Differential phosphorylation, desensitization, and internalization of α1A-adrenoceptors activated by norepinephrine and oxymetazoline.

Loss of response on repetitive drug exposure (i.e., tachyphylaxis) is a particular problem for the vasoconstrictor effects of medications containing oxymetazoline (OXY), an α1-adrenoceptor (AR) agonist of the imidazoline class. One cause of tachyphylaxis is receptor desensitization, usually accompanied by phosphorylation and internalization. It is well established that α1A-ARs are less phosphorylated, desensitized, and internalized on exposure to the phenethylamines norepinephrine (NE), epinephrine, or phenylephrine (PE) than are the α1B and α1D subtypes. However, here we show in human embryonic kidney-293 cells that the low-efficacy agonist OXY induces G protein-coupled receptor kinase 2-dependent α1A-AR phosphorylation, followed by rapid desensitization and internalization (∼40% internalization after 5 minutes of stimulation), whereas phosphorylation of α1A-ARs exposed to NE depends to a large extent on protein kinase C activity and is not followed by desensitization, and the receptors undergo delayed internalization (∼35% after 60 minutes of stimulation). Native α1A-ARs from rat tail artery and vas deferens are also desensitized by OXY, but not by NE or PE, indicating that this property of OXY is not limited to recombinant receptors expressed in cell systems. The results of the present study are clearly indicative of agonist-directed α1A-AR regulation. OXY shows functional selectivity relative to NE and PE at α1A-ARs, leading to significant receptor desensitization and internalization, which is important in view of the therapeutic vasoconstrictor effects of this drug and the varied biologic process regulated by α1A-ARs.

The investigation of the interaction between Oxymetazoline hydrochloride and mucin by spectroscopic approaches.

The fluorescence and ultraviolet spectroscopy were explored to study the interaction between Oxymetazoline hydrochloride (OMZH) and mucin under imitated physiological condition. The results demonstrated that the fluorescence quenching mechanism between OMZH and mucin is a combined quenching process. The binding constants (K(a)), binding sites (n) and the corresponding thermodynamic parameters (ΔG, ΔH, and ΔS) of the interaction system were calculated at different temperatures. The hydrogen bonds and van der Waals forces play a major role in the interaction between OMZH and mucin. According to Förster non-radiation energy transfer theory, the binding distance between OMZH and mucin was calculated.

In vitro metabolism of oxymetazoline: evidence for bioactivation to a reactive metabolite.

Oxymetazoline (6-tert-butyl-3-(2-imidazolin-2-ylmethyl)-2,4-dimethylphenol) has been widely used as a nonprescription nasal vasoconstrictor for >40 years; however, its metabolic pathway has not been investigated. This study describes the in vitro metabolism of oxymetazoline in human, rat, and rabbit liver postmitochondrial supernatant fraction from homogenized tissue (S9) fractions and their microsomes supplemented with NADPH. The metabolites of oxymetazoline identified by liquid chromatography (LC)/UV/tandem mass spectrometry (MS/MS), included M1 (monohydroxylation of the t-butyl group), M2 (oxidative dehydrogenation of the imidazoline to an imidazole moiety), M3 (monohydroxylation of M2), M4 (dihydroxylation of oxymetazoline), and M5 (dihydroxylation of M2). Screening with nine human expressed cytochromes P450 (P450s) identified CYP2C19 as the single P450 isoform catalyzing the formation of M1, M2, and M3. Glutathione conjugates of oxymetazoline (M6) and M2 (M7) were identified in the liver S9 fractions, indicating the capability of oxymetazoline to undergo bioactivation to reactive intermediate species. M6 and M7 were not detected in those liver S9 incubations without NADPH. Cysteine conjugates (M8 and M9) derived from glutathione conjugates and hydroxylated glutathione conjugates (M10 and M11) were also identified. The reactive intermediate of oxymetazoline was trapped with glutathione and N-acetyl cysteine and identified by LC/MS/MS. M6 was isolated and identified by one-dimensional or two-dimensional NMR as the glutathione conjugate of a p-quinone methide. We have shown the tendency of oxymetazoline to form p-quinone methide species via a bioactivation mechanism involving a CYP2C19-catalyzed two-electron oxidation. Nevertheless, we conclude that the formation of this reactive species might not be a safety concern for oxymetazoline nasal products because of the typical low-dose and brief dosage regimen limited to nasal delivery.

Identification and characterization of oxymetazoline glucuronidation in human liver microsomes: evidence for the involvement of UGT1A9.

The incubation of oxymetazoline, a nonprescription nasal decongestant, with human liver microsomes (HLMs) supplemented with uridine-5-diphosphoglucuronic acid (UDPGA) generated glucuronide metabolite as observed by LC/MS/MS. The uridine glucuronosyltransferases (UGTs) responsible for the O-glucuronidation of oxymetazoline remain thus far unidentified. The glucuronide formed in HLMs was identified by LC/MS/MS and characterized by one- and two-dimensional NMR to be the ß-O-glucuronide of oxymetazoline. UGT screening with expressed UGTs identified UGT1A9 as the single UGT isoform catalyzing O-glucuronidation of oxymetazoline. Oxymetazoline O-glucuronidation by using HLMs was best fitted to the allosteric sigmoidal model. The derived S(50) and V(max) values were 2.42 ± 0.40 mM and 8.69 ± 0.58 pmole/(min mg of protein), respectively, and maximum clearance (CL(max)) was 3.61 L/min/mg. Oxymetazoline O-glucuronidation by using expressed UGT1A9 was best fitted to the substrate inhibition model. The derived K(m) and V(max) values were 2.53 ± 1.03 mM and 54.18 ± 16.92 pmole/(min mg of protein), respectively, and intrinsic clearance (CL(int)) was 21.41 L/(min mg). Our studies indicate that oxymetazoline is not glucuronidated at its nanomolar intranasal dose and thus is eliminated unchanged, because UGT1A9 would only contribute to its elimination at the toxic plasma concentrations.

Bulk is a determinant of oxymetazoline affinity for the alpha1A-adrenergic receptor.

The alpha1A-adrenergic receptor (AR) has a higher affinity for several agonists and antagonists compared to alpha1B or alpha1D ARs. Mutagenesis studies were used to determine residues potentially responsible for this subtype selectivity. Oxymetazoline has a 50-fold lower affinity for alpha1D ARs compared to alpha1A ARs and also displayed a significant loss of affinity for an alpha1A Leu-290 to Phe mutant. It was concluded that steric interactions between the alpha1D ARs Phe-360 and the bulkytert-butyl group of oxymetazoline partially accounts for this lower affinity. Thus, the alpha1A AR binding pocket may more easily accommodate bulk at the paraposition of the phenyl ring than the alpha1D AR.

Alpha(2A)-adrenergic versus imidazoline receptor controversy in rilmenidine's action: alpha(2A)-antagonism in humans versus alpha(2A)-agonism in rabbits.

At the alpha(2A)-autoreceptors on the sympathetic nerve terminals of the human atrial appendages and rabbit pulmonary artery, rilmenidine and oxymetazoline exhibit different properties (antagonism and agonism, respectively). These opposite pharmacodynamic properties of alpha(2)-adrenoceptor ligands seem to be due to substantial differences in the nucleotide and amino acid sequences between human and rabbit alpha(2A)-adrenoceptors. Hence, the rabbit alpha(2A)-adrenoceptor is not reliably predictive for the action of ligands at the human alpha(2A)-adrenoceptor.

The stability of oxymetazoline hydrochloride in aqueous solution.

The kinetics of the hydrolysis reaction of oxymetazoline hydrochloride in aqueous solution at three temperatures (343 K, 353 K, 363 K), over the pH-range 0.5-12.5 and ionic strength 0.5 has been investigated. The changes of concentration of oxymetazoline hydrochloride were followed by the HPLC method with UV detection. In the pH range from 0.45 to 12.50, the hydrolysis of oxymetazoline consists of hydrolysis of oxymetazoline molecules catalyzed by hydrogen ions, spontaneous hydrolysis of the dissociated and undissociated oxymetazoline molecules. A minimal rate of the hydrolysis oxymetazoline was observed to occur in the pH range from 2.0 to 5.0. Thermodynamic parameters of the reaction: energy, entropy and enthalpy of activation and the frequency factor for the specific rate constants were determined.

Phe-308 and Phe-312 in transmembrane domain 7 are major sites of alpha 1-adrenergic receptor antagonist binding. Imidazoline agonists bind like antagonists.

Although agonist binding in adrenergic receptors is fairly well understood and involves residues located in transmembrane domains 3 through 6, there are few residues reported that are involved in antagonist binding. In fact, a major docking site for antagonists has never been reported in any G-protein coupled receptor. It has been speculated that antagonist binding is quite diverse depending upon the chemical structure of the antagonist, which can be quite different from agonists. We now report the identification of two phenylalanine residues in transmembrane domain 7 of the alpha(1a)-adrenergic receptor (Phe-312 and Phe-308) that are a major site of antagonist affinity. Mutation of either Phe-308 or Phe-312 resulted in significant losses of affinity (4-1200-fold) for the antagonists prazosin, WB4101, BMY7378, (+) niguldipine, and 5-methylurapidil, with no changes in affinity for phenethylamine-type agonists such as epinephrine, methoxamine, or phenylephrine. Interestingly, both residues are involved in the binding of all imidazoline-type agonists such as oxymetazoline, cirazoline, and clonidine, confirming previous evidence that this class of ligand binds differently than phenethylamine-type agonists and may be more antagonist-like, which may explain their partial agonist properties. In modeling these interactions with previous mutagenesis studies and using the current backbone structure of rhodopsin, we conclude that antagonist binding is docked higher in the pocket closer to the extracellular surface than agonist binding and appears skewed toward transmembrane domain 7.

Effects of N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4) on alpha2-adrenoceptors which regulate the synthesis and release of noradrenaline in the rat brain.

N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4) induces a degeneration of noradrenergic axons originating in the locus coeruleus. The sensitivity of alpha2-adrenoceptors which regulate the synthesis and release of noradrenaline was investigated in three brain regions which receive an unequal innervation from locus coeruleus, 21 days after DSP4 (50 mg/kg) administration. After giving treated rats a dopa decarboxylase inhibitor, the in vivo tyrosine hydroxylase activity and the tissue concentrations of noradrenaline were also evaluated. Relevant reductions of noradrenaline levels were found in hippocampus and parietal cortex (91% and 77.5%, respectively; P<0.001) together with a less pronounced reduction in hypothalamus (32%, P<0.01). The administration of the neurotoxin led to decreases of the basal tyrosine hydroxylase activity, determined as the accumulation of 3,4-dihydroxyphenylalanine, in hippocampus and parietal cortex (75% and 50.5%, respectively; P<0.001), but not in hypothalamus. The inhibitory effect of clonidine on tyrosine hydroxylase activity was markedly reduced in hippocampus of rats treated with DSP4 (10+/-5% vs 57+/-3% in the control group, P<0.001) but was not changed in parietal cortex and hypothalamus. Moreover, in hippocampus, a lack of functionality of the alpha2-adrenoceptors which regulate K(+)-evoked [3H]noradrenaline release was determined. However, in cortical synaptosomes the concentration-effect curve for the oxymetazoline shifted to the right. The administration of the neurotoxin did not modify the inhibitory effects of the agonist in hypothalamus. These results support the previously described selectivity of DSP4 for noradrenergic terminals arising from locus coeruleus and suggest a more severe lesioning of the hippocampus than the parietal cortex.

Sinusitis esfenoidal aguda aislada. presentación de un caso y revisión / Isolated acute sphenoidal sinusitis. Presentation and review of a case

La sinusitis esfenoidal aguda aislada es una enfermedad inflamatoria poco común pero potencialmente peligrosa.Su diagnóstico se realiza raramente de forma inicial debido a la poca especificidad de su sintomatología, sin embargo, dada la gravedad de sus complicaciones, debe ser tenida en cuenta y considerarse como una urgencia terapéutica.El síntoma de presentación más común es una cefalea inespecífica, seguido de las alteraciones visuales y las parálisis de pares craneales. La TAC es actualmente el examen radiológico de elección. Su tratamiento es médico, asociado eventualmente a un drenaje quirúrgico por vía endoscópica.Se presenta un caso de sinusitis esfenoidal aguda aislada diagnosticada y tratada en nuestro servicio y realizamos una revisión de la bibliografía (AU)

Ligand recognition of serine-cysteine amino acid exchanges in transmembrane domain 5 of alpha2-adrenergic receptors by UK 14,304.

Ligand binding of UK 14,304 reveals notable species (i.e., human-rodent) and receptor-subtype differences of alpha2-adrenergic receptors (alpha2-ARs). To study the molecular basis of the selectivity of UK 14,304, we compared a series of conservative serine-cysteine exchange mutants at ligand-accessible positions in transmembrane domain 5 of the human and mouse alpha2A-ARs. UK 14,304 bound with approximately 200-fold higher affinity to the human alpha2A-AR wild-type receptor compared with the human alpha2A-ARSer201 mutant, but only an approximately fivefold difference was seen with the corresponding mouse alpha2A-AR variant. These effects of cysteine-serine exchanges only involved the agonist low-affinity forms of the receptors, as the affinity of [3H]UK 14,304 for the agonist high-affinity receptor populations was not influenced. The apparent affinities of a set of eight structurally diverse alpha2-AR ligands (six agonists and two antagonists) were not influenced significantly by the cysteine-serine exchanges (except for oxymetazoline and yohimbine, with up to nine- and eightfold differences in affinity, respectively). We conclude that position 201 (a) plays a primary role in determining observed subtype/species selectivity of UK 14,304 in competitive antagonist radioligand binding assays and (b) does not determine the subtype selectivity of chlorpromazine.

Benzylimidazolines as h5-HT1B/1D serotonin receptor ligands: a structure-affinity investigation.

Benzylimidazolines may represent a class of 5-HT1D ligands that has yet to be exploited. On the basis of a previous report that the 2-(substituted-benzyl)imidazoline alpha-adrenergic agonist oxymetazoline (8) binds with high affinity at calf brain 5-HT1D receptors, we explored the structure-affinity relationships of a series of related derivatives. Each of the aromatic substituents was removed and then reinstated in a systematic manner to determine the influence of the individual substituents on binding. It was found that all of the aromatic substituents of 8 act in concert to impart high affinity. However, although the 3-hydroxy group could be removed without significantly reducing affinity for h5-HT1D (i.e., human 5-HT1Dalpha) receptors, this modification reduced h5-HT1B (i.e., human 5-HT1Dbeta) receptor affinity by nearly 50-fold. The 2, 6-dimethyl groups also contribute to binding but seem to play a greater role for h5-HT1B binding than h5-HT1D binding. With the appropriate structural modifications, several compounds were identified that display 20- to >100-fold selectivity for h5-HT1D versus h5-HT1B receptors. Preliminary functional data suggest that these compounds behave as agonists. Given that 5-HT1D agonists are currently being explored for their antimigraine action and that activation of h5-HT1B receptors might be associated with cardiovascular side effects, h5-HT1D-selective agents may offer a new lead for the development of therapeutically efficacious agents.

Characterization of alpha2-adrenoceptor subtypes in pregnant human myometrium.

The aim of the present investigation was to determine which subtypes of the alpha2-adrenoceptors are being expressed in the human pregnant myometrium at term pregnancy. In radioligand binding studies, the specific binding of [3H]rauwolscine to human myometrial membranes was specific and of high affinity with Kd of 2.8 +/- 0.6 nMand Bmax of 95 +/- 5 fmol/mg protein. Results from competition for the binding of [3H]rauwolscine using subtype-selective ligands, oxymetazoline (alpha2A-subptype), chlorpromazine (alpha2B-subtype) and prazosin (alpha2B-alpha2C-subtype), suggested that the alpha2A- and alpha2B-subtypes are being co-expressed. In order to examine if also the alpha2C-subtype is being expressed we used an optimal concentration of oxymetazoline or chlorpromazine which would block the high-affinity site, equivalent to the alpha2A- and alpha2B-subtype respectively. Competition curves of both oxymetazoline and chlorpromazine still showed a significantly better fit using a two-site model, suggesting that the alpha2C-subtype also is being expressed. The expression of alpha2C-subtype mRNA was confirmed using reverse transcription-polymerase chain reaction on mRNA isolated from myometrial biopsies. In conclusion, our results suggest that all three subtypes of alpha2-adrenoceptors are being coexpressed in the human myometrium at term pregnancy and that alpha2-expression is dominated by the alpha2A-subtype.

Alterations in the central nervous alpha 2-adrenoceptor system under chronic psychosocial stress.

It has recently been shown for the tree shrew that after 10 days of psychosocial stress, when the central nervous noradrenergic and adrenergic systems are highly activated, alpha 2-adrenoceptors are down-regulated in brain regions which are involved in the regulation of autonomic functions (Flügge G. et al. (1992) Brain Res. 597, 131-137). The present study demonstrates the dynamic changes occurring in the alpha 2-adrenoceptor system during chronic psychosocial stress. Male tree shrews were submitted to psychosocial stress for two, 10, 21 and 28 days, respectively, and alpha 2-adrenoceptor binding characteristics were determined in the locus coeruleus, the prefrontal cortex, the dorsal motor nucleus of the vagus and the solitary tract nucleus by in vitro receptor autoradiography using the antagonist [3H]RX821002 as a ligand. Competition experiments have shown that the radioligand labels the alpha 2-adrenoceptor subtypes A and C in these brain regions. In the locus coeruleus, receptors were already down-regulated two days after the onset of the stress period and stayed low thereafter. In contrast, in the prefrontal cortex, which is innervated by the locus coeruleus, receptor numbers were decreased only on day 10 of psychosocial stress, then returned to control values and were up-regulated from day 28. In the solitary tract nucleus, a down-regulation of alpha 2-adrenoceptors was observed from day 21 of the stress period onwards. The time-course pattern of receptor changes in the dorsal motor nucleus of the vagus followed that of the locus coeruleus. In all cases, changes in receptor numbers did not exceed 17% of the total alpha 2-adrenoceptor number. Besides the changes in receptor numbers, there was a decrease in affinity for the subtype alpha 2A-selective agonists oxymetazoline and UK 14,304, with the strongest effects occurring on day 10 of the stress period. The time-course pattern of these alterations in receptor affinity did not match the time-course pattern in changes of receptor numbers. However, receptor affinities were more or less decreased during the whole period of psychosocial stress. The present findings demonstrate that prolonged periods of recurrent challenging situations induce dynamic changes in the alpha 2-adrenoceptor system leading to region-specific down- or up-regulation of receptors. It is discussed whether the receptor alterations are agonist-mediated or might be due to stress effects on transcription of the receptor genes. The time-dependent alterations indicate that the responsiveness of the receptor system changes with the duration of the stress period, which may have profound consequences for physiological functions subjected to regulation by alpha 2-adrenoceptors.

Buffers differentially alter the binding of [3H]rauwolscine and [3H]RX821002 to the alpha-2 adrenergic receptor subtypes.

3H-antagonists are known to bind to the alpha-2A adrenergic receptor with higher affinity in glycylglycine buffer than in Tris buffer. The purpose of this study was to examine the effect of buffers on the binding of antagonists to all four subtypes of the alpha-2 adrenergic receptor. Our approach was to examine the effects of glycylglycine, Tris, sodium phosphate (NaPO4) and potassium phosphate buffers on the binding of [3H]rauwolscine, [3H]RX821002, prazosin and oxymetazoline. We found that the affinities for the different subtypes varied with the buffer and the ligands used. Although the Bmax values varied somewhat with the buffers, they were similar for both radioligands for a specific subtype. The highest affinities and Bmax values for both radioligands were generally obtained with NaPO4 buffer. The affinities of antagonists in Tris buffer were always significantly lower than in either NaPO4 or glycylglycine buffer, and the affinities decreased as the concentration of Tris increased. In contrast, the affinity of norepinephrine for the alpha-2B subtype was higher in Tris than in NaPO4 buffer. The buffer effects did not appear to be dependent on the cell membrane composition. There appeared to be some species differences in the effects of buffers on the alpha-2C subtype. These results indicate that buffers affect the binding of antagonists to alpha-2 adrenergic receptors, that not all subtypes are altered in the same manner and that buffers alter the binding of different antagonists differently. It is generally recommended that NaPO4 buffer be used, and that Tris be avoided, when measuring the binding of antagonists to the alpha-2 adrenergic receptor.

Molecular and pharmacological identity of the alpha 2D-adrenergic receptor subtype in bovine retina and its photoreceptors.

The rat cA2-47 gene encodes the pharmacologically defined alpha 2D-adrenergic receptor (alpha 2D-AR) subtype. Previously, the expression of its mRNA was shown in bovine retina by amplification through the reverse transcription-polymerase chain reaction (RT-PCR) of a region corresponding to the rat alpha 2D-AR, amino acid (aa) residues 382-439, indicating the presence of this subtype in this neural tissue. In the present study, the structure of this gene has been probed and the encoded receptor subtype has been characterized in bovine retina and its photoreceptor cells. The deduced aa sequence of the two bovine gene fragments, aa residues 290-375 and aa residues 392-434, demonstrates 77% overall identity with the rat alpha 2D-AR subtype and 80% overall identity with the mouse alpha 2D-AR. The receptor encoded by the bovine gene was expressed in the retina and its photoreceptors with the typical pharmacological characteristics established for the rat alpha 2D-AR subtype: The receptor bound rauwolscine with a KD of 14 nM in the retina and with that of 19 nM in the photoreceptor cells; the binding association rate constant, k+1, for the ligand was 0.012 min-1, the dissociation rate constant, k-1, was 0.14 min-1 and the half-time for dissociation was 5 min. Oxymetazoline displaced the bound [3H]-rauwolscine with an EC50 value of 85 nM, while SK & F 104078, and prazosin displaced the bound [3H]-rauwolscine with the respective IC50 values of 900 nM and 3000 nM. The other alpha 2-AR subtypes -alpha 2A-AR, alpha 2B-AR, alpha 2C-AR-were not detected in the retina and its photoreceptors. Thus, this study shows that the bovine alpha 2D-AR gene is a structural variant of the rat and mouse genes, that the bovine gene encodes the typical pharmacologically defined alpha 2D-AR subtype, that this subtype is present in its exclusive form in the bovine retina and its photoreceptors, where it may be presynaptic in nature.

Evidence for presynaptic location of inhibitory 5-HT1D beta-like autoreceptors in the guinea-pig brain cortex.

The effects of 5-hydroxytryptamine (5-HT) receptor agonists and antagonists on tritium overflow evoked by high K+ were determined in superfused synaptosomes and slices, preincubated with [3H]5-HT, from guinea-pig brain cortex. In addition, we estimated the potencies of 5-HT receptor ligands in inhibiting specific [3H]5-HT binding (in the presence of 8-hydroxy-2(di-n-propylamino)tetralin and mesulergine to prevent binding to 5-HT1A and 5-HT2C sites) to guinea-pig cortical synaptosomes and membranes. 5-HT receptor agonists inhibited the K(+)-evoked tritium overflow from synaptosomes and slices. In synaptosomes the rank order of potencies was 2-[5-[3-(4-methylsulphonylamino)benzyl-1,2,4-oxadiazol-5-yl] -1H-indole-3-yl] ethylamine (L-694,247) > 5-carboxamidotryptamine (5-CT) > oxymetazoline (in the presence of idazoxan) > or = 5-HT > sumatriptan > or = 5-methoxy-3(1,2,3,6-tetrahydropyridin-4-yl)-1H-indole (RU 24969). The potencies of the agonists in inhibiting tritium overflow from slices correlated with those in synaptosomes, suggesting that the same site of action is involved in both preparations. In synaptosomes the nonselective antagonist at cloned human 5-HT1D alpha and 5-HT1D beta receptors, methiothepin, shifted the concentration-response curve for 5-CT to the right (apparent pA2: 7.87). In contrast, ketanserin at a concentration which should block the 5-HT1D alpha, but not the 5-HT1D beta, receptor did not alter the inhibitory effect of 5-CT on tritium overflow. In cortical synaptosomes and membranes, [3H]5-HT bound to a single site with high affinity. In competition experiments, 5-HT receptor agonists and antagonists inhibited specific [3H]5-HT binding. In synaptosomes the rank order was L-694,247 > methiothepin > 5-CT > 5-methoxytryptamine > 5-HT > or = sumatriptan > or = oxymetazoline > RU 24969 > ketanserin > ritanserin. A very similar rank order was obtained in cerebral cortical membranes. The potencies of the 5-HT receptor agonists in inhibiting tritium overflow from synaptosomes and slices correlated with their potencies in inhibiting [3H]5-HT binding to synaptosomes and membranes. In conclusion, the 5-HT receptors mediating inhibition of 5-HT release in the guinea-pig cortex are located on the serotoninergic axon terminals and, hence, represent presynaptic inhibitory autoreceptors. The [3H]5-HT binding sites in cerebral cortical synaptosomes and membranes exhibit the pharmacological properties of 5-HT1D receptors. The correlation between the functional responses and the binding data confirms the 5-HT1D character of the presynaptic 5-HT autoreceptors. According to the results of the interaction experiment of ketanserin and methiothepin with 5-CT on 5-HT release, the presynaptic 5-HT autoreceptors can be subclassified as 5-HT1D beta-like.

Selectivity of the imidazoline alpha-adrenoceptor agonists (oxymetazoline and cirazoline) for human cloned alpha 1-adrenoceptor subtypes.

1. To investigate the structure-activity relationships of alpha-adrenoceptor agonists for the alpha 1-adrenoceptor subtypes, we have compared the imidazoline class of compounds, oxymetazoline and cirazoline, with the phenethylamine, noradrenaline, in their affinities and also in their intrinsic activities in Chinese hamster ovary (CHO) cells stably expressing the cloned human alpha 1-adrenoceptor subtypes (alpha 1a-, alpha 1b-, and alpha 1d-subtypes). 2. Radioligand binding studies with [125I]-HEAT showed that cirazoline and oxymetazoline had higher affinities at alpha 1a-subtype than at alpha 1b- and alpha 1d-subtypes, while noradrenaline had higher affinity at the alpha 1d-subtype than at alpha 1a- and alpha 1b-subtypes. 3. In functional studies, cirazoline caused transients of cytosolic Ca2+ concentrations ([Ca2+]i response) in a concentration-dependent manner and developed a maximal response similar to that to noradrenaline in CHO cells expressing the alpha 1a-subtype, while it acted as a partial agonist at alpha 1b- and alpha 1d-adrenoceptors. Oxymetazoline, on the other hand, was a weak agonist at alpha 1a-adrenoceptors, and has no intrinsic activity at the other subtypes. 4. Using the phenoxybenzamine inactivation method, the relationships between receptor occupancy and noradrenaline-induced [Ca2+]i response for alpha 1a- and alpha 1d-subtypes were found to be linear, whereas it was moderately hyperbolic for the alpha 1b-subtype, indicating the absence of receptor reserves in CHO cells expressing alpha 1a- and alpha 1d-subtypes while there exists a small receptor reserve for CHO cells expressing the alpha 1b-subtype. 5 In summary, our data obtained in cells exclusively expressing a single receptor subtype support the idea that the relative role of agonist affinity and intrinsic activity may vary depending on the subtype of alphal-adrenoceptor.

[Evidence for two alpha 2B-adrenoreceptor isoforms in the renal cortex of salt-sensitive and salt resistant Sabra rats. Effect of salt loading]. / Distinction de deux isofromes de récepteurs alpha 2B-adrénergiques dans le cortex rénal des rats Sabra sensibles et résistants au sel. Effet d'une surcharge en sel.

alpha 2-adrenoceptors are involved in various renal functions regulating blood pressure. They were classified in subtypes whom genes were identified in both humans and rats. In rat renal cortex it was evidenced that the alpha 2B isoform is predominant. This result was confirmed in Sabra rats. However, the renal cortex alpha 2B density is higher in salt-sensitive (SBH) than in salt-resistant (SBN) Sabra rats. alpha 2B-adrenoceptors were recently subclassified in two pharmacologically distinct subtypes exhibiting high and low affinity for guanoxabenz and respectively called alpha 2B1 and alpha 2B2. We studied sodium loading effect on alpha 2B1 and alpha 2B2 distribution in Sabra rat renal cortex using competition experiments between [3H]-yohimbine and guanoxabenz. The rats were submitted to normal (0.2%) or high sodium diet (8%) for six weeks. Under normal diet, proportion alpha 2B1 and alpha 2B2 was similar in SBH and SBN. Nevertheless, their respective densities were significantly higher in SBH as compared to SBN (alpha 2B1: 90.6 +/- 4.1 vs 57.4 +/- 2.5 fmoles/mg prot, p < 0.0001; n = 5; alpha 2B2: 102.7 +/- 4.0 vs 66.4 +/- 4.6 fmoles/mg prot; p < 0.0001; n = 5). Under high sodium diet the distribution of these two isoforms was altered. The densities of alpha 2B1 were decreased by 27.0 +/- 5.9% in SBH (68.0 +/- 4.0 fmoles/mg prot; p < 0.0001, n = 5) and by 47.3 +/- 7.4% for SBN (29.2 +/- 3.1 fmoles/mg prot; p < 0.0001; n = 5). Conversely, the densities of alpha 2B2 were increased by 28.3 +/- 5.4% in SBH (131.1 +/- 9.5 fmoles/mg prot; p < 0.001; n = 5) and by 75.0 +/- 17% in SBN (123.2 +/- 9.1 fmoles/mg prot; p < 0.0001; n = 5). In conclusion, alpha 2B1- and alpha 2B2-adrenoceptor subtypes are found in renal cortex of both SBH and SBN. Our data demonstrated an equal distribution of these two isoforms between SBH and SBN under normal salt diet. This distribution is largely altered, especially in SBN, by the high sodium diet. From these modifications might result differential renal responses to activation of alpha 2B-adrenoceptors between SBH and SBN, and consequently responsible for normal or high blood pressure after high sodium diet.

A thermodynamic analysis of estrogen regulation of alpha 2-adrenoceptor binding.

We previously demonstrated that in vivo estradiol treatment markedly attenuates alpha 2-adrenoceptor function and coupling to G-proteins in the hypothalamus of female rats. Ligand binding studies indicated that 48 h exposure to estradiol decreases the number of alpha 2-adrenergic receptors in the agonist high affinity state. In the present studies, when [3H]RX821002 was used to label brain alpha 2-adrenoceptors, the density of binding sites significantly increased in the hypothalamus and preoptic area 48 h after estrogen treatment. Moreover, the thermodynamics of ligand binding to alpha 2-adrenergic receptors in membranes of female rat hypothalamus were modified by the same estradiol treatments that reduce alpha 2-adrenoceptor function. In hypothalamic membranes from ovariectomized control rats, antagonist (RX821002)-receptor binding was primarily entropy-driven while agonist (oxymetazoline) binding had a higher enthalpy component. In membranes from estradiol-exposed animals, the entropic contribution to both agonist and antagonist bindings was markedly increased, and the enthalpy component was reduced. Since the thermodynamic characteristics of ligand-receptor binding are strongly correlated with efficacy in activating signal transduction [36], these data raise the intriguing possibility that steroids regulate transmembrane signaling by stabilization of a receptor conformation with reduced intrinsic efficacy.