Autoradiographic demonstration of increased serotonin 5-HT2 and beta-adrenergic receptor binding sites in the brain of suicide victims.

Suicidal behavior has been linked to a deficiency in serotonin neurotransmission, but it is not known which brain regions are involved. We determined the pattern of alteration in serotonin 5-HT2 (5-HT2) receptor binding sites in suicide victims in prefrontal cortex compared with temporal cortex using a matched-pairs design to study 11 suicide victims and 11 matched controls, by both membrane binding and quantitative receptor autoradiography. Since a relationship between the serotonergic and noradrenergic systems has been proposed, we also examined beta-adrenergic receptor binding sites. Binding to 5-HT2 and beta-adrenergic sites in slide-mounted sections correlated strongly with binding site number in membrane preparations. A specific laminar distribution of 5-HT2 binding sites was found in both the control and suicide groups, whereas beta-adrenergic binding sites did not differ across cortical layers. A significant increase was found in suicide victims across all cortical layers in both receptor subpopulations in the prefrontal cortex, but only beta-adrenergic sites were increased in the temporal cortex. We conclude that suicide is associated with a localized increase in 5-HT2 binding sites.

A novel angiotensin receptor subtype in rat mesangium. Coupling to adenylyl cyclase.

The diversity of angiotensin II (Ang II) actions implies multiple receptor subtypes. To characterize these subtypes in rat mesangial cells, we used the angiotensin subtype 1A (AT1A) antagonist losartan (DuP 753), the subtype 2/1B (AT2/AT1B) antagonist PD 123319, and the AT2 antagonist CGP 42112A in radioreceptor and adenylyl cyclase assays. In radioligand binding competition experiments, approximately 25% of the specific binding sites labeled by 125I-[Sar1]Ang II were inhibited by low concentrations of PD 123319 (0.1 to 10 nM), whereas the AT2 antagonist CGP 42112A was inactive at concentrations less than 0.1 microM. Conversely, losartan inhibited 75% of the binding at low concentrations (0.1 nM to 0.1 microM), but higher concentrations (up to 10 microM) were required to inhibit the second component of 125I-[Sar1]Ang II binding. The effects of the different antagonists on the inhibition by Ang II of forskolin-stimulated cyclic AMP production were also analyzed. Ang II inhibited forskolin-stimulated adenylyl cyclase in a concentration-dependent fashion (IC50, 35 +/- 7 nM), and the maximal inhibition of adenylyl cyclase was 44 +/- 2%. In the radioligand binding experiments, both losartan and PD 123319 antagonized the inhibition of adenylyl cyclase elicited by 0.1 microM Ang II (IC50, 0.5 +/- 0.2 and 1.2 +/- 0.4 microM, respectively), whereas CGP 42112A was less potent (IC50, 5.7 +/- 1.6 microM). Comparison of binding affinities at AT1B receptor sites with antagonist potencies in the adenylyl cyclase assay show good agreement for losartan and CGP 42112A, whereas PD 123319 is less potent than expected from membrane binding assays, possibly because of partial agonist properties.(ABSTRACT TRUNCATED AT 250 WORDS)

Refeeding hypertension in obese spontaneously hypertensive rats.

Very-low-calorie diets lower blood pressure acutely in obese humans and rats. However, refeeding after dietary restriction produces mild hypertension in rats. Refeeding hypertension was characterized in genetically obese spontaneously hypertensive rats (obese SHR, Koletsky rat), a model of genetic obesity and hypertension. Obese SHR were fed a restricted diet (Optifast) for 12 days, refed ad libitum for 28 days, dieted again for 12 days, and then refed 4 days and killed. Control obese SHR and lean SHR littermates were fed ad libitum continuously. Dietary restriction led to rapid weight loss followed by prompt regain to baseline weight after return to unrestricted food intake. Heart rate fell with institution of the low-calorie diet and returned to baseline on refeeding. Blood pressure became elevated during refeeding in dieted obese SHR relative to ad libitum fed obese SHR controls. The fall in blood pressure after ganglionic blockade with chlorisondamine was exaggerated in refed obese SHR, and cardiac beta-adrenergic receptors were downregulated. Both of these findings imply increased sympathetic tone. The left ventricular wall was thicker in the refed obese SHR than in the ad libitum fed obese SHR. Shorter cycles of weight loss and regain in lean SHR led to transient increases in blood pressure and heart rate. Cycles of dietary restriction and refeeding in obese SHR elicit sustained blood pressure elevation via sympathetic activation and exacerbate cardiac hypertrophy. Drastic fluctuations in nutrient intake may not be advantageous in hypertension.

Renal angiotensin receptor mapping in obese spontaneously hypertensive rats.

Obese spontaneously hypertensive rats (SHR) develop nephropathy with severe proteinuria, but lean littermates do not develop renal disease. Intrarenal angiotensin has been suggested to contribute to nephropathy in other experimental models. We examined the regulation of angiotensin receptors as a reflection of target tissue response to possible changes in the renin-angiotensin system. We visualized angiotensin receptors in kidneys of 6-8-month-old obese SHR and their lean littermates. Both obese and lean rats were hypertensive as determined by tail-cuff or by direct measurement. Histologic studies showed early glomerular sclerosis in obese but not lean rats. Autoradiographic visualization of angiotensin receptor binding sites in both obese and lean SHR showed glomeruli and medullary rays having the highest levels of binding with additional diffuse labeling in cortex and outer medulla. In obese rats, binding was reduced relative to lean littermates, particularly in the medulla, while intense binding in glomeruli was preserved. Loss of receptors did not reflect tissue damage, since the medulla showed no pathological changes. Biochemical assays of the binding of subtype-selective antagonists to 125I-angiotensin sites in intact sections showed that both losartan-sensitive and PD 123319-sensitive sites were decreased in nephrotic obese rats. We conclude that specific binding sites for angiotensin are decreased in obese SHR with early glomerular sclerosis, suggesting that angiotensin receptors may be regulated by pathogenic processes in this model of renal disease.

Clonidine-specific antisera recognize an endogenous clonidine-displacing substance in brain.

An endogenous substance in brain, clonidine-displacing substance, binds to the same receptor populations as clonidine and is biologically active. Since receptor binding sites can be modeled by using specific antiligand antibodies, we tested the hypothesis that polyclonal antibodies raised in rat and rabbit against the clonidine analog p-aminoclonidine coupled to hemocyanin would recognize compounds structurally related to clonidine, including clonidine-displacing substance. Binding to anti-p-aminoclonidine antibodies was examined by using a competitive radioimmunoassay with tritiated p-aminoclonidine as the radioligand. Central vasodepressor agents that, like clonidine, are known to bind with high affinity to both imidazole sites and alpha 2-adrenergic receptors in brain inhibited radioligand binding to anti-p-aminoclonidine antibodies. All of these agents contain imidazol(in)e and phenyl ring moieties as part of their chemical structures (e.g., oxymetazoline); a number of other compounds without one or both of these rings failed to cross-react with the antisera. Clonidine-displacing substance, partially purified from bovine brain, also inhibited specific radioligand binding to anti-p-aminoclonidine antibodies. The inhibition was dose dependent and high affinity (IC50, 4 Units). The endogenous substance had no effect on the apparent affinity of the antibodies for the radioligand, but blocked a specific number of binding sites. Immunoprecipitation experiments showed that authentic clonidine-displacing substance, that which displaces tritiated p-aminoclonidine binding to membrane receptors, is recognized by anti-p-aminoclonidine antibodies. We conclude that a unique subset of structural determinants required for ligand interaction with both imidazole and alpha 2-adrenergic receptors is critical for binding to anti-p-aminoclonidine antibodies, and that since clonidine-displacing substance is recognized by highly clonidine-specific antisera, it may also contain these determinants within its structure, namely the imidazol(in)e and phenyl ring systems.

Effects of weight cycling on urinary catecholamines: sympathoadrenal role in refeeding hypertension.

OBJECTIVES: We sought to determine whether the sympathetic nervous system plays a role in the hypertensive response to refeeding from a very low-calorie diet (VLCD). DESIGN: Cycles of weight loss and regain were induced in the obese spontaneously hypertensive rat (SHROB) model of genetic obese hypertension. METHODS: A 12-day VLCD (1/6 of baseline calories) was alternated with 4-6 weeks of ad libitum chow refeeding for three cycles. Control SHROB ate chow ad libitum. Urine was collected for 24 h before and after each period of VLCD, and catecholamines were measured radioenzymatically. Tail cuff blood pressures and body weight were measured in parallel with urine collections. Kidneys were collected for assay of alpha2-adrenergic receptor density. RESULTS: VLCD induced rapid weight loss, but all the lost weight was regained during refeeding. Blood pressure fell during caloric restriction, but rose above baseline during refeeding. Urinary excretion of norepinephrine, epinephrine and dopamine changed several fold during weight cycling. Urinary catecholamines paralleled the changes in blood pressure, falling during caloric restriction and rebounding during refeeding. Dopamine showed the greatest decreases during weight loss and rises during weight regain, whereas epinephrine changed the least and norepinephrine was intermediate. Weight cycling elevated blood pressure above the initial baseline throughout the rapid weight gain phase of refeeding. The density of alpha2-adrenergic receptors was decreased in both the renal medulla and cortex of weight cycled SHROB, consistent with receptor down-regulation owing to overstimulation. CONCLUSIONS: The exacerbations of hypertension during weight regain in SHROB coincide with sustained activation of sympathoadrenal activity, as reflected in urinary catecholamine excretion and adrenergic receptor down regulation.

Synthesis, binding properties, and 18F labeling of fluorocarazolol, a high-affinity beta-adrenergic receptor antagonist.

New beta-adrenergic receptor antagonists, 2-(R)-(+)- and 2-(S)-(-)-1-(9H-carbazol-4-yl-oxy)-3-[[1-(fluoromethyl)ethyl]amino ]-2-propanol ((S)- and (R)-fluorocarazolols), were labeled with fluorine-18 at the no-carrier-added level by reductive alkylation of desisopropylcarazolol (4-(2-hydroxy-3-amino-1-propoxy)carbazole) with [18F]fluoroacetone. The latter was prepared by nucleophilic substitution of fluoride on acetol tosylate and may serve as a useful synthetic precursor for other radiotracers. The radiochemical yield of [18F]fluorocarazolol (500-1200 Ci/mmol) from [18F]fluoride was 40 +/- 10% at the end of the 45 min synthesis. Chiral HPLC showed > 99% enantiomeric purity of 2-(S)- and 2-(R)-[18F]fluorocarazolols. The log P of fluorocarazolol was 2.2 at pH 7.4. The in vitro KD values of (S)- and (R)-fluorocarazolol for the beta-adrenergic receptor were measured in a rat heart preparation to be KD = 68 and 1128 pM, respectively. Biodistribution experiments in mice demonstrated specific beta-adrenergic receptor binding of (S)-[18F]fluorocarazolol. (R)-[18F]fluorocarazolol showed no observable specific binding to beta-receptors in vivo. The uptake of (R)-[18F]fluorocarazolol may therefore be used as an estimation of nonspecific binding. Positron emission tomography images of pigs showed receptor-specific uptake of (S)-[18F]fluorocarazolol in the heart and lung. Washout of dissociated ligand from the tissue was observed only after 70 min postinjection. The maximum ratio of specific to nonspecific uptake in pig heart and lung was ca. 10 at 150 min postinjection. Observed levels of fluorocarazolol metabolites in mouse and pig blood were relatively low and remained fairly constant during the period from 10 to 180 min postinjection. These results indicate that (S)-(-)-[18F]fluorocarazolol is of interest for use as a radiopharmaceutical for estimation of beta-adrenergic receptors with positron tomography.

Psychopharmacology of imidazoline and alpha 2-adrenergic receptors: implications for depression.

The literature on alpha 2-adrenoceptors in depression is replete with seemingly contradictory findings, including reports of both hypersensitive and hyposensitive alterations. Now, with the discovery of nonadrenergic imidazoline receptors (I receptors) and an endogenous I receptor ligand (agmatine), new light is being shed on this controversy. Specifically, those studies that had utilized allegedly "alpha 2-selective" imidazoline radioligands, i.e., 3H-clonidine, could be reinterpreted in terms of increased I receptors in depression. Although the molecular identity of the I1 binding site remains unknown, an I2 receptive site has been reported to be encoded by monoamine oxidase genes (both MAO-A and MAO-B), suggesting a novel explanation for the antidepressant efficacy of idazoxan, a prototypic I2 ligand. Platelet I1 binding sites are also reported to be elevated in patients with unipolar depression and are lowered by antidepressant treatments. Furthermore, clonidine challenge and animal studies of the behavioral effects of imidazolines may be reinterpreted to support a role for I1 sites in the central control of behavior. A hypothesis for depletion of brain clonidine-displacing substance (CDS) in depression is presented. Lowered concentrations of CDS could account for an elevation of I receptors, via compensatory upregulation. Our model offers an explanation for a number of previously discrepant observations as well as testable hypotheses for the study of imidazoline receptors in depression.

Evidence for a bioactive clonidine-displacing substance in peripheral tissues and serum.

Clonidine-displacing substance (CDS) from brain is biologically active in the kidney and stomach and on platelets. To determine whether CDS is contained in these and other peripheral tissues, homogenates of fresh brain, eight other organs and serum from rat were ultrafiltered (less than 10,000 mol. wt only), dried and extracted with methanol. Evaluation by radioimmunoassay (RIA) using antibodies to p-aminoclonidine showed that adrenal gland and gastric fundus (GF) contained significantly greater amounts of CDS-like radioimmunoactivity than brain; intermediate-to-low activity was present in heart, small intestine, serum, kidney and liver; lung and skeletal muscle values were near-background. RIA-positive extracts elicited well-correlated contractile activity in a GF smooth muscle bioassay; contractions persisted in the presence of antagonists of various transmitters and modulators, but were abolished by low concentrations of the calcium channel blocker verapamil. Serum levels of CDS were profoundly reduced following removal of the adrenal glands. We conclude that a CDS-like substance is present not only in brain as previously reported, but also in peripheral organs and in the circulation.

Effects of fasting and refeeding on blood pressure are determined by nutritional state, not by body weight change.

It is commonly assumed that caloric restriction is effective in lowering blood pressure because of the accompanying weight loss and reversal of obesity. However, clinical trials of caloric restriction reporting the greatest falls in blood pressure were those that produced the most rapid weight loss on diets allowing the fewest calories, but the amount of weight loss was unrelated to antihypertensive effect. In obese rats undergoing a supplemented fast, blood pressure fell almost immediately but then stabilized despite continuing weight loss. The depressor effect of fasting was reversed within 2 days of refeeding. Body weight change was no longer correlated with blood pressure change after nutritional state was controlled for. Nutritional state (fed, fasted, refed), but not body weight, has important effects on blood pressure.

Characterization and visualization of clonidine-sensitive imidazole sites in rat kidney which recognize clonidine-displacing substance.

In the ventrolateral medulla oblongata, clonidine binds not only to alpha 2-adrenergic receptors but also to a novel class of nonadrenergic sites that are specific for imidazoles. Since clonidine has direct actions on the kidney, we sought to determine whether imidazole binding sites could be detected in renal cell membranes. Adrenergic agents having an imidazole ring, like clonidine, completely displaced the specific binding of the high-affinity clonidine analog 3H-p-aminoclonidine (3H-PAC) to kidney membranes. Nonimidazole adrenergic agents inhibited 3H-PAC binding by only 75%, indicating that the remaining 25% of the sites were nonadrenergic. Cimetidine, an imidazole compound lacking adrenergic potency, showed a high affinity for approximately 25% of the sites. 3H-PAC binding to imidazole sites was high-affinity (KD = 11 +/- 3 nmol/L) and saturable (Bmax = 41 +/- 10 fmol/mg protein). Like clonidine, an endogenous clonidine-displacing substance (CDS) completely inhibited 3H-PAC binding to renal cortex membranes. Quantitative receptor autoradiography revealed that imidazole receptors exhibited a specific regional distribution within the kidney that was unique, and distinct from that of alpha 2-adrenergic receptors. We conclude that clonidine binds to specific imidazole sites in the renal cortex of the rat and that CDS may be the endogenous ligand at these sites. Thus, in addition to their role in central nervous system control of arterial pressure, imidazole receptors may be involved in the regulation of renal function.

Clonidine-displacing substance is present in peripheral tissues of the rat.

Clonidine-displacing substance (CDS) is biologically active in the brain, as well as the gastric fundus, platelets and vas deferens. We sought to determine whether CDS is contained within peripheral tissues in the rat. Using competitive radioimmunoassay with a clonidine-specific antiserum and 3H-p-aminoclonidine rat adrenal gland and gastric fundus were shown to contain significantly greater amounts of CDS-like radioimmunoactivity than the brain; intermediate-to-low activity was present in the heart, small intestine, serum, kidney and liver. Lung and skeletal muscle exhibited near-background levels. CDS may not be unique to the brain, but also may be synthesized and stored in peripheral organs.

The in vitro pharmacology of the beta-adrenergic receptor pet ligand (s)-fluorocarazolol reveals high affinity for cloned beta-adrenergic receptors and moderate affinity for the human 5-HT1A receptor.

RATIONALE: s-Fluorocarazolol [(S)-FCZ] is the major positron emission tomography (PET) ligand currently used to visualize central beta-adrenergic receptors in vivo, although its pharmacology is incompletely known. OBJECTIVE: Our objective was to comprehensively characterize the in vitro pharmacology of (S)- and (R)-FCZ to determine its suitability for study of central and peripheral beta-adrenergic receptors. METHODS: We characterized the in vitro pharmacology of (S)-FCZ at 42 biogenic amine receptors and transporters in vitro using the resources of the National Institute of Mental Health Psychoactive Drug Screening Program. RESULTS: As expected (R)- and (S)-FCZ had high affinities for beta-adrenergic receptors (Ki values=0.08-0.45 nM) and negligible affinities (Ki values>100 nM) for nearly all other tested receptors and transporters with the exception of the h5-HT1A receptor for which (S)-FCZ had high affinity (Ki=34 nM). Interestingly, (R)-FCZ had low affinity for the h5-HT1A receptor (Ki=342 nM). CONCLUSION: The high affinity of (S)-FCZ for the h5-HT1A receptor is not likely to interfere with studies of peripheral beta-adrenergic receptors, since 5-HT1A receptors are expressed at very low levels outside the central nervous system. Indeed, computer simulations predict that even at low ligand concentrations, 5-HT1A binding in brain regions like hippocampus are likely to be substantial. Thus, (S)-FCZ may not be a suitable PET ligand for studies of central nervous system beta-adrenergic receptors unless the contribution by 5-HT1A sites can be shown to be negligible.

Membrane localization and guanine nucleotide sensitivity of medullary I1-imidazoline binding sites.

Imidazoline binding sites are labeled by [3H]clonidine (I1) or by [3H]idazoxan (I2). I2-sites are mitochondrial. The subcellular localization of I1-sites in brain is unknown. Crude membranes from bovine rostral ventrolateral medulla (RVLM) were further purified by discontinuous sucrose density gradient. Fractions were assayed for I1-site density (Bmax) with [125I]p-iodoclonidine. Nonspecific binding was defined by 10 microM BDF-6143, and alpha 2-adrenergic binding was defined by 10 microM epinephrine. The proportions of I1 and alpha 2 in mitochondrial fractions were similar (28 +/- 3 and 24 +/- 4%, respectively), and both I1 and alpha 2 showed the greatest enrichment within the membrane-enriched fraction (58 +/- 13 and 38 +/- 4%). The myelin fraction contained a higher proportion of alpha 2 than I1 (38 +/- 4 and 15 +/- 2%), consistent with expression of alpha 2, but not I1, by glia. The enrichment of I1 and alpha 2 in cellular membranes and alpha 2 in myelin was confirmed by further purification of these fractions over a second discontinuous gradient. Following irreversible inactivation of alpha 2, the remaining I1 sites in RVLM crude membranes were inhibited by Gpp(NH)p but not by ATP. We conclude that I1-imidazoline sites are non-mitochondrial membrane proteins sensitive to guanine nucleotide and may be functional receptors.

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.

The I1-imidazoline receptor and its cellular signaling pathways.

Two primary questions are addressed. First, do I1-imidazoline binding sites fulfill all the essential criteria for identification as a true receptor? Second, what are the cellular signaling pathways coupled to this novel receptor? I1-imidazoline binding sites show specificity in binding assays, linkage to physiologic functions, appropriate anatomic, and cellular and subcellular localization. Most important, binding affinities correlate with functional drug responses. I1-imidazoline binding sites meet several additional criteria identified with functional receptors: they show physiologic regulation and endogenous ligands and, most crucially, are coupled to cellular signaling events. A series of studies have identified cellular events triggered by I1-imidazoline receptor occupancy. This receptor is not coupled to conventional pathways downstream of heterotrimeric G-proteins, such as activation or inhibition of adenylyl or guanylyl cyclases, stimulation of inositol phospholipid hydrolysis, or induction of rapid calcium fluxes. The I1-imidazoline receptor is coupled to choline phospholipid hydrolysis, leading to the generation of diacylglyceride, arachidonic acid, and eicosanoids. Additional cellular responses include inhibition of Na+/H+ exchange and induction of genes for catecholamine synthetic enzymes. The signaling pathways linked to the I1-imidazoline receptor are similar to those of the interleukin family, implying that I1-receptors may belong to the family of neurocytokine receptors.

Molecular pathology in the obese spontaneous hypertensive Koletsky rat: a model of syndrome X.

The SHROB rat is a unique strain with genetic obesity, hypertriglyceridemia, hyperinsulinemia, renal disease with proteinuria, and genetically determined hypertension, characteristics paralleling human Syndrome X. The obese phenotype results from a single homozygous recessive trait, designated faK, and is allelic with the Zucker fatty trait (fa), but of distinct origin. The faK mutation is a premature stop codon in the extracellular domain of the leptin receptor, resulting in a natural receptor knockout. The SHROB are glucose intolerant compared to heterozygous or wild-type SHR, but retain fasting euglycemia even on a high sucrose diet, suggesting that diabetes requires polygenic interaction with additional modifier genes. Insulin-stimulated phosphorylation of tyrosine residues on the insulin receptor and on the associated docking protein IRS-1 are reduced in skeletal muscle and liver compared to SHR, due mainly to diminished expression of insulin receptor and IRS-1 proteins. Despite multiple metabolic derangements and severe insulin resistance, hypertension is not exacerbated in SHROB compared to SHR. Thus, insulin resistance and hypertension are independent in this model. Increased activity of the sympathetic nervous system may be a common factor leading by separate pathways to hypertension and to insulin resistance. We studied the chronic effects of sympathetic inhibition with moxonidine on glucose metabolism in SHROB. Moxonidine (8 mg/kg/day), a selective I1-imidazoline receptor agonist, not only reduced blood pressure but also ameliorated glucose intolerance. Moxonidine reduced fasting insulin by 47% and plasma free fatty acids by 30%. Moxonidine enhanced expression and insulin-stimulated phosphorylation of IRS-1 in skeletal muscle by 74 and 27%, respectively. Thus, central sympatholytic therapy not only counters hypertension but also insulin resistance, glucose tolerance, and hyperlipidemia in the SHROB model of Syndrome X.

IRAS splice variants.

The human I(1)-imidazoline receptor candidate gene, iras, has previously been cloned and mapped to locus 3p21.1-9 (also known as Nischarin; accession No. AC006208). By comparison to a database of expressed sequence tags (ESTs), three alternatively spliced transcripts have been deduced. A map of 21 exons was constructed for the medium-length transcript (IRAS-M) containing 5,232 base pairs (bp) and encoding 1,504 amino acids (aas). Introns 13B and 13C are inserted into the two alternative transcripts, forming IRAS-S and IRAS-L mRNA (short and long isoforms). Northern blots confirmed the existence of these mRNA isoforms. In most brain regions the order of mRNA abundance was IRAS-M > IRAS-L > IRAS-S mRNA. Although aas 1 through 510 are theoretically identical, truncated proteins could be derived from IRAS-S (2,678 bp transcript yields 515 aas) and IRAS-L (9,457 bp transcript yields 583 aas). Because exon-16 of the iras gene has been proposed to encode the functional domains of imidazoline and a-5 integrin binding, only IRAS-M is expected to possess I(1) receptor properties. Subtype-selective cDNA expression constructs were therefore generated and used to transfect CHO cells. High-affinity I(1) binding was endowed by IRAS-M and IRAS-L, but not by IRAS-S transfection.

Effect of I1-imidazoline receptor activation on responses of hypoglossal and phrenic nerve to chemical stimulation.

Sedation elicited by some centrally acting antihypertensive agents may interfere with respiratory control, and by selectively inhibiting upper airway dilating muscle activity it may facilitate obstructive sleep apnea. Autoradiographic studies with [125I]p-iodoclonidine in the presence of 10 microM epinephrine to block alpha 2-adrenergic sites or 100 nM moxonidine to mask I1-imidazoline sites show that both I1- as well as alpha 2-sites are localized in putative chemosensory areas of the rostral ventrolateral medulla in the cat. We sought to determine the effect of activating I1 and alpha 2-receptors on central chemosensitivity by using moxonidine as a selective I1 agonist, clonidine as a mixed I1/alpha 2 agonist, SK&F-86466 as a specific alpha 2-antagonist, and efaroxan as a mixed I1/alpha 2 antagonist. We recorded responses of phrenic, hypoglossal, and cervical sympathetic nerve activities to progressive hypercapnia after hyperventilation to apnea. Moxonidine (3-100 micrograms/kg i.v.) caused dose-dependent decreases in tonic cervical sympathetic nerve activity and blood pressure, but had no effect on the CO2 threshold (after 30 or 100 micrograms/kg moxonidine, phrenic nerve activity reappeared at 5.8 +/- 0.2% CO2 versus 5.6 +/- 0.3% CO2 in control). Following moxonidine, the slope of the steep portion of the CO2 response tended to increase (10.3 +/- 1.8 versus 7.3 +/- 0.9). Peak phrenic nerve activity was comparable to control at 7.5% CO2 (20 +/- 2 U in control) and at 9.5% CO2 (30 +/- 3 versus 27. +/- 2 U). Similarly, the response of hypoglossal and inspiratory phasic cervical sympathetic nerve activity to a progressive CO2 rise was not affected by moxonidine. By contrast, clonidine in the same doses decreased CO2 sensitivity, because the CO2 threshold was elevated from 5.3 +/- 0.5% to 6.7 +/- 0.4% (p < 0.001). The slope of the CO2 response was decreased from 9.7 +/- 1.9 to 7.4 +/- 1.3 (p = 0.05). Peak phrenic nerve activity was reduced at 7.5% CO2 (11 +/- 5 versus 25 +/- 2 U; p < 0.05) and at 9.5% CO2 (21 +/- 4 versus 33 +/- 2 U; p = 0.06). Clonidine selectively inhibited the response of hypoglossal nerve activity to CO2. The depressive effects of clonidine were reversed by alpha 2-blockade with SK&F-86466 (0.5 or 1 mg/kg). Inspiratory phasic cervical sympathetic nerve activity increased after SK&F-86466 in parallel with phrenic and hypoglossal nerve activity, but the tonic component of cervical sympathetic nerve activity and blood pressure increased only transiently.(ABSTRACT TRUNCATED AT 400 WORDS)

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.