β-Adrenoceptor subtypes in sections of rat and guinea-pig kidney

Summary The present autoradiographical study examines the distribution of the two -adrenoceptor subtypes in sections of rat and guinea-pig kidney. The radioligand [¹²⁵Iodo]-(-)-cyanopindolol was used for the labelling of -adrenoceptors and the selective -adrenoceptor blocking agents ICI 89-406 (₁-antagonist) and ICI 118-551 (₂-antagonist) were utilized to differentiate both subclasses unequivocally. -Adrenoceptors in rat kidney were found to be almost exclusively ₁. They were located mainly on glomeruli and to a lesser extent on the straight part of the distal tubules and on the cortical portion of the collecting ducts. Some ₂-adrenoceptors were localized around the corticomedullary junction. Grain localization in the autoradiograms was absent in the inner medulla and papilla. Glomeruli and distal tubules of the guinea-pig kidney also possess only ₁-adrenoceptors, but, in contrast to the rat, extremely high concentrations of ₂-adrenoceptors were associated with the straight part of the proximal tubules in the cortex and possibly with the cortical portion of the collecting duct. Labelling was not detected on the proximal convoluted tubule in either species.     

β1-Adrenoceptor stimulation suppresses endothelial IKCa-channel hyperpolarization and associated dilatation in resistance arteries

Background and Purpose

In small arteries, small conductance Ca²⁺-activated K⁺ channels (SK_Ca) and intermediate conductance Ca²⁺-activated K⁺ channels (IK_Ca) restricted to the vascular endothelium generate hyperpolarization that underpins the NO- and PGI₂-independent, endothelium-derived hyperpolarizing factor response that is the predominate endothelial mechanism for vasodilatation. As neuronal IK_Ca channels can be negatively regulated by PKA, we investigated whether β-adrenoceptor stimulation, which signals through cAMP/PKA, might influence endothelial cell hyperpolarization and as a result modify the associated vasodilatation.

Experimental Approach

Rat isolated small mesenteric arteries were pressurized to measure vasodilatation and endothelial cell [Ca²⁺]_i, mounted in a wire myograph to measure smooth muscle membrane potential or dispersed into endothelial cell sheets for membrane potential recording.

Key Results

Intraluminal perfusion of β-adrenoceptor agonists inhibited endothelium-dependent dilatation to ACh (1 nM–10 μM) without modifying the associated changes in endothelial cell [Ca²⁺]_i. The inhibitory effect of β-adrenoceptor agonists was mimicked by direct activation of adenylyl cyclase with forskolin, blocked by the β-adrenoceptor antagonists propranolol (non-selective), atenolol (β₁) or the PKA inhibitor KT-5720, but remained unaffected by ICI 118 551 (β₂) or glibenclamide (ATP-sensitive K⁺ channels channel blocker). Endothelium-dependent hyperpolarization to ACh was also inhibited by β-adrenoceptor stimulation in both intact arteries and in endothelial cells sheets. Blocking IK_Ca {with 1 μM 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34)}, but not SK_Ca (50 nM apamin) channels prevented β-adrenoceptor agonists from suppressing either hyperpolarization or vasodilatation to ACh.

Conclusions and Implications

In resistance arteries, endothelial cell β₁-adrenoceptors link to inhibit endothelium-dependent hyperpolarization and the resulting vasodilatation to ACh. This effect appears to reflect inhibition of endothelial IK_Ca channels and may be one consequence of raised circulating catecholamines.     

β-Adrenoceptor activates endothelium-dependent release of nitric oxide in rat aorta

AIM:To examine the possible role of agents elevating cAMP to release NO from aortic en-dothelial cells. METHODS:NG-nitro-L-arg inine methylester (L-NAME) , an inhibitor of NO synthase, partially inhibited endothelium-dependent relaxation evoked in phenylephrine-precontracted rings by isoproterenol and abolished relaxation mediated by forskolin 0. 2 umol L-1.RESULTS: In rings without en-dothelium, isoproterenol and forskolin were less effective relaxants and L-NAME had no effect on the responses. In methylene blue-treated rings isoproterenol- and forskolin-induced relaxation were prevented in both en-dothelium-intact and -denuded rings, but the inhibitory effects of methylene blue were significantly more in rings with endothelium than in those without. On the other hand, relaxation induced by sodium nitroprusside was not inhibited by L-NAME, but was inhibited by methylene blue in both the endothelium-intact and -denuded rings. The concentration relaxation curves to sodium nitroprusside after methylene bl     

Regulation of gap-junction protein connexin 43 by β-adrenergic receptor stimulation in rat cardiomyocytes

Aim:

β-adrenergic receptor (β-AR) agonists are among the most potent factors regulating cardiac electrophysiological properties. Connexin 43 (Cx43), the predominant gap-junction protein in the heart, has an indispensable role in modulating cardiac electric activities by affecting gap-junction function. The present study investigates the effects of short-term stimulation of β-AR subtypes on Cx43 expression and gap junction intercellular communication (GJIC) function.

Methods:

The level of Cx43 expression in neonatal rat cardiomyocytes (NRCM) was detected by a Western blotting assay. The GJIC function was evaluated by scrape loading/dye transfer assay.

Results:

Stimulation of β-AR by the agonist isoproterenol for 5 min induces the up-regulation of nonphosphorylated Cx43 protein level, but not total Cx43. Selective β₂-AR inhibitor ICI 118551, but not β₁-AR inhibitor {"type":"entrez-protein","attrs":{"text":"CGP20712","term_id":"874704353","term_text":"CGP20712"}}CGP20712, could fully abolish the effect. Moreover, pretreatment with both protein kinase A inhibitor H89 and G_i protein inhibitor pertussis toxin also inhibited the isoproterenol-induced increase of nonphosphorylated Cx43 expression. Isoproterenol-induced up-regulation of nonphosphorylated Cx43 is accompanied with enhanced GJIC function.

Conclusion:

Taken together, β₂-AR stimulation increases the expression of nonphosphorylated Cx43, thereby enhancing the gating function of gap junctions in cardiac myocytes in both a protein kinase A- and G_i-dependent manner.     

PKCɛ mediates serine phosphorylation of connexin43 induced by lysophosphatidylcholine in neonatal rat cardiomyocytes

Lysophosphatidylcholine (LPC) is a potent pro-arrhythmic derivative of the membrane phosphotidylcholine, which is accumulated in heart tissues during cardiac ischemia. However, the cellular mechanism underlying LPC-induced cardiomyocyte damage remains to be elucidated. This study focuses on the effects of LPC on cardiomyocyte gap junction. At 30 μM, LPC decreased the spontaneous contraction rates of cardiomyocytes, and caused arrhythmic contraction without affecting cell viability. Connexin43 (Cx43) was seen as large plaques at cell junctions in control cells, whereas upon LPC treatment, the intensity of Cx43 staining was decreased in a concentration-sensitive manner and Cx43 staining appeared as tiny dots at cell junctions with a corresponding increase in cytoplasmic punctate staining. This distributional change of Cx43 was accompanied by an impairment of the gap junction intercellular communication (GJIC). Further, LPC treatment induced protein kinase C (PKC) activation, and PKC-dependent Cx43 phosphorylation at serine (Ser) 368. Pre-treatment with a specific PKC? inhibitor, eV1-2, prevented the LPC-induced Cx43 phosphorylation at Ser368 and the loss of Cx43 from gap junctions, both of which may disturb GJIC functions. Furthermore, siRNA knockdown of PKC? in H9c2 cells prevented LPC-induced serine phosphorylation of Cx43, confirming the role of PKC? in Cx43 serine phosphorylation. Double labeling immunofluorescence showed that LPC increased the colocalization of Cx43 with ubiquitin, and pretreatment with MG132 effectively prevented LPC-induced gap junction disassembly. LPC increased the ubiquitination of Cx43, which was blocked by eV1-2 pretreatment, suggesting that LPC accelerated the intracellular degradation of Cx43 via the ubiquitin-proteasomal pathway. It can be concluded that LPC destroyed the structure and function of gap junctions via PKC?-mediated serine phosphorylation of Cx43. PKC? inhibitors might therefore be effective in prevention of LPC-related diseases.     

β-Adrenoceptor blocking agents as partial agonists in isolated heart muscle: Dissociation of stimulation and blockade

Summary The cardiac stimulant actions of nine -adrenoceptor blocking agents were examined in kitten papillary muscles and in isolated atria of kittens and guinea pigs to determine to what extent these drugs behaved as classical partial agonists. In many ways the agents do appear to comprise a spectrum of partial agonists with widely differing efficacies. However, in one respect the actions of some of the -blockers did not fit into the classical mold. Several -blockers were found to exert stimulant effects only in concentrations appreciably higher than those required for substantial -adrenoceptor blockade. These observations suggest that more than one type of -adrenoceptor may be involved in the production of sympathomimetic effects on cardiac muscle.     

α1-Adrenoceptor subtypes mediating stimulation of Na+,K+-ATPase activity in rat renal proximal tubules

Although both α_1A- and α_1B-adrenoceptors are present in renal proximal tubules, the involvement of these receptor subtypes in the stimulation of Na⁺,K⁺-ATPase activity is not known. This study was undertaken to delineate the receptor subtype(s) involved in α₁-adrenoceptor-mediated increase in Na⁺,K⁺-ATPase activity and to identify the cellular signaling mechanisms such as stimulation of inositol triphosphate formation (IP₃) and protein kinase C activation in this phenomenon. It was found that norepinephrine-induced increase in Na⁺,K⁺-ATPase activity was attenuated by prazosin, but not by rauwolscine, indicating the involvement of α₁-adrenoceptors. Furthermore, this response was selectively inhibited by the α_1B-adrenoceptor inactivator, chloroethylclonidine (100 μM), but not by the α_1A-adrenoceptor antagonist, WB4101 (0.01 μM). We examined whether these effects on Na⁺,K⁺-ATPase activity are mediated via the activation of IP₃ and protein kinase C, Phenylephrine-induced increase in IP₃ levels was abolished by prazosin, and significantly inhibited by WB4101, but not by chloroethylclonidine. similarly, phenylephrine-induced activation of protein kinase C was sensitive to blockade by WB4101, but not by chloroethylclonidine. These results suggest that whereas both α_1A- and α_1B-adrenoceptors are present in proximal tubules, α_1B-adrenoceptors are involved in stimulating Na⁺,K⁺-ATPase activity and α_1A-adrenoceptors are predominantly linked to renal tubular IP₃ production and protein kinase C activation. Therefore, it appears that norepinephrine-induced stimulation of Na⁺,K⁺-ATPase activity does not involve phospholipase-C-coupled protein kinase C pathway.     

β-Adrenoceptor blocking effects and plasma levels of bornaprolol and propranolol in man

Summary The -adrenoceptor blocking effects and pharmacokinetics of bornaprolol (FM 24), a new -adrenoceptor blocking agent, have been compared with those of propranolol and a placebo in a double-blind trial in 6 healthy volunteers. Heart rate, systolic and diastolic blood pressures and peak expiratory flow rate were measured at rest and at the end of 3 min vigorous exercise on a bicycle ergometer, before and 2, 24 and 48 h after single oral doses of bornaprolol (120, 240 and 480 mg) and propranolol (40, 80 and 160 mg). Plasma renin activity at rest and the plasma concentrations of the two drugs were determined. Bornaprolol significantly reduced resting heart rate, dose-dependently lowered exercise-induced tachycardia and decreased peak expiratory flow rate and plasma renin activity. In addition, exercise-induced tachycardia was significantly reduced by bornaprolol up to 48 hours after drug intake (pharmacodynamic half-life approximately 63–86 h) and there was a correlation between this reduction and the log plasma bornaprolol concentration over the 48-h period. Thus, bornaprolol behaved in man as a non-cardioselective and long-lasting -adrenoceptor blocking drug, probably devoid of intrinsic sympathomimetic activity.     

Chronic muscarinic cholinoceptor stimulation increases adenylyl cyclase responsiveness in rat cardiomyocytes by a decrease in the level of inhibitory G-protein α-subunits

Exposure of neonatal rat cardiomyocytes for 3 days to the muscarinic cholinoceptor agonist carbachol led to a concentration-dependent increase in adenylyl cyclase stimulation by the \-adrenoceptor agonist isoproterenol by up to 115% (at 1 mmol/l carbachol). In addition, direct adenylyl cyclase stimulation by forskolin was increased in carbachol (1 mmol/l)-treated cells by 32010. Pretreatment of the rat cardiomyocytes with pertussis toxin, which enhances adenylyl cyclase activity by a functional inactivation of the inhibitory G-protein (G_i), was performed to investigate the possible role of G_i proteins in carbachol-induced sensitization of adenylyl cyclase stimulation. After pretreatment of the cells with pertussis toxin, the carbachol-mediated increase in forskolin-stimulated adenylyl cyclase activity was lost and the carbachol-mediated increase in \-adrenoceptor-stimulated adenylyl cyclase activity was attenuated. Labelling of the 40 kDa pertussis toxin substrates in cardiomyocyte membranes was decreased by carbachol in a concentration-dependent manner by up to 34010 (at 1 mmol/l carbachol). The number and affinity of \₁-adrenoceptors was unaltered following the chronic carbachol treatment.The specific protein synthesis inhibitor Pseudomonas exotoxin A was used to study whether the carbachol-induced decrease in the level of pertussis toxin-sensitive G-proteins and increase in adenylyl cyclase activity depend on de-novo protein synthesis. Pseudomonas exotoxin A inhibits peptide chain elongation by ADP-ribosylating elongation factor 2. Treatment of the cells with 1 ng/ml Pseudomonas exotoxin A for 3 days led to a reduction in the subsequent ADP-ribosylation of elongation factor 2 in the cytosol of the heart muscle cells by 57%. Exposure of the cells to 1 mmol/l carbachol for 3 days increased ADP-ribosylation of elongation factor 2 by 40% concomitant with a slight (about 20%) increase in the total protein content of the cardiomyocytes. The partial protein synthesis inhibition by Pseudomonas exotoxin A had no influence on the carbachol-induced decrease in the level of pertussis toxin-sensitive G-proteins. Similarly, the carbachol-induced increase in adenylyl cyclase responsiveness also remained unaltered by Pseudomonas exotoxin A.The data presented indicate that chronic muscarinic cholinoceptor agonist treatment decreases the level of -subunits of G_i- proteins. This decrease in G_i- subunits is apparently at least in part responsible for the observed increase in adenylyl cyclase responsiveness after chronic carbachol treatment.     

β-Adrenoceptor mediated responses in rat pulmonary artery: putative role of TASK-1 related K channels

The effect of isoprenaline on tone, cyclic adenosine 3′:5′ monophosphate (cAMP), and smooth muscle membrane potential (E _m ) were assessed in rat isolated pulmonary arteries. N^ω-nitro-L-arginine methyl ester (10.0 μM) or removal of endothelium partially inhibited relaxant responses to isoprenaline, but glibenclamide (10.0 μM) and indomethacin (10.0 μM) did not. While Rp-8-Br-cAMP (30.0 μM), tetraethylammonium (0.3 & 1.0 mM), 4-aminopyridine (100 μM), anandamide (10.0 μM), charybdotoxin (0.1 μM), ouabain (100 μM), and barium chloride (100 μM), incompletely blocked relaxation to isoprenaline, cyclopiazonic acid (1.0 μM), apamin (3.0 μM) and zinc acetate (300 μM) were without effect. Increasing extracellular K⁺ ([K⁺]_e) inhibited relaxant responses to isoprenaline, completely abolishing the response at 30 mM [K+]_e. Vasorelaxant effects of isoprenaline were significantly attenuated in buffer pH 6.4, and concomitant presence of Rp-8-Br-cAMP (30.0 μM) in pH 6.4 produced significant additive inhibition when compared to pH 6.4 without Rp-8-Br-cAMP. Isoprenaline increased cAMP turnover (1.55±0.24 fold; mean ± SEM), which was inhibited by propranolol (1.0 μM). Resting E _m of smooth muscle cells was -63.0±0.50 mV, and isoprenaline (1.0 μM) produced hyperpolarisation (−73.3±0.80 mV). While glibenclamide failed to affect isoprenaline-induced hyperpolarisation, ICI 118,551 (1.0 μM), anandamide or buffer pH 6.4 prevented it, and barium chloride and oubain combined caused partial inhibition. Isoprenaline-mediated relaxation seems to arise from several processes, including the generation of nitric oxide, the cAMP-cascade and, more importantly, a hyperpolarisation that is not due to activation of ATP-sensitive K channels but possibly of two-pore domain K channels of the TASK family.     

Tumor necrosis factor α up-regulates Giα and Gß proteins and adenylyl cyclase responsiveness in rat cardiomyocytes

Treatment of cultured rat cardiomyocytes in serum-free medium for 48 h with recombinant human tumor necrosis factor α (TNFα) led to a concentration-dependent increase in the level of membrane-inhibitory guanine nucleotide-binding protein (G₁) α-subunits and in pertussis toxin-catalyzed [³²P]ADP ribosylation of 40 kDa proteins. Both G_iα protein subtypes present in rat cardiac myocyte membranes, G_iα40 and G_iα41, were up-regulated by the cytokine, with the maximal increase occurring at 10 U/ml TNFα. In contrast to noradrenaline exposure, which causes a similar, but apparently exclusive, increase in α₁-subunits, treatment with TNFα in addition increased the level of membrane G protein ß₃₆-subunits. Furthermore, while noradrenaline exposure led to a decrease in receptor-dependent and -independent adenylyl cyclase activity, treatment of cardiomyocytes with TNFα caused a concentration-dependent increase in cyclase responsiveness to either forskolin, guanosine 5′-O-(3-thiotriphosphate) or isoproterenol, even though ß-adrenoceptor density was decreased by TNFα. The increase in adenylyl cyclase activity induced by TNFα was completely suppressed when the cells were cocultured with noradrenaline, a condition leading to an additive increase in G_iα level. The data indicate that the cytokine TNFα can potently modulate G protein-mediated signal transduction in rat cardiac myocytes. Although TNFα, like noradrenaline, exposure of the cells increased the level of membrane G_iα proteins, it did not decrease but rather caused an increase in adenylyl cyclase responsiveness. This hypertensitivity may be due to concomitant alterations of other components, e.g. G_ß, G_sα and/or the cyclase itself, of this multi-subunit signal transduction system following TNFα exposure.     

Changes of phosphorylation of cAMP response element binding protein in rat nucleus accumbens after chronic ethanol intake： naloxone reversal

目的:研究慢性酒精摄取和戒断后大鼠伏核内cAMP反应元件结合蛋白(CREB)表达和磷酸化的变化。方法:给大鼠饮用含低浓度乙醇的水溶液(6％,v/v)1个月,采用免疫组织化学的方法检测大鼠伏核内CREB和p-CREB蛋白表达。结果:给大鼠长期饮酒显著降低伏核组织内p-cREB蛋白含量(-75％),撤除酒精24h、72h后伏核内p-cREB蛋白含量仍较低,与对照组比分别降低35％和28％;戒断7d后恢复到正常水平,但慢性酒精摄取和戒断不改变伏核内CREB蛋白含量,单独应用纳洛酮对大鼠伏核组织内CREB和p-CREB含量亦无影响,然而,当纳洛酮与酒精同时应用时,能拮抗酒精摄取大鼠伏核内p-CREB含量的下降(142％)。结论:长期酒精摄取降低伏核组织内CREB磷酸化,此作用可被纳洛酮翻转,这可能是酒精依赖形成的分子机制之一。     

More PKA independent beta-adrenergic signalling via cAMP: is Rap1-mediated glucose uptake in vascular smooth cells physiologically important?

The proteome characterising a specific cell type makes up a unique intracellular signalling network and signalling has to be studied in a cell specific manner. Beta-adrenergic receptors are coupled to production of cAMP and PKA was initially believed to be the only protein activated by cAMP. However, cAMP-mediated signalling via Epac and Rap1 has emerged as an important contributor to cAMP signalling. In the current issue of the British Journal of Pharmacology, Kanda and Watanabe report that adrenaline stimulates glucose uptake in vascular smooth muscle cells. With pharmacological methods, supplemented with small interfering RNA against Rap1, the authors demonstrate that adrenaline increases glucose uptake via G(s), adenylate cyclase, cAMP and Rap1 activation. The authors could document neither PKA nor Epac as the receptor for cAMP mediating the effect. Although there is no doubt that Rap1 mediates adrenaline-stimulated glucose uptake in vascular smooth muscle cells, it may be too early to exclude PKA and Epac.     

Tumor necrosis factor α decreases inositol phosphate formation and phosphatidylinositol-bisphosphate (PIP2) synthesis in rat cardiomyocytes

Treatment of neonatal rat cardiomyocytes for 72 h in the presence of tumor necrosis factor (TNF) (10 U/ml) lead to a decrease in basal and ₁-adrenoceptor-induced formation of the calcium-mobilizing second messenger inositol trisphosphate (IP₃) and its metabolites, IP₂ and IP₁, by 35 and 26%, respectively. The synthesis of phosphatidylinositol bisphosphate (PIP₂), the substrate of PI-specific phospholipase C, was decreased by 45% following the TNF (10 U/ml) exposure. Time courses of TNF (10 U/ml)-induced alterations in rat cardiomyocytes showed a parallel decline of basal inositol phosphate formation and PIP₂ synthesis suggesting that the decrease in inositol phosphate formation was due to the reduction in PIP₂ synthesis. As the TNF-induced decrease of PIP₂ synthesis was associated with a decreased synthesis of the phospholipid phosphatidylinositol (PI), the precursor of PIP₂, by 33%, the decreased availability of PIP₂ is apparently, at least in part, the result of the decreased synthesis of PI. As an apparent functional consequence of the decrease in IP₃ formation following the TNF exposure, the ₁-adrenoceptor-mediated induction of arrhythmias by 100 mol/l noradrenaline + 10 mol/l timolol was abolished in TNF-pretreated rat cardiomyocytes.To investigate one of the possible mechanisms of the TNF-induced decrease of PIP₂ formation, the effect of TNF pretreatment on glycerol-3-phosphate dehydrogenase (GDH), a key enzyme of lipogenesis, was studied: Exposure of the rat cardiomyocytes for 72 h to TNF induced a concentration-dependent decrease in GDH activity by maximally 55%.The result presented are consistent with the hypothesis that the decreased basal and ₁-adrenoceptor-induced formation of the second messenger IP₃ observed in chronic endotoxinemia and sepsis may be mediated by a TNF-induced decrease in the synthesis of PIP₂, the substrate of PI-specific phospholipase C. This mechanism occurs following long-term exposure to low TNF/ha concentrations and is apparently distinct from the short-term cardiac effects induced by high concentrations of TNF. Correspondence to: C. Reithmann at the above address     

Metformin exaggerates phenylephrine-induced AMPK phosphorylation independent of CaMKKβ and attenuates contractile response in endothelium-denuded rat aorta

Metformin, a widely prescribed antidiabetic drug, has been shown to reduce the risk of cardiovascular disease, including hypertension. Its beneficial effect toward improved vasodilation results from its ability to activate AMPK and enhance nitric oxide formation in the endothelium. To date, metformin regulation of AMPK has not been fully studied in intact arterial smooth muscle, especially during contraction evoked by G protein-coupled receptor (GPCR) agonists. In the present study, ex vivo incubation of endothelium-denuded rat aortic rings with 3 mM metformin for 2 h resulted in significant accumulation of metformin (∼600 pmoles/mg tissue), as revealed by LC–MS/MS MRM analysis. However, metformin did not show significant increase in AMPK phosphorylation under these conditions. Exposure of aortic rings to a GPCR agonist (e.g., phenylephrine) resulted in enhanced AMPK phosphorylation by ∼2.5-fold. Importantly, in metformin-treated aortic rings, phenylephrine challenge showed an exaggerated increase in AMPK phosphorylation by ∼9.7-fold, which was associated with an increase in AMP/ATP ratio. Pretreatment with compound C (AMPK inhibitor) prevented AMPK phosphorylation induced by phenylephrine alone and also that induced by phenylephrine after metformin treatment. However, pretreatment with STO-609 (CaMKKβ inhibitor) diminished AMPK phosphorylation induced by phenylephrine alone but not that induced by phenylephrine after metformin treatment. Furthermore, attenuation of phenylephrine-induced contraction (observed after metformin treatment) was prevented by AMPK inhibition but not by CaMKKβ inhibition. Together, these findings suggest that, upon endothelial damage in the vessel wall, metformin uptake by the underlying vascular smooth muscle would accentuate AMPK phosphorylation by GPCR agonists independent of CaMKKβ to promote vasorelaxation.     

β-Adrenoceptor mediated facilitation of noradrenaline and adenosine 5′-triphosphate release from sympathetic nerves supplying the rat tail artery

1. The effects of prejunctional β-adrenoceptor activation on electrically evoked noradrenaline (NA) and adenosine 5′-triphosphate (ATP) were studied by use of continuous amperometry and conventional intracellular recording techniques. Excitatory junction potentials (e.j.ps) were used as a measure of ATP release, and NA-induced slow depolarizations and oxidation currents as measures of NA release, from postganglionic sympathetic nerves innervating the rat tail artery in vitro.
2. Isoprenaline (0.1 μM) increased the amplitude of e.j.ps, slow depolarizations and oxidation currents evoked by short trains of stimuli at 1 to 4 Hz. The facilitatory effect of isoprenaline on e.j.ps and oxidation currents was most pronounced on responses evoked by the first stimulus in a train.
3. Isoprenaline (0.1 μM) did not detectably alter the amplitude-frequency distribution of spontaneous e.j.ps.
4. The facilitatory effect of isoprenaline on e.j.ps, slow depolarizations and oxidation currents was abolished by the β-adrenoceptor antagonist, propranolol (0.1 μM). Propranolol alone had no effect on e.j.ps, slow depolarizations or oxidation currents.
5. Thus, activation of prejunctional β-adrenoceptors increases the release of both NA and ATP from postganglionic sympathetic nerves. The findings are consistent with the hypothesis that NA and ATP are released from the same population of nerve terminals and presumably from the same vesicles.

     

Methoxychlor and its metabolite HPTE inhibit cAMP production and expression of estrogen receptors α and β in the rat granulosa cell in vitro

The major metabolite of the estrogenic pesticide methoxychlor (MXC) HPTE is a stronger ESR1 agonist than MXC and acts also as an ESR2 antagonist. In granulosa cells (GCs), FSH stimulates estradiol via the second messenger cAMP. HPTE inhibits estradiol biosynthesis, and this effect is greater in FSH-treated GCs than in cAMP-treated GCs. Therefore; we examined the effect of MXC/HPTE on FSH-stimulated cAMP production in cultured GCs. To test involvement of ESR-signaling, we used the ESR1 and ESR2 antagonist ICI 182,780, ESR2 selective antagonist PHTPP, and ESR2 selective agonist DPN. ESR1 and ESR2 mRNA and protein levels were quantified. Both HPTE and MXC inhibited the FSH-induced cAMP production. ICI 182,780 and PHTPP mimicked the inhibitory action of HPTE. MXC/HPTE reduced FSH-stimulated Esr2 mRNA and protein to basal levels. MXC/HPTE also inhibited FSH-stimulated Esr1. The greater inhibition on FSH-stimulated GCs is likely due to reduced cAMP level that involves ESR-signaling, through ESR2.