Autonomic and Autacoid Pharmacology最新文献

1 Increased renal vascular resistance is one renal functional abnormality that contributes to hypertension, and α₁-adrenoceptors play a pivotal role in modulating this renal vascular resistance. This study investigates the functional contribution of α₁-adrenoceptor subtypes in the renal cortical vasculature of Wistar–Kyoto rats on a normal sodium diet (WKYNNa) compared with those given saline to drink for 6 weeks (WKYHNa).

2 The renal cortical vascular responses to the adrenergic agonists noradrenaline (NA), methoxamine (ME) and phenylephrine (PE) were measured in WKYHNa and WKYNNa rats either in the absence (the control phase) or presence of chloroethylclonidine (CEC), an α_1B-adrenoceptor antagonist, 5-methylurapidil (5-MeU), an α_1A antagonist, or BMY7378, an α_1D antagonist.

3 Results showed a greater renal cortical vascular sensitivity to NA, PE and ME in the WKYHNa compared with WKYNNa rats (P < 0.05). Moreover, 5-MeU and BMY7378 attenuated adrenergically induced renal cortical vasoconstriction in WKYHNa and WKYNNa rats; this response was largely blunted in CEC-treated WKYHNa rats (all P < 0.05) but not in CEC-treated WKYNNa rats.

4 The data suggest that irrespective of dietary sodium content, in Wistar–Kyoto rats α_1A- and α_1D-subtypes are the major α₁-adrenoceptors in renal cortical vasculature; however, there appears to be a functional involvement of α_1B-adrenoceptors in the WKYHNa rats.

1 This study examined the role of 20-hydroxyeicosatetraenoic (20-HETE) in altering vascular function in streptozotocin (STZ)-induced diabetic rats.

2 The expression of CYP4A protein and the formation of 20-HETE were elevated in the kidney, but not in the renal or mesenteric vasculature, of diabetic animals. The vasoconstrictor responses to norepinephrine (NE), endothelin-1 (ET-1), and angiotensin II (Ang II) were significantly enhanced in the isolated perfused mesenteric vascular bed and renal artery segments of diabetic rats. Chronic treatment of the diabetic rats with 1-aminobenzotriazole (ABT, 50 mg kg⁻¹ alt⁻¹ diem) or N-hydroxy-N’-(4-butyl-2-methylphenyl) formamidine (HET0016, 2.5 mg kg⁻¹ day⁻¹) attenuated the responses to these vasoconstrictors in both vascular beds.

3 The synthesis of 20-HETE in renal microsomes was reduced by >80% confirming that the doses of ABT and HET0016 were sufficient to achieve system blockade. Addition of HET0016 (1 μM) in vitro also normalized the enhanced vascular responsiveness of renal and mesenteric vessels obtained from diabetic animals to NE and inhibited the formation of 20-HETE by >90% while having no effect on the formation of epoxides. Vasodilator responses to carbachol and histamine were reduced in the mesenteric vasculature, but not in renal arteries, of diabetic rats. Treatment of the diabetic animals with HET0016 improved vasodilator responses in both vascular beds. Vascular sensitivity to exogenous 20-HETE was elevated in the mesenteric bed of diabetic animals compared to controls.

4 These results suggest that 20-HETE contributes to the elevation in vascular reactivity in diabetic animals. This effect is not due to increased vascular expression of CYP4A but may be related to either enhanced agonist-induced release of substrate (arachidonic acid) by the CaMKII/Ras-GTPase system and/or elevated vascular responsiveness to 20-HETE.

1 It has been hypothesized that craniovascular 5-HT receptors mediating dilatation of cranial vessels undergo sensitization on decreased serotonergic transmission in migraine. This study analysed the effect of chemical lesion of the 5-HT system in the brain with 5,7-dihydroxytryptamine (5,7-DHT) on 5-HT receptor-mediated dilator responses to 5-carboxamidotryptamine (5-CT) in the middle meningeal artery of anaesthetized rats. 5-CT has recently been shown to elicit dilator responses in this cranial vessel via 5-HT₇ receptors and, to a much lesser extent, 5-HT_1B/1D receptors.

2 Pretreatment with 5,7-DHT produced a drastic and selective decrease of 5-HT levels in the brain (78 ± 6% and 94 ± 2% in dorsal raphe and hypothalamic paraventricular nuclei, respectively) compared with controls (1% ascorbic acid).

3 Topical application of 5-CT (1–1000 μm) to exposed dura mater encephali produced concentration-dependent decreases in diastolic blood pressure and dilator responses in the middle meningeal artery that were similar in vehicle- and 5,7-DHT-pretreaed animals.

4 Hypotensive and meningeal dilator responses to 5-CT were unaltered by the 5-HT_1B/1D receptor antagonist, GR-127935 (1 mg kg⁻¹, i.v.), but were strongly inhibited by the 5-HT₇ receptor antagonist, SB-269970 (1 mg kg⁻¹, i.v.), with similar efficacy, in both groups of animals. Treatment with GR-127935 + SB-269970 (1 mg kg⁻¹, i.v. each), produced a stronger inhibitory effect than individual treatments on hypotensive but not on meningeal responses to 5-CT. Meningeal 5-HT₇ receptor-mediated responses (i.e. in GR-127935-pretreated animals) were unchanged by 5,7-DHT pretreatment.

5 Results suggest that the sensitivity of craniovascular 5-HT₇ receptors mediating dilatation is unaffected by a decrease of 5-HT levels in the brain. A neuronal involvement of 5-HT in migraine seems more likely, therefore.

1 Mitogen-activated protein kinases mediate hormone/neurotransmitter action on proliferation and differentiation and participate in receptor regulation. The effect of inhibitors of mitogen-activated kinase kinase (MEK) on α_1B-adrenoceptor phosphorylation state and function was studied using different cell lines. It was observed that at nanomolar concentrations the MEK inhibitors, PD98059 (2′-amino-3′-methoxyflavone) and UO126 [1,4-(diamino-2,3-dicyano/1,4-bis-(2-aminophenylthio)-butadiene], increased α_1B-adrenoceptor phosphorylation and diminished the functional response of this receptor to noradrenaline. These agents did not alter the action of lysophosphatidic acid.

2 Staurosporine (IC₅₀ ≈ 0.8 nm) (a general protein kinase inhibitor) and bis-indolyl-maleimide I (IC₅₀ ≈ 200 nm) (a selective protein kinase C inhibitor) inhibited PD98059-induced α_1B-adrenoceptor phosphorylation. In contrast, neither wortmannin (phosphoinositide 3-kinase inhibitor) nor genistein (protein tyrosine kinase inhibitor) had any effect. The data suggest the possibility that MEK might exert control on the activity of the enzymes that regulate receptor phosphorylation, such as G-protein-coupled receptor kinases, protein kinase C or serine/threonine protein phosphatases.

3 Coimmunoprecipitation studies showed a constant association of total extracellular signal-regulated kinase 2 (ERK2) with α_1B-adrenoceptors. Association of phospho-ERK 1/2 to α_1B-adrenoceptors increased not only in response to agonist but also in response to agents that increase α_1B-adrenoceptor and ERK1/2 phosphorylation [such as endothelin-1, phorbol 12-myristate-13-acetate (PMA) and epidermal growth factor (EGF)]; not surprisingly, PD98059 decreased this effect.

4 Our data show that blockade of MEK activity results in increased α_1B-adrenoceptor phosphorylation, diminished adrenoceptor function and perturbation of receptor–ERK1/2 interaction.

1 This study examined the contribution of cytochrome P450 metabolites of arachidonic acid in mediating ischaemia/reperfusion (I/R)-induced cardiac dysfunction in normal and diabetic rats.

2 We first compared the metabolism of arachidonic acid in microsomes prepared from the hearts of control rats and rats treated with streptozotocin (55 mg kg⁻¹) to induce diabetes. The production of dihydroxyeicosatrienoic acids and epoxyeicosatrienoic acids (EETs) were similar in microsomes prepared from the hearts of control and diabetic rats, but the production of 20-hydroxyeicosatetraenoic acid (20-HETE) was two-fold higher in diabetic hearts than in control animals.

3 We then compared the change in left ventricular pressure (P_max), left ventricular end-diastolic pressure, coronary flow and coronary vascular resistance in isolated perfused hearts obtained from control and diabetic animals after 40 min of global ischaemia (I) followed by 30 min of reperfusion (R). The decline in cardiac function was three- to five-fold greater in the hearts obtained from diabetic vs. control animals.

4 Pretreatment of the hearts with N-hydroxy-N′-(4-butyl-2-methyl-phenyl)-formamidine (HET0016, 1 μm), a selective inhibitor of the synthesis of 20-HETE, for 30 min before I/R resulted in significant improvement in the recovery of cardiac function in the hearts obtained from diabetic but not in control rats. Perfusion with an inhibitor of soluble epoxide hydrolase, 1-cyclohexyl-3-dodecyl urea (CDU), before I/R improved the recovery of cardiac function in hearts obtained from both control and diabetic animals. Perfusion with both HET0016 and CDU resulted in significantly better recovery of cardiac function of diabetic hearts following I/R than that seen using either drug alone. Pretreatment of the hearts with glibenclamide (1 μm), an inhibitor of ATP-sensitive potassium channels, attenuated the cardioprotective effects of both CDU and HET0016.

5 This is the first study to suggest that acute blockade of the formation of 20-HETE and/or reduced inactivation of EETs could be an important strategy to reduce cardiac dysfunction following I/R events in diabetes.

1 The effect of the flavonoids genistein (3–100 μM), kaempferol (3–60 μM) and quercetin (1–100 μM) on KCI (60 mM)-induced tonic contraction in rat smooth muscle was assayed. In the same way, the modification of these effects in the presence of an inhibitor of protein kinase (PKA) (Rp-cAMPS), an inhibitor of phosphodiesterase (papaverine) and β-adrenoreceptor blocking agents (propranolol and atenolol) was studied.

2 The flavonoids totally relaxed the KCI-induced tonic contraction (IC₅₀: genistein 20.2 ± 2.0 μM, n = 11; kaempferol 10.1 ± 1.6 μM, n = 8; quercetin 13.2 ± 1.2 μM, n = 8).

3 The incubation with Rp-cAMPS (10 and 100 μM) 30 min prior to KCI shifted the dose-response curve of the flavonoids to the right, increasing their IC₅₀ up to 27.8 ± 3.8 and 31.9 ± 7.3 μM, respectively, for genistein; 24.7 ± 0.2 and 19.6 ± 4.9 μM, respectively, for kaempferol; 18.8 ± 2.2 and 18.4 ± 1.5 μM, respectively, for quercetin.

4 Papaverine (3–100 μM) also relaxed the contraction induced by KCI and this effect was significantly displaced to the right with Rp-cAMPS (10 μM) (IC₅₀ 12.1 ± 2.2 vs. 16.5 ± 3.1 μM). Papaverine (3 μM) added to the organ bath 15 min before the contractile agent increased the relaxing effect of the flavonoids and significantly decreased their IC₅₀ (genistein 20.2 ± 2.0 vs. 9.8 ± 1.4 μM; kaempferol 10.1 ± 1.6 vs. 6.6 ± 0.7 μM; quercetin 13.2 ± 1.2 vs. 7.8 ± 1.4 μM).

5 The incubation with atenolol (10 μM) did not alter the relaxing effect of the flavonoids. In the same experimental conditions, propranolol (10 μM) did not modify the effect of genistein and kaempferol, but shifted the response curve of quercetin significantly to the right (13.2 ± 1.2 vs. 17.7 ± 3.4 μM).

6 The results suggest that genistein, kaempferol and quercetin produced the relaxation of uterine smooth muscle by increasing intracellular cAMP. β-Adrenoceptors could also be involved in the effect of quercetin.

1 Intrathecal (i.t.) injection of baclofen (30, 60 and 100 nmol), a GABA_B receptor agonist, produced a dose-dependent decrease in blood pressure (BP) and heart rate (HR).

2 Pretreatment with 5-aminovaleric acid (50 nmol), a GABA_B receptor antagonist, blocked the depressor and bradycardic effects of baclofen (100 nmol).

3 Pretreatment with 8-bromo-cAMP (10 nmol), a cAMP analogue, attenuated the depressor and bradycardic effects of baclofen (100 nmol), but not with 8-bromo-cGMP (10 nmol), a cGMP analogue.

4 In addition, pretreatment with glipizide (20 nmol), an ATP-sensitive K⁺ channel (K_ATP) blocker, attenuated the depressor and bradycardic effects of baclofen (100 nmol).

5 These results suggest that GABA_B receptors in the spinal cord have an inhibitory role in the central cardiovascular regulation and that these depressive and bradycardic actions are modified by cAMP and by K_ATP channel blockade.

1 There has been considerable debate whether responses mediated via β₁- and β₂-adrenoceptors (β₁ARs and β₂ARs) display the same degree of desensitization after prolonged or repeated exposure to agonists.

2 Examples are provided for selective desensitization of functional responses and loss of binding sites for β₁ARs. Equally, examples are given of selective desensitization and down-regulation involving β₂ARs.

3 This review examines whether receptor subtype-selective desensitization of βAR-mediated responses can occur and whether even within the same subtype, there may be tissue-selective desensitization. Possible reasons why apparent selectivity of desensitization of functional responses may occur are considered and are divided into methodological and non-methodological factors.

4 Methodological factors discussed are: the concentration of agonist used for inducing desensitization and the washout times before construction of the post-incubation concentration-response curve (CRC), the need for correction of CRCs from time-matched controls, and the methods adopted for plotting CRCs.

5 Four non-methodological factors are considered. Firstly, the roles of different receptor reserves for the responses of each tissue can have an important effect on whether desensitization is apparent; a large reserve will make desensitization less likely to be apparent. Secondly, there is more than one site at which desensitization occurs; receptors are uncoupled from adenylyl cyclase activation, there is an additional site at the level of stimulation of cyclic AMP-dependent protein kinase and βARs may ultimately be down-regulated. These processes may differ depending on the tissue and conditions and this may influence whether differential desensitization occurs between tissues. Thirdly, the apparent degree of desensitization after washout of an agonist can depend upon the rate of resensitization. Experiments to overcome this problem are described which demonstrate βAR desensitization in the continued presence of agonist. Finally, the role of up-regulation of PDE in desensitization is discussed.

1 This article gives a short overview of the physiology, pharmacology and the molecular biology of the human Na⁺/CI^- -dependent noradrenaline transporter (hNAT) and its gene.

2 Furthermore, naturally occurring variants of the hNAT are described and new results obtained through site-directed mutagenesis of the hNAT are presented, which increase our understanding about structural domains and amino acids critically involved in substrate, cosubstrate and inhibitor binding to the hNAT.