Δ9-tetrahydrocannabinol modulates the proteasome system in the brain.

Cannabis is the most consumed illicit drug worldwide. Its principal psychoactive component, Δ9-tetrahydrocannabinol (THC), affects multiple brain functions, including cognitive performance, by modulating cannabinoid type-1 (CB1) receptors. These receptors are strongly enriched in presynaptic terminals, where they modulate neurotransmitter release. We analyzed, through a proteomic screening of hippocampal synaptosomal fractions, those proteins and pathways modulated 3 h after a single administration of an amnesic dose of THC (10 mg/kg, i.p.). Using an isobaric labeling approach, we identified 2040 proteins, 1911 of them previously reported in synaptic proteomes, confirming the synaptic content enrichment of the samples. Initial analysis revealed a significant alteration of 122 proteins, where 42 increased and 80 decreased their expression. Gene set enrichment analysis indicated an over-representation of mitochondrial associated functions and cellular metabolic processes. A second analysis focusing on extreme changes revealed 28 proteins with altered expression after THC treatment, 15 of them up-regulated and 13 down-regulated. Using a network topology-based scoring algorithm we identified those proteins in the mouse proteome with the greatest association to the 28 modulated proteins. This analysis pinpointed a significant alteration of the proteasome function, since top scoring proteins were related to the proteasome system (PS), a protein complex involved in ATP-dependent protein degradation. In this regard, we observed that THC decreases 20S proteasome chymotrypsin-like protease activity in the hippocampus. Our data describe for the first time the modulation of the PS in the hippocampus following THC administration under amnesic conditions that may contribute to an aberrant plasticity at synapses.

Neurochemical basis of cannabis addiction.

Cannabis derivatives have become the most widely used illicit substances in developed countries, and constitute a major health concern. The psychoactive compounds contained in cannabis induce their pharmacological effects by the activation of at least two different receptors, CB1 and CB2 cannabinoid receptors. Multiple studies have demonstrated the specific involvement of CB1 cannabinoid receptors in the addictive properties of cannabinoids. Several neurotransmitter systems involved in the addictive effects of other prototypical drugs of abuse, such as the dopaminergic and the opioid system are also involved in cannabis addiction. The participation of other neurochemical systems in behavioural responses of cannabinoids related to their addictive effects has also been reported. This review describes the experimental methods now available to study the pharmacological responses of cannabinoids related to their addictive effects and how these methods have contributed to advance the knowledge of the specific contribution of different neurochemical systems in cannabis addiction.

Differential subcellular localization of the two alternatively spliced isoforms of the Kv3.1 potassium channel subunit in brain.

Voltage-gated K(+) channels containing pore-forming subunits of the Kv3 subfamily have specific roles in the fast repolarization of action potentials and enable neurons to fire repetitively at high frequencies. Each of the four known Kv3 genes encode multiple products by alternative splicing of 3' ends resulting in the expression of K(+) channel subunits differing only in their C-terminal sequence. The alternative splicing does not affect the electrophysiological properties of the channels, and its physiological role is unknown. It has been proposed that one of the functions of the alternative splicing of Kv3 genes is to produce subunit isoforms with differential subcellular membrane localizations in neurons and differential modulation by signaling pathways. We investigated the role of the alternative splicing of Kv3 subunits in subcellular localization by examining the brain distribution of the two alternatively spliced versions of the Kv3.1 gene (Kv3.1a and Kv3.1b) with antibodies specific for the alternative spliced C-termini. Kv3.1b proteins were prominently expressed in the somatic and proximal dendritic membrane of specific neuronal populations in the mouse brain. The axons of most of these neurons also expressed Kv3.1b protein. In contrast, Kv3.1a proteins were prominently expressed in the axons of some of the same neuronal populations, but there was little to no Kv3.1a protein expression in somatodendritic membrane. Exceptions to this pattern were seen in two neuronal populations with unusual targeting of axonal proteins, mitral cells of the olfactory bulb, and mesencephalic trigeminal neurons, which expressed Kv3.1a protein in dendritic and somatic membrane, respectively. The results support the hypothesis that the alternative spliced C-termini of Kv3 subunits regulate their subcellular targeting in neurons.

A role for frequenin, a Ca2+-binding protein, as a regulator of Kv4 K+-currents.

Frequenin, a Ca(2+)-binding protein, has previously been implicated in the regulation of neurotransmission, possibly by affecting ion channel function. Here, we provide direct evidence that frequenin is a potent and specific modulator of Kv4 channels, the principal molecular components of subthreshold activating A-type K(+) currents. Frequenin increases Kv4.2 current amplitudes (partly by enhancing surface expression of Kv4.2 proteins) and it slows the inactivation time course in a Ca(2+)-dependent manner. It also accelerates recovery from inactivation. Closely related Ca(2+)-binding proteins, such as neurocalcin and visinin-like protein (VILIP)-1 have no such effects. Specificity for Kv4 currents is suggested because frequenin does not modulate Kv1.4 or Kv3.4 currents. Frequenin has negligible effects on Kv4.1 current inactivation time course. By using chimeras made from Kv4.2 and Kv4.1 subunits, we determined that the differential effects of frequenin are mediated by means of the Kv4 N terminus. Immunohistochemical analysis demonstrates that frequenin and Kv4.2 channel proteins are coexpressed in similar neuronal populations and have overlapping subcellular localizations in brain. Coimmunoprecipitation experiments demonstrate that a physical interaction occurs between these two proteins in brain membranes. Together, our data provide strong support for the concept that frequenin may be an important Ca(2+)-sensitive regulatory component of native A-type K(+) currents.

Impaired fast-spiking, suppressed cortical inhibition, and increased susceptibility to seizures in mice lacking Kv3.2 K+ channel proteins.

Voltage-gated K(+) channels of the Kv3 subfamily have unusual electrophysiological properties, including activation at very depolarized voltages (positive to -10 mV) and very fast deactivation rates, suggesting special roles in neuronal excitability. In the brain, Kv3 channels are prominently expressed in select neuronal populations, which include fast-spiking (FS) GABAergic interneurons of the neocortex, hippocampus, and caudate, as well as other high-frequency firing neurons. Although evidence points to a key role in high-frequency firing, a definitive understanding of the function of these channels has been hampered by a lack of selective pharmacological tools. We therefore generated mouse lines in which one of the Kv3 genes, Kv3.2, was disrupted by gene-targeting methods. Whole-cell electrophysiological recording showed that the ability to fire spikes at high frequencies was impaired in immunocytochemically identified FS interneurons of deep cortical layers (5-6) in which Kv3.2 proteins are normally prominent. No such impairment was found for FS neurons of superficial layers (2-4) in which Kv3.2 proteins are normally only weakly expressed. These data directly support the hypothesis that Kv3 channels are necessary for high-frequency firing. Moreover, we found that Kv3.2 -/- mice showed specific alterations in their cortical EEG patterns and an increased susceptibility to epileptic seizures consistent with an impairment of cortical inhibitory mechanisms. This implies that, rather than producing hyperexcitability of the inhibitory interneurons, Kv3.2 channel elimination suppresses their activity. These data suggest that normal cortical operations depend on the ability of inhibitory interneurons to generate high-frequency firing.

H2 histamine receptor-phosphorylation of Kv3.2 modulates interneuron fast spiking.

Histamine-containing neurons of the tuberomammilary nucleus project to the hippocampal formation to innervate H1 and H2 receptors on both principal and inhibitory interneurons. Here we show that H2 receptor activation negatively modulates outward currents through Kv3.2-containing potassium channels by a mechanism involving PKA phosphorylation in inhibitory interneurons. PKA phosphorylation of Kv3.2 lowered the maximum firing frequency of inhibitory neurons, which in turn negatively modulated high-frequency population oscillations recorded in principal cell layers. All these effects were absent in a Kv3.2 knockout mouse. These data reveal a novel pathway for histamine-dependent regulation of high-frequency oscillations within the hippocampal formation.

K(+) channel expression distinguishes subpopulations of parvalbumin- and somatostatin-containing neocortical interneurons.

Kv3.1 and Kv3.2 K(+) channel proteins form similar voltage-gated K(+) channels with unusual properties, including fast activation at voltages positive to -10 mV and very fast deactivation rates. These properties are thought to facilitate sustained high-frequency firing. Kv3.1 subunits are specifically found in fast-spiking, parvalbumin (PV)-containing cortical interneurons, and recent studies have provided support for a crucial role in the generation of the fast-spiking phenotype. Kv3.2 mRNAs are also found in a small subset of neocortical neurons, although the distribution of these neurons is different. We raised antibodies directed against Kv3.2 proteins and used dual-labeling methods to identify the neocortical neurons expressing Kv3.2 proteins and to determine their subcellular localization. Kv3.2 proteins are prominently expressed in patches in somatic and proximal dendritic membrane as well as in axons and presynaptic terminals of GABAergic interneurons. Kv3.2 subunits are found in all PV-containing neurons in deep cortical layers where they probably form heteromultimeric channels with Kv3.1 subunits. In contrast, in superficial layer PV-positive neurons Kv3.2 immunoreactivity is low, but Kv3.1 is still prominently expressed. Because Kv3.1 and Kv3.2 channels are differentially modulated by protein kinases, these results raise the possibility that the fast-spiking properties of superficial- and deep-layer PV neurons are differentially regulated by neuromodulators. Interestingly, Kv3. 2 but not Kv3.1 proteins are also prominent in a subset of seemingly non-fast-spiking, somatostatin- and calbindin-containing interneurons, suggesting that the Kv3.1-Kv3.2 current type can have functions other than facilitating high-frequency firing.

The effects of Shaker beta-subunits on the human lymphocyte K+ channel Kv1.3.

The activation of T-lymphocytes is dependent upon, and accompanied by, an increase in voltage-gated K+ conductance. Kv1.3, a Shaker family K+ channel protein, appears to play an essential role in the activation of peripheral human T cells. Although Kv1.3-mediated K+ currents increase markedly during the activation process in mice, and to a lesser degree in humans, Kv1.3 mRNA levels in these organisms do not, indicating post-transcriptional regulation. In other tissues Shaker K+ channel proteins physically associate with cytoplasmic beta-subunits (Kvbeta1-3). Recently it has been shown that Kvbeta1 and Kvbeta2 are expressed in mouse T cells and that they are up-regulated during mitogen-stimulated activation. In this study, we show that the human Kvbeta subunits substantially increase K+ current amplitudes when coexpressed with their Kv1.3 counterpart, and that unlike in mouse, protein levels of human Kvbeta2 remain constant upon activation. Differences in Kvbeta2 expression between mice and humans may explain the differential K+ conductance increases which accompany T-cell proliferation in these organisms.

Molecular diversity of K+ channels.

K+ channel principal subunits are by far the largest and most diverse of the ion channels. This diversity originates partly from the large number of genes coding for K+ channel principal subunits, but also from other processes such as alternative splicing, generating multiple mRNA transcripts from a single gene, heteromeric assembly of different principal subunits, as well as possible RNA editing and posttranslational modifications. In this chapter, we attempt to give an overview (mostly in tabular format) of the different genes coding for K+ channel principal and accessory subunits and their genealogical relationships. We discuss the possible correlation of different principal subunits with native K+ channels, the biophysical and pharmacological properties of channels formed when principal subunits are expressed in heterologous expression systems, and their patterns of tissue expression. In addition, we devote a section to describing how diversity of K+ channels can be conferred by heteromultimer formation, accessory subunits, alternative splicing, RNA editing and posttranslational modifications. We trust that this collection of facts will be of use to those attempting to compare the properties of new subunits to the properties of others already known or to those interested in a comparison between native channels and cloned candidates.

Contributions of Kv3 channels to neuronal excitability.

Four mammalian Kv3 genes have been identified, each of which generates, by alternative splicing, multiple protein products differing in their C-terminal sequence. Products of the Kv3.1 and Kv3.2 genes express similar delayed-rectifier type currents in heterologous expression systems, while Kv3.3 and Kv3.4 proteins express A-type currents. All Kv3 currents activate relatively fast at voltages more positive than -10 mV, and deactivate very fast. The distribution of Kv3 mRNAs in the rodent CNS was studied by in situ hybridization, and the localization of Kv3.1 and Kv3.2 proteins has been studied by immunohistochemistry. Most Kv3.2 mRNAs (approximately 90%) are present in thalamic-relay neurons throughout the dorsal thalamus. The protein is expressed mainly in the axons and terminals of these neurons. Kv3.2 channels are thought to be important for thalamocortical signal transmission. Kv3.1 and Kv3.2 proteins are coexpressed in some neuronal populations such as in fast-spiking interneurons of the cortex and hippocampus, and neurons in the globus pallidus. Coprecipitation studies suggest that in these cells the two types of protein form heteromeric channels. Kv3 proteins appear to mediate, in native neurons, similar currents to those seen in heterologous expression systems. The activation voltage and fast deactivation rates are believed to allow these channels to help repolarize action potentials fast without affecting the threshold for action potential generation. The fast deactivating current generates a quickly recovering after hyperpolarization, thus maximizing the rate of recovery of Na+ channel inactivation without contributing to an increase in the duration of the refractory period. These properties are believed to contribute to the ability of neurons to fire at high frequencies and to help regulate the fidelity of synaptic transmission. Experimental evidence has now become available showing that Kv3.1-Kv3.2 channels play critical roles in the generation of fast-spiking properties in cortical GABAergic interneurons.

Pharmacologic characterization of imidazoline receptor proteins identified by immunologic techniques and other methods.

Biochemical and pharmacologic evidence supports the heterogeneous nature of imidazoline receptors (IRs). However, only monoamine oxidase (MAO) (55- and 61-kD) isozymes have been identified as imidazoline binding site-containing proteins. Idazoxan-binding proteins of approximately 70- and approximately 45-kD of unknown amino acid sequences have been isolated from chromaffin cells and rat brain, respectively. Other proteins of approximately 27-30 to > 80 kD have been visualized by immunologic and photoaffinity labeling techniques in different tissues and species. The specific antiserum that recognizes the approximately 70-, approximately 45-, and approximately 29-kD IR proteins, but not MAO, was used to quantitate these proteins in the rat brain cortex. Treatments (7 days) with the I2-selective imidazoline drugs idazoxan (10 mg/kg), cirazoline (1 mg/kg), and LSL 60101 ([2-(2-benzofuranyl) imidazole; 10 mg/kg]) induced differential changes in these proteins: levels of the approximately 29-kD IR were increased by idazoxan and LSL 60101 (23%), levels of the approximately 45-kD protein only by cirazoline (44%), and those of the approximately 66-kD protein only by idazoxan (50%). These treatments also increased the densities of [3H]-idazoxan (I2) binding sites (32-42%). Chronic treatment with efaroxan, RX821002, and yohimbine (10 mg/kg), which possess very low affinity for I2-IRs, did not alter either their immunoreactivities or the density of I2 sites. Chronic treatment with MAO inhibitors clorgyline and phenelzine (10 mg/kg) and acute treatment with EEDQ (1.6 mg/kg, 6 h) induced decreases in the levels of these IR proteins (17-47%) and I2 sites (31-57%). Significant correlations were found when the mean percentage changes in immunoreactivity of IR proteins were related to the mean percentage changes in the density of I2 sites after treatment with the foregoing drug (r = 0.92, r = 0.69, and r = 0.75 for the approximately 29-, approximately 45-, and approximately 66-kD proteins, respectively). These results indicate that in the rat cerebral cortex, the I2 sites labeled by [3H]idazoxan are heterogeneous and that the related immunoreactive IR proteins contribute differently to the modulation of I2 sites after drug treatment.

The alkylating agent EEDQ facilitates protease-mediated degradation of the human brain alpha2A-adrenoceptor as revealed by a sequence-specific antibody.

An antibody against a sequence from the divergent third intracellular loop of the human alpha2A-adrenoceptor, amino acids 262-276, was produced. The antiserum was tested, along with the preimmune serum, by means of ELISA and dot blot assays which demonstrated that the alpha2A-peptide used for the antibody production was recognized by the immune serum. The antibody also recognized the alpha2A-adrenoceptor protein in the human brain (immunoblot analysis). In cortical membranes a major immunoreactive peptide of approximately 70 kDa (mature glycosylated receptor) was detected and after treatment with N-glycosidase F only a approximately 50 kDa peptide (non-glycosylated receptor) was immunodetected. This antibody was used to demonstrate that a chemical modification of the alpha2A-adrenoceptor induced by the alkylating agent EEDQ facilitates the protease-mediated receptor degradation. Thus in vitro, normal receptor degradation (24-44% at 2-4 h) was enhanced by EEDQ (10(-6) M) (38-71% at 2-4 h) but in the presence of protease inhibitors this effect was almost abolished.

Up-regulation of immunolabeled alpha2A-adrenoceptors, Gi coupling proteins, and regulatory receptor kinases in the prefrontal cortex of depressed suicides.

Suicide and depression are associated with an increased density of alpha2-adrenoceptors (radioligand receptor binding) in specific regions of the human brain. The function of these inhibitory receptors involves various regulatory proteins (Gi coupling proteins and G protein-coupled receptor kinases, GRKs), which work in concert with the receptors. In this study we quantitated in parallel the levels of immunolabeled alpha2A-adrenoceptors and associated regulatory proteins in brains of suicide and depressed suicide victims. Specimens of the prefrontal cortex (Brodmann area 9) were collected from 51 suicide victims and 31 control subjects. Levels of alpha2A-adrenoceptors, Galphai1/2 proteins, and GRK 2/3 were assessed by immunoblotting techniques by using specific polyclonal antisera and the immunoreactive proteins were quantitated by densitometry. Increased levels of alpha2A-adrenoceptors (31-40%), Galphai1/2 proteins (42-63%), and membrane-associated GRK 2/3 (24-32%) were found in the prefrontal cortex of suicide victims and antidepressant-free depressed suicide victims. There were significant correlations between the levels of GRK 2/3 (dependent variable) and those of alpha2A-adrenoceptors and Galphai1/2 proteins (independent variables) in the same brain samples of suicide victims (r = 0.56, p = 0.008) and depressed suicide victims (r = 0.54, p = 0.041). Antemortem antidepressant treatment was associated with a significant reduction in the levels of Galphai1/2 proteins (32%), but with modest decreases in the levels of alpha2A-adrenoceptors (6%) and GRK 2/3 (18%) in brains of depressed suicide victims. The increased levels in concert of alpha2A-adrenoceptors, Galphai1/2 proteins, and GRK 2/3 in brains of depressed suicide victims support the existence of supersensitive alpha2A-adrenoceptors in subjects with major depression.

Density of imidazoline receptors in platelets of euthymic patients with bipolar affective disorder and in brains of lithium-treated rats.

BACKGROUND: Platelet imidazoline receptors have been shown to be up-regulated in patients with unipolar major depression. This study examines the status of imidazoline receptor proteins in platelets of euthymic bipolar patients and in brains of lithium-treated rats. METHODS: Platelets were collected from 12 bipolar patients (lithium-treated or drug-free) and brains from chronic lithium-treated rats. Imidazoline receptors were quantitated by immunoblotting, using a specific antiserum, and/or radioligand binding. RESULTS: No changes in platelet imidazoline receptors (35-kDa and 45-kDa proteins) were found. Lithium treatment did not alter brain imidazoline receptors (29/30-kDa, 45-kDa, and 66-kDa proteins or density/affinity of [3H]-idazoxan binding sites). CONCLUSIONS: Imidazoline receptor proteins are not altered in platelets of euthymic patients with bipolar affective disorder.

Regulation of G protein-coupled receptor kinase 2 in brains of opiate-treated rats and human opiate addicts.

The effects of opiate drugs (heroin, morphine, and methadone) on the levels of G protein-coupled receptor kinase 2 (GRK2) were studied in rat and human brain frontal cortices. The density of brain GRK2 was measured by immunoblot assays in acute and chronic opiate-treated rats as well as in opiate-dependent rats after spontaneous or naloxone-precipitated withdrawal and in human opiate addicts who had died of an opiate overdose. In postmortem brains from human addicts, total GRK2 immunoreactivity was not changed significantly, but the level of the membrane-associated kinase was modestly but significantly increased (12%) compared with matched controls. In rats treated chronically with morphine or methadone modest increases of the enzyme levels (only significant after methadone) were observed. Acute treatments with morphine and methadone induced dose- and time-dependent increases (8-22%) in total GRK2 concentrations [higher increases were observed for the membrane-associated enzyme (46%)]. Spontaneous and naloxone-precipitated withdrawal after chronic morphine or methadone induced a marked up-regulation in the levels of total GRK2 in the rat frontal cortex (18-25%). These results suggest that GRK2 is involved in the short-term regulation of mu-opioid receptors in vivo and that the activity of this regulatory kinase in brain could have a relevant role in opiate tolerance, dependence, and withdrawal.

Role of lipid polymorphism in G protein-membrane interactions: nonlamellar-prone phospholipids and peripheral protein binding to membranes.

Heterotrimeric G proteins (peripheral proteins) conduct signals from membrane receptors (integral proteins) to regulatory proteins localized to various cellular compartments. They are in excess over any G protein-coupled receptor type on the cell membrane, which is necessary for signal amplification. These facts account for the large number of G protein molecules bound to membrane lipids. Thus, the protein-lipid interactions are crucial for their cellular localization, and consequently for signal transduction. In this work, the binding of G protein subunits to model membranes (liposomes), formed with defined membrane lipids, has been studied. It is shown that although G protein alpha-subunits were able to bind to lipid bilayers, the presence of nonlamellar-prone phospholipids (phosphatidylethanolamines) enhanced their binding to model membranes. This mechanism also appears to be used by other (structurally and functionally unrelated) peripheral proteins, such as protein kinase C and the insect protein apolipophorin III, indicating that it could constitute a general mode of protein-lipid interactions, relevant in the activity and translocation of some peripheral (amphitropic) proteins from soluble to particulate compartments. Other factors, such as the presence of cholesterol or the vesicle surface charge, also modulated the binding of the G protein subunits to lipid bilayers. Conversely, the binding of G protein-coupled receptor kinase 2 and the G protein beta-subunit to liposomes was not increased by hexagonally prone lipids. Their distinct interactions with membrane lipids may, in part, explain the different cellular localizations of all of these proteins during the signaling process.

Inhibition of monoamine oxidase A and B activities by imidazol(ine)/guanidine drugs, nature of the interaction and distinction from I2-imidazoline receptors in rat liver.

1. I2-Imidazoline sites ([3H]-idazoxan binding) have been identified on monoamine oxidase (MAO) and proposed to modulate the activity of the enzyme through an allosteric inhibitory mechanism (Tesson et al., 1995). The main aim of this study was to assess the inhibitory effects and nature of the inhibition of imidazol(ine)/guanidine drugs on rat liver MAO-A and MAO-B isoforms and to compare their inhibitory potencies with their affinities for the sites labelled by [3H]-clonidine in the same tissue. 2. Competition for [3H]-clonidine binding in rat liver mitochondrial fractions by imidazol(ine)/guanidine compounds revealed that the pharmacological profile of the interaction (2-styryl-2-imidazoline, LSL 61112 > idazoxan > 2-benzofuranyl-2-imidazoline, 2-BFI = cirazoline > guanabenz > oxymetazoline > > clonidine) was typical of that for I2-sites. 3. Clonidine inhibited rat liver MAO-A and MAO-B activities with very low potency (IC50S: 700 microM and 6 mM, respectively) and displayed the typical pattern of competitive enzyme inhibition (lineweaver-Burk plots: increased K(m) and unchanged Vmax values). Other imidazol(ine)/guanidine drugs also were weak MAO inhibitors with the exception of guanabenz, 2-BFI and cirazoline on MAO-A (IC50S: 4-11 microM) and 2-benzofuranyl-2-imidazol (LSL 60101) on MAO-B (IC50: 16 microM). Idazoxan was a full inhibitor although with rather low potency, on both MAO-A and MAO-B isoenzymes (IC50S: 280 microM and 624 microM, respectively). Kinetic analyses of MAO-A inhibition by these drugs revealed that the interactions were competitive. For the same drugs acting on MAO-B the interactions were of the mixed type inhibition (increased K(m) and decreased Vmax values), although the greater inhibitory effects on the apparent value of Vmax/K(m) than on the Vmax value indicated that the competitive element of the MAO-B inhibition predominated. 4. Competition for [3H]-Ro 41-1049 binding to MAO-A or [3H]-Ro 19-6327 binding to MAO-B in rat liver mitochondrial fractions by imidazol(ine)/guanidine compounds revealed that the drug inhibition constants (Ki values) were similar to the IC50 values displayed for the inhibition of MAO-A or MAO-B activities In fact, very good correlations were obtained when the affinities of drugs at MAO-A or MAO-B catalytic sites were correlated with their potencies in inhibiting MAO-A (r = 0.92) or MAO-B (r = 0.99) activity. This further suggested a direct drug interaction with the catalytic sites of MAO-A and MAO-B isoforms. 5. No significant correlations were found when the potencies of imidazol(ine)/guanidine drugs at the high affinity site (pKiH, nanomolar range) or the low-affinity site (pKiL, micromolar range) of I2-imidazoline receptors labelled with [3H]-clonidine were correlated with the pIC50 values of the same drugs for inhibition of MAO-A or MAO-B activity. These discrepancies indicated that I2-imidazoline receptors are not directly related to the site of action of these drugs on MAO activity in rat liver mitochondrial fractions. 6. Although these studies cannot exclude the presence of additional binding sites on MAO that do not affect the activity of the enzyme, they would suggest that I2-imidazoline receptors represent molecular species that are distinct from MAO.

Pharmacological modulation of immunoreactive imidazoline receptor proteins in rat brain: relationship with non-adrenoceptor [3H]-idazoxan binding sites.

1. The densities of various imidazoline receptor proteins (with apparent molecular masses of approximately 29/30-45- and 66-kDa) were quantitated by immunoblotting in the rat cerebral cortex after various drug treatments. The modulation of these imidazoline receptor proteins was then compared with the changes in the density of non-adrenoceptor [3H]-idazoxan binding sites (I2-sites) induced by the same drug treatments. 2. Chronic treatment (7 days) with the I2-selective imidazol(in)e drugs idazoxan (10 mg kg-1), cirazoline (1 mg kg-1) and LSL 60101 (10 mg kg-1) differentially increased the immunoreactivity of imidazoline receptor proteins. The levels of the 29/30-kDa protein were increased by idazoxan and LSL 60101 (23%), the levels of the 45-kDa protein only by cirazoline (44%) and those of the 66-kDa protein only by idazoxan (50%). These drug treatments also increased the density of I2-sites (32-42%). 3. Chronic treatment (7 days) with efaroxan (10 mg kg-1), RX821002 (10 mg kg-1) and yohimbine (10 mg kg-1), which possess very low affinity for I2-imidazoline receptors, did not alter either the immunoreactivity of imidazoline receptor proteins or the density of I2-sites. 4. Chronic treatment (7 days) with the monoamine oxidase (MAO) inhibitors clorgyline (10 mg kg-1) and phenelzine (10 mg kg-1) decreased the immunoreactivity of the 29/30-kDa (17-24%), 45-kDa (19%) and 66-kDa (23-31%) imidazoline receptor proteins. The alkylating agent N-ethoxycarbonyl-2-ethoxy-1, 2-dihydroquinoline (1.6 mg kg-1, 6 h) also decreased the levels of the three imidazoline receptor proteins (20-47%). These drug treatments consistently decreased the density of I2-sites (31-57%). 5. Significant correlations were found when the mean percentage changes in immunoreactivity of imidazoline receptor proteins were related to the mean percentage changes in the density of I2-sites after the various drug treatments (r = 0.92 for the 29/30-kDa protein, r = 0.69 for the 45-kDa protein and r = 0.75 for the 66-kDa protein). 6. In the rat cerebral cortex the I2-imidazoline receptor labelled by [3H]-idazoxan is heterogeneous in nature and the related imidazoline receptor proteins (29/30-, 45- and 66-kDa) detected by immunoblotting contribute differentially to the modulation of I2-sites after drug treatment.