Phosphorylation of μ-opioid receptors — a putative mechanism of selective uncoupling of receptor — Gi interaction, measured with low-Km CTPase and nucleotide-sensitive agonist binding

The μ-opioid receptor agonist stimulation of low-K_m GTPase in rat striatal membranes was abolished by islet-activating protein (IAP) treatment, and recovered by Gi reconstitution. When the IAP-treated membranes were phosphorylated with a cAMP-dependent protein kinase, there was no such recovery by Gi. The agonist binding was not affected with respect to K_d, B_max and sensitivity to guanine nucleotides in the phosphorylated membranes. These findings suggest that phosphorylation of μ-opioid receptors dissociates the agonist change in G-protein activity from the guanine nucleotide-sensitive agonist binding.     

Activation of μ-opioid receptor modulates GABAA receptor-mediated currents in isolated spinal dorsal horn neurons

Whole-cell voltage-clamp technique was used to examine the effects of a μ-opioid receptor agonist DAGO (Tyr-D-Ala-Gly-Me-Phe-Gly-ol-enkephalin) on GABA-induced currents in acutely isolated spinal dorsal horn (DH) neurons from laminae I-IV of young rats. We found that a bicuculline-sensitive GABA-induced current was potentiated by DAGO (0.5–500 nM), in a dose-dependent manner, in 62% of the tested cells. The elevated GABA responses outlasted the period of DAGO application, and either recovered within 10 min after the removal of the peptide or persisted for up to 50 min. The potentiating effect of DAGO was reduced or prevented by naloxone and the μ-opioid receptor-selective antagonist β-funaltrexamine. A similar enhancing effect on the membrane currents activated by administration of muscimol, a GABA_A receptor-specific agonist, was produced by DAGO. In addition, a transient depression of GABA responses was observed in 25% of the cells tested. These results indicate that the μ-opioid agonist DAGO modulates the sensitivity of postsynaptic GABA_A receptors in a large proportion of spinal neurons from laminae I–IV, with the major effect being facilitation. The DAGO action could contribute to the regulation of the strength of primary afferent neurotransmission, including nociception.     

GABAA and GABAB antagonists prevent the opioid inhibition of endogenous acetylcholine release evoked by glutamate from rat neostriatal slices

We have examined the role of the GABAergic system in the opioid inhibition of endogenous acetylcholine (ACh) release from rat neostriatal slices by blocking either γ-aminobutyric acid-A (GABA_A) or GABA_B receptors. GABAergic antagonists (bicuculline or phaclofen) completely blocked μ- (morphine or DAGO) and δ-opioid (DPDPE) inhibition of glutamate-evoked endogenous ACh release in a concentration-dependent manner. However, GABA antagonists were ineffective in blocking the opioid inhibition of potassium-evoked endogenous ACh release. These findings point to the important role of the GABAergic system in the regulation of μ- and δ-opioid inhibition of cholinergic neurons stimulated by glutamate.     

C1 adrenergic neurons are contacted by presynaptic profiles containing DELTA-opioid receptor immunoreactivity

Ligands of the δ-opioid receptor tonically influence sympathetic outflow. Some of the actions of δ-opioid receptor agonists may be mediated through C1 adrenergic neurons in the rostral ventrolateral medulla. The goal of this study was to determine whether C1 adrenergic neurons or their afferents contain δ-opioid receptors. Single sections through the rostral ventrolateral medulla were labeled for δ-opioid receptor using the immunoperoxidase method and the epinephrine synthesizing enzyme phenylethanolamine N-methyltransferase (PNMT) using the immunogold method, and examined at the light and electron microscopic level. Few (5% of 903) profiles dually labeled for PNMT and δ-opioid receptor were detected; most of these were dendrites with diameters <1.5 μm. δ-Opioid receptor immunoreactivity was affiliated with multivesicular bodies in dually labeled perikarya, whereas δ-opioid receptor immunoperoxidase labeling appeared as isolated clusters within both singly and dually labeled dendrites. The majority (83% of 338) of δ-opioid receptor-immunoreactive profiles were axons and axon terminals. δ-Opioid receptor-immunoreactive terminals averaged 0.75 μm in diameter, contained numerous large dense-core vesicles and usually formed appositions or asymmetric (excitatory-type) synapses with their targets. The majority (>50% of 250) of δ-opioid receptor-immunoreactive axons and axon terminals contacted PNMT-immunoreactive profiles. Most of the contacts formed by δ-opioid receptor-immunoreactive profiles (75% of 132) were on single-labeled PNMT-immunoreactive dendrites with diameters <1.5 μm.

The prominent localization of δ-opioid receptors to dense-core vesicle-rich presynaptic profiles suggests that δ-opioid receptor activation by endogenous or exogenous agonists may modulate neuropeptide release. Furthermore, the presence of δ-opioid receptors on axon terminals that form excitatory-type synapses with PNMT-immunoreactive dendrites suggests that δ-opioid receptor ligands may modulate afferent activity to C1 adrenergic neurons. The observation that some PNMT-immunoreactive neurons contain δ-opioid receptor immunoreactivity associated with multivesicular bodies and other intracellular organelles suggests that some C1 adrenergic neurons may present, endocytose and/or recycle δ-opioid receptors.     

Endogenous protein phosphorylation in chick and rat brain synaptic membranes

The protein kinase activities endogenous to synaptic membranes prepared by an identical procedure from avian (chick) and mammalian (rat) brains were compared. Both species showed similar responses towards both protein kinase effector molecules cyclic adenosine monophosphate and Ca²⁺. K_app for cyclic adenosine monophosphate-dependent protein kinase activity occurred at 0.4–0.8μM cAMP and K_app for Ca²⁺-dependent, calmodulin-requiring protein kinase activity occurred at 1–2μM Ca²⁺ (free ion concentration) both in the absence or presence of calmodulin added to the reaction mixture. This suggests that endogenous calmodulin in these membranes was able to modulate the Ca²⁺-dependent, calmodulin requiring protein kinase activity. After EGTA-treatment of the membranes to remove endogenous Ca²⁺ and calmodulin, no significant response towards Ca²⁺ on the phosphorylation of the membrane polypeptides was measured unless exogenous calmodulin was added after which the K_app for Ca²⁺ was increased to 15μM Ca²⁺ (free ion concentration). There was a difference in the maximal levels of kinase activity in these membranes with chick membranes containing 57% less cyclic adenosine monophosphate-dependent protein kinase activity, but 65% more Ca²⁺-dependent, calmodulin-requiring protein kinase activity than the rat membranes. Similar results were determined when either low (5 μM) or high (5.8mM) concentrations of adenosine 5′-triphosphate were added to the reaction mixtures.

Besides certain species differences in the molecular weights of the resulting phosphoproteins, we observed several major differences with respect to the absence or presence of some of the phosphoproteins. Chick synaptic membranes may lack the cyclic adenosine monophosphate-requiring, microtubule-associated phosphoprotein, MAP₂, one of the 2 neurospecific, cyclic adenosine monophosphate-requiring and Ca²⁺, calmodulin-requiring phosphoproteins (Protein Ib, although Protein Ia apparently is present), and the Ca²⁺-requiring, calmodulin-independent, ACTH-sensitive phosphoprotein, B50.

The phenothiazines, trifluoperazine, fluphenazine and chlorpromazine were found to inhibit the Ca²⁺-dependent, calmodulin-requiring protein kinase activities of both the chick and rat synaptic membranes. This inhibition appeared to be specific for calmodulin because at the same concentrations the phenothiazine analogue, chlorpromazine-sulfoxide, had no effect on this activity. Also found to inhibit Ca²⁺-dependent calmodulin-requiring protein kinase activity were dibucaine and adrenocorticotropin.

These data suggest that rat forebrain synaptic plasma membranes are activated by cyclic adenosine monophosphate to a greater extent than are chick forebrain synaptic plasma membranes while the chick membranes are activated to a greater extent by Ca²⁺, calmodulin than are the rat membranes, though similarities dominate comparison of the two membrane systems.     

Differential involvement of μ1-opioid receptors in endomorphin- and β-endorphin-induced G-protein activation in the mouse pons/medulla

Several genetic mouse models of differential sensitivity to opioids have been used to investigate the mechanisms underlying individual variation in responses to opioids. The CXBK mice are inbred recombinant mice which have a lower level of μ₁-opioid receptors than their parental strain. Endomorphin-1 and endomorphin-2 are endogenous opioid peptides that are highly selective for μ-opioid receptors, while β-endorphin, which is also an endogenous opioid peptide, is non-selective for μ-, δ- and putative -opioid receptors. The present study was designed to investigate the effects of these endogenous opioid peptides on G-protein activation by monitoring guanosine-5′-o-(3-[³⁵S]thio)triphosphate binding to pons/medulla membranes of CXBK mice and their parental strain C57BL/6ByJ mice. Endomorphin-1 (0.1–10 μM), endomorphin-2 (0.1–10 μM) and β-endorphin (0.1–10 μM) increased guanosine-5′-o-(3-[³⁵S]thio)triphosphate binding to the pons/medulla membranes from C57BL/6ByJ and CXBK mice in a concentration-dependent manner. However, the increases of guanosine-5′-o-(3-[³⁵S]thio)triphosphate binding induced by either endomorphin-1 or endomorphin-2 in CXBK mice were significantly much lower than those in C57BL/6ByJ mice. However, no significant difference was found in the increases of the guanosine-5′-o-(3-[³⁵S]thio)triphosphate binding induced by β-endorphin in C57BL/6ByJ and CXBK mice. Moreover, whereas the increase of guanosine-5′-o-(3-[³⁵S]thio)triphosphate binding induced by 10 μM endomorphin-1 or endomorphin-2 were almost completely blocked by a μ-opioid receptor antagonist β-funaltrexamine (10 μM) in both strains, the increase of guanosine-5′-o-(3-[³⁵S]thio)triphosphate binding induced by 10 μM β-endorphin was attenuated to approximately 70% of stimulation by co-incubation with 10 μM β-funaltrexamine in both strains. The residual stimulation of [³⁵S]guanosine-5′-o-(3-thio)triphosphate binding by 10 μM β-endorphin in the presence of 10 μM β-funaltrexamine was further attenuated by the addition of putative -opioid receptor partial agonist β-endorphin (1–27) (1 μM) in both strains. Like the endomorphins, the synthetic μ-opioid receptor agonist [ -Ala²,N-MePhe⁴,Gly-ol⁵]enkephalin at 10 μM showed lower increases of guanosine-5′-o-(3-[³⁵S]thio)triphosphate binding in CXBK mice than those in C57BL/6ByJ mice. However, there was no strain difference in the stimulation of guanosine-5′-o-(3-[³⁵S]thio)triphosphate binding induced by 10 μM of the selective δ₁-opioid receptor agonist [ -Pen^2,5]enkephalin, δ₂-opioid receptor agonist [ -Ala²]deltorphin II or κ-opioid receptor agonist U50,488H.The results indicate that the G-protein activation by endomorphin-1 and endomorphin-2 in the mouse pons/medulla is mediated by both μ₁- and μ₂-opioid receptors. Moreover, β-endorphin-induced G-protein activation in the mouse pons/medulla is, in part, mediated by μ₂- and putative -, but not by μ₁-opioid receptors.     

Binding and GTPgammaS autoradiographic analysis of preproorphanin precursor peptide products at the ORL1 and opioid receptors

Utilizing agonist-stimulated GTPγS autoradiography, we analyzed the ability of preproorphanin FQ (ppOFQ) peptides to stimulate [³⁵S]-GTPγS binding in adult rat brain. Orphanin FQ (OFQ) stimulated [³⁵S]-GTPγS binding in a pattern similar to that described for [¹²⁵I]-OFQ at the endogenous opioid receptor-like (ORL1) receptor. The ppOFQ peptides nocistatin and orphanin FQ2 (OFQ II_1–17) had no effect, suggesting that they do not mediate their reported analgesic effects via a G_i/o-coupled receptor (i.e. opioid or ORL1). Unlike OFQ II_1–17, high concentrations of its C-terminal extension, OFQ II_1–28, stimulated [³⁵S]-GTPγS binding in a mu (μ) opioid receptor-like distribution and the effect was blocked by naloxone. To explore these observations, we evaluated the receptor binding profile of OFQ II_1–28 at the cloned ORL1 and μ opioid receptors. OFQ II_1–28 had no specific binding at either ORL1 or μ opioid receptors at concentrations up to 50 μM. This lack of affinity was not consistent with a μ-mediated effect, as suggested by preliminary observation using functional autoradiography in rat brain sections. Although behavioral studies suggest that OFQ II_1–28 possesses analgesic activity, this effect does not appear to be mediated via direct binding at the μ opioid receptor. Taken together, these findings support the view that (1) OFQ is the only ppOFQ peptide that binds to and activates the ORL1 receptor and (2) OFQ II_1–28 does not bind or stimulate [³⁵S]-GTPγS binding in cells expressing the μ opioid receptor.     

Ultra-low dose naloxone restores the antinociceptive effect of morphine in pertussis toxin-treated rats by reversing the coupling of μ-opioid receptors from Gs-protein to coupling to Gi-protein

Pertussis toxin (PTX) treatment results in ADP-ribosylation of Gi-protein and thus in disruption of μ-opioid receptor signal transduction and loss of the antinociceptive effect of morphine. We have previously demonstrated that pretreatment with ultra-low dose naloxone preserves the antinociceptive effect of morphine in PTX-treated rats. The present study further examined the effect of ultra-low dose naloxone on μ-opioid receptor signaling in PTX-treated rats and the underlying mechanism. Male Wistar rats implanted with an intrathecal catheter received an intrathecal injection of saline or PTX (1 μg in 5 μl of saline), then, 4 days later, were pretreated by intrathecal injection with either saline or ultra-low dose naloxone (15 ng in 5 μl of saline), followed, 30 min later, by saline or morphine (10 μg in 5 μl of saline). Four days after PTX injection, thermal hyperalgesia was observed, together with increased coupling of excitatory Gs-protein to μ-opioid receptors in the spinal cord. Ultra-low dose naloxone pretreatment preserved the antinociceptive effect of morphine, and this effect was completely blocked by the μ-opioid receptor antagonist CTOP, but not by the κ-opioid receptor antagonist nor-BNI or the δ-opioid receptor antagonist naltrindole. Moreover, a co-immunoprecipitation study showed that ultra-low dose naloxone restored μ-opioid receptor/Gi-protein coupling and inhibited the PTX-induced μ-opioid receptor/Gs-protein coupling. In addition to the anti-neuroinflammatory effect and glutamate transporter modulation previously observed in PTX-treated rats, the re-establishment of μ-opioid receptor Gi/Go-protein coupling is involved in the restoration of the antinociceptive effect of morphine by ultra-low dose naloxone pretreatment by normalizing the balance between the excitatory and inhibitory signaling pathways. These results show that ultra-low dose naloxone preserves the antinociceptive effect of morphine, suppresses spinal neuroinflammation, and reduces PTX-elevated excitatory Gs-coupled opioid receptors in PTX-treated rats. We suggest that ultra-low dose naloxone might be clinically valuable in pain management.     

Different metabolic regulation by adenosine in omental and subcutaneous adipose tissue

Adenosine content was higher in omental adipose tissue (0.91 +/- 0.13 nmol g-1 of wet weight; mean +/- S.E.M.) than in abdominal subcutaneous adipose tissue (0.42 +/- 0.08 nmol g-1 of wet weight) in rapidly frozen surgical biopsy samples taken from ten patients undergoing elective abdominal surgery. This difference was statistically significant (P less than 0.002). The sensitivity of isoprenaline-stimulated lipolysis to inhibition by N6-(phenylisopropyl)adenosine was studied in omental and abdominal subcutaneous adipocytes isolated from nine patients. The effect of this adenosine Ri-site agonist was less pronounced in omental than in abdominal subcutaneous adipocytes which could be due to a desensitization phenomenon. This difference was statistically significant (P = 0.012). The ratio of the inhibitory guanine nucleotide binding proteins Gi1 and Gi2 to the corresponding stimulatory protein Gs was the same in plasma membranes prepared from omental and abdominal subcutaneous adipocytes. In conclusion, in omental adipose tissue, adenosine content is higher and the response to this nucleoside is less pronounced than in subcutaneous adipocytes. This difference cannot be explained by a different (Gi1 + Gi2)/Gs ratio.     

Desensitization of acetylcholine induced inositol 1,4,5-trisphosphate formation in neuroblastoma SH-SY5Y cells following repetitive acetylcholine stimulations

In this study, the desensitization of acetylcholine-induced inositol 1,4,5-trisphosphate [I(1,4,5)P₃] formation, upon short-time prestimulations, was investigated in cultures of human neuroblastoma SH-SY5Y cells. Four repeated stimulations for 10 seconds with 10 μM acetylcholine were necessary to induce a desensitization of the I(1,4,5)P₃ formation. The desensitization was observed 4 hours after the initiation of repetitive stimulations. The same effect was obtained by a single prestimulation with 1 mM acetylcholine. Preincubation of the cells with phorbol 12-myristate 13-acetate (PMA) markedly down-regulated the acetylcholine-induced I(1,4,5)P₃ formation. However, the protein kinase C (PKC) inhibitors H7 and staurosporine did not influence the desensitization induced by four repeated stimulations with 20 μM acetylcholine. These results indicate that the signal transduction can be desensitized following repeated stimulations with sub-maximal concentrations of receptor agonist and although activation of PKC can induce the same down-regulation, PKC is most likely not involved in the desensitization induced by repetitive acetylcholine-stimulations.     

Anatomical and affinity state comparisons between dopamine D1 and D2 receptors in the rat central nervous system

The anatomical distributions and affinity states of dopamine D₁ and D₂ receptors were compared in the rat central nervous system using quantitative autoradiography. [³H]SCH23390 and [³H]spiperone (in the presence of 100 nM mianserin) were used to label the D₁, and D₂ receptors, respectively. The densities of D₁ and D₂ receptors displayed a positive correlation among 21 brain regions (Pearson correlation coefficient, r = 0.80, P < 0.001).

The affinity states for the D₁ and D₂ receptors were found to be quite different from each other, and different from the results obtained by others using homogenate preparations. Both the D₁ and D₂ receptors were best modeled using a two-state model. In the absence of exogenous guanine nucleotides and using the nonselective agonist dopamine as the competitor, the D₁ receptor was primarily in a low affinity agonist state (R_H = 21 ± 6%), whereas the D₂ receptor was primarily in the high affinity agonist state (R_H = 77 ± 3%). In the presence of 10 μM guanylyl-imidodiphosphate orguanosine-5'-O-(2-thiophosphate) both the D₁ and the D₂ receptor were completely in a low affinity agonist state (R_L = 100%). These affinity states were found both in the nucleus accumbens and olfactory tubercle using dopamine as the competitor and in the striatum using selective D₁ or D₂ agonists as competitors.

Receptor occupancy of the D₂ receptor with either an agonist or antagonist did not alter the affinity states of the D₁ receptor, and conversely, receptor occupancy of the D₁ receptor did not alter the affinity states of the D₂ receptor.

The correlation between densities of D₁ and D₂ receptors provides an anatomical framework for evaluating behavioral and electrophysiological evidence of an interaction between the two dopamine receptor subtypes. This interaction does not appear to be due to a sharing or coupling of G-proteins in such a way that binding to one dopamine receptor subtype alters the affinity state of the other receptor subtype. The differences between dopamine receptor distributions described by labeled agonists and antagonists may be due in part to differences in their affinity states. The low proportion of high affinity state D₁ receptors may explain some of the difficulties in assigning specific behavioral roles to the D₁ receptor.     

The role of carbohydrate in the assembly and function of polymeric IgG

The carbohydrate present on glycoprotein can influence their biologic and functional properties. In the present paper we have assessed the role of oligosaccharides in the polymerization and effector functions of IgG with the 18 amino acid extension of IgM added to its carboxy terminus (IgGμtp). We found that IgG1μtp and IgG3μtp lacking the carbohydrate addition site in C_H2, in the tail-piece or both assembled into polymers as well as the glycosylated versions. Aglycosylated polymers retained the ability to activate complement as assayed by C1q binding and hemolysis, although they were not as effective as their wild type polymer counterparts. Although IgGμtp lacking the carbohydrate in the tail-piece was able to bind to FcγRII, completely aglycosylated polymers lost the ability to bind to both FcγRI and FcγRII, suggesting a critical role for the C_H2 sugar in FcR binding. Absence of the μtp carbohydrate increased the half life of polymeric IgG1, whereas absence of the carbohydrate in C_H2 accelerated the clearance rate.     

Modulation of the dihydropyridine-insensitive Ca influx by 8-bromo-guanosine-3′:5′-monophosphate, cyclic (8-Br-cGMP) in bovine adrenal chromaffin cells

Pretreatment of chromaffin cells with the permeable analogue of cGMP, 8-Br-cGMP (100 μM), leads to a reduction (35%) of depolarization-evoked intracellular calcium concentration ([Ca²⁺]_i) increases. There is evidence that bovine adrenal chromaffin cells are provided with both dihydropyridine-sensitive and -resistant voltage-sensitive Ca²⁺ influx pathways. Combined incubations with nifedipine 10 μM and 8-Br-cGMP reduced KCl-evoked intracellular Ca²⁺ concentration to a greater extent that each compound separately. Moreover, 8-Br-cGMP failed to affect the [Ca²⁺]_i transient induced by the L-type Ca²⁺ channel agonist Bay K 8644 (1μM) under conditions of low depolarization. Neomycin (0.2 mM) and θ-Aga Toxin-IVA (AgTx) (1μM) inhibited the calcium transient to a similar extent, and this inhibition was not enhanced by the presence of 8-Br-cGMP. It is concluded that 8-Br-cGMP modulated the dihydropyridine-insensitive Ca²⁺ influx pathway in the chromaffin cell.     

Lithium discriminates between muscarinic receptor subtypes on guinea pig hippocampal neurons in vitro

In CA3 pyramidal neurons of guinea pig hippocampal slices an outward current activated by the GABA_B agonist, baclofen (0.3 μM, I_bac) was reduced by low concentrations of carbachol (Cch, 0.1–0.3 μM). The effect of Cch desensitized suggesting that the receptor subtype involved in this muscarinic effect of Cch was of the M₁ subtype. The receptor subtype was also characterized by its equilibrium dissociation constant for pirenzepine (10 nM) as an M₁ receptor. Li⁺ applied extracellularly (1 mM) or intracellularly blocked the suppression of I_bac by Cch without affecting the Cch blockade of a current termed I_AHP, which is mediated by M₂ receptors. While the effect of intracellular Li⁺ application was immediate, it developed very slowly with extracellular application. Since Li⁺-salts are used effectively in the treatment of mania and depression, the selective effect of Li⁺ on M₁-mediated muscarinic neurotransmission might be important for the cholinergic hypothesis of affective disorders.     

Oocytes from Xenopus laevis contain an intrinsic σ2-like binding site

In preparation for expression studies for rat brain σ-binding sites, Xenopus oocytes were tested for the presence of [³H]di-o-tolylguanidine (DTG)-binding sites. Native oocytes were found to contain two intrinsic [³H]DTG-binding sites, a high-affinity site (K_d = 32 ± 6 nM, B_max of 45.7 ± 19 pmol/mg protein) and a low-affinity binding site (K_d = 1.3 ± 0.7 μM, B_max of 3.2 ± 0.7 nmol/mg protein). In a series of radioligand-binding-displacement studies, the high-affinity binding sites were found to have a binding profile which has a similar K_d to that of the mammalian σ₂-binding site (32 vs. 38 nM). Comparison of the IC₅₀ values for inhibition of [³H]DTG binding in rat liver and oocytes for DTG, haloperidol (HAL), (−)-pentazocine, (+)-3-(3-hydroxyphenyl)-N-propylpiperidine hydrochloride ((+)-3-PPP), (+(-pentazocine and Zn²⁺, showed similarity in rank (r² = 0.913) but a 7-fold lower potency in oocytes. These results suggest that the high-affinity [³H]DTG-binding site in oocytes represents a σ₂-like binding site.     

Immunocytochemical mapping of endomorphin-2-immunoreactivity in rat brain

Endomorphin-2 (Tyr-Pro-Phe-Phe-NH₂) is a novel endogenous opioid with high affinity and selectivity for the μ-opioid receptor. Immunocytochemical studies have located this peptide in spinal cord, brainstem and selected brain regions. However, there are disagreements regarding its distribution between published reports. Furthermore, the distributions reported for the endomorphins resemble that of neuropeptide FF, suggesting that some of the previous findings might be due to cross-reactivity with the latter substance. In the present study, the distribution of endomorphin-2-immunoreactivity (ir) was examined throughout the entire rat brain using an affinity-purified antiserum that appeared not to cross-react with neuropeptide FF. Endomorphin-2-ir cell somata were most prominent in the hypothalamus and the nucleus of the solitary tract (NTS). Endomorphin-2-ir varicose fibers were observed in such areas as the bed nucleus of the stria terminalis, the septal nuclei, the periaqueductal gray, the locus coeruleus, the lateral parabrachial nucleus, the NTS, and the substantia gelatinosa of the medulla. More modest immunoreactivity was seen in substantia nigra, nucleus raphe magnus, the ventral tegmental area, the pontine nuclei and the amygdala. Fibers were also observed in the ventral cerebellum. Of note was the negligible immunoreactivity in the striatum, a region known to express high levels of μ-opioid receptors. Thus, endomorphin-2-ir was widely, but not uniformly, distributed throughout the central nervous system and was associated largely, but not exclusively, with regions expressing μ-opioid receptors. Based on its distribution, it may have a role in the control of neuroendocrine, cardiovascular and respiratory functions, and mood, feeding, sexual behavior and pain.     

Differential involvement of mu(1)-opioid receptors in endomorphin- and beta-endorphin-induced G-protein activation in the mouse pons/medulla

Several genetic mouse models of differential sensitivity to opioids have been used to investigate the mechanisms underlying individual variation in responses to opioids. The CXBK mice are inbred recombinant mice which have a lower level of mu(1)-opioid receptors than their parental strain. Endomorphin-1 and endomorphin-2 are endogenous opioid peptides that are highly selective for mu-opioid receptors, while beta-endorphin, which is also an endogenous opioid peptide, is non-selective for mu-, delta- and putative epsilon-opioid receptors. The present study was designed to investigate the effects of these endogenous opioid peptides on G-protein activation by monitoring guanosine-5'-o-(3-[35S]thio)triphosphate binding to pons/medulla membranes of CXBK mice and their parental strain C57BL/6 ByJ mice. Endomorphin-1 (0.1-10 microM), endomorphin-2 (0.1-10 microM) and beta-endorphin (0.1-10 microM) increased guanosine-5'-o-(3-[35S]thio)triphosphate binding to the pons/medulla membranes from C57BL/6 ByJ and CXBK mice in a concentration-dependent manner. However, the increases of guanosine-5'-o-(3-[35S]thio)triphosphate binding induced by either endomorphin-1 or endomorphin-2 in CXBK mice were significantly much lower than those in C57BL/6ByJ mice. However, no significant difference was found in the increases of the guanosine-5'-o-(3-[35S]thio)triphosphate binding induced by beta-endorphin in C57BL/6 ByJ and CXBK mice. Moreover, whereas the increase of guanosine-5'-o-(3-[35S]thio)triphosphate binding induced by 10 microM endomorphin-1 or endomorphin-2 were almost completely blocked by a mu-opioid receptor antagonist beta-funaltrexamine (10 microM) in both strains, the increase of guanosine-5'-o-(3-[35S]thio)triphosphate binding induced by 10 microM beta-endorphin was attenuated to approximately 70% of stimulation by co-incubation with 10 microM beta-funaltrexamine in both strains. The residual stimulation of [35S]guanosine-5'-o-(3-thio)triphosphate binding by 10 microM beta-endorphin in the presence of 10 microM beta-funaltrexamine was further attenuated by the addition of putative epsilon-opioid receptor partial agonist beta-endorphin (1-27) (1 microM) in both strains. Like the endomorphins, the synthetic mu-opioid receptor agonist [D-Ala(2),N-MePhe(4), Gly-ol(5)]enkephalin at 10 microM showed lower increases of guanosine-5'-o-(3-[35S]thio)triphosphate binding in CXBK mice than those in C57BL/6ByJ mice. However, there was no strain difference in the stimulation of guanosine-5'-o-(3-[35S]thio)triphosphate binding induced by 10 microM of the selective delta(1)-opioid receptor agonist [D-Pen(2,5)]enkephalin, delta(2)-opioid receptor agonist [D-Ala(2)]deltorphin II or kappa-opioid receptor agonist U50,488H. The results indicate that the G-protein activation by endomorphin-1 and endomorphin-2 in the mouse pons/medulla is mediated by both mu(1)- and mu(2)-opioid receptors. Moreover, beta-endorphin-induced G-protein activation in the mouse pons/medulla is, in part, mediated by mu(2)- and putative epsilon-, but not by mu(1)-opioid receptors.     

High-affinity agonist binding to rat brainstem muscarinic receptors is eliminated by low pH

The effects of hydrogen ion concentration on high-affinity agonist binding to muscarinic receptors were determined in rat brainstem membranes using [3H]oxotremorine-M as a probe. [3H]Oxotremorine-M bound with high affinity to a small subpopulation of brainstem muscarinic receptors (10% of the receptors labelled by [3H]methylscopolamine). [3H]Oxotremorine-M binding was constant between pH 7.0 and 9.0; the number of high-affinity sites decreased below pH 7.0 and at pH 5.0 no binding was detected. This decrease was irreversible; when brainstem membranes were incubated for 1 h at low pH and then returned to pH 8.0, agonist binding was not restored. In contrast, the total number of receptors, i.e. the number of [3H]antagonist binding sites, was not affected by prolonged incubation at low pH. Agonist affinity for the surviving [3H]oxotremorine binding sites and the sensitivity of agonist binding to guanine nucleotides were not altered in media of low pH (pH 5.5). These findings suggest that [3H]oxotremorine-M binds only to receptors which are functionally coupled to guanine nucleotide-dependent regulatory proteins and that this coupling is irreversibly inactivated in media of low pH.     

Distribution of [H]kainic acid and binding sites in the rat brain: in vivo and in vitro receptor autoradiography

Brain sections incubated in vitro with a-[³H]kainic acid (KA; spec. act. 62.5 Ci/mmol), reveal a heterogenous distribution of low and high affinity KA binding sites in the brain. The highest density of KA binding sites was localised to the hippocampus CA₃ region and to superficial layers of the entorhinal cortex (3.8 6.0 μCi/g tissue). Intravenous injection of [³H]KA (1 μCi/g) reveals limited overall penetration of [³H]KA across the blood-brain barrier. However, a dense labelling of the hippocampus, entorhinal cortex and lateral septal regions (2.5–3.8 μCi/g tissue) was observed. Behaviourally, these rats exhibited mild limbic seizure activity possibly as a result of a direct action of KA in the hippocampus or entorhinal cortex.     

Similar kinetic characteristics of 5-hydroxytryptamine binding in blood platelets and brain membranes of rats

Blood platelets and brain membranes of SIV and Lewis rats both exhibited two saturable binding sites for 5-hydroxytryptamine (5-HT) in the concentration range 1–100 nM. The K_d values for the high-affinity sites were 4–6 nM and for the low-affinity sites 20–40 nM in both tissues of both rat strains. Blood platelets had 100–200 times more binding sites per mg protein (B_max) than brain membranes. Thus, 5-HT receptors of platelets may be used as models for those of cerebral 5-HT-neurons.