Jacareubin inhibits FcεRI-induced extracellular calcium entry and production of reactive oxygen species required for anaphylactic degranulation of mast cells.

Mast cells (MCs) are important effectors in allergic reactions since they produce a number of pre-formed and de novo synthesized pro-inflammatory compounds in response to the high affinity IgE receptor (FcεRI) crosslinking. IgE/Antigen-dependent degranulation and cytokine synthesis in MCs have been recognized as relevant pharmacological targets for the control of deleterious inflammatory reactions. Despite the relevance of allergic diseases worldwide, efficient pharmacological control of mast cell degranulation has been elusive. In this work, the xanthone jacareubin was isolated from the heartwood of the tropical tree Callophyllum brasilense, and its tridimensional structure was determined for the first time by X-ray diffraction. Also, its effects on the main activation parameters of bone marrow-derived mast cells (BMMCs) were evaluated. Jacareubin inhibited IgE/Ag-induced degranulation in a dose-response manner with an IC50 = 46 nM. It also blocked extracellular calcium influx triggered by IgE/Ag complexes and by the SERCA ATPase inhibitor thapsigargin (Thap). Inhibition of calcium entry correlated with a blockage on the reactive oxygen species (ROS) accumulation. Antioxidant capacity of jacareubin was higher than the showed by α-tocopherol and caffeic acid, but similar to trolox. Jacareubin shown inhibitory actions on xanthine oxidase, but not on NADPH oxidase (NOX) activities. In vivo, jacareubin inhibited passive anaphylactic reactions and TPA-induced edema in mice. Our data demonstrate that jacareubin is a potent natural compound able to inhibit anaphylactic degranualtion in mast cells by blunting FcεRI-induced calcium flux needed for secretion of granule content, and suggest that xanthones could be efficient anti-oxidant, antiallergic, and antiinflammatory molecules.

Stimulation of nAchRα7 Receptor Inhibits TNF Synthesis and Secretion in Response to LPS Treatment of Mast Cells by Targeting ERK1/2 and TACE Activation.

The cholinergic anti-inflammatory pathway is recognized as one of the main mechanisms of neuromodulation of the immune system. Activation of the α7 nicotinic acetylcholine receptor (nAchRα7) suppresses cytokine synthesis in distinct immune cells but the molecular mechanisms behind this effect remain to be fully described. Mast cells (MCs) are essential players of allergic reactions and innate immunity responses related to chronic inflammation. Activation of TLR4 receptor in MCs leads to the rapid secretion of pre-synthesized TNF from intracellular pools and to the activation of NFκB, necessary for de novo synthesis of TNF and other cytokines. Here we report that the nAchRα7 receptor specific agonist GTS-21 inhibits TLR4-induced secretion of preformed TNF from MCs in vivo and in vitro. Utilizing bone marrow-derived mast cells (BMMCs) it was found that GTS-21 also diminished secretion of de novo synthesized TNF, TNF mRNA accumulation and IKK-dependent p65-NFκB phosphorylation in response to LPS. nAchRα7 triggering prevented TLR4-induced ERK1/2 phosphorylation, which resulted an essential step for TNF secretion due to the phosphorylation of the metallopeptidase responsible for TNF maturation (TACE). Main inhibitory actions of GTS-21 were prevented by AG490, an inhibitor of JAK-2 kinase. Our results show for the first time, that besides the prevention of NFκB-dependent transcription, inhibitory actions of nAchRα7 triggering include the blockade of pathways leading to exocytosis of granule-stored cytokines in MCs.

Role of main neuroendocrine pathways activated by swim stress on mast cell-dependent peritoneal TNF production after LPS administration in mice.

OBJECTIVE AND DESIGN: To characterize the effects of swim stress on the early mast cell (MC)-dependent peritoneal production of TNF in response to lipopolysaccharide (LPS) administration in mice, identifying the neuroendocrine mediators involved. SUBJECTS: Ten to twelve-week-old Swiss Webster, C57BL/6 J or c-Kit (Wsh/Wsh) mice were used. TREATMENT: Animals were intraperitoneally challenged with LPS at different times after forced swimming (FS) and peak TNF production was determined in peritoneal washes at optimal time after LPS administration. Selective blockage of main neuroendocrine pathways was performed before swim stress. METHODS: TNF concentrations were determined by ELISA. RESULTS: FS provoked an immediate and transient inhibition of LPS-elicited, MC-dependent TNF accumulation in peritoneum, which lasted around 30 min. Suppresive effects of FS were absent on MC-deficient c-Kit (Wsh/Wsh) mice but were recovered after reconstitution with MC. Adrenalectomy or DSP4 administration increased basal ip TNF levels and enhanced LPS-induced TNF release without any effect on stress-induced inhibitory effects, mifepristone did not produce any change on stress-induced inhibition, whereas mecamylamine administration increased basals and attenuated stress effects. CONCLUSIONS: Swim stress transiently inhibits the canonical MC-dependent response of TNF production in response to LPS in murine peritoneal cavity with the main participation of the cholinergic anti-inflammatory reflex.

Time-course of 5-HT(6) receptor mRNA expression during memory consolidation and amnesia.

Growing evidence indicates that antagonists of the 5-hydroxytryptamine (serotonin) receptor(6) (5-HT(6)) improve memory and reverse amnesia although the mechanisms involved are poorly understood. Hence, in this paper RT-PCR was used to evaluate changes in mRNA expression of 5-HT(6) receptor in trained and untrained rats treated with the 5-HT(6) receptor antagonist SB-399885 and amnesic drugs scopolamine or dizocilpine. Changes in mRNA expression of 5-HT(6) receptor were investigated at different times in prefrontal cortex, hippocampus and striatum. Data indicated that memory in the Pavlovian/instrumental autoshaping task was a progressive process associated to reduced mRNA expression of 5-HT(6) receptor in the three structures examined. SB-399885 improved long-term memory at 48h, while the muscarinic receptor antagonist scopolamine or the non-competitive NMDA receptor antagonist dizocilpine impaired it at 24h. Autoshaping training and treatment with SB-399885 increased 5-HT(6) receptor mRNA expression in (maximum increase) prefrontal cortex and striatum, 24 or 48h. The scopolamine-induced amnesia suppressed 5-HT(6) receptor mRNA expression while the dizocilpine-induced amnesia did not modify 5-HT(6) receptor mRNA expression. SB-399885 and scopolamine or dizocilpine were able to reestablish memory and 5-HT(6) receptor mRNA expression. These data confirmed previous memory evidence and of more interest is the observation that training, SB-399885 and amnesic drugs modulated 5-HT(6) receptor mRNA expression in prefrontal cortex, hippocampus and striatum. Further investigation in different memory tasks, times and amnesia models together with more complex control groups might provide further clues.

Super-oxidized solution inhibits IgE-antigen-induced degranulation and cytokine release in mast cells.

Activation of the high affinity IgE receptor (Fc epsilonRI) through IgE-antigen complexes induces mast cell degranulation, synthesis of lipid mediators and cytokine production. These effects are involved in Type I hypersensitivity reactions and controlling them has been the main objective of many anti-allergic therapies. Here we report that pretreatment of murine bone marrow derived mast cells (BMMC) with super-oxidized solution (SOS) inhibits Fc epsilonRI dependent-beta hexosaminidase and cytokine release. This effect is exerted without altering total protein tyrosine phosphorylation, MAPK activation, cytokine mRNA accumulation or calcium mobilization after Fc epsilonRI triggering. Our data suggest that this neutral pH-SOS acts like a mast cell-membrane stabilizer inhibiting the cell machinery for granule secretion without altering the signal transduction pathways induced by IgE-antigen receptor crosslinking.

[Wandering spleen torsion]. / Bazo ectópico torsionado.

The ectopic spleen characterizes for absence of its suspensory ligaments and a long pedicle that are predisposed to complicate it for a torsion with commitment of the venous drainage at first and arterial at a later time himself, producing increase of its volume and infarct. This anomalous situation, it can be had to a congenital malformation of the development of its suspensory elements and fall toward the inferior abdomen or else to an inferior growth of the mesodermic yolk of that this organ originates itself. The suitable treatment is the laparoscopic splenopexy, but when it exists infarction, it is no possible avoid the splenectomy.

Molecular cloning and functional expression of the guinea pig alpha(1a)-adrenoceptor.

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.

Vav1 regulates phospholipase cgamma activation and calcium responses in mast cells.

The hematopoietic cell-specific protein Vav1 is a substrate of tyrosine kinases activated following engagement of many receptors, including FcepsilonRI. Vav1-deficient mice contain normal numbers of mast cells but respond more weakly than their normal counterparts to a passive systemic anaphylaxis challenge. Vav1-deficient bone marrow-derived mast cells also exhibited reduced degranulation and cytokine production, although tyrosine phosphorylation of FcepsilonRI, Syk, and LAT (linker for activation of T cells) was normal. In contrast, tyrosine phosphorylation of phospholipase Cgamma1 (PLCgamma1) and PLCgamma2 and calcium mobilization were markedly inhibited. Reconstitution of deficient mast cells with Vav1 restored normal tyrosine phosphorylation of PLCgamma1 and PLCgamma2 and calcium responses. Thus, Vav1 is essential to FcepsilonRI-mediated activation of PLCgamma and calcium mobilization in mast cells. In addition to its known role as an activator of Rac1 GTPases, these findings demonstrate a novel function for Vav1 as a regulator of PLCgamma-activated calcium signals.

A perspective: regulation of IgE receptor-mediated mast cell responses by a LAT-organized plasma membrane-localized signaling complex.

BACKGROUND: To understand how the high-affinity IgE receptor (FcepsilonRI) communicates with downstream effectors, we focused on exploring the functional importance of the FcepsilonRI-mediated formation and localization of a signaling complex that contains the hematopoietic cell-specific scaffolding protein linker for activation of T cells (LAT) and the guanine nucleotide exchange factor Vav1. METHODS: Using the mast cell line RBL-2H3, we explored the localization of these proteins by confocal microscopy and cell fractionation. Additionally, the mechanism of function and the importance of LAT and Vav1 to mast cells was studied in genetically disrupted mice and in mast cells derived from their bone marrow. RESULTS: We found that LAT, Vav1 and the adapter molecule SLP-76 associated in detergent-resistant microdomains (lipid rafts) found in the plasma membrane upon FcepsilonRI stimulation. In the absence of LAT, mast cells showed a remarkable loss of the secretory response and reduced cytokine responses. Vav1 deficiency also affected secretion, although not to the extent of LAT deficiency, and inhibited IL-2 and IFN-gamma production. LAT- and Vav1-deficient mice showed reduced blood histamine levels after a systemic anaphylaxis challenge as compared to their normal counterparts. CONCLUSIONS: The results demonstrate that LAT is a central mediator in IgE receptor signaling by regulating multiple signaling pathways that affect mast cell degranulation and cytokine production. Vav1, a component of this LAT-containing signaling complex, regulates a specific subset of these responses.

Norepinephrine- and phorbol ester-induced phosphorylation of alpha(1a)-adrenergic receptors. Functional aspects.

Maximal adrenergic responses in Rat-1 fibroblasts expressing alpha(1a)-adrenergic receptors are not blocked by activation of protein kinase C. In contrast, activation of protein kinase C induces the phosphorylation of alpha(1b)-adrenoreceptors and blocks their actions. The effect of norepinephrine and phorbol esters on alpha(1a)-adrenoreceptor phosphorylation and coupling to G proteins were studied. Both stimuli lead to dose-dependent receptor phosphorylation. Interestingly, protein kinase C activation affected to a much lesser extent the actions of alpha(1a)-adrenergic receptors than those of the alpha(1b) subtype (norepinephrine elicited increases in calcium in whole cells and [(35)S]GTPgammaS binding to membranes). Basal phosphorylation of alpha(1a)-adrenergic receptors was much less than that observed with the alpha(1b) subtype. The carboxyl terminus seems to be the main domain for receptor phosphorylation. Therefore, chimeric receptors, where the carboxyl-terminal tails of alpha(1a) and alpha(1b) adrenergic receptors were exchanged, were constructed and expressed. alpha(1a)-Adrenoreceptors wearing the carboxyl tail of the alpha(1b) subtype had a high basal phosphorylation and displayed a strong phosphorylation in response to norepinephrine and phorbol esters. Our results demonstrate that stimulation of alpha(1a)-adrenergic receptor, or activation of protein kinase C, leads to alpha(1a)-adrenergic receptor phosphorylation. alpha(1a)-Adrenoreceptors are affected to a much lesser extent than alpha(1b)-adrenoreceptors by protein kinase C activation.

Inverse alpha(1A) and alpha(1D) adrenoceptor mRNA expression during isolation of hepatocytes.

It is now well documented that changes in gene expression take place during cell isolation and culture. Here, we report the change in the expression of the mRNAs for alpha(1)-adrenoceptor subtypes, during dissociation of guinea pig liver cells with collagenase. Using Reverse Transcription-Polymerase Chain Reaction (RT-PCR) assays, it was observed that during the isolation procedure, the mRNA for the alpha(1A)-adrenoceptor, normally expressed in whole liver, was degraded and the mRNA for alpha(1D) subtype, barely expressed in whole liver, increased in an actinomycin D-sensitive manner. When the isolation procedure was performed in the presence of cycloheximide, the mRNA for the alpha(1A)-adrenoceptor did not diminish and the induction of the alpha(1D)-adrenoceptor mRNA was even more evident. Our data indicate that cell isolation alters alpha(1)-adrenoceptor mRNA expression.

Angiotensin AT1 receptors in Clone 9 rat liver cells: Ca2+ signaling and c-fos expression.

In C9 (Clone 9) liver cells, angiotensin 11 increased the intracellular Ca2+ content, inositol phosphate production and c-fos mRNA expression. Other angiotensins were also active with the order of potency being angiotensin II = angiotensin III >> angiotensin I > angiotensin IV. Losartan, but not PD 123177 (1-(4-amino-3-methyl)-5-diphenylacetyl-4,5,6,7-tetrahydro-1H-imida zo [4,5c]pyridine-6-carboxylic acid), blocked the effects of angiotensin II. Pertussis toxin did not alter these actions of angiotensin II. These data indicate that the effects were mediated through angiotensin AT1 receptors involving pertussis toxin-insensitive G-proteins. Phorbol myristate acetate was also able to increase c-fos mRNA expression. The action of angiotensin II was consistently greater than that of the active phorbol ester. Staurosporine but not genistein inhibited this effect of angiotensin II. Angiotensin II- and phorbol myristate acetate-induced proto-oncogene mRNA expression was attenuated in cells incubated overnight with the active phorbol ester, which suggests a major role of protein kinase C.

Characterization of the AT1 angiotensin II receptor expressed in guinea pig liver.

In guinea pig hepatocytes angiotensin II induced phosphorylase a activation. This effect was mimicked by other angiotensins with the potency order: angiotensin II (EC50 approximately 1 nM) > angiotensin III (EC50 approximately nM) > angiotensin I (EC50 approximately 300 nM). The effect of 10 nM angiotensin II was blocked by the angiotensin II receptor AT1-selective antagonists irbesartan and losartan (Ki values of approximately 1 nM and approximately 10 nM for irbesartan and losartan respectively) but not by the AT2-selective antagonist PD123177. Similar data were obtained when the production of [3H]IP3 from [3H]myo-inositol-labeled cells was studied Angiotensin II induced a dose-dependent increase in [3H]IP3 production; the maximal effect (approximately 3-fold) was observed at a concentration of 10 microM. This effect of angiotensin II was completely blocked by the AT1-selective antagonists irbesartan and losartan, but only in a very limited fashion by PD123177. [125I][Sar1-Ile8]angiotensin II bound with high affinity (approximately 3.8 nM) to a moderately abundant number of sites (approximately 660 fmol/mg protein) in guinea pig liver membranes. Binding competition experiments indicate the following orders of potency for agonists: angiotensin II (approximately 1.5 nM) > angiotensin III (approximately 7 nM) > angiotensin I (approximately 176 nM), and for antagonists: irbesartan (approximately 0.5 nM) > losartan (approximately 36 nM) > > PD123177 (> > 10000 nM). The functional and binding data strongly indicate that the effects of angiotensin II were mediated through AT1 receptors. Expression of the mRNA for these receptors was confirmed by RT-PCR and hybridization of the reaction product with a radiolabeled rat AT1 receptor cDNA probe.

Coexpression of alpha 1A- and alpha 1B-adrenoceptors in the liver of the rhesus monkey (Macaca mulatta).

The alpha 1-adrenoceptors present in the liver of rhesus monkeys was characterized using [3H]prazosin. This radioligand binds to monkey liver membranes with high affinity (KD 0.33 nM) to a moderately abundant number of sites (97 fmol/mg of protein). These sites were characterized pharmacologically, by binding competition, observing two affinities for most ligands. The order of potency for agonists was: (a) for the high affinity sites: oximetazoline > epinephrine = norepinephrine > methoxamine; and (b) for the other sites (low affinity for the alpha 1A-adrenoceptor-selective agonists): oximetazoline > or = epinephrine = norepinephrine > > methoxamine. For antagonists the orders of potency were: (a) for the high affinity sites: R-(-)-5[2-[[2-(ethoxyphenoxy)ethyl]amino]propyl]-2-metoxybenzen esulfonamide HCl (tamsulosin) > or = 2-(2,6-dimethoxyphenoxyethyl)-aminomethyl-1,4-benzodioxane (WB4101) > or = prazosin > or = (+)-niguldipine > 5-methylurapidil = benoxathian > phentolamine > 8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4,5]deca ne-7,9- dione dihydrochloride (BMY 7378); (b) for the other sites (low affinity for the alpha 1A-adrenoceptor-selective antagonists): prazosin > tamsulosin > phentolamine = WB4101 > (+)-niguldipine > or = 5-methyl-urapidil = benoxathian > BMY 7378. These data strongly suggest that Macaca mulatta liver cells coexpress alpha 1A- and alpha 1B-adrenoceptors. Expression of the mRNA for these receptors was confirmed by reverse transcriptase-polymerase chain reactions.

Hormonal modulation of c-fos expression in isolated hepatocytes. Effects of angiotensin II and phorbol myristate acetate on transcription and mRNA degradation.

It has been shown that angiotensin II and PMA increase the expression of proto-oncogenes (c-fos, c-myc and c-mos) in liver cells. In this study the effects of angiotensin II and PMA on c-fos transcription and mRNA stability were investigated. Using nuclear run-off transcription assays, it was observed that PMA and angiotensin II induced a rapid increase in c-fos transcription. The transcription rate of the GAPDH gene did not change, indicating that the effects were not general on gene transcription. The ability of these agents to modulate proto-oncogene mRNA stability was tested by measuring c-fos mRNA half-life. It was observed that c-fos mRNA half-life was relatively short (approximately 14-18 min) and that angiotensin II and PMA markedly stabilized mRNA, increasing its half-life (approximately 4-fold and approximately 2-fold, respectively). The protein synthesis inhibitor cycloheximide increased mRNA stability to a much greater extent. Our results clearly demonstrate that angiotensin II and PMA increased c-fos mRNA accumulation in liver cells through two actions: induction of c-fos gene transcription and increase in mRNA stability.

Characterization of the alpha 1-adrenoceptors of cat liver. Predominance of the alpha 1A-adrenergic subtype.

The alpha 1-adrenoceptors present in the liver of cats were characterized using [3H]prazosin. This radioligand binds to cat liver membranes with high affinity ((KD 0.79 nM) to a moderately abundant number of sites (160 fmol/mg of protein). This sites were characterized pharmacologically, by binding competition, observing the following orders of potency: a) for agonists: oxymetazoline > epinephrine = norepinephrine >> methoxamine, and b) for antagonists: WB4101 > or = prazosin > or = (+) niguldipine > or = benoxathian > or = spiperone = 5-methyl-urapidil > phentolamine > BMY 7378. These data suggested that cat liver expresses alpha 1A-adrenoceptors. Expression of the mRNA for this receptor was confirmed by RT-PCR.

Characterization of the alpha 1-adrenoceptors of dog liver: predominance of the alpha 1A-subtype.

Using dog liver membranes we observed that [125I]HEAT ((+/-)-beta-([125I]iodo-4-hydroxyphenyl)-ethyl-aminomethyl-tetralone) binds with high affinity (KD 97 pM) to a discrete number of sites (Bmax 40 fmol/mg protein) with the pharmacological characteristics expected for alpha 1-adrenoceptors. Such sites were inactivated by pretreatment with chloroethylclonidine. Binding competition experiments indicated the following order of potency: (a) for agonists: oxymetazoline > epinephrine > or = norepinephrine > methoxamine and (b) for antagonists: WB4101 > or = 5-methyl-urapidil = prazosin > or = benoxathian > or = (+)-niguldipine > phentolamine. Northern analysis indicated that total RNA isolated from dog liver hybridized with an alpha 1c selective probe (bovine brain). The orders of potency for agonists and antagonists, their Ki values and the Northern analysis suggest that dog liver expresses alpha 1A-adrenoceptors.

Protein kinases and phosphatases modulate c-fos expression in rat hepatocytes. Effects of angiotensin II and phorbol myristate acetate.

In isolated rat hepatocytes angiotensin II and phorbol 12-myristate 13-acetate (PMA) induce the expression of c-fos. We studied the possible transduction pathway(s) involved in this effect using inhibitors of serine-threonine and tyrosine protein kinases. Calphostin and staurosporine, inhibitors of protein kinase C and other serine-threonine protein kinases, block in a dose-dependent manner the effect of angiotensin II and PMA. Interestingly, genistein also blocks the induction of this proto-oncogene, suggesting a role for tyrosine protein kinases. Inhibitors of serine-threonine protein phosphatases, such as okadaic acid, microcystin LR and calyculin also induce c-fos expression. These data suggest that protein phosphatases exert a tonic inhibitory control of c-fos expression. The effect of these phosphatase inhibitors were not blocked by staurosporine, calphostin or genistein. Our results suggest that the expression of c-fos in rat hepatocytes is regulated by complex phosphorylation-dephosphorylation cascade(s) probably involving serine/threonine and tyrosine protein kinase and protein phosphatase activities.

Differences between rapid and longer-term actions of angiotensin II in isolated rat hepatocytes. Effects on phosphorylase a activity and c-fos expression.

Angiotensin II stimulates phosphorylase a activity and c-fos expression in isolated rat hepatocytes. Both effects are mediated through AT1 receptors. However, the time-courses and the dose-dependencies of these responses were different. These effects of angiotensin II were blocked by Losartan when the antagonist was added to the cells before the hormone or when both the hormone and the antagonist were added simultaneously. Interestingly, when the antagonist was added 2 or 3 min after the peptide hormone, the action on phosphorylase a activity was markedly decreased but, in contrast, that on c-fos expression was not affected. The data suggest that angiotensin II-mediated activation of phosphorylase a was reversible and dependent on continuous receptor activation by the hormone whereas c-fos expression did not seem to have these characteristics, i.e., our data suggest that once protooncogene expression is triggered, the process becomes independent of the hormone-receptor interaction.

Characterization of the hepatic alpha 1B-adrenoceptors of rats, mice and hamsters.

The alpha 1-adrenoceptors present in liver membranes from rats, hamsters and mice were characterized using [3H]prazosin. In the liver membranes from the three species a relatively large number of receptors was observed (500-900 fmol/mg of protein) and the affinities for [3H]prazosin were very similar (0.2-0.3 nM). Membrane preincubation with 10 microM chloroethylclonidine markedly decreased [3H]prazosin binding and higher concentrations essentially abolished specific binding of this radioligand. Binding competition experiments indicated the following orders of potency: a) for agonists: oxymetazoline > epinephrine > or = norepinephrine >> methoxamine and b) for antagonists: prazosin > WB 4101 > or = phentolamine = benoxathian > 5-methyl urapidil. The affinity for (+)niguldipine was also low but there was variation between the three species. Total RNA obtained from the liver of these species hybridized with the alpha 1B-adrenergic cDNA probe. The data suggest that these receptors correspond to the alpha 1B subtype.