Rita Jabr

Prominent role of intracellular Ca2+ release in hypoxic vasoconstriction of canine pulmonary artery

1 The possible role of sarcoplasmic reticulum (SR) Ca2+ stores in hypoxic pulmonary vasoconstriction (HPV) is not well understood. In order to assess the possible role of intracellular Ca2+ release from SR Ca2+ stores in HPV, we examined the effects of: (1) ryanodine (10 μM) depletion of intracellular Ca2+ stores, and (2) thapsigargin (THAPS, 2 μM) or cyclopiazonic acid (CPA, 10 μM) depletion of intracellular Ca2+ stores on HPV in canine pulmonary artery. 2 Isometric tension was measured from arterial ring suspended in Krebs‐Henseliet solution (K‐H) bubbled with 95%O2/5%CO2. Hypoxia was induced by bubbling phenylephrine (PE, 1 μM) precontracted rings with 95%N2/5%CO2. HPV was observed in both intact and endothelial‐denuded arteries and expressed as % of maximal KCl contraction (%Tkmax)=21.3±3.2%; n=13 and 21.7±4%; n=4, respectively. 3 When SR caffeine sensitive Ca2+ stores were depleted by pretreatment with ryanodine and brief caffeine (15 mM) exposure, the hypoxic response was signifcantly reduced to 19.1±9.2% of the control hypoxic contraction (n=7; P<0.001) with little or no effect on PE or KCl contractions. On the other hand, in normoxic rings pretreated with THAPS or CPA, the PE responses were significantly reduced (%Tkmax=18.2±3.1% compared to 39.0±3.9% in control; n=16; P<0.001; %Tkmax=3.4±1.6% compared to 49.9±7.9% in control; n=6; P<0.001; respectively) with no significant effect on caffeine‐induced contractions, suggesting that both THAPS and CPA preferentially deplete InsP3‐sensitive Ca2+ stores, without affecting the caffeine‐sensitive Ca2+ store; consistent with the existence of separate and independent InsP3 and caffeine‐sensitive Ca2+ stores in this preparation. 4 When hypoxia was induced in the presence of THAPS or CPA, developed tension was significantly larger than control (%Tkmax=64.5±6.0%; n=16; P<0.05%; %Tkmax=78.2±15%; n=6; P<0.05; respectively), was partially blocked by nisoldipine (10 μM) and ryanodine (%Tkmax=20.3±3.7%; n=6), and nearly completely blocked by SK&F 96365 (50 μM). However, the actions of SK&F 96365 appeared to be nonselective since this compound also significantly reduced contractions elicited by KCl, PE and caffeine. 5 Finally, evidence was obtained suggesting: (a) that at least some of the Ca2+ released from the caffeine‐ and ryanodine‐sensitive Ca2+ stores by hypoxia may be taken up and buffered by the InsP3‐sensitive Ca2+ stores, and (b) the apparent dependence of HPV on extracellular Ca2+ entry pathways may be partially due to the dependence of the Ca2+ content of intracellular SR Ca2+ stores on sarcolemmal Ca2+ entry pathways. 6 These data suggest that caffeine‐ and ryanodine‐sensitive SR Ca2+ stores contribute significantly to HPV under normal conditions and, in the presence of THAPS or CPA, an additional nisoldipine‐ and ryanodine‐insensitive Ca2+ entry pathway is evoked by hypoxia.

Calcium modulation of vascular smooth muscle ATP-sensitive K+ channels - Role of protein phosphatase-2B

ATP-sensitive K+ (KATP) channels are broadly distributed in the vasculature and regulate arterial tone. These channels are inhibited by intracellular ATP ([ATP]i) and vasoconstrictor agents and can be activated by vasodilators. It is widely assumed that KATP channels are insensitive to Ca2+, although regulation has not been examined in the intact cell where cytosolic regulatory processes may be important. Thus we investigated the effects of Ca2+ on whole-cell KATP current in rat aortic smooth muscle cells recorded in a physiological [ATP]i and K+ gradient. Under control recording conditions, cells had a resting potential of ≈−40 mV when bathed in 1.8 mmol/L Ca2+. The KATP channel inhibitor glibenclamide caused membrane depolarization (9 mV) and inhibited a small, time-independent background current. Reducing [ATP]i from 3 to 0.1 mmol/L hyperpolarized cells to ≈−60 mV and increased glibenclamide-sensitive current by 2- to 4-fold. Similar effects were observed when Ca2+ levels were decreased either externally or internally by increasing EGTA from 1 to 10 mmol/L. Dialysis with solutions containing different free [Ca2+]i showed that KATP current was maximally activated at 10 nmol/L [Ca2+]i and almost totally inhibited at 300 nmol/L. Moreover, under control conditions, when rat aortic smooth muscle cells were dialyzed with either cyclosporin A, FK-506, or calcineurin autoinhibitory peptide (structurally unrelated inhibitors of Ca2+-dependent protein phosphatase, type 2B), glibenclamide-sensitive currents were large and the resting potential was hyperpolarized by ≈20 to 25 mV. We report for the first time that KATP channels can be modulated by Ca2+ at physiological [ATP]i and conclude that modulation occurs via protein phosphatase type 2B.

p42/p44-MAPK and PI3K are sufficient for IL-6 family cytokines/gp130 to signal to hypertrophy and survival in cardiomyocytes in the absence of JAK/STAT activation.

The effect of differential signalling by IL-6 and leukaemia inhibitory factor (LIF) which signal by gp130 homodimerisation or LIFRβ/gp130 heterodimerisation on survival and hypertrophy was studied in neonatal rat cardiomyocytes. Both LIF and IL-6 [in the absence of soluble IL-6 receptor (sIL-6Rα)] activated Erk1/2, JNK1/2, p38-MAPK and PI3K signalling peaking at 20 min and induced cytoprotection against simulated ischemia-reperfusion injury which was blocked by the MEK1/2 inhibitor PD98059 but not the p38-MAPK inhibitor SB203580. In the absence of sIL-6R, IL-6 did not induce STAT1/3 phosphorylation, whereas IL-6/sIL-6R and LIF induced STAT1 and STAT3 phosphorylation. Furthermore, IL-6/sIL-6R induced phosphorylation of STAT1 Tyr701 and STAT3 Tyr705 were enhanced by SB203580. IL-6 and pheneylephrine (PE), but not LIF, induced cardiomyocyte iNOS expression and nitric oxide (NO) production. IL-6, LIF and PE induced cardiomyocyte hypertrophy, but with phenotypic differences in ANF and SERCA2 expression and myofilament organisation with IL-6 more resembling PE than LIF. Transfection of cardiomyocytes with full length or truncated chimaeric gp130 cytoplasmic domain/Erythropoietin receptor (EpoR) extracellular domain fusion constructs showed that the membrane proximal Box 1 and Box 2 containing region of gp130 was necessary and sufficient for MAPK and PI3K activation; hypertrophy; SERCA2 expression and iNOS/NO induction in the absence of JAK/STAT activation. In conclusion, IL-6 can signal in cardiomyocytes independent of sIL-6R and STAT1/3 and furthermore, that Erk1/2 and PI3K activation by IL-6 are both necessary and sufficient for induced cardioprotection. In addition, p38-MAPK may act as a negative feedback regulator of JAK/STAT activation in cardiomyocytes.

Paradoxical resistance to myocardial ischemia and age-related cardiomyopathy in NHE1 transgenic mice: A role for ER stress?

Sarcolemmal Na(+)/H(+) exchanger (NHE) activity, which is provided by the NHE isoform 1 (NHE1), has been implicated in ischemia/reperfusion-induced myocardial injury in animal models and humans, on the basis of studies with pharmacological NHE1 inhibitors. We generated a transgenic (TG) mouse model with cardiac-specific over-expression of NHE1 to determine whether this would be sufficient to increase myocardial susceptibility to ischemia/reperfusion-induced injury. TG mouse hearts exhibited increased sarcolemmal NHE activity and normal morphology and function. Surprisingly, they also showed reduced susceptibility to ischemia/reperfusion-induced injury, as reflected by improved functional recovery and smaller infarcts. Such protection was sustained in the presence of NHE1 inhibition with zoniporide, indicating a mechanism that is independent of sarcolemmal NHE activity. Immunoblot analysis revealed accumulation of immature NHE1 protein as well as marked upregulation of both cytoprotective (78/94 kDa glucose-regulated proteins, calreticulin, protein disulfide isomerase) and pro-apoptotic (C/EBP homologous protein) components of the endoplasmic reticulum (ER) stress response in TG myocardium. With increasing age, NHE1 TG mice exhibited increased myocyte apoptosis, developed left ventricular contractile dysfunction, underwent cardiac remodelling and died prematurely. Our findings indicate that: (1) Cardiac-specific NHE1 over-expression induces the ER stress response in mouse myocardium, which may afford protection against ischemia/reperfusion-induced injury despite increased NHE activity; (2) Ageing NHE1 TG mice exhibit myocyte apoptosis, cardiac remodelling and failure, likely as a result of sustained ER stress; (3) The pluripotent effects of the ER stress response may confound studies that are based on the chronic over-expression of complex proteins in myocardium.

Relationship Between Gap-Junctional Conductance and Conduction Velocity in Mammalian Myocardium

Background—Gap junction resistivity, Rj, has been proposed as a key determinant of conduction velocity (CV). However, studies in connexin-gene knockout mice demonstrated significant CV slowing only with near-complete connexin deletion, and these findings led to the concept of a significant redundancy of myocardial gap junctions. We challenged this prevailing concept and addressed the hypothesis that there is a continuous relationship between Rj and CV, each independently measured in human and guinea-pig myocardium. Methods and Results—Rj and CV were directly measured by oil-gap impedance and microelectrode techniques in human left ventricular myocardium from patients with hypertrophic cardiomyopathy and in guinea-pig atrial and ventricular myocardium before and during pharmacological uncoupling with 20-µmol/L carbenoxolone. There was a continuous relationship between Rj and CV in human and guinea-pig myocardium, pre- and post-carbenoxolone (r2=0.946; P<0.01). In guinea-pig left ventricle, left atrium, and right atrium, carbenoxolone increased Rj by 28±9%, 26±16%, and 25±14% and slowed CV by 17±3%, 23±8%, and 11±4% respectively (all P<0.05 versus control). As a clinically accessible measure of local microscopic myocardial conduction slowing in vivo in the intact human heart, carbenoxolone prolonged electrogram duration in the right atrium (39.7±4.2 to 42.3±4.3 ms; P=0.01) and right ventricle (48.1±2.5 to 53.3±5.3 ms; P<0.01). Conclusions—There is a continuous relationship between Rj and CV that is consistent between cardiac chambers and across species, indicating that naturally occurring variations in cellular coupling can account for variations in CV, and that the concept that there is massive redundancy of coupling is not tenable.

Modulation of spontaneous activity in the overactive bladder: the role of P2Y agonists

Spinal cord transection (SCT) leads to an increase in spontaneous contractile activity in the isolated bladder that is reminiscent of an overactive bladder syndrome in patients with similar damage to the central nervous system. An increase in interstitial cell number in the suburothelial space between the urothelium and detrusor smooth muscle layer occurs in SCT bladders, and these cells elicit excitatory responses to purines and pyrimidines such as ATP, ADP, and UTP. We have investigated the hypothesis that these agents underlie the increase in spontaneous activity. Rats underwent lower thoracic spinal cord transection, and their bladder sheets or strips, with intact mucosa except where specified, were used for experiments. Isometric tension was recorded and propagating Ca(2+) and membrane potential (E(m)) waves were recorded by fluorescence imaging using photodiode arrays. SCT bladders were associated with regular spontaneous contractions (2.9 ± 0.4/min); ADP, UTP, and UDP augmented the amplitude but not their frequency. With strips from such bladders, a P2Y(6)-selective agonist (PSB0474) exerted similar effects. Fluorescence imaging of bladder sheets showed that ADP or UTP increased the conduction velocity of Ca(2+)/E(m) waves that were confined to regions of the bladder wall with an intact mucosa. When transverse bladder sections were used, Ca(2+)/E(m) waves originated in the suburothelial space and propagated to the detrusor and urothelium. Analysis of wave propagation showed that the suburothelial space exhibited properties of an electrical syncitium. These experiments are consistent with the hypothesis that P2Y-receptor agonists increase spontaneous contractile activity by augmenting functional activity of the cellular syncitium in the suburothelial space.

Bladder Compliance What Does it Represent: Can We Measure it, and is it Clinically Relevant?

To report the conclusion of the Think Thank 8 on Compliance Discussions during the second ICI‐RS meeting in 2010.

Protein Kinase Cε-Calcineurin Cosignaling Downstream of Toll-Like Receptor 4 Downregulates Fibrosis and Induces Wound Healing Gene Expression in Cardiac Myofibroblasts

ABSTRACT The pathways which regulate resolution of inflammation and contribute to positive remodeling of the myocardium following injury are poorly understood. Here we show that protein kinase C epsilon (PKCε) cooperates with the phosphatase calcineurin (CN) to potentiate induction of cardioprotective gene expression while suppressing expression of fibrosis markers. This was achieved by detailed analysis of the regulation of cyclooxygenase 2 (COX-2) expression as a marker gene and by using gene expression profiling to identify genes regulated by coexpression of CN-Aα/PKCε in adult rat cardiac myofibroblasts (ARVFs) on a larger scale. GeneChip analysis of CN-Aα/PKCε-coexpressing ARVFs showed that COX-2 provides a signature for wound healing and is associated with downregulation of fibrosis markers, including connective tissue growth factor (CTGF), fibronectin, and collagens Col1a1, Col3a1, Col6a3, Col11a1, Col12a1, and Col14a1, with concomitant upregulation of cardioprotection markers, including COX-2 itself, lipocalin 2 (LCN2), tissue inhibitor of metalloproteinase 1 (TIMP-1), interleukin-6 (IL-6), and inducible nitric oxide synthase (iNOS). In primary rat cardiomyocyte cultures Toll-like receptor 4 (TLR4) agonist- or PKCε/CN-dependent COX-2 induction occurred in coresident fibroblasts and was blocked by selective inhibition of CN or PKC α/ε or elimination of fibroblasts. Furthermore, ectopic expression of PKCε and CN in ARVFs showed that the effects on COX-2 expression are mediated by specific NFAT sites within the COX-2 promoter as confirmed by site-directed mutagenesis and chromatin immunoprecipitation (ChIP). Therefore, PKCε may negatively regulate adverse myocardial remodeling by cooperating with CN to downregulate fibrosis and induce transcription of cardioprotective wound healing genes, including COX-2.

Accurate quantification of apoptosis progression and toxicity using a dielectrophoretic approach

A loss of ability of cells to undergo apoptosis (programmed cell death, whereby the cell ceases to function and destroys itself) is commonly associated with cancer, and many anti-cancer interventions aim to restart the process. Consequently, the accurate quantification of apoptosis is essential in understanding the function and performance of new anti-cancer drugs. Dielectrophoresis has previously been demonstrated to detect apoptosis more rapidly than other methods, and is low-cost, label-free and rapid, but has previously been unable to accurately quantify cells through the apoptotic process because cells in late apoptosis disintegrate, making cell tracking impossible. In this paper we use a novel method based on light absorbance and multi-population tracking to quantify the progress of apoptosis, benchmarking against conventional assays including MTT, trypan blue and Annexin-V. Analyses are performed on suspension and adherent cells, and using two apoptosis-inducing agents. IC50 measurements compared favourably to MTT and were superior to trypan blue, whilst also detecting apoptotic progression faster than Annexin-V.

Cytoplasm Resistivity of Mammalian Atrial Myocardium Determined by Dielectrophoresis and Impedance Methods

Many cardiac arrhythmias are caused by slowed conduction of action potentials, which in turn can be due to an abnormal increase of intracellular myocardial resistance. Intracellular resistivity is a linear sum of that offered by gap junctions between contiguous cells and the cytoplasm of the myocytes themselves. However, the relative contribution of the two components is unclear, especially in atrial myocardium, as there are no precise measurements of cytoplasmic resistivity, R(c). In this study, R(c) was measured in atrial tissue using several methods: a dielectrophoresis technique with isolated cells and impedance measurements with both isolated cells and multicellular preparations. All methods yielded similar values for R(c), with a mean of 138 ± 5 Ω·cm at 23°C, and a Q(10) value of 1.20. This value is about half that of total intracellular resistivity and thus will be a significant determinant of the actual value of action potential conduction velocity. The dielectrophoresis experiments demonstrated the importance of including divalent cations (Ca(2+) and Mg(2+)) in the suspension medium, as their omission reduced cell integrity by lowering membrane resistivity and increasing cytoplasm resistivity. Accurate measurement of R(c) is essential to develop quantitative computational models that determine the key factors contributing to the development of cardiac arrhythmias.