Oestrogen receptors interact with the α-catalytic subunit of AMP-activated protein kinase

Normal and pathological stressors engage the AMP-activated protein kinase (AMPK) signalling axis to protect the cell from energetic pressures. Sex steroid hormones also play a critical role in energy metabolism and significantly modify pathological progression of cardiac disease, diabetes/obesity and cancer. AMPK is targeted by 17β-oestradiol (E2), the main circulating oestrogen, but the mechanism by which E2 activates AMPK is currently unknown. Using an oestrogen receptor α/β (ERα/β) positive (T47D) breast cancer cell line, we validated E2-dependent activation of AMPK that was mediated through ERα (not ERβ) by using three experimental strategies. A series of co-immunoprecipitation experiments showed that both ERs associated with AMPK in cancer and striated (skeletal and cardiac) muscle cells. We further demonstrated direct binding of ERs to the α-catalytic subunit of AMPK within the βγ-subunit-binding domain. Finally, both ERs interacted with the upstream liver kinase B 1 (LKB1) kinase complex, which is required for E2-dependent activation of AMPK. We conclude that E2 activates AMPK through ERα by direct interaction with the βγ-binding domain of AMPKα.     

Characterization of interactions of 4-nitrophenylpropyl-N-alkylamine with ς receptors

Sigma receptors are small membrane proteins implicated in a number of pathophysiological conditions, including drug addiction, psychosis, and cancer; thus, small molecule inhibitors of sigma receptors have been proposed as potential pharmacotherapeutics for these diseases. We previously discovered that endogenous monochain N-alkyl sphingolipids, including d-erythro-sphingosine, sphinganine, and N,N-dimethylsphingosine, bind to the sigma-1 receptor at physiologically relevant concentrations [Ramachandran, S., et al. (2009) Eur. J. Pharmacol. 609, 19-26]. Here, we investigated several N-alkylamines of varying chain lengths as sigma receptor ligands. Although the K(I) values for N-alkylamines were found to be in the micromolar range, when N-3-phenylpropyl and N-3-(4-nitrophenyl)propyl derivatives of butylamine (1a and 1b, respectively), heptylamine (2a and 2b, respectively), dodecylamine (3a and 3b, respectively), and octadecylamine (4a and 4b, respectively) were evaluated as sigma receptor ligands, we found that these compounds exhibited nanomolar affinities with both sigma-1 and sigma-2 receptors. A screen of high-affinity ligands 2a, 2b, 3a, and 3b against a variety of other receptors and/or transporters confirmed these four compounds to be highly selective mixed sigma-1 and sigma-2 ligands. Additionally, in HEK-293 cells reconstituted with K(v)1.4 potassium channel and the sigma-1 receptor, these derivatives were able to inhibit the outward current from the channel, consistent with sigma receptor modulation. Finally, cytotoxicity assays showed that 2a, 2b, 3a, and 3b were highly potent against a number of cancer cell lines, demonstrating their potential utility as mixed sigma-1 and sigma-2 receptor anticancer agents.     

Evidence for the presence of novel β-melatonin receptors along with classical α-melatonin receptors in the fish Rasbora daniconius (Ham.)

Abstract

The effects of melatonin (MT) were examined on the isolated scale melanophores from dorso-lateral (D-L) and band regions of a tropical fish Rasbora daniconius. Our study primarily aimed for further depiction of the signaling receptors involved in MT mediated pigment translocations in the fish. Melanophore Size Index (MSI) was employed as a recording parameter for the responses of melanophores to MT and various antagonists. MT has induced aggregation as well as dispersion in D-L region and aggregation in band region melanophores during summer season. During winter, MT-induced responses were only of aggregatory type in D-L region, while in the band region there was an increase in the sensitivity. The responses of the melanophores to MT were reversible. The aggregation of innervated melanophores induced by MT on the D-L and band regions was partially mediated through the neurotransmitters released under the influence of MT and partially by the specific MT receptors. Luzindole and K185 have completely blocked the aggregatory responses of D-L and band region melanophores. Aggregatory receptors may be of the conventional α-MT type. Dispersion of D-L and band region melanophores induced by MT in the presence of various antagonists and on denervated band region could be the result of activation of β-MT receptors of dispersive nature. Presence of α and β MT receptors is thus indicated in this fish melanophores.     

Molecular orbital calculation for the model compounds of kainoid amino acids, agonists of excitatory amino acid receptors. Does the kainoid C4-substituent directly interact with the receptors?

Kainoid amino acids are agonists of the AMPA/kainate receptors and exhibit highly potent neuroexcitatory activity. From the results of extensive structure--activity relationship studies, we previously postulated that the C4-substituent of the kainoid amino acids interacts with an allosteric site of the glutamate receptor with electron-donating character. In order to investigate the mode of action in more detail, molecular orbital calculation for model compounds of the kainoid were performed. The results indicated that the HOMO energy level of the C4-substituent is involved in the potent neuroexcitatory activity, thus supporting our hypothesis.     

Do C cells of the thyroid gland of the baboon contain estrogen receptors?

The uptake and retention of radiolabelled estradiol was studied in the thyroid gland of the female baboon. Four baboons were injected intravenously with 1 micron/kg body weight of 3H-estradiol. One animal, which served as a control, received an additional injection of 100 micrograms/kg body weight of unlabelled hormone. One hour after the injections, the animals were killed and the thyroid glands removed and processed for either autoradiography or autoradiography in combination with immunocytochemical staining for C cells. Localization of estradiol was observed in the nuclei of interstitial cells, but not in those of the follicular cells. Nuclei of immunostained calcitonin-containing cells in both the walls of the follicles and the interfollicular compartment were not radiolabelled. This study suggests that estrogen does not regulate calcitonin secretion by the C cells of the thyroid via a classical receptor system.     

Local and global calcium signals associated with the opening of neuronal α7 nicotinic acetylcholine receptors

Neuronal nicotinic acetylcholine receptors (nAChRs) are Ca²⁺-permeable ligand-gated channels widely expressed in the central and peripheral nervous system. One of the most Ca²⁺ selective isoform is the homopentameric α7-nAChR implicated in schizophrenia. The activity of α7-nAChRs is usually recorded electrophysiologically, which limits the amount of information obtained. Here, we used fluorescence imaging to record Ca²⁺ transients associated with activation of the α7-nAChR in neuroblastoma cells stably expressing human α7-nAChRs. Application of nicotine (50 μM) consistently evoked transient (30 s), stereotyped Ca²⁺ responses that were inhibited by the selective α7-nAChRs antagonists methyllycaconitine (MLA) and α-bungarotoxin, and greatly increased and prolonged by the allosteric modulator PNU-120596 (1 μM). Unexpectedly, brief (1–5 s), repetitive Ca²⁺ transients of sub-micrometric dimension were observed in filopodia of cells expressing α7-nAChR. PNU-120596 increased the frequency and slowed the decay kinetics of these miniature Ca²⁺ elevations, which were insensitive to ryanodine, preserved during hyperpolarisation, and prevented by MLA, α-bungarotoxin, or Ca²⁺ removal. Global Ca²⁺ responses were also recorded in ganglion cells of embryo chicken retina during co-application of PNU-120596 and nicotine, together with whole-cell currents and brief current bursts. These data demonstrate that Ca²⁺ signals generated by α7-nAChRs can be recorded optically both in cell lines and in intact tissues. The possibility to image miniature Ca²⁺ signals enables to map the location of functional α7-nAChR channel clusters within cells and to analyze their single channel properties optically. Deciphering the rich pattern of intracellular Ca²⁺ signals generated by the activity of the α7-nAChRs will reveal the physiological role of these receptor-channels.     

Characterization of postsynaptic β-adrenergic receptors and presynaptic muscarinic cholinergic receptors in the rat spleen

The β-adrenergic and muscarinic cholinergic receptors in the splenic homogenates of control and 6-hydroxydopamine (6-OHDA) treated rats were characterized. The specific binding of [³H]dihydroalprenolol (DHA) and [³H]quinuclidinyl benzilate (QNB) in the rat spleen were saturable and of high affinity and showed pharmacological specificity of splenic β-adrenergic and muscarinic cholinergic receptors. Following 6-OHDA treatment, the Bmax value for specific [³H](-)DHA binding to the rat spleen was significantly increased by 26 percent and 22 percent compared to control at 2 and 3 weeks without a change in the Kd. In contrast, there was a 38 percent decrease in the Bmax for [³H](-)QNB in the 6-OHDA treated rat spleen at 2 and 3 weeks respectively without a change in the Kd. The Bmax value at 5 weeks was significantly greater than that at 2 or 3 weeks. The splenic norepinephrine (NE) concentration was markedly reduced by the 6-OHDA treatment at 1 to 3 weeks, while there was a significant recovery in the splenic NE concentration at 5 weeks. Thus, our results strongly suggest that we are biochemically localizing muscarinic cholinergic receptors on the sympathetic nerves of the rat spleen and that the β-adrenergic receptors of the spleen are localized postsynaptically.     

Contribution of nicotinic receptors to the function of synapses in the central nervous system: The action of choline as a selective agonist of α7 receptors

The α7-nicotinic receptor (nAChR)-selective agonist choline and nAChR-subtype-selective antagonists led to the discovery that activation of both α7 and α4β2 nAChRs located in CA1 interneurons in slices taken from the rat hippocampus facilitates the tetrodotoxin (TTX)-sensitive release of γ-aminobutyric acid (GABA). Experiments carried out in cultured hippocampal neurons not only confirmed that preterminal α7 and α4β2 nAChRs modulate the TTX-sensitive release of GABA, but also demonstrated that evoked release of GABA is reduced by rapid exposure of the neurons to acetylcholine (ACh, 10 μM-1 mM) in the presence of the muscarinic receptor antagonist atropine (1 μM). This effect of ACh, which is fully reversible and concentration-dependent, is partially blocked by superfusion of the cultured neurons with external solution containing either the α7-nAChR-selective antagonist methyllycaconitine (MLA, 1 nM) or the α4β2-nAChR-selective antagonist dihydro-β-erythroidine (DHβE, 100 nM). A complete blockade of ACh-induced reduction of evoked release of GABA was achieved only when the neurons were perfused with external solution containing both MLA and DHβE, suggesting that activation of both α7 and α4β2 nAChRs modulates the evoked release of GABA from hippocampal neurons. Such mechanisms may account for the apparent involvement of nAChRs in the psychological effects of tobacco smoking, in brain disorders (e.g., schizophrenia and epilepsy), and in physiological processes, including cognition and nociception.     

Synthesis of substituted diphenyl sulfones and their structure–activity relationship with the antagonism of 5-НТ6 receptors

Substituted diphenyl sulfones (10a–n) were synthesised, and the structures were confirmed by NMR, LC–MS and X-ray crystallography. Their antagonistic activities towards 5-HT₆ receptor were assessed in a cell-based functional assay. Diphenyl sulfone 10a, in spite of being the smallest and simplest known sulfonyl-containing 5-HT₆R antagonist, showed a strong potency (K_i = 1.6 μM). Its derivative with a methylamine substituent, 10g (N-methyl-2-(phenylsulfonyl)aniline), was ～66-times as active as diphenyl sulfone (K_i = 24.3 nM). Addition of a piperazinyl moiety in the para-position relative to the sulfonyl group in compound 10m (N-methyl-2-(phenylsulfonyl)-5-piperazin-1-ylaniline) led to a further 150-fold increase in potency (K_i = 0.16 nM) to block the serotonin-induced response of HEK-293 cells that were stably transfected with the human recombinant 5-HT₆ receptor.     

Up-regulation of Μ-opioid receptors in the spinal cord of morphine-tolerant rats

Though morphine remains the most powerful drug for treating pain, its effectiveness is limited by the development of tolerance and dependence. The mechanism underlying development of tolerance to morphine is still poorly understood. One of the factors could be an alteration in the number of Μ-receptors within specific parts of the nervous system. However, reports on changes in the Μ-opioid receptor density in the spinal cord after chronic morphine administration are conflicting. Most of the studies have used subcutaneously implanted morphine pellets to produce tolerance. However, it does not simulate clinical conditions, where it is more common to administer morphine at intervals, either by injections or orally. In the present study, rats were made tolerant to morphine by injecting increasing doses of morphine (10-50 mg/kg, subcutaneously) for five days.In vitro tissue autoradiography for localization of Μ-receptor in the spinal cord was done using [³H]-DAMGO. As compared to the spinal cord of control rats, the spinal cord of tolerant rats showed an 18.8% increase or up-regulation in the density of Μ-receptors in the superficial layers of the dorsal horn. This up-regulation of Μ-receptors after morphine tolerance suggests that a fraction of the receptors have been rendered desensitized, which in turn could lead to tolerance     

Do benzodiazepine receptors mediate the anticonflict action of pentobarbital?

The effects of the benzodiazepine antagonist CGS 8216 (2-phenylpyrazolo[4,3-c]quinoline-3(5H)-one) were examined in a thirsty rat conflict test in the presence and absence of pentobarbital. CGS 8216 (2.5-10 mg/kg i.p.) did not affect nonpunished responding, but doses of 5 and 10 mg/kg significantly reduced the rate of punished responding (i.e., the number of 3 second drinking episodes in a "shock" contingency). However, a dose of CGS 8216 which did not significantly alter punished responding (2.5 mg/kg) antagonized the anticonflict actions of pentobarbital. These observations suggest that while high doses of CGS 8216 may elicit an "anxiogenic" response in rodents, lower doses of CGS 8216 antagonize the anticonflict actions of a compound which has been shown to enhance benzodiazepine affinity in vitro. These data imply that the anticonflict actions of pentobarbital may be mediated through benzodiazepine receptors.     

The γ subunits of the native GABAA/benzodiazepine receptors

Subunit-specific antibodies to all the γ subunit isoforms described in mammalian brain (γ₁, γ_2S, γ_L, and γ₃) have been made. The proportion of GABA_A receptors containing each γ subunit isoform in various brain regions has been determined by quantitative immunoprecipitation. In all tested regions of the rat brain, the γ₁, and γ₃ subunits are present in considerable smaller proportion of GABA_A receptor than the γ₂ subunit. Immunocytochemistry shows that γ₁ immunoreactivity concentrates in the stratum oriens and stratum radiatum of the CA1 region of the hippocampus. In the dentate gyrus, γ₁ immunoreactivity concentrates on the outer 2/3 of the molecular layer coinciding with the localization of the axospinous synapses of the perforant pathway. In contrast, γ₃ immunoreactivity concentrates on the basket cells and other GABAergic local circuit neurons of the hilus. These cells are also rich in γ_2S. In the cerebellu, γ₁ immunolabeling was localized on the Bergmann glia. The γ_2S and γ_2L subunits are differentially expressed in various brain regions. Thus the γ_2S is highly expressed in the olfactory bulb and hippocampus whereas the γ_2L is very abundant in inferior colliculus and cerebellum, particularly in Purkinje cells, as immunocytochemistry, in situ hybridization and immunoprecipitation techniques have revealed. The γ_2S and γ_2L coexist in some brain areas and cell types. Moreover, the γ_2S and γ_2L subunits can coexist in the same GABA_A receptor pentamer. We have shown that this is the case in some GABA_A receptors expressed in cerebellar granule cells. These GABA_A receptors also have α and β subunits forming the pentamer. Immunoblots have shown that the rat γ₁, γ_2S, γ_2L and γ₃ subunits are peptides of 47, 45, 47 and 44 kDa respectively. Results also indicate that there are aging-related changes in the expression of the γ_2S and γ_2L subunits in various brain regions which suggest the existence of aging-related changes in the subunit composition of the GABA_A receptors which in turn might lead to changes in receptor pharmacology. The results obtained with the various γ subunit isoforms are discussed in terms of the high molecular and binding heterogeneity of the native GABA_A receptors in brain. Special issue dedicated to Dr. Kinya Kuriyama     

Selective interactions of spinophilin with the C-terminal domains of the δ- and μ-opioid receptors and G proteins differentially modulate opioid receptor signaling

Previous studies have shown that the intracellular domains of opioid receptors serve as platforms for the formation of a multi-component signaling complex consisting of various interacting partners (Leontiadis et al., 2009, Cell Signal. 21, 1218-1228; Georganta et al., 2010, Neuropharmacology, 59(3), 139-148). In the present study we demonstrate that spinophilin a dendritic-spine enriched scaffold protein associates with δ- and μ-opioid receptors (δ-ΟR, μ-OR) constitutively in HEK293 an interaction that is altered upon agonist administration and enhanced upon forskolin treatment for both μ-OR and δ-ΟR. Spinophilin association with the opioid receptors is mediated via the third intracellular loop and a conserved region of the C-terminal tails. The portion of spinophilin responsible for interaction with the δ-OR and μ-OR is narrowed to a region encompassing amino acids 151-444. Spinophilin, RGS4, Gα and Gβγ subunits of G proteins form a multi-protein complex using specific regions of spinophilin and a conserved amino acid stretch of the C-terminal tails of both δ-μ-ORs. Expression of spinophilin in HEK293 cells potentiated DPDPE-mediated adenylyl-cyclase inhibition of δ-OR leaving unaffected the levels of cAMP accumulation mediated by the μ-OR. Moreover, measurements of extracellular signal regulated kinase (ERK1,2) phosphorylation indicated that the presence of spinophilin attenuated agonist-driven ERK1,2 phosphorylation mediated upon activation of the δ-OR but not the μ-OR. Collectively, these findings suggest that spinophilin associates with both δ- and μ-ΟR and G protein subunits in HEK293 cells participating in a multimeric signaling complex that displays a differential regulatory role in opioid receptor signaling.     

Tension receptors on the apodemes of muscles in the walking legs of the crab,Cancer magister †

1. Mechanoreceptors monitoring tension in working muscles are described in the Decapoda Crustacea.

2. The receptors are associated with apodemes of muscles in the walking leg and are well‐developed in the extensor and flexor of the meropodite (Figures 1, 2).

3. The unbranched dendrites of the receptor neurones innervate the tissues surrounding the insertions of the muscle fibres (Figures 3, 4, 5(A)).

4. The receptors show spontaneous activity with the M‐C joint at resting position and this activity increases when the muscle is stretched by holding the joint at a different position (Figure 7).

5. Isometric tension increase in the muscle recruits sensory units (Figures 8, 10(A)) and increases the activity of units firing (Figure 9).

6. Apodeme receptors may be an entirely distinct input channel from chordotonal organs (Figure 10(B,C)). Joint movements produced by a standard muscle stimulus against increasing loads reveal very different responses (Figure 11).

7. Attempts to determine whether chordotonal organs (CP1, Figures 5(B), 6) monitor isometric muscle tension (Figure 12) suggest possible complexities in their dynamic responses.

8. Abbreviations used in this paper are FASN flexor apodeme sensory nerve, EASN extensor apodeme sensory nerve, BASN bender apodeme sensory nerve, and OASN opener apodeme sensory nerve.     

Differential response to morphine of the oligomeric state of μ-opioid in the presence of δ-opioid receptors

Prolonged morphine treatment induces extensive desensitization of the μ-opioid receptor (μOR) which is the G-protein-coupled receptor that primarily mediates the cellular response to morphine. To date, the molecular mechanism underlying this process is unknown. Here, we have used live cell fluorescence imaging to investigate whether prolonged morphine treatment affects the physical environment of μOR, or its coupling with G-proteins, in two neuronal cell lines. We find that chronic morphine treatment does not change the amount of enhanced yellow fluorescence protein (eYFP)-tagged μOR on the plasma membrane, and only slightly decreases its association with G-protein subunits. Additionally, morphine treatment does not have a detectable effect on the diffusion coefficient of eYFP-μOR. However, in the presence of another family member, the δ-opioid receptor (δOR), prolonged morphine exposure results in a significant increase in the diffusion rate of μOR. Number and brightness measurements suggest that μOR exists primarily as a dimer that will oligomerize with δOR into tetramers, and morphine promotes the dissociation of these tetramers. To provide a plausible structural context to these data, we used homology modeling techniques to generate putative configurations of μOR-δOR tetramers. Overall, our studies provide a possible rationale for morphine sensitivity.     

Thyroid dependent maturation of β-adrenergic receptors in the rat lung

β-Adrenergic receptors were identified in membranes of fetal and postnatal rat lung with (?)-[³H]dihydroalprenolol, [³H]DHA. β-Receptor number (Bmax) increased 11-fold from day 18 of gestation to adult levels by day 28 of postnatal life. The increase of β-adrenergic receptors occurring between postnatal days 15 and 28 was dependent on thyroxine (T4) in propylthiouracil treated pups. β-Adrenergic receptors on day 28 were identical in euthyroid (PTU + T4) as compared to normal control pups (489±31 and 491±30 femtomoles·mg^?1) however receptors were markedly reduced in 28 day hypothyroid pups (PTU alone), Bmax = 294±21.5, m±S.E. p<0.01. Treatment of the hypothyroid pups with T4 for three days on postnatal day 25 increased β-adrenergic receptors approximately two-fold by day 28. This thyroid hormone dependent increase in lung β-adrenergic receptors occurs between postnatal days 15 and 28 coincident with the known increase in thyroid gland activity in the rat pup.     

“Calcium receptors” on eukaryotic cells with special reference to the osteoclast

There is a growing list of cells that are capable of detecting and responding to changes in the concentration of extracellular calcium. The two classic examples of this behaviour are the calcitonin-secreting parafollicular cells of the thyroid and parathyroid hormone-secreting chief cells of the parathyroid gland. A more recent addition to this list is the renin-secreting juxtaglomerular cell of the kidney. Particularly intriguing has been independently the discovery by two laboratories, that the resorptive cell of bone, the osteoclast, is capable of detecting changes in ambient calcium. A common theme amongst all these so called calcium-responsive cells is that extracellular calcium increases elevate intracellular calcium levels, and this intracellular signal is either stimulatory or inhibitory to the functional response. But how these cells detect changes in the concentration of extracellular calcium, and how these recognition events are subsequently transformed into intracellular signals that regulate cell function are somewhat less clear. The commentary reveals some recent developments that seemingly provide insights into these mechanisms, with special reference to the osteoclast.     

Mechanism for sympathetic modulation of activity of cutaneous receptors (with Aδ fibers)

The afferent flow in A fibers from cutaneous receptors was studied in acute experiments with cats. A decline in the number of fibers activated during adequate receptor stimulation was observed at 5 and 35 sec after the excitation of sympathetic efferents. Similar changes in the afferent flow were recorded in identical time intervals after the preliminary stretching and cooling of the skin. The results obtained indicate that the quantitative characteristics of afferent flow during the first 30 sec after stimulation of the sympathetic chain are primarily due to the change in the mechanical state of the tissues surrounding the receptors.N. I. Lobachevskii Institute of Mathematics and Cybernetics, Gor'kii University. Translated from Neirofiziologiya, Vol. 17, No. 4, pp. 517–521, July–August, 1985.