Time course of liver nitric oxide concentration in early septic shock by cecal ligation and puncture in rats.

An overwhelming nitric oxide (NO) production is a crucial step in the circulatory events as well as in the cellular alterations taking place in septic shock. However, evidences of this role arise from studies assessing the NO production on an intermittent basis precluding any clear evaluation of temporal relationship between NO production and circulatory alterations. We evaluated this relationship by using a NO specific electrode allowing a continuous measurement of NO production. Septic shock was induced by a cecal ligation and puncture (CLP) in a first group of anesthetized rats. After the same CLP, a second group received a selective iNOS inhibitor (L-NIL). Control rats were sham operated or sham operated with L-NIL administration. While NO concentration was measured every 2 min by a NO-sensitive electrode over 7h following CLP, the liver microcirculation was recorded by a laser-Doppler flowmeter. CLP induced a severe septic shock with hypotension occurring at a mean time of 240 min after CLP. At the same time, an increase in liver NO concentration was observed, whereas a decrease in microvascular liver perfusion was noted. In the septic shock group, L-NIL administration induced an increase in arterial pressure whereas the liver NO concentration returned to baseline values. In addition, shock groups experienced an increase in iNOS mRNA. These data showed a close temporal relationship between the increase in liver NO concentration and the microvascular alteration taking place in the early period of septic shock induced by CLP. The iNOS isoform is involved in this NO increase.

[Williams-Beuren syndrome: a multidisciplinary approach]. / Le syndrome de Williams-Beuren: une approche pluridisciplinaire.

Williams-Beuren syndrome (WBS) (OMIM# 194050) is a rare, most often sporadic, genetic disease caused by a chromosomal microdeletion at locus 7q11.23 involving 28 genes. Among these, the elastin gene codes for the essential component of the arterial extracellular matrix. Developmental disorders usually associate an atypical face, cardiovascular malformations (most often supravalvular aortic stenosis and/or pulmonary artery stenosis) and a unique neuropsychological profile. This profile is defined by moderate mental retardation, relatively well-preserved language skills, visuospatial deficits and hypersociability. Other less known or rarer features, such as neonatal hypercalcemia, nutrition problems in infancy, ophthalmological anomalies, hypothyroidism, growth retardation, joint disturbances, dental anomalies and hypertension arising in adolescence or adulthood, should be treated. The aim of this paper is to summarize the major points of WBS regarding: (i) the different genes involved in the deletion and their function, especially the elastin gene and recent reports of rare forms of partial WBS or of an opposite syndrome stemming from a microduplication of the 7q11.23 locus, (ii) the clinical features in children and adults with a focus on cardiovascular injury, and (iii) the specific neuropsychological profile of people with WBS through its characteristics, the brain structures involved, and learning.

A circulating substance cross-reacting with antiimidazoline antibodies. Detection in serum in relation to essential hypertension.

It has been shown in various mammal species that clonidine, a well known centrally acting hypotensive agent, acts through the activation of imidazoline receptors (IRs) in the nucleus reticularis lateralis (NRL) of the brainstem. Specific binding sites sensitive to imidazolines and insensitive to catecholamines have been detected in rat and bovine, as well as human brains. An endogenous ligand, other than catecholamines, should exist for these IRs. Such a ligand could play a role in the pathophysiology of human essential hypertension. Therefore, we developed two RIAs with polyclonal and monoclonal anticlonidine antibodies. These antibodies presented specificity spectra similar to that of the IRs: they bound imidazolines and not catecholamines at all. These RIAs were used to detect imidazoline-like immunoreactivity in the human serum. Immunoreactive substance was measured in 26 normotensive subjects' sera, and specificity of interaction between antibodies and sera was verified. None of the known endogenous substances tested so far were able to interact with the two antibodies. Immunoreactivity in 32 essential hypertensive patients' sera proved higher in approximately 30% of cases. Values of immunoreactivity positively correlated with the mean arterial pressure values. This study demonstrates the existence of an "imidazoline-like" immunoreactive substance in the human serum with high levels in some hypertensive patients.

ICAM-1 deficiency reduces atherosclerotic lesions in double-knockout mice (ApoE(-/-)/ICAM-1(-/-)) fed a fat or a chow diet.

Intercellular adhesion molecule (ICAM)-1, a major adhesion molecule, plays a critical role in the homing of leukocytes to sites of atherosclerotic lesions. However, very little is known on the role of ICAM-1 in initiating and perpetuating vascular lesions in ApoE(-/-) mice fed a chow or a fat diet. This study has investigated the mean aortic lesions in mice (C57BL6 background) with a single-knockout (ApoE(-/-)) or double-knockout (DKO; ApoE(-/-), ICAM-1(-/-)) fed a chow or a fat diet over a period of 3, 6, 15, and 20 weeks. A 3-fold reduction in lesion size was observed at all time points in DKO mice fed a chow diet. However, in DKO mice fed a fat diet, a marked reduction in the aortic lesion was observed at 3 and 15 weeks, which did not reach a significant level at 6 and 20 weeks. This study shows in essence that DKO mice are protected from developing significant lesions for up to 6 weeks when fed a chow diet and from 3 to 6 weeks when fed a fat diet. After 6 weeks, the lesion size of the DKO mice follows that of the single-knockout mice when fed a chow diet and gets to the same level in mice fed a fat diet. Plasma cholesterol levels were not altered as a result of ICAM-1 deficiency. These studies show that ICAM-1 is implicated in the formation and progression of atherosclerotic lesions.

Sense, antisense, nonsense: where's the right way?

The application of small nucleic acids is slowly moving from chemistry and molecular biology laboratories into the clinic. However, the development of a new family of therapeutics is always difficult. Research must be performed to produce chemically compatible compounds in vivo and pharmacological investigations are necessary to precisely define the in vivo mechanisms of action of these potentially new drugs. The "2nd International Conference on Antisense Nucleic Acids: Biology, Pharmacology, Therapy" held in Garmisch-Partenkirchen addressed these questions.

Heat stress-induced resistance to myocardial infarction in the isolated heart from transgenic [(mREN-2)27] hypertensive rats.

OBJECTIVE: Heat stress (HS) is known to confer protection against ischaemia-reperfusion injury, including mechanical dysfunction and myocardial necrosis. However, the effects of disease states on this HS-induced cytoprotective response are less known. Therefore, we investigated the effects of prior heat stress on the infarct size in the isolated rat heart and on the myocardial heat stress protein (HSP) 72 synthesis, in transgenic [(mREN-2)27] hypertensive (TGH) rats or normotensive (NT) controls. METHODS: TGH or NT rats were either heat stressed (42 degrees C for 15 min) or sham anaesthetised. After 24 h, their hearts were isolated, perfused using the Langengorff technique, and subjected to a 35-min occlusion of the left coronary artery followed by 120 min of reperfusion. Myocardial HSP72 content was measured 24 h after HS or sham treatment using electrophoresis coupled with Western blot analysis. RESULTS: Infarct-to-risk (I/R) ratio was significantly reduced in HS (15.5 +/- 1.2%) compared to sham (42.2 +/- 2.1%) hearts of NT rats. This reduction in infarct size was maintained in TGH hearts (I/R: 20.0 +/- 1.0 vs. 48.0 +/- 3.8%). Risk zones were similar between all experimental groups. The incidence of ventricular arrhythmias during ischaemia and reperfusion periods was not different between the four experimental groups. Western blot analysis of the myocardial HSP72 content showed a heat stress-induced increase of this protein, in both TGH and NT animals. CONCLUSION: These results demonstrate that the myocardial protective effect induced by heat stress could extend to a pathological animal model like the transgenic [(mREN-2)27] hypertensive rat and is correlated with a myocardial HSP72 induction.

Can we identify response markers to antihypertensive drugs? First results from the IDEAL Trial.

Current antihypertensive strategies do not take into account that individual characteristics may influence the magnitude of blood pressure (BP) reduction. Guidelines promote trial-and-error approaches with many different drugs. We conducted the Identification of the Determinants of the Efficacy of Arterial blood pressure Lowering drugs (IDEAL) Trial to identify factors associated with BP responses to perindopril and indapamide. IDEAL was a cross-over, double-blind, placebo-controlled trial, involving four 4-week periods: indapamide, perindopril and two placebo. Eligible patients were untreated, hypertensive and aged 25-70 years. The main outcome was systolic BP (SBP) response to drugs. The 112 participants with good compliance had a mean age of 52. One in every three participants was a woman. In middle-aged women, the SBP reduction from drugs was -11.5 mm Hg (indapamide) and -8.3 mm Hg (perindopril). In men, the response was significantly smaller: -4.8 mm Hg (indapamide) and -4.3 (perindopril) (P for sex differences 0.001 and 0.015, respectively). SBP response to perindopril decreased by 2 mm Hg every 10 years of age in both sexes (P=0.01). The response to indapamide increased by 3 mm Hg every 10 years of age gradient in women (P=0.02). Age and sex were important determinants of BP response for antihypertensive drugs in the IDEAL population. This should be taken into account when choosing drugs a priori.

Permanent inhibition of angiotensinogen synthesis by antisense RNA expression.

The renin-angiotensin system plays a pivotal role in blood pressure regulation. Recent molecular biological findings led to the new concept that in addition to the classic endocrine system, local tissue systems may also play an important role in cardiovascular diseases such as hypertension. In particular, the brain renin-angiotensin system was shown to influence the central control of blood pressure and is thought to contribute to the hypertensive phenotype of genetically hypertensive rat models. To identify the physiological role of these local systems, we established an antisense strategy to downregulate the expression of the precursor hormone angiotensinogen (AOGEN) in cell culture, which can also be used to establish transgenic rat lines. Plasmids encoding an RNA sequence complementary to the rat AOGEN mRNA under control of different viral and tissue-specific promoters were constructed and transfected into an AOGEN-expressing cell line. A competitive reverse transcription-polymerase chain reaction method was established for the quantification of AOGEN mRNA. Depending on the level of antisense RNA, the expression of the AOGEN gene was reduced down to 22% of control levels. Furthermore, the secretion of AOGEN protein was totally abolished. These results clearly demonstrate that the antisense constructs used are functional in reducing the AOGEN gene expression in vivo and can be used for the production of transgenic rats.

Thioredoxin reductase 1 is upregulated in atherosclerotic plaques: specific induction of the promoter in human macrophages by oxidized low-density lipoproteins.

Uptake of modified low-density lipoproteins (LDLs) by macrophages in the arterial wall is an important event in atherogenesis. Indeed, oxidatively modified LDLs (oxLDLs) are known to affect various cellular processes by modulating oxidation-sensitive signaling pathways. Here we found that the ubiquitous 55 kDa selenoprotein thioredoxin reductase 1 (TrxR1), which is a key enzyme for cellular redox control and antioxidant defense, was upregulated in human atherosclerotic plaques and expressed in foam cells. Using reverse transcription polymerase chain reaction analysis, we also found that oxLDLs, but not native LDLs (nLDLs), dose-dependently increased TrxR1 mRNA in human monocyte-derived macrophages (HMDMs). This stimulating effect was specific for oxLDLs, as pro-inflammatory factors, such as lipopolysaccharides (LPSs), interleukin-1beta (IL-1beta), interleukin-6 (Il-6), and tumor necrosis factor alpha (TNFalpha), under the same conditions, failed to induce TrxR1 mRNA levels to the same extent. Moreover, phorbol ester-differentiated THP-1 cells or HMDMs transiently transfected with TrxR1 promoter fragments linked to a luciferase reporter gene allowed identification of a defined promoter region as specifically responding to the phospholipid component of oxLDLs (p <.05 vs. phospholipid component of nLDLs). Gel mobility shift analyses identified a short 40-nucleotide stretch of the promoter carrying AP-1 and HoxA5 consensus motifs that responded with an altered shift pattern in THP-1 cells treated with oxLDLs, however, without evident involvement of either the Fos, Jun, Nrf2 or HoxA5 transcription factors.

New concepts on the central regulation of blood pressure. Alpha 2-adrenoceptors and "imidazoline receptors".

The most usual hypothesis to explain the central hypotensive effect of clonidine-like substances was to admit that these drugs stimulated alpha 2-adrenoceptors within the brainstem. Now it has been demonstrated that neither the endogenous ligand to the alpha-adrenoceptors, noradrenaline, nor any other catecholamine or phenylethylamine was hypotensive in the medullary nucleus reticularis lateralis, where all imidazolines proved to be such. Recently, a membrane receptor population sensitive to clonidine and insensitive to catecholamines was described within the nucleus reticularis lateralis; this subgroup of receptors represented 20 to 30 percent of the [3H]clonidine binding sites in the bovine nucleus reticularis lateralis and 100 percent within the human nucleus reticularis lateralis region. Thus, the existence of such imidazoline specific receptors was clearly established and the endogenous ligand for those receptors, which is neither a catecholamine nor likely a peptide, is under processing for purification. Therefore, it appeared that the hypotensive effect of substances with an imidazoline or imidazoline-like structure might be due to their action within medullary receptors specific for this endogenous ligand temporarily named "clonidine displacing substance." Rilmenidine, structurally close to imidazolines, also interfered with these receptors. The central component of its hypotensive effect was recently confirmed in rabbits, where its central cardiovascular effects were antagonized by "the clonidine displacing substance." Although exhibiting a lower affinity than the reference substance for these receptors, rilmenidine might have a higher selectivity, thus explaining its restricted side effects. A structure-activity study with this molecule would bring a confirmation to these first observations.

Imidazoline receptors, non-adrenergic idazoxan binding sites and alpha 2-adrenoceptors in the human central nervous system.

Both [3H]clonidine and [3H]idazoxan bind to alpha 2 adrenoceptors. The former also labels imidazoline receptors, and the latter non-adrenergic idazoxan binding sites. In order to investigate whether the imidazoline receptors and non-adrenergic idazoxan binding sites are identical, we compared the binding characteristics of [3H]clonidine and [3H]idazoxan to these sites by radioligand binding experiments on ultra-thin slices and homogenates of human striatum. A good correlation was found between the effect of different ions on the binding characteristics of [3H]clonidine and [3H]idazoxan, and the affinities of most competing drugs. However, clonidine and rilmenidine displayed a 100- and 10-fold lower affinity, respectively, for the idazoxan binding sites than for the imidazoline receptors. Autoradiography with [3H]clonidine showed that high densities of imidazoline receptors were present in the striatum, pallidum, gyrus dentatus of the hippocampus, amygdala, and substantia nigra. Moderate densities were found throughout the cerebral cortex, thalamus and several brainstem nuclei including the nucleus olivarius inferior. Low densities were seen in the cerebellum, spinal cord and pituitary gland. As for the non-adrenergic sites labelled by [3H]idazoxan, the imidazoline receptors can be found in all major brain areas examined. However, there are some striking differences between the concentrations of imidazoline receptors and non-adrenergic idazoxan binding sites in certain brain regions. To reconcile distribution and pharmacologic data, we propose that imidazoline receptors and non-adrenergic idazoxan binding sites represent different proteins or protein complexes and that at least in the nucleus reticularis lateralis and the striatum, imidazoline receptors and non-adrenergic idazoxan binding sites may be physically associated. The regional distribution of alpha 2 adrenoceptors within the human CNS was determined by quantitative autoradiography with [3H]RX821002. The highest densities of alpha 2 adrenoceptors were found in the cerebral and cerebellar cortex, and certain regions in the medulla oblongata (floor of the IV ventricle, reticular formation, hypoglossal nucleus and nucleus olivarius inferior). No alpha 2 adrenoceptors were detected in the pituitary gland. There exists no relationship between the distribution pattern of imidazoline receptors and alpha 2 adrenoceptors, indicating that these binding sites are independent from each other.

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.

Evidence for the involvement of imidazoline receptors in the central hypotensive effect of rilmenidine in the rabbit.

1. Rilmenidine has recently been introduced as a new centrally-acting antihypertensive agent. We examined its cardiovascular effects after intracerebral injection to anaesthetized rabbits. Cumulative doses of rilmenidine injected intracisternally (1 to 300 micrograms kg-1) led to dose-dependent decreases in arterial blood pressure and heart rate. The effective doses of rilmenidine were lower when injected centrally than when injected intravenously. 2. Pretreatment with the same dose of yohimbine or idazoxan shifted the rilmenidine dose-response curves for its hypotensive and bradycardic effects to the right. Idazoxan, which has an imidazoline structure, proved to be a more active antagonist than yohimbine of rilmenidine centrally-mediated cardiovascular effects. 3. The dose-response curve for the central hypotensive effect of rilmenidine was also shifted to the right after pretreatment with a bovine brain extract. This extract contains the endogenous ligand of the imidazoline-preferring receptors which is not a catecholamine. 4. Rilmenidine, like clonidine, proved to be active when micro-injected into the rabbit nucleus reticularis lateralis region. 5. In conclusion, rilmenidine exhibited in the rabbit a central hypotensive effect which originated in the same area as where clonidine acts. Specific imidazoline-preferring receptors appear to be involved in this hypotensive effect.

Relevance of the use of [3H]-clonidine to identify imidazoline receptors in the rabbit brainstem.

1. [3H]-clonidine binding was investigated in membranes isolated from the ventral medulla oblongata of the rabbit, where clonidine produced a hypotensive effect which was not mediated by adrenoceptors. [3H]-clonidine specific binding, as defined by the difference between the binding of [3H]-clonidine in the presence and in the absence of 10 microM cirazoline, occurred at two sites: a high affinity site with a KD = 2.9 +/- 0.7 nM and a Bmax of 40 +/- 8 fmol mg-1 protein and a low affinity site with a KD = 18.2 +/- 0.4 nM and a Bmax of 66 +/- 14 fmol mg-1 protein. 2. The high affinity sites being catecholamine-sensitive were identified as alpha 2-adrenoceptors. The low affinity binding of [3H]-clonidine was insensitive to catecholamines, as well as to other alpha 2-adrenoceptor specific probes, and could be inhibited with high affinity only by compounds which lowered blood pressure when directly injected in the nucleus reticularis lateralis of the ventral brainstem, or by antagonists. 3. It was concluded that in the ventral medulla of the rabbit, [3H]-clonidine labelled alpha 2-adrenoceptors and imidazoline receptors (IRs). Only the latter were related to the hypotensive effects of clonidine and rilmenidine directly injected into the rostroventrolateral medulla oblongata (RVLM) of the rabbit. The methodological problems regarding the study of IRs with [3H]-clonidine are discussed.

Imidazoline receptors. A new concept in central regulation of the arterial blood pressure.

Clonidine-like antihypertensive substances bind to nonadrenergic imidazoline specific sites within the nucleus reticularis lateralis (NRL), their medullary privileged site of action. Rilmenidine, a new central antihypertensive agent, was tested in the anesthetized rabbit. For the same hypotensive effect, cumulative doses given intracisternally proved 50 times more active than of those given systemically. Idazoxan, an alpha 2-adrenergic antagonist structurally related to the imidazolines, given centrally as pretreatment proved more potent in preventing the hypotensive effects than the same molar dose of yohimbine. When injected within the NRL area of the anesthetized rabbit, rilmenidine, like clonidine, always exhibited a hypotensive effect. The influence of clonidine and rilmenidine upon the NRL noradrenergic neurons involved in the blood pressure regulation, and upon those of the locus coeruleus (LC) involved in the sedative effect was studied by differential voltammetry. It was observed that rilmenidine was two times more selective than clonidine in inhibiting the NRL, as opposed to LC, neuronal activity. In addition, binding experiments of tritiated clonidine to human cortical and NRL membrane preparations showed that rilmenidine, as compared to clonidine, has a two to three times higher selectivity for the imidazoline receptors. In conclusion, we are now able to discriminate between the mechanism of the hypotensive effect of imidazoline-like drugs and that of their sedative action. Rilmenidine is the first example of an hypotensive drug more selective for imidazolin preferring receptors than for classical alpha 2-adrenoceptors.

Characterization of imidazoline binding protein(s) solubilized from human brainstem: studies with [3H]idazoxan and [3H]clonidine.

Imidazoline binding sites from the human brainstem were solubilized with 3-[(3-cholamido-propyl)-dimethylammonio]-1-propane-sulfonate (CHAPS). [3H]idazoxan and [3H]clonidine were used as ligands to characterize the solubilized binding sites. In both the soluble and membrane fractions, [3H]idazoxan binding was saturable, stereoselective, sensitive to imidazolines and insensitive to (-)norepinephrine and to amiloride. The affinities of [3H]idazoxan for the soluble and membrane sites were similar (KD = 25 +/- 11 nM and 20 +/- 3 nM). In both soluble and membrane fractions, the alpha 2-adrenoceptor binding being masked with (-)norepinephrine, [3H]clonidine bound to a low affinity site which was insensitive to (-)norepinephrine and which exhibited the same selectivity for various drugs as the [3H]idazoxan binding site. alpha 2-adrenoceptor binding was present in the membrane and the soluble fractions although it was difficult to detect in the soluble fraction because of inhibition of [3H]rauwolscine binding by the CHAPS detergent.

Heterogeneity of the specific imidazoline binding of [3H]idazoxan in the human cerebral cortex.

The aim of the present study was to verify whether [3H]idazoxan can be considered as a highly selective ligand for imidazoline preferring receptors (IPR). In human frontal cortex membrane preparations [3H]idazoxan at a low concentration (2 nM) only labelled imidazoline sensitive, catecholamine insensitive sites. Binding was of high affinity, saturable and stereospecific. The rank order of potency of different compounds able to inhibit this binding was cirazoline > (+/-)-idazoxan > guanoxan > (-)-idazoxan > tolazoline > UK-14304 > clonidine. Amiloride, imidazol-4-acetic acid and histamine had no significant affinity for IPR labelled by [3H]idazoxan. [3H]idazoxan bound to 2 different sites (KD1 = 1 nM and KD2 = 16.4 nM). Clonidine behaved as a non competitive, non allosteric inhibitor of [3H]idazoxan binding. Both [3H]idazoxan binding sites were equally affected by clonidine. In membrane preparations obtained from the Nucleus Reticularis Lateralis region (NRL) of the brainstem, [3H]idazoxan binding was similar to that in cortical membranes, particularly with regard to specificity and kinetics. However, in the NRL region binding sites were 4-5 times more numerous than in the frontal cortex. Non linear analyses of saturation data obtained with NRL membrane preparations were compatible with both a one site and a two sites model. No significant effects of 1 mM MgCl2 alone or with 100 microM Gpp(NH)p were observed on either [3H]idazoxan binding or the competition with clonidine or rilmenidine. As in the cortical membrane, clonidine was a non competitive inhibitor of [3H]idazoxan binding to membranes from the NRL region. In conclusion, we show that when a low concentration is used, [3H]idazoxan binding to human brain involves sites almost completely insensitive to catecholamines and specific for imidazolines or related compounds. This binding involves two distinct sites. We also report that [3H]idazoxan imidazoline binding sites are not coupled with a G protein. Because of the non competitive interaction between clonidine and [3H]idazoxan for the binding sites of the latter, we are unable to conclude that the binding sites of the two drugs are identical. However, the non competitive, non allosteric interaction suggests a complex model of multiple binding sites.

Postnatal development of the endothelial system of the rat cerebellum using immunohistochemical techniques.

The development of the endothelial system of the rat cerebellum was studied by immunohistochemical techniques with two antibodies specific of two membrane antigens characteristic of 'mature' endothelial cells. It appears that the maturation of endothelial cells is very late in the rat cerebellum. This absence of a fully endothelial barrier in cerebella of young rats could explain the extreme sensitivity of this part of the central nervous system to various agents.

Production and characterization of anti-clonidine antibodies not cross-reacting with catecholamines.

Polyclonal antibodies against clonidine were developed, with para-aminoclonidine coupled to bovine serumalbumin or hemocyanine with glutaraldehyde used as antigens. The selected antibody (from rabbits) cross-reacted with high specificity with clonidine and its structurally closely related analogues but it recognized neither catecholamines nor various endogenous imidazole molecules such as histamine, purine, adenine, and adenosine, thus appearing to be specific for the aminoimidazoline structure. An interesting cross-reactivity was observed with the bovine clonidine displacing substance, the probable endogenous ligand for receptors involved in the hypotensive effect of clonidine-type substances. This suggested that this molecule should contain an aminoimidazoline or guanidine moiety.

The imidazoline preferring receptor: binding studies in bovine, rat and human brainstem.

The binding of [3H]clonidine to brainstem membrane preparations was studied in an attempt to characterize imidazoline-sensitive, catecholamine-insensitive receptors. Human samples and samples from two animal species were used. [3H]Clonidine binding was always saturable, reversible and specific with a KD value of 6-7 nM. The Bmax values were 45.5 +/- 5.5, 145 +/- 34 and 65 +/- 33 fmol/mg protein in the whole rat medulla oblongata, the nucleus reticularis lateralis region of bovine and that of human, respectively. In the whole rat brainstem we could not demonstrate the presence of [3H]clonidine binding sites that were insensitive to catecholamines. In bovine and human nucleus reticularis lateralis (NRL) preparations, the amount of specifically bound labelled clonidine that was not displaced by an excess of (-)-norepinephrine was 25 and 100%, respectively. Substances that had a structure similar to that of clonidine were able to compete with [3H]clonidine binding within the human NRL. Cirazoline was the most potent to inhibit [3H]clonidine binding although yohimbine was also able to displace binding in the human NRL but with lower apparent affinity. Competition assays with idazoxan stereoisomers clearly showed that this binding was stereospecific. Therefore the human NRL region provides the first model of an homogenous population of imidazoline-preferring, non-alpha-adrenergic membrane receptors.