Constitutive nitric oxide modulates the injurious actions of vasopressin on rat intestinal microcirculation in acute endotoxaemia

The administration of the nitric oxide (NO) synthase inhibitor, N^G-nitro-l-arginine methyl ester (L-NAME, 5 mg/kg s.c.) concurrently with Escherichia coli endotoxin (3 mg/kg i.v.) increased vascular permeability and caused mucosal damage in the rat intestine 1 h later. The vasopressin V₁ receptor antagonist, [Mca¹,Tyr(Me)²,Arg⁸]vasopressin (0.01–0.2 μg/kg s.c., 15 min before endotoxin) dose-dependently reduced this damage. These results suggest a beneficial role of NO, counteracting the injurious vascular actions of endogenous vasopressin, in maintaining intestinal mucosal integrity in acute endotoxaemic states.     

The central sympatho-inhibitory effect of 5,6-dihydroxy-2-dimethylaminotetralin (M7) is mediated by α2-adrenoceptors

M7 (5,6-dihydroxy-2-dimethylaminotetralin) produces in anesthetized rats a hypotensive response previously attributed to peripheral dopaminergic mechanisms. We re-examined the effects of this drug on arterial blood pressure, heart rate and sympathetic nerve activity in anesthetized rats and dogs. M7 (1–100 μg/kg i.v.) produced in the rats transient dose-dependent pressor effects, with bradycardia and sympatho-inhibition, followed by long-lasting dose-dependent hypotension, bradycardia and sympatho-inhibition. The sympatho-inhibitory and hypotensive effects were comparable in baroreceptor-denervated rats and were reversed by idazoxan (0.1 mg/kg i.v.). The sympatho-inhibitory response induced by M7 (1–100 μg/kg) was prevented by treatment with the specific α₂-adrenoceptor antagonist, 2-methoxy-idazoxan (0.03 mg/kg i.v.). This central effect of M7 was not altered by treatment with the α₁-adrenoceptor antagonist, prazosin (0.1 mg/kg i.v.) and was reduced by treatment with the α₂-adrenoceptor antagonists, yohimbine (1 mg/kg i.v.) or idazoxan (0.3 mg/kg i.v.), and the dopaminergic antagonists, haloperidol (0.5 mg/kg i.v.) or sulpiride (3 mg/kg i.v.). Bilateral microinjections of M7 (0.3–3 nmol) into the rostroventral medulla in the rat produced dose-dependent hypotension, bradycardia and sympathetic nerve inhibition which were reversed and prevented by bilateral microinjection of 2-methoxy-idazoxan (1 nmol) into the same sites. Microinjections of 2-methoxy-idazoxan into the rostroventral medulla also inhibited the central effects of M7 at 0.03 mg/kg i.v. In anesthetized dogs, M7 administered into the cisterna magna (1–10 μg/kg) reduced arterial blood pressure, heart rate and sympathetic nerve activity; these effects were reversed by administration of 2-methoxy-idazoxan (0.03 mg/kg i.v.). In conclusion, M7, a rigid catecholamine, produces a potent central sympatho-inhibitory and hypotensive effect by activation of α₂-adrenoceptors.     

Postsynaptic α-adrenoceptors in the perfused canine saphenous vein in vitro

To study the relative localization of α₁- and α₂-adrenoceptors in relation to the intima and the adventitia of canine saphenous vein, a comparison was made of the potency of α₁- and α₂-adrenoceptors agonists applied by intraluminal and extraluminal route of perfused segments of that vessel. Noradrenaline was the most potent of the agonists used and was approximately as potent by intraluminal as by extraluminal route. Cocaine (12 μmol/1) caused supersensitivity to noradrenaline which was of about the same magnitude (threefold) irrespective of the route of administration of noradrenaline. The selective α₁-agonist phenylephrine was about 10 times less potent than noradrenaline and was also equieffective by both routes. The selective α₂-agonist UK-14,304, at concentrations lower than 0.3 μmol/l, caused very small responses and only in 3 out of 14 experiments. In all cases it caused responses at concentrations higher than 0.3 μmol/l. Cocaine did not change the sensitivity to either phenylephrine or UK-14,304. Thus, it is concluded that the results obtained with cocaine agreed with expectations for a homogeneously innervated tissue. Furthermore, α₁-adrenoceptors seem to predominate and to be evenly distributed throughout the media. The lack of responses to the low concentrations of UK-14,304—those selectively acting on α₂-adrenoceptors—was ascribed to the very low efficacy of this agonist on the distal part of the canine saphenous vein and to the tone created by the perfusion pressure which might be high enough to mask this small response.     

The rostroventromedial medulla is not involved in α2-adrenoceptor-mediated antinociception in the rat

The aim of the current study was to investigate the role of the rostroventromedial medulla (RVM) in α₂-adrenoceptor-mediated antinociception. Medetomidine or clonidine, selective α₂-adrenoceptor agonists were microinjected into the RVM in unanesthetized rats with a chronic guide cannula. The antinociceptive effects were evaluated using the tail-flick and hot-plate tests. For comparison, medetomidine was microinjected into the cerebellum or the periaqueductal gray (PAG). To study the role of medullospinal pathways, the tail-flick latencies were also measured in spinalized rats. The reversal of the antinociception induced by intracerebral microinjections of medetomidine was attempted by s.c. atipamezole, a selective α₂-adrenoceptor antagonist. The reversal of the antinociception induced by systemic administration of medetomidine was attempted by microinjections of 5% lidocaine or atipamezole into the RVM. When administered into the RVM, medetomidine produced a dose-dependent (1–30 μg) antinociception in the tail-flick and hot-plate tests, which antinociceptive effect was completely reversed by atipamezole (1 mg/kg, s.c.). Also clonidine produced a dose-dependent (3–30 μg) antinociception following microinjection into the RVM. Microinjections of medetomidine into the cerebellum or the PAG produced an identical dose-response curve in the tail-flick test as that obtained following microinjection into the RVM. In spinalized rats the antinociceptive effect (tail-flick test) induced by medetomidine microinjected into the RVM was not less effective than in intact rats. Lidocaine (5%) or atipamezole (5 μg) microinjected into the RVM did not attenuate the antinociception induced by systemically administered medetomidine (100 μg/kg, s.c.). The adapting skin temperature of the tail was increased in a nonmonotonic fashion following medetomidine. The results indicate that the RVM is not a site which is critical for the α₂-adrenergic antinociception. The antinociception following intracerebral microinjections of medetomidine into the RVM, PAG or the cerebellum in the current study can be explained by a spread of the α₂-adrenoceptor agonist into the spinal level to activate directly spinal α₂-adrenoceptors. Also, the antinociception following systemic administration of medetomidine can be explained by spinal α₂-adrenergic mechanisms. The medetomidine-induced increase of the adapting skin temperature may have attenuated the medetomidine-induced increases in the response latencies to noxious heat.     

Some unusual effects of α2-adrenergic drugs on cortical high voltage spindles in rats

The dose-response curves for a number of α-adrenergic drugs were investigated to estimate a possible role of the α₂/α₁ selectivity of these drugs on the incidence of cortical high voltage spindles (HVS), reflecting level of vigilance. The α₂ antagonists yohimbine (0.25–4 mg/kg) and idazoxan (0.5–4 mg/kg), but not atipamezole induced a biphasic effect on the incidence of HVS in rats. This effect of relatively small doses of yohimbine and idazoxan should be taken into consideration when using these drugs as ga₂ antagonists in behavioral and neurophysiological tests. On the other hand the linearity of the dose-response curve for atipamezole (0.01–4 mg/kg) indicates that this drug is a good candidate for use in such tests.     

The role of A3 adenosine receptors in central regulation of arterial blood pressure

1. Pharmacological studies have suggested that A₃ receptors are present on central neurons. Recently this adenosine receptor subtype has been identified in the rat and its presence in the central nervous system has been confirmed.
2. In this study we investigated the effects of acute intracerebroventricular (i.c.v.) injections of N⁶-2-(4-aminophenyl)-ethyladenosine (APNEA), a non-selective A₃ adenosine receptor agonist, on arterial blood pressure (ABP) and heart rate (HR), after treatment with 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), a selective antagonist of A₁ adenosine receptors.
3. Anaesthetized rats, after DPCPX (12 μg⁻¹ kg i.c.v.), were treated with APNEA (0.4–4 μg kg⁻¹ i.c.v.) resulting in a transitory and dose-dependent decrease in arterial blood pressure without a change in heart rate. APNEA also induced hypotensive responses after i.c.v. pretreatment with aminophylline, at a dose of 20 μg kg⁻¹. In contrast, pretreatment 48 h before, with 4 μg kg⁻¹ i.c.v. of pertussis toxin reduced the hypotensive effect induced by APNEA. Administration of APNEA at a higher dose (20 μg kg⁻¹ i.c.v.), after DPCPX, induced a decrease in ABP of −66±5.4 mmHg and after 3 min a decrease in heart rate of −62±6.0 beats min⁻¹. Transection of the spinal cord abolished this significant fall in ABP, but not the decrease of HR.
4. These results suggest that a population of A₃-receptors is present in the CNS, whose activation induces a decrease in blood pressure with no change of heart rate.

     

Modulation of AT1 receptor-mediated contraction of rat uterine artery by AT2 receptors

1. The aim of this study was to characterize the angiotensin II receptors in isolated uterine arteries from non pregnant and pregnant rats, since it has been reported from binding studies that ovine uterine arteries contain AT₂ receptors.
2. Uterine arterial segments were obtained from virgin, non-pregnant and late pregnant (18–21 days) Sprague-Dawley rats and mounted in small vessel myographs. Concentration-response curves were constructed to angiotensin II (1 nM–10 μM) in the absence and presence of various angiotensin II receptor subtype selective compounds. These included losartan (AT₁ antagonist; 1, 10 and 100 nM), PD 123319 (AT₂ antagonist; 1 μM) and CGP 42112 (AT₂ agonist; 1 μM). Responses to angiotensin II were measured as increases in force (mN) and expressed as a per cent of the response to a K⁺ depolarizing solution.
3. Losartan (1, 10 and 100 nM) caused significant concentration-dependent rightward shifts of the angiotensin II concentration-response curve in uterine arteries from non-pregnant and pregnant rats. The pA₂ values calculated from these data were 9.8 and 9.2, respectively, although the slope of the Schild plot in the non-pregnant group was less than unity.
4. PD 123319 (1 μM) caused significant 6- and 3 fold leftward shifts of the angiotensin II concentration-response curve in uterine arteries from non-pregnant and pregnant rats, respectively. In vessels from pregnant rats, PD 123319 also significantly increased the maximum response to angiotensin II.
5. CGP 42112 (1 μM) attenuated the response to angiotensin II of uterine arteries from non-pregnant rats. This was reflected by a 14 fold rightward shift of the angiotensin II concentration-response curve and a decrease in the maximum response. In uterine arteries from pregnant rats, CGP 42112 (1 μM) caused a 3 fold rightward shift of the angiotensin II concentration-response curve, but had no effect on the maximum response.
6. PD 123319 (1 μM) and CGP 42112 (1 μM) had no effect on the concentration-response curves to phenylephrine (PE) of uterine arteries from non-pregnant or pregnant rats. In addition, CGP 42112 (1 nM–1 mM) had no vasodilator effect on tissues precontracted with phenylephrine.
7. These results suggest that the contractile responses of the rat uterine artery are mediated by the AT₁ receptor. Furthermore, in this vascular preparation, the AT₂ receptor appears to inhibit the response mediated by the AT₁ receptor, although, this is not uniform between the non-pregnant and pregnant states.

     

Bolus Intravenous Injection of Phosphorothioate Oligonucleotides Causes Hypotension by Acting as α1-Adrenergic Receptor Antagonists

Bolus intravenous injections of phosphorothioate oligonucleotides (PS-ODN) into primates cause profound hypotension, which has been attributed to complement activation, the biochemical pathway leading to acute inflammatory response. Because the hypotension was not accompanied by peripheral or pulmonary edema and epinephrine was not effective, but administration of 200 ml Ringer's lactate was effective, we examined the possibility that the 15-base PS-ODN interferes with sympathetic tone. We administered doses ranging from 3.3 to 10 mg/kg of a 15-base PS-ODN as a 30–60 s iv bolus into the right atrium of conscious Macaca mulatta. Blood pressure fell to 27 mm Hg following a 5.0 mg/kg dose, but no hypotension was observed after a 3.3 mg/kg dose; 10 mg/kg was lethal. Adrenergic receptor binding was evaluated in radioligand binding assays using rat cerebral cortex membranes with radiolabeled prazosin. The 15-base PS-ODN competes with prazosin for the α₁-adrenergic receptor with an IC50 of 14 μM, which favors binding over serum albumin (K_d = 37 to 48 μM). Admixing serum albumin with 5.0 mg/kg 15-base PS-ODN prior to injection prevented hypotension, suggesting that unbound PS-ODN interferes with sympathetic tone before binding to plasma proteins. Interactions of the 15-base PS-ODN with the α₁-adrenergic receptor in vivo were confirmed by a decreased response to phenylephrine. Reducing the length from 15 to 9 or 5 bases abolished α₁-adrenergic receptor binding in vitro and bolus infusion of 5.0 mg/kg of 9-base PS-ODN no longer produced hypotension. In conclusion, the 15-base PS-ODN shows cooperative binding to the α₁-adrenergic receptor, which produces cardiovascular effects that are oligomer length, dose, and formulation dependent.     

Thromboxane A2-mediated Cl secretion induced by platelet-activating factor in isolated rat colon

Thromboxane A₂ is a novel endogenous secretagogue of Cl⁻ secretion in the distal colon. Here, we examined if the Cl⁻ secretion caused by platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is mediated by thromboxane A₂ production using isolated mucosae of the rat colon. Furosemide (100 μM) and 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB; 300 μM) completely inhibited PAF (10 μM)-induced increase in short-circuit current (Isc) across the mucosa, indicating that PAF caused a Cl⁻ secretion in the rat colon. A selective thromboxane A₂ receptor antagonist (sodium(E)-11-[2-(5,6-dimethyl-1-benzimidazolyl)-ethylidene]-6,11-dihydrobenz[b,e]oxepine-2-carboxylate monohydrate; KW-3635), and a selective thromboxane synthase inhibitor (sodium 4-[α-hydroxy-5-(1-imidazolyl)-2-methylbenzyl]-3,5-dimethylbenzoate dihydrate; Y-20811) inhibited the PAF-induced Cl⁻ current in a concentration-dependent manner. The IC₅₀ values of KW-3635 and Y-20811 were 2.1 and 0.5 μM, respectively. 30 μM KW-3635 and 1 μM Y-20811 inhibited the PAF response by 92% and 83%, respectively. These inhibitors did not affect the prostaglandin E₂-induced increase in Isc. A 5-lipoxygenase-activating protein inhibitor (3-[1-(p-chlorobenzyl)-5-(isopropyl)-3-t-butylthioindol-2-yl]-2,2-dimethyl-propanoic acid sodium; MK-886) (5 μM) did not affect the PAF-induced Cl⁻ current. The present study suggests that the PAF-induced Cl⁻ secretion in the rat colonic mucosa is mainly mediated by a release of thromboxane A₂.     

The activity of the neuronal and extraneuronal catecholamine-metabolizing enzymes of the perfused rat heart

Summary In a comparative study the neuronal and extraneuronal metabolism of several ³H-catecholamines (all of which were tritiated in the C-7 position of the side chain only) was determined in isolated rat hearts perfused at a concentration of the 3H-amines of 50 nmol/1. While the neuronal MAO activity was determined after inhibition of extraneuronal uptake (100 mol/1 OMI) and COMT (10 mol/1 U-0521), the extraneuronal MAO activity was estimated after inhibition of neuronal uptake (30 mol/1 cocaine) and COMT. The extraneuronal COMT activity was determined under conditions of inhibition of both neuronal uptake and MAO (pretreatment with pargyline). Hearts were perfused with the 3H-catecholamines until the rate of appearance of the various ³H-metabolites in the venous effluent has reached a steady state. From these rates (v _st-st) and the steady-state content of the unchanged ³H-catecholamines in the tissue (S _i), the rate constants (V _max/K _m) for the unsaturated intracellular enzymes COMT (_COMT) and MAO (_MAO) were calculated. The _COMTvalues for all four catecholamines, (–)-noradrenaline, dopamine, (–)-adrenaline and (±)-isoprenaline exhibit a range from 0.24 to 0.78 min^–1; the metabolism of the catecholamines by the COMT differs: (-)-noradrenaline = dopamine < (–)-adrenaline < (±)-isoprenaline. The extraneuronal MAO activity was low for all three catecholamines, (–)-adrenaline, (–)-noradrenaline and dopamine (range of _MAOfrom 0.05 to 0.28 min^–1) and declined in the order: (–)-adrenaline < (–)-noradrenaline < dopamine. The neuronal MAO activity for (–)-adrenaline, (–)-noradrenaline and dopamine was slightly higher than that in the extraneuronal cells (range of kMAO from 0.08 to 0.35 min^–1), but the ranking order showed the same pattern: (–)-adrenaline < (–)-noradrenaline = dopamine.Abbreviations MAO monoamine oxidase - COMT catechol-Omethyltransferase - NMN normetanephrine - MN metanephrine - MT 3-methoxytyramine - OMI 3-O-methyl-isoprenaline - DOPEG dihydroxyphenylglycol - DOPET dihydroxyphenylethanol - DOMA dihydroxymandelic acid - DOPAC dihydroxyphenylacetic acid - U-0521 3,4-dihydroxy-2-methyl propiophenone     

Human adenosine A1 receptor and P2Y2-purinoceptor-mediated activation of the mitogen-activated protein kinase cascade in transfected CHO cells

1. The mitogen-activated protein (MAP) kinase signalling pathway can be activated by a variety of heterotrimeric G_i/G_o protein-coupled and G_q/G₁₁ protein-coupled receptors. The aims of the current study were: (i) to investigate whether the G_i/G_o protein-coupled adenosine A₁ receptor activates the MAP kinase pathway in transfected Chinese hamster ovary cells (CHO-A1) and (ii) to determine whether adenosine A1 receptor activation would modulate the MAP kinase response elicited by the endogenous P2Y2 purinoceptor.
2. The selective adenosine A₁ receptor agonist N⁶-cyclopentyladenosine (CPA) stimulated time and concentration-dependent increases in MAP kinase activity in CHO-A1 cells (EC50 7.1±0.4 nM). CPA-mediated increases in MAP kinase activity were blocked by PD 98059 (50 μM; 89±4% inhibition), an inhibitor of MAP kinase kinase 1 (MEKI) activation, and by pre-treating cells with pertussis toxin (to block G_i/G_o-dependent pathways).
3. Adenosine A₁ receptor-mediated activation of MAP kinase was abolished by pre-treatment with the protein tyrosine inhibitor, genistein (100 μM; 6±10% of control). In contrast, daidzein (100 μM), the inactive analogue of genistein had no significant effect (96±12 of control). MAP kinase responses to CPA (1 μM) were also sensitive to the phosphatidylinositol 3-kinase inhibitors wortmannin (100 nM; 55±8% inhibition) and LY 294002 (30 μM; 40±5% inhibition) but not to the protein kinase C (PKC) inhibitor Ro 31-8220 (10 μM).
4. Activation of the endogenous P2Y₂ purinoceptor with UTP also stimulated time and concentration-dependent increases in MAP kinase activity in CHO-A1 cells (EC₅₀=1.6±0.3 μM). The MAP kinase response to UTP was partially blocked by pertussis toxin (67±3% inhibition) and by the PKC inhibitor Ro 31-8220 (10 μM; 45±5% inhibition), indicating the possible involvement of both G_i/G_o protein and G_q protein-dependent pathways in the overall response to UTP.
5. CPA and UTP stimulated concentration-dependent increases in the phosphorylation state of the 42 kDa and 44 kDa forms of MAP kinase as demonstrated by Western blotting.
6. Co-activation of CHO-A1 cells with CPA (10 nM) and UTP (1 μM) produced synergistic increases in MAP kinase activity which were not blocked by the PKC inhibitor Ro 31-8220 (10 μM).
7. Adenosine A1 and P2Y2 purinoceptor activation increased the expression of luciferase in CHO cells transfected with a luciferase reporter gene containing the c-fos promoter. However, co-activating these two receptors produced only additive increases in luciferase expression.
8. In conclusion, our studies have shown that the transfected adenosine A₁ receptor and the endogenous P2Y₂ purinoceptor couple to the MAP kinase signalling pathway in CHO-A1 cells. Furthermore, co-stimulation of the adenosine A1 receptor and the P2Y₂ purinoceptor produced synergistic increases in MAP kinase activity but not c-fos mediated luciferase expression.

     

Effects of immobilization stress on hippocampal monoamine release: Modification by mivazerol, a new α2-adrenoceptor agonist

Mivazerol is a new and selective α₂-adrenoceptor agonist which has demonstrated anti-ischemic effects, both in animals and in patients with myocardial ischemia. In the present study, mivazerol was evaluated for its ability to inhibit the release of catecholamines and serotonin (5-HT) in the hippocampus of freely moving rats, and also was compared to clonidine. In vivo microdialysis in combination with high-performance liquid chromatography (HPLC) was employed. Intravenous administration of mivazerol (8.0 μg/kg) had no effect on basal outflow of norepinephrine (NE), dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC). In contrast, clonidine (8.5 μg/kg, i.v.) attenuated the basal release of DOPAC, which has been proposed to reflect NE biosynthesis, suggesting that clonidine has an inhibitory effect on NE synthesis. In addition, both mivazerol and clonidine decreased the spontaneous release of 5-HT, which provided further evidence that α₂-adrenoceptors in the hippocampus modulate 5-HT. Sixty-min immobilization stress significantly increased the release of NE (177 ± 28%), DA (209 ± 46%) and DOPAC (337 ± 72%). Mivazerol (2.5, 8.0 and 25 μg/kg, i.v.) completely prevented the immobilization stress-induced enhancement of NE, DA and DOPAC, which was equi-effective to clonidine at a dose of 8.5 gmg/kg, i.v. These findings demonstrate that mivazerol has a profound modulatory effect on stress-induced neurotransmitter release in the hippocampus, at dose levels reported to protect against myocardial ischemia.     

Long-term increase of hippocampal excitability by histamine and cyclic AMP

The action of histamine (HA) on rat hippocampal CA1 pyramidal cells in vitro was investigated in slices perfused with solution containing 0.2 mM Ca²⁺/4.0 mM Mg²⁺. Extracellular recordings of the spontaneous discharges occurring under these conditions revealed that HA caused a long-lasting increase in cell firing. The HA-effects were dose-dependent, in that low concentrations of HA (0.1–0.5 μM) exhibited an initial transient depression of cell firing and practically no long-lasting action, whereas higher concentrations of HA (1–10 μM) exerted strong, non-declining increases. The H₁-receptor antagonist mepyramine (1 μM) blocked the initial depression of firing and attenuated the long-lasting HA-mediated excitation. Pure H₁-receptor activation, tested with the H₁-receptor agonist 2-(3-fluorphenyl)histamine (1–10 μM) depressed cell firing, similar to the low dose effects of HA. HA-induced excitations were prevented by the H₂-receptor antagonist cimetidine (10–50 μM), and mimicked by the very potent H₂-receptor agonist impromidine (1 or 3 μM) which was, however, less effective compared to equal concentrations of HA. H₃-receptor activation by R-α-methylhistamine had no significant effect on cell firing. Thus, histamine H₁ and H₂ receptors seem to cooperate in producing this long-lasting augmentation of excitability. 8-Bromo-cyclic AMP monophosphate (8-Br-cAMP, 50–100 μM) mimicked the long-term excitation, whereas the adenylyl-cyclase inhibitor 9-tetrahydro-2-furyladenine (THFA, 100–500 μM) or the PKA-inhibitor Rp-adenosine-3′5′-cyclic monophosphate (Rp-cAMPS, 10 μM) blocked it, indicating that the HA-mediated increase of excitability in the hippocampus is dependent on the adenylate cyclase/PKA-signal transduction cascade. -2-Amino-5-phosphonopentanoic acid (APV, 50 μM) significantly attenuated the magnitude of the HA-induced enhancement, indicating an NMDA receptor-dependent component. Other biogenic amines, acting through receptors positively coupled to adenylyl cyclase, elicited similar responses as HA, indicating common mechanisms by which these substances modulate excitability in CA1 pyramidal cells.     

The discriminative stimulus effects of N-methyl-d-aspartate antagonists in phencyclidine-trained rats

The discriminative stimulus effects of two competitive N-methyl-d-aspartate (NMDA) antagonists, 2-amino-7-phosphonoheptanoate (APH) and 3-[(±)-2-carboxypiperazin-4-yl]propyl-1-phosphonate (CPP), were assessed in rats trained to discriminate phencyclidine from saline. Systemically administered APH (10–60 mg/kg i.p.) failed to elicit phencyclidine-lever responding; however, partial generalization from phencyclidine occurred following intracerebraventricular (i.c.v.) administration of APH (1.5–30/ig). Systemic and central administration of CPP (3–30 mg/kg i.p.; 0.1–10 μg i.c.v.) also resulted in partial generalization from phencyclidine. Partial generalization was also obtained with methohexital (5–30 mg/kg i.p.). However, generalization to APH, CPP and methohexital was usually accompanied by decreased response rates, and response rate decreases frequently occurred without appreciable phencyclidine-lever selection, indicating that these drugs also had no phencyclidine-like behavioral effects. The drug di-ortho-tolyl guanidine (DTG) which binds with high-affinity to sigma receptors failed to elicit phencyclidine-lever responding, even at doses which reduced response rates. These findings suggest that although competitive NMDA antagonists share some discriminative stimulus properties with phencyclidine, there is not a complete overlap in the discriminative stimulus properties of competitive and non-competitive NMDA antagonists. Furthermore, the discriminative stimulus effects of APH and CPP were no more similar to phencyclidine than those of methohexital.     

Involvement of both GABAA and GABAB receptors in tonic inhibitory control of blood pressure at the rostral ventrolateral medulla of the rat

Summary The rostral ventrolateral medulla (RVLM) contains vasopressor neurons which increase vasomotor tone. Endogenous GABA is suggested to be involved in mediation of the tonic inhibition of vasopressor neurons in the RVLM. To obtain more precise information about GABAergic mechanisms in the RVLM, we microinjected GABA agonists and antagonists unilaterally into the RVLM and examined their effects on blood pressure and heart rate. In addition, involvement of the other inhibitory amino acids glycine, -alanine and taurine in blood pressure regulation in the rat RVLM was also investigated. Male Wistar rats were anesthetized with urethane, paralyzed and artificially ventilated. The GABA_A agonist muscimol (3–30 pmol) and the GABA_B agonist baclofen (10–100 pmol) microinjected into the RVLM produced a decrease in blood pressure. The GABA_A antagonist bicuculline (300 pmol) abolished the depressor response to muscimol (10 pmol) but not to baclofen (30 pmol) whereas the GABA_B antagonist 2-hydroxysaclofen (1 nmol) abolished the depressor response to baclofen (30 pmol) but not to muscimol (10 pmol). Either bicuculline or 2-hydroxysaclofen alone produced a pressor response. Both antagonists inhibited depressor response to nipecotic acid (7.7 nmol)and GABA (0.3 nmol). Glycine (0.13 – 4.0 nmol), -alanine (0.11 – 3.4 nmol) and taurine (0.08 – 2.4 nmol) microinjected into the RVLM also produced decreases in blood pressure. The glycine antagonist strychnine (0.58 nmol) abolished the depressor response to glycine, -alanine and taurine but not to GABA. The taurine antagonist (6-aminomethyl-3-methyl-4-H-1,2,4-benzothiadiazine-1,1-diox-ide) (1.3 nmol) inhibited the depressor response to -alanine and taurine but not to glycine and GABA. These results show that in the rat RVLM there exist GABA_A and GABA_B receptors, and that endogenous GABA tonically stimulates both GABA_A and GABA_B receptors to decrease arterial pressure. It seems unlikely that glycine and taurine are involved in mediation of the tonic inhibition of vasopressor neurons in the rat RVLM.Correpondence to T Kubo at the above address     

Role of α2-adrenoceptors in the regulation of intestinal water transport

1. The influence of the sympathetic nervous system on intestinal fluid transport by the jejunum and ileum of the anaesthetized rat was investigated under basal conditions and during active secretion induced by intra-arterial infusion of vasoactive intestinal peptide (VIP).
2. Intra-arterial infusion of noradrenaline (3, 10, 30 nmol min⁻¹, i.a.) and i.v. injection of the selective α₂-adrenoceptor agonist UK 14,304 (1 μmol kg⁻¹, i.v.) increased the rate of basal fluid absorption. The effect of UK 14,304 was blocked by yohimbine (10 μmol kg⁻¹, i.v). However, the selective α₁-adrenoceptor agonist phenylephrine (5 μmol kg⁻¹, i.v.) did not alter either the jejunal or ileal absorption rate.
3. The α₂-adrenoceptor antagonists yohimbine (0.3, 1.0, 3 and 10 μmol kg⁻¹, i.v.) and rauwolscine (10 μmol kg⁻¹, i.v.) decreased the basal absorption rate, while the α₁-adrenoceptor antagonist prazosin (3 μmol kg⁻¹, i.v.) was without effect. Intracerebroventricular injection of yohimbine (3 μmol kg⁻¹) caused a significant antiabsorptive effect in the jejunum but not ileum.
4. Peripheral chemical sympathectomy induced by pretreating animals with 6-hydroxydopamine (150 mg kg⁻¹, i.p., total dose) induced a trend towards impaired absorption in the jejunum and ileum.
5. The findings provide evidence that the sympathetic nervous system exerts tonic control on intestinal fluid transport and that the effect is mainly through peripheral α₂-adrenoceptors.
6. The subtype determination of α₂-adrenoceptors in modulating intestinal fluid transport was assessed by determining the effects of α₂-adrenoceptor agents on intestinal fluid secretion induced by i.a. infusion of VIP (0.8 μg min⁻¹).
7. Intravenous administration of UK 14,304 caused a dose-dependent reversal of the secretory phase of the VIP-induced response, but failed to restore fluid transport to the control level of net absorption. EC₅₀ values were 0.17 μmol kg⁻¹ in the jejunum and 0.22 μmol kg⁻¹ in the ileum.
8. The effect of UK 14,304 was blocked by the selective α_2A/D antagonist BRL 44408 and the non-selective α₂ antagonist yohimbine (each 10 μmol kg⁻¹). The selective α_2B/C antagonist ARC 239 (10 μmol kg⁻¹) did not affect the antisecretory action of UK 14,304. It is suggested that the α₂-adrenoceptors in the rat intestinal epithelium are the α_2D or α_2A-like subtype.

     

Leucocyte recruitment induced by type II phospholipases A2 into the rat pleural cavity

Bothropstoxin-I (BthTX-I) and bothropstoxin-II (BthTX-II) are Lys-49 and Asp-49 phospholipases A₂ (PLA₂s), respectively, isolated from Bothrops jararacussu venom. Piratoxin-I (PrTX-I) is a Lys-49 PLA₂ isolated from Bothrops pirajai venom. In this study, the ability of BthTX-I, BthTX-II and PrTX-I to recruit leucocytes into the rat pleural cavity and potential mechanisms underlying this effect were investigated. Intrapleural injection of either BthTX-I or PrTX-I (10–100 μg/cavity each) caused a significant leucocyte infiltration at 12 h after injection. The maximal cell migration was observed with the dose of 30 μg/cavity (14.9±15.5 and 17.6±1.6×10⁶ cells/cavity, respectively). Leucocyte counts consisted mainly of mononuclear cells, but significant amounts of neutrophils and eosinophils were also observed. Intrapleural injection of BthTX-II (10–100 μg/cavity) caused a marked leucocyte infiltration at 6 and 12 h after injection. The maximal response was observed with the dose of 100 μg/cavity (57.3±3.4×10⁶ cells/cavity, 6 h). The leucocyte counts were mainly composed of neutrophils and mononuclear cells. The treatment of either BthTX-I (30 μg/cavity, 12 h) or BthTX-II (30 μg/cavity, 6 h) with the PLA₂ inhibitor p-bromophenacyl bromide (p-BPB) had no effect on the total and differential leucocyte counts induced by these proteins. The same treatment partially reduced the PrTX-I-induced pleural leucocyte infiltration. In rats depleted of the histamine and 5-hydroxytryptamine (5-HT) stores by chronic treatment with compound 48/80, the total leucocyte counts in response to BthTX-I, BthTX-II and PrTX-I was not significantly affected compared to control animals. In addition, BthTX-I, BthTX-II and PrTX-I (100 μg/ml each) significantly degranulated pleural mast cells in vitro leading to the release of [¹⁴C]5-hydroxytryptamine ([¹⁴C]5-HT). p-BPB and heparin (50 IU/ml) significantly reduced the [¹⁴C]5-HT release induced by these PLA₂s. Our results demonstrate that BthTX-I, BthTX-II and PrTX-I recruit leucocyte into the pleural cavity of the rat by mechanisms unrelated to enzymatic activity and pleural mast cell degranulation.     

A1 and A2 adenosine receptor modulation of contractility in the cauda epididymis of the guinea-pig

1. The effects of adenosine receptor agonists upon phenylephrine-stimulated contractility and [³H]-cyclic adenosine monophosphate ([³H]-cyclic AMP) accumulation in the cauda epididymis of the guinea-pig were investigated. The α₁-adrenoceptor agonist, phenylephrine elicited concentration dependent contractile responses from preparations of epididymis. In the absence or presence of the L-type Ca²⁺ channel blocker, nifedipine (10 μM) the non-selective adenosine receptor agonist, 5′-N-ethylcarboxamido-adenosine (NECA, 1 μM) shifted phenylephrine concentration-response curves to the left (4 and 5 fold respectively). Following the incubation of preparations with pertussis toxin (200 ng ml⁻¹ 24 h) NECA shifted phenylephrine concentration-response curves to the right (5.7±0.9 fold).
2. In the presence of phenylephrine (1 μM), NECA and the A₁ adenosine receptor selective agonists, N⁶-cyclopentyladenosine (CPA) and (2S)-N⁶-[2-endo-norbornyl]adenosine ((S)-ENBA) elicited concentration-responses dependent contractions from preparations of epididymis (pEC₅₀ values 8.18±0.19, 7.79±0.29 and 8.15±0.43 respectively). The A₃ adenosine receptor agonists N⁶-iodobenzyl-5′-N-methyl-carboxamido adenosine (IBMECA) and N⁶-2-(4-aminophenyl) ethyladenosine (APNEA) mimicked this effect (but only at concentrations greater than 10 μM). In the presence of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 30 nM) CPA concentration-response curves were shifted, in parallel to the right (apparent pK_B 8.75±0.88) and the maximal response to NECA was reduced.
3. In the presence of DPCPX (100 nM) the adenosine agonist NECA and the A_2A adenosine receptor selective agonist, CGS 21680 (2-p-(2-carboxyethyl)-phenethylamino-N-ethylcarboxamido adenosine), but not CPA, inhibited phenylephrine (20 μM) stimulated contractions (pIC₅₀ 7.15±0.48). This effect of NECA was blocked by xanthine amine congener (XAC, 1 μM) and the A_2A adenosine receptor-selective antagonist 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385; 30 nM).
4. (S)-ENBA (in the absence and presence of ZM 241385, 100 nM), but not NECA or CPA inhibited the forskolin (30 μM)-stimulated accumulation of [³H]-cyclic AMP in preparations of the epididymis of the guinea-pig (by 17±6% of control). In the presence of DPCPX (100 nM) NECA and CGS 21680, but not (S)-ENBA, increased the accumulation of [³H]-cyclic AMP in preparations of epididymis (pEC₅₀ values 5.35±0.35 and 6.42±0.40 respectively), the NECA-induced elevation of [³H]-cyclic AMP was antagonised by XAC (apparent pK_B 6.88±0.88) and also by the A_2A adenosine receptor antagonist, ZM 241385 (apparent pK_B 8.60± 0.76).
5. These studies are consistent with the action of stable adenosine analogues at post-junctional A₁ and A₂ adenosine receptors in the epididymis of the guinea-pig. A₁ Adenosine receptors potentiate α₁-adrenoceptor contractility, an effect blocked by pertussis toxin, but which may not be dependent upon an inhibition of adenylyl cyclase. The epididymis of the guinea-pig also contains A₂ adenosine receptors, possibly of the A_2A subtype, which both inhibit contractility and also stimulate adenylyl cyclase.

     

Mechanism underlying histamine-induced intracellular Ca movement in PC3 human prostate cancer cells

The effect of histamine on intracellular free Ca ²⁺levels ([Ca ²⁺] _i) in PC3 human prostate cancer cells and the underlying mechanism were evaluated using fura-2 as a Ca ²⁺dye. Histamine at concentrations between 0.1 and 50 μM increased [Ca ²⁺] _iin a concentration-dependent manner with an EC ₅₀value of 1 μM. The [Ca ²⁺] _iresponse comprised an initial rise and a slow decay, which returned to baseline within 3 min. Extracellular Ca ²⁺removal inhibited 50% of the [Ca ²⁺] _isignal. In the absence of extracellular Ca ²⁺, after cells were treated with 1 μM thapsigargin (an endoplasmic reticulum Ca ²⁺pump inhibitor), 10 μM histamine did not increase [Ca ²⁺] _i. After pretreatment with 10 μM histamine in a Ca ²⁺-free medium for several minutes, addition of 3 mM Ca ²⁺induced [Ca ²⁺] _iincreases. Histamine (10 μM)-induced intracellular Ca ²⁺release was abolished by inhibiting phospholipase C with 2 μM 1-(6-((17 β-3- methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122), and by 10 μM pyrilamine but was not altered by 50 μM cimetidine. Collectively, the present study shows that histamine induced [Ca ²⁺] _itransients in PC3 human prostate cancer cells by stimulating H1 histamine receptors leading to Ca ²⁺release from the endoplasmic reticulum in an inositol 1,4,5-trisphosphate-dependent manner, and by inducing Ca ²⁺entry.     

Possible involvement of the locus coeruleus in inhibition by prostanoid EP3 receptor-selective agonists of morphine withdrawal syndrome in rats

We examined the mechanism of the inhibitory effect of prostanoid EP₃ receptor agonists on naloxone-precipitated withdrawal syndrome in morphine-dependent rats. Rats were rendered morphine dependent by subcutaneous (s.c.) implantation of two pellets containing 75 mg morphine for 5 days. Morphine withdrawal syndrome was precipitated by i.p. injection of naloxone (3 mg/kg). Intracerebroventricular (i.c.v.) administration of (±)-15α-hydroxy-9-oxo-16-phenoxy-17,18,19,20-tetranorprost-13-trans-enoic acid (M&B28,767: prostanoid EP₃ receptor agonist) or sulprostone (prostanoid EP₁/EP₃ receptor agonist) significantly suppressed many withdrawal signs. Northern blotting and in situ hybridization studies revealed that i.c.v. administration of M&B28,767 (1 pg/rat) attenuated the elevation of c-fos mRNA during naloxone-precipitated withdrawal in many brain regions, including the cerebral cortex, thalamus, hypothalamus and locus coeruleus. Double in situ hybridization analysis revealed that in the locus coeruleus most of the tyrosine hydroxylase mRNA-positive neurons expressed μ-opioid receptor mRNA and more than half of these neurons were positive for prostanoid EP₃ receptor mRNA. These results indicate that the suppression by prostanoid EP₃ receptor agonists of naloxone-precipitated morphine withdrawal syndrome can be attributed to the inhibition of neuronal activity in several brain regions, including the locus coeruleus, the largest source of central noradrenergic neurons.