Imidazoline receptor proteins are regulated in platelet-precursor MEG-01 cells by agonists and antagonists.

The I1-imidazoline receptor is a novel brainstem modulator of sympathetic outflow that is elevated on platelets and in brains of depressed patients. A positive correlation has been reported (accompanying manuscript) between plasma norepinephrine (NE) concentrations and the densities (Bmax) of platelet I1 binding sites (I1 sites). I1-candidate proteins of 33 kDa and 85 kDa are now identified on Western blots probed with anti-imidazoline receptor antiserum (IRBP antiserum), that correlate with Bmax values for I1 sites. Furthermore, a human megakaryoblastoma cell line (MEG-01) has been used to study the regulation of these proteins on megakaryocytic cells, while bovine adrenal chromaffin cells provide a standard I1 cell type for comparison. Both the 33 kDa and 85 kDa IRBP-immunoreactive bands were enriched in plasma membrane fractions. IRBP antiserum did not cross-react with I2 imidazoline binding sites located on platelet mitochondrial membranes. The 85 kDa band was enhanced under conditions lacking fetal bovine serum (FBS) from the culture medium 6 h prior to harvesting. Conversely, 33 kDa protein was enhanced on MEG-01 cells grown in the presence of 10% FBS; suggesting that a precursor (85 kDa) and product (33 kDa) relationship might be induced by serum. The 85 kDa band was robustly up-regulated in response to imidazoline receptor-sensitive ligands; moxonidine, idazoxan and agmatine (10 microM each for 6 h). NE also up-regulated the 85 kDa IRBP-immunoreactive protein on MEG-01 membranes, but to a lesser extent. Idazoxan, an imidazoline alpha 2-antagonist, off-set its induction of 85 kDa protein by reducing the 33 kDa band. Yohimbine, a non-imidazoline alpha 2-antagonist, was ineffective alone, or in combination with moxonidine (up to 40 microM), but yohimbine blocked NE's induction of the 85 kDa band. Therefore, a rise in either plasma NE and/or endogenous I-site ligands (i.e. agmatine) could explain an elevation of imidazoline receptors observed in depression.

Comparison of ligand binding affinities at human I1-imidazoline binding sites and the high affinity state of alpha-2 adrenoceptor subtypes.

To identify selective compounds for nonadrenergic I1-imidazoline receptors (I1), the affinities of 22 ligands for [125I]p-iodoclonidine binding have been compared at human platelet I1-imidazoline binding sites (analyzed under norepinephrine mask of alpha-2 AR) and at human alpha-2A, alpha-2B and alpha-2C adrenoceptors stably expressed on transfected Chinese hamster ovary cells. Competition curves at the platelet I1-imidazoline binding site were biphasic for most compounds. Only tizanidine and BDF,6143 displayed monophasic I1 competition curves. Agmatine, an endogenous neurotransmitter candidate for the I1-imidazoline receptor, was identified as the most selective agent for a subcomponent of platelet I1 sites. The affinity of agmatine at the high affinity component of platelet I1 sites was 1400-fold selective over alpha-2A adrenoceptors, 5000-fold selective over alpha-2B adrenoceptors and 800-fold selective over alpha-2C adrenoceptors. Moxonidine and tizanidine also displayed selectivities for a high affinity component of the platelet I1 binding sites over alpha-2 adrenoceptors. Naphazoline was the most selective compound for the high affinity state of the alpha-2A adrenoceptor, displaying 7-, 23- and 9-fold higher affinity than alpha-2B, alpha-2C and platelet I1-midazoline binding sites, respectively. No single selective compound was identified for the alpha-2B adrenoceptor. Norepinephrine displayed, respectively, 18- and 31-fold selectivity for the high affinity state of the alpha-2C adrenoceptor as compared to alpha-2A- or alpha-2B adrenoceptors, and was > 100,000- fold selective over platelet I1-imidazoline sites. Thus, human alpha-2 adrenoceptors and the platelet I1-imidazoline binding site can be clearly discriminated based on their affinities for certain compounds.

Platelet I1-imidazoline binding sites are decreased by two dissimilar antidepressant agents in depressed patients.

Previous studies have indicated that there may be a dysregulation of alpha 2-adrenoceptors and imidazoline receptors in depression. This study compares the effects of chronic antidepressant treatment with a serotonin reuptake inhibitor (fluoxetine) versus a noradrenaline reuptake inhibitor (desipramine) on the binding parameters of the platelet imidazoline binding site (subtype I1) and of the platelet alpha 2-adrenoceptor in depressed patients. After 6 weeks of treatment with either antidepressant, platelet I1 binding sites became normalized (i.e. downregulated). A negative correlation was obtained between plasma epinephrine concentrations and platelet alpha 2-adrenoceptor Bmax values within the samples, but no correlation was obtained between any plasma catecholamine and a platelet I1 binding parameter. An additional finding was the increased affinity of alpha 2-adrenoceptors for p125I-clonidine in untreated depressed patients compared to healthy subjects. Because of the density of platelet I1 binding sites was downregulated by both of the antidepressants, we postulate that a decrease in platelet I1 binding site density may be related to an improved state from depression that these antidepressants produce.

Comparison of the properties of agmatine and endogenous clonidine-displacing substance at imidazoline and alpha-2 adrenergic receptors.

Nonadrenergic imidazoline receptors (I receptors) mediate the central antihypertensive effects of clonidine. Agmatine, an arginine metabolite that is synthesized within bovine brain and exhibits clonidine-displacing substance (CDS) activity, might be the endogenous I receptor neurotransmitter. The authors compared the affinity of agmatine versus the potency of endogenous bovine hypothalamic CDS, at bovine adrenomedullary I1 receptors and at human clonidine-binding sites: human alpha-2A, alpha-2B and alpha-2C expressed on transfected cell lines, I1 sites on human platelet plasma membranes and I2b (amiloride-insensitive) sites on human platelet intracellular membranes. The alpha-2 and I1 sites were labeled with [125I]p-iodoclonidine and the I2b sites were labeled with [3H]-idazoxan. Agmatine displayed preferential affinity for I1 receptors, with both high (H) and low (L) affinity components; the rank order was I1(human)(H) > I1(bovine)(H) > I2b = I1(human)(L) = I1(bovine) (L) = alpha-2A = alpha-2B = alpha-2C. By comparison, hypothalamic CDS competition curves modeled to a single site for all receptors, i.e., I1(bovine) > I2b = alpha-2C > I1(human) = alpha-2A = alpha-2B. Thus, agmatine alone cannot explain the rank order of potency of hypothalamic CDS. Moreover, I1 sites on human platelets differ from I1 sites on bovine adrenomedullary cells with respect to CDS potency but not agmatine affinity. These results do not rule out agmatine as an I1 transmitter but suggest that other I-receptor ligands might exist within endogenous CDS.

Delayed desensitization of alpha 2-adrenoceptor-mediated platelet aggregation in depressed patients.

After prolonged exposure to epinephrine, platelets are observed to desensitize alpha 2-adrenoceptor-mediated aggregation responses in vitro. Herein, this phenomenon was studied as a possible in vitro model for alpha 2-adrenoceptor dysregulation in depression. Platelet-rich plasmas obtained from 22 unipolar depressed patients and 25 healthy subjects were preincubated with 20 mumol/L of epinephrine for various lengths of time prior to stirring. By comparing the subsequent extents of aggregation, we observed significantly less desensitization at 4, 20, 30, or 60 minutes postepinephrine exposure (p < or = .05) in depressed patients as compared to healthy controls. This blunted desensitization appeared to be due to a delayed onset of desensitization during the first 0.5 to 2 minutes after epinephrine exposure, since thereafter, the monoexponential desensitization rate did not differ in depressed patients, but the extent of desensitization remained less as compared to healthy subjects. The extent of desensitization was correlated (r = -0.48, p = .02) with the density (Bmax) of the alpha 2-adrenoceptor high-affinity state, as detected in undesensitized platelet membranes by p125I-clonidine binding. An elevation was also observed in the density of nonadrenergic p125I-clonidine-binding sites (putative imidazoline I1 sites) in platelet membranes from depressed patients compared to healthy control subjects. Following treatment with desipramines, the patients (n = 15) displayed more normal (nonblunted) extents of desensitization of aggregation, and the Bmax values for the putative I1 sites were at the levels of healthy controls. If similar aberrations exist in neurons of depressed patients, this may explain a dysregulation of the noradrenergic system believed to underlie depression.