Endothelial NOS and the blockade of LTP by NOS inhibitors in mice lacking neuronal NOS.

Long-term potentiation (LTP) is a persistent increase in synaptic strength implicated in certain forms of learning and memory. In the CA1 region of the hippocampus, LTP is thought to involve the release of one or more retrograde messengers from the postsynaptic cell that act on the presynaptic terminal to enhance transmitter release. One candidate retrograde messenger is the membrane-permeant gas nitric oxide (NO), which in the brain is released after activation of the neuronal-specific NO synthase isoform (nNOS). To assess the importance of NO in hippocampal synaptic plasticity, LTP was examined in mice where the gene encoding nNOS was disrupted by gene targeting. In nNOS- mice, LTP induced by weak intensity tetanic stimulation was normal except for a slight reduction in comparison to that in wild-type mice and was blocked by NOS inhibitors, just as it was in wild-type mice. Immunocytochemical studies indicate that in the nNOS- mice as in wild-type mice, the endothelial form of NOS (eNOS) is expressed in CA1 neurons. These findings suggest that eNOS, rather than nNOS, generates NO within the postsynaptic cell during LTP.

Endothelial, platelet and leukocyte interactions in ischemic heart disease: insights into potential mechanisms and their clinical relevance.

Recent investigations of the complex interactions among vascular endothelium, platelets and leukocytes have relevance to the pathogenesis of atherosclerosis and ischemic heart disease. Perturbations in the hemodynamic equilibrium maintained by these cellular elements may lead to vasospasm, in vivo thrombosis and a reduction in blood flow. Recent advances in the understanding of these interactions in health and disease states are summarized. The effect of pharmacologic agents on these cell-cell interactions are discussed to provide the reader with a general understanding of the relevance of these interactions in cardiovascular disease.

Increased neutrophil elastase release in unstable angina pectoris and acute myocardial infarction.

Neutrophils, a source of proteolytic enzymes and oxygen free radicals, have been shown to participate in animal models of myocardial ischemic injury. To characterize neutrophil activation in human ischemic heart disease, a specific neutrophil elastase-derived fibrinopeptide in plasma was measured in 25 patients with stable angina pectoris, 29 patients with unstable angina pectoris, 17 patients with acute myocardial infarction and 22 control subjects. Mean plasma levels (+/- standard error) of a neutrophil elastase-derived fibrinopeptide (B beta 30-43) measured by a specific radioimmunoassay were fivefold higher in patients with acute myocardial infarction (877 +/- 337 pmol/liter, p less than 0.02) and 13-fold higher in patients with unstable angina (2,277 +/- 613 pmol/liter, p less than 0.006) as compared with control subjects (172 +/- 74 pmol/liter). Mean plasma levels of peptide B beta 30-43 in patients with stable angina (676 +/- 334 pmol/liter), although higher than in control subjects, were not significantly increased (p = 0.64). Total leukocyte counts were 11.0 +/- 0.6 x 10(6)/ml in those with acute myocardial infarction, 9.2 +/- 0.7 x 10(6)/ml in those with unstable angina, 7.1 +/- 0.3 x 10(6)/ml in those with stable angina and 7.7 +/- 0.4 x 10(6)/ml in control subjects. Although total leukocyte counts in patients with unstable angina pectoris and acute myocardial infarction were higher (p less than 0.01) than in patients with stable angina or in control subjects, elevations in peptide B beta 30-43 levels were independent of the differences in both leukocyte count and absolute neutrophil count as well as in history of smoking, hypertension, diabetes mellitus or treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Cyclic nucleotide dependent phosphorylation of neuronal nitric oxide synthase inhibits catalytic activity.

We have examined the regulation of neuronal nitric oxide synthase (NOS) by phosphorylation with cyclic-GMP (PKG) and cyclic-AMP-dependent (PKA) protein kinases. In vitro phosphorylation studies indicate that both PKG and PKA phosphorylate NOS on a single site. Phosphoamino-acid analysis and peptide mapping demonstrate that phosphorylation by either cyclic-nucleotide kinase occurs on a similar serine residue. Phosphorylation of purified NOS by either PKG or PKA diminishes catalytic activity. Stimulation by 8-Br-cGMP of HEK-293 cells stably transfected with the cDNA for neuronal NOS (293.NOS cells) results in phosphorylation of immunoprecipitated NOS. Incubation of 293-NOS cells with 8-bromo-cGMP or dibutyryl-cAMP reduces nitrite release in response to stimulation with calcium ionophore A23187. Phosphorylation-induced decreases in NOS activity may counterbalance and modulate NOS activating signals.

Dispersion of ventricular activation and refractoriness in patients with idiopathic dilated cardiomyopathy.

We sought to evaluate the electrophysiologic substrate for ventricular arrhythmias and sudden cardiac death in patients with idiopathic dilated cardiomyopathy. When compared with controls, patients with cardiomyopathy had prolonged activation times, increased dispersion of activation and recovery, and prolonged duration of monophasic action potential recordings at 70%, but not at 90%, of repolarization.

Interactions between nitroglycerin and endothelium in vascular smooth muscle relaxation.

To evaluate the role of endothelium in nitroglycerin (NTG)-mediated vascular relaxation, epinephrine-contracted rat thoracic aortic segments with and without intact endothelium were exposed to NTG (10(-10) to 10(-5) M). Aortic segments with intact (endo+, n = 15) and denuded endothelium (endo-, n = 9) exhibited typical NTG-induced relaxation. However, the mean effective concentration of NTG was lower for endo- than for endo+ segments (P less than 0.001). To determine if this phenomenon related to nitric oxide (NO) generation by endothelium, six endo+ segments were treated with NG-monomethyl-L-arginine (L-NMMA), an inhibitor of NO production. These endo+ segments exhibited greater (P less than 0.001) relaxation in response to NTG than the untreated endo+ segments. Oxyhemoglobin, an inhibitor of guanylate cyclase activation, greatly diminished NTG-mediated relaxation of all aortic segments. To determine if the enhanced NTG-mediated relaxation of endo- segments was unique to the guanosine 3',5'-cyclic monophosphate-dependent vasodilator NTG, other endo+ and endo- segments were exposed to adenosine 3',5'-cyclic monophosphate-dependent vasodilator papaverine (10(-8) to 10(-4) M), and no difference in EC50 was noted between endo+ and endo- segments. Thus endothelium attenuates NTG-mediated vasorelaxation, and this attenuation is abolished by inhibition of endothelial NO production with L-NMMA. These observations indicate that endothelium is a dynamic modulator of vascular smooth muscle relaxant effects of NTG. This modulation appears to result from a competitive interaction between endothelial NO and NTG.

Temperature monitoring during radiofrequency ablation.

Thermal injury is the primary mechanism of lesion formation during radiofrequency catheter ablation procedures. Irreversible tissue injury requires heating to approximately 50 degrees C. Temperatures above 100 degrees C result in coagulum formation. Because of this importance of temperature during radiofrequency catheter ablation procedures, temperature monitoring has been proposed as a tool to facilitate catheter ablation procedures. The results of recent clinical studies demonstrate that electrode temperatures do not differ at successful and failed ablation sites, electrode temperature does not predict or eliminate the possibility of arrhythmia recurrence, and closed-loop temperature control decreases but does not eliminate the development of coagulum nor guarantees that target temperatures will be achieved. These observations are due in large part to the important distinctions between electrode temperature, the temperature at the electrode-tissue interface, and the temperature at the ablation target. Nonetheless, temperature monitoring and temperature control are valuable tools during radiofrequency ablation procedures as they provide important information regarding the adequacy of tissue heating, minimize the development of coagulum, and maximize lesion size.

Nitric oxide: a physiologic messenger.

PURPOSE: To review the physiologic role of nitric oxide, an unusual messenger molecule that mediates blood vessel relaxation, neurotransmission, and pathogen suppression. DATA SOURCES: A MEDLINE search of articles published from 1987 to 1993 that addressed nitric oxide and the enzyme that synthesizes it, nitric oxide synthase. STUDY SELECTION: Animal and human studies were selected from 3044 articles to analyze the clinical importance of nitric oxide. Descriptions of the structure and function of nitric oxide synthase were selected to show how nitric oxide acts as a biological messenger molecule. DATA EXTRACTION: Biochemical and physiologic studies were analyzed if the same results were found by three or more independent observers. DATA SYNTHESIS: Two major classes of nitric oxide synthase enzymes produce nitric oxide. The constitutive isoforms found in endothelial cells and neurons release small amounts of nitric oxide for brief periods to signal adjacent cells, whereas the inducible isoform found in macrophages releases large amounts of nitric oxide continuously to eliminate bacteria and parasites. By diffusing into adjacent cells and binding to enzymes that contain iron, nitric oxide plays many important physiologic roles. It regulates blood pressure, transmits signals between neurons, and suppresses pathogens. Excess amounts, however, can damage host cells, causing neurotoxicity during strokes and causing the hypotension associated with sepsis. CONCLUSIONS: Nitric oxide is a simple molecule with many physiologic roles in the cardiovascular, neurologic, and immune systems. Although the general principles of nitric oxide synthesis are known, further research is necessary to determine what role it plays in causing disease.

Carbon monoxide: a role in carotid body chemoreception.

Carbon monoxide (CO), produced endogenously by heme oxygenase, has been implicated as a neuronal messenger. Carotid bodies are sensory organs that regulate ventilation by responding to alterations of blood oxygen, CO2, and pH. Changes in blood gases are sensed by glomus cells in the carotid body that synapse on afferent terminals of the carotid sinus nerve that projects to respiratory-related neurons in the brainstem. Using immunocytochemistry, we demonstrate that heme oxygenase 2 is localized to glomus cells in the cat and rat carotid bodies. Physiological studies show that zinc protoporphyrin IX, a potent heme oxygenase inhibitor, markedly increases carotid body sensory activity, while copper protoporphyrin IX, which does not inhibit the enzyme, is inactive. Exogenous CO reverses the stimulatory effects of zinc protoporphyrin IX. These results suggest that glomus cells are capable of synthesizing CO and endogenous CO appears to be a physiologic regulator of carotid body sensory activity.

Endothelial nitric oxide synthase localized to hippocampal pyramidal cells: implications for synaptic plasticity.

Using antibodies that react selectively with peptide sequences unique to endothelial nitric oxide synthase (eNOS), we demonstrate localizations to neuronal populations in the brain. In some brain regions, such as the cerebellum and olfactory bulb, eNOS and neuronal NOS (nNOS) occur in the same cell populations, though in differing proportions. In the hippocampus, localizations of the two enzymes are strikingly different, with eNOS more concentrated in hippocampal pyramidal cells than in any other brain area, whereas nNOS is restricted to occasional interneurons. In many brain regions NADPH diaphorase staining reflects NOS catalytic activity. Hippocampal pyramidal cells do not stain for diaphorase with conventional paraformaldehyde fixation but stain robustly with glutaraldehyde fixatives, presumably reflecting eNOS catalytic activity. eNOS in hippocampal pyramidal cells may generate the NO that has been postulated as a retrograde messenger of long-term potentiation.

Regulation of endothelial nitric-oxide synthase during hypoxia.

The mechanism by which nitric-oxide (NO) production increases during hypoxia is unknown. To explore the effect of hypoxia upon endothelial nitric-oxide synthase (ecNOS) activity and expression, we exposed bovine aortic endothelial cells (BAEC) to hypoxia (1% O2) for 0-24 h and measured levels of ecNOS mRNA, protein, and activity. The amount of ecNOS mRNA increases to more than twice the basal level after 6 h of hypoxia. Incubation of BAEC with actinomycin D during hypoxia prevents this increase, demonstrating that higher levels of mRNA observed during hypoxia are due to increased synthesis, not to increased stability of ecNOS mRNA. Levels of ecNOS protein increase throughout 24 h of hypoxia to more than twice normoxic levels. Although ecNOS expression increases within 2 h of hypoxia, total activity remains unchanged. To explore the transcriptional regulation of ecNOS, we constructed a reporter plasmid containing the ecNOS promoter region upstream of the luc gene and transfected this reporter plasmid into BAEC. In this system, hypoxia induces a linear increase over time in the expression of luciferase driven by the ecNOS promoter. It is concluded that hypoxia induces an increase in transcription of ecNOS in endothelial cells, activating the regulatory region of ecNOS by undefined transcription factors.

Heme oxygenase 2: endothelial and neuronal localization and role in endothelium-dependent relaxation.

Heme oxygenase 2 (HO-2), which synthesizes carbon monoxide (CO), has been localized by immunohistochemistry to endothelial cells and adventitial nerves of blood vessels. HO-2 is also localized to neurons in autonomic ganglia, including the petrosal, superior cervical, and nodose ganglia, as well as ganglia in the myenteric plexus of the intestine. Enzyme studies demonstrated that tin protoporphyrin-9 is a selective inhibitor of HO with approximately 10-fold selectivity for HO over endothelial nitric oxide synthase (NOS) and soluble guanylyl cyclase. Inhibition of HO activity by tin protoporphyrin 9 reverses the component of endothelial-derived relaxation of porcine distal pulmonary arteries not reversed by an inhibitor of NOS. Thus, CO, like NO, may have endothelial-derived relaxing activity. The similarity of NOS and HO-2 localizations and functions in blood vessels and the autonomic nervous system implies complementary and possibly coordinated physiologic roles for these two mediators.

Immunosuppressant FK506 enhances phosphorylation of nitric oxide synthase and protects against glutamate neurotoxicity.

Immunosuppressants FK506 and cyclosporin A inhibit neurotoxicity of N-methyl-D-aspartate in primary cortical cultures, while having no effect on quisqualate- and kainate-mediated neurotoxicity. Rapamycin completely reverses the neuroprotective effect of FK506. Both FK506 and cyclosporin A inhibit NMDA-elicited/nitric oxide-mediated increases in cGMP levels in cortical cultures. FK506 has no effect on sodium nitroprusside-induced increases in cGMP. In a stably transfected human kidney 293 cell line overexpressing the gene encoding nitric oxide synthase [L-arginine, NADPH:oxygen oxidoreductase (nitric oxide-forming), EC 1.14.13.39], FK506 inhibits the calcium ionophore A23187, stimulated increases in nitrite (a breakdown product of nitric oxide), and potentiates phorbol ester-mediated inhibition of nitrite formation. FK506-mediated inhibition of nitric oxide formation is completely reversed by rapamycin. Calcineurin dephosphorylates protein kinase C-mediated phosphorylation of nitric oxide synthase. FK506 prevents the calcineurin-mediated dephosphorylation of nitric oxide synthase and thereby diminishes the enzyme's catalytic activity. These data establish nitric oxide synthase as a calcineurin substrate. Nitric oxide synthase catalytic activity is regulated by the phosphorylation state of the enzyme. Enhanced phosphorylation of nitric oxide synthase diminishes catalytic activity, and dephosphorylation (through activation of calcineurin) enhances catalytic activity. The neuroprotective effect of FK506 and cyclosporin A presumably involves the inhibition of calcineurin, preventing the dephosphorylation of nitric oxide synthase and its subsequent activation.

Analysis of the pattern of initiation of sustained ventricular arrhythmias in patients with implantable defibrillators.

INTRODUCTION: The purpose of this study was to analyze the pattern of initiation of sustained ventricular arrhythmias in patients with varying types of underlying structural heart disease. METHODS AND RESULTS: The study group consisted of 90 patients with an implantable cardioverter defibrillator. Cardiovascular diagnoses included coronary artery disease in 64 patients (71%). The patients were divided into four groups based on the type and severity of structural heart disease. Two hundred sixty episodes of sustained ventricular arrhythmias were analyzed. The mean coupling interval of the initiating beat of all ventricular arrhythmias was 523 +/- 171 msec. The coupling interval of the initiating beat was longer in patients with impaired ventricular function, particularly those with nonischemic dilated cardiomyopathy. The prematurity index was similar regardless of the type of underlying structural heart disease. However, the prematurity index was shorter in patients with polymorphic ventricular tachycardia (VT) compared to those with monomorphic VT. A pause was observed more commonly before the onset of polymorphic VT/ventricular fibrillation than sustained monomorphic VT. Two hundred twenty-two (85%) of the arrhythmia episodes were initiated by a late-coupled premature beat, 33 (13%) were initiated by an early-coupled premature beat, and 5 episodes (2%) were initiated with a short-long-short sequence. The pattern of initiation of the ventricular arrhythmias was similar in all patient groups and for both monomorphic and polymorphic tachycardias. CONCLUSION: These findings demonstrate that sustained ventricular arrhythmias typically are initiated by late-coupled ventricular premature depolarizations, regardless of the type or severity of underlying structural heart disease or resultant arrhythmia.

Acute radiation dermatitis following radiofrequency catheter ablation of atrioventricular nodal reentrant tachycardia.

Radiation exposure during fluoroscopic imaging poses potential risks to patients and physicians, especially during protracted cardiovascular or radiological interventional procedures. We describe a woman with refractory paroxysmal supraventricular tachycardia who underwent radiofrequency catheter ablation of the slow pathway involved in atrioventricular nodal reentrant tachycardia. The patient subsequently returned 4 weeks later with acute radiation dermatitis that was retrospectively attributed to a malfunction in the fluoroscopy unit that lacked a maximum current output cut-off switch. Using dose reconstruction studies and her estimated biological response, we determined that she received between 15 and 20 Gy (1 Gy = 100 rads) to the skin on her back during the procedure. The exposure will result in an increase in her lifelong risk of skin and lung cancer. This article underscores the potential for radiation-induced injury during lengthy therapeutic procedures using x-ray equipment.