C-peptide of preproinsulin-like peptide 7: localization in the rat brain and activity in vitro.

With the use of a rabbit polyclonal antiserum against a conserved region (54-118) of C-peptide of human preproinsulin-like peptide 7, referred to herein as C-INSL7, neurons expressing C-INSL7-immunoreactivity (irC-INSL7) were detected in the pontine nucleus incertus, the lateral or ventrolateral periaqueductal gray, dorsal raphe nuclei and dorsal substantia nigra. Immunoreactive fibers were present in numerous forebrain areas, with a high density in the septum, hypothalamus and thalamus. Pre-absorption of C-INSL7 antiserum with the peptide C-INSL7 (1 microg/ml), but not the insulin-like peptide 7 (INSL7; 1 microg/ml), also known as relaxin 3, abolished the immunoreactivity. Optical imaging with a voltage-sensitive dye bis-[1,3-dibutylbarbituric acid] trimethineoxonol (DiSBAC4(3)) showed that C-INSL7 (100 nM) depolarized or hyperpolarized a small population of cultured rat hypothalamic neurons studied. Ratiometric imaging studies with calcium-sensitive dye fura-2 showed that C-INSL7 (10-1000 nM) produced a dose-dependent increase in cytosolic calcium concentrations [Ca2+]i in cultured hypothalamic neurons with two distinct patterns: (1) a sustained elevation lasting for minutes; and (2) a fast, transitory rise followed by oscillations. In a Ca2+-free Hanks' solution, C-INSL7 again elicited two types of calcium transients: (1) a fast, transitory increase not followed by a plateau phase, and (2) a transitory rise followed by oscillations. INSL7 (100 nM) elicited a depolarization or hyperpolarization in a small population of hypothalamic neurons, and an increase of [Ca2+]i with two patterns that were dissimilar from that of C-INSL7. [125I]C-INSL7 bindings to rat brain membranes were inhibited by C-INSL7 in a dose-dependent manner; the Kd and Bmax. values were 17.7 +/- 8.2 nM and 45.4 +/- 20.5 fmol/mg protein. INSL7 did not inhibit [125I]C-INSL7 binding to rat brain membranes, indicating that C-INSL7 and INSL7 bind to distinct binding sites. Collectively, our result raises the possibility that C-INSL7 acts as a signaling molecule independent from INSL7 in the rat CNS.

An antisense oligodeoxynucleotide to the mu opioid receptor attenuates cocaine-induced behavioral sensitization and reward in mice.

Numerous studies support a role for the endogenous opioid system in cocaine-influenced behavior. Few of these studies, however, selectively delineate a role for the mu opioid receptor (MOR) in this regard. This investigation examined if the MOR modulates cocaine-induced behavior in mice using a 17-base antisense oligodeoxynucleotide (AS ODN) directed against the MOR coding sequence 16-32. Specifically, cocaine-induced behavioral sensitization and conditioned reward were investigated. For the sensitization study, C57BL/6J mice received eight intermittent i.c.v. infusions of saline, mismatch oligodeoxynucleotide (ODN) (20 microg/4 microl) or AS ODN (20 microg/4 microl) over 20 days. Mice also received concomitant once daily i.p. injections of saline (4 ml/kg) or cocaine (15 mg/kg) for 10 days. There was a 7-day withdrawal period, after which all mice were challenged with cocaine (15 mg/kg) to test for behavioral sensitization. For the conditioned place preference (CPP) study, mice received five i.c.v. infusions of mismatch ODN or MOR AS ODN (days 1-5). An unbiased counterbalanced conditioning procedure was used where mice were conditioned with saline (4 ml/kg, i.p.) and cocaine (15 mg/kg, i.p.) on alternate days for four sessions (days 3-6). Mice were tested on day 7 for CPP. Immediately following testing, [3H]DAMGO (D-Ala2, N-Me-Phe4, Gly-ol5-enkephalin) receptor binding to brain homogenates was conducted. MOR AS attenuated cocaine-induced behavioral sensitization and conditioned reward. MOR AS ODN also reduced [3H]DAMGO binding. Collectively, these findings implicate the MOR as playing an important neuromodulatory role in the behavioral effects of cocaine in mice.

Distribution and ultrastructural localization of GEC1 in the rat CNS.

We have previously demonstrated that GEC1 interacts with the kappa opioid receptor and GEC1 expression enhances cell surface expression of the receptor in Chinese hamster ovary cells. In this study, we generated an antiserum (PA629) directed against GEC1 in rabbits, characterized its specificity, and investigated distribution of GEC1 in tissues and in brain regions and spinal cord and its subcellular localization in hypothalamic neurons in the rat. Immunofluorescence staining demonstrated that PA629 recognized HA-GEC1 transfected into Chinese hamster ovary cells, but not HA-GABARAP or HA-GATE-16, although the three share high homology. Pre-incubation of PA629 with GST-GEC1, but not GST, abolished the staining. In immunoblotting, affinity-purified PA629 (PA629p) recognized GEC1, GABARAP and GATE-16. GEC1 migrated slower than GABARAP and GATE-16, with a M(r) of 16 kDa for GEC1 and M(r) of 14 kDa for GABARAP and GATE-16. Immunoblotting results showed that GEC1 level was higher in liver and brain than in lung and heart, and very low in kidney and skeletal muscle. GEC1 was present in all rat brain regions examined and spinal cord. Immunohistochemistry demonstrated that GEC1 immunoreactivity was distributed ubiquitously in the rat CNS with highly intense immunoreactivity in various brain nuclei and motor neurons of the spinal cord. Ultrastructural examination of neurons in the paraventricular nucleus of the hypothalamus showed that GEC1 was associated with endoplasmic reticulum and Golgi apparatus and distributed along plasma membranes and in cytosol. Coupled with our previous observation that GEC1 interacts with N-ethylmaleimide-sensitive factor, these findings strongly suggest that GEC1 functions in intracellular trafficking in the biosynthesis pathway and perhaps also the endocytic pathway. The widespread distribution of GEC1 suggests that GEC1 may be associated with many proteins, in addition to the kappa opioid receptor.

Palmitoylation of the rat mu opioid receptor.

We examined whether the mu opioid receptor was palmitoylated and attempted to determine sites of palmitoylation. Following metabolic labeling with [3H]palmitic acid and immunoaffinity purification of the mu opioid receptor, SDS-PAGE and fluorography revealed a broad labeled band with Mr of approximately 80 kDa in CHO cells stably expressing the rat mu receptor, but not in CHO cells transfected with the vector alone, indicating that the mu receptor is palmitoylated. Activation of the receptor with morphine did not affect the extent of palmitoylation. Hydroxylamine or dithiothreitol treatment removed most of the radioactivity, demonstrating that [3H]palmitic acid is incorporated into Cys residue(s) via thioester bond(s). Surprisingly, mutations of the only two Cys residues in the C-terminal domain did not reduce [3H]palmitic acid incorporation significantly. Thus, unlike many G-protein coupled receptors, the palmitoylation site(s) of the rat mu opioid receptor do(es) not reside in the C-terminal domain.

Functional role of the spatial proximity of Asp114(2.50) in TMH 2 and Asn332(7.49) in TMH 7 of the mu opioid receptor.

We examined whether a proposed spatial proximity between Asp114(2.50) and Asn332(7.49) affected the functional properties of the mu opioid receptor. The D114(2.50)N mutant had reduced binding affinities for morphine, DAMGO and CTAP, but not for naloxone and [3H]diprenorphine; this mutation also abolished agonist-induced increase in [35S]GTPgammaS binding. The N332(7.49)D mutation eliminated detectable binding of either [3H]diprenorphine or [3H]DAMGO. The combined D114(2.50)N-N332(7.49)D mutation restored high affinity binding for [3H]diprenorphine, CTAP and naloxone, and restored partially the binding affinities, potencies and efficacies of morphine and DAMGO. Thus, reciprocal mutations of Asp114(2.50) and Asn332(7.49) compensate for the detrimental effects of the single mutations, indicating that the residues are adjacent in space and that their chemical functionalities are important for ligand binding and receptor activation.

The region in the mu opioid receptor conferring selectivity for sufentanil over the delta receptor is different from that over the kappa receptor.

We determined the binding domains of sufentanil and lofentanil in the mu opioid receptor by comparing their binding affinities to seven mu/delta and six mu/kappa chimeric receptors with those to mu, delta and kappa opioid receptors. TMHs 6 and 7 and the e3 loop of the mu opioid receptor were important for selective binding of sufentanil and lofentanil to the mu over the kappa receptor. TMHs 1-3 and the e1 loop of the mu opioid receptor conferred binding selectivity for sufentanil over the delta receptor. Thus, the region that conferred binding selectivity for sufentanil differs, depending on chimeras used. In addition, the interaction TMHs 1-3 and TMHs 6-7 was crucial for the high affinity binding of these two ligands. These two regions are likely to contain sites of interaction with the ligands or to confer conformations specific to the mu receptor.

Immunolabeling of Mu opioid receptors in the rat nucleus of the solitary tract: extrasynaptic plasmalemmal localization and association with Leu5-enkephalin.

Activation of the mu opioid receptor (MOR) by morphine within the caudal nucleus of the solitary tract (NTS) is known to mediate both cardiorespiratory and gastrointestinal responses. Leu5-enkephalin (LE), a potential endogenous ligand for MOR, is also present within neurons in this region. To determine the cellular sites for the visceral effects of MOR ligands, including LE, we used immunogold-silver and immunoperoxidase methods for light and electron microscopic localization of antisera against MOR (carboxyl terminal domain) and LE in the caudal NTS of rat brain. Light microscopy of coronal sections through the NTS at the level of the area postrema showed MOR-like immunoreactivity (MOR-LI) and LE labeling in punctate processes located within the subpostremal, dorsomedial and medial subnuclei. Electron microscopy of sections through the medial NTS at this level showed gold-silver particles identifying MOR-LI prominently distributed to the cytoplasmic side of the plasma membranes of axons and terminals. MOR labeled terminals formed mostly symmetric (inhibitory-type) synapses but sometimes showed multiple asymmetric junctions, characteristic of excitatory visceral afferents. MOR-LI was also present along extrasynaptic plasma membranes of dendrites receiving afferent input from unlabeled and LE-labeled terminals. We conclude that MOR ligands, possibly including LE, can act at extrasynaptic MORs on the plasma membranes of axons and dendrites in the caudal NTS to modulate the presynaptic release and postsynaptic responses of neurons. These are likely to include local inhibitory neurons and both gastric and cardiorespiratory afferents known to terminate in the subnuclei with the most intense MOR-LI.

Co-localization of retrogradely transported wheat germ agglutinin and the putative neurotransmitter substance P within trigeminal ganglion cells projecting to cat middle cerebral artery.

Neurons containing both wheat germ agglutinin (WGA) and substance P (SP) immunoreactivities were found in the ophthalamic division of the ipsilateral trigeminal ganglia following application of the axonally transported lectin WGA to cat middle cerebral artery. Immunohistochemistry was accomplished by staining for WGA and SP on adjacent sections by using the chromogen diaminobenzidine, or by staining on a single section with sequential application of WGA and SP antisera using the chromogens diaminobenzidine and 4-chloro-1-naphthol, respectively. These observations confirm the results of trigeminal lesion studies indicating that trigeminovascular projections to the middle cerebral artery in the cat contain the neurotransmitter SP. In addition, other neurotransmitters may be present in this pathway since less than 50% of WGA-labeled cells contained SP.

Sequence of kappa-opioid receptor cDNA in the R1.1 thymoma cell line.

To our knowledge, this is the first demonstration of kappa-opioid receptor mRNA in cells of the immune system. While the presence of opioid receptors on cells of the immune system has been controversial, cell-binding analysis has indicated that the kappa-opioid receptor is expressed by the immature T cell line R1.1. We have developed a reverse transcriptase-polymerase chain reaction protocol to amplify the mRNA extracted from R1.1 cells with primers derived from the cDNA sequence of the mouse kappa-opioid receptor. Nucleotide sequences of the amplified products were examined and two populations of cDNA were detected which differ in the 5' region upstream of the ATG start codon. Comparison of these sequences to the previously published kappa-opioid receptor cDNA sequence suggests the presence of an intron-exon junction in the 5' non-coding region.

Characterization of kappa-opioid receptor transcripts expressed by T cells and macrophages.

We have found that the immature T cell lines R1.1 and DPK and the macrophage lines P388D1 and WEHI-3 also express kappa-opioid receptor (KOR) mRNA. Characterization of the KOR transcripts in both brain tissue and these T cells has revealed both the normal full-length as well as a truncated form of the mRNA. Our results show that the truncated transcript lacks the second exon. Primary macrophages express this truncated form of the transcript in the absence of detectable levels of the full-length form. These results suggest a degree of heterogeneity in the expression of the opioid receptors which has not previously been reported.

Pia arachnoid contains substance P originating from trigeminal neurons.

Immunoreactive substance P is present in measurable amounts in pia arachnoid from rat, cat, dog and calf. Levels of substance P in this tissue are comparable to those found in peripheral structures receiving innervation from dorsal root or trigeminal ganglia. Separation and measurement of bovine pia-arachnoid extract by reverse phase high performance liquid chromatography and radioimmunoassay reveals a single peak of activity with a retention time identical to that of substance P. Unilateral lesions of the trigeminal ganglia decrease substance P levels within cat pia arachnoid and accompanying blood vessels ipsilaterally by greater than 50%. These data indicate that most of the substance P surrounding pial blood vessels resides within afferent nerve fibres from trigeminal ganglion cells.

In vitro release of immunoreactive substance P from putative afferent nerve endings in bovine pia arachnoid.

The release of substance P-like immunoreactivity was examined using bovine pia arachnoid and its attendant blood vessels in vitro. At concentrations of 20,51, and 100 mM, potassium ions evoked the release of substance P-like immunoreactivity in a dose-dependent manner. The drug capsaicin released substance P at concentrations greater than 10(-8) M. Both potassium- and capsaicin-induced release were abolished by omitting calcium ions from the superfusion buffer. When subjected to separation by reverse phase high performance liquid chromatography, the superfusate from capsaicin perfused tissues contained a peak of immunoreactivity which migrated at the retention time corresponding to substance P. During basal and stimulated states, the percent endogenous substance P released ranged between 0.4-6.5 X 10(-2) and 1.3-11.6 X 10(-2) per minute, rates comparable to those previously reported by others using slices of dorsal horn or spinal cord segments. The immunoreactivity measurable in the conditioned buffer probably reflected release from afferent nerve endings in as much as most of the substance P immunoreactivity in pia arachnoid arises from trigeminal ganglia. Release of substance P, a cerebrovasodilating peptide from perivascular nerve endings in pia arachnoid suggests a possible role for substance P in the pathophysiology of disorders associated with pain of cerebrovascular origin.

Properties of a kappa-opioid receptor expressed in CHO cells: interaction with multiple G-proteins is not specific for any individual G alpha subunit and is similar to that of other opioid receptors.

The purpose of the present study was to examine the coupling pattern of a recently cloned kappa-opioid receptor stably transfected in CHO cells to individual G alpha subunits with subsequent comparison to that observed previously for delta- and mu-opioid receptors. Data presented in the current study indicate the successful stable expression of a kappa-opioid receptor in CHO cells. This is supported by experiments in which ligands with selectivity for kappa-, but not delta- or mu-opioid receptors demonstrated high affinity for the expressed receptor and were able to potently and efficaciously produce inhibition of adenylyl cyclase activity. In addition, only kappa-opioid agonists were able to induce dose-dependent increases in the incorporation of [32P]azidoanilido-GTP into four G alpha subunits, three of which were identified as Gi3 alpha, Gi2 alpha and Go2 alpha. Further, the amount of kappa-opioid agonists required to induce 50% maximal labeling of any individual G alpha subunit was similar. Although kappa-opioid agonists produced equivalent maximal labelling of Gi3 alpha, Gi2 alpha and Go2 alpha, significantly less agonist-induced labeling was observed for an unknown G-protein designated as G? alpha. Although these results are slightly different than those observed previously for both delta- and mu-opioid receptors, it appears that all opioid receptors stably transfected in CHO cells interact with multiple G-proteins and that this coupling is not selective for any individual G alpha subunit.

Substance P-like immunoreactivity in rat forebrain leptomeninges and cerebral vessels originates from the trigeminal but not sympathetic ganglia.

Substance P-like immunoreactivity (SPLI) is present in the pia mater, arachnoid and pial vessels (leptomeninges) of the rat. Unilateral electrolytic lesions of the trigeminal ganglion decreased levels of SPLI in leptomeninges on the lesioned side. Levels did not change following unilateral or bilateral superior cervical ganglionectomies or after i.c.v. injections of 6-hydroxydopamine, sufficient to deplete norepinephrine in pial arteries by more than 90%. SP levels did not decrease in leptomeninges or in blood vessels within the circle of Willis following treatment of adult or neonatal rats with capsaicin despite the fact that levels were reduced in the cornea and dorsal spinal cord in these same animals. These results suggest that not all substance P-containing sensory fibers are susceptible to the peptide-depleting effects of capsaicin.

Dual ultrastructural immunocytochemical labeling of mu and delta opioid receptors in the superficial layers of the rat cervical spinal cord.

The delta opioid receptor (DOR) and mu opioid receptor (MOR) are abundantly distributed in the dorsal horn of the spinal cord. Simultaneous activation of each receptor by selective opiate agonists has been shown to result in synergistic analgesic effects. To determine the cellular basis for these functional associations, we examined the electron microscopic immunocytochemical localization of DOR and MOR in single sections through the superficial layers of the dorsal horn in the adult rat spinal cord (C2-C4). From a total of 270 DOR-labeled profiles, 49% were soma and dendrites, 46% were axon terminals and small unmyelinated axons, and 5% were glial processes. 6% of the DOR-labeled soma and dendrites, and < 1% of the glial processes also showed MOR-like immunoreactivity (MOR-LI). Of 339 MOR-labeled profiles, 87% were axon terminals and small unmyelinated axons, 12% were soma and dendrites, and 2% were glial processes. 21% of the MOR-labeled soma and dendrites, but none of the axon terminals also contain DOR-LI. The subcellular distributions of MOR and DOR were distinct in axon terminals. In axon terminals, both DOR-LI and MOR-LI were detected along the plasmalemma, but only DOR-LI was associated with large dense core vesicles. DOR-labeled terminals formed synapses with dendrites containing MOR and conversely, MOR-labeled terminals formed synapses with DOR-labeled dendrites. These results suggest that the synergistic actions of selective MOR- and DOR-agonists may be attributed to dual modulation of the same or synaptically linked neurons in the superficial layers of the spinal cord.

Autoradiographic study of irreversible binding of [3H]beta-funaltrexamine to opioid receptors in the rat forebrain: comparison with mu and delta receptor distribution.

beta-Funaltrexamine (beta-FNA) is an irreversible mu antagonist and a reversible kappa agonist in in vivo and in vitro tests. However, whether it produces irreversible delta antagonism is controversial. In binding studies, it is clear that beta-FNA does not bind irreversibly (it does reversibly) to kappa receptors. Yet there is no consensus as to whether beta-FNA binds irreversibly to mu and/or delta receptors. In this study, irreversible binding of [3H]beta-FNA to opioid receptors was examined in rat forebrain sections in the presence of 200 mM NaCl and its distribution compared with those of mu and delta opioid receptors, labeled by [3H][D-Ala2,MePhe4,Gly-ol5]enkephalin ([3H]DAMGO) and [3H][D-Pen2,D-Pen5]enkephalin ([3H]DPDPE), respectively. Irreversible binding of [3H]beta-FNA was determined as the binding that remained following 5 washes at room temp. for 1, 5, 20, 20, and 20 min each. Non-specific binding was defined by including 10 microM naloxone, beta-chlornaltrexamine (beta-CNA), or beta-FNA in the incubation mixture. At 37 degrees C, specific irreversible binding of [3H]beta-FNA to opioid receptors reached a plateau at 10 nM in 60 min, and constituted 50-70% of total irreversible binding. Series of 4 sections of similar anatomical levels were labeled with [3H]DAMGO, [3H]beta-FNA, [3H]beta-FNA + 10 microM naloxone, beta-CNA, or beta-FNA, and [3H]DPDPE, resp., and exposed to [3H]-Ultrofilm. The distribution of [3H]beta-FNA (5 nM) irreversible labeling is very similar to that of [3H]DAMGO, i.e. patches and subcallosal streaks in caudate-putamen, patches in nucleus accumbens, dense labeling in thalamus, and more binding in the rostral than caudal striatum.(ABSTRACT TRUNCATED AT 250 WORDS)

Some cholecystokinin-8 immunoreactive fibers in large pial arteries originate from trigeminal ganglion.

Cholecystokinin-8 immunoreactive (CCK8I) nerve fibers were demonstrated in whole mount preparations and cross-sections of pial blood vessels in the cat, guinea pig and rat using a specific antiserum and the avidin-biotin-peroxidase complex method. Positive fibers were present in nearly all pial arteries examined, and were located in the adventitial layer and at the junction of the adventitia and media. In general, CCK8I fibers were less abundant than substance P immunoreactive (SPI) fibers visualized in the same vessels. A marked depletion of CCK8I was noted in large cerebral arteries following treatment of adult guinea pigs with capsaicin, a drug shown previously to deplete CCK8 in some primary sensory neurons. The density of CCK8I-containing fibers was also decreased in the ipsilateral vessels of the cat circle of Willis following unilateral trigeminal ganglionectomies. These results indicate that CCK8I is contained in afferent fibers within large pial arteries of Willis' circle which project from neurons in the ipsilateral trigeminal ganglion. Whether CCK8 coexists with SP in these fibers remains to be determined.

Intracerebroventricular treatment with an antisense oligodeoxynucleotide to kappa-opioid receptors inhibited kappa-agonist-induced analgesia in rats.

In vivo treatment with an antisense (AS) phosphorothioate oligodeoxynucleotide (oligo) to the rat kappa-opioid receptor selectively inhibited kappa-mediated analgesia in the rat cold-water tail-flick test. Intracerebroventricular (i.c.v.) AS oligo significantly inhibited the analgesic effect of i.c.v. spiradoline, but not that of mu- or delta-opioid agonists. The dose-effect curve for s.c. spiradoline was shifted to the right after AS, but not missense or sense oligo treatment. Thus, AS oligos provide another technique with which to selectively manipulate opioid receptors and further support the role of non-mu opioid receptors in mediating analgesia in rats.

Immunoelectron microscopic study of substance P-containing fibers in feline cerebral arteries.

The ultrastructure of substance P-containing fibers in feline cerebral arteries was examined by combining substance P immunohistochemistry and electron microscopy. At the light and electron microscopic level, positive fibers were observed in the adventitia and at the border between the adventitia and media, but not within the media or the endothelium. The substance P-containing fibers were unmyelinated with diameters consistent with C-fiber caliber. Positive axons were in close contact with Schwann cell processes. Positive axons contained 24 nm microtubules, 10 nm neurofilaments, clear vesicles and scattered mitochondria. The number of mitochondria and organelles resembling vesicles appeared to increase in presumptive axon terminals. No synaptic membrane specializations were observed.

Antisense oligodeoxynucleotides against mu- or kappa-opioid receptors block agonist-induced body temperature changes in rats.

PL017 and dynorphin A1-17 were shown previously to cause a marked increase and a profound decrease in body temperature (Tb), respectively. In this study, we examined whether an antisense (AS) oligodeoxynucleotide (oligo) against cloned mu or kappa opioid receptors could block PL017- or dynorphin A-induced body temperature changes. Treatment with an AS oligo against mu receptors, but not sense (S) oligo, missense (MS) oligo or artificial cerebrospinal fluid (aCSF), abolished PL017-induced hyperthermia. In addition, treatment with an AS oligo against kappa receptors, but not S oligo, MS oligo or aCSF, greatly attenuated dynorphin A-induced hypothermia. This study further supports the notion that mu and kappa receptors mediate Tb regulation.