Calcineurin-dependent regulation of gap junction conductance and connexin phosphorylation in guinea pig left atrium.

Atrial fibrillation (AF) occurs from disordered atrial action potential conduction and is associated with reduced gap junction electrical conductance (Gj). The Ca2+ and calmodulin-dependent phosphatase, calcineurin, reduces Gj in ventricular myocardium via a protein phosphatase-1 (PP1)-dependent pathway culminating in phosphorylation of serine368 on connexin43 (pSer368-Cx43). However, characterisation of corresponding pathways in left atrial myocardium, which have a more complex connexin subtype profile, is undefined and was the aim of this study. Gj was measured in guinea-pig left atrium from the frequency-dependent variation of intracellular impedance; intracellular [Ca2+], ([Ca2+]i) in low-Na solution was measured by Fura-2 fluorescence. Phosphorylation of guinea-pig Ser368-Cx43 residues was measured by Western blot; Cx40 was immunoprecipitated and probed for serine/threonine residue phosphorylation. Low-Na solution reversibly reduced Gj, in turn attenuated or prevented by calcineurin inhibitors cyclosporin-A or CAIP, respectively. Moreover, Ser368-Cx43 phosphorylation in low-Na solution was also prevented by CAIP. Changes were partially prevented by fostreicin (FST), a protein phosphatase-2A (PP2A) inhibitor; but not by tautomycin, a PP1 inhibitor. Serine/threonine residues on Cx40 were also phosphorylated in low-Na solution; prevented by CAIP and attenuated by FST. Reduced Gj with raised [Ca2+]i is paralleled by a changed Cx43/Cx40 phosphorylation status; changes mediated by calcineurin and PP2A-dependent pathways, but not PP1. The pharmacological profile underlying changes to guinea-pig atrial gap junction electrical conductance with raised intracellular [Ca2+]i is fundamentally different from that in ventricular myocardium. This provides a targeted drug model whereby atrial and ventricular myocardium can be selectively targeted to correct conduction defects.

T-type Ca2+ channels and the urinary and male genital tracts.

T-type Ca(2+) channels are widely expressed throughout the urinary and male genital tracts, generally alongside L-type Ca(2+) channels. The use of pharmacological blockers of these channels has suggested functional roles in all regions, with the possible exception of the ureter. Their functional expression is apparent not just in smooth muscle cells but also in interstitial cells that lie in close proximity to muscle, nerve and epithelial components of these tissues. Thus, T-type Ca(2+) channels can contribute directly to modulation of muscle function and indirectly to changes of epithelial and nerve function. T-type Ca(2+) channel activity modulates phasic contractile activity, especially in conjunction with Ca(2+)-activated K(+) channels, and also to agonist-dependent responses in different tissues. Upregulation of channel density occurs in pathological conditions associated with enhanced contractile responses, e.g. overactive bladder, but it is unclear if this is causal or a response to the pathological state. Moreover, T-type Ca(2+) channels may have a role in the development of prostate tumours regulating the secretion of mitogens from neuroendocrine cells. Although a number of selective channel blockers exist, their relative selectivity over L-type Ca(2+) channels is often low and makes evaluation of T-type Ca(2+) channel function in the whole organism difficult.

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.

Modeling the urinary tract-computational, physical, and biological methods.

Models of the lower urinary tract are used to understand better the physiological and pathological functions of the tract and to gain insight into the relative importance of different components. The key requirement of a model is described, namely: to involve a continuous iteration with experiment; whereby experiments provide parameters and validation for components of the model, which is then used to generate hypotheses, which are tested experimentally. Different types of models are described: computational models that describe mathematically the whole urinary tract or components; physical models useful especially in testing medical devices; and tissue-engineered models. The purpose of modeling is first described in terms of the ability of models to predict the properties of the system of interest, using components that have a physiological interpretation, and to gain insight into the relative importance of different components. Examples are used to illustrate the use of modeling the urinary tract with reference to the different categories listed above.

Bladder compliance what does it represent: can we measure it, and is it clinically relevant?

AIMS: To report the conclusion of the Think Thank 8 on Compliance Discussions during the second ICI-RS meeting in 2010. METHODS: During a 3-day meeting a group of specialists discussed bladder compliance, what it represents, how it can be measured and if it is clinically relevant. RESULTS: Bladder compliance is the result of a mathematical calculation of the volume required for a unit rise of pressure measured during a cystometric filling. It gives an indication on how the different mechanisms in the bladder wall react on stretching. There is a need of standardization of measurement and suggestions for this are given in the text. Pitfalls are described and how to avoid them. There is a wide range of compliance values in healthy volunteers and groups of patients. Poor compliance needs to be defined better as it can have significant clinical consequences. Prevention and treatment are discussed. CONCLUSION: If compliance is correctly measured and interpreted, it has importance in urodynamic testing and gives information relevant for clinical management.

Calcium modulation of vascular smooth muscle ATP-sensitive K(+) channels: role of protein phosphatase-2B.

ATP-sensitive K(+) (K(ATP)) 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 K(ATP) channels are insensitive to Ca(2+), although regulation has not been examined in the intact cell where cytosolic regulatory processes may be important. Thus we investigated the effects of Ca(2+) on whole-cell K(ATP) 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 approximately -40 mV when bathed in 1.8 mmol/L Ca(2+). The K(ATP) 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 approximately -60 mV and increased glibenclamide-sensitive current by 2- to 4-fold. Similar effects were observed when Ca(2+) levels were decreased either externally or internally by increasing EGTA from 1 to 10 mmol/L. Dialysis with solutions containing different free [Ca(2+)](i) showed that K(ATP) current was maximally activated at 10 nmol/L [Ca(2+)](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 Ca(2+)-dependent protein phosphatase, type 2B), glibenclamide-sensitive currents were large and the resting potential was hyperpolarized by approximately 20 to 25 mV. We report for the first time that K(ATP) channels can be modulated by Ca(2+) at physiological [ATP](i) and conclude that modulation occurs via protein phosphatase type 2B.

Diagnosing Paroxysmal Atrial Fibrillation: Are Biomarkers the Solution to This Elusive Arrhythmia?

Atrial fibrillation (AF) is the commonest sustained arrhythmia globally and results in significantly increased morbidity and mortality including a fivefold risk of stroke. Paroxysmal atrial fibrillation (PAF) constitutes approximately half of all AF cases and is thought to represent an early stage of the disease. This intermittent form of atrial arrhythmia can be a challenge to identify and as a result many affected individuals are not prescribed appropriate antithrombotic therapy and hence are at risk of stroke and thromboembolism. Despite these adverse outcomes there have been relatively few diagnostic advances in the field since the introduction of the Holter monitor in 1949. This review aims to establish the available evidence for electrophysiological, molecular, and morphological biomarkers to improve the detection of PAF with reference to the underlying mechanisms for the condition.

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

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 microM) depletion of intracellular Ca2+ stores, and (2) thapsigargin (THAPS, 2 microM) or cyclopiazonic acid (CPA, 10 microM) 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 microM) 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 significantly 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 microM) and ryanodine (% Tkmax = 20.3 +/- 3.7%; n = 6), and nearly completely blocked by SK&F 96365 (50 microM). 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.

Recovery of the conduction system from ischemic arrest and cardioplegia in perfused rat heart.

Normothermic ischemic arrest of the perfused rat heart for 1 h was followed by severe reperfusion arrhythmias, depressed excitability, atrioventricular (AV) node and sinoatrial (SA) node conduction blocks and by marked impairment of contractility. Excitability and conduction recovered within 10 to 15 mins whereas contractility remained greatly depressed. This finding is in agreement with a theoretically based assumption of lesser sensitivity of conduction system to ischemia compared to the working myocardium. When the hearts were exposed to ischemia at 20 degrees C, the sinus rhythm recovered instantaneously upon reperfusion; excitability and SA node conduction times were not significantly different from control; and contractility was much less depressed than after 34 degrees C ischemia. However, AV node conduction times did not normalize even after 15 mins of reperfusion. Potassium cardioplegia (34 degrees C) did not prevent post ischemic arrhythmias but evidently protected both SA node and AV node conduction. Optimum recovery of the conduction system and contractility was achieved by hypothermic (20 degrees C) potassium cardioplegia. Other cardioplegic additives did not further improve the hypothermic potassium protection of the conduction system.

Oxygen-derived free radical stress activates nonselective cation current in guinea pig ventricular myocytes. Role of sulfhydryl groups.

Oxygen-derived free radicals (O-Rs) cause alterations in cardiac electrical activity, including sustained depolarization, which may contribute to arrhythmic activity in reperfusion after ischemia. The ionic current(s) and cellular mechanism(s) underlying the sustained depolarization are not well defined. We used the whole-cell variant of the patch-clamp technique to study sustained depolarization in guinea pig ventricular myocytes during the extracellular application of O-Rs (generating system: dihydroxyfumaric acid, 3 to 6 mmol/L; FeCl3/ADP, 0.05:0.5 mmol/L). Myocytes superfused with O-Rs (pipette EGTA, 0.1 mmol/L) showed (1) sustained depolarization to between -40 and -10 mV, (2) oscillations in membrane potential, and (3) triggered activity. The depolarization resulted from an increase in quasi-steady state difference current reversing at approximately -18 mV, and the oscillations were due to transient inward current. The latter were inhibited with ryanodine (10 mumol/L) or high pipette EGTA (5 mmol/L), but the steady state current was unaffected. Nonselective cation current (INSC) (recorded with Cs+, Li+, and Mg2+ replacing K+, Na+, and Ca2+, respectively; 20 mmol/L tetraethylammonium chloride [TEA] and 5 mmol/L BAPTA in the pipette solution and 10 mmol/L TEA, 10 mumol/L tetrodotoxin, and 10 mumol/L nicardipine in the bath solution) was activated by O-Rs; the increase in current was unaffected by preventing changes in [Ca2+]i but was inhibited with dithiothreitol. Oxidizing agents (diamide and thimerosal) or caffeine (pipette EGTA, 0.1 mmol/L) produced a similar increase in membrane conductance. INSC activated with O-Rs, oxidizing agents, or caffeine was sensitive to SK&F 96365. O-R treatment was without effect when INSC was already activated with caffeine. The data suggest that (1) extracellular O-Rs activate a Ca(2+)-sensitive INSC in the absence of changes in [Ca2+]i, (2) oxidative modification of extracellular sulfhydryl groups may be involved, and (3) this mechanism is different from the Ca(2+)-dependent activation of INSC by intracellular O-Rs, indicating that O-Rs may alter ion channel activity by differential mechanisms, depending on the compartment, extracellular or intracellular, in which they are present.

Alterations in electrical activity and membrane currents induced by intracellular oxygen-derived free radical stress in guinea pig ventricular myocytes.

Oxygen-derived free radicals (O-Rs) are thought to induce alterations in cardiac electrical activity; however, the underlying membrane ionic currents affected by O-Rs and the mechanisms by which O-Rs induce their effects on ion channels in the heart are not well defined. In this study, we investigated the time-dependent changes in resting membrane potential and action potential configuration and changes in steady-state membrane currents in guinea pig ventricular myocytes after intracellular application of an O-R-generating system. O-Rs were generated from the combination of dihydroxyfumaric acid (3 mM) and FeCl3:ADP (0.05:0.5 mM) added to the pipette solution that was used to record membrane potential and currents via the whole-cell variant of the patch-clamp technique. Intracellular exposure of myocytes to the O-R-generating solution induced three stages of changes: 1) an early depolarization (5-10 mV) and an increase in action potential duration accompanied by a decrease in resting inward rectifying K+ current conductance, 2) delayed afterdepolarizations and triggered activity caused by the activation of transient inward current mediated by Na(+)-Ca2+ exchange, with failure to repolarize and sustained depolarization between -35 and -20 mV, reflecting the stimulation of nonselective cation current, and 3) a late stage of marked decline in action potential duration, hyperpolarization, and loss of excitability accompanied by activation of the outward current through ATP-sensitive K+ channels. These alterations in electrical activity and membrane currents could be prevented by pretreatment with N-(2-mercaptopropionyl)glycine (500 microM), a scavenger of hydroxyl free radicals. The alterations associated with stages 1 and 2 but not stage 3 were completely abolished on intracellular Ca2+ chelation (5 mM EGTA in the pipette solution) or disruption of sarcoplasmic reticulum Ca2+ handling with ryanodine (10 microM). This study shows that intracellular O-R stress causes specific alterations in membrane ionic currents, leading to changes in resting membrane potential and action potential configuration. Moreover, the data indicate that an elevation in intracellular Ca2+ due to abnormal Ca2+ handling by the sarcoplasmic reticulum is a cause of some of the alterations in membrane currents during O-R stress.

Lysophosphatidylcholine triggers intracellular calcium release and activation of non-selective cation channels in renal arterial smooth muscle cells.

The effects of a lipid component of oxidized low-density lipoproteins (ox-LDL), L-alpha-palmitoyl-lysophosphatidylcholine (LPC), on membrane currents of isolated canine renal artery smooth muscle cells (RASMC) were examined using the whole-cell configuration of the patch-clamp technique. In RASMC exposed to nominally Ca2+-free solutions and dialyzed with 0.1 mM EGTA and 140 mM K+, superfusion with LPC (10 microM) elicited spontaneous transient outward currents (STOCs) and/or spontaneous transient inward currents (STICs), followed by the activation of a large voltage-independent current with a reversal potential (Er) close to 0 mV. Buffering intracellular Ca2+ with 10 mM BAPTA prevented the appearance of STOCs and STICs, but not the activation of the voltage-independent current. Er of the LPC-induced voltage-independent current exhibited sensitivity to changes in [K+]o and [Na+]o in a manner consistent with a non-selective cation current (I(NSC)) and was blocked by gadolinium (Gd3+; 10 microM). Shifts in Er of the LPC-induced I(NSC) in response to changes in [Ca2+]o were used to estimate a relative Ca2+ to Na+ permeability ratio (P(Ca)/P(Na)) of 1.67. These results suggest that LPC causes abnormal sarcoplasmic reticulum Ca2+ regulation, leading to the appearance of STOCs and STICs, and the activation of I(NSC) in vascular smooth muscle cells. These effects may explain the ability of ox-LDLs to elevate [Ca2+]i in vascular smooth muscle and inhibit endothelium-dependent relaxation.

Arrhythmia and delayed recovery of cardiac action potential during reperfusion after ischemia. Role of oxygen radical-induced no-reflow phenomenon.

The role of reactive metabolites of oxygen, oxygen radicals (O-Rs), as mediators of potentially arrhythmogenic alterations in cellular electrical properties and contractile dysfunction of cardiac muscle during reperfusion after ischemia was investigated. Electrical and mechanical activities of arterially perfused guinea pig right ventricular walls were recorded simultaneously with intracellular microelectrodes and a force transducer. Preparations were maintained in Krebs-Henseleit solution (perfusion rate, 1.5 mL/min) and subjected to 30 minutes of no-flow ischemia followed by 60 minutes of reperfusion or pretreated with O-R scavengers (superoxide dismutase, 50 U/mL; catalase, 600 U/mL; and mannitol, 2 mmol/L) for 10 to 20 minutes, followed by 30 minutes of ischemia and 60 minutes of reperfusion. Reperfusion in untreated preparations caused (1) depolarization of resting membrane potential by 8 to 10 mV and slow recovery of action potential duration requiring 60 minutes to attain the preischemic duration, (2) tachyarrhythmias and premature action potentials, (3) postischemic contractile dysfunction, and (4) increased coronary perfusion pressure in untreated preparations. Pretreatment with scavenger cocktail affected neither electrical nor contractile activity before or during no-flow ischemia, but it (1) accelerated recovery of resting membrane potential and action potential duration, (2) reduced the incidence of tachyarrhythmia, (3) improved contractile function, and (4) inhibited the rise in perfusion pressure on reflow. Reperfusion with an exogenous O-R-generating system containing xanthine/xanthine oxidase (X/XO, 2 mmol/L:10 mU/mL) inhibited recovery of action potential duration and contractility. Treatment of normoxic arterially perfused right ventricular walls with X/XO caused a decline in action potential duration by approximately 20% within 30 minutes. In contrast, X/XO caused a 30% increase in the duration of action potentials in superfused papillary muscles or small strips of right ventricular walls over the same time period. Pretreatment with sodium nitroprusside (10 mumol/L) inhibited the decline in duration induced by X/XO in normoxic right ventricular walls but was without effect on prolongation due to X/XO in papillary muscles. Reperfusion with nitroprusside after no-flow ischemia caused (1) accelerated recovery of preischemic action potential configuration, (2) a significant decline in the incidence of reperfusion arrhythmias, (3) improved postischemic contractile performance, and (4) inhibition of the increase in perfusion pressure associated with reflow. The data indicate that slow recovery of the action potential duration caused by O-Rs in reperfusion cannot be explained by the direct effects of O-Rs on cardiac myocytes.(ABSTRACT TRUNCATED AT 400 WORDS)