Nitrite is a positive modulator of the Frank-Starling response in the vertebrate heart

Evidence from both mammalian and nonmammalian vertebrates indicates that intracardiac nitric oxide (NO) facilitates myocardial relaxation, ventricular diastolic distensibility, and, consequently, the Frank-Starling response, i.e., the preload-induced increase of cardiac output. Since nitrite ion (NO(2)(-)), the major storage pool of bioactive NO, recently emerged as a cardioprotective endogenous modulator, we explored its influence on the Frank-Starling response in eel, frog, and rat hearts, used as paradigms of fish, amphibians, and mammals, respectively. We demonstrated that, like NO, exogenous nitrite improves the Frank-Starling response in all species, as indicated by an increase of stroke volume and stroke work (eel and frog) and of left ventricular (LV) pressure and LVdP/dt max (rat), used as indexes of inotropism. Unlike in frog and rat, in eel, the positive influence of nitrite appeared to be dependent on NO synthase inhibition. In all species, the effect was sensitive to NO scavengers, independent on nitroxyl anion, and mediated by a cGMP/PKG-dependent pathway. Moreover, the nitrite treatment increased S-nitrosylation of lower-molecular-weight proteins in cytosolic and membrane fractions. These results suggest that nitrite acts as a physiological source of NO, modulating through different species-specific mechanisms, the stretch-induced intrinsic regulation of the vertebrate heart.     

Cardiac heterometric response: the interplay between Catestatin and nitric oxide deciphered by the frog heart

The length-active tension relation or heterometric regulation (Frank-Starling mechanism) is modulated by nitric oxide (NO) which, released in pulsatile fashion from the beating heart, improves myocardial relaxation and diastolic distensibility. The NO signaling is also implicated in the homeometric regulation exerted by extrinsic factors such as autonomic nervous system, endocrine and humoral agents. In the in vitro working frog heart, the Chromogranin A (CGA)-derived peptide, Catestatin (CTS; bovine CGA344-364), exerts a direct cardio-suppressive action through a NOS-NO-cGMP-mediated mechanism which requires the functional integrity of the endocardial endothelium (EE) and its endothelin-1 B type (ETB) receptor. However, functional interplay between NO and CTS and their role in the Frank-Starling response of the frog heart are lacking. Here we show that CTS improves the sensitivity to preload increases similar to that exerted by NO. This effect is abolished by inhibition of NO synthase (L-NAME), guanylate cyclase (ODQ), protein kinase G (KT5823), PI3K (Wortmannin), as well as by the functional damage of EE (Triton X-100) suggesting that CTS operates through an EE-dependent NO release. On the whole, the use of the avascular frog heart revealed the EE as major sensor-transducer interface between the physical (volume load) and chemical (CTS) stimuli, NO functioning as a connector between heterometric and homeometric regulation.     

Acute hypothyroidism slows the rate of left ventricular diastolic relaxation

The effect of acute thyroid hormone deficiency on left ventricular diastolic filling was studied by radionuclide ventriculography with simultaneous right heart catheterization in nine athyreotic patients without cardiovascular disease. The patients were studied when they were hypothyroid and when they were euthyroid on replacement therapy. Peak filling rate and the time to peak filling were used to characterize diastolic function. The time to peak filling was defined as the interval from end-systole on the radionuclide time-volume curve to the time of occurrence of peak filling. The peak filling rate was determined in absolute terms from the normalized radionuclide peak filling rate and from the end-diastolic volume, which was derived from the radionuclide ejection fraction and from the thermodilution stroke volume. In all patients, the values for peak filling rate were lower in the hypothyroid than in the euthyroid state (287 +/- 91 mL/s vs. 400 +/- 118 mL/s, delta = 41 +/- 13%, p less than 0.01). Peak filling always occurred during the first half of the diastolic interval. The time to peak filling was not significantly affected by the thyroid state (170 +/- 10 ms vs. 159 +/- 21 ms, delta = 7 +/- 10%). Left ventricular filling pressure as reflected by the pulmonary capillary wedge pressure and end-systolic volume were similar in both thyroid states (6 +/- 2 mmHg vs. 8 +/- 2 mmHg (1 mmHg = 133.32 Pa) and 32 +/- 11 mL vs. 32 +/- 7 mL, respectively). The data suggest that the rate of active diastolic relaxation is decreased in short-duration hypothyroidism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nitric oxide-compromised hypertension: facts and enigmas

NO concentration in the femoral artery and femoral vein of anesthetized dogs was found to be 154.2+/-5.6 nM and 90.0+/-12 nM, respectively. Inhibition of NO synthase (NOS) slightly decreased the basal NO concentration in femoral artery from 154.2+/-5.6 to 137.2+/-3.3 nM. Acetylcholine-induced increase in NO concentration was slightly but still significantly attenuated, suggesting that very probably L-NAME did not inhibit all sources of nitric oxide (NO). Local NOS inhibition in the posterior hypothalamus dose-dependently increased systemic blood pressure (BP) in rats. Short-term general NOS inhibition in anesthetized dogs increased diastolic BP but not systolic BP. The heart rate after one-hour down-fluctuation returned to initial values. Proteosynthesis in the myocardium and both branches of the left coronary artery increased, but this was not supported by polyamines, since the activity of ornithine decarboxylase declined. Long-term general NOS inhibition elicited a sustained BP increase, a decrease in heart rate, cardiac hypertrophy and an increase in wall thickness of the coronary and carotid artery. The results indicate that NO deficiency itself plays a role in proteosynthesis and cardiac hypertrophy, in spite of relatively small increase in diastolic blood pressure and no change in systolic blood pressure, at least after an acute L-NAME administration. The hypotension response to acetylcholine and bradykinin studied in anesthetized NO-compromised rats, was unexpectedly enhanced. The elucidation of this paradoxical phenomenon will require further experiments.     

Inhaled nitric oxide does not reduce systemic vascular resistance in mice

Inhaled nitric oxide (NO) is a highly selective pulmonary vasodilator. It was recently reported that inhaled NO causes peripheral vasodilatation after treatment with a NO synthase (NOS) inhibitor. These findings suggested the possibility that inhibition of endogenous NOS uncovered the systemic vasodilating effect of NO or NO adducts absorbed via the lungs during NO inhalation. To learn whether inhaled NO reduces systemic vascular resistance in the absence of endothelial NOS, we studied the systemic vascular effects of NO breathing in wild-type mice treated without and with the NOS inhibitor N(omega)-nitro-l-arginine methyl ester and in NOS3-deficient (NOS3(-/-)) mice. During general anesthesia, the cardiac output, left ventricular function, and systemic vascular resistance were not altered by NO breathing at 80 parts/million in both genotypes. Breathing NO in air did not alter blood pressure and heart rate, as measured by tail-cuff and telemetric methods, in either awake wild-type mice (whether or not they were treated with N(omega)-nitro-l-arginine methyl ester), or in awake NOS3(-/-) mice. Our findings suggest that absorption of NO or adducts during NO breathing is insufficient to cause systemic vasodilation in mice, even when endogenous endothelial NO production is congenitally absent.     

Impact of gender on basal and insulin-like growth factor I-regulated nitric oxide synthase activity in adult rat left ventricular myocytes

Cardiovascular morbidity and mortality are far less in pre-menopausal women compared to age-matched men. Ovarian hormones are believed to be mainly responsible for this "female advantage" in cardiovascular function although the underlying mechanism has not been fully elucidated. A gender difference exists in vascular nitric oxide (NO) synthesis, which may play a key role in ventricular function and cardiac remodeling. This study was designed to compare NO production, basal NO synthase (NOS) expression and activity, as well as insulin-like growth factor I (IGF-1)-induced response on NOS activity in left ventricular myocytes from age-matched adult male and female Sprague-Dawley rats. NO production and protein expression of NOS, IGF-1 receptor (IGF-1R) and IGF binding protein-3 (IGFBP-3) were measured by Griess assay and Western blot analysis, respectively. NOS activity was evaluated by conversion of (3)H-arginine to (3)H-citrulline. Basal NO production, endothelial NOS expression and NOS activity were both significantly higher in female left ventricular myocytes than their male counterparts. However, protein expression of inducible and neuronal NOS as well as IGFBP-3 was comparable between the two genders. IGF-1R expression was less in female than male group. IGF-1 (10(-10)-10(-6) m) induced a concentration-dependent inhibition of NOS activity in male myocytes with a maximal inhibition of 22.2%. However, the IGF-1-induced inhibition in NOS activity was not present in left ventricular myocytes from female rats. These data revealed a gender difference in myocardial basal NO levels, endothelial NOS expression, basal NOS activity and IGF-1-induced inhibition on NOS activity, which may contribute to the gender-related difference of cardiac function.     

Elucidation of spatially distinct compensatory mechanisms in diastole: radial compensation for impaired longitudinal filling in left ventricular hypertrophy.

Cardiac output maintenance is so fundamental that, when regional systolic function is impaired, as during ischemia, nonischemic segments compensate by becoming hypercontractile. By analogy, diastolic compensatory mechanisms that maintain filling volume must exist but remain to be fully elucidated. Viewing filling in spatially distinct (longitudinal, radial) mechanistic terms facilitates elucidation of diastolic compensatory mechanisms. Because impairment of longitudinal (long axis) diastolic function (DF) in left ventricular hypertrophy (LVH) is established, we hypothesized that to maintain filling volume, radial (short-axis) filling function would compensate. In 20 normal left ventricular ejection fraction (LVEF) subjects (10 with LVH, 10 without LVH), we analyzed longitudinal function via Doppler tissue imaging of mitral annular motion and radial function as change in short-axis endocardial dimension via M-mode. The spatial (long axis, short axis) endocardial LV dimensions and their changes allowed assignment of E-wave filling volume into (cylindrical geometry-based) longitudinal and radial components. Despite indistinguishable (P = 0.70) E-wave velocity-time integrals (E-wave filling volume surrogate), systolic stroke volumes, and end-diastolic volumes in the LVH and control groups, longitudinal volume in absolute terms and the percent of E-wave volume accommodated longitudinally were reduced in the LVH group (P < 0.05 and P < 0.01, respectively), whereas the percent of E-wave volume accommodated radially was enhanced (P < 0.01). We conclude that, in normal LVEF (decreased longitudinal volume accommodation) LVH subjects vs. controls, spatially distinct compensatory mechanisms in diastole manifest as increased radial volume accommodation per unit of E-wave filling volume. Assessment of spatially distinct diastolic compensatory mechanisms in other pathophysiological subsets is warranted.     

Prostaglandin E2 inhibits vasotocin-induced osmotic water permeability in the frog urinary bladder by EP1-receptor-mediated activation of NO/cGMP pathway

PGE(2) is a well-known inhibitor of the antidiuretic hormone-induced increase of osmotic water permeability (OWP) in different osmoregulatory epithelia; however, the mechanisms underlying this effect of PGE(2) are not completely understood. Here, we report that, in the frog Rana temporaria urinary bladder, EP(1)-receptor-mediated inhibition of arginine-vasotocin (AVT)-induced OWP by PGE(2) is attributed to increased generation of nitric oxide (NO) in epithelial cells. It was shown that the inhibitory effect of 17-phenyl-trinor-PGE(2) (17-ph-PGE(2)), an EP(1) agonist, on AVT-induced OWP was significantly reduced in the presence of 7-nitroindazole (7-NI), a neuronal NO synthase (nNOS) inhibitor. NO synthase (NOS) activity in both lysed and intact epithelial cells measured as a rate of conversion of l-[(3)H]arginine to l-[(3)H]citrulline was Ca(2+) dependent and inhibited by 7-NI. PGE(2) and 17-ph-PGE(2), but not M&B-28767 (EP(3) agonist) or butaprost (EP(2) agonist), stimulated NOS activity in epithelial cells. The above effect of PGE(2) was abolished in the presence of SC-19220, an EP(1) antagonist. 7-NI reduced the stimulatory effect of 17-ph-PGE(2) on NOS activity. 17-ph-PGE(2) increased intracellular Ca(2+) concentration and cGMP in epithelial cells. Western blot analysis revealed an nNOS expression in epithelial cells. These results show that the inhibitory effect of PGE(2) on AVT-induced OWP in the frog urinary bladder is based at least partly on EP(1)-receptor-mediated activation of the NO/cGMP pathway, suggesting a novel cross talk between AVT, PGE(2), and nNOS that may be important in the regulation of water transport.     

Nebivolol improves diastolic dysfunction and myocardial remodeling through reductions in oxidative stress in the transgenic (mRen2) rat

Angiotensin II contributes to myocardial tissue remodeling and interstitial fibrosis through NADPH oxidase-mediated generation of oxidative stress in the progression of heart failure. Recent data have suggested that nebivolol, a third-generation β-blocker, improves diastolic dysfunction by targeting nitric oxide (NO) and metabolic pathways that decrease interstitial fibrosis. We sought to determine if targeting NO would improve diastolic function in a model of tissue renin-angiotensin system overactivation. We used the transgenic (TG) (mRen2)27 rat, which overexpresses the murine renin transgene and manifests insulin resistance and left ventricular dysfunction. We treated 6- to 7-wk-old TG (mRen2)27 rats and age-matched Sprague-Dawley control rats with nebivolol (10 mg·kg(-1)·day(-1)) or placebo via osmotic minipumps for a period of 21 days. Compared with Sprague-Dawley control rats, TG (mRen2)27 rats displayed a prolonged diastolic relaxation time and reduced initial filling rate associated with increased interstitial fibrosis and left ventricular hypertrophy. These findings were temporally related to increased NADPH oxidase activity and subunits p47(phox) and Rac1 and increased total ROS and peroxynitrite formation in parallel with reductions in the antioxidant heme oxygenase as well as the phosphorylation/activation of endothelial NO synthase and PKB/Akt. Treatment with nebivolol restored diastolic function and interstitial fibrosis through increases in the phosphorylation of 5'-AMP-activated protein kinase, Akt, and endothelial NO synthase and reductions in oxidant stress. These results support that targeting NO with nebivolol treatment improves diastolic dysfunction through reducing myocardial oxidative stress by enhancing 5'-AMP-activated protein kinase and Akt activation of NO biosynthesis.     

Species- and tissue-dependent effects of NO and cyclic GMP on cardiac ion channels

Biochemical studies have established the presence of a NO pathway in the heart, including sources of NO and various effectors. Several cardiac ion channels have been shown to be modified by NO, such as L-type Ca(2+), ATP-sensitive K(+), and pacemaker f-channels. Some of these effects are mediated by cGMP, through the activity of three main proteins: the cGMP-dependent protein kinase (PKG), the cGMP-stimulated phosphodiesterase (PDE2) and the cGMP-inhibited PDE (PDE3). Other effects appear independent of cGMP, as for instance the NO modulation of the ryanodine receptor-Ca(2+) channel. In the case of the cardiac L-type Ca(2+) channel current (I(Ca,L)), both cGMP-dependent and cGMP-independent effects have been reported, with important tissue and species specificity. For instance, in rabbit sinoatrial myocytes, NO inhibits the beta-adrenergic stimulation of I(Ca,L) through activation of PDE2. In cat and human atrial myocytes, NO potentiates the cAMP-dependent stimulation of I(Ca,L) through inhibition of PDE3. In rabbit atrial myocytes, NO enhances I(Ca,L) in a cAMP-independent manner through the activation of PKG. In ventricular myocytes, NO exerts opposite effects on I(Ca,L): an inhibition mediated by PKG in mammalian myocytes but by PDE2 in frog myocytes; a stimulation attributed to PDE3 inhibition in frog ventricular myocytes but to a direct effect of NO in ferret ventricular myocytes. Finally, NO can also regulate cardiac ion channels by a direct action on G-proteins and adenylyl cyclase.     

Role of inducible nitric oxide synthase in cardiac function and remodeling in mice with heart failure due to myocardial infarction

Using inducible nitric oxide (NO) synthase (iNOS) knockout mice (iNOS-/-), we tested the hypotheses that 1) lack of iNOS attenuates cardiac remodeling and dysfunction and improves cardiac reserve postmyocardial infarction (MI), an effect that is partially mediated by reduction of oxidative stress due to reduced interaction between NO and reactive oxygen species (ROS); and 2) the cardioprotection afforded by iNOS deletion is eliminated by Nomega-nitro-L-arginine methyl ester (L-NAME) due to inhibition of endothelial NOS (eNOS) and neuronal NOS (nNOS). MI was induced by ligating the left anterior descending coronary artery. Male iNOS-/- mice and wild-type controls (WT, C57BL/6J) were divided into sham MI, MI+vehicle, and MI+l-NAME (100 mg.kg(-1).day(-1) in drinking water for 8 wk). Cardiac function was evaluated by echocardiography. Left ventricular (LV) maximum rate of rise of ventricular pressure divided by pressure at the moment such maximum occurs (dP/dt/instant pressure) in response to isoproterenol (100 ng.kg(-1).min(-1) iv) was measured with a Millar catheter. Collagen deposition, myocyte cross-sectional area, and expression of nitrotyrosine and 4-hydroxy-2-nonenal (4-HNE), markers for ROS, were determined by histopathological and immunohistochemical staining. We found that the MI-induced increase in LV chamber dimension and the decrease in ejection fraction, an index of systolic function, were less severe in iNOS-/- compared with WT mice. L-NAME worsened LV remodeling and dysfunction further, and these detrimental effects were also attenuated in iNOS-/- mice, associated with better preservation of cardiac function. Lack of iNOS also reduced nitrotyrosine and 4-HNE expression after MI, indicating reduced oxidative stress. We conclude that iNOS does not seem to be a pathological mediator of heart failure; however, the lack of iNOS improves cardiac reserve post-MI, particularly when constitutive NOS isoforms are blocked. Decreased oxidative stress and other adaptive mechanisms independent of NOS may be partially responsible for such an effect, which needs to be studied further.     

Effect of acute high-intensity interval exercise on postexercise biventricular function in mild heart failure

We studied the acute effect of high-intensity interval exercise on biventricular function using cardiac magnetic resonance imaging in nine patients [age: 49 ± 16 yr; left ventricular (LV) ejection fraction (EF): 35.8 ± 7.2%] with nonischemic mild heart failure (HF). We hypothesized that a significant impairment in the immediate postexercise end-systolic volume (ESV) and end-diastolic volume (EDV) would contribute to a reduction in EF. We found that immediately following acute high-intensity interval exercise, LV ESV decreased by 6% and LV systolic annular velocity increased by 21% (both P < 0.05). Thirty minutes following exercise (+30 min), there was an absolute increase in LV EF of 2.4% (P < 0.05). Measures of preload, left atrial volume and LV EDV, were reduced immediately following exercise. Similar responses were observed for right ventricular volumes. Early filling velocity, filling rate, and diastolic annular velocity remained unchanged, while LV untwisting rate increased 24% immediately following exercise (P < 0.05) and remained 18% above baseline at +30 min (P < 0.05). The major novel findings of this investigation are 1) that acute high-intensity interval exercise decreases the immediate postexercise LV ESV and increases LV EF at +30 min in patients with mild HF, and this is associated with a reduction in LV afterload and maintenance of contractility, and 2) that despite a reduction in left atrial volume and LV EDV immediately postexercise, diastolic function is preserved and may be modulated by enhanced LV peak untwisting rate. Acute high-intensity interval exercise does not impair postexercise biventricular function in patients with nonischemic mild HF.     

NO modulation of myocardial performance in fish hearts

In the mammalian heart, intracardiac nitric oxide (NO) regulates in an autocrine-paracrine manner cardiac function in the beat-to-beat response (Starling's law of the heart), short-term response (phasic control, e.g. excitation-contraction coupling, responses to neurotransmitters and endocrines) and long-term response (tonic control by altering gene expression). This trio of NO temporal-dependent actions has a long evolutionary history, as we have documented in the prototypic vertebrate heart, the teleost heart. This heart shares a common structural and functional scenario with higher vertebrate hearts exhibiting, at the same time, differences in myoarchitecture (trabecular vs. compact type), blood supply (lacunary vs. vascular) and pumping performance (sensitivity to filling pressure), thus providing challenging opportunities for revealing aspects of unity and diversity of cardiac NO in vertebrates. Using in vitro working teleost heart preparations we have shown that, under basal conditions, NO through a cGMP-mediated mechanism modulates ventricular performance (negative inotropism) and remarkably increases the sensitivity to filling pressure (i.e. the Frank-Starling response). NO-cGMP mechanism also influences the short-term response elicited by inotropic agents such as acetylcholine and angiotensin II. A role of NO in long-term cardiac adaptation is illustrated by morphologic evidence (e.g. NOS immuno-localization in phylogenetically distant species) which emphasizes the importance of NO in reshaping the angio-myoarchitecture of the fish heart ventricle (i.e. compensation for regional heterogeneity). Finally, by studying the avascular hearts of teleosts and amphibians that lack vascular endothelium, a relevant role of endocardial endothelium-NO signalling in intracavitary regulation of myocardial performance has been firmly established, thus revealing its early evolutionary role in non-mammalian vertebrates.     

Interactions between angiotensin II and nitric oxide during exercise in normal and heart failure rats.

We hypothesized that nitric oxide (NO) opposes ANG II-induced increases in arterial pressure and reductions in renal, splanchnic, and skeletal muscle vascular conductance during dynamic exercise in normal and heart failure rats. Regional blood flow and vascular conductance were measured during treadmill running before (unblocked exercise) and after 1) ANG II AT(1)-receptor blockade (losartan, 20 mg/kg ia), 2) NO synthase (NOS) inhibition [N(G)-nitro-L-arginine methyl ester (L-NAME); 10 mg/kg ia], or 3) ANG II AT(1)-receptor blockade + NOS inhibition (combined blockade). Renal conductance during unblocked exercise (4.79 +/- 0.31 ml x 100 g(-1) x min(-1) x mmHg(-1)) was increased after ANG II AT(1)-receptor blockade (6.53 +/- 0.51 ml x 100 g(-1) x min(-1) x mmHg(-1)) and decreased by NOS inhibition (2.12 +/- 0.20 ml x 100 g(-1) x min(-1) x mmHg(-1)) and combined inhibition (3.96 +/- 0.57 ml x 100 g(-1) x min(-1) x mmHg(-1); all P < 0.05 vs. unblocked). In heart failure rats, renal conductance during unblocked exercise (5.50 +/- 0.66 ml x 100 g(-1) x min(-1) x mmHg(-1)) was increased by ANG II AT(1)-receptor blockade (8.48 +/- 0.83 ml x 100 g(-1) x min(-1) x mmHg(-1)) and decreased by NOS inhibition (2.68 +/- 0.22 ml x 100 g(-1) x min(-1) x mmHg(-1); both P < 0.05 vs. unblocked), but it was unaltered during combined inhibition (4.65 +/- 0.51 ml x 100 g(-1) x min(-1) x mmHg(-1)). Because our findings during combined blockade could be predicted from the independent actions of NO and ANG II, no interaction was apparent between these two substances in control or heart failure animals. In skeletal muscle, L-NAME-induced reductions in conductance, compared with unblocked exercise (P < 0.05), were abolished during combined inhibition in heart failure but not in control rats. These observations suggest that ANG II causes vasoconstriction in skeletal muscle that is masked by NO-evoked dilation in animals with heart failure. Because reductions in vascular conductance between unblocked exercise and combined inhibition were less than would be predicted from the independent actions of NO and ANG II, an interaction exists between these two substances in heart failure rats. L-NAME-induced increases in arterial pressure during treadmill running were attenuated (P < 0.05) similarly in both groups by combined inhibition. These findings indicate that NO opposes ANG II-induced increases in arterial pressure and in renal and skeletal muscle resistance during dynamic exercise.     

Cardiac function of the leopard shark,Triakis semifasciata

Summary The pressure difference between the cardinal sinus and the pericardium, and the transmural ventricular diastolic pressure at rest and during swimming in the leopard shark, Triakis semifasciata, was measured to characterize the mechanism of cardiac filling in chronically-instrumented fish and to evaluate cardiac responses to swimming. Echo-Doppler and radiographic imaging were also used to fully describe the cardiac cycle. Swimming induces an increase in preload as manifested by a large increment of cardinal sinus pressure (0.26/0.20 [systolic/diastolic] to 0.49/0.32 kPa) which always exceeds pericardial pressure. Increases in both mean ventricular diastolic transmural pressure (0.30–0.77 kPa) and cardinal sinus pressure during swimming suggest increased cardiac filling by vis a tergo as the mechanism for augmenting cardiac output. In contrast to mammals, the fluid-filled pericardial space of elasmobranchs is considerably larger and the pericardium itself does not move in concert with the heart throughout the cardiac cycle. Also, modest increases in heart rate drastically curtail the duration of diastole, which becomes much less than that of systole, a phenomenon not found in mammals. In the absence of tachycardia (<40 bpm), ventricular filling is characterized by a period of early rapid filling, and a late period of filling owing to atrial systole, separated by a period of diastasis. Ventricular filling in elasmobranchs is thus biphasic and is not solely dependent on atrial systole. Atrial diastole is characterized by three filling periods associated with atrial relaxation, ventricular ejection, and sinus venosus contraction. The estimated ventricular ejection fraction of Triakis (80%) exceeds that of the mammalian left ventricle.     

Localization and characterization of nitric oxide synthase at the frog neuromuscular junction

Summary. The frog neuromuscular junction is sensitive to nitric oxide (NO), since exogenously applied NO reduces the release of transmitter by presynaptic terminals and the size of ATP-induced Ca²⁺ responses in perisynaptic Schwann cells. This study aimed at determining whether an NO synthase (NOS) is present at the neuromuscular junction, notably in perisynaptic Schwann cells, the glial cells at this synapse. The NADPH-diaphorase (NADPH-d) histochemical technique revealed the presence of NOS in cell bodies and presumed processes of perisynaptic Schwann cells. Incubation with NOS inhibitors, N^G-nitro-L-arginine methyl ester or N^G-monomethyl-L-arginine-acetate, abolished the NADPH-d staining. Moreover, L-arginine, the precursor of NO, impeded the blockade by NOS inhibitors, establishing the NOS specificity of NADPH-d staining in frog tissue. The pattern of labelling with a polyclonal antibody against the neuronal form of NOS was similar to the NADPH-d staining, also suggesting the presence of a neuronal NOS in perisynaptic Schwann cells. Using electron microscopy, the NOS immunostaining was found at the membrane and occasionally in the cytoplasm of perisynaptic Schwann cells and was not detected in the nerve terminal or muscle. There was no enzymatic or immunocytochemical labelling of NOS 6 days after denervation. It is concluded that NOS is present in frog perisynaptic Schwann cells. The presence of this endogenous NOS suggests that NO may act as a diffusible glial messenger to modulate synaptic activity and synapse formation at the neuromuscular junction.     

Angiotensin-(1-7) increases neuronal potassium current via a nitric oxide-dependent mechanism

Actions of angiotensin-(1-7) [Ang-(1-7)], a heptapeptide of the renin-angiotensin system, in the periphery are mediated, at least in part, by activation of nitric oxide (NO) synthase (NOS) and generation NO(·). Studies of the central nervous system have shown that NO(·) acts as a sympathoinhibitory molecule and thus may play a protective role in neurocardiovascular diseases associated with sympathoexcitation, such as hypertension and heart failure. However, the contribution of NO in the intraneuronal signaling pathway of Ang-(1-7) and the subsequent modulation of neuronal activity remains unclear. Here, we tested the hypothesis that neuronal NOS (nNOS)-derived NO(·) mediates changes in neuronal activity following Ang-(1-7) stimulation. For these studies, we used differentiated catecholaminergic (CATH.a) neurons, which we show express the Ang-(1-7) receptor (Mas R) and nNOS. Stimulation of CATH.a neurons with Ang-(1-7) (100 nM) increased intracellular NO levels, as measured by 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM) fluorescence and confocal microscopy. This response was significantly attenuated in neurons pretreated with the Mas R antagonist (A-779), a nonspecific NOS inhibitor (nitro-L-arginine methyl ester), or an nNOS inhibitor (S-methyl-L-thiocitrulline, SMTC), but not by endothelial NOS (eNOS) or inhibitory NOS (iNOS) inhibition {L-N-5-(1-iminoethyl)ornithine (L-NIO) and 1400W, respectively}. To examine the effect of Ang-(1-7)-NO(·) signaling on neuronal activity, we recorded voltage-gated outward K(+) current (I(Kv)) in CATH.a neurons using the whole cell configuration of the patch-clamp technique. Ang-(1-7) significantly increased I(Kv), and this response was inhibited by A-779 or S-methyl-L-thiocitrulline, but not L-NIO or 1400W. These findings indicate that Ang-(1-7) is capable of increasing nNOS-derived NO(·) levels, which in turn, activates hyperpolarizing I(Kv) in catecholaminergic neurons.     

Control of cardiovascular function in the icefish Chionodraco hamatus: involvement of serotonin and nitric oxide

The involvement of nitric oxide (NO) in the branchial circulation and cardiac performance of the Antarctic hemoglobinless icefish Chionodraco hamatus was investigated using isolated and perfused head and working heart preparations. In the branchial vasculature under basal (i.e. unstimulated conditions), the nitric oxide synthase (NOS) inhibitor L-NIO (L-N(5)-(1-iminoethyl) ornithine, 10(-5) and 10(-4) M), caused a marked vasoconstriction (20%), indicating a basal nitrergic vasodilator tone, while the dose-response curve of the NO donor SIN-1 (3-morpholinosydnonimine) showed a dose-dependent vasodilator effect. Acetylcholine induced a dose-dependent branchial vasoconstriction mediated by muscarinic receptors, since the effects were abolished by pre-treatment with atropine (10(-4) M). Serotonin (5-HT) induced a dose-dependent branchial methysergide-sensitive vasoconstriction which was abolished by pre-treatment with L-NIO, indicating a NO-dependent mechanism. On the isolated heart, the NOS inhibitor L-NMMA (N(G)-monomethyl-L-arginine) 10(-4) M produced a small, but significant decrease of heart rate and, as a consequence, a decrease of power output, while the NO donor sodium nitroprusside (SNP) 10(-4) M elicited increases of stroke volume, stroke work and power output, suggesting an exogenous NO-dependent positive inotropism. Exposure of the bulbus arteriosus to L-NMMA was without any appreciable effect. In contrast, SNP produced a notable relaxation of the bulbus wall with a marked increase of its stiffness, as indicated by the increase of systolic and diastolic dP/dt max (23 and 104%, respectively).     

Sepiapterin enhances angiogenesis and functional recovery in mice after myocardial infarction

Uncoupling of nitric oxide synthase (NOS) has been implicated in left ventricular (LV) remodeling and dysfunction after myocardial infarction (MI). We hypothesized that inducible NOS (iNOS) plays a crucial role in LV remodeling after MI, depending on its coupling status. MI was created in wild-type, iNOS-knockout (iNOS(-/-)), endothelial NOS-knockout (eNOS(-/-)), and neuronal NOS-knockout (nNOS(-/-)) mice. iNOS and nNOS expressions were increased after MI associated with an increase in nitrotyrosine formation. The area of myocardial fibrosis and LV end-diastolic volume and ejection fraction were more deteriorated in eNOS(-/-) mice compared with other genotypes of mice 4 wk after MI. The expression of GTP cyclohydrolase was reduced, and tetrahydrobiopterin (BH(4)) was depleted in the heart after MI. Oral administration of sepiapterin after MI increased dihydrobiopterin (BH(2)), BH(4), and BH(4)-to-BH(2) ratio in the infarcted but not sham-operated heart. The increase in BH(4)-to-BH(2) ratio was associated with inhibition of nitrotyrosine formation and an increase in nitrite plus nitrate. However, this inhibition of NOS uncoupling was blunted in iNOS(-/-) mice. Sepiapterin increased capillary density and prevented LV remodeling and dysfunction after MI in wild-type, eNOS(-/-), and nNOS(-/-) but not iNOS(-/-) mice. N(ω)-nitro-L-arginine methyl ester abrogated sepiapterin-induced increase in nitrite plus nitrate and angiogenesis and blocked the beneficial effects of sepiapterin on LV remodeling and function. These results suggest that sepiapterin enhances angiogenesis and functional recovery after MI by activating the salvage pathway for BH(4) synthesis and increasing bioavailable nitric oxide predominantly derived from iNOS.     

Cardiac performance in Turner's syndrome patients on growth hormone therapy.

OBJECTIVES: To investigate possible cardiac morphofunctional alterations observed in 26 Turner's syndrome (TS) patients on prolonged high-dose growth hormone (GH) therapy. STUDY DESIGN: We examined 26 TS subjects treated with rhGH (1 U/kg/week) for a mean period of 4.9 years (range 1-7.8) and 37 age-, weight- and height-matched healthy girls. Left ventricular volume, mass, systolic function, cardiac index, systemic vascular resistance and diastolic function were evaluated by two-dimensional and Doppler echocardiography. RESULTS: Heart rate and systolic blood pressure (BP) were higher in TS patients than in controls, while diastolic BP was lower. Left ventricular volumes, ejection fraction, mass index, M/V ratio and cardiac index did not differ significantly; systemic vascular resistance was slightly decreased. Left ventricular fractional shortening and mean velocity of circumferential shortening were slightly increased while end-systolic meridional stress was decreased in TS. Contractile state was normal in TS. Diastolic function assessment showed a shortening of isovolumetric relaxation and diastolic filling times with an increased atrial contribution and a normal pulmonary venous flow. CONCLUSION: Cardiac morphology in TS patients on GH therapy is similar to controls. The observed changes in left ventricular systolic and diastolic function should be interpreted as an adaptation to the higher heart rate and reduced peripheral vascular resistance induced by GH therapy.