Bryan L. Roth

Differential postnatal development of mu and delta opiate receptors

We found a differential postnatal development of mu and delta opiate receptors. Mu receptors labelled with low concentrations of [3H]naloxone appeared to develop earlier than did delta receptors labelled with [3H]D-Ala2-D-Leu5-enkephalin (0.5 nM). Competition binding studies also revealed a delayed appearance of delta receptors (day 12 postnatal).

The postnatal development of VIP binding sites in rat forebrain and hindbrain

The specific binding of 125I-labeled vasoactive intestinal peptide (VIP) to brain membranes from the forebrain and hindbrain regions of 2 to 37-day postnatal rats was measured. In both regions of the brain, VIP binding was low but detectable two days after birth and rose markedly between postnatal days 7 and 17. This increase in VIP binding with age correlates well with observed increases in VIP-stimulated adenylate cyclase activity and increases in VIP content as determined by radioimmunoassay. In hindbrain, the density of VIP binding sites was substantially higher than the forebrain at two days, while at 37 days, they were about equal, suggesting that the hindbrain regions may mature neurochemically prior to the forebrain. Total binding sites for forebrain and hindbrain were about equal at birth for both brain regions, while forebrain had a substantially greater number of sites at 37 days postnatal. The presence of VIP binding sites in both forebrain and hindbrain early in postnatal development suggests that VIP may play a role in development of the brain.

Microsomal opiate receptors: Characterization of smooth microsomal and synaptic membrane opiate receptors

Abstract: In continuing studies on smooth microsomal and synaptic membranes from rat forebrain, we compared the binding properties of opiate receptors in these two discrete subcellular populations. Receptors in both preparations were saturable and stereospecific. Scatchard and Hill plots of [3H]naloxone binding to microsomes and synaptic membranes were similar to plots for crude membranes. Both synaptic membranes and smooth microsomes contained similar enrichments of low‐ and high‐affinity [3H]naloxone binding sites. No change in the affinity of the receptors was observed. When [3H]D‐ala2‐D‐leu5‐enkephalin was used as ligand, microsomes possessed 60% fewer high‐affinity sites than did synaptic membranes, and a large number of low‐affinity sites. In competition binding experiments microsomal opiate receptors lacked the sensitivity to (guanyl‐5′‐yl)imidodiphosphate [Gpp(NH)p] shown by synaptic and crude membrane preparations. In this respect microsomal opiate receptors resembled membranes that were experimentally guanosine triphosphate (GTP)‐uncoupled with N‐ethylmaleimide (NEM). Agonist binding to microsomal and synaptic membrane opiate receptors was decreased by 100 mM NaCl. Like NEM‐treated crude membranes, microsomal receptors were capable of differentiating agonist and antagonists in the presence of 100 mM NaCl. MnCl2 (50–100 μM) reversed the effects of 100 mM NaCl and 50 μM GTP on binding of the μ‐specific agonist [3H]dihydromorphine in both membrane populations. Since microsomal receptors are unable to distinguish agonists from antagonists in the presence of Gpp(NH)p, they are a convenient source of guanine nucleotide‐uncoupled opiate receptors.

Secretin Receptors on Neuroblastoma Cell Membranes: Characterization of 125I-Labeled Secretin Binding and Association with Adenylate Cyclase

Abstract: Secretin, a gut‐brain peptide, elicited cyclic AMP production in a clone of neuroblastoma cells derived from the C1300 mouse tumor. Adenylate cyclase (EC 4.6.1.1) in plasma membranes from these cells was stimulated by secretin > vasoactive intestinal peptide > peptide histidine isoleucine amide, but not by the related peptides glucagon, gastric inhibitory polypeptide, or human growth hormone releasing factor. Hill coefficients for stimulation approximated one and the response to submaximal peptide concentrations was additive, as expected for hormones competing for a single receptor associated with the enzyme. Binding of 125I‐labeled secretin to the neuroblastoma plasma membranes was saturable, time‐dependent, and reversible. The KD determined from kinetic and equilibrium binding studies approximated 1 nM. The binding site displayed marked ligand specificity that paralleled that for stimulation of adenylate cyclase. The secretin receptor was regulated by guanine nucleotides, with guanosine 5′‐(β,γ‐imino)‐triphosphate being the most potent to accelerate the rate of dissociation of bound secretin. These findings demonstrate the functional association of the secretin receptor with adenylate cyclase in neuronally derived cells.

The effects of morphine on catecholamine metabolism during postnatal development

In an attempt to delineate the mechanism of action of opiates at the molecular level, recent studies have focused on the effects of morphine on catecholamine (CA) metabolism 8. Both morphine and endogenous opiates cause increased turnover of CA, particularly in the striatuml,4,17 and adrenal medulla 16. This effect on turnover is reflected by increased levels of homovanillic acid and dihydroxyphenylacetic acid 17 (DOPAC) in the striatum and elevated dopamine (DA) in the adrenal medulla 16. One approach to studying the mechanism of action of morphine in eliciting these changes is to assess its effects during postnatal development. Correlation of the appearance of effects with the development of other neurochemical functions may aid in the elucidation of the role of endogenous opiates as well as the mode of action of morphine. Accordingly, we have examined the effects of morphine on CA metabolism in rats during postnatal development. For these studies, timed-pregnant female Sprague-Dawley rats (ARS) were obtained one week prior to parturition and housed in separate cages at a constant temperature with 12 h alternating light and darkness. At birth litters were adjusted to 8-10 pups and all experiments were performed between I 1.00 and 14.00 h to minimize diurnal CA fluctuations. At various days after birth animals were given intraperitoneal injections of test substances dissolved in 0.1 ml sterile saline. Animals were decapitated 1.5 h later and brains and adrenals rapidly removed. Striata were dissected according to established techniques 11. For CA determinations, tissues were homogenized in 0.4 N perchloric acid and centrifuged to obtain the acid-soluble supernatant. An aliquot of the supernatant was then directly injected onto a Waters Associates high pressure liquid chromatograph to separate CA and metabolites 15 with quantitation by electrochemical detection 9.

Demonstration of a slowly dissociating form of bovine hippocampal synaptic membrane opiate receptors

We studied the binding characteristics of opiate receptors in synaptic plasma membranes isolated from bovine hippocampus. On the basis of kinetic binding studies the interaction of [3H][D-Ala2,D-Leu5]enkephalin (DADL) with its receptor was an extremely slow process. Rates of association and dissociation were determined and a pseudo-first order rate constant for association calculated to be 5.68 x 10(5) l/mol . s at 25 degrees C. The rate of dissociation (t 1/2 = 70 min) was accelerated by GTP and changed from linear to biphasic. The kinetically derived equilibrium dissociation constant (0.29 nM) was considerably lower than the KD obtained from Scatchard and Hill plots (1.24 nM). Measurement of DADL association as a function of temperature yielded a linear Arrhenius plot. Finally, competition binding assays revealed that delta-specific agonists exhibited relatively high potency in displacing [3H]DADL from synaptic plasma membranes receptors whereas mu-specific agonists and antagonists were less effective. These results with bovine hippocampus may be explained by the formation of a slow-dissociating, high affinity agonist conformation of the delta-opiate receptor which has been predicted for the system by the cyclic-allosteric and ternary complex models.

Ontogeny of benzomorphan-selective (κ) sites: A computerized analysis

Abstract In an investigation of the postnatal development of κ opiate receptors, the affinity and capacity of 0.5 nM [3H]-ethylketocyclazocine (EKC) binding in crude rat brain homogenates was measured by displacement with unlabeled EKC, morphine, or D-ala2-D-leu5-enkephalin (DADL). Displacement curves were analyzed using a weighted, non-linear regression, curve fitting computer program. At all stages of development, [3H]-EKC binding fit a two site model significantly better than a one site model. Affinities of EKC, morphine, or DADL for the high affinity [3H]-EKC binding site did not change during the postnatal period. The density of the high affinity [3H]-EKC binding site increased linearly with age, whereas the levels of the low affinity site rose more rapidly during the second week.

Microsomal opiate receptors differ from synaptic membrane receptors in proteolytic sensitivity.

We have found that opiate receptors in smooth microsomal fractions differ from synaptic membrane-associated receptors in proteolytic sensitivity. With 3 proteases of different substrate specificities (trypsin, chymotrypsin and S. griseus protease) smooth microsomal opiate receptors from rat brain were consistently less sensitive to limited proteolysis than were synaptic membrane receptors. Thiamine pyrophosphatase, a luminal Golgi membrane marker enzyme, exhibited a similar resistance to S. griseus protease in microsomal preparations, while microsomal Na+/K+-ATPase (ouabain-sensitive) was readily destroyed by trypsin. We also discovered that smooth microsomal opiate receptors co-migrate with both Golgi membrane and endoplasmic reticulum marker proteins on equilibrium density gradients under isopycnic conditions. Electron microscopic examination of the Golgi-enriched fraction showed the typical cisternae frequently associated with isolated Golgi membranes. Synaptic junctions, presynaptic membranes, myelin and mitochondria were conspicuously absent from this fraction. Since the microsomes isolated in vitro showed similar topography to those in vivo, the binding sites for opiates could be localized on the luminal surface membranes of the microsomal fractions. The exquisite sensitivity of synaptic membrane opiate receptors to proteolysis suggests that these receptors are found on the extracellular surface of the synaptic junction.

The effects of tetrahydroisoquinolinecarboxylic acids on tyrosine 3-monooxygenase☆

Abstract The effects of tetrahydroisoquinolinecarboxylic acids, derived from dopamine and various phenylpyruvates, on the enzyme tyrosine 3-monooxygenase have been investigated. Using a partially purified tyrosine 3-monooxygenase from bovine adrenal medulla, 3′,4′-deoxynorlaudanosolinecarboxylic acid was found to be a mixed inhibitor against the cofactor ( K i = 122 μM), equipotent with norepinephrine. Norlaudanosolinecarboxylic acid inhibited tyrosine 3-monooxygenase competitively with respect to the cofactor ( K i = 126 μM). When tyrosine 3-monooxygenase activity in catecholamine-free striatal homogenates was studied, again 3′,4′-deoxynorlaudanosolinecarboxylic acid ( K i = 40 μM) behaved as a mixed inhibitor whereas norlaudanosolinecarboxylic acid ( K i = 136 μM) was competitive. When the rat striatal tyrosine 3-monooxygenase was subjected to phosphorylating conditions in vitro , decreases in the K i of norlaudanosolinecarboxylic acid and in that of 3′,4′-deoxynorlaudanosolinecarboxylic acid were observed, whereas the K i of dopamine was increased. Tyrosine 3-monooxygenase activity in rat striatal synaptosomes was also inhibited by 3′,4′-deoxynorlaudanosolinecarboxylic acid (IC 50 = 100 μ m ) and phosphorylating conditions affected only that inhibition produced by dopamine, but not that by the tetrahydroisoquinolinecarboxylic acids. The results are discussed in relation to the structure of the tetrahydroisoquinolinecarboxylic acids and their possible role in vivo .

A Slowly Dissociating form of Bovine Hippocampal Synaptic Membrane Opiate Receptors

Since 1973 considerable effort has been devoted to the study of opiate receptors in nervous tissue. Most of these studies have utilized crude membrane preparations with few attempts to localize receptors within the cell. We have recently investigated the subcellular location of opiate receptors in rat forebrain. In our initial experiments we found receptor enrichment in highly purified synaptic plasma membrane (s.p.m.) fractions [1] and in microsomes [2] which mainly contain vesicles derived from Golgi apparatus and endoplasmic reticulum. We have since attempted to characterize the receptors in each organelle population.