The cardiac sympathetic co-transmitter galanin reduces acetylcholine release and vagal bradycardia: implications for neural control of cardiac excitability

The autonomic phenotype of congestive cardiac failure is characterised by high sympathetic drive and impaired vagal tone, which are independent predictors of mortality. We hypothesize that impaired bradycardia to peripheral vagal stimulation following high-level sympathetic drive is due to sympatho-vagal crosstalk by the adrenergic co-transmitters galanin and neuropeptide-Y (NPY). Moreover we hypothesize that galanin acts similarly to NPY by reducing vagal acetylcholine release via a receptor mediated, protein kinase-dependent pathway. Prolonged right stellate ganglion stimulation (10 Hz, 2 min, in the presence of 10 μM metoprolol) in an isolated guinea pig atrial preparation with dual autonomic innervation leads to a significant (p<0.05) reduction in the magnitude of vagal bradycardia (5 Hz) maintained over the subsequent 20 min (n=6). Immunohistochemistry demonstrated the presence of galanin in a small number of tyrosine hydroxylase positive neurons from freshly dissected stellate ganglion tissue sections. Following 3 days of tissue culture however, most stellate neurons expressed galanin. Stellate stimulation caused the release of low levels of galanin and significantly higher levels of NPY into the surrounding perfusate (n=6, using ELISA). The reduction in vagal bradycardia post sympathetic stimulation was partially reversed by the galanin receptor antagonist M40 after 10 min (1 μM, n=5), and completely reversed with the NPY Y(2) receptor antagonist BIIE 0246 at all time points (1 μM, n=6). Exogenous galanin (n=6, 50-500 nM) also reduced the heart rate response to vagal stimulation but had no effect on the response to carbamylcholine that produced similar degrees of bradycardia (n=6). Galanin (500 nM) also significantly attenuated the release of (3)H-acetylcholine from isolated atria during field stimulation (5 Hz, n=5). The effect of galanin on vagal bradycardia could be abolished by the galanin receptor antagonist M40 (n=5). Importantly the GalR(1) receptor was immunofluorescently co-localised with choline acetyl-transferase containing neurons at the sinoatrial node. The protein kinase C inhibitor calphostin (100 nM, n=6) abolished the effect of galanin on vagal bradycardia whilst the protein kinase A inhibitor H89 (500 nM, n=6) had no effect. These results demonstrate that prolonged sympathetic activation releases the slowly diffusing adrenergic co-transmitter galanin in addition to NPY, and that this contributes to the attenuation in vagal bradycardia via a reduction in acetylcholine release. This effect is mediated by GalR(1) receptors on vagal neurons coupled to protein kinase C dependent signalling pathways. The role of galanin may become more important following an acute injury response where galanin expression is increased.     

l-arginine supplementation reduces cardiac noradrenergic neurotransmission in spontaneously hypertensive rats

Spontaneously hypertensive rats (SHR) are known to have cardiac noradrenergic hyperactivity due to an impaired nitric oxide (NO)-cGMP pathway. We hypothesized that dietary l-arginine supplementation may correct this autonomic phenotype. Male SHR and Wistar Kyoto rats (WKY) aged 16-18 weeks were given l-arginine (10 g/L in drinking water) for 1 week. Separate control groups received no supplementation. The SHR control had a significantly lower plasma l-arginine than WKY control, but this was increased to a comparable level following l-arginine. Atrial cGMP was lower in the SHR control compared with the WKY control (2.4 ± 0.4 pmol/mg vs 3.9 ± 0.5 pmol/mg, p < 0.05), but increased to 4.1 ± 0.5 pmol/mg protein (n = 8, p < 0.05) with l-arginine. Evoked [³H]norepinephrine release in isolated spontaneously beating right atria from the SHR control (328 ± 19%, n = 19) was 28% higher than the WKY control (256 ± 20%, n = 14, p < 0.05), but was reduced to 258 ± 11% with l-arginine feeding (n = 24, p < 0.01). Soluble guanylyl cyclase (sGC) inhibition caused a greater increase of evoked norepinephrine release in the l-arginine fed SHR compared with the non-fed SHR. l-arginine feeding did not reduce evoked norepinephrine release in the WKY. In-vitro heart rate response to exogenous norepinephrine (0.1-5 μmol/L) was similar between l-arginine fed (n = 13) and non-fed SHR (n = 10), suggesting that l-arginine supplementation worked pre-synaptically. Myocardial tyrosine hydroxylase protein was decreased in SHR following l-arginine supplementation, providing a link to reduced synthesis of norepinephrine. In conclusion, l-arginine supplementation corrects local cardiac noradrenergic hyperactivity in the SHR, probably via increased pre-synaptic substrate availability of NOS-sGC-cGMP pathway and reduced tyrosine hydroxylase levels.     

Differential KATP channel pharmacology in intact mouse heart

Classically, cardiac sarcolemmal K_ATP channels have been thought to be composed of Kir6.2 (KCNJ11) and SUR2A (ABCC9) subunits. However, the evidence is strong that SUR1 (sulfonylurea receptor type 1, ABCC8) subunits are also expressed in the heart and that they play a significant functional role in the atria. To examine this further, we have assessed the effects of isotype-specific potassium channel-opening drugs, diazoxide (specific to SUR1 > SUR2A) and pinacidil (SUR2A > SUR1), in intact hearts from wild-type mice (WT, n =   6), SUR1^−/− (n = 6), and Kir6.2^−/− mice (n = 5). Action potential durations (APDs) in both atria and ventricles were estimated by optical mapping of the posterior surface of Langendorff-perfused hearts. To confirm the atrial effect of both openers, isolated atrial preparations were mapped in both WT (n = 4) and SUR1^−/− (n =   3) mice. The glass microelectrode technique was also used to validate optical action potentials. In WT hearts, diazoxide (300 μM) decreased APD in atria (from 33.8 ± 1.9 ms to 24.2 ± 1.1 ms, p < 0.001) but was without effect in ventricles (APD 60.0 ± 7.6 ms vs. 60.8 ± 7.5 ms, respectively, NS), consistent with an atrial-specific role for SUR1. The absence of SUR1 resulted in loss of efficacy of diazoxide in SUR1^−/− atria (APD 36.8 ± 1.9 ms vs. 36.8 ± 2.8 ms, respectively, NS). In contrast, pinacidil (300 μM) significantly decreased ventricular APD in both WT and SUR1^−/− hearts (from 60.0 ± 7.6 ms to 29.8 ± 3.5 ms in WT, p < 0.001, and from 63.5 ± 2.1 ms to 24.8 ± 3.8 ms in SUR1^−/−, p < 0.001), but did not decrease atrial APD in either WT or SUR1^−/− hearts. Glibenclamide (10 μM) reversed the effect of pinacidil in ventricles and restored APD to control values. The absence of Kir6.2 subunits in Kir6.2^−/− hearts resulted in loss of efficacy of both openers (APD 47.2 ± 2.2 ms vs. 47.6 ± 2.1 ms and 50.8 ± 2.4 ms, and 90.6 ± 5.7 ms vs. 93.2 ± 6.5 ms and 117.3 ± 6.4 ms, for atria and ventricle in control versus diazoxide and pinacidil, respectively). Collectively, these results indicate that in the same mouse heart, significant differential K_ATP pharmacology in atria and ventricles, resulting from SUR1 predominance in forming the atrial channel, leads to differential effects of potassium channel openers on APD in the two chambers.     

Stimulatory effects of neuropeptide Y on the growth of orange-spotted grouper (Epinephelus coioides)

Neuropeptide Y (NPY) is a member of the pancreatic polypeptide family which is a potent orexigenic peptide known to date in mammals and teleost. This study was carried out to investigate the effects of NPY on food intake and growth of orange-spotted grouper (Epinephelus coioides). Synthetic grouper NPY (gNPY) was given orally at the dose of 0.5, 1.0 and 2.0 μg/g feed for 50 days, results showed that NPY treatment (1.0 and 2.0 μg/g feed) significantly increased growth rate, weight gain, feed conversion efficiency (FCE) and pituitary growth hormone (GH) mRNA level than the control group (p < 0.05). Furthermore, high level secretion of gNPY was expressed and purified in the Pichia pastoris expression system. The bioactivity of recombinant gNPY was confirmed by its ability to up-regulate GH mRNA expression in vivo and in vitro and down-regulate preprosomatostatin I (PSSI) mRNA expression in vivo. These results demonstrate that NPY has stimulatory effects on food intake as well as growth of grouper as in other teleost fish, also indicate that recombinant gNPY from P. pastoris has the same bioactivity as synthetic gNPY and has the potential to be used as a feed additive for both research and aquatic application.     

Ghrelin-induced growth hormone release from goldfish pituitary cells involves voltage-sensitive calcium channels

Ghrelin (GRL) is a stimulator of growth hormone (GH) release in many organisms, including goldfish. As a first study to examine the signalling mechanisms mediating GRL action on GH release in goldfish, we tested the hypothesis that GLR induces GH release from goldfish pituitary cells by enhancing Ca²⁺ entry through L-type voltage-sensitive Ca²⁺ channels (LVSCCs) using perifusion GH release and fura-2/AM Ca²⁺-imaging experiments. Goldfish (g)GRL₁₉ at 1 nM elicited reversible and repeatable GH responses from dispersed goldfish mixed pituitary cultures. However, the lack of a dose-response relationship in sequential treatments with decreasing concentrations of gGRL₁₉ (ranging from 10 to 0.01 nM) implicated rapid desensitization of the GH response. Sequential applications of gGRL₁₉ (1 nM) and salmon GnRH (100 nM), a known Ca²⁺-dependent stimulator of GH release, increased intracellular free Ca²⁺ levels ([Ca²⁺]_i) from the same identified somatotropes, suggesting co-expression of GRL and GnRH receptors on single cells. In contrast, 1 nM gGRL₁₉ failed to elicit GH release and elevation in [Ca²⁺]_i when the cells are incubated with nominally Ca²⁺-free media. When GH release and [Ca²⁺]_i increases were already stimulated by the LVSCC agonist Bay K8644 (10 μM), addition of 1 nM gGRL₁₉ did not further elevate these responses. Finally, the LVSCC inhibitors nifedipine (1 μM) and verapamil (1 μM) abolished 1 nM gGRL₁₉-induced GH release responses while nifedipine eliminated gGRL₁₉-induced [Ca²⁺]_i increase. Taken together, the results of this study provide evidence that entry of extracellular Ca²⁺ through LVSCCs is a key component of the GRL signalling pathway leading to GH release in the goldfish pituitary.     

Effects of galantamine on β-amyloid release and beta-site cleaving enzyme 1 expression in differentiated human neuroblastoma SH-SY5Y cells

Galantamine (Gal) is an acetylcholinesterase inhibitor and used to treat the symptoms of Alzheimer's disease (AD). Recent studies show that Gal may affect amyloid precursor protein (APP) metabolism and increase release of secretory APPα (sAPPα). However the effect of Gal on amyloid-β peptide (Aβ) release and β-site cleaving enzyme 1 (BACE1) expression is still unknown. Consequently, we investigated the effect of Gal on the level of Aβ and BACE1. In a differentiated human neuroblastoma cell line (SH-SY5Y), Gal (0.3 μM) was found to significantly decrease Aβ release and BACE1 expression following treatment for 6, 12, and 24 h. Increasing Gal to 0.9 μM or 10 μM had no further effect. The effect of Gal (0.3 μM for 18 h) was maximal on BACE1 expression but not on Aβ secretion. At higher concentration (0.9 μM and 10 μM), Gal had no effect on the level of full-length APP but could still stimulate further decrease in Aβ secretion and release of sAPPα. These observations suggested that 0.3 μM Gal exerts its effect on Aβ production by inhibiting BACE1 expression, while 0.9 μM or 10 μM Gal mainly reduces Aβ production by stimulating the non-amyloidogenic pathway to decrease the amount of APP substrate available for β-secretase cleavage. In addition, α7 nicotinic acetylcholine receptor (α7nAChR) and multiple second messengers (including PKC, MEK, and p38MAPK) were found to be involved in the regulation of Gal-inhibited Aβ release and BACE1 expression.     

Up-regulation of A 2B adenosine receptor in A 2A adenosine receptor knockout mouse coronary artery

In this study, we looked into possible compensatory changes of other adenosine receptors (ARs) in A_2A genetic knockout mice (A2AKO) as well as the functional role of nitric oxide (NO) in A_2A AR-mediated vasodilation. Gene expression of ARs from coronary arteries of A_2A AR wild type mice (A2AWT) and A2AKO was studied using real-time PCR. Functional studies were carried out in isolated heart and isolated coronary artery preparations. A_2B AR was found to be 4.5 fold higher in A2AKO than in A2AWT, while A_2A AR expression was absent in A2AKO. There was no difference in A₁ and A₃ ARs between WT and KO animals. The concentration-relaxation curve for adenosine-5′-N-ethylcarboxamide (NECA, non-selective AR agonist) in isolated coronary arterial rings in A2AKO was shifted to the left when compared to A2AWT. The concentration-response curve for A_2B selective agonist (BAY 60-6583) was also shifted to the left in A2AKO hearts. L-NAME, a non-specific NO synthase inhibitor, did not affect baseline coronary flow (CF) until the concentration reached 10 µM in A2AWT (76.32 ± 11.35% from baseline, n = 5). In A2AKO, the CF decreased significantly by L-NAME only at a higher concentration (100 µM, 93.32 ± 5.8% from baseline, n = 5). L-NMA (1 µM, n = 4), another non-specific NO synthase inhibitor, also demonstrated similar results in decreasing CF (59.66 ± 3.23% from baseline in A2AWT, while 81.76 ± 8.91% in A2AKO). It was further demonstrated that the increase in CF by 100 µM NECA was significantly blunted with 10 µM L-NAME (377.08 ± 25.23% to 305.41 ± 30.73%, n = 9) in A2AWT but not in A2AKO (153.66 ± 22.7% to 143.88 ± 36.65%, n = 5). Similar results were also found using 50 nM of CGS-21680 instead of NECA in A2AWT (346 ± 22.85 to 277 ± 31.39, n = 6). No change in CF to CGS-21680 was noted in A_2AAKO. Our data demonstrate, for the first time, that coronary A_2B AR was up-regulated in mice deficient in A_2A AR. We also provide direct evidence supporting a role for NO in A_2A AR-mediated coronary vasodilation. The data further support the role for A_2A AR in the regulation of basal coronary tone through the release of NO.     

Qo site of mitochondrial complex III is the source of increased superoxide after transient exposure to hydrogen peroxide

Transient exposure of cardiac myocytes to hydrogen peroxide (H₂O₂) results in further production of superoxide by the mitochondria as a result of increased influx of calcium through the L-type Ca²⁺ channel and increased calcium uptake by the mitochondria. The response persists as a result of positive feedback on the channel and induces alterations in protein synthesis and cell size consistent with the development of myocyte hypertrophy. The aim of this study was to investigate the site of increased superoxide production within the mitochondria. Exposure of myocytes to 30 μM H₂O₂ (5 min) then 10 U/mL catalase (5 min) increased dihydroethidium (DHE) signal by 58.7 ± 12.0% (n = 4) compared to myocytes exposed to 0 μM H₂O₂ for 5 min followed by 10 U/mL catalase (n = 9). Complex I inhibitors DPI (n = 5) and rotenone (n = 7) attenuated the increase in DHE signal due to H₂O₂. Complex III inhibitors myxothiazol (n = 16) and stigmatellin (n = 5) also attenuated the increase in DHE signal due to H₂O₂. However, antimycin A (inhibitor of Q_i site of complex III) had no effect. We “isolated” complex III in the intact cell by applying succinate in the patch pipette and exposing the cell to rotenone and antimycin A. Myxothiazol and TCA cycle inhibitors α-keto-β-methyl-n-valeric acid (KMV) and 4-hydroxynonenal (4-HNE) completely attenuated the increase in DHE signal. Direct activation of the L-type Ca²⁺ channel by voltage-step mimicked the increase in DHE signal after transient exposure to H₂O₂ (47.6 ± 17.8%, n = 6) while intracellular application of catalase attenuated the increase in DHE signal due to H₂O₂ (n = 6). We propose that elevated superoxide production after transient exposure to H₂O₂ occurs at the Q_o superoxide generation site of complex III in cardiac myocytes and that an increase in TCA cycle activity plays a significant role in mediating the response.     

Effects of neuropeptide Y on collateral development in a swine model of chronic myocardial ischemia

We investigated the role of neuropeptide Y (NPY), abundant in the myocardial sympathetic nervous system and endothelial cells, in angiogenesis during chronic myocardial ischemia. Adult male Yorkshire swine underwent ameroid constrictor placement on the proximal left circumflex coronary artery. After 3 weeks, an osmotic pump was placed to deliver either placebo (control, n = 8) or NPY_3–36 (NPY, n = 8) to the collateral dependent region. Five weeks after pump placement, after cardiac catheterization and hemodynamic assessment, the heart was harvested for analysis. NPY treated animals demonstrated increased mean arterial pressures and improved left ventricular function (+ dP/dt). Cardiac catheterization demonstrated a significant increase in the blush score in the NPY group (p < 0.001). Blood flow to the ischemic myocardium was not different between groups at rest or during ventricular pacing. Immunohistochemical double staining for CD-31 and smooth muscle actin demonstrated an increase in capillary and arteriole formation in NPY treated animals (p = 0.02 and p < 0.001). Immunoblotting showed a significant upregulation of DPPIV (p = 0.009) and NPY receptors 1 (p = 0.008), 2 (p = 0.02) and 5 (p = 0.03) in the NPY treated group. Additionally, there was significant upregulation of VEGF (p = 0.04), eNOS (p = 0.014), phospho-eNOS (ser1177) (p = 0.02), and PDGF (p < 0.001) in NPY treated group. The anti-angiogenic factors endostatin and angiostatin were significantly decreased in NPY treated animals (endostatin, p = 0.03; angiostatin, p = 0.04). Exogenous NPY_3-36 resulted in improved myocardial function and increased angiogenesis and arteriogenesis by stimulating growth factor, pro-angiogenic receptor upregulation, and decreasing anti-angiogenic expression, but did not increase blood flow to the ischemic myocardium. NPY may act as a good adjunct to primary agents of therapeutic angiogenesis.     

Epoxyeicosatrienoic acid prevents postischemic electrocardiogram abnormalities in an isolated heart model

Cytochrome P450 epoxygenases metabolize arachidonic acid (AA) to epoxyeicosatrienoic acids (EETs) which are in turn converted to dihydroxyeicosatrienoic acids (DHETs) by soluble epoxide hydrolase (sEH). The main objective of this study was to investigate the protective effects of EETs following ischemic injury using an ex vivo electrocardiogram (EKG) model. Hearts from C57Bl/6, transgenic mice with cardiomyocyte-specific overexpression of CYP2J2 (Tr) and wildtype (WT) littermates were excised and perfused with constant pressure in a Langendorff apparatus. Electrodes were placed superficially at the right atrium and left ventricle to assess EKG waveforms. In ischemic reperfusion experiments hearts were subjected to 20 min of global no-flow ischemia followed by 20 min of reperfusion (R20). The EKG from C57Bl/6 hearts perfused with 1 μM 14,15-EET showed less QT prolongation (QTc) and ST elevation (STE) (QTc = 41 ± 3, STE = 2.3 ± 0.3; R20: QTc = 42 ± 2 ms, STE = 1.2 ± 0.2mv) than control hearts (QTc = 36 ± 2, STE = 2.3 ± 0.2; R20: QTc = 53 ± 3 ms; STE = 3.6 ± 0.4mv). Similar results of reduced QT prolongation and ST elevation were observed in EKG recording from CYP2J2 Tr mice (QTc = 35 ± 1, STE = 1.9 ± 0.1; R20: QTc = 38 ± 4 ms, STE = 1.3 ± 0.2mv) compared to WT hearts. The putative epoxygenase inhibitor MS-PPOH (50 μM) and EET antagonist 14,15-EEZE (10 μM) both abolished the cardioprotective response, implicating EETs in this process. In addition, separate exposure to the K_ATP channel blockers glibenclamide (1 μM) and HMR1098 (10 μM), or the PKA protein inhibitor H89 (50 nM) during reperfusion abolished the improved repolarization in both the models. Consistent with a role of PKA, CYP2J2 Tr mice had an enhanced activation of the PKAα regulatory II subunit in plasma membrane following IR injury. The present data demonstrate that EETs can enhance the recovery of ventricular repolarization following ischemia, potentially by facilitating activation of K⁺ channels and PKA-dependent signaling.     

Role of protein kinase C in facilitation of luteinizing hormone (LH)-releasing hormone-induced LH surges by neuropeptide Y.

In female rats, neuropeptide Y (NPY) facilitates LHRH-induced LH surges without affecting basal LH release. The signal transduction mechanisms mediating this facilitation are unknown. Here, the involvement of PKC in this process was investigated. Anterior pituitaries (APs) were removed from rats at 1400 h proestrus and perifused in vitro with M199 for 5 h. After an equilibration and baseline period, tissue received hourly 5-minute pulses of the PKC inhibitor GF109203X (GFX), 2.5 microM, followed 15 min later by a 5-minute pulse of LHRH (10(-8) M), NPY (10(-6) M), or phorbol 12-myristate 13-acetate (PMA, 50 nM), or some combination. This regimen was repeated hourly for 3 h. As shown previously, NPY had no effect on basal LH release but greatly facilitated LHRH-induced LH release. Treatment with PMA also facilitated LHRH-induced LH release, to approximately the same degree as NPY. Inhibition of PKC activity with GFX completely prevented NPY's and PMA's facilitation of LH release but did not inhibit LH release stimulated by LHRH alone. Because previous work suggested involvement of both NPY and PKC in alterations of LHRH receptor affinity or number, the in vivo effects of NPY on LHRH binding characteristics were also investigated. Although NPY treatment reliably enhanced LHRH-induced LH and FSH surges in proestrous rats, this action was not accompanied by any detectable change in the affinity or concentration of LHRH receptors in anterior pituitary cell membranes. In summary, we have found that NPY's actions are blocked by PKC inhibition, mimicked by PKC stimulation, and not associated with any overt alterations in LHRH receptor affinity or number. We conclude that PKC activation is required for NPY's facilitation of LHRH-induced LH surges, and that this mechanism likely involves PKC targets other than those which may alter LHRH receptor number or affinity.     

Influence of polymeric microspheres on the myocardial oxygen partial pressure in the beating heart of pigs

Injection of labeled microspheres is an established method in animal models to analyze the capillary organ blood flow at different time points. However, the microspheres can lead to stenoses of the capillary lumen, which might affect tissue oxygen supply.Our study aimed to investigate the influence of repeated injections of microspheres into the left coronary artery on the tissue oxygen partial pressure (pO₂) in the downstream supplied myocardium of Göttingen minipigs. Tests (n = 6 pigs each) were performed with two differently sized microspheres (ø = 10 ± 0.1 μm (M10) or ø = 15 ± 0.15 μm (M15)) from polystyrene. The pO₂ was measured in the midmyocardium of the left and right ventricle for 6 min continuously after each of five injections (1 × 10⁶ microspheres each). There was a time laps of 12 min between each injection. In addition, the influence of the carrier solution was analyzed solely in the identical time frame.pO₂ decreased significantly in the myocardial area supplied by the ramus interventricularis paraconalis after injection of M15 microspheres. In contrast, the application of the M10 microspheres did not change the myocardial pO₂. This finding suggests to use microspheres with diameters not exceeding 10 μm for the coronary blood flow assessment.     

Expression of functional Y1 receptors for neuropeptide Y in human Ewing's sarcoma cell lines

Summary In the human Ewing's sarcoma cell line WE-68, saturation analysis using³H-labelled neuropeptide Y ([³H]NPY) as the radioligand disclosed a homogeneous population of binding sites with a dissociation constant (K _d ) of 4.5 nM and maximal binding capacity (B _max) of 712 fmol/mg cell protein. Besides the WE-68 cell line, ten other human Ewing's sarcoma cell lines (FM-62, HS-80, HT-78, HT-M1-78, NT-68, RM-82, RS-63, VH-64, WE-M1-68, WE-M2-68) were also found to display NPY receptors withK _d varying from 3.5 nM to 10.7 nM andB _max=247–3744 fmol/mg cell protein. NPY, its natural analogues and the Y₁-receptor-specific peptide ligand [Leu³¹, Pro³⁴]NPY inhibited [³H]NPY binding in the potency order: [Leu³¹,Pro³⁴]NPYhuman NPYpeptide YY (PYY)>> salmon pancreatic polypeptide (PP) > human PP>porcine NPY_13–36NPY_22–36. In the Ewing's sarcoma cell lines NPY provoked inhibition of forskolin-stimulated cyclic AMP formation by up to 98%. Pertussis toxin alleviated the cyclic-AMP-inhibitory response to NPY. In isolated Ewing's sarcoma plasma membranes pertussis toxin [³²P]ADP-ribosylated a 41-kDa protein. The ability of NPY and analogues to inhibit cyclic AMP accumulation paralleled their potencies in displacing radioligand binding. By contrast, a cell line derived from an atypical form of Ewing's sarcoma did not express specific and functional NPY receptors. These results demonstrate that conventional Ewing's sarcoma cells possess G_i-potein-coupled NPY receptors of the Y₁ type, which upon interaction with NPY, PYY, and PP mediate inhibition of cyclic AMP generation.Abbreviations NPY neuropeptide Y - PP pancreatic polypeptide - PYY peptide YY - VIP vasoactive intestinal peptide     

Distinct contractile and molecular differences between two goat models of atrial dysfunction: AV block-induced atrial dilatation and atrial fibrillation

Atrial dilatation is an independent risk factor for thromboembolism in patients with and without atrial fibrillation (AF). In many patients, atrial dilatation goes along with depressed contractile function of the dilated atria. While some mechanisms causing atrial contractile dysfunction in fibrillating atria have been addressed previously, the cellular and molecular mechanisms of atrial contractile remodeling in dilated atria are unknown. This study characterized in vivo atrial contractile function in a goat model of atrial dilatation and compared it to a goat model of AF. Differences in the underlying mechanisms were elucidated by studying contractile function, electrophysiology and sarcoplasmic reticulum (SR) Ca²⁺ load in atrial muscle bundles and by analyzing expression and phosphorylation levels of key Ca²⁺-handling proteins, myofilaments and the expression and activity of their upstream regulators. In 7 chronically instrumented, awake goats atrial contractile dysfunction was monitored during 3 weeks of progressive atrial dilatation after AV-node ablation (AV block goats (AVB)). In open chest experiments atrial work index (AWI) and refractoriness were measured (10 goats with AVB, 5 goats with ten days of AF induced by repetitive atrial burst pacing (AF), 10 controls). Isometric force of contraction (FC), transmembrane action potentials (APs) and rapid cooling contractures (RCC, a measure of SR Ca²⁺ load) were studied in right atrial muscle bundles. Total and phosphorylated Ca²⁺-handling and myofilament protein levels were quantified by Western blot. In AVB goats, atrial size increased by 18% (from 26.6 ± 4.4 to 31.6 ± 5.5 mm, n = 7 p < 0.01) while atrial fractional shortening (AFS) decreased (from 18.4 ± 1.7 to 12.8 ± 4.0% at 400 ms, n = 7, p < 0.01). In open chest experiments, AWI was reduced in AVB and in AF goats compared to controls (at 400 ms: 8.4 ±0.9, n = 7, and 3.2 ± 1.8, n = 5, vs 18.9 ± 5.3 mm×mmHg, n = 7, respectively, p < 0.05 vs control). FC of isolated right atrial muscle bundles was reduced in AVB (n = 8) and in AF (n = 5) goats compared to controls (n = 9) (at 2 Hz: 2.3 ± 0.5 and 0.7 ± 0.2 vs 5.5 ± 1.0 mN/mm², respectively, p < 0.05). APs were shorter in AF, but unchanged in AVB goats. RCCs were reduced in AVB and AF versus control (AVB, 3.4 ± 0.5 and AF, 4.1 ± 1.4 vs 12.2 ± 3.2 mN/mm², p < 0.05). Protein levels of protein kinase A (PKA) phosphorylated phospholamban (PLB) were reduced in AVB (n = 8) and AF (n = 8) vs control (n = 7) by 37.9 ± 12.4% and 29.7 ± 10.1%, respectively (p < 0.01), whereas calmodulin-dependent protein kinase II (CaMKII) phosphorylated ryanodine channels (RyR2) were increased by 166 ± 55% in AVB (n = 8) and by 146 ± 56% in AF (n = 8) goats (p < 0.01). PKA-phosphorylated myosin-binding protein-C and troponin-I were reduced exclusively in AVB goat atria (by 75 ± 10% and 55 ± 15%, respectively, n = 8, p < 0.05). Atrial dilatation developing during slow ventricular rhythm after complete AV block as well as AF-induced remodeling are associated with atrial contractile dysfunction. Both AVB and AF goat atria show decreased SR Ca²⁺ load, likely caused by PLB dephosphorylation and RYR2 hyperphosphorylation. While shorter APs further compromise contractility in AF goat atria, reduced myofilament phosphorylation may impair contractility in AVB goat atria. Thus, atrial hypocontractility appears to have distinct molecular contributors in different types of atrial remodeling.     

Inhibition of cardiac vagal effects by neurally released and exogenous neuropeptide Y

Neuropeptide Y (NPY) attenuates vagal effects on cardiac cycle length, presumably by inhibiting the release of acetylcholine from vagal nerve endings. We sought to determine if NPY inhibited the vagal effects on atrioventricular (AV) interval and atrial contraction in a manner similar to its inhibition of the vagal effects on cycle length. In 19 anesthetized dogs we measured the vagal effects on cycle length, AV interval, and atrial contraction before and after 3-minute trains of sympathetic stimulation or before and after exogenous NPY (20 micrograms/kg i.v.). Three minutes after 10-Hz sympathetic stimulation, the vagal effects on cycle length and AV interval were attenuated by 52 +/- 9% and 63 +/- 8%, respectively. Phentolamine significantly augmented this attenuation, but propranolol had no appreciable effect. In the control group of animals or in the group that received phentolamine, the vagal effects on atrial contraction measured before and after sympathetic stimulation were not significantly different. In these two groups, however, the basal atrial contraction was reduced substantially after the cessation of sympathetic stimulation. Propranolol prevented this reduction in atrial contraction. After propranolol, the vagal effects on atrial contraction 3 minutes after sympathetic stimulation were attenuated by 31 +/- 6%. Exogenous NPY had no direct effect on cycle length, AV interval, or atrial contraction, but exogenous NPY did persistently inhibit the vagal effects on each of these cardiac processes. Three minutes after NPY was given, the vagal effects on cycle length, AV interval, and atrial contraction were inhibited by 62 +/- 7%, 69 +/- 5%, and 68 +/- 5%, respectively. We conclude that NPY attenuates the vagal effects on the atrial myocardium and on the sinus and AV nodes. In the absence of beta-blockade, the inhibitory effect of neurally released NPY on the vagally induced decreases in atrial contraction may be masked by the reduction in the atrial contraction that occurs after sympathetic stimulation.     

Mesure des mycotoxines aéroportées

The aim of this study was to apply a new methodological approach to estimate the exposure of individuals to airborne moulds. We have developed a method that allows us to measure three mycotoxins (sterigmatocystin, deoxynivalenol and ochratoxin A) present in the indoor environment. The method we developed allows us to identify and to quantify these toxins at concentrations of 30 μg/L (N = 30, σ/m = 3,9%), 20 μg/L (N = 10, σ/m = 5,5%) et 20 μg/L (N = 10, σ/m = 7,6%, respectively. Using this analytical method and a rotating foam air sampler, previously validated, it was possible to measure the concentrations of the three airborne mycotoxins with a detection limit of 60 pg/m³. The results of this study of the transfer of mycotoxins from their substrate into the air showed that the quantity that was aerosolized was not proportional to that present on contaminated materials. In addition, measurement of airborne mycotoxins appears be a more relevant means to evaluate the level of exposure in populations.     

Protein kinase C mediated inhibition of endothelial l-arginine transport is mediated by MARCKS protein

The endothelium plays a vital role in the maintenance of vascular tone and structural vascular integrity, principally mediated via the actions of nitric oxide (NO). l-arginine is the immediate substrate for NO synthesis, and the availability of extracellular l-arginine is critical for the production of NO. Activation of protein kinase C (PKC) dependent signalling pathways are a feature of a number of cardiovascular disease states, and in this study we aimed to systematically evaluate the mechanism by which PKC regulates l-arginine transport in endothelial cells. In response to PKC activation (PMA 100 nM, 30 min), [³H]l-arginine uptake by bovine aortic endothelial cells (BAEC) was reduced to 45 + 4% of control (p < 0.05). This resulted from a 53% reduction in the Vmax (p < 0.05), with no change in the K_m for l-arginine. Western blot analysis and confocal microscopy revealed no change in the expression or membrane distribution of CAT-1, the principal BAEC l-arginine transporter. Moreover in ³²P-labeling studies, PMA exposure did not result in CAT-1 phosphorylation. We therefore explored the possibility that PKC altered and interaction with MARCKS protein, a candidate membrane associated protein. By co-immunoprecipitation we show that CAT-1 interacts with, a membrane associated protein, that was significantly inhibited by PKC activation (p < 0.05). Moreover antisense inhibition of MARCKS abolished the PMA effect on l-arginine transport. PKC dependent mechanisms regulate the transport of l-arginine, mediated via process involving MARCKS.     

Distinctive sodium and calcium regulation associated with sex differences in atrial electrophysiology of rabbits

Background

Sex and sodium/calcium regulation play critical roles in cardiac electrophysiology and atrial arrhythmogenesis. We investigated whether sodium and calcium contributed to sex differences in atrial electrophysiology.

Methods

Whole-cell patch clamp techniques and the indo-1 fluorometric ratio technique were used to investigate the ionic current and intracellular calcium in single isolated male and female rabbit myocytes from the left atrium posterior wall (LAPW) and right atrium (RA).

Results

Female LAPW (n = 95) and RA (n = 49) myocytes had larger cell widths (15.1 ± 0.4 vs. 13.8 ± 0.4 μm, p < 0.05; 14.9 ± 0.6 vs. 13.5 ± 0.4 μm, p < 0.05) than male LAPW (n = 142) and RA (n = 57) myocytes. Male LAPW myocytes (n = 26) had a higher incidence (57 vs. 16%, p < 0.05) of delayed afterdepolarizations (DADs) than female LAPW myocytes (n = 24) but there were similar incidences (20 vs. 20%, p > 0.05) of DADs in male and female RA myocytes. The late sodium current, calcium transients, and sarcoplasmic reticulum calcium contents were larger in male than female LAPW myocytes but were similar in male and female RA myocytes. However, the I_Ca-L and nickel-sensitive sodium/calcium exchanger currents were similar between two groups. Different from those in female myocytes, ouabain (10 μM) only induced repeated atrial beats (0 to 45%, p < 0.05) in male myocytes (n = 11). Moreover, ranolazine (3 μM) perfusion (4.5 ± 0.6 vs. 1 min, p < 0.05) was required to decrease the amplitude of DADs in male but not female LAPW myocytes.

Conclusions

Increased late sodium currents and calcium contents may contribute to higher arrhythmogenesis in male LAPW myocytes.     

Cardiac arrhythmias induced by glutathione oxidation can be inhibited by preventing mitochondrial depolarization

We have previously proposed that the heterogeneous collapse of mitochondrial inner membrane potential (ΔΨ_m) during ischemia and reperfusion contributes to arrhythmogenesis through the formation of metabolic sinks in the myocardium, wherein clusters of myocytes with uncoupled mitochondria and high K_ATP current levels alter electrical propagation to promote reentry. Single myocyte studies have also shown that cell-wide ΔΨ_m depolarization, through a reactive oxygen species (ROS)-induced ROS release mechanism, can be triggered by global depletion of the antioxidant pool with diamide, a glutathione oxidant. Here we examine whether diamide causes mitochondrial depolarization and promotes arrhythmias in normoxic isolated perfused guinea pig hearts. We also investigate whether stabilization of ΔΨ_m with a ligand of the mitochondrial benzodiazepine receptor (4′-chlorodiazepam; 4-ClDzp) prevents the formation of metabolic sinks and, consequently, precludes arrhythmias. Oxidation of the GSH pool was initiated by treatment with 200 μM diamide for 35 min, followed by washout. This treatment increased GSSG and decreased both total GSH and the GSH/GSSG ratio. All hearts receiving diamide transitioned from sinus rhythm into ventricular tachycardia and/or ventricular fibrillation during the diamide exposure: arrhythmia scores were 5.5 ± 0.5; n = 6 hearts. These arrhythmias and impaired LV function were significantly inhibited by co-administration of 4-ClDzp (64 μM): arrhythmia scores with diamide + 4-ClDzp were 0.4 ± 0.2 (n = 5; P < 0.05 vs. diamide alone). Imaging ΔΨ_m in intact hearts revealed the heterogeneous collapse of ΔΨ_m beginning 20 min into diamide, paralleling the timeframe for the onset of arrhythmias. Loss of ΔΨ_m was prevented by 4-ClDzp treatment, as was the increase in myocardial GSSG. These findings show that oxidative stress induced by oxidation of GSH with diamide can cause electromechanical dysfunction under normoxic conditions. Analogous to ischemia-reperfusion injury, the dysfunction depends on the mitochondrial energy state. Targeting the mitochondrial benzodiazepine receptor can prevent electrical and mechanical dysfunction in both models of oxidative stress.     

Effets cardiovasculaires des auto-anticorps anti-récepteurs béta1 et béta3-adrénergiques chez le rat Lewis

Purpose

To analyze vascular reactivity changes in response to immunization protocols with antigens corresponding to the second extracellular loop of –β₃ and –β_1 and 3 adrenergic receptors (AR).

Methods

Lewis rats were immunized for 3 months with peptidic sequences corresponding to the second extracellular loop of β₃–AR or β_1 and 3–AR. Specific β₃–AR antibodies were characterized by Elisa and purified using “Proteus Protein G” kit. Their functionality were tested in rabbit isolated ventricular cardiomyocytes. Aortic and mesenteric artery rings isolated from control or immunized rats were mounted in organ baths and precontracted with phenylephrine. Then, relaxant curves were established.

Results

SR58611A (10 nM), a preferential β₃–AR agonist and purified β₃–AR antibodies (25 μg/mL) induced a decrease of cell shortening (−39.56 ± 4.4% [n = 11] and −18.45 ± 3.9% [n = 10] respectively) in isolated cardiomyocytes. This decrease was significantly inhibited when the cardiomyocytes were pre-incubated with the L–748337 (1 μM), a selective β₃–AR antagonist (P < 0.05). In contrast with what was observed in rats immunized against the β₁–AR, vasorelaxations induced by acetylcholine and SR58611A in both aorta and mesenteric arteries were unaltered in rats immunized against the β₃–AR and β_1 and 3–AR.

Conclusion

These results show, for the first time, that β₃–AR antibodies induced a β₃–AR agonist-like activity. They would not have a vascular pathogenic action but would offset the endothelial dysfunction caused by β₁–AR antibodies.