Heterogeneous Expression of NO-Activated Soluble Guanylyl Cyclase in Mammalian Heart: Implications for NO- and Redox-Mediated Indirect Versus Direct Regulation of Cardiac Ion Channel Function

Nitrogen oxides exert significant but diverse regulatory effects on cardiac myocytes. Many of these effects are due to modulation of voltage-sensitive ion channel function. The redox-status of NO-related compounds is a critical factor in determining whether indirect (cGMP-dependent) versus direct (cGMP-independent) effects are dominant. However, molecular mechanisms by which different cardiac myocyte types, and associated different ion channel types expressed within them, could achieve selectivity between NO-related indirect versus direct effects are unclear We have previously demonstrated heterogeneous expression gradients of Type III NO synthase (eNOS) and sarcolemmal superoxide dismutase (ECSOD) in ferret and human ventricle, with both enzymes being highly expressed in right ventricle and left ventricular subepicardium but markedly reduced in left ventricular subendocardium. In this study we extend this previous analysis by analyzing NO-activated soluble guanylyl cyclase (sGC) expression in the heart (ferret and human). We demonstrate that, at both tissue and single myocyte levels, sGC protein expression is heterogeneous, being high in sinoatrial node, right atrium, right ventricle and left ventricular subepicardium, but markedly reduced to absent in left atrium and left ventricular subendocardium. Thus, there is a significant overlap in expression gradients of sGC, eNOS, and ECSOD among distinct cardiac tissue and myocyte types. These gradients positively correlate with both: i) experimentally measured basal NO production levels; and ii) expression gradients of specific voltage-gated ion channels (particularly Kv1 and Kv4 channels). Our results provide the first demonstration in the heart of an expressed coupled multienzymatic system for selective regulation of indirect (sGC-dependent) versus direct (sGC-independent) NO- and redox-related modulation of voltage-gated ion channel function in different myocyte types. Our results also have functional implications for NO. / redox - related modulation of ion channels expressed in other cell types, including neurons, skeletal muscle and smooth muscle.     

Apoptosis in severe, compensated pressure overload predominates in nonmyocytes and is related to the hypertrophy but not function

It is widely held that myocyte apoptosis in left ventricular hypertrophy (LVH) contributes to left ventricle (LV) dysfunction and heart failure. The main goal of this investigation was to determine if there is a statistical relationship among LV hypertrophy, apoptosis and LV function, and importantly whether the apoptosis occurs in myocytes or nonmyocytes in the heart. We used both rat and canine models of severe LVH induced by chronic thoracic aortic banding with resultant LV-aortic pressure gradients 145-155 mmHg and increases in LV/body weight of 58 and 70%. These models also provided the ability to examine transmural apoptosis in LVH. In both models, the overwhelming majority (88%) of apoptotic cells were nonmyocytes. The regressions for apoptosis vs. LVH were stronger for nonmyocytes than myocytes and also stronger in the subendocardium than the subepicardium. Importantly, LV systolic and diastolic wall stresses were normal, indicating that the apoptosis could not be attributed to LV stretch or heart failure. In addition, there was no relationship between the extent of apoptosis and LV ejection fraction, which actually increased (P < 0.05), in the face of elevated LV systolic pressure, indicating that greater apoptosis did not result in a decrease in LV function. Thus, in response to chronic, severe pressure overload, LVH in the absence of LV dilation, and elevated LV wall stress, apoptosis occurred predominantly in nonmyocytes in the myocardial interstitium, more in the subendocardium than the subepicardium. The extent of apoptosis was linearly related to the amount of LV hypertrophy, but not to LV function.     

Effects of ectopic pacing on repolarization of the chicken left ventricle

Effects of ectopic pacing on left ventricular repolarization were studied in six anesthetized open-chest chickens. In each animal, unipolar electrograms were acquired from as many as 98 sites with 14 plunge needles (seven transmural locations between epicardium and endocardium in each needle). Activation-recovery intervals (ARIs), corrected to the cycle length, were used for estimating repolarization. At baseline, the nonuniform ARI distribution in the left ventricle resulted in the apicobasal differences being greater than the transmural gradient. Nonuniform ARI prolongation caused by ectopic pacing resulted in decreasing the transmural repolarization gradient and increasing the differences in the apex-to-base direction. The basal, but not apical transmural differences contributed to the total left ventricular transmural gradient. The total left ventricular apicobasal gradient was contributed by the apicobasal differences in mid-myocardial and subendocardial layers more than in subepicardial ones. Thus, in in situ chicken hearts, the transmural and apicobasal ARI gradients exist within the left ventricle with the shortest ARIs in the basal subepicardium and the longest ARIs in the subendocardium of the apical and middle parts of the left ventricle. Apicobasal compared to transmural heterogeneity of local repolarization properties contributes more to the total left ventricular repolarization gradient.     

Morphometric data on the lymph capillaries of the ventricles of the rabbit heart

A morphometric analysis was done on the lymph capillaries of both left and right ventricles from the rabbit heart. The measurements were made on the lymphatics identified in the subepicardium, myocardium and subendocardium of the ventricular walls. Quantitative evaluations were carried out on light and electron microscopic sections by a computerized image analysis system. The following parameters were selected and measured: (1) the diameter (of area-equivalent circle) of lymph capillaries, (2) the diameter of the uncoated micropinocytotic vesicles (located on the abluminal and adluminal side and in the cytoplasm of the endothelial cell) and the area occupied by the vesicles per unit area of cytoplasm. Differences in the size of the lymph capillaries were found in the three layers (subepicardium, myocardium and subendocardium) of the ventricular walls. The largest vessels were present in the subepicardium both in the left ventricle and in the right one. No significant variations were found in the lymphatics of corresponding regions on both ventricles. Little variations on the mean diameter of the uncoated micropinocytotic vesicles are present in the three regions of the endothelial wall. In the left ventricle only, the subendocardial vesicles are significantly larger than the subepicardial and the myocardial ones (p less than 0.05). The areal density occupied by vesicular system in the three layers of the ventricular wall showed significant differences in both ventricles (p less than 0.05). The vesicles present in the subepicardial vessels occupied the smallest areal density. No significant variations existed in the vesicular areal density between the two ventricles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of syntaxin 1A with the N-terminus of Kv4.2 modulates channel surface expression and gating

Kv4.2 channels are responsible in the heart for the Ca2+-independent transient outward currents and are important in regulating myocardial excitability and Ca2+ homeostasis. We have identified previously the expression of syntaxin 1A (STX1A) on the cardiac ventricular myocyte plasma membranes, and its modulation of cardiac ATP-sensitive K+ channels. We speculated that STX1A interacts with other cardiac ion channels, thus we examined the interaction of STX1A with Kv4.2 channels. Co-immunoprecipitation and GST pulldown assays demonstrated a direct interaction of STX1A with the Kv4.2 N-terminus. We next investigated the functional alterations of Kv4.2 gating by STX1A in Xenopus oocytes. Coexpression of Kv4.2 with STX1A (1) resulted in a reduction of Kv4.2 current amplitude; (2) caused a depolarizing shift of the steady-state inactivation curve; (3) enhanced the rate of current decay; and (4) accelerated the rate of recovery from inactivation. Additional coexpression of botulinum neurotoxin C, which cleaves STX1A, reversed the effects of STX1A on Kv4.2. STX1A inhibited partially the gating changes by KChIP2, suggesting a competitive interaction of these proteins for an overlapping binding region on the N-terminus of Kv4.2. Indeed, the N-terminal truncation mutants of Kv4.2 (Kv4.2Delta2-40 and Kv4.2Delta7-11), which form part of the KChIP2 binding site, displayed reduced sensitivity to STX1A modulation. Our study suggests that STX1A directly modulates Kv4.2 current amplitude and gating through its interaction with an overlapping region of the KChIP binding motif domain on the Kv4.2 N-terminus.     

Intramural chronotopography of myocardial depolarization in the heart ventricles of the pig (Sus scrofa domesticus)

Multichannel synchronous cardioelectrotopography is used to analyze the sequence of myocardial depolarization in the ventricles of the pig heart. Formation of early depolarization areas has been established in subendocardium of interventricular septum and in the base of papillary muscles of the left ventricle; of multiple foci-in the thickness of myocardial walls; of areas of late depolarization-in subepicardium of the dorsolateral side of the left ventricle. In pig heart ventricles, as compared with other ungulates (reindeer and sheep) there are revealed differences in location of areas of the early and late depolarization areas and the breakthrough of excitation wave onto subepicardium.     

Myoglobin content and citrate synthase activity in different parts of the normal human heart

Myoglobin (Mb) content and citrate synthase (CS) activity were determined in myocardial samples from nine human brain-dead organ donors with normal hearts. Six regions of each heart were analyzed: right and left atria, right ventricle, left ventricular subepicardium, subendocardium, and anterior papillary muscle. The Mb content was similar, whereas the CS activity was higher in the left than in the right heart at both atrial and ventricular levels. Mb content and CS activity were higher in ventricles than in atria. The subendocardial layer and papillary muscle of the left ventricle had a higher Mb content than the subepicardial layer, whereas CS activity was similar in these three locations. The results suggested a closer relationship between CS activity (oxidative potential) and work load than between Mb content and work load. Mb content may, instead, be related to intramuscular oxygen tension (PO2) on the basis of a comparison between our Mb data and those of others on regional variations in myocardial PO2.     

Inhomogeneity in the response to mechanical stimulation: cardiac muscle function and gene expression

Mechanical stimulation has important consequences for myocardial function. However, this stimulation and the response to it, is not uniform. The right ventricle is thinner walled and operates at lower pressure than the left ventricle. Within the ventricles, differences in the orientation of myocardial fibres exist. These differences produce inhomogeneity in the stress and strain between and across the ventricles. Possibly as a result of these variations in mechanical stimulation, there are well characterised inhomogeneities in gene expression and protein function within the ventricular myocardium, for example in the transient outward K+ current and its associated Kv channels. Perhaps not surprisingly, it is becoming apparent that gradients of expression and function exist for proteins that are intimately involved in the response to mechanical stimulation in the heart, for example in the left ventricle of the rat there is a transmural gradient in mRNA and current density of the mechanosensitive two-pore domain K+ channel TREK-1 (ENDO>EPI). In healthy hearts it is assumed that these gradients are important for normal function and therefore that their disruption in diseased myocardium is involved in the dysfunction that occurs.     

Transmural sheet strains in the lateral wall of the ovine left ventricle

In an attempt to provide a better understanding of our finding that regions with contracting left ventricular myofibers need not develop a significant transmural systolic wall thickening gradient, the analytic approach of Costa et al. was applied to the four-dimensional dynamic data obtained 1 and 8 wk after surgical implantation of transmural radiopaque beads in the lateral equatorial left ventricular wall in seven ovine hearts. Quantitative histology of tissue blocks demonstrated that fiber angles varied linearly across the wall in this region from -37 degrees in the subepicardium to +18 degrees in the subendocardium. Sheet angles exhibited a pleated-sheet behavior, alternating sign from subepicardium to subendocardium. From end diastole (reference configuration) to end systole (deformed configuration), fiber strain was uniformly negative, sheet extension and sheet thickening were uniformly positive, and sheet-normal shear contributed to wall thickening at all wall depths. Subepicardial radial wall thickening increased significantly from week 1 to week 8, with significant increases in the contributions from subepicardial sheet extension and sheet-normal shear. At 1 and 8 wk, the contribution of sheet-normal shear to wall thickening was substantial at all transmural depths; the contribution of sheet extension to wall thickening was greatest in the subepicardium and least in the subendocardium, and the contribution of sheet thickening to wall thickening was greatest in the subendocardium and least in the subepicardium. A mechanistic model is proposed that provides a working hypothesis that a selective decrease in subepicardial intercellular matrix stiffness is responsible for elimination of the transmural wall thickening gradient 1-8 wk after marker implantation surgery.     

Expression and localization of TRPC proteins in rat ventricular myocytes at various developmental stages

Growing evidence indicates that transient receptor potential canonical (TRPC) channels play important roles in various Ca²⁺-mediated physiological and pathophysiological processes, including development. Many types of TRPC proteins are expressed in the heart. However, limited data are available comparing the expression and localization among TRPC proteins in the ventricular myocyte at various developmental stages. Our purpose is to investigate the expression and localization profile of TRPC proteins in ventricular myocytes of fetal (18.5 days), neonatal (< 24 h after birth) and adult (8 week old) rats. Western blotting, immunofluorescence and confocal laser scanning microscopy were employed. TRPC1/3-6 proteins were expressed in the rat ventricle throughout the three developmental stages. The expression profile of TRPC1/3/4 in the ventricle followed an upward trend from the fetus to the adult. By contrast, TRPC6 in the ventricle was expressed at the highest level in the fetal group and was sharply down-regulated immediately after birth. TRPC5 expression in the ventricle did not change significantly during the three stages. TRPC1/3/5/6 proteins were localized to the T-tubule and TRPC1/3/4/6 to intercalated disks in adult myocytes. The wide spatiotemporal overlap and dynamic regulation of TRPC expression in ventricular myocytes indicates potential complex combinations and redundancy of native TRPC proteins in the heart and gives important clues for further investigations into the exact subunit compositions and functional properties of native TRPC channels in the heart.     

Effects of training and anabolic steroids on collagen synthesis in dog heart.

The effects of endurance training and anabolic steroid (Methandienone 1.5 mg.kg-1 p. o. daily) and their combination on regional collagen biosynthesis and concentration in the hearts of male beagle dogs were studied by measuring prolyl 4-hydroxylase (PH) activity and hydroxyproline (HYP) concentration. The PH (P less than 0.05) and HYP (P less than 0.05) were both greater in the subendocardinal layer than in the subepicardium (EPI) of the left ventricular wall in controls, whereas opposite gradients (P less than 0.05) were observed in the right ventricle. Endurance exercise caused an increase of PH activity in EPI of the left ventricular wall (P less than 0.01). The HYP concentration increased in both layers of the right ventricle in the exercise plus steroid group (P less than 0.05). The results suggest that transmural differences exist in the rate of collagen synthesis and concentration in canine cardiac ventricles and that endurance exercise may accelerate collagen synthesis in EPI of the left ventricle and the combination of exercise and anabolic steroid causes an increase in collagen concentration in the right ventricular wall.     

Distinct transient outward potassium current (Ito) phenotypes and distribution of fast-inactivating potassium channel alpha subunits in ferret left ventricular myocytes

The biophysical characteristics and alpha subunits underlying calcium-independent transient outward potassium current (Ito) phenotypes expressed in ferret left ventricular epicardial (LV epi) and endocardial (LV endo) myocytes were analyzed using patch clamp, fluorescent in situ hybridization (FISH), and immunofluorescent (IF) techniques. Two distinct Ito phenotypes were measured (21-22 degrees C) in the majority of LV epi and LV endo myocytes studied. The two Ito phenotypes displayed marked differences in peak current densities, activation thresholds, inactivation characteristics, and recovery kinetics. Ito,epi recovered rapidly [taurec, -70 mV = 51 +/- 3 ms] with minimal cumulative inactivation, while Ito,endo recovered slowly [taurec, -70 mV = 3,002 +/- 447 ms] with marked cumulative inactivation. Heteropoda toxin 2 (150 nM) blocked Ito,epi in a voltage-dependent manner, but had no effect on Ito,endo. Parallel FISH and IF measurements conducted on isolated LV epi and LV endo myocytes demonstrated that Kv1.4, Kv4.2, and Kv4.3 alpha subunit expression in LV myocyte types was quite heterogenous: (a) Kv4.2 and Kv4.3 were more predominantly expressed in LV epi than LV endo myocytes, and (b) Kv1.4 was expressed in the majority of LV endo myocytes but was essentially absent in LV epi myocytes. In combination with previous measurements on recovery kinetics (Kv1.4, slow; Kv4.2/4.3, relatively rapid) and Heteropoda toxin block (Kv1.4, insensitive; Kv4.2, sensitive), our results strongly support the hypothesis that, in ferret heart, Kv4.2/Kv4.3 and Kv1.4 alpha subunits, respectively, are the molecular substrates underlying the Ito,epi and Ito,endo phenotypes. FISH and IF measurements were also conducted on ferret ventricular tissue sections. The three Ito alpha subunits again showed distinct patterns of distribution: (a) Kv1.4 was localized primarily to the apical portion of the LV septum, LV endocardium, and approximate inner 75% of the LV free wall; (b) Kv4. 2 was localized primarily to the right ventricular free wall, epicardial layers of the LV, and base of the heart; and (c) Kv4.3 was localized primarily to epicardial layers of the LV apex and diffusely distributed in the LV free wall and septum. Therefore, in intact ventricular tissue, a heterogeneous distribution of candidate Ito alpha subunits not only exists from LV epicardium to endocardium but also from apex to base.     

Kv11.1 (ERG1) K+ channels localize in cholesterol and sphingolipid enriched membranes and are modulated by membrane cholesterol

The localization of ion channels to specific membrane microdomains can impact the functional properties of channels and their role in cellular physiology. We determined the membrane localization of human Kv11.1 (hERG1) alpha-subunit protein, which underlies the rapidly activating, delayed rectifier K(+) current (I(Kr)) in the heart. Immunocytochemistry and membrane fractionation using discontinuous sucrose density gradients of adult canine ventricular tissue showed that Kv11.1 channel protein localized to both the cell surface and T-tubular sarcolemma. Furthermore, density gradient membrane fractionation using detergent (Triton X-100) and non-detergent (OptiPrep) methods from canine ventricular myocytes or HEK293 cells demonstrated that Kv11.1 protein, along with MiRP1 and Kv7.1 (KCNQ1) proteins, localize in cholesterol and sphingolipid enriched membrane fractions. In HEK293 cells, Kv11.1 channels, but not long QT-associated mutant G601S-Kv11.1 channels, also localized to cholesterol and sphingolipid enriched membrane fractions. Depletion of membrane cholesterol from HEK293 cells expressing Kv11.1 channels using methyl-beta-cyclodextrin (MbetaCD) caused a positive shift of the voltage dependence of activation and an acceleration of deactivation kinetics of Kv11.1 current (I(Kv11.1)). Cholesterol loading of HEK293 cells reduced the steep voltage dependence of I(Kv11.1) activation and accelerated the inactivation kinetics of I(Kv11.1). Incubation of neonatal mouse myocytes in MbetaCD also accelerated the deactivation kinetics of I(Kr). We conclude that Kv11.1 protein localizes in cholesterol and sphingolipid enriched membranes and that membrane cholesterol can modulate I(Kv11.1) and I(Kr).     

Cortactin is required for N-cadherin regulation of Kv1.5 channel function

The intercalated disc serves as an organizing center for various cell surface components at the termini of the cardiomyocyte, thus ensuring proper mechanoelectrical coupling throughout the myocardium. The cell adhesion molecule, N-cadherin, is an essential component of the intercalated disc. Cardiac-specific deletion of N-cadherin leads to abnormal electrical conduction and sudden arrhythmic death in mice. The mechanisms linking the loss of N-cadherin in the heart and spontaneous malignant ventricular arrhythmias are poorly understood. To investigate whether ion channel remodeling contributes to arrhythmogenesis in N-cadherin conditional knock-out (N-cad CKO) mice, cardiac myocyte excitability and voltage-gated potassium channel (Kv), as well as inwardly rectifying K(+) channel remodeling, were investigated in N-cad CKO cardiomyocytes by whole cell patch clamp recordings. Action potential duration was prolonged in N-cad CKO ventricle myocytes compared with wild type. Relative to wild type, I(K,slow) density was significantly reduced consistent with decreased expression of Kv1.5 and Kv accessory protein, Kcne2, in the N-cad CKO myocytes. The decreased Kv1.5/Kcne2 expression correlated with disruption of the actin cytoskeleton and reduced cortactin at the sarcolemma. Biochemical experiments revealed that cortactin co-immunoprecipitates with Kv1.5. Finally, cortactin was required for N-cadherin-mediated enhancement of Kv1.5 channel activity in a heterologous expression system. Our results demonstrate a novel mechanistic link among the cell adhesion molecule, N-cadherin, the actin-binding scaffold protein, cortactin, and Kv channel remodeling in the heart. These data suggest that in addition to gap junction remodeling, aberrant Kv1.5 channel function contributes to the arrhythmogenic phenotype in N-cad CKO mice.     

In vivo gene transfer of Kv1.5 normalizes action potential duration and shortens QT interval in mice with long QT phenotype

Mutations in cardiac voltage-gated K+ channels cause long QT syndrome (LQTS) and sudden death. We created a transgenic mouse with a long QT phenotype (Kv1DN) by overexpression of a truncated K+ channel in the heart and investigated whether the dominant negative effect of the transgene would be overcome by the direct injection of adenoviral vectors expressing wild-type Kv1.5 (AV-Kv1.5) into the myocardium. End points at 3-10 days included electrophysiology in isolated cardiomyocytes, surface ECG, programmed stimulation of the right ventricle, and in vivo optical mapping of action potentials and repolarization gradients in Langendorff-perfused hearts. Overexpression of Kv1.5 reconstituted a 4-aminopyridine-sensitive outward K+ current, shortened the action potential duration, eliminated early afterdepolarizations, shortened the QT interval, decreased dispersion of repolarization, and increased the heart rate. Each of these changes is consistent with a physiologically significant primary effect of adenoviral expression of Kv1.5 on ventricular repolarization of Kv1DN mice.     

sGC{alpha}1 mediates the negative inotropic effects of NO in cardiac myocytes independent of changes in calcium handling

In the heart, nitric oxide (NO) modulates contractile function; however, the mechanisms responsible for this effect are incompletely understood. NO can elicit effects via a variety of mechanisms including S-nitrosylation and stimulation of cGMP synthesis by soluble guanylate cyclase (sGC). sGC is a heterodimer comprised of a β(1)- and an α(1)- or α(2)-subunit. sGCα(1)β(1) is the predominant isoform in the heart. To characterize the role of sGC in the regulation of cardiac contractile function by NO, we compared left ventricular cardiac myocytes (CM) isolated from adult mice deficient in the sGC α(1)-subunit (sGCα(1)(-/-)) and from wild-type (WT) mice. Sarcomere shortening under basal conditions was less in sGCα(1)(-/-) CM than in WT CM. To activate endogenous NO synthesis from NO synthase 3, CM were incubated with the β(3)-adrenergic receptor (β(3)-AR) agonist BRL 37344. BRL 37344 decreased cardiac contractility in WT CM but not in sGCα(1)(-/-) myocytes. Administration of spermine NONOate, an NO donor compound, did not affect sarcomeric shortening in CM of either genotype; however, in the presence of isoproterenol, addition of spermine NONOate reduced sarcomere shortening in WT but not in sGCα(1)(-/-) CM. Neither BRL 37344 nor spermine NONOate altered calcium handling in CM of either genotype. These findings suggest that sGCα(1) exerts a positive inotropic effect under basal conditions, as well as mediates the negative inotropic effect of β(3)-AR signaling. Additionally, our work demonstrates that sGCα(1)β(1) is required for NO to depress β(1)/β(2)-AR-stimulated cardiac contractility and that this modulation is independent of changes in calcium handling.     

Nitric oxide signalling augments neuronal voltage-gated L-type (Ca(v)1) and P/q-type (Ca(v)2.1) channels in the mouse medial nucleus of the trapezoid body

Nitric Oxide (NO) is a diffusible second messenger that modulates ion channels, intrinsic excitability and mediates synaptic plasticity. In light of its activity-dependent generation in the principal neurons of the medial nucleus of the trapezoid body (MNTB), we have investigated its potential modulatory effects on native voltage-gated calcium channels (Ca(V)) within this nucleus. Whole-cell patch recordings were made from brain slices from P13-15 CBA mice. Slices were incubated with the inhibitor of neuronal nitric oxide synthase (nNOS) 7-nitroindazole (10 μM) and pharmacological blockers used to isolate Ca(2+) current subtypes. Unpaired observations in the presence and absence of the NO-donors sodium nitroprusside (SNP, 100 μM) or Diethyl-ammonium-nonoate (DEA, 100 μM) were made to elucidate NO-dependent modulation of the expressed Ca(V) subtypes. A differential effect of NO on the calcium channel subtypes was observed: Ca(V)1 and Ca(V)2.1 (L+R- and P/Q+R-type) conductances were potentiated, whereas N+R-type (Ca(V)2.2) and R-type (Ca(V)2.3) current amplitudes were unaffected. L+R-type currents increased from 0.36 ± 0.04 nA to 0.64 ± 0.11 nA and P/Q+R-type from 0.55 ± 0.09 nA to 0.94 ± 0.05 nA, thereby changing the balance and relative contribution of each subtype to the whole cell calcium current. In addition, N+R-type half-activation voltage was left shifted following NO exposure. NO-dependent modulation of P/Q+R and N+R-type, but not L+R-type, channels was removed by inhibition of soluble guanylyl cyclase (sGC) activity. This data demonstrates a differential effect of NO signalling on voltage-gated calcium entry, by distinct NO-dependent pathways.