Biomedical subjects

D K Grandy

Publications and source records attributed to D K Grandy.

At least 73 records · Page 4Linked to original sources

PubMed1997

Pituitary lactotroph hyperplasia and chronic hyperprolactinemia in dopamine D2 receptor-deficient mice.

Dopamine secreted from hypophysial hypothalamic neurons is a principal inhibitory regulator of pituitary hormone secretion. Mice with a disrupted D2 dopamine receptor gene had chronic hyperprolactinemia and developed anterior lobe lactotroph hyperplasia without evidence of adenomatous transformation. Unexpectedly, the mutant mice had no hyperplasia of the intermediate lobe melanotrophs. Aged female D2 receptor -/- mice developed uterine adenomyosis in response to prolonged prolactin exposure. These data reveal a critical role of hypothalamic dopamine in controlling pituitary growth and support a multistep mechanism for the induction and perpetuation of lactotroph hyperplasia, involving the lack of dopamine signaling, a low androgen/estrogen ratio, and a final autocrine or paracrine "feed-forward" stimulation of mitogenesis, probably by prolactin itself.

Animals↗

PubMed1997

Bidirectional modulatory effect of orphanin FQ on morphine-induced analgesia: antagonism in brain and potentiation in spinal cord of the rat.

1. The present study was designed to investigate further the effects of the newly discovered orphanin FQ (OFQ)-the endogenous ligand for the orphan opioid receptor (called, e.g., ORL, and LC132)-on pain modulation in the rat. We used the tail-flick assay as a nociceptive index. 2. When injected into a cerebral ventricle, OFQ (4 fmol-10 nmol) has no effect on basal tail-flick latency by itself at any dose, but dose-dependently antagonizes systemic morphine analgesia (400 fmol 50 nmol). 3. Injected intrathecally, OFQ (3 and 10 nmol) displayed an analgesic effect without producing motor dysfunction, and potentiated morphine analgesia (1 and 10 nmol). 4. The anti-opioid effect of OFQ in rat brain and the high level of expression of LC132/ORL, receptor in the locus coeruleus indicated a possible role of OFQ in the precipitation of opiate withdrawal symptoms. However, no such precipitation was observed by OFQ in morphine-dependent rats.

Analgesia↗

PubMed1997

The effects of recombinant rat mu-opioid receptor activation in CHO cells on phospholipase C, [Ca2+]i and adenylyl cyclase.

1. The rat mu-opioid receptor has recently been cloned yet its second messenger coupling remains unclear. The endogenous mu-opioid receptor in SH-SY5Y cells couples to phospholipase C (PLC), increases [Ca2+]i and inhibits adenylyl cyclase (AC). We have examined the effects of mu-opioid agonists on inositol(1,4,5)trisphosphate (Ins(1,4,5)P3), [Ca2+]i and adenosine 3':5'-cyclic monophosphate (cyclic AMP) formation in Chinese hamster ovarian (CHO) cells transfected with the cloned mu-opioid receptor. 2. Opioid receptor binding was assessed with [3H]-diprenorphine ([3H]-DPN) as a radiolabel. Ins(1,4,5)P3 and cyclic AMP were measured by specific radioreceptor assays. [Ca2+]i was measured fluorimetrically with Fura-2. 3. Scatchard analysis of [3H]-DPN binding revealed that the Bmax varied between passages. Fentanyl (10 pM 1 microM) dose-dependently displaced [3H]-DPN, yielding a curve which had a Hill slope of less than unity (0.6 +/- 0.1), and was best fit to a two site model, with pK1 values (% of sites) of 9.97 +/- 0.4 (27 +/- 4.8%) and 7.68 +/- 0.07 (73 +/- 4.8%). In the presence of GppNHp (100 microM) and Na+ (100 mM), the curve was shifted to the right and became steeper (Hill slope = 0.9 +/- 0.1) with a pK1 value of 6.76 +/- 0.04. 4. Fentanyl (0.1 nM-1 microM) had no effect on basal, but dose-dependently inhibited forskolin (1 microM)-stimulated, cyclic AMP formation (pIC50 -7.42 +/- 0.23), in a pertussis toxin (PTX; 100 ng ml-1 for 24 h)-sensitive and naloxone-reversible manner (K1 = 1.7 nM). Morphine (1 microM) and [D-Ala2, MePhe4, gly(ol)5]-enkephalin (DAMGO, 1 microM) also inhibited forskolin (1 microM)-stimulated cyclic AMP formation, whilst [D-Pen2, D-Pen5], enkephalin (DPDPE, 1 microM) did not. 5. Fentanyl (0.1 nM-10 microM) caused a naloxone (1 microM)-reversible, dose-dependent stimulation of Ins(1,4,5)P3 formation, with a pEC50 of 7.95 +/- 0.15 (n-5), PTX (100 ng ml-1 for 24 h) abolished, whilst Ni2 (2.5 mM) inhibited (by 52%), the fentanyl-induced Ins(1,4,5)P3 response. Morphine (1 microM) and DAMGO (1 microM), but not DPDPE (1 microM), also stimulated Ins(1,4,5)P3 formation. Fentanyl (1 microM) also caused an increase in [Ca2+]i (80 +/- 16.4 nM, n-6), reaching a maximum at 26.8 +/- 2.5 s. The increase in [Ca2+]i remained elevated until sampling ended (200 s) and was essentially abolished by the addition of naloxone (1 microM). Pre-incubation with naloxone (1 microM, 3 min) completely abolished fentanyl-induced increases in [Ca2+]i. 6. In conclusion, the cloned mu-opioid receptor when expressed in CHO cells stimulates PLC and inhibits AC, both effects being mediated by a PTX-sensitive G-protein. In addition, the receptor couples to an increase in [Ca2+]i. These findings are consistent with the previously described effector-second messenger coupling of the endogenous mu-opioid receptor.

Adenylyl Cyclases↗

PubMed1997

Circuitry underlying antiopioid actions of orphanin FQ in the rostral ventromedial medulla.

Several laboratories recently identified a 17 amino-acid peptide, termed "nociceptin" or "orphanin FQ (OFQ)", as the endogenous ligand for the LC132 (or "opioid receptor-like1") receptor. Taken together with the fact that the cellular effects of OFQ are to a large extent opioid-like, the close relationship between the LC132 receptor and known opioid receptors raised expectations that the behavioral effects of this peptide would resemble those of opioids. However studies of the role of OFQ in nociception have not provided a unified view. The aim of the present study was to use a combination of electrophysiological and pharmacological techniques to characterize the actions of OFQ in a brain region in which the circuitry mediating the analgesic actions of opioids has been relatively well characterized, the rostral ventromedial medulla (RVM). Single-cell recording was combined with opioid administration and local infusion of OFQ in the RVM of rats lightlyanesthetized with barbiturates. The tail flick reflex was used as a behavioral index of nociceptive responsiveness. Two classes of physiologically identifiable RVM neurons with distinct responses to opioids have been characterized. -cells are activated, although indirectly, by opioids, and there is strong evidence that this activation is crucial to opioid antinociception. -cells, thought to enable nociception, are directly inhibited by opioids. Cells of a third class, cells, do not respond to opioids and whether or not they have any role in nociceptive modulation remains an open question. OFQ infused within the RVM profoundly suppressed the firing of all classes of RVM neurons, blocking opioid-induced activation of -cells. The antinociceptive effects of a micro-opioid agonist infused at the same site were significantly attenuated in these animals. Those of systemically administered morphine, which can produce its antinociceptive effects by acting at a number of CNS sites, were not blocked by RVM OFQ. Inasmuch as activation of -cells can account for the antinociceptive action of opioids within the RVM, these results demonstrate that, at least within the medulla, OFQ can exert a functional "antiopioid" effect by suppressing firing of this cell class. However to the extent that antinociceptive and pronociceptive outflows from various brain regions involved in both transmission and modulation of nociception are active under different conditions, focal application of OFQ in different regions could potentially produce either hypalgesia or hyperalgesia.

Analgesics, Opioid↗

PubMed1996

Downregulation of mu-opioid receptor mRNA in the mediobasal hypothalamus of the female guinea pig following morphine treatment.

Ribonuclease protection assays were performed on the basal hypothalamus (BH), the preoptic area (POA) and the thalamus (THAL) from female guinea pigs that were ovariectomized and implanted with morphine (n = 8) or placebo (n = 8) pellets for 1 week. An antisense [32P]rUTP labeled riboprobe, representing a 280 bp fragment of the guinea pig mu-opioid receptor gene (spanning putative TM II through eight residues of TM IV), protected a single RNA band of 280 bp. In contrast to the rat, the guinea pig THAL expressed less mu-opioid receptor mRNA than both POA and BH. Morphine treatment caused a significant decrease (15.6 +/- 5.8%) in mu-opioid receptor mRNA expression in the BH, while POA and THAL were not different from placebo controls. Therefore, in conjunction with our previous findings of a downregulation of mu-opioid receptors, mu-opioid receptor mRNA is downregulated in the mediobasal hypothalamus of female guinea pigs following chronic morphine treatment.

Analgesics, Opioid↗

PubMed1996

Orphanin FQ acts as a supraspinal, but not a spinal, anti-opioid peptide.

Orphanin FQ (OFQ), the endogenous ligand for the orphan opioid receptor, LC132, was recently isolated and characterized. The anti-opioid role of OFQ in supraspinal pain modulation was demonstrated by our previous observations that intracerebroventricular (i.c.v.) OFQ administration dose-dependently reverses systemic morphine antinociception and opioid-mediated stress-induced antinociception. The present study was designed to evaluate whether OFQ also modulates the antinociceptive actions of morphine in the spinal cord. Immediately after assessment of baseline nociceptive sensitivity on the 49 degrees C tail-withdrawal assay, mice of both sexes were given i.c.v. or intrathecal (i.t.) cocktails of morphine (0, 1, 10 or 50 micrograms [0-135 nmol]) and OFQ (0 or 10 nmol), and re-tested 15, 30 and 60 min later. OFQ alone did not affect nociceptive sensitivity when administered by either route. Following i.c.v. administration, the antinociception produced by 10 micrograms morphine was completely reversed by 10 nmol OFQ; antinociception induced by 50 micrograms morphine was significantly antagonized. In contrast, OFQ was completely ineffective against antinociception induced by i.t. morphine. These findings indicate that the anti-opioid actions of OFQ are restricted to supraspinal central nervous system sites.

Animals↗

PubMed1996

Functional antagonism of mu-, delta- and kappa-opioid antinociception by orphanin FQ.

Orphanin FQ (OFQ) is the recently isolated endogenous ligand for the orphan opioid-like receptor, LC132. Initial reports suggested that OFQ increased pain sensitivity when injected intracerebroventricularly (i.c.v.) in mice. However, we have recently demonstrated that OFQ is instead an anti-opioid peptide that reverses morphine- and opioid-mediated stress-induced antinociception. Morphine binds to multiple opioid receptor types (mu, delta, and kappa). The present study was designed to examine specific interactions of OFQ with antinociception mediated by each receptor type. To this end, mice were administered i.c.v. cocktails containing either vehicle or OFQ (10 nmol) and a mu-specific ([D-Ala2, N-Me-Phe4-Gly-ol]enkephalin; DAMGO; 0-0.1 nmol), delta-specific ([D-Pen2, D-Pen5]enkephalin; DPDPE; 0-50 nmol), or kappa-specific (U-50,488H; 0-1000 nmol) agonist. As we have shown previously, OFQ alone had no effect on nociceptive sensitivity. OFQ was, however, able to completely block supraspinal antinociception produced by all three receptor type-selective agonists. We conclude, therefore, that OFQ functionally antagonizes mu (and (opioid receptors, and may play a general role in opioid modulation.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗

PubMed1996

Orphanin FQ is a functional anti-opioid peptide.

The heptadecapeptide orphanin FQ has recently been shown to be the endogenous agonist for the orphan opioid-like receptor, LC132. The molecular evidence that LC132 and orphanin FQ are evolutionarily related to other opioid receptors and their ligands suggests that these proteins may also play a role in modulating opiate actions. We now report that orphanin FQ (0.5-10 nmol), injected intracerebroventricularly in mice, does not produce hyperalgesia as suggested previously but rather reverses opioid-mediated (i.e. naloxone-sensitive) stress-induced antinociception in three different algesiometric assays. In addition to its antagonism of endogenous opioid antinociception, orphanin FQ dose-dependently (2.5-25 nmol) reverses systemic morphine antinociception (5 mg/kg, s.c.). Based on these data, we propose that orphanin FQ is a functional anti-opioid peptide.

Acetates↗

PubMed1995

Orphanin FQ: a neuropeptide that activates an opioidlike G protein-coupled receptor.

A heptadecapeptide was identified and purified from porcine brain tissue as a ligand for an orphan heterotrimeric GTP-binding protein (G protein)-coupled receptor (LC132) that is similar in sequence to opioid receptors. This peptide, orphanin FQ, has a primary structure reminiscent of that of opioid peptides. Nanomolar concentrations of orphanin FQ inhibited forskolin-stimulated adenylyl cyclase activity in cells transfected with LC132. This inhibitory activity was not affected by the addition of opioid ligands, nor did the peptide activate opioid receptors. Orphanin FQ bound to its receptor in a saturable manner and with high affinity. When injected intracerebroventricularly into mice, orphanin FQ caused a decrease in locomotor activity but did not induce analgesia in the hot-plate test. However, the peptide produced hyperalgesia in the tail-flick assay. Thus, orphanin FQ may act as a transmitter in the brain by modulating nociceptive and locomotor behavior.

Adenylyl Cyclase Inhibitors↗

PubMed1995

Functional characterization of the human dopamine D4.2 receptor using vaccinia virus as an expression system.

Recombinant vaccinia viruses harboring the human dopamine D4 receptor cDNA containing two 48 base pair-repeats (D4.2) or the rat dopamine D2 short (D2s) receptor cDNA were used to infect rat-1 fibroblasts. Heterologous expression of both dopamine receptors was demonstrated in binding assays. The affinity constants of these receptors were consistent with values previously reported, including D4.2's higher affinity for the antipsychotic clozapine and raclopride's selectivity for D2 receptors. In the presence of 200 microM 5'-guanylyl-imidodiphosphate (Gpp[NH]p) both receptors exhibited reduced affinities for dopamine. Furthermore, when rat-1 cells were infected with the D2s or the D4.2 recombinant vaccinia viruses and exposed to dopamine agonists, the inhibition of adenylyl cyclase activity was prevented in pertussis toxin-treated cells. This study demonstrates the utility of recombinant receptor-vaccinia viruses in studies of expression, pharmacology and functional coupling of inhibitory G protein-coupled receptors.

Adenylyl Cyclase Inhibitors↗

PubMed1995

Characterization and distribution of a cloned rat mu-opioid receptor.

We have cloned and expressed a rat brain cDNA, TS11, that encodes a mu-opioid receptor based on pharmacological, physiological, and anatomical criteria. Membranes were prepared from COS-7 cells transiently expressing TS11 bound [3H]diprenorphine with high affinity (KD = 0.23 +/- 0.04 nM). The rank order potency of drugs competing with [3H]diprenorphine was as follows: levorphanol (Ki = 0.6 +/- 0.2 nM) approximately beta-endorphin (Ki = 0.7 +/- 0.05 nM) approximately morphine (Ki = 0.8 +/- 0.5 nM) approximately [D-Ala2, N-Me-Phe4,Gly-ol5]-enkephalin (DAMGO; Ki = 1.6 +/- 0.5 nM) uch much greater than U50,488 (Ki = 910 +/- 0.78 nM) > [D-Pen2,5]- enkephalin (Ki = 3,170 +/- 98 nM) > dextrorphan (Ki = 4,100 +/- 68 nM). The rank order potencies of these ligands, the stereospecificity of levorphanol, and morphine's subnanomolar Ki are consistent with a mu-opioid binding site. Two additional experiments provided evidence that this opioid-binding site is functionally coupled to G proteins: (a) in COS-7 cells 50 microM 5'-guanylylimidodiphosphate shifted a fraction of receptors with high affinity for DAMGO (IC50 = 3.4 +/- 0.5 nM) to a lower-affinity state (IC50 = 89.0 +/- 19.0 nM), and (b) exposure of Chinese hamster ovary cells stably expressing the cloned mu-opioid receptor to DAMGO resulted in a dose-dependent, naloxone-sensitive inhibition of forskolin-stimulated cyclic AMP production. The distribution of mRNA corresponding to the mu-opioid receptor encoded by TS11 was determined by in situ hybridization to brain sections prepared from adult female rats. The highest levels of mu-receptor mRNA were detected in the thalamus, medial habenula, and the caudate putamen; however, significant hybridization was also observed in many other brain regions, including the hypothalamus.

Amino Acid Sequence↗

PubMed1995

Differential coupling of D1 and D5 dopamine receptors to guanine nucleotide binding proteins in transfected GH4C1 rat somatomammotrophic cells.

D1 and D5 dopamine receptor genes, stably expressed in GH4C1 rat somatomammotrophic cells, display identical binding values and stimulate adenylate cyclase. Approximately 60% of D1 receptors were in the agonist high-affinity state and were converted to the low-affinity state by 100 microM guanyl-5'-ylimidodiphosphate [Gpp(NH)p]. Of the 48% of D5 receptors in the high-affinity state, only half were modulated by 100 microM Gpp(NH)p; in the presence of the G protein activator, AIF4-, the high-affinity sites of D5 receptors were abolished by Gpp(NH)p, suggesting tight coupling between D5 receptors and G proteins. The high-affinity sites of D1, but not D5, receptors were reduced after pertussis toxin treatment of cells. Thus, whereas D1 receptors in GH4C1 cells couple to both Gs, the G stimulatory protein, and a pertussis toxin-sensitive G protein, D5 receptors couple to Gs and a pertussis toxin-insensitive G protein. Neither D1 nor D5 receptors were able to stimulate phosphoinositide metabolism in these cells. The ability of D5, but not D1, receptors to couple to novel G proteins may be significant in assigning a functional role for these receptors.

Adenylate Cyclase Toxin↗

PubMed1995

Analysis of selective binding epitopes for the kappa-opioid receptor antagonist nor-binaltorphimine.

The structural determinants for the selective binding of the nonpeptide opioid receptor antagonist nor-binaltorphimine (nor-BNI) to the kappa-opioid receptor were characterized using a systematic series of chimeras between the kappa receptor and the homologous mu-opioid receptor. All 10 chimeric constructs bound the nonselective antagonists (-)-naloxone and diprenorphine with similar affinities, as did the two wild-type receptors. Introduction of amino-terminal segments of increasing length, extending to and including transmembrane segment VI, from the mu receptor into the kappa receptor did not impair the high affinity binding of nor-BNI, and neither did introduction of the intracellular carboxyl-terminal extension of the mu receptor. In contrast, nor-BNI binding was impaired > or = 600-fold in constructs in which extracellular loop 3 and transmembrane segment VII originated from the mu receptor. The exchange of a single residue within this region, Glu297, for lysine, the corresponding residue from the mu receptor, reduced the binding affinity of nor-BNI 142-fold, without affecting the binding the nonselective compounds (-)-naloxone and diprenorphine. It is concluded that the selective binding of nor-BNI to the kappa-opioid receptor is determined by nonconserved residues located in extracellular loop 3 and transmembrane segment VII and that Glu297, located just outside transmembrane segment VI, plays a major role in the kappa-selective binding characteristics of nor-BNI.

Amino Acid Sequence↗

PubMed1995

Kappa-opioid receptors couple to inwardly rectifying potassium channels when coexpressed by Xenopus oocytes.

Xenopus oocytes expressed kappa-opioid specific binding sites after injection of cRNA prepared from a clone of the rat kappa-opioid receptor. Coinjection of kappa receptor cRNA with cRNA coding for a G protein-linked, inwardly rectifying, K+ channel (GIRK1, or KGA) resulted in oocytes that responded to the kappa agonist U-69593 by activating a large (1.0-1.5-microA) K+ current. U-69593 exhibited an EC50 of 260 +/- 50 nM and was blocked by the opioid antagonists norbinaltorphimine and naloxone. The kappa agonist bremazocine was 200-fold more potent than U-69593 in eliciting K+ current but exhibited a partial agonist profile in this expression system. The present results indicate that stimulation of inwardly rectifying K+ channels may be a potential effector mechanism for kappa-opioid receptors.

Amino Acid Sequence↗

PubMed1995

Evolution of haplotypes at the DRD2 locus.

We present here the first evolutionary perspective on haplotypes at DRD2, the locus for the dopamine D2 receptor. The dopamine D2 receptor plays a critical role in the functioning of many neural circuits in the human brain. If functionally relevant variation at the DRD2 locus exists, understanding the evolution of haplotypes on the basis of polymorphic sites encompassing the gene should provide a powerful framework for identifying that variation. Three DRD2 polymorphisms (TaqI "A" and "B" RFLPs and the (CA)n short tandem repeat polymorphic in all the populations studied, and they display strong and significant linkage disequilibria with each other. The common haplotypes for the two TaqI RFLPs are separately derived from the ancestral haplotype but predate the spread of modern humans around the world. The knowledge of how the various haplotypes have evolved, the allele frequencies of the haplotypes in human populations, and the physical relationships of the polymorphisms to each other and to the functional parts of the gene should now allow proper design and interpretation of association studies.

Base Sequence↗

PubMed1994

Molecular cloning and tissue distribution of a putative member of the rat opioid receptor gene family that is not a mu, delta or kappa opioid receptor type.

A novel G protein-coupled receptor was cloned by PCR and homology screening. Its deduced amino acid sequence is 47% identical overall to the mu, delta and kappa opioid receptors and 64% identical in the putative transmembrane domains. When transiently expressed in COS-7 cells this receptor did not bind any of the typical mu, delta or kappa opioid receptor ligands with high affinity. In situ hybridization analysis revealed that LC132 mRNA is highly expressed in several rat brain areas, including the cerebral cortex, thalamus, subfornical organ, habenula, hypothalamus, central gray, dorsal raphe, locus coeruleus and the dorsal horn of the spinal cord. Based on this distribution and its high homology with the mu, delta and kappa opioid receptors, it is proposed that LC132 is a new member of the opioid receptor family that is involved in analgesia and the perception of pain.

Amino Acid Sequence↗

PubMed1994

D2 dopamine receptor gene and obesity.

The prevalence of Taql A D2 dopamine receptor (DRD2) alleles was determined in 73 obese women and men. In this sample with a mean body mass index of 35.1, the A1 (minor) allele of the DRD2 gene was present in 45.2% of these nonalcohol, nondrug abusing subjects. The DRD2 A1 allele was not associated with a number of cardiovascular risk factors examined, including blood lipids (cholesterol, high-density lipoprotein [HDL]- and low-density lipoprotein [LDL]-cholesterol, and triglycerides). However, phenotypic factors characterized by the presence of parental history and postpuberty onset of obesity as well as carbohydrate preference were associated with obese subjects carrying the A1 allele. The cumulative number of these three factors was positively and significantly (p < .0002) related to A1 allelic prevalence. The data showing an association of the minor allele of the DRD2 gene with phenotypic characteristics suggest that this gene, located on q22-q23 region of chromosome 11, confers susceptibility to a subtype of this disorder.

Adolescent↗

PubMed1994

Dopamine receptor gene expression in the human medial temporal lobe.

The distributions of the messenger RNA molecules encoding the five known dopamine receptors have been determined in the medial temporal lobe of postmortem human brain. All five receptor mRNAs are present in temporal lobe structures, although their distributions are heterogeneous. The D1-like receptors, D1 and D5, have strikingly dissimilar distributions. D1 receptor mRNA is abundant in temporal neocortex but is rare elsewhere. D5 receptor message, however, is seen in the hippocampus, subicular complex, and in temporal cortex. The D2-like receptors have similar distributions: D2, D3, and D4 receptor mRNAs are all identifiable in the hippocampal formation and in the cortical regions of the medial temporal lobe. Distinct patterns of relative regional concentrations for each message are observed, however, suggesting a neuroanatomical substrate for potential differences in dopaminergic regulation within discrete regions of the medial temporal lobe. These results provide a description of the distribution of these receptor mRNAs in normal humans and suggest multiple levels of complexity as well as regulation of the medial temporal lobe dopamine projection.

Adult↗