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C J Evans

Publications and source records attributed to C J Evans.

At least 73 records · Page 4Linked to original sources
PubMed1996

Irreversible binding of cis-(+)-3-methylfentanyl isothiocyanate to the delta opioid receptor and determination of its binding domain.

Binding of cis-(+)-3-methylfentanyl isothiocyanate (SUPERFIT) to cloned opioid receptors stably expressed in Chinese hamster ovary cells was characterized. SUPERFIT inhibited [3H]diprenorphine binding with much higher affinity for the delta than the mu or kappa receptor. Pretreatment with SUPERFIT followed by extensive washing reduced delta binding with an IC50 value of 7.1 nM, yet it did not affect mu and kappa binding up to 0.1 microns. The reduction in delta binding by SUPERFIT pretreatment was due to a decrease in Bmax with no change in Kd. These results indicate that SUPERFIT is a highly selective delta irreversible ligand. We then determined the region in the delta receptor that confered binding selectivity for SUPERFIT by examining its binding to six mu/delta chimeric receptors. SUPERFIT bound to delta, mu/delta 1 (amino acids mu 1-94/delta 76-372), delta/mu 3 (delta 1-134/mu 154-398), and delta/mu 4 (delta 1-187/mu 207-398) receptors with high affinity but to mu, delta/mu 1 (delta 1-75/mu 95-398), mu/delta 3 (mu 1-153/delta 135-372), and mu/delta 4 (mu 1-206/delta 188-372) receptors with low affinity. Pretreatment with SUPERFIT potently inhibited [3H]diprenorphine binding to delta, mu/delta 1, delta/mu 3, and delta/mu 4 but affected binding to mu, delta/mu 1, mu/delta 3, and mu/delta 4 only at much higher concentrations. Thus, the segment from the beginning of the first intracellular loop to the middle of the third transmembrane helix of the delta receptor is important for selective binding of SUPERFIT.

Amino Acid Sequence↗
PubMed1996

Mu opioid receptor-like sequences are present throughout vertebrate evolution.

The sequence of the mu opioid receptor is highly conserved among human, rat, and mouse. In order to gain insights into the evolution of the mu opioid receptor, polymerase chain reaction (PCR) was used to screen genomic DNA from a number of different species using degenerate oligonucleotides which recognize a highly conserved region. DNA was assayed from representative species of both the protostome and deuterostome branches of the metazoan phylogenetic tree. Mu opioid receptor-like sequences were found in all vertebrate species that were analyzed. These species included bovine, chicken, bullfrog, striped bass, thresher shark, and Pacific hagfish. However, no mu opioid receptor-like sequences were detected from protostomes or from any invertebrates. The PCR results demonstrate that the region of the mu opioid receptor gene between the first intracellular loop and the third transmembrane domain (TM3) has been highly conserved during evolution and that mu opioid receptor-like sequences are present in the earliest stages of vertebrate evolution. Additional opioid receptor-like sequence was obtained from mRNA isolated from Pacific hagfish brain using rapid amplification of cDNA ends (RACE). The sequence of the Pacific hagfish was most homologous with the human mu opioid receptor (72% at the amino acid level between intracellular loop 1 and transmembrane domain 6) although over the same region high homology was also observed with the delta opioid receptor (69%), the kappa receptor (63%), and opioid receptor-like (ORL1) (59%). The hagfish sequence showed low conservation with the mammalian opioid receptors in the first and second extracellular loops but high conservation in the transmembrane and intracellular domains.

Amino Acid Sequence↗
PubMed1996

Functional analysis of cloned opioid receptors in transfected cell lines.

Opioids modulate numerous central and peripheral processes including pain perception neuroendocrine secretion and the immune response. The opioid signal is transduced from receptors through G proteins to various different effectors. Heterogeneity exists at all levels of the transduction process. There are numerous endogenous ligands with differing selectivities for at least three distinct opioid receptors (mu, delta, kappa). G proteins activated by opioid receptors are generally of the pertussis toxin-sensitive Gi/Go class, but there are also opioid actions that are thought to involve Gq and cholera toxin-sensitive G proteins. To further complicate the issue, the actions of opioid receptors may be mediated by G-protein alpha subunits and/or beta gamma subunits. Subsequent to G protein activation several effectors are known to orchestrate the opioid signal. For example activation of opioid receptors increases phosphatidyl inositol turnover, activates K+ channels and reduces adenylyl cyclase and Ca2+ channel activities. Each of these effectors shows considerable heterogeneity. In this review we examine the opioid signal transduction mechanism. Several important questions arise: Why do opioid ligands with similar binding affinities have different potencies in functional assays? To which Ca2+ channel subtypes do opioid receptors couple? Do opioid receptors couple to Ca2+ channels through direct G protein interactions? Does the opioid-induced inhibition of vesicular release occur through modulation of multiple effectors? We are attempting to answer these questions by expressing cloned opioid receptors in GH3 cells. Using this well characterized system we can study the entire opioid signal transduction process from ligand-receptor interaction to G protein-effector coupling and subsequent inhibition of vesicular release.

Animals↗
PubMed1996

Pentylenetetrazol-induced kindling: early involvement of excitatory and inhibitory systems.

Alterations in the brain of rats receiving a single non-convulsive administration pentylenetetrazol (PTZ), 30 mig/kg, i.p. (single PTZ group) were investigated and compared with those detected in fully PTZ kindled rats (chronic PTZ group). In vitro receptor autoradiography experiments showed that both single and chronic PTZ groups presented mu opioid and benzodiazepine (BDZ) receptor binding in specific brain areas. Using an antibody generated against the delta opioid receptor (DOR-1), it was found that DOR-1 like immunoreactivity was reduced in cortex and amygdala in mice following single and chronic PTZ administration. Microdialysis experiments revealed that the administration of PTZ 30 mg/kg, i.p. in freely moving rats without previous experience with the drug, induces a rise in glutamate release, detected in the first and second 10 min dialysates collected from amygdala (138% and 50%, respectively) and frontal cortex (70% and 45%, respectively) as well as aspartate in frontal cortex in the first and second PTZ-dialysates (143% and 80%, respectively). Subsequently, values returned to basal conditions. It may be speculated that decreased BDZ receptor binding results from enhanced release of GABA. On the other hand, the decrease of mu receptor binding and DOR-1 immunoreactivity observed after PTZ administration may be the result of enhanced levels of opioid peptides probably released over the kindling procedure. In conclusion, the present study indicates that PTZ-kindling is associated with an imbalance between excitatory and inhibitory systems which is apparent early in the epileptogenic process.

Amygdala↗
PubMed1996

Opioid receptor types and subtypes: the delta receptor as a model.

Since the discovery of opioid receptors over two decades ago, an increasing body of work has emerged supporting the concept of multiple opioid receptors. Molecular cloning has identified three opioid receptor types--mu, delta, and kappa--confirming pharmacological studies that previously postulated the existence of these three receptors. The cloned opioid receptors are highly homologous and belong to the family of seven-transmembrane, G protein-coupled receptors. With the development of novel opioid ligands, subtypes of the mu, delta, and kappa receptors have been proposed, although the molecular basis of these subtypes has not been elucidated. In this review, we present the pharmacological data supporting the concept of multiple delta opioid receptor subtypes and offer hypothetical mechanisms which might generate these "subtypes."

Alternative Splicing↗
PubMed1996

Voltage-dependent inhibition of Ca2+ channels in GH3 cells by cloned mu- and delta-opioid receptors.

To study cloned opioid receptor binding and modulation of both adenylyl cyclase and ion channel activity, we stably expressed mu- and delta-opioid receptors in the rodent pituitary-derived GH3 cell line. GH3 cells express G proteins and voltage-activated Ca2+ channels (predominantly of the L-type). Activation of cloned rat mu-opioid receptors expressed in GH3 cells (termed GH3MOR cells) inhibits L-type Ca2+ channel activity. GH3MOR cells, further transfected with mouse delta receptor cDNA (termed GH3MORDOR cells), bound both [D-Ala2,N-MePhe4,Gly-ol5]enkephalin (DAMGO) and [D-Pen2,D-Pen5]enkephalin (DPDPE). These opioid ligands inhibited adenylyl cyclase activity (IC50 = 174 and 0.53 nM, respectively). This action of DAMGO and DPDPE was attenuated selectively by mu- and delta-opioid receptor-specific antagonists. Activation of both opioid receptors also led to inhibition of Ca2+ channel activity, measured with Ba2+ as the charge carrier using the whole-cell patch-clamp technique. Both DAMGO (1 microM) and DPDPE (1 microM) reversibly inhibited Ba2+ currents (by 17.0 +/- 1.4% and 20.7 +/- 1.3%, respectively) in GH3MORDOR cells. The inhibitory action of DPDPE was dose dependent (IC50 = 1.6 nM) and was attenuated by pretreatment with pertussis toxin (200 ng/ml) or by the inclusion of guanosine-5'-O-(2-thio)diphosphate (2 mM) in the recording electrode. Ba2+ current inhibitions by both DAMGO and DPDPE were completely reversed by depolarizing (to > 50 mV) prepulses in GH3MORDOR cells. In summary, cloned mu- and delta-opioid receptors expressed in GH3 cells voltage-dependently couple through Gi/G(o) proteins to L-type Ca2+ channels.

Adenylate Cyclase Toxin↗
PubMed1995

Induction of opioid receptor-mediated macrophage chemotactic activity after neonatal brain injury.

Macrophages have a prominent role in the injury response of the brain, yet the molecular mechanisms that control their invasion to the site of neuronal degeneration is unknown. After removal of the posterior cortex at birth, there is massive and specific targeting of nonresident macrophages to axotomized neurons in the lateral thalamus. The present study has identified an injury-induced, brain-derived chemotactic factor (BDCF) capable of eliciting chemotactic responses from resident peritoneal macrophages and brain macrophages. Conditioned media collected from tissue slices containing the axotomized central nervous system neurons exhibit BDCF activity. Initial experiments indicated that BDCF is a small peptide and, thus, we used specific pharmacologic reagents to characterize further BDCF activity. Naloxone, a pan opioid receptor antagonist, completely blocks BDCF activity. Although both kappa and mu opioid receptor antagonists failed to modify BDCF-induced macrophage chemotaxis, two specific delta receptor antagonists blocked BDCF. Analysis of BDCF by reverse phase HPLC and RIA revealed peak chemotactic activity in fractions consistent with the presence of an opioid peptide. The results suggest that cells in the brain respond to neuronal injury by producing and releasing opioids that can initiate a specific macrophage response.

Animals↗
PubMed1995

Microdialysis reveals a morphine-induced increase in pallidal opioid peptide release.

Freely moving rats were implanted with microdialysis probes in the globus pallidus/ventral pallidum, nucleus accumbens or caudate nucleus. Morphine (10 mg kg-1 i.p.) induced an average 128% increase in extracellular opioid peptide levels in the pallidum over a 2 h period peaking 1 h after injection. No change was observed in the nucleus accumbens or caudate nucleus. Dose-response analysis showed a smaller (26%), non-significant, increase at a lower dose of 2 mg kg-1 and no effect at a higher dose of 40 mg kg-1. No evidence of significant acute tolerance was apparent following repeated administration of morphine (10 mg kg-1, i.p.) at 3 h intervals. The magnitude of the morphine effect varied greatly between animals and was dependent on the pre-injection baseline opioid peptide levels.

Animals↗
PubMed1995

Postpartum home care in the United States.

Shorter postpartum hospital stays have increased the need for postpartum home care. Health-care reform in the United States must include postpartum follow-up care as a basic maternity benefit. This article explores the variety of options for postpartum home care, including telephone calls, home visits, information lines, lactation consultations, mother-infant outpatient clinics, support groups, and a model program for childbearing women in Washington state.

Female↗
PubMed1995

Ca2+ channel and adenylyl cyclase modulation by cloned mu-opioid receptors in GH3 cells.

Members of the three classes of opioid receptors (mu, delta, and kappa) have been cloned and characterized in unexcitable cell lines using biochemical techniques. However, an important function of these cloned receptors, their coupling to voltage-activated Ca2+ channels, remains untested. We stably transfected cloned rat mu-opioid receptor cDNAs into clonal pituitary GH3 cells. GH3 cells expressing mu-opioid receptors (GH3MOR cells) bound the receptor-specific ligands [D-Ala2,Me-Phe4,Gly-ol5]-enkephalin (DAMGO) and morphine with high affinity (Ki = 1.0 and 7.2 nM, respectively), and these ligands also potently inhibited adenylyl cyclase activity (IC50 = 21.9 and 55.2 nM, respectively). Functional coupling of mu-opioid receptors to voltage-activated Ca2+ channels was compared with that of endogenous somatostatin (SRIF) receptors in GH3MOR cells, using the patch-clamp technique, with Ba2+ as the charge carrier. DAMGO (1 microM) and SRIF (1 microM) inhibited Ba2+ currents by 23.8 +/- 1.0% and 22.9 +/- 2.5%, respectively. DAMGO (0.1 nM to 10 microM) dose-dependently inhibited Ba2+ currents, with an IC50 of 105 nM. The mu-opioid receptor agonist morphine (1 microM) inhibited currents by 13.5 +/- 1.1% and the delta-opioid receptor-selective ligand [D-Pen2,5]-enkephalin (1 microM) caused only 3.5 +/- 2.1% inhibition. The inhibitory actions of DAMGO, morphine, and [D-Pen2,5]-enkephalin were reversed by naloxone. Ba2+ current inhibitions by DAMGO and SRIF were attenuated by pertussis toxin pretreatment. Nimodipine reduced the amplitude of Ba2+ current inhibition by DAMGO, suggesting that mu-opioid receptors couple to L-type Ca2+ channels in GH3MOR cells.

Adenylate Cyclase Toxin↗
PubMed1994

Ability of delta-opioid receptors to interact with multiple G-proteins is independent of receptor density.

To determine whether the previously demonstrated ability of delta-opioid receptors to interact simultaneously with multiple G-proteins was a function of high receptor levels, this interaction was investigated in Chinese hamster ovary cells stably expressing 10 different levels of cloned delta-opioid receptors, ranging from 18,000 to 1.6 x 10(6) receptors/cell. The opioid agonist D-Ala2,D-Leu5-enkephalin (DADLE) inhibited forskolin-stimulated adenylyl cyclase activity in all 10 clones with variable maximal inhibitory levels. Furthermore, opioid agonists altered incorporation of [alpha-32P]azidoanilido-GTP into at least four G-protein alpha-subunits in all 10 clones, three of which were determined to be Gi3 alpha, Gi2 alpha and Go2 alpha. This effect was concentration-dependent, naloxone-reversible, and delta-opioid agonist-specific and was blocked by pretreatment with pertussis toxin. Although DADLE induced an increase in the incorporation of [alpha-32P]azidoanilido-GTP into three of the four G alpha proteins that was independent of receptor density, the magnitude of this response was greater as receptor density increased. In addition, concentrations of DADLE required to promote 50% maximal labeling were similar for all four G alpha proteins within each clone and did not appear to be affected by receptor density. Therefore, the ability of delta-opioid receptors to interact with multiple G-proteins is independent of receptor density and there is also no apparent correlation between the amount of G-protein activated and the maximal effect of an agonist.

Amino Acid Sequence↗
PubMed1994

Microdialysis reveals changes in extracellular opioid peptide levels in the amygdala induced by amygdaloid kindling stimulation.

Enkephalins released in basolateral nucleus of the amygdala in response to electrical stimulation were determined in amygdala kindled and nonkindled freely moving rats using microdialysis. Enkephalin release was enhanced after a single and repetitive electrical stimulation (233 and 130% above control levels, respectively) in nonkindled rats. In fully kindled rats, the extracellular enkephalin levels decreased (35% below the control levels) within the first 20 min after onset of stage V kindled seizures, reaching baseline level 40 to 60 min following the generalized seizure activity. HPLC analysis identified the majority of recovered immunoreactive material from the amygdala as Met-enkephalin. On the basis of our results it is suggested that the enhanced enkephalin release in the amygdala during the early kindling stages might have a suppressive effect which may represent a homeostatic mechanism to avoid the spread of the afterdischarge to other structures. The decreased extracellular level of enkephalin in the amygdala after stage V kindled seizures could reflect a general impairment of inhibitory mechanisms in this structure with subsequent production of seizure activity.

Amygdala↗
PubMed1994

Antipeptide antibodies against a Torpedo cysteine-string protein.

An antipeptide antiserum was raised against the C-terminal undecapeptide of a Torpedo cysteine-string protein (csp), a putative subunit or modulator of presynaptic calcium channels. This antiserum was shown to identify selectively the 27-kDa in vitro translation product of the csp cRNA both by immunoprecipitation and on immunoblots. When affinity-purified anti-csp antibodies were used to probe immunoblots of membrane proteins from Torpedo electric organ or liver, specific immunoreactivity was detected only in electric organ. This immunoreactivity was associated principally with a single protein species of about 34 kDa. These results indicate that csp immunoreactivity is detectably expressed in electroplax, a heavily innervated tissue, but not in liver, which should have an appreciably lower abundance of presynaptic calcium channel proteins. Moreover, the increased relative molecular mass of csp in electric organ (compared with in vitro translated material) implies that csp is posttranslationally modified. Finally, immunoblot analysis of either intact, alkali-treated, or solubilized membrane fractions of electric organ reveals that csp is predominantly a membrane protein.

Amino Acid Sequence↗