Candace B. Pert

Opiate receptor: Demonstration in nervous tissue

Tritiated naloxone, a powerful opiate antagonist, specifically binds to an opiate receptor of mammalian brain and guinea pig intestine. Competition for the opiate receptor by various opiates and their antagonists closely parallels their pharmacological potency. The opiate receptor is confined to nervous tissue.

Opiate receptor binding in primate spinal cord: distribution and changes after dorsal root section

Pharmacological receptor sites for opiates in vertebrate nervous tissue are associated with synaptic membrane fractions 16. The density of opiate receptors varies in different regions of the central nervous system with relatively low levels in the spinal cord of rhesus monkey xl. After labeling opiate receptors in intact rats by intravenous administration of [aH]diprenorphine, a potent opiate antagonist, autoradiographic studies reveal a dense localization of opiate receptor associated grains in the substantia gelatinosa of the spinal cord (laminae II-III of Rexed 15) which is a major site for early integration of sensory signals~L In the present study we have measured opiate receptor binding in discrete areas of rhesus monkey spinal cord. We report a localization of receptor binding to the upper dorsal horn and describe a fall in opiate receptor binding in this area following dorsal root section. In three juvenile rhesus monkeys (3-5 kg), the cervical spinal cord from C2 to C8 was exposed by a dorsal laminectomy and the dorsal roots of C2-C8 were sectioned on the left side with the right side left intact. Following initial tranquilizing with ketamine hydrochloride (Ketaset) (i.m., 10 mg/kg), each animal was given one dose each of sodium thiamylal (Surital) (i.v., 3 mg/kg) and atropine sulfate (i.m., 0.5 mg/kg) to allow tracheal intubation, and then maintained throughout the surgery under methyoxyflurane (Metofane) anesthesia using a gas anesthetic-O2 machine. Sterile surgical procedures were followed and both the laminectomy and the rhizotomy were performed using a Zeiss surgical microscope. After removal of the spinous processes and interspinal ligaments, the dura was slit with iris scissors to expose the rootlets. Eachrootletwasgentlyliftedfromthecordwithafineglassrod and cut withirisscissors at all times being careful to avoid cord trauma or hemorrhage. If a blood vessel (e.g. a radicular artery) was intimately associated with a rootlet and could not be separated, that rootlet was not cut. Following the root sections, the cord was covered with Gelfoam and the muscles repositioned and stitched. No attempt was made to sew the dura as this frequently results in spinal cord compression. Vascular shock and res-

Opiate agonists and antagonists discriminated by receptor binding in brain.

Receptor binding of opiate agonists and antagonists can be differentiated in vivo and in vitro. Administration of either rapidly elevates stereospecific [3H]dihydromorphine binding to mouse brain extracts by 40 to 100 percent, but antagonists are 10 to 1000 times more potent than agonists; as little as 0.02 milligram of naloxone per kilogram of body weight significantly enhances opiate receptor binding. Sodium enhances antagonist binding in vitro but decreases agonist binding, a qualitative difference that may be relevant to the divergent pharmacological properties of opiate agonists and antagonists.

Autoradiographic localization of the opiate receptor in rat brain

Abstract One hour after injection of the potent opiate antagonist 3 H-diprenorphine (125 μCi, 13 Ci/mmole) 75–85% of the drug is associated with opiate receptor sites. Autoradiography of fresh frozen unfixed brain has been carried out to visualize receptor distribution. Dense clusters of autoradiographic grains are highly localized in the caudate-putamen, locus coeruleus, zona compacta of the substantia nigra and the substantia gelatinosa.

Autoradiographic distribution of nicotine receptors in rat brain.

The autoradiographic visualisation of 90%-specific tritiated nicotine binding to slide-mounted sections of rat brain is reported. Tritiated nicotine bound with high affinity (nanomolar Kd) and was selectively displaced by nicotinic agonists (e.g. L-nicotine approximately ACh greater than D-nicotine). The strikingly discrete distribution pattern obtained deviates from that of alpha-bungarotoxin, and suggests several possible roles for nicotinic transmission in the brain.

Autoradiographic distribution of substance P receptors in rat central nervous system

Among various neuropeptides present in the central nervous system (CNS), substance P, an undecapeptide1, is of great interest as a putative pain neurotransmitter2–4. Substance P is present within numerous intrinsic neural pathways throughout the CNS5,6,36. Several groups have attempted to label substance P receptors on brain membranes by ligand binding techniques7–12; only one study10 used native 3H-labelled substance P as the ligand and the precise anatomical distribution of substance P receptors has not yet been described. Here we report the autoradiographic localization of 3H-labelled substance P receptors in rat brain using the in vitro autoradiographic technique developed recently13,14. 3H-substance P binds specifically to an apparently single class of sites on slide-mounted brain sections (Kd = 0.52 nM; Bmax = 21.6 fmol per mg protein). The ligand selectivity pattern suggests that 3H-substance P binding sites are similar to those found in other assays11,15. 3H-substance P receptors are highly concentrated in the external layers of the olfactory bulb, medial amygdala, dentate gyrus, superior colliculus, dorsal parabrachial nucleus and locus coeruleus, with moderate densities being found in the nucleus accumbens, striatum, periaqueductal grey and subiculum. The distribution of 3H-substance P receptors suggests that substance P is probably involved in the control of sensory processes such as pain, vision, audition and olfaction.

Ontogenetic development of [3H]naloxone binding in rat brain.

To study the ontogenesis of an apparent opiate receptor, the high affinity, stereospecific binding of [3H]naloxone was measured in whole brains and several brain regions of the developing rat. Stereospecific binding of [3H]naloxone was first detected in whole rat brain at 15 days of gestation and by birth attained 40% of adult specific concentration of binding sites. Scatchard analysis indicated that developmental changes in stereospecific binding reflect increases in the number of sites with the equilibrium dissociation constant for [3H]naloxone remaining constant. At birth, caudal regions of the brain attained relatively higher levels of the binding site than the cortical regions. Treatment of pregnant rats with morphine or naloxone during the last week of pregnancy did not significantly affect the stereospecific binding of [3H]naloxone in the brains of their offspring.

Ontogeny of opiate receptors in rat forebrain: Visualization by in vitro autoradiography

The embryonic and postnatal ontogeny of opiate receptors in rat telencephalon was mapped by in vitro autoradiographic localization of [3H]naloxone and [3H]enkephalin binding. Opiate receptors marked by naloxone binding first appear at embryonic day 14 in the striatum, rapidly proliferate to adult densities and at the time of birth, gradually become reorganized into the adult heterogeneous pattern of receptor-rich patches surrounded by sparse, diffuse labeling in the rest of the striatum. The enkephalin binding in the striatum appears later in embryonic development and gradually increases in density to form the rather homogeneous adult pattern. Naloxone binding in the paleocortical olfactory areas appears early also, densely within the molecular layer as soon as it is formed at E16. This density is only temporary, as labeling just after birth falls to low adult levels in all areas except portions of the amygdala. Receptors disappear also in the islands of Calleja and the pallidum. Naloxone binding in the septum and neocortex appears gradually in development. The early appearance of striatal and paleocortical [3H]naloxone-labeled opiate receptors and their localization within the subependymal zones suggest that receptors appear on immature neurons before and during migration and, therefore, may influence the intricate patterns of connections that later form. The delayed appearance of the [3H]enkephalin-labeled receptors may reflect the dependence of the peptide binding on later developing molecules of adenylate cyclase.

Opiate receptor affinities and behavioral effects of enkephalin: structure-activity relationship of ten synthetic peptide analogues.

Abstract Synthetic met- and leu-enkephalin bind to rat brain opiate receptors with 1 2 and 1 7 the affinity of morphine. The aromatic hydroxyl moiety of the tyrosine residue is critical for receptor binding. Intracranial microinjection of met-enkephalin requires very high doses to produce an evanescent, naloxone reversible analgesia and stuperous immobility, presumably because of its rapid enzymatic degradation. Leu-enkephalin fails to elicit analgesia.

Substance P receptor-mediated chemotaxis of human monocytes

The undecapeptide substance P (SP) was tested for its ability to promote human monocyte chemotaxis in a modified Boyden chamber assay. Substance P was found to be active in this assay system with an ED50 for chemotactic effect of approximately 10(-13) M. This response was shown to be chemotactic in nature since a concentration gradient of attractant was required for maximal effect. Other substance P analogs tested showed a rank order of potency of substance P greater than or equal to SP(3-11) greater than SP(8-11) approximately equal to SP(9-11) much greater than SP(1-9), SP, free acid. These results suggest that chemotactic responsiveness is largely encoded in the C-terminus of the molecule. The relative potency order for SP and its analogs in promoting monocyte chemotaxis correlates well with their potencies in displacing labeled SP when binding sites are directly measured in other tissues, such as rat brain or human lymphocytes. Additionally, the chemotactic effects of SP could be partially reversed by the weak antagonist [D-Arg1, D-Pro2, D-Trp7,9, Leu11]-SP. The N-formyl peptide receptor antagonist, t-Boc-Phe-Leu-Phe-Leu-Phe, did not block SP-mediated chemotaxis, further indicating the specificity of these effects. These results suggest the existence of a specific substance P receptor on human monocytes which directs this chemotactic response. The ability of monocytes to respond chemotactically to SP may be relevant to the enhancing effects of SP in arthritis or other inflammatory diseases.