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Rilmenidine, a ligand for imidazoline and α 2 -adrenergic receptors, is neuroprotective following focal cerebral ischemia. We investigated the effects of rilmenidine on cytosolic free Ca 2+ concentration ([Ca 2+ ] i ) in rat astrocytes. Rilmenidine caused concentration-dependent elevation of [Ca 2+ ] i , consisting of a transient increase (1-100 μM rilmenidine) or a transient increase followed by sustained elevation above basal levels (1-10 mM rilmenidine). A similar elevation in [Ca 2+ ] i was induced by the imidazoline ligand cirazoline. The transient response to rilmenidine was observed in Ca 2+ -free medium, indicating that rilmenidine evokes release of Ca 2+ from intracellular stores. However, the sustained elevation of Ca 2+ was completely dependent on extracellular Ca 2+ , consistent with rilmenidine activating Ca 2+ influx. Pretreatment with thapsigargin, an inhibitor of the endoplasmic reticulum Ca 2+ -ATPase, abolished the response to rilmenidine, confirming the involvement of intracellular stores and suggesting that rilmenidine and thapsigargin activate a common Ca 2+ influx pathway. The α 2 -adrenergic antagonist rauwolscine attenuated the increase in [Ca 2+ ] i induced by clonidine (a selective α 2 agonist), but not the response to rilmenidine. These results indicate that rilmenidine stimulates both Ca 2+ release from intracellular stores and Ca 2+ influx by a mechanism independent of α 2 -adrenergic receptors. In vivo, rilmenidine may enhance uptake of Ca 2+ from the extracellular fluid by astrocytes, a process that may contribute to the neuroprotective effects of this agent.

There is evidence to suggest that niflumic acid is capable of selectively inhibiting Ca(2+)-dependent Cl(-) channels. Furthermore, it has been demonstrated that niflumic acid is capable of antagonizing contractile responses due to activation of alpha(1)-adrenoceptor in mesenteric vasculature. Here, we have examined the effects of three Cl(-) channel blockers, niflumic acid, indanyloxyacetic acid 94 (IAA-94) and diphenylamine-2-carboxylic acid (DPC) on cirazoline-mediated vasoconstriction in mesenteric blood vessel in vivo. Infusion of cirazoline produced a dose-dependent increase in blood pressure, decrease in superior mesenteric blood flow, mesenteric vascular conductance and heart rate. While niflumic acid and IAA-94 did not have any impact on cirazoline-induced changes in blood pressure, DPC accentuated the pressor effect of cirazoline. Neither agent affected cirazoline-mediated reflex reduction in the heart rate. Niflumic acid, IAA-94 and DPC attenuated alpha(1)-adrenoceptor mediated decrease in mesenteric blood flow and vascular conductance. Based on the profile of the actions of these compounds, it may be suggested that IAA-94 did not appear to act as selective inhibitor of Ca(2+)-activated Cl(-) channels when compared to niflumic acid in the mesenteric blood vessels. In addition, while DPC seems to be as effective as niflumic acid in its effects on mesenteric blood vessels, its actions may be attributed to other pharmacological effects.

Excitatory responses evoked by N -methyl- d -aspartate (NMDA) in the vestibular nuclei (VN) of the rat were studied in vivo during microiontophoretic application of noradrenaline (NA) and/or its agonists and antagonists. Ejection of NA-modified excitatory responses mediated by NMDA receptors (NMDAR) in all neurons tested; the effect was enhancement in 59% of cases and depression in the remaining 41%. Enhancements prevailed in all VN with the exception of the lateral vestibular nucleus, where both effects were recorded in an equal number of cases. The enhancing action of NA on NMDAR-mediated responses was mimicked by the noradrenergic beta-receptor agonist isoproterenol, the beta 1 specific agonist denopamine and the alpha 2 agonist clonidine. These effects were blocked respectively by the generic beta-receptor antagonist timolol, the beta 1 antagonist atenolol and the alpha 2 antagonist yohimbine. In contrast, application of the alpha 1 receptor agonist cirazoline and the specific alpha 1 antagonist prazosin respectively mimicked and partially antagonized the depression of NMDAR-mediated excitations induced by NA. Double-labeling immunohistochemical techniques demonstrated broad colocalization of NMDAR (specifically NR1 and NR2 subunits) with noradrenergic receptors (alpha 1 , alpha 2 and beta 1 ) in many VN neurons; only minor differences were found between nuclei. These results indicate that NA can produce generalized modulation of NMDAR-mediated excitatory neurotransmission in VN, which may in turn modify synaptic plasticity within the nuclei.

Stretch of vascular smooth muscle stimulates growth and proliferation as well as contraction and expression of contractile/cytoskeletal proteins, all of which are also regulated by calcium‐dependent signals. We studied the role of the calcium‐ and integrin‐activated proline‐rich tyrosine kinase 2 (PYK2) in stretch‐induced responses of the rat portal vein loaded by a hanging weight ex vivo. PYK2 phosphorylation at Tyr‐402 was increased both by a 10‐min stretch and by organ culture with load over several days. Protein and DNA synthesis were reduced by the novel PYK2 inhibitor PF‐4594755 (0.5–1 μmol/L), while still sensitive to stretch. In 3‐day organ culture, PF‐4594755 caused maintained myogenic spontaneous activity but did not affect contraction in response to high‐K+ (60 mmol/L) or to α1‐adrenergic stimulation by cirazoline. Basal and stretch‐induced PYK2 phosphorylation in culture were inhibited by PF‐4594755, closely mimicking inhibition of non‐voltage‐dependent calcium influx by 2‐APB (30 μmol/L). In contrast, the L‐type calcium channel blocker, nifedipine (1 μmol/L) eliminated stretch‐induced but not basal PYK2 phosphorylation. Stretch‐induced Akt and ERK1/2 phosphorylation was eliminated by PF‐4594755. PYK2 inhibition had no effect on mRNA expression of several smooth muscle markers, and stretch‐sensitive SM22α synthesis was preserved. Culture of portal vein with the Ang II inhibitor losartan (1 μmol/L) eliminated stretch sensitivity of PYK2 and Akt phosphorylation, but did not affect mRNA expression of smooth muscle markers. The results suggest that PYK2 signaling functionally distinguishes effects of voltage‐ and non‐voltage‐dependent calcium influx. A small‐molecule inhibitor of PYK2 reduces growth and DNA synthesis but does not affect contractile differentiation of vascular smooth muscle., We studied the role of the calcium‐ and integrin‐activated proline‐rich tyrosine kinase 2 (PYK2) in stretch‐induced responses of the rat portal ex vivo. PYK2 phosphorylation at Tyr‐402 was increased both by a 10‐min stretch and by organ culture under stretch for 3 days. Protein and DNA synthesis were reduced by the novel PYK2 inhibitor PF‐4594755 (0.5–1 μmol/L), but contractile differentiation was not affected. Basal and stretch‐induced PYK2 phosphorylation in culture were inhibited by PF‐4594755, closely mimicking inhibition of non‐voltage‐dependent calcium influx by 2‐APB (30 μmol/L). In contrast, the L‐type calcium channel blocker, nifedipine (1 μmol/L) eliminated stretch‐induced but not basal PYK2 phosphorylation. The results suggest that PYK2 signaling functionally distinguishes effects of voltage‐ and non‐voltage‐dependent calcium influx.

In sheep, injection of noradrenaline suppresses prolactin secretion by a direct effect at the pituitary gland. The aims of this study were to use primary cultures of ovine pituitary cells to examine the receptor subtypes that mediate the inhibitory effect of noradrenaline on prolactin secretion and, by using receptor antagonists in vivo, determine whether noradrenaline acts as a prolactin release-inhibiting factor (PIF). Noradrenaline and dopamine suppressed prolactin secretion from ovine pituitary cells with ED50s of 60.9 +/- 46.6 and 1.5 +/- 1.0 x 10(-9) mol/l, respectively (P < 0.05). The in-vitro prolactin release-inhibiting effect of noradrenaline (10(-7) mol/l) was not blocked by the dopamine antagonists pimozide (D-2) or SCH23390 (D-1) but was blocked by each of the adrenoceptor antagonists (alpha 1-adrenoceptor antagonists prazosin and WB4101, the alpha 2-adrenoceptor antagonist yohimbine and the beta-adrenoceptor antagonist propranolol). The response to adrenoceptor agonists was also tested in vitro. The alpha 1-adrenoceptor agonists phenylephrine and cirazoline significantly suppressed prolactin. Of the alpha 2-agonists, clonidine had no effect whereas oxymetazoline and p-aminoclonidine both suppressed prolactin. The beta-adrenoceptor agonist isoproterenol also suppressed prolactin while the specific beta 3-antagonist BRL37344 had no effect. When the adrenoceptor antagonists were tested in vivo in ewes manipulated to be in the luteal phase, only WB4101 significantly (P

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 α1a-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.

Objectives: The effects of chloride-free buffer in the absence or presence of the nitric oxide synthase inhibitor, N ω-nitro-l-arginine methyl ester (l-NAME), or chloride channel antagonist, indanyloxyacetic acid 94 (IAA-94) on α1-adrenoceptor mediated contractions were investigated in aortic rings from Dahl salt-resistant normotensive (SRN) and salt-sensitive hypertensive (SSH) rats on a 4% salt diet. Methods: Systolic and diastolic blood pressure were measured via intra-arterial catheters under halothane anesthesia. Subsequently, the aorta was removed and cirazoline-induced contractions were recorded in normal Krebs and chloride-free buffer using ring preparation. Guanosine 3′,5′-cyclic monophosphate (cyclic GMP) content of aortic rings was also determined by scintillation proximity assay kits. Results: Systolic and diastolic blood pressure of SSH (180/130±1/1, mean±S.E.; n = 14) were significantly higher than those of SRN (101/76±1/1, mean±S.E.; n = 14) 7 weeks after they were placed on a salt diet. While the presence of l-NAME failed to have any impact on cirazoline-induced contractions in aortic rings from SSH rats, it significantly accentuated the effects of cirazoline in tissues from SRN rats. On the other hand, IAA-94 was able to inhibit cirazoline-mediated contractions in aortic rings from both SRN and SSH rats. The removal of chloride ions potentiated contractions produced by cirazoline in tissues from SRN but not SSH rats. Moreover, cirazoline-evoked responses in tissues from SRN were not further accentuated by the inclusion of l-NAME in chloride-free buffer. Cirazoline-mediated contractions in tissues from SSH rats were not influenced by absence of chloride ions, and the presence of l-NAME. It was also apparent that the inclusion of IAA-94 in absence of chloride ions did not prevent the potentiation of responses to cirazoline. Removal of chloride ions did not significantly decrease basal cyclic GMP levels in aortic rings from either strain. Conclusions: Basal release of nitric oxide seems to make a greater contribution in the suppression of cirazoline-evoked contractions in vessels from SRN as opposed to SSH rats. Chloride channels appear to contribute to cirazoline-evoked contractions in normal Krebs but not in chloride-free buffer.

In the presence of L-NNA (100 microM), indomethacin (10 microM) and ODQ (10 microM), acetylcholine induced a concentration-dependent vasorelaxation of guinea-pig mesenteric and middle cerebral arteries precontracted with cirazoline or histamine, but not with high K(+), indicating the contribution of an endothelium-derived hyperpolarizing factor (EDHF). In cerebral arteries, charybdotoxin (ChTX; 0.1 microM) completely inhibited the indomethacin, L-NNA and ODQ-insensitive relaxation; iberiotoxin (IbTX, 0.1 microM), 4-aminopyridine (4-AP, 1 mM), or barium (30 microM) significantly reduced the response; in the mesenteric artery, ChTX and IbTX also reduced this relaxation. Glibenclamide (10 microM) had no affect in either the mesenteric or cerebral artery. Neither clotrimazole (1 microM) nor 7-ethoxyresorufin (3 microM) affected EDHF-mediated relaxation in the mesenteric artery, but abolished or attenuated EDHF-mediated relaxations in the cerebral artery. AM404 (30 microM), a selective anandamide transport inhibitor, did not affect the vasorelaxation response to acetylcholine in the cerebral artery, but in the mesenteric artery potentiated the vasorelaxation response to acetylcholine in an IbTX, and apamin-sensitive, but SR 141816A-insensitive manner. Ouabain (100 microM) almost abolished EDHF-mediated relaxation in the mesenteric artery, but enhanced the relaxation in the cerebral artery whereas the addition of K(+) (5 - 20 mM) to precontracted guinea-pig cerebral or mesenteric artery induced further vasoconstriction. These data suggest that in the guinea-pig mesenteric and cerebral arteries different EDHFs mediate acetylcholine-induced relaxation, however, EDHF is unlikely to be mediated by K(+).

In isolated, perfused mesenteric vascular beds from female rats, it was assessed whether the constrictor response to cirazoline, an alpha(1)-adrenergic agonist, or acetylcholine (ACh)-induced relaxation was altered by oestrous cycle or pregnancy and the ability of nitric oxide (NO), prostanoids and endothelium-derived hyperpolarizing factor (EDHF) to modulate these responses. Mesenteries, removed from female rats on each oestrous cycle day and gestation day 16, were perfused with physiological salt solution. Tone was induced with cirazoline (1 micromol/l), and concentration-response curves to ACh generated. Responsiveness to ACh was tested in the presence of N(omega)-nitro-L-arginine (L-NA), ibuprofen (IBU) and tetrabutylammonium (TBA), to inhibit nitric oxide synthase (NOS), cyclo-oxygenase and K(+) channels respectively. Cirazoline-induced tone was smaller in pro-oestrous and pregnant groups, but the increase in tone to L-NA was larger in pregnant compared with oestrous and dioestrous groups. Control responses to ACh were not different, but L-NA attenuated the response in virgin groups only. IBU did not affect the ACh response, but TBA attenuated it in all groups. When TBA was introduced first, ACh-induced dilatation was significantly reduced and not altered by L-NA addition. These results suggest that in the mesenteric vascular bed from cycling and pregnant rats, EDHF is the major mediator of ACh-induced dilatation and NOS may be up-regulated in pregnant and pro-oestrous rats.

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 effects of the T-type and L-type Ca2+ channel antagonists, mibefradil and nifedipine, respectively, and those of a Cl- channel antagonist, indanyloxyacetic acid 94, on mechanical responses elicited by selective activation of alpha1-adrenoceptors using cirazoline were examined in rat isolated aortic rings. The presence of mibefradil (300 nM), indanyloxyacetic acid, 94 (30 microM) and nifedipine (300 nM) alone inhibited mechanical responses elicited by cirazoline. The concentration-response curves to cirazoline were displaced to the right with significant increases in the EC50 and significant depressions of the maximal responses in the presence of the individual agents mibefradil, indanyloxyacetic acid 94, or nifedipine. A combination of mibefradil and indanyloxyacetic acid 94 further inhibited the mechanical activity produced by cirazoline. The further reduction in the maximal response to cirazoline, in the presence of mibefradil and nifedipine, was insignificant when compared with the effects of nifedipine alone. In addition, maximal mechanical responses produced by cirazoline were not significantly affected by a combination of nifedipine and indanyloxyacetic acid 94 when compared with either nifedipine alone or mibefradil and indanyloxyacetic acid 94 combined. Our current findings indicate that mibefradil, indanyloxyacetic acid 94, and nifedipine can inhibit cirazoline-induced contractions to a varying degree. Moreover, based on our present data it would be reasonable to suggest that the contribution of T-type versus L-type Ca2+ channels to contractile responses obtained with cirazoline are approximately 21% and 35%, respectively, of the Emax. It would appear that L-type Ca2+ channels play a greater role in processes that are involved in excitation-contraction coupling subsequent to stimulation of alpha1-adrenoceptors. In addition, Cl- channels also appear to be involved in the process of contraction following alpha1-adrenoceptor activation.

Imidazoline-binding sites are non-adrenergic receptors and classified into I11/I2 subtypes. There is strong evidence that I1-binding sites, located in the rostro-ventrolateral medulla, are involved in regulation of blood pressure. However, less is known about the peripheral participation of I1-binding sites in cardiovascular reactions. Therefore, the aim of this study was to investigate whether specific imidazoline derivatives influence myocardial contractility and whether imidazoline binding sites are expressed in rat heart. Agmatine, clonidine and idazoxan failed to alter inotropy in left atria within the whole concentration range tested (1 nM - 100 microM), whereas cirazoline (1- 100 microM) and moxonidine (100 microM) increase inotropy by about 20-30%. After preincubation with the alpha1-adrenoceptor antagonist prazosin, the cirazoline and moxonidine stimulated inotropy was antagonized, indicating more an alpha1-adrenergic and less an imidazoline binding site mediated mechanism. Radioligand-binding studies in membranes of left ventricles using [3H]-clonidine to specify I1-binding yielded KD values of 12.7 microM, confirming the functional results of an absence of I1-binding sites in ventricles of rats. However, the existence of low affinity I2-binding sites determined by [3H]-idazoxan labeling could not be excluded since a KD of 0.5 microM was calculated and since competition studies with guanabenz (Ki = 0.1 microM), clonidine (Ki = 58.1 microM) and moxonidine (Ki = 129 microM) confirmed the specificity of the I2-binding.

1. The electroencephalographic (EEG) effects of drugs interacting with dopaminergic and noradrenergic systems were studied in conscious rats. Power spectra (0 - 30 Hz) were recorded from electrodes implanted bilaterally in the prefrontal cortex. Drug effects on EEG power were calculated as the spectral power following drug administration divided by the spectral power after vehicle administration. 2. Dopaminergic agonists at low doses, (apomorphine 0. 01 mg kg-1 s.c., quinpirole 0.01 mg kg-1 i.p.) and dopaminergic antagonists (haloperidol 1 mg kg-1 i.p., raclopride 2.5 mg kg-1 s.c. ), which decrease dopaminergic transmission, induced an increase of EEG power. Conversely, dopaminergic agonists at higher doses (apomorphine 0.5 mg kg-1 s.c., quinpirole 0.5 mg kg-1 i.p.) which increase activation of postsynaptic D2 and D3 receptors, induced a decrease of EEG power. 3. The alpha1-adrenoceptor antagonists (phenoxybenzamine 0.64 mg kg-1 s.c., prazosin 0.32 mg kg-1 s.c.) and the alpha2-adrenoceptor agonists (UK 14304 0.05 mg kg-1 s.c., clonidine 0.025 mg kg-1 i.p.), which decrease noradrenergic transmission, induced an increase of EEG power. Conversely, the alpha1-adrenoceptor agonist, cirazoline (0.05 mg kg-1 s.c.), the adrenergic agent modafinil (250, 350 mg kg-1 i.p.) and alpha2-adrenoceptor antagonists (RX 821002 0.01 mg kg-1 s.c., yohimbine 0.5 mg kg-1 i.p.), which increase noradrenergic transmission, induced a decrease of EEG power. The effects of prazosin (0.64 mg kg-1 s.c.) were dose-dependently antagonized by co-administration with modafinil and cirazoline, but not by apomorphine. 4. In conclusion, pharmacological modulation of dopaminergic and noradrenergic transmission may result in consistent EEG changes: decreased dopaminergic or noradrenergic activity induces an increase of EEG spectral power; while increased dopaminergic or noradrenergic activity decreases EEG spectral power.

2-(2-Benzofuranyl)-2-imidazoline (BFI) is a highly selective ligand for imidazoline-type 2 (I2) binding sites that are known to be associated with monoamine oxidase (MAO). Recently we demonstrated a potentiation of 3H-BFI binding in human but not in rat brain by the non-selective MAO inhibitor tranylcypromine. In the present studies, we evaluated the effect of tranylcypromine on the binding of 3H-BFI to human platelet inner membranes. Membranes were incubated with 3H-BFI at 22 °C in 50 mM Tris, 1.5 mM EDTA, pH 7.5. Saturation experiments with 3H-BFI (0.5–80 nM) were analyzed using non-linear curve fitting. Addition of tranylcypromine (0.1 µM) increased the number of 3H-BFI binding sites (B max=0.35±0.06 vs. 1.87±0.15 pmol/mg protein for vehicle and tranylcypromine, respectively) and increased 3H-BFI affinity slightly (K D =16.0±4.1 vs. 6.5±0.3 nM for vehicle and tranylcypromine, respectively). In competitive binding experiments using the less selective I2 ligand, 3H-idazoxan, tranylcypromine only weakly inhibited binding. Preincubation of platelet membranes with tranylcypromine (1 nM–10 µM) enhanced the B max of 3H-BFI binding in a concentration-dependent manner peaking at 1 µM (13×control) and returning to near baseline at 100 µM. 3H-BFI binding was displaced monophasically (in order of decreasing potency) by BFI > 2-(4,5-dihydroimidazol-2-yl)quinoline (BU224) > cirazoline >idazoxan >>(1,4-benzodioxan-2-methoxy-2-yl)-2-imidazoline (RX821002)=moxonidine. Amiloride, clorgyline, guanabenz and clonidine displayed biphasic curves with nanomolar high affinity components. Tranylcypromine altered the competition curves for all ligands (except BFI) by increasing the affinities for clonidine and RX821002 and decreasing affinities for BU224, cirazoline, guanabenz, idazoxan, clorgyline, moxonidine, and amiloride. Thus, in human platelets tranylcypromine exposes a high capacity 3H-BFI binding site distinct from previously described I2 sites that retains high affintiy for BFI but not other I2 ligands. Our results suggest that 3H-BFI and 3H-idazoxan may not be considered as interchangeable probes for the I2 binding site.

The present study was conducted in order to examine the influence of catecholaminergic afferents on the release of serotonin in the median raphe nucleus in vivo. To this aim, selective dopamine D1 and D2, and α1- and α2-adrenergic agonists and antagonists were administered locally (1, 10 and 100 μm) through a dialysis probe implanted in the median raphe nucleus of freely moving rats. The D1 and D2 agonists, (±)-1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol (SKF-38393) and quinpirole, respectively, and the D1 and D2 antagonists, r-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SCH-23390) and raclopride, respectively, did not alter the release of serotonin in the median raphe nucleus. The α1-adrenoceptor agonist phenylephrine did not modify the release of serotonin in this nucleus, although an increased release was observed when the more potent α1-adrenoceptor agonist cirazoline was used. In contrast, the α1-adrenoceptor antagonist prazosin reduced the release of 5-hydroxytryptamine (5-HT) in a concentration-dependent manner. The release of 5-HT was also reduced by the α2-adrenoceptor agonist clonidine and increased by the α2-adrenoceptor antagonist 2-methoxy-idazoxan (RX821002). These results indicate that the release of serotonin in the median raphe nucleus does not appear to be regulated by dopaminergic afferents through the activation of dopamine D1 or D2 receptors. On the contrary, it is suggested that endogenous noradrenaline exerts a direct tonic stimulatory control on the release of serotonin through α1-adrenoceptors, and an indirect tonic inhibitory influence through α2-adrenoceptors located probably in noradrenergic nerve terminals within the median raphe nucleus.

1. To illuminate the controversy on alpha 1A- or alpha 1L-adrenoceptor involvement in noradrenaline-mediated contractions of rat small mesenteric artery (SMA), we have studied the effects of subtype-selective alpha 1-adrenoceptor agonists and antagonists under different experimental conditions. 2. The agonist potency order in rat SMA was: A61603 >> SKF89748-A > cirazoline > noradrenaline > ST-587 > methoxamine. Prazosin antagonized all agonists with a low potency (pA2: 8.29-8.80) indicating the involvement of alpha 1L-rather than alpha 1A-adrenoceptors. 3. The putative alpha 1L-adrenoceptor antagonist JTH-601, but not the alpha 1B-adrenoceptor antagonist chloroethylclonidine (10 microM) antagonized noradrenaline-induced contractions of SMA. The potency of the selective alpha 1D-adrenoceptor antagonist BMY 7378 against noradrenaline (pA2 = 6.16 +/- 0.13) and of the selective alpha 1A-adrenoceptor antagonist RS-17053 against noradrenaline (pKB = 8.35 +/- 0.10) and against the selective alpha 1A-adrenoceptor agonist A-61603 (pKB = 8.40 +/- 0.09) were too low to account for alpha 1D- and alpha 1A-adrenoceptor involvement. 4. The potency of RS-17053 (pKB/pA2's = 7.72-8.46) was not affected by lowering temperature, changing experimental protocol or inducing myogenic tone via KCl or U46619. 5. Selective protection of a putative alpha 1A-adrenoceptor population against the irreversible action of phenoxybenzamine also failed to increase the potency of RS-17053 (pA2 = 8.25 +/- 0.06 against A61603). 6. Combined concentration-ratio analysis demonstrated that tamsulosin, which does not discriminate between alpha 1A- and alpha 1L-adrenoceptors, and RS-17053 competed for binding at the same site in the SMA. 7. In summary, data obtained in our experiments in rat SMA indicate that the alpha 1-adrenoceptor mediating noradrenaline-induced contraction displays a distinct alpha 1L-adrenoceptor pharmacology. This study does not provide evidence for the hypothesis that alpha 1L-adrenoceptors represent an affinity state of the alpha 1A-adrenoceptor in functional assays. Furthermore, there is no co-existing alpha 1A-adrenoceptor in the SMA.

In a recent study using Wistar rats, the serotonergic 5-HT2 receptor antagonists ketanserin and risperidone reduced the disruptive effects of the noncompetitive N-methyl-D-aspartate (NMDA) antagonist dizocilpine on prepulse inhibition (PPI), suggesting that there is an interaction between serotonin and glutamate in the modulation of PPI. In contrast, studies using the noncompetitive NMDA antagonist phencyclidine (PCP) in Sprague–Dawley rats found no effect with 5-HT2 antagonists. To test the hypothesis that strain differences might explain the discrepancy in these findings, risperidone was tested for its ability to reduce the PPI-disruptive effects of dizocilpine in Wistar and Sprague–Dawley rats. Furthermore, to determine which serotonergic receptor subtype may mediate this effect, the 5-HT2A receptor antagonist M100907 (formerly MDL 100,907) and the 5-HT2C receptor antagonist SDZ SER 082 were tested against dizocilpine. Recent studies have found that the PPI-disruptive effects of PCP are reduced by the α1 adrenergic receptor antagonist prazosin. Furthermore, the α1 receptor agonist cirazoline disrupts PPI. As risperidone and M100907 have affinity at the α1 receptor, a final study examined whether M100907 would block the effects of cirazoline on PPI. Risperidone partially, but nonsignificantly, reduced the effects of dizocilpine in Wistar rats, although this effect was smaller than previously reported. Consistent with previous studies, risperidone did not alter the effects of dizocilpine in Sprague–Dawley rats. Most importantly, M100907 pretreatment fully blocked the effect of dizocilpine in both strains; whereas SDZ SER 082 had no effect. M100907 had no influence on PPI by itself and did not reduce the effects of cirazoline on PPI. These studies confirm the suggestion that serotonin and glutamate interact in modulating PPI and indicate that the 5-HT2A receptor subtype mediates this interaction. Furthermore, this interaction occurs in at least two rat strains. [Neuropsychopharmacology 20:311–321, 1999]

1. This study examines whether positive inotropy via alpha-adrenoceptors could be observed in vivo in pithed rats. Cardiac contractility was measured as the maximum rate of rise of left ventricular pressure (dP/dt(max)). Heart rate and aortic blood pressure were also recorded. 2. The selective alpha1-adrenoceptor agonists, methoxamine, cirazoline, amidephrine and phenylephrine caused dose-related increases in dP/dt(max). This response was progressively reduced by increasing doses of the alpha1-adrenoceptor antagonist prazosin. However, since the concomitant increase in diastolic blood pressure (DBP) was also blocked, the changes in dP/dt(max) may have been a consequence of increased after load. 3. Adrenaline and noradrenaline also increased dP/dt(max), accompanied by pressor responses. Propranolol (1 mg kg(-1)) antagonized the increase in dP/dt(max) in response to noradrenaline, suggesting beta-adrenoceptor involvement, but not that to adrenaline. The additional presence of prazosin (1 mg kg(-1)) further shifted the dose-response curves for both noradrenaline and adrenaline to the right. 4. Analysis of the increases in dP/dt(max) at predetermined increases in DBP by each agonist revealed three groups of regression lines. Adrenaline in the presence of propranolol and the four selective alpha1-adrenoceptor agonists occupied a common central position. Above this group were adrenaline and noradrenaline in the absence of antagonists; their additional effects on contractility were beta-adrenoceptor-mediated since the regression lines were lowered by propranolol. Clearly below the main group of agonists was noradrenaline in the presence of propranolol. 5. Thus, for a given increase in DBP, adrenaline (in the presence of beta-blockade) and the alpha1-adrenoceptor agonists exert an additional inotropic effect to noradrenaline (also in the presence of beta-blockade). This is concluded to be an alpha-adrenoceptor-mediated increase in cardiac contractility which is not shared by noradrenaline.

The alpha 1-adrenergic blocking activity of nicergoline was re-examined in rats, with a particular emphasis on alpha 1-adrenoceptor subtypes. In pithed rats, nicergoline and prazosin infused at a single small dose (0.5 microgram/kg/min i.v.) produced a substantial and identical shift to the right of the control dose pressor response curve to the specific alpha 1-agonist cirazoline (ED50 = 4.0 +/- 0.1, 4.0 +/- 0.1 and 0.9 +/- 0.01 microgram/kg i.v. for nicergoline, prazosin and vehicle respectively). In the isolated perfused mesenteric vascular bed, nicergoline strongly inhibited the pressor responses elicited by cirazoline, with approximately 40-fold higher potency (pA2 = 11.1 +/- 0.3) than prazosin (pA2 = 9.5 +/- 0.3). Conversely, nicergoline was 20-fold less potent than prazosin to antagonize the contractile effects of cirazoline in isolated endothelium-denuded aorta (pA2 = 8.6 +/- 0.2 and 9.9 +/- 0.2 for nicergoline and prazosin respectively). Pretreatment of mesenteric vascular beds with chloroethylclonidine did not significantly modify nicergoline antagonistic potency (pA2 = 10.6 +/- 0.2). Nicergoline displaced [3H]-prazosin bound to rat forebrain membranes pretreated with chloroethylclonidine (pKi = 9.9 +/- 0.2) at concentrations 60-fold lower than in rat liver membranes (pKi = 8.1 +/- 0.2). Finally, of the nicergoline metabolites studied, lumilysergol acted as a modest alpha 1 antagonist (bromonicotinic acid was devoid of alpha 1 antagonist activity). In conclusion, nicergoline is a potent and selective alpha 1A-adrenoceptor subtype antagonist, an alpha 1-adrenoceptor subtype which is mainly represented in resistance arteries.

In rat vena cava and aorta preincubated with [ 3 H]noradrenaline the involvement of imidazoline receptors in modulation of [ 3 H]noradrenaline release from sympathetic nerves was investigated. In the vena cava, the guanidine 1,3-di(2-tolyl)guanidine (DTG) inhibited the electrically evoked [ 3 H]noradrenaline release; the inhibitory effect was more pronounced in the presence than in the absence of the α 2 -adrenoceptor antagonist rauwolscine. The concentration-response curves of BDF 6143 [4-chloro-2-(2-imidazolin-2-ylamino)-isoindoline], and idazoxan for their facilitatory effect on electrically evoked [ 3 H]noradrenaline release was bell-shaped; in the presence of rauwolscine, BDF 6143 inhibited the evoked [ 3 H]noradrenaline release, whereas idazoxan did not. After blockade of α 2 -autoreceptors by rauwolscine, the electrically evoked [ 3 H]noradrenaline release from vena cava was inhibited not only by DTG and BDF 6143 but also by aganodine, clonidine and cirazoline; the rank order of potency of most of the drugs was similar to that found at the presynaptic imidazoline receptors in the rabbit aorta and pulmonary artery as well as in human atrial appendages. In the presence of rauwolscine, clonidine-induced inhibition of electrically evoked [ 3 H]noradrenaline release was counteracted by 1 μM of the selective CB 1 receptor antagonist SR141716A (N-[piperidin-1-yl]-5-[4-chlorophenyl]-1-[2,4-dichlorophenyl]-4-methyl-1H-pyrazole-3-carboxamide). In the aorta, BDF 6143 and cirazoline did not modify [ 3 H]noradrenaline release in the absence of α 2 -adrenoceptor blockade; in the presence of rauwolscine, the electrically evoked [ 3 H]noradrenaline release from aorta was inhibited by BDF 6143, cirazoline, aganodine and clonidine with a rank order of potency similar to that in the vena cava. SR141716A 1 μM antagonized the inhibitory effect of BDF 6143 and clonidine (in the presence of rauwolscine). In conclusion, noradrenaline release in rat vena cava and aorta is inhibited via presynaptic imidazoline receptors which appear to be related to those previously characterized in rabbit and human cardiovascular tissue.

Clozapine and the novel putative, antipsychotic S 16924 ((1-(benzodioxane-5-yl)-3-[3-(4-fluorophenacyl)pyrrolidine]-1-o xapropane HCl) share significant affinity for alpha1-adrenoceptors and 5-HT1A autoreceptors in vitro and display an 'atypical' behavioural profile in in vivo models used for detecting potential neuroleptic effects. In the present study, in vivo microdialysis was used to examine the effect of clozapine and S 16924 on 5-HT overflow in the rat ventral hippocampus, and to assess the relative role of putative alpha1-adrenoceptor antagonist and 5-HT1A autoreceptor agonist properties of the drugs in this regard. S 16924 (0.1-3 mg/kg, s.c.) reduced dialysate 5-HT in a dose- and time-dependent fashion by maximally approximately 70% from baseline 40-60 min after injection. Clozapine (0.1-10 mg/kg, s.c.) reduced 5-HT overflow in the same manner, with a maximum effect of approximately 60% from baseline, obtained after 60-80 min. The 5-HT decrease elicited by S 16924 (1.0 mg/kg, s.c.) was significantly, though only partially, antagonized by pretreatment with the selective 5-HT1A receptor antagonist WAY 100635 (0.3 mg/kg, s.c.). The selective alpha1-adrenoceptor agonist cirazoline (0.02 mg/kg, i.p.) alone did not significantly attenuate the effect of S 16924 (1.0 mg/kg, s.c.) on 5-HT overflow. Combined treatment with both WAY 100635 and cirazoline, however, totally reversed the 5-HT-suppressing effect of S 16924 (1.0 mg/kg, s.c.). By comparison, when given separately, neither WAY 100635 (0.3 mg/kg, s.c.) nor cirazoline (0.02 mg/kg, i.p.) antagonized the clozapine (0.3 mg/kg, s.c.)-induced decrease of 5-HT in ventral hippocampus dialysates. In the presence of both WAY 100635 and cirazoline, the response to this dose of clozapine was however significantly, though modestly, attenuated. In contrast, the WAY 100635/cirazoline combination failed to antagonise the 5-HT decrease resulting from a higher dose (3.0 mg/kg, s.c.) of clozapine. We conclude that both alpha1-adrenoceptor antagonist and 5-HT1A receptor agonist properties of clozapine and S 16924 contribute to the 5-HT release-reducing action of these drugs. Whereas these factors apparently explain the effect of S 16924 fully, additional mechanism(s) appear to be involved in the case of clozapine. With regard to the interplay between alpha1-adrenoceptor and 5-HT1A (auto)receptor mechanisms in the control of 5-HT release in the rat forebrain, the present data suggest that an excitation mediated by the former is outweighed by the simultaneous activation of the latter-inhibitory-receptors.

The relationship between cAMP levels and thermogenesis was investigated in brown fat cells from Syrian hamsters. Irrespective of whether the selective beta3-, beta2-, and beta1-agonists BRL 37344, salbutamol, and dobutamine or the physiological agonist norepinephrine was used to stimulate the cells, increases in cAMP levels were mediated via the beta3-receptor, as were the thermogenic effects. However, the relationship "thermogenesis per cAMP" was much lower for agents other than norepinephrine. Similarly, forskolin, although more potent than norepinephrine in elevating cAMP, was less potent in inducing thermogenesis. The selective alpha1-agonist cirazoline was in itself without effect on cAMP levels or thermogenesis, but when added to forskolin-stimulated cells, potentiated thermogenesis, up to the norepinephrine level, without affecting cAMP. This potentiation could not be inhibited by chelerythrine, but could be mimicked by Ca2+ ionophores. It was apparently not mediated via calmodulin-dependent protein kinase and was not an effect on mitochondrial respiratory control. The ability of all cAMP-elevating agents to induce thermogenesis in brown fat cells has earlier been interpreted to mean that it is only through the beta-receptors and the resulting increase in cAMP levels that thermogenesis is induced. However, it is here concluded that the thermogenic response to norepinephrine involves two interacting parts, one mediated via beta-receptors and cAMP and the other via alpha1-receptors and increases in cytosolic Ca2+ levels.

The influence of niflumic acid (3 and 10 μM), a Cl − channel antagonist, on cirazoline-induced vasoconstriction in isolated perfused mesenteric artery (5 ml/min) from two-kidney one-clip (2K1C) hypertensive and sham normotensive rats was examined. In addition, the effect of a single i.v. bolus injection of niflumic acid (3 mg/kg) on cirazoline-mediated reduction in vascular conductance in superior mesenteric artery was determined in pentobarbital-anaesthetized hypertensive and normotensive rats. Bolus injections of cirazoline induced a dose-dependent transient increase in the perfusion pressure in vitro. In the presence of niflumic acid, cirazoline-mediated vasoconstriction was significantly inhibited. Cirazoline-induced vasoconstriction in isolated mesenteric beds was also significantly inhibited following perfusion with Cl − -free buffer. Pre-perfusion of mesenteric blood vessels with Cl − -free buffer resulted in a significantly greater inhibition of cirazoline-mediated vasoconstriction in sham normotensive rats than in hypertensive rats. We found that in Cl − -free buffer, cirazoline-mediated vasoconstriction could be further inhibited by niflumic acid. Intravenous infusion of cumulative doses of cirazoline in vivo caused a dose-dependent decrease in superior mesenteric vascular conductance. Pretreatment with niflumic acid significantly impaired cirazoline-mediated decreases in vascular conductance. Our results indicate that chloride ions play an important role in α 1 -adrenoceptor-mediated vasoconstriction in mesenteric blood vessels. In addition, the contribution of chloride ions in α 1 -adrenoceptor-mediated vasoconstriction in blood vessels from hypertensive rats appears to be reduced.

1 In segments of human right atrial appendages and pulmonary arteries preincubated with [3H]-noradrenaline and superfused with physiological salt solution containing desipramine and corticosterone, the involvement of imidazoline receptors in the modulation of [3H]-noradrenaline release was investigated. 2 In human atrial appendages, the guanidines aganodine and DTG (1,3-di(2-tolyl)guanidine) which activate presynaptic imidazoline receptors, inhibited electrically-evoked [3H]-noradrenaline release. The inhibition was not affected by blockade of α2-adrenoceptors with 1 μM rauwolscine, but antagonized by extremely high concentrations of this drug (10 and/or 30 μM; apparent pA2 against aganodine and DTG: 5.55 and 5.21, respectively). 3 In the presence of 1 μM rauwolscine, [3H]-noradrenaline release in human atrial appendages was also inhibited by the imidazolines idazoxan and cirazoline, but not by agmatine and noradrenaline. The inhibitory effects of 100 μM idazoxan and 30 μM cirazoline were abolished by 30 μM rauwolscine. 4 In the atrial appendages, the rank order of potency of all guanidines and imidazolines for their inhibitory effect on electrically-evoked [3H]-noradrenaline release in the presence of 1 μM rauwolscine was: aganodineBDF 6143 [4-chloro-2-(2-imidazolin-2-yl-amino)-isoindoline]>DTGclonidine>cirazoline>idazoxan (BDF 6143 and clonidine were previously studied under identical conditions). This potency order corresponded to that previously determined at the presynaptic imidazoline receptors in the rabbit aorta. 5 When, in the experiments in the human pulmonary artery, rauwolscine was absent from the superfusion fluid, the concentration-response curve for BDF 6143 (a mixed α2-adrenoceptor antagonist/imidazoline receptor agonist) for its facilitatory effect on electrically-evoked [3H]-noradrenaline release was bell-shaped. In the presence of 1 μM rauwolscine, BDF 6143 and cirazoline concentration-dependently inhibited the evoked [3H]-noradrenaline release. 6 In human atrial appendages, non-adrenoceptor [3H]-idazoxan binding sites were identified and characterized. The binding of [3H]-idazoxan was specific, reversible, saturable and of high affinity (KD: 25.5 nM). The specific binding of [3H]-idazoxan (defined by cirazoline 0.1 mM) to membranes of human atrial appendages was concentration-dependently inhibited by several imidazolines and guanidines, but not by rauwolscine and agmatine. In most cases, the competition curves were best fitted to a two-site model. 7 The rank order of affinity for the high affinity site (in a few cases for the only detectable site; cirazoline=idazoxan>BDF 6143>DTGclonidine) is compatible with the pharmacological properties of I2-imidazoline binding sites, but is clearly different from the rank order of potency for inhibiting evoked noradrenaline release from sympathetic nerves in the same tissue. 8 It is concluded that noradrenaline release in the human atrium and, less well established, in the pulmonary artery is inhibited via presynaptic imidazoline receptors. These presynaptic imidazoline receptors appear to be related to those previously characterized in rabbit aorta and pulmonary artery, but differ clearly from I1 and I2 imidazoline binding sites. British Journal of Pharmacology (1997) 122, 43–50; doi:10.1038/sj.bjp.0701343

Mouse brown adipocytes in primary culture were shown to contain high levels of mRNA for interleukin-1alpha (IL-1alpha) which could be further stimulated up to 9-fold by norepinephrine (NE). Even higher stimulation by NE, up to 40-fold, was found in case of interleukin-6 (IL-6). Time-course of activation of both genes was biphasic, but the response of IL-6 gene was slower than of IL-1alpha gene. IL-1alpha mRNA level reached the maximum after 1 h and the second, lower increase, occurred after 8 h. IL-6 mRNA level showed first maximum after 2 h, but the highest level was found after 8 h. Similarly to NE, the expression of IL-1alpha and IL-6 genes was stimulated by selective beta-adrenergic agonist isoproterenol, beta3-selective agonist CGP-12117, forskoline and db-cAMP. The activation of both genes by CGP-12177 was dose-dependent with the optimum at 100 nM concentration. Stimulation of alpha-adrenergic receptors by cirazoline and oxymetazoline was without any effect. When the expression of IL-6 was studied at the protein level, the stimulation of IL-6 gene via beta3-receptors resulted in secretion of IL-6 up to the concentration 10 ng/ml culture media in 24 h. The results indicate a new type of regulation of expression of IL-1alpha and IL-6 genes in brown adipocytes by catecholamines acting via beta3-adrenergic receptors. The resulting increase in IL-6 production by brown adipocytes could significantly contribute to systemic levels of IL-6.

The novel selective imidazoline radioligand [3H]2-(2-benzofuranyl)-2-imidazoline (2-BFI) was used to characterize and assess further the nature of I2-imidazoline receptors in rat brain and liver. In the cerebral cortex, 2-BFI displayed high affinity (K i= 9.8 nM) for a single class of [3H]2-BFI binding sites. Other imidazoline/guanidine compounds (e.g. aganodine, cirazoline and idazoxan) displayed biphasic competition curves, indicating the existence of high (K iH= 2.9-78 nM; R H= 61-83%) and low (K iL= 4.7-158 μM) affinity sites. The pharmacological profile for [3H]2-BFI binding (aganodine > cirazoline > 2-BFI >> clonidine > amiloride >> efaroxan) was typical of that for I2-sites. This profile was almost identical to that obtained against [3H]idazoxan (correlation between pK i values, r = 0.97) which indicated that the sites characterized with [3H]2-BFI in brain corresponded to I2-imidazoline receptors. The low affinity of amiloride against [3H]2-BFI (K i= 900 nM) further indicated that these brain I2-sites belong to the I2B-subtype. [3H]2-BFI binding sites (B max= 72 fmol/mg protein) in brain were differentially modulated by treatment (7 days) with cirazoline (up-regulation: 25%) and the MAO inhibitor phenelzine (down-regulation: 31%), indicating that these I2-sites are regulated in vivo, as is the case for those labelled by [3H]idazoxan. Chronic treatment with 2-phenylethylamine, a phenelzine metabolite and endogenous amine, did not alter the density of brain of I2-imidazoline receptors labelled by [3H]idazoxan. Preincubation of liver membranes with the MAO inhibitor clorgyline (10-7 M) abolished the binding of [3H]Ro 41-1049 (N-(2-aminoethyl)-5-(m-fluorophenyl)-4-thiazole carboxamide) to MAO-A, but it did not alter the binding of [3H]Ro 19-6327 (N-(2-aminoethyl)-5-chloro-2-pyridine carboxamide) to MAO-B or that of [3H]2-BFI to I2-sites. At 10-4 M it also abolished MAO-B sites, but a substantial proportion of I2-sites (40%) remained intact. Preincubation of liver membranes at 60 °C also abolished MAO-A/B sites, whereas still 22% of I2-sites remained. The results indicate that [3H]2-BFI is a good tool for the identification of I2-imidazoline receptors and suggest further that certain I2-sites and MAO are different proteins.

Although many of the behavioral effects of cocaine are widely believed to be mediated by blockade of dopamine transporters, recent studies suggest that norepinephrine (NE) may play a modulatory role. In this study, selective and nonselective beta-adrenergic receptor antagonists were administered alone or in combination with cocaine (2.5 mg/kg, i.p.) to rats trained to discriminate a low dose (2.5 mg/kg) from a high dose of cocaine (10 mg/kg) in a two-lever, FR10 drug discrimination procedure. The central beta 2/beta 1-adrenergic antagonists (-)-propranolol and tertatolol, and the beta 2-adrenergic antagonist, ICI 118,551, produced high-dose appropriate responding in a dose-related manner when administered (i.p.) in combination with the low training dose of cocaine. In contrast, neither the peripheral beta 2/beta 1-adrenergic antagonist, nadolol, nor the central beta 1-adrenergic antagonist, beta-xolol enhanced the behavioral effects of the low dose of cocaine in a manner comparable with that produced by compounds with central beta 2-adrenergic antagonist properties. Also in contrast to findings obtained using beta-adrenergic antagonists, neither the alpha 1-adrenergic agonist cirazoline, nor the alpha 2-adrenergic ligands (+/-)-efaroxan and UK-14304 enhanced the behavioral effects of the low dose of cocaine. Overall, these results suggest that central beta 2-adrenergic receptors may play a modulatory role in the discriminative stimulus effects of cocaine.

Activation of alpha 1-Adrenergic receptors via systemic administration of drugs such as phenylpropanolamine (PPA) and cirazoline results in the suppression of feeding in rats. Whether PPA acts via activation of the three currently identified alpha 1-Adrenoceptor subtypes is unknown. The intent of the present study was thus to examine the effects of systemic administration of the novel alpha 1a-Adrenoceptor antagonist RS-17053 on PPA-induced anorexia. Adult male rats (n = 6 to 8 per group) were pretreated (IP) with either 0, 0.1, 0.5, 2.5, or 10.0 mg/kg RS-17053 or with 2.0 mg/kg of the prototypical alpha 1-Adrenoceptor antagonist prazosin. Five minutes later, each rat was treated (IP) with either 0, 5, 10 or 15 mg/kg PPA. Food and water intakes were recorded for a 30 min period starting 10 min after the the treatment injection. Rats pretreated with vehicle and then treated with PPA exhibited a dose-dependent suppression of feeding with a maximal effect evident at the 15 mg/kg dose of PPA. Pretreatment with 2.0 mg/kg prazosin reversed the anorexic activity of PPA. Pretreatment with RS-17053 (0.1-2.5 mg/kg) did not alter either baseline feeding or the anorexic action of PPA. These results suggest that PPA does not act via the alpha 1a-Adrenergic receptor subtype to suppress food intake.

A polyclonal antibody was generated in rabbits to an idazoxan-albumin antigen. The anti-idazoxan antiserum had high affinity for unconjugated 3 H-idazoxan ( K d of 19.8 nM) in a radio-immunoassay (RIA). Of various drugs and native molecules only idazoxan potently ( K i of 24 nM) inhibited 3 H-idazoxan binding to the anti-idazoxan antibody. A few drugs weakly inhibited 3H-idazoxan binding (IC50 > 605 μM) with rank order of UK 14304 > guanabenz > cirazoline > amiloride > naphazoline. Neither agmatine, an endogenous clonidine displacing substance (CDS), catecholamines or imidazoles inhibited the binding of 3 H-idazoxan to the anti-idazoxan antibody. The anti-idazoxan RIA was 4–6 fold more sensitive than an antibody to para -amino clonidine. The CDS detected by ligand displacement from bovine brain dose-dependently inhibited 3 H-idazoxan binding. This immunoreactive (ir-) CDS activity was present in human (0.9–4.1 U/ml) and rat sera (1–2 U/ml) and in the cerebro-spinal fluid of eight patients with serious disease of the central nervous system, but not in controls. We conclude: (1) an anti-idazoxan RIA is a sensitive, selective and clinically applicable RIA for measuring ir-CDS; (2) ir-CDS is not agmatine; (3) CDS represents a family of endogenous ligands for imidazoline receptors including ir-CDS and agmatine.

The physiological and pharmacological effects of imidazoli(di)ne derivatives, such as clonidine, have been related not only to the interaction with alpha 2-adrenoceptors but also to their activity on non-adrenoceptor sites termed imidazoline receptors. The modulation of catecholamine release by imidazoline drugs was studied by monitoring extracellular levels of norepinephrine (NE), dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) with microdialysis in cingulate cortex of rats, with or without irreversible alpha 2-adrenoceptor blockade. NE and DA levels were in the 1 nM range whereas DOPAC and HIVA levels were approximately equal to 100 nM. NE and DA levels were increased when the uptake blocker desipramine (1 microM) or KCl (100 mM) were added to the perfusion medium. Clonidine induced a dose-dependent (0.3-1.2 mg/kg i.p.) decrease in NE (max 61%) and DA (max 40+) levels that was reversed by the alpha 2-adrenoceptor antagonist RX821002. After alpha 2-adrenoceptor irreversible blockade with the alkylating agent N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), [3H]clonidine binding to alpha 2-adrenoceptors was reduced by 94 +/- 1%. Under such conditions, clonidine elicited a paradoxical dose-dependent (0.6-2.4 mg/kg i.p.) increase of NE (max 56%) without modifications in DA, DOPAC and HVA levels. The stimulatory effect of clonidine was prevented by the imidazoline receptor antagonist idazoxan (10 mg/kg i.p.) but not by RX821002 (5 mg/kg i.p.). In rats pretreated with EEDQ, cirazoline (I1/I2-imidazoline receptor agonist), moxonidine (I1-imidazoline receptor agonist), but not guanabenz (I2-imidazoline receptor agonist) (1.2-2.4 mg/kg i.p.) elicited an increase of NE levels in a similar manner to clonidine (11-82%). Idazoxan also abolished these responses to cirazoline or moxonidine. In contrast to systemic administration, local perfusion of clonidine (10-100 microM) through the microdialysis probe under alpha 2-adrenoceptor alkylating conditions, did not modify extracellular levels of NE and DA suggesting an indirect mechanism. The results demonstrate that clonidine and related imidazoli(di)ne drugs are able not only to inhibit NE release in rat cerebral cortex involving an alpha 2-adrenoceptor mechanism, but also to induce a paradoxical NE release through an indirect extracortical mechanism. The findings evidence that the indirect modulation of NE levels by imidazoline drugs is mainly due to a functional activity on I1-imidazoline receptors.

Imidazoline compounds are known to interact with alpha 2-adrenoceptors as well as with specific non-adrenergic binding sites. Such binding sites are present in the brain and in peripheral tissues. Hypotensive effects of imidazolines were shown to be related to specific interaction with imidazoline binding sites within the brainstem. Heterogeneity of these sites based on differences in selectivities was reported. In order to facilitate the characterization of human brain imidazoline receptors, we synthetized new ligands by substitutions on the cirazoline phenyl ring. Affinities of these cirazoline derivatives were determined in two imidazoline binding site models, namely the human brain and the rabbit kidney. Interaction of these compounds with imidazoline binding sites from the human brain appeared more sensitive to structural variations of the imidazoline than those with rabbit kidney sites. Moreover, no correlation was found between affinities for imidazoline binding sites and those for alpha 2-adrenoceptors of the rat brain. Arylazide derivative of 2-(5-amino-2-methyl-phenoxymethyl)-imidazoline exhibited a higher affinity for human brain imidazoline binding sites than for human brain alpha 2-adrenoceptors. Photoincorporation of this azido-compound in human brain imidazoline binding sites was achieved and blockade of [3H]idazoxan imidazoline specific binding observed. These new tools may allow fine characterization of the different subtypes of imidazoline binding proteins.

Skin fibroblasts modulate tissue repair, wound healing and immunological responses. Adrenergic receptors (ARs) mediate important physiological functions, such as endocrine, metabolic and neuronal activity. In this study, the expression α1A-ARs in human skin fibroblasts is examined and verified. Regulatory effects of α1-agonist cirazoline on cell migration and the production of transforming growth factor β1 (TGF-β1), insulin-like growth factor 1 (IGF-1), hyaluronan (HA), fibronectin and procollagen type I carboxy-terminal peptide (PIP) by human skin fibroblasts are assessed and validated. α1A-AR mRNA and protein were found in human skin fibroblasts WS1. Exposure of cirazoline doubled skin fibroblast migration and the increase in cell migration was attenuated by α1-antagonist prazosin. TGF-β1 mRNA and production were enhanced after exposure to cirazoline and IGF-1 production was also increased after treatment with cirazoline. Exposure to cirazoline also enhanced HA and PIP production. The increases in TGF-β1, IGF-1, HA and PIP production were partially abolished in fibroblasts transfected with α1A-AR short interfering RNAs, indicating that α1A-ARs are involved in the cirazoline-induced increases in TGF-β1, IGF-1, HA and PIP production. Thus, α1A-ARs are stably expressed and stimulate cell migration and TGF-β1, IGF-1, HA and PIP production in human skin fibroblasts. Moreover, TGF-β1, IGF-1, HA and PIP production and the cell migration of human skin fibroblasts are possibly modulated by natural catecholamines produced by the endocrine system or sympathetic innervation, which could directly or indirectly participate in cytokine secretion, fibroblast migration and matrix production of wound healing in the skin.

Histaminergic neurons within the tuberomammillary nucleus (TMN) play an important role in the regulation of sleep-wakefulness. Here, we report the adrenergic modulation of GABAergic transmission in rat TMN histaminergic neurons using a conventional whole-cell patch clamp technique. Norepinephrine (NE) reversibly decreased the amplitude of action potential-dependent GABAergic inhibitory post-synaptic currents (IPSCs) and increased the paired pulse ratio. The NE-induced inhibition of GABAergic IPSCs was mimicked by clonidine, a selective α2 adrenoceptor agonist. However, cirazoline and isoproterenol, nonselective α1 and β adrenoceptor agonists, respectively, had no effect on GABAergic IPSCs. The NE-induced inhibition of GABAergic IPSCs was significantly blocked by BRL44408, a selective α2A adrenoceptor antagonist, but not imiloxan or JP1302, a selective α2B and α2C adrenoceptor antagonists. The extent of NE-induced inhibition of GABAergic IPSCs was inversely proportional to the extracellular Ca(2+) concentration. Pharmacological agents affecting the activities of adenylyl cyclase or G-protein-coupled inwardly rectifying K(+) channels did not affect the NE-induced inhibition of GABAergic IPSCs. However, NE had no effect on the frequency and amplitude of GABAergic miniature IPSCs. These results suggest that NE acts on presynaptic α2A adrenoceptor to inhibit action potential-dependent GABA release via the inhibition of Ca(2+) influx from the extracellular space to GABAergic nerve terminals, and that this α2A adrenoceptor-mediated modulation of GABAergic transmission may be involved in regulating the excitability of TMN histaminergic neurons.

"Non-adrenoceptor'-binding sites for [3H]clonidine (I1-sites) and [3H]idazoxan (I2-sites) are identified in calf striatum membranes. The pharmacological profile of both subtypes was investigated by competition binding with the imidazolines idazoxan, cirazoline, Bu 224 (2-(4,5-dihydroimidaz-2-yl)-quinoline) and Bu 239 (2-(4,5-dihydroimidaz-2-yl)-quinoxaline); the guanidino derivatives clonidine, moxonidine, guanabenz, amiloride and agmatine; the oxazoline rilmenidine and the imidazole histamine. The competition experiments indicate that both populations of imidazoline-binding sites in calf striatum consist of a high- (H) and a low-affinity (L) compartment. The monoamine oxidase (MAO) inhibitors pargyline (non-selective) and deprenyl (MAO-B-selective) have micromolar affinity for the I1-sites and much lower affinity for the I2-sites. The venom of the marine snail Conus geographus is the most potent of the 13 tested Conus venom preparations. None of the tested venoms is able to discriminate between both sites.

Imidazoline/guanidinium receptive sites (IGRS) are shown to be present in the subfornical organ and hypothalamic arcuate nucleus by a derivative of cirazoline, 2-(3-amino-4-[125I]iodophenoxy)methylimidazoline ([125I]AMIPI). Because many of the nonpeptide angiotensin II (Ang II) receptor antagonists contain imidazole ring structures, they may interact with IGRS. Therefore, we studied competitive activity of Ang II and several nonpeptide Ang II receptor antagonists [DuP 753 (losartan), EXP 3174, CV11974, and PD123319] at IGRS in rat forebrain. The results showed specific binding of 944 +/- 169 fmol/mg protein in the subfornical organ (n = 11) and of 367 +/- 27 fmol/mg protein in the arcuate nucleus (n = 6) at 0.4 nM [125I]AMIPI, as defined by competition with 10 microM cirazoline. Specific [125I]AMIPI binding was competed for completely by 10 microM idazoxan or clonidine as further characterization of IGRS. Ang II and the nonpeptide AT1 and AT2 antagonists did not significantly compete for specific [125I]AMIPI binding in either brain region at concentrations of 10 microM (< 20% competition with each compound), which is 10- to 100-fold higher than the concentration necessary to compete completely for their respective Ang II receptor subtypes. Only at the highest concentration (100 microM) did losartan compete significantly for binding (56 +/- 8%). Therefore, Ang II receptor antagonists interact with IGRS in rat forebrain cardiovascular areas only at high concentrations.

1. The densities of various imidazoline receptor proteins (with apparent molecular masses of approximately 29/30-45- and 66-kDa) were quantitated by immunoblotting in the rat cerebral cortex after various drug treatments. The modulation of these imidazoline receptor proteins was then compared with the changes in the density of non-adrenoceptor [3H]-idazoxan binding sites (I2-sites) induced by the same drug treatments. 2. Chronic treatment (7 days) with the I2-selective imidazol(in)e drugs idazoxan (10 mg kg-1), cirazoline (1 mg kg-1) and LSL 60101 (10 mg kg-1) differentially increased the immunoreactivity of imidazoline receptor proteins. The levels of the 29/30-kDa protein were increased by idazoxan and LSL 60101 (23%), the levels of the 45-kDa protein only by cirazoline (44%) and those of the 66-kDa protein only by idazoxan (50%). These drug treatments also increased the density of I2-sites (32-42%). 3. Chronic treatment (7 days) with efaroxan (10 mg kg-1), RX821002 (10 mg kg-1) and yohimbine (10 mg kg-1), which possess very low affinity for I2-imidazoline receptors, did not alter either the immunoreactivity of imidazoline receptor proteins or the density of I2-sites. 4. Chronic treatment (7 days) with the monoamine oxidase (MAO) inhibitors clorgyline (10 mg kg-1) and phenelzine (10 mg kg-1) decreased the immunoreactivity of the 29/30-kDa (17-24%), 45-kDa (19%) and 66-kDa (23-31%) imidazoline receptor proteins. The alkylating agent N-ethoxycarbonyl-2-ethoxy-1, 2-dihydroquinoline (1.6 mg kg-1, 6 h) also decreased the levels of the three imidazoline receptor proteins (20-47%). These drug treatments consistently decreased the density of I2-sites (31-57%). 5. Significant correlations were found when the mean percentage changes in immunoreactivity of imidazoline receptor proteins were related to the mean percentage changes in the density of I2-sites after the various drug treatments (r = 0.92 for the 29/30-kDa protein, r = 0.69 for the 45-kDa protein and r = 0.75 for the 66-kDa protein). 6. In the rat cerebral cortex the I2-imidazoline receptor labelled by [3H]-idazoxan is heterogeneous in nature and the related imidazoline receptor proteins (29/30-, 45- and 66-kDa) detected by immunoblotting contribute differentially to the modulation of I2-sites after drug treatment.

Adrenoceptor agonists were used to characterize the alpha 1-adrenoceptor subtype responsible for mediating tension (phasic and tonic combined) in the denuded rat aorta and compared with radioligand binding at alpha 1-adrenoceptor subtypes. The rank order of potency at the rat aorta was the same as that obtained for binding affinity at the rat clonal alpha 1d-adrenoceptor: norepinephrine > epinephrine > cirazoline > phenylephrine > oxymetazoline > A-61603 > methoxamine. Correlation coefficients comparing rat aortic contraction (pD2) to binding (pKi) were 0.09-0.21 for alpha 1A/a receptors, 0.66 for clonal alpha 1b and 0.94 for clonal alpha 1d-adrenoceptors. Correlation coefficients comparing the clonal alpha 1d-adrenoceptor binding affinity to in vitro contractile responses were 0.03 and 0.10 for the rat vas deferens and canine prostate alpha 1A-adrenoceptor responses, respectively, 0.09 for the rat spleen alpha 1B and as noted, 0.94 for the rat aorta. The agreement observed between agonist potency at the rat aorta and affinity for the alpha 1d binding site provide new evidence that the alpha 1D-adrenoceptor subtype is responsible for mediating contractions in the rat aorta.

Clonidine, idazoxan, and related imidazoline adrenergic drugs bind to non-adrenergic sites in brain and several peripheral tissues. These sites, termed imidazoline receptors, appear to exist in two major subclasses, I1 sites labeled by clonidine and I2 sites labeled by idazoxan. In this study, we investigated whether rat prostate expresses imidazoline receptors and, if so, whether their expression can be regulated by circulating testosterone. Studies in rat ventral prostate membrane revealed that [3H]idazoxan, but not [3H]p-aminoclonidine, bound to non-adrenergic sites. The binding of [3H]idazoxan was saturable (Bmax: 941 +/- 105 fmol/mg protein) and high affinity (KD: 16.4 +/- 2.3 nM). The rank order of the inhibition of binding by imidazoline ligands was cirazoline > clonidine > UK 14,304 > guanabenz, indicating an I2 subclass of imidazoline receptors. Bilateral orchiectomy increased the number of binding sites (Bmax) for [3H]idazoxan without changing the affinity (KD). Testosterone replacement, while completely restoring the plasma testosterone levels, only partially reversed the increase in Bmax. In contrast, the binding of [3H]idazoxan to prostate membranes of rats in different age groups (4, 7, and 16 months) revealed a progressive decrease in the Bmax without any change in KD. We conclude that the rat prostate expresses the I2 subclass of imidazoline receptors and that the expression is regulated by circulating testosterone.

In the present report, we investigated the potential involvement of imidazoline I 1 and I 2 binding sites in the inhibition of the Ca 2+ -activated K + channel (Gardos channel) by clotrimazole in human red cells. Ca 2+ -activated 86 Rb influx was inhibited by clotrimazole and efaroxan but not by the imidazoline binding site ligands clonidine, moxonidine, cirazoline and idazoxan (100 μM). Binding studies with [ 3 H]idazoxan and [ 3 H] p -aminoclonidine did not reveal the expression of I 1 and I 2 binding sites in erythrocytes. These data indicate that the effects of clotrimazole and efaroxan on the erythrocyte Ca 2+ -activated K + channel may be mediated by a ‘non-I 1 /non-I 2 ’ binding site.

Recent studies have shown that cirazoline, an alpha 1-adrenoceptor agonist, has greater affinity than do other imidazoline or guanidinium compounds at imidazoline recognition sites. In this report we used [3H]cirazoline as a probe to characterize imidazoline recognition sites present in membrane homogenates of rat brain and kidney as well as pancreatic beta HIT T15 cells. Specific binding of [3H]cirazoline to these various homogenates was saturable and reversible and was resolved into two classes of high affinity binding sites. Competition inhibition studies of [3H]cirazoline binding to these different membrane preparations were performed with alkaloid, phenylethylamine, imidazoline, and guanidinium compounds. Catecholamines and non-imidazoline adrenoceptor ligands such as epinephrine, benextramine, prazosin, propranolol, rauwolscine, or adrenoceptor ligands such as epinephrine, benextramine, prazosin, propranolol, rauwolscine, or yohimbine did not compete with [3H]cirazoline (Ki > 10 microM). Under our experimental conditions, only guanidinium and imidazoline derivatives had high affinities for [3H]cirazoline binding sites. Unlabeled cirazoline, clonidine, bromoxidine, idazoxan, and amiloride had the highest affinities with this respective rank order. These results suggest that [3H]cirazoline is a novel high affinity radioligand that specifically labels nonadrenergic imidazoline-guanidinium sites in the brain, kidney, and beta cells. Furthermore, the obtained rank order of inhibition suggests that [3H]cirazoline binding does not distinguish between I1 and I2 sites. In addition, we compared the specific binding of [3H]cirazoline with that of the alpha 2-adrenoceptor antagonist [3H]rauwolscine in chinese hamster ovary (CHO) cell lines stably expressing human alpha 2C2-, alpha 2C4-, and alpha 2C10-adrenoceptor subtypes. Using [3H]rauwolscine as a probe, each of these transfected cell lines expressed high levels for the three different alpha 2-adrenoceptor subtypes (Bmax values were between 2 and 7 pmol.mg-1 protein). In contrast, none of these cell lines displayed measurable imidazoline recognition sites. In summary, [3H]cirazoline is a novel high affinity radioligand that specifically labels imidazoline recognition sites without significant alpha- or beta-adrenoceptor binding. Furthermore, our results using alpha 2-adrenoceptor transfected cells confirm that the imidazoline recognition sites and each of the cloned alpha 2-adrenoceptor subtypes represent distinct macromolecular entities.

Two classes of high-affinity sites for [3H]cirazoline were characterized in rat brain and kidney membranes. In both tissues, the binding parameters for the high- and low-affinity sites are similar with Bmax values of approximately 50 fmol/mg protein, Kd approximately 0.6 nM and Bmax approximately 470 fmol/mg protein, Kd approximately 11 nM respectively. Inhibition studies of [3H]cirazoline binding to the lower affinity site revealed that only guanidinium or imidazoline derivatives compete with the specific binding of this radioligand. Our results suggest that [3H]cirazoline could be used as a novel ligand to label the non-adrenergic imidazoline-preferring sites.

The role of adrenergic stimulation on pulmonary veins (PV) ectopy and atrial fibrillation initiation is unclear. In the rat, left atrium (LA) and PV cardiomyocytes (CM) have different reactions to α-adrenergic receptor activation. Here, we examine the functional consequences of α-adrenergic receptors activation by cirazoline in rat LA and PV. PV, LA and right and left atria-PV preparations dissected from male Wistar rats were superfused with a physiological solution at 37°C. Electrical conduction within the LA and the PV was recorded with a linear array of 8 extracellular electrodes. Dual intracellular microelectrode recording used a WPI Duo 773 electrometer amplifier. Calcium transients (CaT) were recorded in isolated CM loaded with Fluo-4. Confocal microscopy was used to visualize the distribution of α-adrenergic receptors labeled with BODIPY-prazosin. In PV but not in LA strips stimulated at 0.1Hz, cirazoline induced a concentration-dependent negative inotropic effect (-79±5% at 1μM, p The author hereby declares no conflict of interest

The effects of various inotropic interventions on post-rest potentiation and its decay were investigated in isolated cardiac muscle. The inotropic interventions studied were: reduced extracellular Na+ and elevated extracellular Ca2+ concentration; exposure to ouabain, monensin, isoprenaline, phenylephrine and cirazoline. Force of contraction (stimulation frequency 2 Hz) was measured isometrically in left atria and right ventricular strips of rat hearts. Maximum post-rest potentiation was reached after 10 sec. of rest and amounted to 245 +/- 26% of pre-rest control in ventricle and 192 +/- 15% in atria. Ca(2+)-recirculation fraction was calculated from the decay of post-rest potentiation after resumption of regular stimulation, it was 0.77 +/- 0.01 in 11 control ventricular strips. High concentrations of caffeine (3 mmol/l) completely abolished post-rest potentiation in both tissues. The development of post-rest potentiation was accelerated in the presence of most of the inotropic agents. However, with the exception of ouabain and only in atrial muscle, none of the inotropic interventions produced higher post-rest contraction amplitudes than during controls. In rat heart muscle, the inotropic interventions studied are not any more effective in augmenting force of contraction than prolonged stimulation intervals. This suggests that (1) the distribution of Ca2+ into the sarcoplasmic reticulum is at a maximum during post-rest potentiation; (2) modifications of signal transduction pathways cannot further increase post-rest potentiation; and therefore that (3) shifts in Ca2+ distribution act as a limiting factor.

The technique of drug discrimination was used to examine the ability of the highly selective α2-adrenoceptor antagonist ethoxy idazoxan, which has negligible affinity for α 1-adrenoceptors or I2 imidazoline receptors, to produce an interoceptive discriminable stimulus or cue in rats. Rats were trained to respond on one lever after receiving α-ethoxy idazoxan (2.5 mg/kg, i.p.) and on the opposite lever after saline vehicle. The ethoxy idazoxan cue appeared to be mediated by antagonists of central α2-adrenoceptors, on the basis that dose- related substitution was produced by the highly selective α2-adrenoceptor antagonists idazoxan (imidazoline), fluparoxan and 1-(2-pyrimidinyl) piperazine (1-PP) (both non-imidazoline) but not by clonidine, which acts as an agonist at this receptor, nor by the peripherally acting α2-adrenoceptor antagonist L659,066. However, the α2-adrenoceptor antagonists yohimbine and atipamezole showed partial and non-dose-dependent substitution for ethoxy idazoxan over a wide range of doses. 2-BFI [2-(2-benzfuranyl)-2-imidazoline, RX801077], an imidazoline which is highly selective for I2 imidazoline receptors over α2-adrenoceptors, showed dose- dependent substitution for ethoxy idazoxan, although the maximum effect (73% responding on the ethoxy idazoxan lever) fell short of criteria adopted for full substitution. Among other agents which bind to I2 receptors, only idazoxan and 2-phenyl-2-imidazoline exhibited significant substitution; cirazoline could only be tested at very low doses because it powerfully inhibited responding in general, probably due to its α1-adrenoceptor agonist properties. It is suggested that the ability of 2-BFI to substitute partially for ethoxy idazoxan might be due to the ability of both agents to increase extracellular concentrations of noradrenaline.

The effect on insulin release of efaroxan, an alpha 2-adrenoceptor antagonist and a highly potent drug at imidazoline I1 receptors, and the effects of seven other imidazoline compounds selective for the imidazoline I1 or I2 receptors, were studied in the rat insulinoma cell line RIN-5AH. The cells released insulin in response to glucose (0.3-10 mM), and efaroxan (100 microM) potentiated glucose-induced insulin release. (-)-Adrenaline completely displaced the binding of [125I]p-iodoclonidine to membranes of RIN-5AH cells, indicating that these cells do not express imidazoline I1 receptors. Cirazoline and idazoxan (100 microM), both highly potent drugs at imidazoline I2 receptors, and the guanidines guanoxan and amiloride (200 microM), also promoted insulin release from RIN-5AH cells. Irreversible blockade of imidazoline I2 receptors with 10 microM clorgyline did not prevent the stimulatory effects of cirazoline or idazoxan; however, these compounds completely reversed the inhibition by diazoxide (250 microM), an opener of ATP-dependent K+ channels (K+ATP channels), of glucose-induced insulin release. These data indicate that the imidazoline/guanidine compounds promote insulin release from RIN-5AH cells, by interacting with a novel binding site related to K+ATP channels that does not represent any of the known imidazoline I1 or I2 receptors.

A series of alpha 1-adrenoceptor agonists evoked concentration-dependent contraction of isolated guinea-pig spleen strips ((-)-adrenaline(-)-noradrenalineL-phenylephrine(-)-(4aR, 10aR)-3, 4,4a,5,10,10a-hexahydro-6-methoxy-4-methyl-9-methylthio-2H-naphth [2,3-b]-1,4-oxazine (SDZ NVI 085)cirazoline), whereas indanidine, methoxamine, oxymetazoline and UK-14.304 were ineffective. (-)-Noradrenaline-induced contractions were inhibited by chloroethylclonidine (3 x 10(-6)-6 x 10(-5) M) and partially attenuated by SZL-49 (10(-7)-10(-6) M), but remained resistant to (+/-)-isradipine (10(-9)-10(-7) M). The contractions of the splenic strips were competitively antagonized by low concentrations of the alpha 1B-adrenoceptor-selective antagonist, spiperone (pA2 8.05), but by relatively high concentrations of the alpha 1A-adrenoceptor-selective antagonists, (+)-niguldipine (pA2 6.32) and 5-methyl-urapidil (pA2 6.95). The affinities of subtype-selective antagonists determined at guinea-pig spleen alpha-adrenoceptors significantly correlated with pKi values at rat liver alpha 1B binding sites (r = 0.96) and pA2 values at putative alpha 1B-adrenoceptors in rat aorta (r = 0.95), but differed from pKi values at rat cortical alpha 1A binding sites and pA2 values at alpha 1A-adrenoceptors in rat vas deferens. Also no correlation was obtained between antagonist affinities at guinea-pig spleen alpha-adrenoceptors and alpha 1C binding sites in rabbit liver. Thus, from the (1) potencies of agonists, (2) affinities of subtype-selective antagonists and (3) differential sensitivity of the contractions to alpha 1-adrenoceptor inactivating agents and their resistance to Ca2+ channel blockade, the alpha 1-adrenoceptor mediating smooth muscle contraction of guinea-pig spleen can be best characterized as being of the B subtype.

Recent studies have indicated that α 1 -adrenoceptor agonists such as phenylpropanolamine (PPA), cirazoline, amidephrine, and SK&F-89748 suppress food intake in rats. These compounds activate α 1 -adrenoceptors within the paraventricular hypothalamic nucleus (PVN) and may excite efferent fibers that inhibit feeding. Studies of the effects of α 1 -agonists suggest a specificity for feeding behavior, but no study to date has evaluated whether these agonists may suppress feeding behavior by the induction of malaise. Accordingly, the present experiment examined the ability of systemically administered cirazoline (0.1, 0.2, and 0.4 mg/kg, IP) to induce conditioned taste aversion (CTA) to a saccharin solution. Significant CTA was noted for 0.2 and 0.4 mg/kg cirazoline but not for 0.1 mg/kg cirazoline, compared to a vehicle treatment. The ED 50 for cirazoline-induced aversion was computed to be 0.3 mg/kg, which contrasts with an ED 50 value of 0.09 mg/kg for the effect of cirazoline on food intake (computed in other studies). More importantly, a 0.1 mg/kg dose of cirazoline, which is slightly greater than that of the ED 50 value for suppression of feeding, did not induce significant CTA in the present study. These results suggest that malaise is not a prominent factor in the suppressive activity of cirazoline on food intake and advocate the use of cirazoline as an effective appetite suppressant.

1 The concentration of the astrocytic marker, glial fibrillary acidic protein (GFAP) was quantitated by immunoblotting (western blotting) in the rat brain after treatment with various imidazoline drugs and other agents. 2 Chronic (7 days) but not acute (1 day) treatment with the imidazoline drugs, cirazoline (1 mg kg−1, i.p.) and idazoxan (10 mg kg−1, i.p.), but not with the structurally related α2-adrenoceptor antagonists, RX821002 (2-methoxy idazoxan) (10 mg kg−1, i.p.) and efaroxan (10 mg kg−1, i.p.), markedly increased (45%) GFAP immunoreactivity in the rat cerebral cortex. Chronic treatment (7 days) with yohimbine (10 mg kg−1, i.p.), a non-imidazoline α2-adrenoceptor antagonist, did not significantly modify GFAP immunoreactivity in the cerebral cortex. 3 Chronic treatment (7 days) with cirazoline and idazoxan did not alter the density of brain monoamine oxidase (MAO)-B sites labelled by [3H]-Ro 19–6327 (lazabemide), another relevant astroglial marker. Moreover, these imidazoline drug treatments did not modify the levels of α-tubulin in the cerebral cortex. These negative results reinforced the specificity of the effects of imidazoline drugs on GFAP. 4 Irreversible inactivation of brain α2-adrenoceptors (and other neurotransmitters receptors) after treatment with an optimal dose of the peptide-coupling agent EEDQ (1.6 mg kg−1, i.p., for 6–24 h) did not alter GFAP immunoreactivity in the cerebral cortex. These results further disproved the involvement of these receptors on astroglial cells in the tonic control of GFAP levels. 5 The binding of [3H]-idazoxan in the presence of 10−6 m (–)-adrenaline was used to quantitate in parallel I2-imidazoline preferring sites in the rat brain after the same treatments. Chronic treatment (7 days) with cirazoline and idazoxan, but not with RX821002, efaroxan or yohimbine, significantly increased (25%) the density of I2-sites in the cerebral cortex. The up-regulation of I2-sites induced by cirazoline was not observed in the liver, a tissue that also expresses I2-sites but lacks glial cells. 6 A strong correlation (r = 0.97) was found when the mean percentage changes in GFAP immunoreactivity were related to the mean percentage changes in I2 imidazoline sites after the various drug treatments. 7 Together the results suggest a direct physiological function of glial I2-imidazoline preferring sites in the regulation of GFAP levels.

We have examined the effects of captopril on pressor responses to the selective alpha 1-adrenoceptor agonist cirazoline in the pithed rat preparation following treatment with phenoxybenzamine and/or nifedipine. Pretreatment with captopril reduced the pressor responses to cirazoline and displaced the dose-response curve for this agonist to the right, significantly increasing the ED50 without altering the maximum response. Pretreatment with phenoxybenzamine accentuated the inhibitory actions of captopril and a combination of phenoxybenzamine and captopril significantly increased the ED50 without altering the maximum response. Administration of nifedipine in animals, which had already received phenoxybenzamine and captopril, led to a further displacement to the right of the cirazoline dose-response curve. The ED50 was found to be significantly increased and the maximum response was now significantly depressed. Captopril produced further additive inhibition with nifedipine and phenoxybenzamine of the vasoconstrictor effects of cirazoline. These data indicate, perhaps not surprisingly, that the cellular basis for the inhibitory effects of captopril is different from that of nifedipine and phenoxybenzamine, however, more importantly, that captopril may directly, or indirectly, inhibit receptor-operated cation channel mediated pressor responses.

Amphetamine (AMPH) increases adult rat 50-kHz ultrasonic vocalizations, preferentially promoting frequency-modulated (FM) calls that have been proposed to reflect positive affect. The main objective of this study was to investigate a possible noradrenergic contribution to AMPH-induced calling. Adult male Long-Evans rats were tested with AMPH (1 mg/kg intraperitoneal) or saline combined with various systemic pretreatments: clonidine (α2 adrenergic agonist), prazosin (α1 antagonist), atipamezole (α2 antagonist), propranolol, betaxolol, and/or ICI 118,551 (β1/β2, β1, and β2 antagonists, respectively), nadolol (β1/β2 antagonist, peripheral only), or NAD-299 (5HT(1A) antagonist). In addition, effects of cirazoline (α1 adrenergic agonist) and cocaine (0.25-1.5 mg/kg intravenous) were studied alone. AMPH-induced calling was suppressed by low-dose clonidine and prazosin. Cirazoline and atipamezole did not significantly affect calling rate. Propranolol, without affecting the call rate, dose dependently promoted 'flat' calls under AMPH while suppressing 'trills,' thus reversing the effects of AMPH on the 'call subtype profile.' This effect of propranolol seemed to be mediated by simultaneous inhibition of CNS β1 and β2 rather than by 5HT(1A) receptors. Finally, cocaine elicited fewer calls than did AMPH, but produced the same shift in the call subtype profile. Taken together, these results reveal differential drug effects on flat vs trill vs other FM 50-kHz calls. These findings highlight the value of detailed call subtype analyses, and show that 50-kHz calls are associated with adrenergic α1- and β-receptor mechanisms. These preclinical findings suggest that noradrenergic contributions to psychostimulant subjective effects may warrant further investigation.