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Psychostimulants, including methylphenidate (MPH), improve cognitive processes dependent on the prefrontal cortex (PFC) and extended frontostriatal circuitry. In both humans and animals, systemic MPH improves certain cognitive processes, such as working memory, in a narrow inverted-U-shaped manner. In contrast, other processes, including attention-related, are improved over a broader/right-shifted dose range. The current studies sought to elucidate the potential circuit and receptor mechanisms underlying the divergent dose-dependent procognitive effects of psychostimulants. We first observed that, as with working memory, although sustained attention testing was highly dependent on multiple frontostriatal regions, only MPH infusion into the dorsomedial PFC improved task performance. Importantly, the dose-response curve for this action was right-shifted relative to working memory, as seen with systemic administration. Additional studies examined the receptor mechanisms within the PFC associated with the procognitive actions of MPH across working memory and sustained attention tasks. We observed that PFC α2 and D1 receptors contributed to the beneficial effects of MPH across both cognitive tasks. However, α1 receptors only contributed to MPH-induced improvement in sustained attention. Moreover, activation of PFC α1 receptors was sufficient to improve sustained attention. This latter action contrasts with the impairing actions of PFC α1 receptors reported previously for working memory. These results provide further evidence for a prominent role of the PFC in the procognitive actions of MPH and demonstrate the divergent dose sensitivity across cognitive processes aligns with the differential involvement of PFC α1 receptors.

At present, it is not clear whether α 1 -adrenoceptors in the prelimbic cortex (PrL) are involved in Parkinson's disease-related depression. Here we examined effects of PrL α 1 -adrenoceptors on depressive-like behaviors in rats with unilateral 6-hydroxydopamine lesions of the medial forebrain bundle. The lesion induced depressive-like responses as measured by the sucrose preference and forced swim tests compared to sham-operated rats. Intra-PrL injection of α 1 -adrenoceptor agonist phenylephrine induced or increased the expression of depressive-like behaviors in sham-operated and the lesioned rats. Further, intra-PrL injection of α 1 -adrenoceptor antagonist benoxathian produced antidepressant effects in two groups of rats. Intra-PrL injection of phenylephrine increased the mean firing rate of PrL pyramidal neurons in both sham-operated and the lesioned rats, while benoxathian decreased the mean firing rate of the neurons. Compared to sham-operated rats, the duration of phenylephrine and benoxathian action on the firing rate of the pyramidal neurons was shortened in the lesioned rats. Neurochemical results showed that intra-PrL injection of phenylephrine or benoxathian increased or decreased dopamine and noradrenaline and serotonin levels in the medial prefrontal cortex, ventral hippocampus and habenula in sham-operated and the lesioned rats, respectively. Altogether, these results suggest that activation and blockade of α 1 -adrenoceptors in the PrL change the firing activity of the pyramidal neurons, and then increase or decrease levels of three monoamines in the limbic and limbic-related brain regions, which are involved in the regulation of depressive-like behaviors. Additionally, the results also suggest that the dopaminergic lesion leads to hypofunctionality of α 1 -adrenoceptors on pyramidal neurons of the PrL., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Trigeminal neuropathic pain is described as constant excruciating facial pain. The study goal was to investigate the role of nucleus locus coeruleus (LC) in a model of chronic orofacial neuropathic pain (CCI-ION). The study examines LC's relationship to both the medullary dorsal horn receiving trigeminal nerve sensory innervation and the medial prefrontal cortex (mPFC). LC is a major source of CNS noradrenaline (NA) and a primary nucleus involved in pain modulation. Although descending inhibition of acute pain by LC is well established, contribution of the LC to facilitation of chronic neuropathic pain is also reported. In the present study, a rat orofacial pain model of trigeminal neuropathy was induced by chronic constrictive injury of the infraorbital nerve (CCI-ION). Orofacial neuropathic pain was indicated by development of whisker pad mechanical hypersensitivity. Hypersensitivity was alleviated by selective elimination of NA neurons, including LC (A6 cell group), with the neurotoxin anti-dopamine-β-hydroxylase saporin (anti-DβH-saporin) microinjected either intracerebroventricularly (i.c.v.) or into trigeminal spinal nucleus caudalis (spVc). The GABA A receptor antagonist, bicuculline, administered directly into LC (week 8) inhibited hypersensitivity. This indicates a valence shift in which increased GABA A signaling ongoing in LC after trigeminal nerve injury paradoxically produces excitatory facilitation of the chronic pain state. Microinjection of NAα1 receptor antagonist, benoxathian, into mPFC attenuated whisker pad hypersensitivity, while NAα2 receptor antagonist, idazoxan, was ineffective. Thus, GABA A -mediated activation of NA neurons during CCI-ION can facilitate hypersensitivity through NAα1 receptors in the mPFC. These data indicate LC is a chronic pain generator., (Published by Elsevier Ltd.)

The aim of the present study was to investigate the effect of microinjection of benoxathian, selective α1 adrenoceptor antagonist, into the ventrolateral orbital cortex (VLO) on morphine-induced behavioral sensitization and its underlying molecular mechanism in rats. A single morphine treatment protocol was used in establishing the behavioral sensitization model. The effect of bilateral intra-VLO benoxathian injection on locomotor activity was examined and the protein expression levels of α1 adrenoceptors and activation of extracellular signal-regulated kinase (ERK) in the VLO were detected after locomotor test. The results showed that a single injection of morphine could induce behavioral sensitization by a low challenge dosage of morphine after a 7-days drug free period. Benoxathian significantly suppressed the expression but not the development of morphine-induced behavioral sensitization. Morphine treatment significantly elicited ERK phosphorylation and downregulated the expression level of α1 adrenoceptors in the VLO. In addition, intra-VLO benoxathian injection enhanced the expression levels of α1 adrenoceptors and phosphorylated ERK. These results suggest that α1 adrenoceptors in the VLO are involved in regulating the expression of morphine-induced behavioral sensitization. The effect of decreased locomotor activity by blocking α1 adrenoceptors might be associated with activation of ERK in the VLO., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

A series of 6-substituted 2-benzoxathiine-2,2-dioxides were synthesized starting from 2,5-dihydroxybenzaldehyde, and then screened in vitro for their inhibition properties against five human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms. All the compounds showed excellent selectivity against the mitochondrial (hCA VA) and the tumor associated (hCA IX and XII) enzymes.

Neonatal ventral hippocampus (nVH) lesion in rats is a useful model to study developmental origins of adult cognitive deficits and certain features of schizophrenia. nVH lesion-induced reorganization of excitatory and inhibitory neurotransmissions within prefrontal cortical (PFC) circuits is widely believed to be responsible for many of the behavioral abnormalities in these animals. Here we provide evidence that development of an aberrant medial PFC (mPFC) α-1 adrenergic receptor (α-1AR) function following neonatal lesion markedly affects glutamatergic synaptic plasticity within PFC microcircuits and contributes to PFC-related behavior abnormalities. Using whole-cell patch-clamp recording, we report that norepinephrine-induced α-1AR-dependent long-term depression (LTD) in a subset of cortico-cortical glutamatergic inputs is strikingly diminished in mPFC slices from nVH-lesioned rats. The LTD impairment occurs in conjunction with completely blunted α-1AR signaling through extracellular signal-regulated kinase 1/2. These α-1AR abnormalities have functional significance in a mPFC-related function, that is, extinction of conditioned fear memory. Post-pubertal animals with nVH lesion show significant resistance to extinction of fear by repeated presentations of the conditioned tone stimulus. mPFC infusion of an α-1AR antagonist (benoxathian) or LTD blocking peptide (Tat-GluR23Y) impaired fear extinction in sham controls, but had no significant effect in the lesioned animals. The data suggest that impaired α-1 adrenergic regulation of cortical glutamatergic synaptic plasticity may be an important mechanism in cognitive dysfunctions reported in neurodevelopmental psychiatric disorders.

Most clinically-used antidepressants acutely increase monoamine levels in synaptic clefts, while their therapeutic effects often require several weeks of administration. Slow neuroadaptive changes in serotonergic neurons are considered to underlie this delayed onset of beneficial actions. Recently, we reported that sustained exposure of rat organotypic raphe slice cultures containing abundant serotonergic neurons to selective serotonin (5-HT) reuptake inhibitors (citalopram, fluoxetine and paroxetine) caused the augmentation of exocytotic serotonin release. However, the ability of other classes of antidepressants to evoke a similar outcome has not been clarified. In this study, we investigated the sustained actions of two tricyclic antidepressants (imipramine and desipramine), one tetracyclic antidepressant (mianserin), three 5-HT and noradrenaline reuptake inhibitors (milnacipran, duloxetine and venlafaxine) and one noradrenergic and specific serotonergic antidepressant (mirtazapine) on serotonin release in the slice cultures. For seven of nine antidepressants, sustained exposure to the agents at concentrations of 0.1-100 μ m augmented the level of increase in extracellular serotonin. The rank order of their potency was as follows: milnacipran>duloxetine>citalopram>venlafaxine>imipramine>fluoxetine>desipramine. Neither mirtazapine nor mianserin caused any augmentation. The highest augmentation by sustained exposure to milnacipran was partially attenuated by an α 1-adrenoceptor antagonist, benoxathian, while the duloxetine-, venlafaxine- and citalopram-mediated increases were not affected. These results suggest that inhibition of the 5-HT transporter is required for the enhancement of serotonin release. Furthermore, the potent augmentation by milnacipran is apparently due to the accompanied activation of the α 1-adrenoceptor.

As part of our efforts to develop safer selective estrogen receptor modulators (SERMs), compound I {(2S,3R)-(+)-3-(3-hydroxyphenyl)-2-[4-(2-pyrrolidin-1-ylethoxy)-phenyl]-2,3-dihydro-1,4-benzoxathiin-6-ol} was previously identified as a lead for further development. Subsequent studies showed that compound I is genotoxic in both in vitro Chinese hamster ovary (CHO) cells and in vivo mouse studies. To better understand the possible mechanisms for the observed genetoxicity effects, in vitro incubations of I with liver microsomes of human, monkey, and mouse in the presence of adenine were performed, which led to the detection of five adenine adducts. The formation of these adducts was NADPH-dependent, suggesting the involvement of oxidative bioactivation catalyzed by cytochrome P450 enzymes. The mechanism for the formation of the major adenine adduct (A1) involves the formation of a reactive ring-opened para-quinone intermediate. The formation of four other adenine adducts may involve the formation of a reactive epoxide or ortho-quinone intermediate. Furthermore, incubations of compound I with human hepatocytes showed dose-dependent DNA damages in Comet assays. All of the above suggest that some reactive metabolites of compound I, formed through bioactivation mechanisms, have a potential to interact with DNA molecules in vitro and in vivo. This may be one of the causes of the genotoxicity observed preclinically both in vitro and in vivo. This case study demonstrated an approach using in vitro DNA trapping assays for assessing the genotoxicity potential of drug candidates.

N-methyl-D-aspartate (NMDA) glutamate receptors mediate fast neurotransmission and regulate synaptic plasticity in the brain. Disruption of NMDA receptor-mediated signaling by noncompetitive antagonists, such as PCP or ketamine, evokes psychotomimetic behaviors, although the cellular mechanisms by which hypofunctional NMDA receptor signaling drives behavioral pathology are still unclear. Activation of glycogen synthase kinase-3 (GSK-3) has been implicated in the cellular neurotoxicity of NMDA receptor antagonists. Accordingly, in the present study we examined the ability of GSK-3 inhibitors, SB216763 and 1-azakenpaullone, to reverse the behavioral aberrations induced by ketamine. Male NMRI mice received intracerebroventricular (i.c.v.) injection of the GSK-3 inhibitors, SB216763 and 1-azakenpaullone, 5 min prior to ketamine administration. Locomotor activity, rotarod performance, prepulse inhibition, novel object recognition, and duration of loss of righting reflex were monitored. GSK-3 inhibitors attenuated ketamine-induced locomotor hyperactivity, motor incoordination, sensorimotor impairment, and cognitive deficits, but did not affect ketamine anesthesia. These data support an important role of GSK-3 in the expression of behavioral aberrations associated with NMDA receptor hypofunction, and suggest that GSK-3 inhibitors may ameliorate certain behavioral and cognitive dysfunctions in patients with schizophrenia., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Molecular dynamics simulations were performed to investigate the distinct uterine activity of ten dihydrobenzoxathiin diastereomers against human estrogen receptor (ER) α. These diastereomers share similar binding mode to ER α ligand binding domain (LBD). Dihydrobenzoxathiin diastereomers with full antagonistic activity form more stable hydrogen bonds with Glu353 and His524 of ER α LBD than corresponding diastereomers. The molecular mechanics based generalized born surface area (MM-GBSA) analysis revealed that van der Waals interactions are predominant to the binding of dihydrobenzoxathiin diastereomers to ER α LBD. The per-residue free energy decomposition analysis revealed that the uterine activity difference is contributed mainly by electrostatic interactions. Our study provides mechanistic insights into the difference of uterine activity for dihydrobenzoxathiin diastereomers., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Preliminary data showed that α1-adrenergic antagonists induce apoptosis and a switch towards megakaryocytic differentiation in human erythroleukemia cells. To test the hypothesis whether survival and differentiation of erythroleukemia cells are under control of α1-adrenergic signalling, we examined α1-adrenoceptor expression of erythroleukemia cells and compared the in vitro effects of α-adrenergic antagonists with those of agonists. We discovered that α1-adrenergic agonists suppress both erythroid differentiation and growth of erythroleukemia cells concomitant with lipofuscin accumulation, autophagy and necrotic cell death. α1-adrenergic agonists also inhibit the in vitro growth of physiologic hematopoietic progenitors obtained from umbilical cord blood with high selectivity for the erythroid lineage. Interestingly, the observed effects could not be related to α1-adrenoceptors, even though agonists and antagonists displayed opposing effects regarding cellular growth and differentiation of erythroleukemia cells. Our data suggest that the effects of α1-adrenergic drugs are related to a non-adrenoceptor binding site, controlling the fate of erythroid progenitor cells towards differentiation and cell death. Since the observed effects are not mediated through adrenoceptors, the physiologic relevance of our data remains unclear, so far. Nevertheless, the identification of the still unknown binding site(s) might disclose new insights into regulation of erythroid differentiation and cell death., (Copyright © 2011 Elsevier Inc. All rights reserved.)

While stressors are known to increase medial prefrontal cortex (PFC) glutamate (GLU) levels, the mechanism(s) subserving this response remain to be elucidated. We used microdialysis and local drug applications to investigate, in male Long-Evans rats, whether the PFC GLU stress response might reflect increased interhemispheric communication by callosal projection neurons. We report here that tail-pinch stress (20 min) elicited comparable increases in GLU in the left and right PFC that were sodium and calcium dependent and insensitive to local glial cystine-GLU exchanger blockade. Unilateral ibotenate-induced PFC lesions abolished the GLU stress response in the opposite hemisphere, as did contralateral mGlu(2/3) receptor activation. Local dopamine (DA) D(1) receptor blockade in the left PFC potently enhanced the right PFC GLU stress response, whereas the same treatment applied to the right PFC had a much weaker effect on the left PFC GLU response. Finally, the PFC GLU stress response was attenuated and potentiated, respectively, following alpha(1)-adrenoreceptor blockade and GABA(B) receptor activation in the opposite hemisphere. These findings indicate that the PFC GLU stress response reflects, at least in part, activation of callosal neurons located in the opposite hemisphere and that stress-induced activation of these neurons is regulated by GLU-, DA-, norepinephrine-, and GABA-sensitive mechanisms. In the case of DA, this control is asymmetrical, with a marked regulatory bias of the left PFC DA input over the right PFC GLU stress response. Together, these findings suggest that callosal neurons and their afferentation play an important role in the hemispheric specialization of PFC-mediated responses to stressors.

2,3-dihydrobenzo[b][1,4]oxathiine represents a valuable pharmacophoric heterocyclic nucleus known since very long time. Initially, together with some patents reporting the use of these compounds as herbicides or lipogenesis inhibitors, several papers reported their ability as melatonin, histamine and serotonin receptor ligands, alpha-adrenoreceptor blockers as well as non-glycoside sweeteners. This wide range of biological activities has been recently further improved by studies stating their activity as antimycotics, multi-defense antioxidants and estrogen receptor ligands. The last insights regarding the preparation, the biological activity and the structure activity relationship (SAR) of derivatives containing the dihydrobenzoxathiine skeleton will be discussed in this review.

The erythroleukemia cell lines K562 and human erythroleukemia (HEL) are established models to study erythroid and megakaryocytic differentiation in vitro. In this study, we show that the alpha1-adrenergic antagonists, benoxathian and prazosin, inhibit the proliferation and induce apoptosis in K562 and HEL cells. Furthermore, both tested substances induced the expression of the megakaryocytic marker CD41a, whereas the expression of the erythroid marker glycophorin-a was decreased or unchanged. Even though the expression of differentiation markers was similar after benoxathian and prazosin treatment in both cell lines, endomitosis of erythroleukemia cells was observed only after prazosin treatment. So far, benoxathian and prazosin are the first described extracellular ligands, which cause megakaryocytic differentiation in K562 and HEL cells. In summary, these results indicate a possible role of alpha1-adrenergic receptor signaling in the regulation of erythroid and megakaryocytic differentiation, even though the receptor dependence of the observed effects needs further investigation.

Considering the data on the low level of self-organization (self-synchronization) of protein synthesis rhythm in aging, we studied the possible interference of the signaling factors of self-organization, gangliosides and catecholamines, as well as catecholamine reception. Experiments were carried out on primary cultures of rat hepatocytes on slides. Inhibited ganglioside synthesis did not prevent the organization of protein synthesis rhythm by the alpha-adrenomimetic agent phenylephrine. Upon the blockade of alpha-receptors by prazosin, the protein synthesis rhythm was observed after the exposure to gangliosides. Alpha-adrenolytic agents prazosin and benoxathian abolished the synchronizing effect of the beta-adrenomimetic isoproterenol. A mixture of alpha- and beta-adrenomimetic agents inhibited the protein synthesis rhythm-organizing effect of noradrenaline. Thus, the signaling molecules of self-organization of protein synthesis function independently via specific receptors.

A series of eleven 2- and 6-substituted (R,S)-9-(2,3-dihydro-1,4-benzoxathiin-3-ylmethyl)-9H-purine derivatives was obtained by applying a standard Mitsunobu protocol that led to a six-membered ring contraction from (R,S)-3,4-dihydro-2H-1,5-benzoxathiepin-3-ol via an episulfonium intermediate. The signal approximately delta=151 ppm, which corresponds to the C4' carbon atom, is unequivocal proof of the N9' regioisomer. The potential of the target molecules as anticancer agents is reflected in their activity against the MCF-7 cancer cell line. The most active compounds have IC(50) values of (6.18+/-1.70) and (8.97+/-0.83) microM. The results indicate that the anticancer activity for the most active compounds is correlated with their capacity to induce apoptosis.

Rationale: The medial prefrontal cortex (PFC) receives stress-sensitive dopamine (DA) and noradrenergic (NE) projections from the ventral tegmental area and locus coeruleus, respectively, and evidence from various sources point to a complex functional interaction between these two systems. Stress will also stimulate DA transmission in the nucleus accumbens (NAcc), and our previous work has shown that this response is under the indirect inhibitory control of a DA-sensitive mechanism in PFC., Objective: We examined the possibility that the NAcc DA stress response is also modulated by prefrontal cortical NE., Materials and Methods: We used voltammetry to study in freely behaving rats the effects of local applications of alpha(1) (benoxathian 0.1, 1, 10 nmol), alpha(2) (SKF86466), and beta(1/2) (alprenolol) receptor selective antagonists into the PFC on the NAcc DA response to tail-pinch stress., Results: The NAcc DA stress response was dose-dependently inhibited by local PFC blockade of alpha(1) receptors. Additional tests revealed, however, that the DA stress response in NAcc is unaffected after local alpha(1) receptor activation with cirazoline. Furthermore, at equivalent doses, neither alpha(2) nor beta(1/2) receptor blockade significantly affected the NAcc DA stress response., Conclusions: These data indicate that stress-induced activation of subcortical DA transmission is modulated by the NE input to PFC acting at alpha(1) receptors. They suggest that, under normal circumstances, this system exerts a facilitatory or enabling influence on the NAcc DA stress response.

The biological effects of catecholamines in mammalian pigment cells are poorly understood, but in poikilothermic vertebrates they regulate the translocation of pigment granules. We have previously demonstrated in SK-Mel 23-human melanoma cells the presence of low affinity alpha(1)-adrenoceptors, which mediate a decrease in cell proliferation and increase in tyrosinase activity, with no change of tyrosinase expression. In this report, we investigated the signalling pathways involved in these responses. Calcium mobilization in response to phenylephrine (PHE), an alpha(1)-adrenergic agonist, was investigated by confocal microscopy, and no change of fluorescence during the treatment was observed, suggesting that calcium is not involved in the signalling pathway activated by alpha(1)-adrenoceptors in SK-Mel 23 cells. cAMP levels, determined by enzyme-immunoassay, were significantly increased by PHE (10(-5)-10(-4)M), that could be blocked by the alpha(1)-adrenergic antagonist benoxathian (10(-5)-10(-4)M). Several biological assays were then performed with PHE, for 72 h, in the absence or presence of various signalling pathway inhibitors, in an attempt to determine the intracellular messengers involved in the responses of proliferation and tyrosinase activity. Our results suggest the participation of p38 and ERKs in PHE-induced decrease of proliferation, and possibly also of cAMP and protein kinase A. Regarding PHE-induced increase of tyrosinase activity, it is suggested that the following signalling components are involved: cAMP/PKA, PKC, PI3K, p38 and ERKs., (Copyright 2005 John Wiley & Sons, Ltd.)

The role of adrenoceptor subtypes was studied in rat brown adipose tissue (BAT). The type II 5'-deiodinase (5'DII) was activated in response to simultaneous stimulation by beta3- and alpha1-adrenergic agonists, BRL 37344 or CGP 12177, and cirazoline, in brown adipocytes. Inhibition of the alpha1- and beta-adrenergic phenylephrine-stimulated 5'DII activity was obtained by the alpha1-adrenergic antagonists in the order of prazosin >/= wb 4101 > 5-methylurapidil. In comparison, the binding of [3H]prazosin to rat BAT plasma membranes was inhibited by alpha1-adrenergic antagonists in the order of prazosin > WB 4101 = benoxathian > 5-methylurapidil. Although the order of the alpha1-adrenergic competition seemed to be rather typical for the alpha1B-adrenergic receptors, a molecular analysis on adrenoceptor mRNAs should be made to confirm the exact alpha1-adrenergic subtypes at the level of brown adipocytes, since the possibility of a mixture of different receptor subtypes in brown fat cells and/or tissue may interact with the pharmacological characterization. Thus, specific alpha1- and beta-adrenoceptor subtypes participate in the regulation of 5'DII activity in the rat brown adipocytes, and therefore, an impaired alpha1- and beta-adrenergic co-work may be involved in a defective BAT function, e.g., in obese Zucker rats, too. An interesting possibility is that the decreased number of alpha1-adrenoceptors in the BAT of obese Zucker rats is due to the decrease in the alpha1B-adrenoceptor subtype which would further be involved especially in the regulation of BAT 5'DII activity.

Chronic intermittent cold stress sensitises activation of the hypothalamic-pituitary-adrenal (HPA) axis by novel acute stress. We have shown that enhanced noradrenergic function in limbic forebrain contributes to HPA sensitisation. In the present study, we investigated whether chronic intermittent cold also induced changes in noradrenergic function in the paraventricular nucleus (PVN), the primary mediator of the HPA stress response. Rats were exposed to chronic intermittent cold (7 days, 6 h per day, 4 degrees C). On the day after final cold exposure, there were no differences in baseline plasma ACTH, but the peak ACTH response to 30 min of acute immobilisation stress was greater in cold-stressed rats compared to controls. Bilateral microinjection of the alpha(1)-adrenergic receptor antagonist benoxathian into the PVN reduced acute stress-induced adrenocorticotrophic hormone (ACTH) levels by approximately 25% in controls. Furthermore, in cold-stressed rats, all of the sensitisation of the ACTH response was blocked by benoxathian, to a level comparable to benoxathian-treated controls. In a second study using microdialysis to measure norepinephrine release in the PVN, there were no differences in either baseline or acute stress-induced increases in norepinephrine release in the PVN of cold-stressed rats compared to controls. Thus, in a third study, we tested potential alterations in postsynaptic alpha(1)-receptor sensitivity after chronic cold stress. Dose-dependent activation of ACTH secretion by microinjection of the alpha(1)-adrenergic receptor agonist, phenylephrine, into the PVN was significantly enhanced in cold-stressed rats compared to controls. Thus, the sensitised HPA response to acute stress after chronic intermittent cold exposure is at least partly attributable to an enhanced response to alpha1-adrenergic receptor activation in the PVN. Chronic stress-induced plasticity in the acute stress response may be important for stress adaptation, but may also contribute to pathophysiological conditions associated with stress. Thus, understanding the neural mechanisms underlying such adaptations may help us understand the aetiology of such disorders, and contribute to the future development of more effective treatment or prevention strategies.

An optimized side chain for dihydrobenzoxathiin SERAMs was discovered and attached to four dihydrobenzoxathiin platforms. The novel SERAMs show exceptional estrogen antagonist activity in uterine tissue and an MCF-7 breast cancer cell assay.

[reaction: see text] During the course of drug metabolism studies, a major metabolite of compound 1 was detected in rhesus monkeys and assigned structure 4. The intriguing biotransformation of 1 leading to 4 was confirmed by a 19-step total synthesis starting from resorcinol (11), the key feature of which was the construction of the oxygen bridge utilizing a phenolic oxidation and trapping sequence. In addition, the synthesis of a related metabolite (5) is described.

A series of dihydrobenzoxathiin SERAMs with alkylated pyrrolidine side chains or alkylated linkers was prepared. Minor modifications in the side chain or linker resulted in significant effects on biological activity, especially in uterine tissue.

Activation of the brain noradrenergic system during stress plays an important integrative function in coping and stress adaptation by facilitating transmission in many brain regions involved in regulating behavioural and physiological components of the stress response. The medial amygdala (MeA) has been implicated in modulation of stress-induced activation of the hypothalamic-pituitary-adrenal (HPA) axis, and MeA is a target of innervation from brainstem noradrenergic neurones. However, it is not known whether, and to what extent, activation of the ascending noradrenergic innervation of MeA might modulate stress-induced adrenocorticotropic hormone (ACTH) secretion. In the first experiment in this study, we measured extracellular norepinephrine (NE) levels in MeA using in vivo microdialysis. The concentration of NE in dialysate samples collected in MeA was elevated by more than three-fold over baseline in response to acute immobilisation stress, providing evidence of a possible modulatory role for NE in the MeA during stress. This potential role was then assessed in the second experiment by measuring changes in the elevation of plasma ACTH concentration induced by acute immobilisation stress immediately following bilateral microinjections of alpha1- or beta-adrenergic receptor antagonists directly into MeA. Compared to vehicle-injected controls, the alpha1-receptor antagonist benoxathian dose-dependently and significantly attenuated the ACTH response to acute stress, whereas combined beta1/beta2-receptor blockade in MeA had only a modest effect. These results indicate that MeA does play a role in the stress response, and support the hypothesis that stress-induced activation of NE release in MeA, acting primarily through alpha1 receptors, facilitates activation of the HPA axis in response to acute stress.

We investigated whether changes in noradrenaline neurotransmission in the hypothalamus could explain the hyporesponsiveness of the hypothalamic-pituitary-adrenal (HPA) axis in late pregnancy. Noradrenaline release within the hypothalamic paraventricular nucleus in response to swim stress, as estimated by microdialysis and high-performance liquid chromatography, was lower in 20-day pregnant rats compared to virgin rats. Driving a central noradrenergic pathway using intravenous cholecystokinin increased adrenocorticotropic hormone (ACTH) secretion in virgin rats, but the response was significantly less in 16-day and 20-day pregnant rats. Thus, the activity of noradrenergic inputs to the paraventricular nucleus and the HPA axis is attenuated in late pregnancy. The sensitivity of the HPA axis to noradrenaline in pregnancy was investigated by intracerebroventricular administration of an alpha1-receptor antagonist, benoxathian, before and during exposure to swim stress. In virgin rats, benoxathian increased basal and stress-induced ACTH secretion, but in late pregnant rats the benoxathian effects were attenuated, indicating reduced sensitivity of the HPA axis to noradrenaline neurotransmission and/or the inability of the system to become disinhibited at this time. alpha1A-adrenoreceptor mRNA expression in the parvocellular and magnocellular paraventricular nucleus, measured by in situ hybridisation, was decreased in late pregnant compared to virgin rats. Additionally, blocking endogenous opioid inhibition with naloxone pretreatment restored the ACTH secretory response to cholecystokinin in pregnant rats. Thus, in late pregnancy, there is reduced noradrenergic input to the paraventricular nucleus and reduced alpha1A-receptor expression in the paraventricular nucleus, both of which may contribute to the reduced responsiveness of the HPA axis in pregnancy.

Norepinephrine (NE) plays a complex role in the behavioral state-dependent regulation of sensory processing. However, the role of forebrain NE action in modulating high-order sensory activity has not been directly addressed. In this study, we take advantage of the discrete, feedforward organization of the avian song system to identify a site and mechanism of NE action underlying state-dependent modulation of sensory processing. We have developed an experimental paradigm in which brief arousal repeatedly suppresses song system auditory responsiveness. Using pharmacological manipulations in vivo, we show that infusion of alpha-adrenergic antagonists into the NIf (nucleus interfacialis of the nidopallium), an auditory forebrain area, blocks this state-dependent modulation. We also demonstrate dose-dependent enhancement and suppression of song system auditory response properties by NE and adrenergic agonists. Our results demonstrate that noradrenergic release in a single forebrain area is a mechanism underlying behavioral state-dependent regulation of auditory processing in a neural system specialized for vocal learning.

Dihydrobenzoxathiin analogs (1-11) with modifications on the basic side chain region were prepared and evaluated for estrogen/anti-estrogen activity in both in vitro and in vivo models. The compounds generally maintained a high degree of selectivity for ERalpha over ERbeta, similar to the original lead compound I. Many of the compounds also maintained high potency in the inhibition of human carcinoma MCF-7 cell growth. However, all were less potent in the inhibition of estradiol-triggered uterine growth. This work demonstrates the sensitive nature of modification to the antagonist basic side chain region.

A series of estrogen receptor ligands based on a dihydrobenzoxathiin scaffold is described and evaluated for estrogen/anti-estrogen activity in both in vitro and in vivo models. The most active analogue, 22, was found to be 40-fold ERalpha selective in a competitive binding assay, and 22 demonstrated very potent in vivo antagonism of estradiol driven proliferation in an immature rat uterine weight gain assay.

The discovery and synthesis of dihydrobenzoxathiins as potent, ERalpha subtype selective ligands are described. The most active analogue, 4-D, was found to be 50-fold selective in a competitive binding assay and 100-fold selective in a transactivation assay in HEK-293 cells. The alpha selectivity was postulated to lie in the interaction of the sulfur atom of the benzoxathiin ring with the two discriminating residues in the binding pocket of the receptor isoforms.

Many psychiatric disorders, including depression, post-traumatic stress disorder and other anxiety disorders, result from an interaction between genetic factors and exposure to a sufficiently sensitizing environmental stressor. The inbred Wistar Kyoto (WKY) rat strain has been proposed as a model of stress vulnerability, exhibiting an exaggerated hypothalamic-pituitary-adrenal (HPA) response to stress and susceptibility to gastric ulceration. Previously, we showed that stress-activation of the brain noradrenergic system was deficient in WKY rats, and they lacked noradrenergic facilitation of the HPA response in the lateral bed nucleus of the stria terminalis (BSTL), compared to outbred Sprague-Dawley (SD) controls. Deficient modulatory function of the noradrenergic system may contribute to the stress susceptibility of WKY rats. Thus, we investigated the influence of a sensitizing stimulus, chronic intermittent cold exposure, on neuroendocrine and noradrenergic stress reactivity, and on noradrenergic facilitation of the HPA response in these two strains. Chronic cold exposure (7 days, 4 h/day, 4 degrees C) potentiated activation of the HPA axis by acute immobilization stress, assessed by measuring plasma adrenocorticotropic hormone (ACTH), in both strains, although to a greater extent in WKY rats, and enhanced stress-induced norepinephrine (NE) release in BSTL of WKY but not SD rats. We then compared the influence of chronic cold exposure on noradrenergic modulation of the HPA stress response in BSTL, by measuring changes in acute stress-induced elevation of plasma ACTH after microinjecting the alpha(1)-adrenoreceptor antagonist benoxathian into the BSTL. As shown previously, benoxathian attenuated stress-induced ACTH secretion in control SD but not control WKY rats. After chronic cold, the ACTH response to acute stress was attenuated by benoxathian administration into BSTL of both strains, such that the WKY response was not different from that of SD rats. Thus, chronic cold not only sensitized the release of NE in BSTL of WKY rats, but also restored noradrenergic facilitation of their already-elevated HPA response. Such functional sensitization of a previously-deficient facilitatory system may be one mechanism whereby exposure to repeated or severe stress may induce pathologic dysregulation of the stress response in susceptible individuals, resulting in psychiatric illness.

The synthesis of benzoxathiins bearing a retroamide function is described from 8-hydroxythiochroman, the key step involving the synthesis of the benzoxathiin ring through a sulfonium salt. These new melatonin analogues were evaluated on human receptors MT1 and MT2 and have a similar affinity to that of melatonin itself.

Previous studies suggesting that norepinephrine is directly trophic for the vascular wall have been confounded by concomitant hemodynamic disturbances. Herein, a microcatheter connected to an osmotic minipump was implanted adjacent to the rat carotid for 2-wk perivascular suffusion of agents at systemic levels ~1,000 times below the threshold for altering arterial pressure. Norepinephrine decreased lumen and adventitial areas and circumference by 10, 14, and 5%, respectively (all P < 0.05); a nonsubtype-specific alpha(1)-adrenoceptor (AR) antagonist had no effect. When begun at the time of balloon injury, 2-wk norepinephrine increased lumen loss by 45%, increased neointimal area by 64% and collagen content by 33%, and reduced vessel circumference by 5% (all P < 0.05). alpha(1)-AR antagonists decreased neointimal area by 33% (all P < 0.05). alpha(1)A-AR antagonist reduced lumen loss by 70%, neointimal area by 54%, circumference decline by 84%, and adventitial thickening by 87% (all P < 0.05), whereas alpha(1B)-, alpha(1D)-, alpha(2)- and beta-AR antagonists were without effect. These are the first in vivo studies demonstrating that norepinephrine is directly trophic for the vascular wall and augments injury-induced intimal lesion growth.

Previous research has shown that the effect of exposure to uncontrollable stressors on conditioned fear responding and escape behavior in rats is dependent on serotonergic neural activity in the dorsal raphe nucleus (DRN). The role that norepinephrine released in the DRN plays in producing the behavioral consequences of exposure to inescapable tail shock in rats was investigated in the present study. The selective alpha1 adrenoreceptor antagonist benoxathian was injected into the DRN before exposure to inescapable tail shock or before behavioral testing conducted 24 h later. Benoxathian prevented the impairment of escape responding produced by inescapable shock, but did not reverse this effect when given before testing. The enhancement of conditioned fear produced by prior inescapable shock was attenuated by benoxathian administered before inescapable shock or before behavioral testing. These results support the view that noradrenergic input to the DRN is necessary to produce the behavioral effects of inescapable tail shock., (Copyright 2002 Elsevier Science B.V.)

In the present paper, the cloning and expression of the guinea pig alpha(1A)-adrenoceptor is presented. The nucleotide sequence had an open reading frame of 1401 bp that encoded a 466 amino-acid protein with an estimated molecular mass of approximately 51.5 kDa. When the clone was expressed in Cos-1 cells, specific high-affinity binding of [(3)H]prazosin and [(3)H]tamsulosin was observed. Chloroethylclonidine treatment of membranes slightly decreased the total binding with both radioligands. Binding competition experiments using [(3)H]tamsulosin showed the following potency order: (a) for agonists: oxymetazoline >>epinephrine>norepinephrine>methoxamine, and (b) for antagonists: prazosin> or 5-methyl-urapidil=benoxathian>phentolamine>>BMY 7378 (8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4,5]decane-7,9-dione). Photoaffinity labeling using [(125)I-aryl]azido-prazosin revealed a major broad band with a molecular mass between 70 and 80 kDa. The receptor was functional, as evidenced by an epinephrine-increased production of [(3)H]inositol phosphates that was blocked by prazosin.

Oxytocin secretion is important for the normal progress of parturition in the rat. We tested the hypotheses that contractions of the uterus before pup delivery activate oxytocin neurons, and that they do so via a noradrenergic projection. In anesthetized 22-day (term) pregnant rats, i.v. oxytocin pulses enhanced both uterine contractile activity and the firing rate of oxytocin and vasopressin neurons in the supraoptic nucleus, and these were significantly correlated. The same oxytocin treatment also increased the expression of Fos in both the supraoptic nucleus and the nucleus of the tractus solitarius, but not in 21-day pregnant or virgin rats. In five of eight rats on the day of expected parturition, noradrenaline release in the supraoptic nucleus (sampled by microdialysis) exhibited sudden peaks during oxytocin administration, seen in only one of nine rats given vehicle pulses. Noradrenaline release was significantly greater in rats that went into labor or gave birth to a pup than in rats not in labor. In rats infused with the alpha(1)-noradrenergic receptor antagonist, benoxathian, into the supraoptic nucleus before and during iv oxytocin administration, Fos expression in supraoptic neurons was significantly less than that in vehicle controls. Thus, at term pregnancy, uterine contractions activate both oxytocin and vasopressin neurons in the SON, and this activation involves a noradrenergic pathway.

The alpha(1)-adrenoceptor subtype(s) mediating contractions of the rat mesenteric artery were investigated using the agonists methoxamine, cirazoline, P7480 (N-(4-pyridinyl)-1H-indol-1-amine) and subtype-selective antagonists including BMY 7378 (8-(-2(-4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-8-azaspiro(4, 5)decane-7,9,dione dihydrochloride). pA(2) or apparent pK(B) values of antagonists against methoxamine contractions correlated best with its pK(i) values at the cloned alpha(1b)-(0.88), with cirazoline, antagonists affinities correlated equally well with those at alpha(1a)-(0.79) or the alpha(1b)-(0.81) while with P7480 antagonist affinities correlated best with the alpha(1d)-adrenoceptor subtype (0.94). The low affinity estimate for 5-methylurapidil (7.5) against the alpha(1a)-selective cirazoline suggests an alpha(1A)-subtype mediating contraction is unlikely. Shallow Schild plot slopes of subtype selective antagonists against all three agonists are consistent with heterogeneity of alpha(1)-adrenoceptors. P7480 (putative alpha(1D)-adrenoceptor-selective) acts primarily at this subtype and at another which is more likely to be an alpha(1B)- than an alpha(1A)-adrenoceptor. The results with both agonists and antagonists are consistent with contractions of the rat mesenteric artery being mediated via the alpha(1D)- and possibly alpha(1B)-adrenoceptor.

The primary goal of the study was to identify the types of catecholamines and the associated receptors which might be involved in the recall of the conditioned NK cell response. Specific catecholamine receptor antagonists were selected to block the conditioned NK cell response at the recall step. The regional contents of dopamine (DA), norepinephrine (NE), and epinephrine were determined in the brain of the conditioned animals by using the high performance liquid chromatography with electrochemical detection (HPLC/ED). Results showed that pre-disruption of the central alpha1-, alpha2-, beta1-, beta2-, D1-, or D2-receptors at the conditioned recall stage, interrupted the conditioned enhancement in NK cell activity. The NE contents at the cerebellum, and DA contents at the striatum and hippocampus, were significantly higher in the brain of the conditioned animals when compared to that of the control animals. These information indicated the possible roles of the central noradrenergic and dopaminergic systems in regulating the recall of the conditioned NK cell response.

Conditioned fear or novel environmental stimuli suppress vasopressin (VP) and augment oxytocin (OT) and prolactin (PRL) release in rats. We examined the effects of intracerebroventricular (i.c.v.) injections of adrenoceptor antagonists on these neuroendocrine responses to conditioned fear or novel environmental stimuli in male rats. A beta1 antagonist, metoprolol, blocked the VP but not the OT or PRL response to conditioned fear stimuli, but did not abolish neuroendocrine responses to novel environmental stimuli. A beta2 antagonist, ICI118551, impaired the PRL but not the VP or OT response to fear or novel environmental stimuli. In rats injected with a alpha1 adrenoceptor antagonist, benoxathian, conditioned fear stimuli did not significantly induce the VP, OT or PRL responses. The effects of benoxathian were not due to a general reduction of arousal, since benoxathian did not prevent the VP, OT or PRL response to novel environmental stimuli. These data suggest that beta1 adrenoceptors play a selective role in the VP response to conditioned fear stimuli, as do beta2 adrenoceptors in the prolactin response to conditioned fear and novel environmental stimuli. We conclude that alpha1 adrenoceptors play a facilitative role in VP, OT, PRL responses to conditioned fear stimuli.

1. We have tested the hypothesis that morphine withdrawal excitation of oxytocin neurones that follows from administration of naloxone to morphine-dependent rats is a consequence of excitation of noradrenergic neurones. 2. Female rats were made morphine dependent by intracerebroventricular (i.c.v.) infusion of the opioid at increasing doses over 5 days. On the sixth day, the rats were anaesthetized with urethane or pentobarbitone and prepared for blood sampling to determine plasma oxytocin by radioimmunoassay or for in vivo extracellular recording of the firing rate of identified oxytocin neurones from the supraoptic nucleus. Morphine withdrawal was induced by intravenous (i.v.) injection of the opioid antagonist naloxone (5 mg kg-1). 3. In one group of rats the noradrenergic projections to the hypothalamus were lesioned by i.c.v. injection of 6-hydroxydopamine immediately prior to the induction of morphine dependence. In these rats the oxytocin secretion induced by i.v. cholecystokinin was reduced to 9 % of that seen in sham-lesioned rats but in contrast, no attenuation of morphine withdrawal-induced oxytocin secretion was observed. 4. i.c.v. infusion of the alpha1-adrenoreceptor antagonist benoxathian, at up to 5.3 microg min-1, dose- dependently inhibited the withdrawal excitation of oxytocin neurones in morphine-dependent rats under urethane anaesthesia, and benoxathian reduced withdrawal-induced oxytocin secretion to 37 % of that of vehicle-infused rats. i.c.v. benoxathian also inhibited the activity of oxytocin neurones in morphine-naïve rats. Similarly, microdialysis administration of 2 mM benoxathian directly onto the surface of the supraoptic nucleus reduced the activity of oxytocin neurones by 53 %. 5. Thus noradrenergic systems are not essential for the expression of morphine withdrawal excitation, since chronic neurotoxic destruction of the noradrenergic inputs to the hypothalamus did not affect the magnitude of withdrawal-induced oxytocin secretion. However, tonically active noradrenergic inputs influence the excitability of oxytocin neurones, and acute antagonism of this noradrenergic tone can powerfully impair the ability of oxytocin neurones to exhibit morphine withdrawal excitation.

1. The subtype of alpha1-adrenoceptor mediating contractions to phenylephrine of the rat thoracic aorta, mesenteric artery and pulmonary artery were investigated by use of antagonists which show selectivity between the cloned alpha1-adrenoceptor subtypes in binding studies. 2. Cumulative concentration-contraction curves for phenylephrine were competitively antagonized in the rat thoracic aorta by prazosin (pA2 9.9), WB4101 (pA2 9.6), 5-methylurapidil (pA2 8.1), benoxathian (pA2 9.2) and indoramin (pA2 7.4). These compounds were also competitive antagonists in the mesenteric and pulmonary arteries (except for 5-methylurapidil in the pulmonary artery), (prazosin pA2 9.9 and 9.7; WB4101 pA2 9.8 and 9.6; 5-methylurapidil pA2 7.9 and pK(B) estimate 8.0; benoxathian pA2 8.8 and 9.3; indoramin pA2 7.2 and 7.5, respectively). 3. RS 17053 was not a competitive antagonist in any blood vessel as Schild plot slopes were greater than unity. The pK(B) estimates for RS 17053 were 7.1 in aorta, 7.0 in the mesenteric artery and 7.7 in the pulmonary artery. 4. The alpha1D-subtype selective antagonist BMY 7378 appeared to be non-competitive with shallow Schild plot slopes. The data were better fitted with two lines in all tissues, with Schild plot slopes that were no longer different from unity, except in the pulmonary artery. The higher affinity site for BMY 7378 in the aorta had a pA2 of 9.0, while it was 8.8 and 8.9 in the mesenteric and pulmonary arteries, respectively. 5. MDL73005EF acted in a non-competitive manner in all three blood vessels, with shallow Schild plot slopes. The pK(B) estimates for MDL73005EF were 8.4 in aorta, 7.5 in the mesenteric artery and 8.0 in the pulmonary artery. 6. In all three blood vessels the functionally determined antagonist affinity estimates correlated best with published pKi values for their displacement of [3H]-prazosin binding on membranes expressing cloned alpha1d-adrenoceptors compared with alpha1a- or alpha1b-adrenoceptors. The antagonist affinity estimates in the aorta, mesenteric and pulmonary arteries correlated highly with their previously published pA2 values in rat aorta (alpha1D) and less well with those for alpha1A- and alpha1B-adrenoceptors mediating contraction of the rat epididymal vas deferens and rat spleen, respectively. 7. The results of this study suggest that the contraction to phenylephrine of the rat thoracic aorta, mesenteric artery and pulmonary artery are mediated in part via the alpha1D-subtype of adrenoceptor. The data for both BMY 7378 and MDL73005EF in all three blood vessels are consistent with receptor heterogeneity. However, the identity of the second site is unclear.

1. The selectivity of action of boldine and the related aporphine alkaloids, predicentrine (9-O-methylboldine) and glaucine (2,9-O-dimethylboldine) and alpha 1-adrenoceptor subtypes was studied by examining [3H]-prazosin competition binding in rat cerebral cortex. WB 4101 and benoxathian were used as selective alpha 1A-adrenoceptor antagonists. 2. In the competition experiments [3H]-prazosin (0.2 nM) binding was inhibited by WB 4101 and benoxathian. The inhibition curves displayed shallow slopes which could be subdivided into high and low affinity components (pKi = 9.92 and 8.29 for WB 4101, 9.35 and 7.94 for benoxathian). The two antagonists recognized approximately 37% of the sites with high affinity from among the total [3H]-prazosin specific binding sites. 3. Boldine, predicentrine and glaucine also competed for [3H]-prazosin (0.2 nM) binding with shallow and biphasic curves recognizing 30-40% of the sites with high affinity. Drug affinities (pKi) at the high and low affinity sites were, 8.31 and 6.50, respectively, for boldine, 8.13 and 6.39 for predicentrine, and 7.12 and 5.92 for glaucine. The relative order of selectivity for alpha 1A-adrenoceptors was boldine (70 fold alpha 1A-selective) = predicentrine (60 fold, alpha 1A-selective) > glaucine (15 fold, alpha 1A-selective). 4. Pretreatment of rat cerebral cortex membranes with chloroethylclonidine (CEC, 10 microM) for 30 min at 37 degrees C followed by thorough washing out reduced specific [3H]-prazosin binding by approximately 70%. The CEC-insensitive [3H]-prazosin binding was inhibited by boldine monophasically (Hill slope = 0.93) with a single pKi value (7.76). 5. These results suggest that whereas the aporphine structure shared by these alkaloids is responsible for their selectively of action for the alpha 1A-adrenoceptor subtype in rat cerebral cortex, defined functional groups, namely the 2-hydroxy function, induces a significant increase in alpha 1A-subtype selectivity and affinity.

1. In the present study, depletion of internal Ca2+ stores sensitive to noradrenaline (1 microM) in rat aorta, is the signal for the entry of extracellular Ca2+, not only to refill the stores but also, in our experimental conditions, to activate the contractile proteins. This induces an increase in the resting tone that constitutes, the first functional evidence of this Ca2+ entry. 2. The fact that methoxamine (100 microM) reproduces the same processes as noradrenaline but clonidine (1 microM) does not, indicates that alpha(1)-adrenoceptor activation is related to the increase in the resting tone observed after depletion of adrenoceptor-sensitive internal Ca2+-stores. 3. Benoxathian and WB 4101 (alpha(1A)- and alpha(1D)-adrenoceptor antagonists) selectively inhibit, in a concentration-dependent manner, this mechanical response observed in absence of the agonist, which suggests that these agents can act as inverse agonists and provide a functional model for studying this phenomenon. Since chloroethylclonidine (100 microM) has no effect on this response, the participation of alpha(1B)-adrenoceptors can be ruled out. 4. Contractile responses to noradrenaline (1 microM) in Ca2+-free medium were selectively blocked by chloroethylclonidine. This suggests that the response to noradrenaline in Ca2+-free medium mainly depends on the activation of the alpha(1B)-adrenoceptor subtype.

Pharmacological techniques have defined the existence of two different alpha 1-adrenoceptors, the alpha 1A- and alpha 1B-adrenoceptor subtypes and both of these receptors have been cloned in addition to a cloned alpha 1d-adrenoceptor. A subpopulation of interneurons in layer III of the rat piriform cortex that are excited by 5-hydroxytryptamine (5-HT) via 5-HT2A receptors are also excited by norepinephrine via alpha 1-adrenoceptors. In the present study we determined the pA2 values against the norepinephrine-mediated excitation of piriform cortical interneurons for a number of antagonists that are (1) not selective for alpha 1A- or alpha 1B-adrenoceptors (prazosin), (2) selective for alpha 1A-adrenoceptors (5-methyl urapidil, 2-(2,6-dimethoxy-phenoxyethyl)- aminomethyl-1,4-benzodioxane hydrochloride (WB 4101), benoxathian, phentolamine) and (3) selective for alpha 1B-adrenoceptors (spiperone and risperidone). The pA2 values for the antagonist blockade of norepinephrine-mediated interneuron excitation were significantly correlated to literature values for the pKi values of antagonist binding to the alpha 1B-adrenoceptor (r = 0.919) and the cloned alpha 1b-adrenoceptor (r = 0.849) but were not correlated to the pKi values of antagonist binding to the alpha 1A-adrenoceptor or the cloned alpha 1a- and alpha 1d-adrenoceptor. Thus, we conclude that this population of piriform cortical interneurons is excited by norepinephrine via alpha 1B-adrenoceptors.

Noxious stimuli facilitate oxytocin release from the neurohypophysis. Oxytocin-secreting hypothalamic magnocellular neurosecretory neurons receive excitatory synaptic inputs from noradrenergic neurons in the medulla oblongata. The medulla oblongata includes the A2 noradrenergic and the A1 noradrenergic cells. Here we investigated whether medullary noradrenergic neurons mediate oxytocin release after noxious stimuli in male rats. 5-Amino-2,4-dihydroxy-alpha-methylphenylethylamine, a neurotoxin selective for noradrenergic fibers, was injected into the lateral cerebral ventricle or the medulla. Seven days after the injection, the hypothalamic content of noradrenaline was decreased. In the rats injected with the neurotoxin, the release of oxytocin but not vasopressin after footshocks was impaired. Surgical ablation by suction of the caudal dorsomedial medulla including the A2 cell region did not significantly affect oxytocin release after footshocks, though the surgery abolished oxytocin release after i.v. injection of cholecystokinin octapeptide. In the rats whose A2 cell region had been ablated, an i.c.v. injected alpha 1 adrenoreceptor antagonist, benoxathian, blocked oxytocin release after footshocks. These results demonstrate that brainstem noradrenergic neurons mediate oxytocin release following noxious stimuli in the rat and suggest that responsible noradrenergic neurons are the A1 cells in the caudal ventrolateral medulla.

Four antagonists were examined for their ability to differentiate alpha 2A-from the orthologous alpha 2D-adrenoceptors. The antagonists were (2S,12bS)1',3'-dimethylspiro(1,3,4,5',6,6',7,12b-octah ydro-2H- benzo[b]furo[2,3-a]quinolizine)-2,4'-pyrimidin-2'-one (MK912), 2-[2-(methoxy-1,4-benzodioxanyl)imidazoline (RX 821002), efaroxan and benoxathian. The alpha 2-autoreceptors in rabbit brain cortex were chosen as alpha 2A-and the alpha 2-autoreceptors in guinea-pig brain cortex as alpha 2D-adrenoceptors. Slices of the brain cortex were preincubated with 3H-noradrenaline and then superfused and stimulated electrically by brief pulse trains (4 pulses, 100 Hz) that led to little, if any, alpha 2-autoinhibition. 5-Bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14,304) was used as an alpha 2-adrenoceptor agonist. UK 14, 304 decreased the stimulation-evoked overflow of tritium. The antagonists shifted the concentration-inhibition curve of UK 14, 304 to the right in an apparently competitive manner. Dissociation constants of the antagonists were calculated from the shifts. MK 912, RX 821002 and efaroxan had markedly higher affinity for (guinea-pig) alpha 2D-adrenoceptors (pKd values 10.0, 9.7 and 9.1, respectively) than for (rabbit) alpha 2A-adrenoceptors (pKd 8.9, 8.2 and 7.6, respectively). Benoxathian had higher affinity for alpha 2A-(pKd 7.4) than for alpha 2D-adrenoceptors (pKd 6.9). Ratios calculated from the Kd values of the four compounds differentiated between alpha 2A and alpha 2D up to 100 fold. It is concluded that MK 912, RX 821002, efaroxan and benoxathian are antagonists with high power to differentiate alpha 2A-from alpha 2D-adrenoceptors.

1. The interaction between chloroethylclonidine (CEC) and noradrenaline (NA) has been examined at alpha-adrenoceptors mediating contractions of rat aorta. 2. In rat aorta, the competitive antagonist prazosin, over the concentration-range 0.01-10 microM, produced concentration-dependent shifts in the contractile potency of NA, so that there was no component of the NA contraction resistant to prazosin. 3. The irreversible alpha 1-adrenoceptor antagonists, phenoxybenzamine (PBZ) (1-10 microM) and benextramine (10 microM) produced shifts in potency of NA and reduced the maximum response in a concentration-dependent manner. 4. The irreversible alpha 1-adrenoceptor antagonist, CEC (100 microM), produced a non-parallel shift in the NA concentration-response curve so that low concentrations of NA produced relatively small contractions but relatively high concentrations produced further contractions, so that the maximum response was not significantly reduced. 5. The combination of CEC pretreatment and subsequent prazosin (0.1 microM) produced a parallel shift in the potency of NA. However, prazosin (10 microM) failed to produce any further effect on the response to high concentrations of NA following CEC pretreatment. Hence, a component of the contraction to NA in the presence of CEC was resistant to subsequent prazosin. Likewise, this component was resistant to a combination of prazosin (10 microM) and yohimbine (10 microM). 6. Receptor protection experiments were carried out in which tissues were exposed to NA (100 microM), yohimbine (10 microM) or prazosin (0.1 microM) prior to and during exposure to CEC. Receptor protection with NA, yohimbine or prazosin (0.1 microM), followed by washout prevented the shift in potency of NA produced by CEC. 7. Further experiments examined the effects of prazosin (10 microM) on responses to NA following receptor protection with NA (100 microM), yohimbine (10 microM), prazosin (10 microM), or xylazine (100 microM). In receptor protection studies with NA, subsequent prazosin (10 microM) produced a shift in response to NA following CEC which was not signficantly different from the shift produced by prazosin alone in the absence of receptor protection. In receptor protection studies with prazosin, yohimbine or xylazine, subsequent prazosin (10 microM) produced shifts in the response to NA following CEC which were significantly less than the shift produced by prazosin alone in the absence of receptor protection.8. It is concluded that CEC has two actions in the rat aorta. Firstly, it behaves as an irreversible a,-adrenoceptor antagonist, reducing the response to low concentrations of NA (up to 10 microM). However,after exposure to CEC, concentrations of NA of 10 microM and above produced contractions resistant toprazosin. This resistant component was still present following receptor protection with alpha1,- or alpha2-adrenoceptor antagonists, but absent following receptor protection with NA. Hence, the latter response may represent an irreversible agonist interaction between CEC, NA and alpha-adrenoceptors which cannot be affected by subsequent competitive antagonism, but which can be prevented by receptor protection with the agonist NA prior to CEC.

The effects of adrenaline and adrenoreceptor antagonists on ovulation and follicle wall contraction were investigated in brook trout (Salvelinus fontinalis) follicles using in vitro incubation systems. Adrenaline significantly stimulated a dose-dependent increase in ovulation and follicle contraction at concentrations between 1.0 and 100 mumol l-1. The ovulatory and contractile effects of 10 mumol adrenaline l-1 could be blocked by the alpha 1-adrenoreceptor antagonists WB-4101 and benoxathian, and by the alpha 2-antagonist yohimbine. WB-4101 was the most potent blocker, significantly inhibiting ovulation and contraction at 1.0 mumol l-1. In contrast, the beta-antagonist propranolol (100-0.001 mumol l-1) was totally ineffective in blocking adrenaline-induced ovulation and follicle contraction. The results indicate that there is a strong correlation between the effects of adrenaline on ovulation and contraction. In addition, the antagonist studies indicate that adrenaline stimulates ovulation and follicle contraction of brook trout follicles through alpha-adrenoreceptors.

1. The alpha 1-adrenoceptor subtypes of the prostatic and epididymal portion of rat vas deferens were characterized in binding and functional experiments. 2. In saturation experiments, [3H]-prazosin bound to two distinct affinity sites in the epididymal portion of rat vas deferens (pKD = 10.1 +/- 0.13 and 9.01 +/- 0.15, Bmax = 507 and 1231 fmol mg-1 protein, respectively). In the prostatic portion [3H]-prazosin bound to a single affinity site (pKD = 9.82 +/- 0.04, Bmax = 924 fmol mg-1 protein). 3. In the displacement experiments, unlabelled prazosin displaced biphasically the binding of 200 pM [3H]-prazosin to the epididymal portion; the resulting two pKI values were consistent with the affinity constants obtained in the saturation experiments. WB4101 (2-(2,6-dimethoxy-phenoxyethyl)-amino-methyl-1,4-benzodioxane) and benoxathian also discriminated the two affinity sites in the epididymal portion and the population of low affinity sites for the three antagonists was approximately 40%. On the other hand, the prostatic portion predominantly showed a single affinity site for prazosin, WB4101 and benoxathian, although the presence of a small proportion (less than 10%) of the low affinity site could be detected. HV723 (alpha-ethyl-3,4,5-trimethoxy-alpha-(3-((2-(2-methoxyphenoxy)ethyl)-a min o)- propyl) benzeneacetonitrile fumarate) displaced the [3H]-prazosin binding monophasically with a low affinity in both halves. 4. Pretreatment with chlorethylclonidine (CEC) at concentrations higher than 1 microM inhibited 700 pM [3H]-prazosin binding to the prostatic portion by approximately 50%. However, the inhibition in the epididymal portion was much less (approximately 21% at 50 microM CEC).5. In the functional study, the contractile response to noradrenaline was competitively inhibited by prazosin, WB4101, benoxathian and HV723 with similar and low affinities (pKB value ranging from 8.0to 9.0) in the epididymal portion of rat vas deferens. In the prostatic portion of rat vas deferens,noradrenaline also produced a contraction, but the maximal amplitude of contraction developed was approximately one-fourth of that in the epididymal portion. Prazosin and WB4101 also inhibited the contractile response of the prostatic portion with the pKB values similar to those obtained in the epididymal portion. The contractions to noradrenaline in both portions were potently attenuated by 1 LM nifedipine but were not affected by pretreatment with 1O LM CEC.6. Under conditions where P2x-purinoceptors and prejunctional M2-adrenoceptors were blocked, electrical transmural stimulation produced a rapidly developing phasic contraction and a subsequent tonic contraction in the epididymal portion of rat vas deferens. The phasic and tonic contractions were inhibited in a concentration-dependent manner by prazosin (ICs = 25.7 and 25.9 nm, respectively),WB4101 (ICo= 7.27 and 7.58 nM), benoxathian (ICs = 10.9 and 8.66 nM) and HV723 (ICs = 15.9 and 14.9 nM). Nifedipine selectively attenuated the tonic contraction induced by electrical stimulation, and the residual phasic response was inhibited by the antagonists mentioned above with similar affinities to those in the absence of nifedipine. CEC (10 gM) had little effect on the adrenergic neurogenic contractions.7. The present results indicate the presence of two distinct alpha&-adrenoceptor subtypes in the rat vas deferens, which show respectively high and low affinities for each of prazosin, WB4101 and benoxathian,and presumably correspond to putative MIA and alL subtypes according to the recent am-adrenoceptorsubclassifications. The contractions induced by exogenous and endogenous noradrenaline seem to be predominantly mediated through the alL subtype. The heterogeneous distribution of the low affinity sites(alL subtype) may well explain differences in functional responsiveness between the two portions of rat vas deferens.

Static incubations of dispersed goldfish pituitary cells with 1-100 nM norepinephrine (NE) stimulated gonadotropin (GTH) release. Additions of the alpha-agonist phenylephrine, and the alpha 1-agonist 6-fluoronorepinephrine, but not the alpha 2-agonist clonidine, nor the beta-agonist isoproterenol, also enhanced GTH secretion. The GTH responses to 1 nM NE was significantly inhibited by coincubations with 1 microM of the alpha-antagonist phentolamine, the alpha 1-antagonists prazosine and benoxathian, but not the alpha 2-antagonist yohimbine nor the beta-antagonist propranolol. The GTH responses to NE and phenylephrine were also additive to salmon GTH-releasing hormone (sGnRH)-induced GTH release. These results suggests that NE directly stimulates GTH secretion independent of sGnRH receptors via alpha 1-like adrenergic receptors.