an antiserum directed against one of the proenkephalin products, methionine enkephalin-arg-gly-leu (m-Enk). To identify immunoreactive dynorphin we used an antiserum directed against the prodynorphin product, dynorphin B (Dyn B). The PAP method was used on both colchicine and normal animals. Caudal to the obex, within the commissural nucleus, there is extensive overlap of both immunoreactive m-Enk and Dyn B terminals and cells. While the cells are morphologically similar, the immunoreactive dynorphin cells are somewhat larger. Rostral to the obex, there is a marked difference in the distribution of the two compounds. Immunoreactive m-Enk terminals are concentrated medial to the solitary tract; there is minimal staining laterally. In contrast, immunoreactive Dyn B terminals are concentrated lateral to the solitary tract. The rostral cellular distribution of the two opioid peptides follows a similar pattern. The morphology of the medially located m-Enk and laterally located Dyn B cells is also readily distinguished. The former are small, round cells with minimal dendritic labelling; the latter are larger, pyramidal neurons with prominent apical and basal dendrites. Since the medial and lateral nuclei of the solitary tract have been associated with cardiovascular and respiratory control, respectively, these data suggest that different endorphin families have different functional actions within the nucleus of the solitary tract.

Distribution of dynorphin and enkephalin peptides in the rat brain.

The neuroanatomical distribution of dynorphin B-like immunoreactivity (DYN-B) was studied in the adult male and female albino rat. The distribution of DYN B in colchicine- and noncolchicine-treated animals was also compared to that of another opioid peptide derived from the prodynorphin precursor dynorphin A (1-8) (DYN 1-8), and an opioid peptide derived from the proenkephalin precursor met-enkephalin-arg-gly-leu (MERGL). DYN B cell bodies were present in nonpyramidal cells of neo- and allocortices, medium-sized cells of the caudate-putamen, nucleus accumbens, lateral part of the central nucleus of the amygdala, bed nucleus of the stria terminalis, preoptic area, and in sectors of nearly every hypothalamic nucleus and area, medial pretectal area, and nucleus of the optic tract, periaqueductal gray, raphe nuclei, cuneiform nucleus, sagulum, retrorubral nucleus, peripeduncular nucleus, lateral terminal nucleus, pedunculopontine nucleus, mesencephalic trigeminal nucleus, parabigeminal nucleus, dorsal nucleus of the lateral lemniscus, lateral superior olivary nucleus, superior paraolivary nucleus, medial superior olivary nucleus, ventral nucleus of the trapezoid body, lateral dorsal tegmental nucleus, accessory trigeminal nucleus, solitary nucleus, nucleus ambiguus, paratrigeminal nucleus, area postrema, lateral reticular nucleus, and ventrolateral region of the reticular formation. Fiber systems are present that conform to many of the known output systems of these nuclei, including major descending pathways (e.g., striatonigral, striatopallidal, reticulospinal, hypothalamospinal pathways), short projection systems (e.g., mossy fibers in hippocampus, hypothalamo-hypophyseal pathways), and local circuit pathways (e.g., in cortex, hypothalamus). The distribution of MERGL was, with a few notable exceptions, in the same nuclei as DYN B. From these neuroanatomical data, it appears that the dynorphin and enkephalin peptides are strategically located in brain regions that regulate extrapyramidal motor function, cardiovascular and water balance systems, eating, sensory processing, and pain perception.

Distribution of opioid peptides in the preoptic region: immunohistochemical evidence for a steroid-sensitive enkephalin sexual dimorphism.

The distribution of cells and fibers that contain opioid peptides within the preoptic region of the rat was examined immunohistochemically. Cells and/or fibers that contain peptides derived from each of the three major opioid peptide families were differentially stained by using antisera that recognize unique derivatives of each precursor molecule and do not cross-react with members of the other opioid peptide families. A beta-endorphin (beta E) antiserum was used to stain fibers that contain peptides derived from the proopiomelanocortin molecule, and dynorphin-containing cells were identified by using an antiserum directed toward dynorphin B (Dyn B) that does not show detectable cross-reactivity with enkephalin-related peptides. An antiserum raised against peptide E (PE), which does not appear to cross-react significantly with dynorphin peptides, was used to localize enkephalin cells and fibers. Each family of opioid peptides showed a unique distribution in the preoptic region. beta E-immunoreactive fibers were primarily localized to the preoptic part of the periventricular nucleus, with moderate densities of fibers contained in the anteroventral periventricular nucleus (AVPv) and medial preoptic nucleus (MPN). Dyn B-immunoreactive fibers showed a somewhat more uniform distribution throughout the region, and only a few Dyn B-stained cells bodies were found within the medial preoptic area. In contrast, the preoptic region contained hundreds of PE-immunoreactive cells, which were particularly numerous within the AVPv, MPN, and anterodorsal preoptic nucleus. The AVPv and MPN also contained discretely localized plexuses of PE-stained fibers. Although the overall distributions of opioid peptide-containing fibers within the preoptic region were quite similar in male and female rats, differential distributions of fibers were found in certain nuclei such as the AVPv and MPN, and they were correlated with previously identified cytoarchitectonic sexual dimorphisms. Such differential distributions were particularly distinct for enkephalin-containing fibers. Although the AVPv is larger in female rats, it contained more PE-immunoreactive cell bodies in male rats, and we have shown here that this sexual dimorphism appears to be at least partially dependent on perinatal levels of gonadal steroids. In contrast, no difference in the number of PE-stained cells was found within the anterodorsal preoptic nucleus of male and female animals, indicating that sexual differences are not a general characteristic of enkephalinergic cells in the preoptic region of the rat.(ABSTRACT TRUNCATED AT 400 WORDS)

Levels of dynorphin peptides, substance P and CGRP in the spinal cord after subchronic administration of morphine in the rat.

Rats were rendered dependent on morphine by repeated injections of morphine, in increasing doses for 14 days and sacrificed. Levels of peptides in the dorsal spinal cord and dorsal root ganglia were analyzed in rats decapitated 2 hr, 24 hr (acute abstinent) or 7 days (late abstinent) respectively, after the last injection of drug. Dynorphin A was significantly decreased in rats abstinent for 24 hr, while dynorphin B remained unaffected. Substance P and CGRP, both putative transmitters in nociceptive primary afferent neurones, and partly existing together in the same neurone, were affected differently. Significantly less substance P but unchanged levels of CGRP were detected in rats abstinent for 24 hr, while on the other hand, CGRP but not levels of substance P, were increased 2 hr after the final injection. In dorsal root ganglia, levels of substance P were lower at 2 hr, while levels of CGRP were unaffected. In late (7 days) abstinence, no effect of opiate on any peptide was detected.

Blockade of morphine reward through the activation of kappa-opioid receptors in mice.

The effects of systemic (s.c.) treatment with the kappa-agonists U-50,488H and E-2078 (a stable dynorphin analog) on the morphine-induced place preference were examined in mice. Morphine (s.c.) caused a dose-related preference for the drug-associated place; the effects at doses of 3 and 5 mg/kg were significant. On the other hand, U-50,488H or E-2078 produced a dose-related conditioned place aversion. Both U-50,488H (1 mg/kg, s.c.) and E-2078 (0.1 mg/kg, s.c.) induced a slight, nonsignificant place aversion. Pretreatment with U-50,488H (1 mg/kg) abolished the morphine (3 mg/kg)-induced place preference. The morphine-induced place preference was also significantly decreased by pretreatment with E-2078 (0.1 mg/kg). The inhibitory effects of the kappa-agonists were antagonized by the kappa-antagonist nor-binaltorphimine (nor-BNI; 3 mg/kg, s.c.). In contrast, pretreatment with U-50,488H did not affect the place preference induced by the dopamine (DA) receptor agonist apomorphine (1 mg/kg, s.c.). In addition, morphine (3 mg/kg, s.c.) significantly increased the levels of the DA metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the limbic forebrain (nucleus accumbens and olfactory tubercle) but not in the striatum, implying that activation of the mesolimbic DA system may play an important role in the morphine-induced place preference in mice. Pretreatment with U-50,488H significantly reduced the morphine-induced elevation of DA metabolites in the limbic forebrain.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of modification of the basic residues of dynorphin A-(1-13) amide on kappa opioid receptor selectivity and opioid activity.

A series of dynorphin A-(1-13) amide (Dyn A-(1-13)NH2) analogues containing lysine or N epsilon-acetyllysine (Lys(Ac)) was prepared by solid-phase peptide synthesis and evaluated for opioid receptor affinity in radioligand binding assays and for opioid activity in the guinea pig ileum (GPI). Substitutions were made at positions 6, 7, 9, 11, and 13, the basic amino acids in the C-terminus of the peptide, in order to assess the individual contributions of these residues to the kappa opioid receptor affinity and selectivity of Dyn A-(1-13)NH2. While substitutions of Lys(Ac) for Arg in position 6 did not affect kappa receptor affinity, it enhanced affinity for mu and delta receptors and therefore caused a loss of kappa receptor selectivity. When Lys(Ac) was substituted for Arg9, kappa opioid receptor affinity was enhanced and kappa receptor selectivity was retained. Replacement for Arg7, Lys11, or Lys13 by Lys(Ac) resulted in both decreased affinity and selectivity for kappa receptors. These results demonstrate the importance of Arg6 to the receptor selectivity profile of Dyn A-(1-13)NH2 and indicate that, of the five basic residues in the C-terminus, only Arg9 can be replaced by a nonbasic residue without substantial loss of kappa opioid receptor selectivity.

Design and synthesis of highly potent and selective cyclic dynorphin A analogs. 2. New analogs.

We have designed and synthesized several cyclic disulfide-containing peptide analogs of dynorphin A (Dyn A) which are conformationally constrained in the putative "address" segment of the opioid ligand. Several of these Dyn A analogs exhibit unexpected apparent selectivities for the kappa and mu opioid receptors(s) of the central vs peripheral nervous systems. Thus, incorporation of conformational constraint in the putative "address" segment of Dyn A analogs has resulted in the kappa/mu opioid receptor ligands [L-Pen5,Cys11]Dyn A1-11-NH2 (4), [Cys5,Cys10]Dyn A1-11-NH2 (5), [Cys5,Cys9]DynA1-11-NH2 (6), and [Cys4,Cys9,Arg10]DynA1-11-NH2(7). All of these analogs possess high kappa and mu opioid receptor affinities for the central receptor (guinea pig brain), but effect only weak potency at peripheral kappa and mu opioid receptors (GPI). In fact cyclic dynorphin A analog 4 shows > 19,000-fold differences between central kappa opioid affinity and potency in the guinea pig ileum (GPI). Additionally analog 4 is not an antagonist in the GPI, suggesting possible receptor differences between these sites. Substitution of Tyr1 by Phe1 in the cyclic 1-11 series gave the analog [Phe1,Cys5,Cys11]Dyn A1-11-NH2 (1) that was surprisingly potent in the guinea pig brain binding assay (IC50 = 15.1 nM) at the kappa receptor, but was inactive in the GPI and mouse vas deferens bioassays. D-Ala2 and Tic4 analogs of 1 had lower affinity at brain kappa receptors and had very weak potencies in the GPI and MVD bioassays. On the other hand, [Cys6,Cys10]DynA1-11-NH2 (8), [Cys8,D-Cys13]DynA1-13-NH2 (9), [D-Cys8,D-Cys12]DynA1-13-NH2 (10), and [D-Pro10,Cys5,Cys13]-Dyn A1-13-NH2 (11) were surprisingly potent in the GPI bioassay, though considerable apparent selectivity for central receptors is still retained. The apparent lack of correlation between the pharmacological profiles observed in smooth muscle and in the brain binding assays, particularly with 1 and 4, may suggest the existence of different subtypes of the kappa and mu opioid receptors in the brain and peripheral systems.

Design and synthesis of highly potent and selective cyclic dynorphin A analogues.

We have designed and synthesized several cyclic disulfide-containing peptide analogues of dynorphin A (Dyn A) which are conformationally constrained in the putative "address" segment of the opioid ligand. Several of these Dyn A analogues exhibit unexpected selectivities for the kappa and mu opioid receptors(s) of the central vs peripheral nervous systems. Thus, incorporation of conformational constraint in the putative "address" segment of Dyn A analogues has resulted in the kappa/mu opioid receptor ligands [Cys5,Cys11]Dyn A1-11-NH2 (1) and [Cys5,Cys11,D-Ala8]Dyn A1-11-NH2 (2), which possess high kappa and mu opioid receptor affinities centrally (guinea pig brain, GPB), but only weak activity at peripheral kappa and mu opioid receptors (guinea pig ileum, GPI). On the other hand, [Cys8,Cys13]Dyn A1-13-NH2 and [D-Cys8,D-Cys13]Dyn A1-13-NH2 (5) display high kappa potencies and selectivities at the peripheral (GPI) but not at the central (GPB) kappa opioid receptor. The lack of correlation between the pharmacological profiles observed in smooth muscle and in the brain binding assays suggests the existence of different subtypes of the kappa and mu opioid receptors in the brain and peripheral nervous systems.

Localization of dynorphin gene product-immunoreactivity in neurons from spinal cord and dorsal root ganglia.

Using spinal cord and dorsal root ganglion cell cultures, we have studied the immuno-histochemical distribution of several peptide products of the dynorphin gene. With antibody directed toward the midregion of dynorphin A, peptide-immunoreactivity was found exclusively in the cell bodies of spinal cord neurons. Antibody directed toward the amino- or carboxy-terminus of dynorphin A revealed peptide-immunoreactivity in the neurites, as well as perikarya. Spinal cord neurons also expressed dynorphin B- and alpha-neo-endorphin-immunoreactivities in both cell bodies and neurites. Dorsal root ganglion neurons cultured from embryonic tissue expressed dynorphin A-(1-13)-, dynorphin A-(9-17)- and dynorphin B-immunoreactivities in their perikarya. Sensory neurons obtained from dissociated adult ganglia similarly expressed dynorphin-immunoreactivity immediately upon inoculation into culture. Embryonic and adult murine sensory ganglia from the sacral region more frequently expressed dynorphin than did cells obtained from other spinal levels. Expression of dynorphin-immunoreactivity by sensory neurons was not influenced by elevated levels of Nerve Growth Factor or spinal cord conditioned medium. These data indicate that intrinsic spinal cord neurons may modulate sensory and spinal function in rather subtle ways via the expression of several different opioid peptide products of the dynorphin gene, in addition to the opioid peptides produced by the proenkephalin A gene. Beyond this, the observation of dynorphin-related peptides in dorsal root ganglion neurons suggests that these opioid peptides may have a specialized role in primary afferent neurotransmission.

Electrophysiological evidence for a non-opioid interaction between dynorphin and GABA in the substantia nigra of the rat.

Interactions between neuronal responses mediated by dynorphin A1-8 and GABA were investigated in the substantia nigra zona reticulata. Extracellular recordings and microiontophoresis were performed using five-barrel microelectrodes in chloral hydrate-anesthetized male rats. When iontophoresed alone, dynorphin A1-Q significantly inhibited the firing of 22% of the neurons tested. The inhibition was rapid in onset and recovery and was dose-dependent. In another 22% of the cells, iontophoretic dynorphin produced an increase in the baseline firing rate which was slow in both onset and offset; the remaining 56% were unaffected by dynorphin. When GABA and dynorphin A1-8 were applied in conjunction, the inhibitory action of GABA was attenuated in 61% of the cells; whereas, when dynorphin and GABA were ejected simultaneously onto the cells that were inhibited by dynorphin A1-8, the respective inhibitory effects of dynorphin and GABA appeared to be additive. The kappa antagonist, MR-2266, failed to block the ability of dynorphin A1-8 to attenuate the action of GABA. In addition, the non-opiate peptide des-tyr-dynorphin A2-17, produced effects similar to that of dynorphin A1-8. The role of dynorphin in the basal ganglia and its interaction with the other major transmitter in the substantia nigra zona reticulata, GABA, is discussed.

Generalization tests with intraventricularly applied pro-enkephalin B-derived peptides in rats trained to discriminate the opioid kappa receptor agonist ethylketocyclazocine.

Rats were trained in a two-lever food-reinforced procedure to discriminate between the effects of saline and the opioid kappa receptor agonist ethylketocyclazocine. After acquisition of this discrimination, generalization tests with opioid peptides such as beta-endorphin, alpha-neoendorphin, dynorphin A and some dynorphin-derived peptides were conducted. The rats dose-dependently generalized the effects of intracerebroventricularly injected ethylketocyclazocine but not beta-endorphin, alpha-neoendorphin, dynorphin A1-8, dynorphin A1-13, D-Cys2-L-Cys5-dynorphin A1-13 or dynorphin A. D-Cys2-L-Cys5-dynorphin A1-13, in contrast to dynorphin A itself, dose-dependently caused analgesia and catatonia that was reversible with naloxone. Studies into the receptor preference of this derivative, using the technique of "selective tolerance", revealed that this dynorphin derivative is almost devoid of kappa-receptor activity.

Analgesic and discriminative stimulus properties of U-62,066E, the selective kappa-opioid receptor agonist, in the rat.

The analgesic and discriminative stimulus properties of U-62,066E, a selective kappa-opioid receptor agonist, were investigated in the rat and compared with those of morphine. In the hot-plate test, U-62,066E produced a potent analgesic effect almost comparable to that of morphine. U-62,066E-induced analgesia was far less sensitive to antagonism by naloxone than was morphine-induced analgesia, but was potently reversed by MR-2266, a kappa-receptor antagonist. Although tolerance occurred to both U-62,066E and morphine analgesia, there was no cross-tolerance between these drugs. U-62,066E did show cross-tolerance to U-50,488H, another selective kappa-receptor agonist. Rats were trained to discriminate either 1.0 mg/kg U-62,066E or 3.2 mg/kg morphine from saline in a two-level food-reinforced procedure. The stimulus effect of U-62,066E was substituted for by U-50,488H and E-2078 a stable dynorphin derivative, but not by morphine. None of the kappa-agonists substituted for the morphine stimulus. Although U-62,066E stimulus by itself was not antagonized by MR-2266 or naloxone up to as high a dose as 10 mg/kg, the U-62,066E-like stimulus effect of U-50,488H was markedly blocked by MR-2266. The dopamine antagonists haloperidol and sulpiride substituted for the U-62,066E stimulus cue that was, however, not attenuated by the dopamine agonist lisuride. Lisuride reversed the U-62,066E-like stimulus induced by U-50,488H.(ABSTRACT TRUNCATED AT 250 WORDS)