Mechanisms underlying kainate receptor-mediated disinhibition in the hippocampus

Kainate (KA) receptor activation depresses stimulus-evoked γ-aminobutyric acid (GABA-mediated) synaptic transmission onto CA1 pyramidal cells of the hippocampus and simultaneously increases the frequency of spontaneous GABA release through an increase in interneuronal spiking. To determine whether these two effects are independent, we examined the mechanism by which KA receptor activation depresses the stimulus-evoked, inhibitory postsynaptic current (IPSC). Bath application of the α-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA)/KA receptor agonist KA in the presence of the AMPA receptor antagonist GYKI 53655 caused a large increase in spontaneous GABA release and a coincident depression of the evoked IPSC. The depressant action on the evoked IPSC was reduced, but not abolished, by the GABAB receptor antagonist SCH 50911, suggesting that the KA-induced increase in spontaneous GABA release depresses the evoked IPSC through activation of presynaptic GABAB receptors. KA had no resolvable effect on the potassium-induced increase in miniature IPSC frequency, suggesting that KA does not act through a direct effect on the release machinery or presynaptic calcium influx. KA caused a decrease in pyramidal cell input resistance, which was reduced by GABAA receptor antagonists. KA also caused a reduction in the size of responses to iontophoretically applied GABA, which was indistinguishable from the SCH 50911-resistant, residual depression of the evoked IPSC. These results suggest that KA receptor activation depresses the evoked IPSC indirectly by increasing interneuronal spiking and GABA release, leading to activation of presynaptic GABAB receptors, which depress GABA release, and postsynaptic GABAA receptors, which increase passive shunting.

Peptide analogue studies of the hypothalamic neuropeptide Y receptor mediating pituitary adrenocorticotrophic hormone release

Hypothalamic neuropeptide Y (NPY) is thought to be important in the regulation of feeding and also in the release of Adrenocorticotrophic hormone (ACTH). Intracerebroventricular administration of NPY to male rats significantly increased plasma ACTH 10 min after injection and stimulated 2-h food intake. A series of analogues of NPY that have a greatly reduced affinity for the Y1 [human pancreatic polypeptide (human PP), NPY(3–36)], the Y2 ([Pro34]NPY, human PP), the Y3 (peptide YY), and the Y6 (human PP) receptor, all markedly stimulated ACTH release. Rat PP, which binds with high affinity to the Y4 receptor, was unable to stimulate ACTH release. A novel analogue fragment [Pro34]NPY(13–36) was synthesized as a ligand with low Y1 and Y2 receptor affinity. Interestingly, neither [Pro34]NPY(13–36) nor the selective Y5 receptor agonist [d-Trp32]NPY stimulated food intake, whereas both significantly increased plasma ACTH. Thus the hypothalamic NPY receptor mediating increases in plasma ACTH has a fragment activation profile unlike the Y1–Y4 or Y6 receptors and appears distinct from the NPY receptor controlling food intake.

Electrophysiological evidence for a hyperpolarizing, galanin (1-15)-selective receptor on hippocampal CA3 pyramidal neurons

The effects of the 29-amino acid neuropeptide galanin [GAL (1–29)], GAL(1–15), GAL(1–16), and the GAL subtype 2 receptor agonist d-tryptophan2-GAL(1–29) were studied in the dorsal hippocampus in vitro with intracellular recording techniques. GAL(1–15) induced, in the presence of tetrodotoxin, a dose-dependent hyperpolarization in hippocampal CA3 neurons. Most of the GAL(1–15)-sensitive neurons did not respond to GAL(1–29), GAL(1–16), or d-tryptophan2-GAL(1–29). These results indicate the presence of a distinct, yet-to-be cloned GAL(1–15)-selective receptor on CA3 neurons in the dorsal hippocampus.

Elevated anxiety and antidepressant-like responses in serotonin 5-HT1A receptor mutant mice

The brain serotonin (5-hydroxytryptamine; 5-HT) system is a powerful modulator of emotional processes and a target of medications used in the treatment of psychiatric disorders. To evaluate the contribution of serotonin 5-HT1A receptors to the regulation of these processes, we have used gene-targeting technology to generate 5-HT1A receptor-mutant mice. These animals lack functional 5-HT1A receptors as indicated by receptor autoradiography and by resistance to the hypothermic effects of the 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT). Homozygous mutants display a consistent pattern of responses indicative of elevated anxiety levels in open-field, elevated-zero maze, and novel-object assays. Moreover, they exhibit antidepressant-like responses in a tail-suspension assay. These results indicate that the targeted disruption of the 5-HT1A receptor gene leads to heritable perturbations in the serotonergic regulation of emotional state. 5-HT1A receptor-null mutant mice have potential as a model for investigating mechanisms through which serotonergic systems modulate affective state and mediate the actions of psychiatric drugs.

Retinoic acid alters the intracellular trafficking of the mannose-6-phosphate/insulin-like growth factor II receptor and lysosomal enzymes

Previously, we showed that retinoic acid (RA) binds to the mannose-6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R) with high affinity, suggesting that M6P/IGF2R may be a receptor for RA. Here, we show that RA, after 2–3 h of incubation with cultured neonatal-rat cardiac fibroblasts, dramatically alters the intracellular distribution of M6P/IGF2R as well as that of cathepsin B (a lysosomal protease bearing M6P). Immunofluorescence techniques indicate that this change in intracellular distribution is characterized by a shift of the proteins from the perinuclear area to cytoplasmic vesicles. The effect of RA was neither blocked by an RA nuclear receptor antagonist (AGN193109) nor mimicked by a selective RA nuclear-receptor agonist (TTNPB). Furthermore, the RA-induced translocation of cathepsin B was not observed in M6P/IGF2R-deficient P388D1 cells but occurred in stably transfected P388D1 cells expressing the receptor, suggesting that the effect of RA might be the result of direct interaction with M6P/IGF2R, rather than the result of binding to the nuclear receptors. These observations not only support the idea that M6P/IGF2R mediates an RA-response pathway but also indicate a role for RA in control of intracellular trafficking of lysosomal enzymes. Therefore, our observations may have important implications for the understanding of the diverse biological effects of retinoids.

Receptors induce chemotaxis by releasing the βγ subunit of Gi, not by activating Gq or Gs

Many chemoattractants cause chemotaxis of leukocytes by stimulating a structurally distinct class of G protein-coupled receptors. To identify receptor functions required for chemotaxis, we studied chemotaxis in HEK293 cells transfected with receptors for nonchemokine ligands or for interleukin 8 (IL-8), a classical chemokine. In gradients of the appropriate agonist, three nonchemokine Gi-coupled receptors (the D2 dopamine receptor and opioid μ and δ receptors) mediated chemotaxis; the β2-adrenoreceptor and the M3-muscarinic receptor, which couple respectively to Gs and Gq, did not mediate chemotaxis. A mutation deleting 31 C-terminal amino acids from the IL-8 receptor type B quantitatively impaired chemotaxis and agonist-induced receptor internalization, but not inhibition of adenylyl cyclase or stimulation of mitogen-activated protein kinase. To probe the possible relation between receptor internalization and chemotaxis, we used two agonists of the μ-opioid receptor. Morphine and etorphine elicited quantitatively similar chemotaxis, but only etorphine induced receptor internalization. Overexpression of two βγ sequestering proteins (βARK-ct and αt) prevented IL-8 receptor type B-mediated chemotaxis but did not affect inhibition of adenylyl cyclase by IL-8. We conclude that: (i) Nonchemokine Gi-coupled receptors can mediate chemotaxis. (ii) Gi activation is necessary but probably not sufficient for chemotaxis. (iii) Chemotaxis does not require receptor internalization. (iv) Chemotaxis requires the release of free βγ subunits.

Circadian modulation of dopamine receptor responsiveness in Drosophila melanogaster

We investigated the circadian function of Drosophila dopamine receptors by using a behaviorally active decapitated preparation that allows for direct application of drugs to the nerve cord. Quinpirole, a D2-like dopamine receptor agonist, induces reflexive locomotion in decapitated flies. We show that the amount of locomotion induced changes as a function of the time of day, with the highest responsiveness to quinpirole during the subjective night. Furthermore, dopamine receptor responsiveness is under circadian control and depends on the normal function of the period gene. The head pacemaker is at least partly dispensable for the circadian modulation of quinpirole-induced locomotion, because changes in agonist responsiveness persist in decapitated flies that are aged for 12 h. This finding suggests a role for the period-dependent molecular oscillators in the body in the modulation of amine receptor responsiveness.

Inositol 1,4,5-tris-phosphate activation of inositol tris-phosphate receptor Ca2+ channel by ligand tuning of Ca2+ inhibition

Inositol 1,4,5-tris-phosphate (IP3) binding to its receptors (IP3R) in the endoplasmic reticulum (ER) activates Ca2+ release from the ER lumen to the cytoplasm, generating complex cytoplasmic Ca2+ concentration signals including temporal oscillations and propagating waves. IP3-mediated Ca2+ release is also controlled by cytoplasmic Ca2+ concentration with both positive and negative feedback. Single-channel properties of the IP3R in its native ER membrane were investigated by patch clamp electrophysiology of isolated Xenopus oocyte nuclei to determine the dependencies of IP3R on cytoplasmic Ca2+ and IP3 concentrations under rigorously defined conditions. Instead of the expected narrow bell-shaped cytoplasmic free Ca2+ concentration ([Ca2+]i) response centered at ≈300 nM–1 μM, the open probability remained elevated (≈0.8) in the presence of saturating levels (10 μM) of IP3, even as [Ca2+]i was raised to high concentrations, displaying two distinct types of functional Ca2+ binding sites: activating sites with half-maximal activating [Ca2+]i (Kact) of 210 nM and Hill coefficient (Hact) ≈2; and inhibitory sites with half-maximal inhibitory [Ca2+]i (Kinh) of 54 μM and Hill coefficient (Hinh) ≈4. Lowering IP3 concentration was without effect on Ca2+ activation parameters or Hinh, but decreased Kinh with a functional half-maximal activating IP3 concentration (KIP3) of 50 nM and Hill coefficient (HIP3) of 4 for IP3. These results demonstrate that Ca2+ is a true receptor agonist, whereas the sole function of IP3 is to relieve Ca2+ inhibition of IP3R. Allosteric tuning of Ca2+ inhibition by IP3 enables the individual IP3R Ca2+ channel to respond in a graded fashion, which has implications for localized and global cytoplasmic Ca2+ concentration signaling and quantal Ca2+ release.

GTP-binding protein βγ subunits mediate presynaptic calcium current inhibition by GABAB receptor

A variety of GTP-binding protein (G protein)-coupled receptors are expressed at the nerve terminals of central synapses and play modulatory roles in transmitter release. At the calyx of Held, a rat auditory brainstem synapse, activation of presynaptic γ-aminobutyric acid type B receptors (GABAB receptors) or metabotropic glutamate receptors inhibits presynaptic P/Q-type Ca2+ channel currents via activation of G proteins, thereby attenuating transmitter release. To identify the heterotrimeric G protein subunits involved in this presynaptic inhibition, we loaded G protein βγ subunits (Gβγ) directly into the calyceal nerve terminal through whole-cell patch pipettes. Gβγ slowed the activation of presynaptic Ca2+ currents (IpCa) and attenuated its amplitude in a manner similar to the externally applied baclofen, a GABAB receptor agonist. The effects of both Gβγ and baclofen were relieved after strong depolarization of the nerve terminal. In addition, Gβγ partially occluded the inhibitory effect of baclofen on IpCa. In contrast, guanosine 5′-O-(3-thiotriphosphate)-bound Goα loaded into the calyx had no effect. Immunocytochemical examination revealed that the subtype of G proteins Go, but not the Gi, subtype, is expressed in the calyceal nerve terminal. These results suggest that presynaptic inhibition mediated by G protein-coupled receptors occurs primarily by means of the direct interaction of Go βγ subunits with presynaptic Ca2+ channels.

Structure, tissue distribution, and chromosomal localization of the prepronociceptin gene.

Nociceptin (orphanin FQ), the newly discovered natural agonist of opioid receptor-like (ORL1) receptor, is a neuropeptide that is endowed with pronociceptive activity in vivo. Nociceptin is derived from a larger precursor, prepronociceptin (PPNOC), whose human, mouse, and rat genes we have now isolated. The PPNOC gene is highly conserved in the three species and displays organizational features that are strikingly similar to those of the genes of preproenkephalin, preprodynorphin, and preproopiomelanocortin, the precursors to endogenous opioid peptides, suggesting the four genes belong to the same family-i.e., have a common evolutionary origin. The PPNOC gene encodes a single copy of nociceptin as well as of other peptides whose sequence is strictly conserved across murine and human species; hence it is likely to be neurophysiologically significant. Northern blot analysis shows that the PPNOC gene is predominantly transcribed in the central nervous system (brain and spinal cord) and, albeit weakly, in the ovary, the sole peripheral organ expressing the gene. By using a radiation hybrid cell line panel, the PPNOC gene was mapped to the short arm of human chromosome 8 (8p21), between sequence-tagged site markers WI-5833 and WI-1172, in close proximity of the locus encoding the neurofilament light chain NEFL. Analysis of yeast artificial chromosome clones belonging to the WC8.4 contig covering the 8p21 region did not allow to detect the presence of the gene on these yeast artificial chromosomes, suggesting a gap in the coverage within this contig.

Mutation of a conserved serine in TM4 of opioid receptors confers full agonistic properties to classical antagonists.

The involvement of a conserved serine (Ser196 at the mu-, Ser177 at the delta-, and Ser187 at the kappa-opioid receptor) in receptor activation is demonstrated by site-directed mutagenesis. It was initially observed during our functional screening of a mu/delta-opioid chimeric receptor, mu delta2, that classical opioid antagonists such as naloxone, naltrexone, naltriben, and H-Tyr-Tic[psi,CH2NH]Phe-Phe-OH (TIPPpsi; Tic = 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) could inhibit forskolin-stimulated adenylyl cyclase activity in CHO cells stably expressing the chimeric receptor. Antagonists also activated the G protein-coupled inward rectifying potassium channel (GIRK1) in Xenopus oocytes coexpressing the mu delta2 opioid receptor and the GIRK1 channel. By sequence analysis and back mutation, it was determined that the observed antagonist activity was due to the mutation of a conserved serine to leucine in the fourth transmembrane domain (S196L). The importance of this serine was further demonstrated by analogous mutations created in the mu-opioid receptor (MORS196L) and delta-opioid receptor (DORS177L), in which classical opioid antagonists could inhibit forskolin-stimulated adenylyl cyclase activity in CHO cells stably expressing either MORS196L or DORS177L. Again, antagonists could activate the GIRK1 channel coexpressed with either MORS196L or DORS177L in Xenopus oocytes. These data taken together suggest a crucial role for this serine residue in opioid receptor activation.

Expression of corticotropin-releasing factor in inflamed tissue is required for intrinsic peripheral opioid analgesia.

Immune cell-derived opioid peptides can activate opioid receptors on peripheral sensory nerves to inhibit inflammatory pain. The intrinsic mechanisms triggering this neuroimmune interaction are unknown. This study investigates the involvement of endogenous corticotropin-releasing factor (CRF) and interleukin-1beta (IL-1). A specific stress paradigm, cold water swim (CWS), produces potent opioid receptor-specific antinociception in inflamed paws of rats. This effect is dose-dependently attenuated by intraplantar but not by intravenous alpha-helical CRF. IL-1 receptor antagonist is ineffective. Similarly, local injection of antiserum against CRF, but not to IL-1, dose-dependently reverses this effect. Intravenous anti-CRF is only inhibitory at 10(4)-fold higher concentrations and intravenous CRF does not produce analgesia. Pretreatment of inflamed paws with an 18-mer 3'-3'-end inverted CRF-antisense oligodeoxynucleotide abolishes CWS-induced antinociception. The same treatment significantly reduces the amount of CRF extracted from inflamed paws and the number of CRF-immunostained cells without affecting gross inflammatory signs. A mismatch oligodeoxynucleotide alters neither the CWS effect nor CRF immunoreactivity. These findings identify locally expressed CRF as the predominant agent to trigger opioid release within inflamed tissue. Endogenous IL-1, circulating CRF or antiinflammatory effects, are not involved. Thus, an intact immune system plays an essential role in pain control, which is important for the understanding of pain in immunosuppressed patients with cancer or AIDS.

Determinants of the G protein-dependent opioid modulation of neuronal calcium channels.

The modulation of a family of cloned neuronal calcium channels by stimulation of a coexpressed mu opioid receptor was studied by transient expression in Xenopus oocytes. Activation of the morphine receptor with the synthetic enkephalin [D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin (DAMGO) resulted in a rapid inhibition of alpha1A (by approximately 20%) and alpha1B (by approximately 55%) currents while alpha1C and alpha1E currents were not significantly affected. The opioid-induced effects on alpha1A and alpha1B currents were blocked by pertussis toxin and the GTP analogue guanosine 5'-[beta-thio]diphosphate. Similar to modulation of native calcium currents, DAMGO induced a slowing of the activation kinetics and exhibited a voltage-dependent inhibition that was partially relieved by application of strong depolarizing pulses. alpha1A currents were still inhibited in the absence of coexpressed Ca channel alpha2 and beta subunits, suggesting that the response is mediated by the alpha1 subunit. Furthermore, the sensitivity of alpha1A currents to DAMGO-induced inhibition was increased approximately 3-fold in the absence of a beta subunit. Overall, the results show that the alpha1A (P/Q type) and the alpha1B (N type) calcium channels are selectively modulated by a GTP-binding protein (G protein). The results raise the possibility of competitive interactions between beta subunit and G protein binding to the alpha1 subunit, shifting gating in opposite directions. At presynaptic terminals, the G protein-dependent inhibition may result in decreased synaptic transmission and play a key role in the analgesic effect of opioids and morphine.

Evidence for the presence of a protease-activated receptor distinct from the thrombin receptor in human keratinocytes.

Thrombin receptor activation was explored in human epidermal keratinocytes and human dermal fibroblasts, cells that are actively involved in skin tissue repair. The effects of thrombin, trypsin, and the receptor agonist peptides SFLLRN and TFRIFD were assessed in inositolphospholipid hydrolysis and calcium mobilization studies. Thrombin and SFLLRN stimulated fibroblasts in both assays to a similar extent, whereas TFRIFD was less potent. Trypsin demonstrated weak efficacy in these assays in comparison with thrombin. Results in fibroblasts were consistent with human platelet thrombin receptor activation. Keratinocytes, however, exhibited a distinct profile, with trypsin being a far better activator of inositolphospholipid hydrolysis and calcium mobilization than thrombin. Furthermore, SFLLRN was more efficacious than thrombin, whereas no response was observed with TFRIFD. Since our data indicated that keratinocytes possess a trypsin-sensitive receptor, we addressed the possibility that these cells express the human homologue of the newly described murine protease-activated receptor, PAR-2 [Nystedt, S., Emilsson, K., Wahlestedt, C. & Sundelin, J. (1994) Proc. Natl. Acad. Sci. USA 91, 9208-9212]. PAR-2 is activated by nanomolar concentrations of trypsin and possesses the tethered ligand sequence SLIGRL. SLIGRL was found to be equipotent with SFLLRN in activating keratinocyte inositolphospholipid hydrolysis and calcium mobilization. Desensitization studies indicated that SFLLRN, SLIGRL, and trypsin activate a common receptor, PAR-2. Northern blot analyses detected a transcript of PAR-2 in total RNA from keratinocytes but not fibroblasts. Levels of thrombin receptor message were equivalent in the two cell types. Our results indicate that human keratinocytes possess PAR-2, suggesting a potential role for this receptor in tissue repair and/or skin-related disorders.

Depolarisation and suppression of burst firing activity in the mouse subthalamic nucleus by dopamine D1/D5 receptor activation of a cyclic-nucleotide gated non-specific cation conductance

Neuronal burst firing in the subthalamic nucleus (STN) is one of the hallmarks of dopamine depletion in Parkinson's disease. Here, we have determined the postsynaptic effects of dopamine in the STN and the functional consequences of dopamine receptor modulation on burst firing in vitro. STN cells displayed regular spiking activity at a rate of 7.9 +/- 0.5 Hz. Application of dopamine (30 mu M) induced membrane depolarisations accompanied by an increase in firing rate of mean 12.0 +/- 0.6 Hz in all 69 cells. The dopamine effect was mimicked by the dopamine D1/D5 receptor agonist SKF38393 (10 mu M, 17 cells) and the dopamine D2-like receptor agonist quinpirole (10 mu M, 35 cells), partly reduced by D1/D5 antagonist SCH23390 (2 mu M, seven cells), but unaffected by the D2 antagonists sulpiride (10 mu M, seven cells) or eticlopride (10 mu M, six cells). Using voltage ramps, dopamine induced an inward current of 69 +/- 9.4 pA at a holding potential of -60 mV (n = 17). This current was accompanied by an increase in input conductance of 1.55 +/- 0.35 nS which reversed at -30.6 +/- 2.3 mV, an effect mimicked by SKF38393 (10 AM, nine cells). Similar responses were observed when measuring instantaneous current evoked by voltage steps and in the presence of the I-h blocker, ZD7288, indicating effects independent of I-h. The increase in conductance was blocked by SCH23390 (2 mu M, n = 4), mimicked by the activator of adenylyl cyclase forskolin (10 mu M, n = 7) and blocked by H-89, an inhibitor of cyclic AMP dependent protein kinase A (10 PM, n = 6). These results indicate that the dopamine depolarisation is in part mediated by D1/D5 receptor mediated activation of a cyclic-nucleotide gated (CNG) non-specific cation conductance. This conductance contributes to the membrane depolarisation that changes STN neuronal bursting to more regular activity by significantly increasing burst duration and number of spikes per burst.