Localization of sulfonylurea receptor subunits, SUR2A and SUR2B, in rat heart.

To understand the possible functions and subcellular localizations of sulfonylurea receptors (SURs) in cardiac muscle, polyclonal anti-SUR2A and anti-SUR2B antisera were raised. Immunoblots revealed both SUR2A and SUR2B expression in mitochondrial fractions of rat heart and other cellular fractions such as microsomes and cell membranes. Immunostaining detected ubiquitous expression of both SUR2A and SUR2B in rat heart in the atria, ventricles, interatrial and interventricular septa, and smooth muscles and endothelia of the coronary arteries. Electron microscopy revealed SUR2A immunoreactivity in the cell membrane, endoplasmic reticulum (ER), and mitochondria. SUR2B immunoreactivity was mainly localized in the mitochondria as well as in the ER and cell membrane. Thus, SUR2A and SUR2B are not only the regulatory subunits of sarcolemmal K(ATP) channels but may also function as regulatory subunits in mitochondrial K(ATP) channels and play important roles in cardioprotection.

Mobility of lymphocyte surface membrane concanavalin A receptors of normal and feline leukemia virus-infected viremic felines.

The virus-associated depression of concanavalin A mitogenesis which accompanies feline leukemia virus-induced cat lymphoma was investigated by comparing lymphocyte surface receptor mobility of normal cats to that of viremic diseased animals. The mechanics of feline lymphocyte receptor mobility were studied using fluorescein-conjugated concanavalin A to quantitate lymphocyte capping. The results of a study of 21 disease-free animals showed that cat lymphocytes undergo appreciable concanavalin A capping, with a mean capping rate of 17% under conditions developed in this study. In contrast, morphologically normal peripheral blood lymphocytes of six feline leukemia virus-infected viremic cats, with or without lymphoma, exhibited a mean capping of only 7%, significantly less than that of the control animals (p less than 0.005). These findings suggest that a membrane-related lymphocyte deficiency accompanies the development of virus-induced lymphoma in the cat.

Resiniferatoxin induces paradoxical changes in thermal and mechanical sensitivities in rats: mechanism of action.

Resiniferatoxin (RTX), an ultrapotent analog of capsaicin, has been used as a tool to study the role of capsaicin-sensitive C fibers in pain. Recently, we found that RTX diminished the thermal sensitivity but unexpectedly increased the sensitivity to tactile stimulation in adult rats. In this study, we explored the potential mechanisms involved in RTX-induced changes in somatosensory function. An intraperitoneal injection of 200 microg/kg RTX, but not its vehicle, rapidly produced an increase in the paw withdrawal latency to a heat stimulus. Also, profound tactile allodynia developed in all the RTX-treated rats in 3 weeks. This paradoxical change in thermal and mechanical sensitivities lasted for at least 6 weeks. Electron microscopic examination of the sciatic nerve revealed a loss of unmyelinated fibers and extensive ultrastructural damage of myelinated fibers in RTX-treated rats. Immunofluorescence labeling showed a diminished vanilloid receptor 1 immunoreactivity in dorsal root ganglia neurons and the spinal dorsal horn of RTX-treated rats. Furthermore, two transganglionic tracers, horseradish peroxidase conjugates of cholera toxin B subunit (CTB) and isolectin-B(4) of Bandeiraea simplicifolia (IB(4)), were injected into the opposite sides of the sciatic nerve to trace myelinated and unmyelinated afferent terminations, respectively, in the spinal dorsal horn. In RTX-treated rats, IB(4)-labeled terminals in the dorsal horn were significantly reduced, and CTB-labeled terminals appeared to sprout into lamina II of the spinal dorsal horn. Thus, this study demonstrates that systemic RTX diminishes the thermal pain sensitivity by depletion of unmyelinated afferent neurons. The delayed tactile allodynia induced by RTX is likely attributable to damage to myelinated afferent fibers and their abnormal sprouting in lamina II of the spinal dorsal horn. These data provide new insights into the potential mechanisms of postherpetic neuralgia.

Regulation of the cAMP cascade, gene expression and immune function by cannabinoid receptors.

The objective of this article is to discuss the putative role of cannabinoid receptors in immune modulation by cannabinoid compounds. The primary focus is on the signal transduction events that are initiated following ligand binding to cannabinoid receptors and how these events lead to detrimental effects on the normal responsiveness of immunocompetent cells. Toward this end, signalling events are traced from the cannabinoid receptor to the transcription factors which are adversely regulated in the presence of cannabinoid compounds during leukocyte activation. Moreover, this aberrant regulation of transcription factors is discussed in the context of altered gene expression and the impact this has on leukocyte function. Lastly, an important goal of this article is to dispel a long standing myth that the cyclic adenosine 3':5'-monophosphate (cAMP) cascade is a negative regulatory pathway for immunocompetent cells. This chapter examines two major immunologic cell-types which are well established as exhibiting altered function following cannabinoid treatment, helper T-cells and the macrophage. Not discussed are the effects of cannabinoids on B-cell function. This is primarily due to the rather refractory nature of B-cells to inhibition by cannabinoids in spite of the fact that this cell-type expresses functional cannabinoid receptors [Schatz, A.R., Koh, W.S., Kaminski, N.E., 1993. Delta9-tetrahydrocannabinol selectively inhibits T-cell dependent humoral immune responses through direct inhibition of accessory T-cell function. Immunopharmacol., 26, pp. 129-137.]. One cautionary note, although the focus of this article is on cannabinoid receptor mediated signalling events, immune modulation by cannabinoid compounds is likely multi-factorial presumably involving receptor as well as receptor-nonrelated events. Effects on leukocytes by cannabinoids which are believed to be mediated by receptor-nonrelated events are outside the scope of this paper and will not be discussed. One last introductory point is that even though their is presumably little overlap in the genes which are regulated by cannabinoids in leukocytes as compared to other cell-types (e.g., neural cells), the major signalling pathways involved in cellular regulation are ubiquitous. With that in mind, it is likely that their is a considerable amount of similarity in the signalling pathways regulated by cannabinoids in cell-types of different lineage, given that they express cannabinoid receptors. In this context, signalling events observed in leukocytes can provide important insight into which genes may be modulated by cannabinoid in other cell types.

Expression of the CB1 cannabinoid receptor in macrophage-like cells from brain tissue: immunochemical characterization by fusion protein antibodies.

Antibodies designed to recognize a 74 amino acid sequence of the N- or C-terminal domain of the rat CB1 cannabinoid receptor detected a 58 kDa protein in immunoblots of brain and various cells known to express the CB1 cannabinoid receptor. A human B-lymphoblastoid cell line and macrophage-like cells from neonatal rat brain were also positive for CB1 receptor-like immunoreactivity. Immunocytochemical analysis performed with isolated Fab fragments showed surface staining in NG108-15 cells and brain macrophage like cells which also express MHC class II antigens. The data suggest a plausible role for CB1 receptors in the immune function of brain.

In vivo and in vitro treatment with the synthetic cannabinoid CP55, 940 decreases the in vitro migration of macrophages in the rat: involvement of both CB1 and CB2 receptors.

Cannabinoids have been shown to affect immune responses, acting on different populations of immune cells. In the present paper we analyze the ability of in vivo and in vitro treatment with the potent synthetic cannabinoid CP55,940 to interfere with an important function of rat peritoneal macrophages, i.e. spontaneous migration and formyl-metionyl-leucine-phenylalanine (fMLP)-induced chemotaxis, that were assessed by the use of a Boyden-modified microchemotaxis chamber. When added in vitro, CP55,940 induced a significant and dose-dependent inhibition of both spontaneous migration and fMLP-induced chemotaxis. Both the Cannabinoid Receptor 1 (CB1) and the Cannabinoid Receptor 2 (CB2) antagonists were able to block the CP55,940-induced inhibition of spontaneous migration, although the CB2 antagonist was more potent and only the CB2 antagonist was able to reverse the effect of CP55,940 on fMLP-induced chemotaxis. Similarly, in the in vivo experiments, 1 h after the acute subcutaneous administration of 0.4 mg/kg of CP55,940, both spontaneous motility and chemotaxis were reduced. The pretreatment with the CB2 antagonist, but not with the CB1 antagonist, was able to prevent this effect. Our data confirm that cannabinoids can affect some macrophage functions, mainly throughout CB2 receptors, and suggest that the development of specific CB2 ligands may lead to an interesting new class of anti-inflammatory drugs.

Anti-CD40, anti-CD3, and IL-2 stimulation induce contrasting changes in CB1 mRNA expression in mouse splenocytes.

The expression and function of cannabinoid receptor 1 (CB1) in mouse immune cells is unclear. Here we show that splenic B cells express more CB1 mRNA than T cells. Furthermore, splenocytes stimulated with the T cell mitogens, PMA/Io and anti-CD3, showed a decrease in CB1 message while cultures stimulated with the B cell mitogen, anti-CD40 antibody, showed an increase in message. In addition, co-treatment with mitogens and IL-2 uniformly caused an increase in CB1 mRNA. It is suggested that signaling pathways activated by T cell mitogens lead to decreased CB1 gene activation while pathways activated by B cell mitogens and IL-2 lead to increased CB1.

Delta 9-Tetrahydrocannabinol regulates Th1/Th2 cytokine balance in activated human T cells.

Human leukocytes express cannabinoid (CB) receptors, suggesting a role for both endogenous ligands and Delta 9-tetrahydrocannabinol (THC) as immune modulators. To evaluate this, human T cells were stimulated with allogeneic dendritic cells (DC) in the presence or absence of THC (0.625-5 microg/ml). THC suppressed T cell proliferation, inhibited the production of interferon-gamma and shifted the balance of T helper 1 (Th1)/T helper 2 (Th2) cytokines. Intracellular cytokine staining demonstrated that THC reduced both the percentage and mean fluorescence intensity of activated T cells capable of producing interferon-gamma, with variable effects on the number of T cells capable of producing interleukin-4. Exposure to THC also decreased steady-state levels of mRNA encoding for Th1 cytokines, while increasing mRNA levels for Th2 cytokines. The CB2 receptor antagonist, SR144528, abrogated the majority of these effects. We conclude that cannabinoids have the potential to regulate the activation and balance of human Th1/Th2 cells by a CB2 receptor-dependent pathway.

Localization of the CB1 type cannabinoid receptor in the rat basolateral amygdala: high concentrations in a subpopulation of cholecystokinin-containing interneurons.

The neuronal localization of the CB1 cannabinoid receptor in the rat basolateral amygdala was studied using peroxidase and fluorescence immunohistochemical techniques. All nuclei of the basolateral amygdala contained a large number of lightly stained pyramidal neurons and a small number of more intensely stained non-pyramidal neurons. Most of the latter cells had medium-sized to large multipolar somata and three to four aspiny dendrites, but some exhibited smaller oval somata. The axon initial segments of some of these non-pyramidal neurons exhibited large swollen varicosities in colchicine-injected animals, suggesting that much of the CB1 receptor protein is transported down the axons of these cells. Double-labeling studies using immunofluorescence histochemistry combined with confocal laser scanning microscopy revealed that the great majority of non-pyramidal neurons with CB1 receptor immunoreactivity belonged to a cholecystokinin-containing subpopulation. Whereas none of the other subpopulations of non-pyramidal neurons (exhibiting immunoreactivity for calretinin, parvalbumin, or somatostatin) expressed high levels of CB1 receptor immunoreactivity, a small percentage of these cells exhibited low levels of immunoreactivity. The results indicate that cannabinoids may modulate the activity of pyramidal projection neurons as well as a subpopulation of cholecystokinin-containing non-pyramidal neurons in the basolateral amygdala. Previous studies indicate that most of the latter are inhibitory interneurons that utilize GABA as a neurotransmitter. The intense staining of the cholecystokinin-containing interneurons and the evidence that large amounts of CB1 receptor protein are transported down the axons of these cells suggests that, as in the hippocampus, cannabinoids may inhibit the release of GABA from the axon terminals of these neurons.

Role of imidazoline receptors in cardiovascular regulation.

The involvement of nonadrenergic imidazoline specific receptors in the central control of the vasomotor tone and in the mechanism of action of drugs bearing an imidazoline structure, or analogs, is now well documented. Imidazoline-specific binding sites were found in many tissues and species. Moreover, until now, it is only in the brainstem that such binding sites are associated with a function: the hypotensive effect of imidazoline-like drugs. Rilmenidine, which is an oxazoline structurally related to the reference imidazolines, exerts a central hypotensive effect of central origin involving imidazoline receptors. The selectivity of rilmenidine for the imidazoline receptors compared to alpha 2-adrenergic receptors could explain the low incidence of sedative side effects observed with this antihypertensive drug. A specific anti-imidazoline radioimmunoassay allowed us to detect the presence of an immunoreactive imidazoline-like substance in human sera. High levels of this immunoreactive substance are associated with high blood pressure in 20-30% of the hypertensive patients. This observation indicates that high levels of this immunoreactive substance in the serum can be associated with some kinds of primary hypertension. The cause-and-effect relation between these 2 phenomena has not yet been determined. This substance is in process of purification; it could be a candidate to be an endogenous ligand of the imidazoline receptors.

Co-detection by two imidazoline receptor protein antisera of a novel 85 kilodalton protein.

Imidazoline receptors (I-receptors) are considered as potential therapeutic targets for a spectrum of stress-induced illnesses. Yet, I-receptors remain poorly defined at the molecular level. In this study, candidate imidazoline receptor proteins were compared using two imidazoline receptor-selective antisera of diverse origins. One antiserum was derived from affinity-purified imidazoline-binding protein. The second antiserum was produced as an anti-idiotypic antiserum, from purified IgG selective for imidazolines. Despite such diverse origins, both antisera co-identified an 85 kDa band on western blots from a variety of tissues. The integrity of the 85 kDa band was dependent on protection by eight different protease inhibitors. Other proteolytic breakdown products (obtained after homogenization with only one protease inhibitor) were comparable in size to previously reported smaller immunoreactive bands. The full-size 85 kDa band was also enriched in plasma membrane fractions and abundant in rat PC12 cells and brain regions known to be abundant in I1 binding sites. Furthermore, the immunodensity of the 85 kDa band, against anti-idiotypic antiserum, was linearly correlated with reported I1 site radioligand Bmax values (r2 = 0.8736, P = 0.0002) across nine rat tissues. Therefore, a possible candidate for the full-length imidazoline receptor(s) appears to be an 85 kDa protein.

Evidence for an endocannabinoid system in the central nervous system of the leech Hirudo medicinalis.

In invertebrates, like Hydra and sea urchins, evidence for a functional cannabinoid system was described. The partial characterization of a putative CB1 cannabinoid receptor in the leech Hirudo medicinalis led us to investigate the presence of a complete endogenous cannabinoid system in this organism. By using gas chromatography-mass spectrometry, we demonstrate the presence of the endocannabinoids anandamide (N-arachidonoylethanolamine, 21.5+/-0.7 pmol/g) and 2-arachidonoyl-glycerol (147.4+/-42.7 pmol/g), and of the biosynthetic precursor of anandamide, N-arachidonylphosphatidyl-ethanolamine (16.5+/-3.3 pmol/g), in the leech central nervous system (CNS). Anandamide-related molecules such as N-palmitoylethanolamine (32.4+/-1.6 pmol/g) and N-linolenoylethanolamine (5.8 pmol/g) were also detected. We also found an anandamide amidase activity in the leech CNS cytosolic fraction with a maximal activity at pH 7 and little sensitivity to typical fatty acid amide hydrolase (FAAH) inhibitors. Using an antiserum directed against the amidase signature sequence, we focused on the identification and the localization of the leech amidase. Firstly, leech nervous system protein extract was subjected to Western blot analysis, which showed three immunoreactive bands at ca. approximately 42, approximately 46 and approximately 66 kDa. The former and latter bands were very faint and were also detected in whole homogenates from the coelenterate Hydra vulgaris, where the presence of CB1-like receptors, endocannabinoids and a FAAH-like activity was reported previously. Secondly, amidase immunocytochemical detection revealed numerous immunoreactive neurons in the CNS of three species of leeches. In addition, we observed that leech amidase-like immunoreactivity matches to a certain extent with CB1-like immunoreactivity. Finally, we also found that stimulation by anandamide of this receptor leads, as in mammals, to inhibition of cAMP formation, although this effect appeared to be occurring through the previously described anandamide-induced and CB1-mediated activation of nitric oxide release. Taken together, these results suggest the existence of a complete and functional cannabinoid system in leeches.

Imidazoline receptor antisera-selected (IRAS) cDNA: cloning and characterization.

The imidazoline-1 receptor (IR1) is considered a novel target for drug discovery. Toward cloning an IR1, a truncated cDNA clone was isolated from a human hippocampal lambda gt11 cDNA expression library by relying on the selectivity of two antisera directed against candidate IR proteins. Amplification reactions were performed to extend the 5' and 3' ends of this cDNA, followed by end-to-end PCR and conventional cloning. The resultant 5131-basepair molecule, designated imidazoline receptor-antisera-selected (IRAS) cDNA, was shown to encode a 1504-amino acid protein (IRAS-1). No relation exists between the amino acid sequence of IRAS-1 and proteins known to bind imidazolines (e.g., it is not an alpha2-adrenoceptor or monoamine oxidase subtype). However, certain sequences within IRAS-1 are consistent with signaling motifs found in cytokine receptors, as previously suggested for an IR1. An acidic region in IRAS-1 having an amino acid sequence nearly identical to that of ryanodine receptors led to the demonstration that ruthenium red, a dye that binds the acidic region in ryanodine receptors, also stained IRAS-1 as a 167-kD band on SDS gels and inhibited radioligand binding of native I1 sites in untransfected PC-12 cells (a source of authentic I1 binding sites). Two epitope-selective antisera were also generated against IRAS-1, and both reacted with the same 167-kD band on Western blots. In a host-cell-specific manner, transfection of IRAS cDNA into Chinese hamster ovary cells led to high-affinity I1 binding sites by criteria of nanomolar affinity for moxonidine and rilmenidine. Thus, IRAS-1 is the first protein discovered with characteristics of an IR1.

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.

Imidazoline receptors in vascular smooth muscle and endothelial cells.

We sought to determine if smooth muscle and endothelial cells of blood vessels express imidazoline receptors. Membranes of cultured smooth muscle cells specifically bind with high affinity to alpha 2-adrenergic ligands, [3H]p-aminoclonidine, [3H]rauwolscine, and [3H]idazoxan. All of [3H]rauwolscine and [3H]p-aminoclonidine but less than 10% of [3H]idazoxan binding was displaced by 10 microM epinephrine, indicating a nonadrenergic binding site for [3H]idazoxan. [3H]Idazoxan binding was inhibited with a rank order of potency: cirazoline > idazoxan > naphazoline >> guanabenz > amiloride > clonidine = phentolamine. Agmatine, an endogenous ligand for I-receptors, inhibited binding with a Ki of 240 +/- 25 nM. The binding of [3H]idazoxan to membranes of pulmonary artery endothelial cells was to both alpha 2-adrenergic and imidazoline receptors. Cultured smooth muscle cells, as well as rat carotid arterioles, were specifically immunostained by antibodies to an I-receptor-associated protein. We conclude that vascular smooth muscle and endothelial cells express not only alpha 2-adrenergic receptors but also I-receptors of the I2 subclass with high affinity for agmatine. Since serum contains an endogenous ligand for I-receptors, possibly agmatine, the results suggest the presence of a novel receptor mechanism on vascular smooth muscle which may regulate vascular tone.

Imidazoline receptor antisera-selected cDNA clone and mRNA distribution.

A novel cDNA, designated Imidazoline Receptor Antisera-Selected cDNA-1 (iras-1), encodes a 167-kD protein. Two of its predicted peptides (42-43 kD) are immunologically consistent with a previously reported 1(1)-imidazoline binding protein. In the present study, two forms of iras mRNA (6.0 and 9.5 kb) were quantified across fresh rat tissues. Highest levels were found in brain (almost exclusively 6.0 kb in size), followed by liver and lung (9.5 > or = 6.0 kb iras mRNA), kidney (6.0 > 9.5 kb), heart (6.0 kb), spleen (6.0 > or = 9.5 kb), testes (6.0 > 9.5 kb), and skeletal muscle (6.0 > 9.5 kb). A correlation exists (p = 0.71, p = 0.05) between total (6.0 + 9.5 kb) iras mRNA and I1 BMAX values across rat tissues, corrected for housekeeping gene expression. Thus, total iras mRNA appears to be roughly proportional to the density of I1-imidazoline binding sites.

VR1-immunoreactive primary sensory neurons in the rat trigeminal ganglion.

Immunohistochemistry for VR1, a nociceptive transducer for vanilloid compounds, protons and heat (>43 degrees C), was performed on the rat trigeminal ganglion (TG). The immunoreactivity (IR) was detected in 20% of TG cells and these neurons were mostly small- to medium-sized (mean+/-S.D. 427+/-189 microm(2)). Twenty-six percent of the TG neurons retrogradely labeled from the facial skin exhibited VR1-IR, while the IR was detected in only 8% of those labeled from the tooth pulp. Co-expression of VR1 was common among the calcitonin gene-related peptide-immunoreactive cutaneous neurons (63%) but not among the similar tooth pulp neurons (20%). The present study indicates that primary nociceptive neurons which respond to vanilloid compounds, protons and heat are abundant in the facial skin but not in the tooth pulp.

Immunocytochemical localization of an imidazoline receptor protein in the central nervous system.

Imidazoline (I) receptors have been implicated in the regulation of arterial blood pressure and behavior although their distribution in the central nervous system (CNS) remains in question. Presumptive I- receptor sites were detected in the rat central nervous system with a polyclonal antibody to an imidazoline receptor protein (IRP) with binding characteristics of the native receptor. IRP-like immunoreactivity (LI) was detected in neurons and glia by light and electron microscopy. Spinal cord: processes were heavily labeled in superficial laminae I and II of the dorsal horn, lateral-cervical and -spinal nuclei and sympathetic cell column. Medulla: label was concentrated in the area postrema, rostral, subpostremal and central subnuclei of nucleus tractus solitarii, spinal trigeminal nucleus caudalis, and inferior olivary subnuclei. Visceromotor neurons in the dorsal vagal and ambigual nuclei were surrounded by high concentrations of immunoreactive processes. In reticular formation, label was light, though predominant in the intermediate reticular zone and ventrolateral medulla. Pons: label was detected in the neuropil of the periventricular gray, concentrated in the dorsal- and external-lateral subnuclei of lateral parabrachial nucleus, and present intracellularly in the mesencephalic trigeminal nucleus. Midbrain: IRP-LI was most heavily concentrated in the interpeduncular nucleus, nuclei interfascicularis and rostral-linearis, the subcommissural organ, central gray, and in glia surrounding the cerebral aqueduct. Diencephalon: high densities were detected in the medial habenular nucleus, nucleus paraventricularis thalami, other midline-intralaminar thalamic nuclei, the supramammillary and mediobasal hypothalamic nuclei. In the median eminence, immunolabeled processes were restricted to the lamina interna and lateral subependymal zone. Telencephalon: IRP-LI was concentrated in the central amygdaloid nucleus, bed nucleus of stria terminalis and globus pallidus, followed by moderate labeling of the medial amygdaloid nucleus, amygdalostriatal zone and caudoputamen, the hilus of the dentate gyrus, and stratum lacunosum-moleculare of field CA1 of Ammon's horn. The subfornical organ and organum vasculosum lamina terminalis were filled with diffuse granular immunoreactivity. Ultrastructural studies identified IRP-LI within glia and neurons including presynaptic processes. I-receptor(s) localize to a highly restricted network of neurons in the CNS and circumventricular regions lying outside of the blood-brain barrier. Putative imidazoline receptors have a unique distribution pattern, show partial overlap with alpha 2 adrenoreceptors and are heavily represented in sensory processing centers and the visceral nervous system.

Polyclonal anti-idazoxan antibodies: characterization and purification.

Amino-idazoxan coupled to hemocyanine was used to raise anti-idazoxan antibodies in the rabbit. The antibodies were affinity purified with an amino-idazoxan affinity column. Binding studies with [3H]idazoxan showed a dissociation constant of 2.2 +/- 1.4 nM. The specificity spectrum of these antibodies indicates that the imidazoline part of idazoxan is more important for recognition than the benzodioxan ring as imidazoline substances (clonidine, cirazoline) are powerful competitors of [3H]idazoxan binding on the antibodies. Catecholamines or imidazoles were unable to displace [3H]idazoxan from the antibodies. These anti-idazoxan antibodies present specificity similarities with the imidazoline receptor as did our previously obtained anti-clonidine antibodies. Affinity-purified antibodies represent useful tools for studying the imidazoline receptors particularly with an anti-idiotypic approach.

Role of vanilloid VR1 receptor in thermal allodynia and hyperalgesia in diabetic mice.

We examined the role of the vanilloid VR1 receptor in the thermal hyperalgesia and allodynia seen in diabetic mice. Tail-flick latencies at source voltages of 35 and 50 V for a 50-W projection bulb in diabetic mice were significantly shorter than those in non-diabetic mice. Tail-flick latencies at 35 and 50 V in diabetic mice were increased by pretreatment with anti-vanilloid VR1 receptor serum. Intrathecal (i.t.) injection of anti-VR1 serum resulted in a significant increase in the tail-flick latency at 50 V in non-diabetic mice. However, i.t. pretreatment with anti-vanilloid VR1 receptor serum did not affect the tail-flick latency at a heat intensity of 35 V in non-diabetic mice. Thus, it seems likely that thermal allodynia and hyperalgesia in diabetic mice may be due to the sensitization of vanilloid VR1 receptors in primary sensory neurons in the spinal cord.