beta -Adrenergic regulation of constitutive nitric oxide synthase in cardiac myocytes

Anthony J. Kanai1, Stephan Mesaros2, Mitchell S. Finkel3, Carmine V. Oddis3, Lori A. Birder4, and Tadeusz Malinski4

1 Department of Biomedical Engineering, Duke University, Durham, North Carolina 27708; 3 Division of Cardiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213; 4 Department of Physiology, University of North Carolina-Chapel Hill School of Medicine, Chapel Hill, North Carolina 27599; and 2 Department of Chemistry, Oakland University, Rochester, Michigan 48309

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

Nitric oxide (NO) has been implicated in endogenous control of myocardial contractility. However, NO release has not yet been demonstrated in cardiac myocytes. Accordingly, endogenous NO production was measured with a porphyrinic microsensor positioned on the surface of individual neonatal or adult rat ventricular myocytes (n > 6 neonatal and adult cells per experiment). In beating neonatal myocytes, there was no detectable spontaneous NO release with each contraction. However, norepinephrine (NE; 0.25-1 µM) elicited transient NO release from beating neonatal (149 ± 11 to 767 ± 83 nM NO) and noncontracting adult (157 ± 13 to 791 ± 89 nM NO) cells. NO was released by adrenergic agonists with the following rank order of potency: isoproterenol (beta 1beta 2) > NE (alpha /beta 1) > dobutamine (beta 1approx  epinephrine (alpha /beta 1beta 2) > tertbutylene (beta 2); NO was not released by phenylephrine (alpha ). NE-evoked NO release was reversibly blocked by NG-monomethyl-L-arginine, trifluoperazine, guanosine 5'-O-(2-thiodiphosphate), and nifedipine but was enhanced by 3-isobutyl-1-methylxanthine (0.5 mM = 14.5 ± 1.6%) and BAY K 8644 (10 µM = 11.9 ± 1%). NO was also released by A-23187 (10 µM = 884 ± 88 nM NO), guanosine 5'-O-(3-thiotriphosphate) (1 µM = 334 ± 56 nM NO), and dibutyryl adenosine 3',5'-cyclic monophosphate (10-100 µM = 35 ± 9 to 284 ± 49 nM NO) but not by ATP, bradykinin, carbachol, 8-bromoguanosine 3',5'-cyclic monophosphate, or shear stress. This first functional demonstration of a constitutive NO synthase in cardiac myocytes suggests its regulation by a beta -adrenergic signaling pathway and may provide a novel mechanism for the coronary artery vasodilatation and enhanced diastolic relaxation observed with adrenergic stimulation.

norepinephrine; beta -adrenergic agonists; nitric oxide-selective porphyrinic microsensor

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

THE PRESENCE OF a constitutive nitric oxide synthase (cNOS) in cardiac myocytes was first suggested by two studies investigating Ca2+-independent inducible NOS (iNOS) activity. One study noted negative inotropic effects within minutes of the application of inflammatory cytokines to isolated papillary muscles, before iNOS upregulation (7). The other study observed Ca2+-dependent conversion of L-[14C]arginine to L-[14C]citrulline in the cytosolic homogenates of myocytes (25). However, several contradictions arise when considering the presence of a cardiac cNOS. Because cholinergic stimulation (acetylcholine and carbachol) decreases myocardial contractility and nitric oxide (NO) has similarly been reported to decrease contractility (4, 7), one might predict that cholinergic stimulation would evoke NO release. On the other hand, adrenergic stimulation [norepinephrine (NE) and epinephrine] increases myocardial contractility by elevating cytoplasmic Ca2+ levels, which should also stimulate NO release. In addition, since cardiac myocytes undergo cyclic elevations in cytoplasmic Ca2+ as a prerequisite to contraction, beat-to-beat activation of a Ca2+-dependent cNOS might also be predicted, but beat-to-beat NO release might also inhibit adrenergically evoked increases in myocardial contractility.

Several studies, not directly measuring NO, have reported that cardiac myocytes produce NO in response to acetylcholine (1, 3, 9). In one study, this conclusion was based on experiments in open-chest dogs, in which the vagus nerve innervating the heart was stimulated to release acetylcholine (9). Because decreases in contractility due to vagal stimulation could be opposed by systemic administration of NG-monomethyl-L-arginine (L-NMMA), a competitive inhibitor of NO production, it was interpreted that acetylcholine was evoking, and L-NMMA inhibiting, NO production by cardiac myocytes. However, it is equally plausible that the observed effects were due to acetylcholine-evoking and L-NMMA-inhibiting NO release from vascular endothelial cells in the heart. Acetylcholine-evoked NO release from vascular endothelium is well established. In another study, from isolated rat ventricular myocytes, L-NMMA was reported to prevent cholinergic agonists from opposing adrenergically mediated increases in voltage-dependent Ca2+ currents (3). However, similar studies in frog ventricular myocytes reported no effects of L-NMMA on cholinergic modulation of voltage-dependent Ca2+ currents (16). Finally, transgenic mice that overexpress beta 2-adrenergic receptors have been shown to exhibit enhanced myocardial relaxation (24), similar to the observed effects of NO on cardiac function (22). This raises the possibility that cardiac myocytes may produce NO in response to adrenergic agonists.

To test the hypothesis that cardiac myocytes contain a cNOS and to characterize its regulatory pathway, we used a highly selective porphyrinic microsensor to directly measure endogenous NO production by individual myocytes exposed to a variety of putative agonists, antagonists, and second messenger intermediates of NO release.

    METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References

NO electrode preparation. Nafion-coated porphyrinic microsensors (tip diameter, 5-15 µm; NO detection limit, 5 nM; response time, 1 ms) were prepared, with modifications, as previously described (12). Carbon strands (1-5 fibers, 5 µm diameter each; Amoco, Greenville, SC) were inserted into glass capillary tubes (1 mm internal diameter) pulled to a 100-µm opening at one end. Copper wire was connected to the carbon fiber, with electrically conductive epoxy at the blunt end, while the strand protruded at the pulled end. The opening at the pulled end was sealed with molten wax, and the protruding fiber was cut to the desired length (2 mm). The 2-mm-long carbon fiber was coated with tetrakis(3-methoxy-4-hydroxyphenyl)-nickel(II)porphyrin (TMHPPNi), synthesized according to a procedure previously described (15). Monomeric TMHPPNi was dissolved in 0.1 N NaOH and deposited, as a polymeric film, on the carbon fiber using a multiple-potential scanning cyclic voltameter (-0.2 to +1.0 V, model 283 Potentiostat/Galvanostat; EG&G Instruments, Princeton Applied Research, Princeton, NJ). Polymeric TMHPPNi catalyzes the oxidation of NO to NO+. After the porphyrin dried (2-3 h), the cation exchanger Nafion (Aldrich Chemical) was applied by dipping the electrode into a 1% solution made up in ethanol. The negative charges of the SO<SUP>−</SUP><SUB>3</SUB> functional group of Nafion keeps NO<SUP>−</SUP><SUB>2</SUB> (nitrite) and NO<SUP>−</SUP><SUB>3</SUB> (nitrate) anions from gaining access to the active porphyrin surface, preventing overestimation of the NO response. The Nafion-coated electrodes were characterized by differential pulse voltametry to determine the redox potential of the oxidation of NO to NO+. Chronoamperometry, performed at a constant potential 50 mV more positive than the redox potential, was used to determine the NO concentration. High purity (>99.99%) NO standards (12) were prepared daily to accurately calibrate the electrodes. The currents generated by the oxidation of NO to NO+ at the porphyrinic interface (0.5-1.5 nA/cm2 for 1 µM NO under static conditions) were amplified and converted to voltages (model 283 Potentiostat/Galvanostat) and then were digitized for real-time viewing on a monitor or stored on a computer hard disk for later retrieval and analysis.

Isolation of neonatal and adult cardiac ventricular myocytes. Neonatal myocytes were prepared from the ventricles of 1- to 2-day-old halothane (1%) anesthetized rats as we previously described (18). Noncontracting Ca2+-tolerant adult myocytes were prepared from Sprague-Dawley rats (250-300 g, either sex). After deep anesthesia with pentobarbital sodium (40 mg/kg ip), the thoracic cavity was opened and the heart was rapidly excised, cannulated at the aorta, and retrogradely perfused (37°C; 120 mmHg) in a modified Langendorff preparation. Hearts were first perfused for 5 min with a solution containing (in mM) 144 NaCl, 5.4 KCl, 0.4 NaH2PO4, 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, 1 MgCl2, and 1.8 CaCl2, pH 7.4, and then for 5 min with a Ca2+-free solution additionally containing (in mM) 0.1 ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 5 creatine, and 10 taurine, and, finally, for 15 min with a Ca2+-free solution without EGTA but additionally containing (in mg/ml) 1.5 collagenase, 0.5 protease, and 0.01 elastase. After perfusion, the atria and right ventricle were removed and the remaining left ventricle was minced and incubated, with stirring, in the collected enzyme mix additionally containing bovine serum albumin (10 mg/ml). Aliquots (10 ml) of the stirring enzyme mix containing suspended dissociated cells were centrifuged at 1,000 revolutions/min for 5 min. The supernatant was returned to the stirring enzyme mix, and the pellet was resuspended in medium 199 (Sigma Chemical). The cycle was repeated until the tissue was completely dissociated. The cells in medium 199 were kept at 25°C for immediate use.

Measurements of NO release from neonatal and adult cardiac ventricular myocytes. Neonatal myocytes were cultured on 10-mm-diameter collagen-coated glass coverslips; a suspension of adult myocytes was gently applied, dropwise, to collagen-coated glass coverslips and allowed to settle out and attach. The glass coverslip with cells was placed in a temperature-regulated bath (30 mm diameter, 2 ml vol, 37°C; Fig. 1). A glass disk forms the floor of the bath, which was mounted on the stage of an inverted microscope (Diaphot; Nikon). Perfusate [containing (in mM) 144 NaCl, 5.4 KCl, 1 MgCl2, 1.8 CaCl2, 10 glucose, and 10 phosphate; pH 7.4] entered the chamber from one end (1 ml/min) and drained out the other. Drugs were added to the bath along with the perfusate or were locally applied to a cell via a remote-controlled positive displacement nanoejector (Drummond Scientific, Broomall, PA). The nanoejector was mounted on a remote-controlled x-y-z micromanipulator with 1-µm resolution, which allows the glass tip of the ejector to be positioned upstream and proximal to the cell. Inert dye was added to the solution in the capillary tube of the ejector to visually guarantee that the drug was delivered on demand but did not otherwise diffuse out of the capillary tube. A three-electrode recording system was used, consisting of a working electrode (porphyrinic microsensor), saturated calomel reference electrode, and platinum counter electrode. The porphyrinic microsensor was mounted on a remote-controlled x-y-z ultramicromanipulator with 0.2-µm resolution, which allowed the tip of the microsensor to be positioned onto the surface of the cell.


View larger version (46K):
[in this window]
[in a new window]
 
Fig. 1.   Nitric oxide measurements were made in a temperature-regulated perfusion bath (30 mm diameter, 2 ml vol, 37°C). A glass disk forms the floor of the bath, which was mounted on the stage of an inverted microscope. Perfusate entered the chamber from one end (1 ml/min) and drained out the other. Drugs were added to the bath along with the perfusate or were locally applied to a cell via a remote-controlled nanoejector. The nanoejector was mounted on a micromanipulator that allows the glass tip of the ejector to be positioned upstream and proximal to a cell. A three-electrode recording system was used, consisting of a working electrode (porphyrinic microsensor), saturated calomel reference electrode, and platinum counter electrode. An ultramicromanipulator allowed the tip of the porphyrinic microsensor to be positioned directly on the surface of a cell.

Immunohistochemical staining for iNOS protein and semiquantitative reverse transcriptase-polymerase chain reaction analysis for iNOS mRNA. Immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR) were performed as we have previously described (18).

Statistical methods. Data are means ± SE for determinations in at least six different cells for microsensor recordings. Analysis of variance and the Student-Newman-Keuls test were used for multigroup comparisons. Values of P < 0.05 were considered statistically significant.

    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Endogenous NO release was measured with a porphyrinic microsensor placed directly on the surface of spontaneously beating neonatal or noncontracting adult rat ventricular myocytes. The presence of a functional Ca2+-dependent cNOS was definitively demonstrated, for the first time, with the Ca2+ ionophore A-23187 (Fig. 2A). NO release began 1-2 s after the application of ionophore and continued for 3-4 s. Transient release of NO was also elicited by the adrenergic agonist NE (Fig. 2B). NO release began 100-200 ms after the application of NE and lasted for 1-6 s, depending on the concentration of agonist applied. The participation of beta -adrenergic receptors in this response was indicated by the rank order of potency of selective adrenergic agonists: isoproterenol (beta 1beta 2) > NE (alpha /beta 1) > dobutamine (beta 1approx epinephrine (alpha /beta 1beta 2) > tertbutylene (beta 2); NO was not released by the alpha -agonist, phenylephrine (alpha ) (data not shown). NO release was blocked by the beta -adrenergic antagonists, propranolol (beta 1beta 2) > atenolol (beta 1) > butoxamine (beta 2) but not by the alpha -adrenergic antagonists, prazosin (alpha 1) or yohimbine (alpha 2) (data not shown).


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 2.   A: Ca2+ ionophore A-23187 elicited transient nitric oxide (NO) release from a spontaneously beating neonatal myocyte (873 ± 84 nM, n = 8 neonatal cells). Similar results were obtained from nonbeating adult myocytes (895 ± 91 nM, n = 6 adult cells, data not shown). Control, application of A-23187 (10 µM) under the NO electrode in absence of a cell; application of Ringer solution in presence of a cell also failed to elicit a response. B: norepinephrine (NE; 0.25-1 µM, left 4 arrows, same cell) also evoked transient NO release (149 ± 11 to 767 ± 83 nM, n = 8 neonatal cells; and 157 ± 13 to 791 ± 89 nM, n = 6 adult cells, not shown) from a beating neonatal myocyte. Control, application of NE (1 µM, right arrow) under the NO electrode without a cell present. C: NO response was solely from myocyte on which porphyrinic microsensor was placed, as demonstrated by raising microsensor in 5-µm increments and reapplying NE. Response was abolished at distance of 30 µm from surface of a nonbeating adult myocyte. All NO recordings in this report were made from individual neonatal or adult myocytes with a >= 30-µm cell-free radius surrounding them. D: results from C were graphically depicted by plotting peak NO concentrations as a function of electrode distance from cell. Similar results were obtained from beating neonatal myocytes (not shown).

Verification that a measured NO response was from an individual myocyte was accomplished by raising the tip of the microsensor from the cell surface and applying NE (Fig. 2C). As the distance from the cell surface increased, there was a gradual decrease in the measured NO concentration. NO was undetectable beyond a distance of 30 µm (Fig. 2D). Mathematical modeling of the diffusion (12, 27) and breakdown (13) of NO indicated that the decline in concentration with distance was primarily due to degradation. NE increased the beating rate of neonatal cells (70 ± 6 to 128 ± 11 beats/min at 37°C; n = 7) and induced noncontracting adult cells to contract (98 ± 18 beats/min at 37°C; n = 8) as predicted (23). Positive chronotropy began within 15-30 s after the application of NE and lasted several minutes. However, only a single transient release of NO, beginning in 100-200 ms and lasting 1-6 s, followed each application of NE rather than a beat-to-beat release. Repetitive applications of agonist caused repetitive transient releases of NO.

Endothelial and neuronal cells possess cNOS, which are inhibited by L-NMMA (8) and are Ca2+/calmodulin dependent (5, 6). Cardiac cNOS was similarly inhibited by L-NMMA (Fig. 3A) and the calmodulin antagonist trifluoperazine (Fig. 3B). Evidence supporting the dependence of cardiac cNOS on extracellular Ca2+ was suggested by the inhibition of agonist-induced NO release with the L-type Ca2+ channel blockers, verapamil (Fig. 3C) and nifedipine (data not shown). Further evidence for a dependence on extracellular Ca2+ was provided by the enhancement of NE-evoked NO release with the L-type Ca2+ channel activator, BAY K 8644 (Fig. 3D).


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 3.   Effects of NO synthase (NOS) inhibitor NG-monomethyl-L-arginine (L-NMMA, 2.5-10 µM; A), calmodulin antagonist trifluoperazine (TFP, 2.5-10 mM; B), Ca2+ channel antagonist verapamil (VPM, 5-10 µM; C), and Ca2+ channel agonist BAY K 8644 (10 µM; D) on NE (1 µM, arrows)-elicited NO release are shown. Cardiac constitutive NOS (cNOS) was completely inhibited by 10 µM L-NMMA (n = 7 neonatal/6 adult cells; neonatal cell shown), 10 mM TFP (n = 6 neonatal/6 adult cells; neonatal cell shown), and 10 µM VPM (n = 7 neonatal/6 adult cells; neonatal cell shown) but was enhanced by 10 µM BAY K 8644 (787 ± 93 to 881 ± 101 nM; n = 6 neonatal/6 adult cells, P < 0.05; neonatal cell shown). All effects were reversible on washout (A-C; not shown for D).

The release of NO by the nonhydrolyzable GTP analog, guanosine 5'-O-(3-thiotriphosphate) (GTPgamma S), and blockade by the nonhydrolyzable GDP analog, guanosine 5'-O-(2-thiodiphosphate) (GDPbeta S), suggest that cardiac cNOS is regulated by a G protein-dependent mechanism (Fig. 4A). The regulation of cardiac cNOS by a adenosine 3',5'-cyclic monophosphate (cAMP)-dependent mechanism (10, 14) was also suggested by NO release with the membrane-permeant cAMP analog dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP; Fig. 4B). NO release due to NE was enhanced by 3-isobutyl-1-methylxanthine (IBMX), which increases intracellular cAMP and guanosine 3',5'-cyclic monophosphate (cGMP) levels by inhibiting phosphodiesterases (Fig. 4C). The release of NO was not induced or enhanced by 8-bromoguanosine 3',5'-cyclic monophosphate (1 mM; n = 7). Thus cGMP was not involved and IBMX-enhanced NE evoked NO release by prolonging the half-life of cAMP. In sharp contrast to endothelial cells (12), NO release was not elicited or enhanced by shear stress (0.2-10 dyn/cm2; n = 6), ATP (10 µM; n = 6), bradykinin (10 µM; n = 7), or the cholinergic agonist carbachol (10 µM; n = 7).


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 4.   A: removal and chelation (100 µM EGTA) of extracellular Ca2+ diminished the response to 1 µM NE (from 769 ± 87 to 302 ± 28 nM NO). Loading cells (in presence of saponin, 25 µg/ml) with nonhydrolyzable GDP analog guanosine 5'-O-(2-thiodiphosphate) (GDPbeta S; 1 µM) blocked response to NE. After washout, nonhydrolyzable GTP analog guanosine 5'-O-(3-thiotriphosphate) (GTPgamma S; 1 µM) evoked direct NO release (334 ± 56 nM, n = 6 adult cells). B: dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP; 10-100 µM, arrows) elicited NO release (35 ± 9 to 284 ± 49 nM; n = 6 neonatal/6 adult cells; adult cell shown). C: phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX; 0.5 mM) did not elicit NO release. However, IBMX (0.5 mM) enhanced NE (1 µM, arrow)-evoked NO release (760 ± 79 to 870 ± 91 nM; n = 6 neonatal/6 adult cells, P < 0.05; neonatal cell shown).

iNOS has previously been reported in cardiac myocytes after exposure to inflammatory cytokines (2, 18, 25). The absence of an inducible enzyme in our cells was confirmed by immunohistochemical staining that was negative for iNOS (Fig. 5A). In contrast, cells treated with the proinflamatory cytokine, interleukin-1beta (IL-1), stained positive for iNOS protein (Fig. 5B) as previously reported (18). The absence of iNOS mRNA was also confirmed by RT-PCR. Only IL-1-treated cells exhibited an RT-PCR iNOS product (Fig. 5C).


View larger version (42K):
[in this window]
[in a new window]
 
Fig. 5.   Measured NO release from myocytes resulted solely from cNOS activity as demonstrated by absence of inducible NOS (iNOS) protein by immunohistochemistry (A) and iNOS mRNA by reverse transcriptase-polymerase chain reaction (RT-PCR; C). In contrast, interleukin-1beta (IL-1; 500 U/ml)-stimulated cells stained positive for iNOS protein (B) and exhibited an RT-PCR iNOS product (C), as we previously described (18). bp, Base pairs.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

Pharmacological and molecular studies have suggested the existence of a cNOS in cardiac myocytes (1, 7, 25). However, the present study definitively demonstrates, for the first time, the presence of a functional cNOS in both neonatal and adult cardiac ventricular myocytes. Cardiac cNOS shares inhibition by L-NMMA and Ca2+/calmodulin dependence with both endothelial and neuronal cNOS. However, cardiac cells differ from endothelial cells by releasing NO in response to adrenergic rather than cholinergic agonists. Reports of cholinergic-mediated NO release from cardiac myocytes are controversial and based on studies where NO was not measured. In vivo studies involved blocking, with NOS inhibitors, the decreases in myocardial contractility that resulted from vagal stimulation (9). However, the released NO being blocked may have come from vascular endothelial cells that are known to produce NO in response to cholinergic agonists. In vitro studies involved opposing, with NOS inhibitors, cholinergic inhibition of adrenergically evoked increases in voltage-dependent Ca2+ currents (3). However, although these findings are interesting, other studies have found no effects of NOS inhibitors on cholinergic modulation of voltage-dependent Ca2+ currents (16).

beta -Adrenergic receptor regulation of cNOS was demonstrated by the release of NO with selective beta -adrenergic (isoproterenol, dobutamine, and tertbutylene) but not selective alpha -adrenergic (phenylephrine) agonists. Because GTPgamma S directly evoked NO release and GDPbeta S blocked adrenergically mediated NO release, a G protein-dependent pathway is indicated in myocyte NO release (Fig. 4A). In addition, the responses of myocytes to DBcAMP (Fig. 4B) and the phosphodiesterase inhibitor IBMX (Fig. 4C) suggest that a cAMP-dependent adrenergic signaling pathway is also present (10, 19). The enhancement of NE-evoked NO release by BAY K 8644 (Fig. 3D) and the blockade of release with verapamil (Fig. 3C) further suggest that these pathways are operating through L-type Ca2+ channels.

It is generally accepted that beta -adrenergic receptors are coupled, via G proteins, to adenylyl cyclase. The formation of cAMP and the effects that it brings about through the phosphorylation of proteins typically take from seconds to tens of seconds to develop. An example of this time lag is the 15-30 s required for increases in chronotropy and inotropy to develop in beating neonatal myocytes after the addition of beta -adrenergic agonists. However, the onset of NE-evoked NO release began within 100-200 ms of the addition of agonist, suggesting the presence of a "fast" beta -adrenergic/G protein pathway where G proteins may be directly coupled to L-type Ca2+ channels (14, 28). If a fast G protein-dependent pathway is responsible for NO release, it is interesting to speculate that the "slow" cAMP-dependent pathway is responsible for modulation of NO levels. Finally, NO was not spontaneously released beat-to-beat from contracting cells. This suggests that myocyte cNOS is compartmentalized and isolated from the transient elevations in intracellular Ca2+ responsible for excitation-contraction coupling.

NO serves a paracrine function in a variety of cell types (17, 26). For example, NO released by endothelial cells acts on underlying smooth muscle cells to induce vasodilatation (11, 20, 21). Similarly, NO released from cardiac myocytes may be responsible for dilation of adjacent coronary resistance vessels. The resultant increase in coronary perfusion is necessary to support adrenergically induced increases in myocardial contractility. It is interesting to speculate on an autocrine role for this free radical in cardiac myocytes. NO produced by myocytes may enhance myocardial relaxation and regulate contractility as well as coronary perfusion. Consequently, deficiencies in cardiac myocyte cNOS and NO production may play a role in such diverse cardiac syndromes as coronary artery vasospasm and myocardial diastolic dysfunction.

    ACKNOWLEDGEMENTS

This work was supported by National Heart, Lung, and Blood Institute National Research Service Award Grant 1F32-HL-09072 and Grant-in-Aid NC-95-GB-08 from the American Heart Association, North Carolina Affiliate (A. J. Kanai); by Grant RO1-HL-53372 (M. S. Finkel); and by a grant from the Biotechnology Research Program of Oakland University (T. Malinski).

    FOOTNOTES

Address for reprint requests: A. J. Kanai, Box 90281, Dept. of Biomedical Engineering, Duke Univ., Durham, NC 27708.

Received 21 August 1996; accepted in final form 15 July 1997.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

1.   Balligand, J. L., L. Kobzik, X. Han, D. M. Kaye, L. Belhassen, D. S. O'Hara, R. A. Kelly, T. W. Smith, and T. Michel. Nitric oxide-dependent parasympathetic signaling is due to activation of constitutive endothelial (type III) nitric oxide synthase in cardiac myocytes. J. Biol. Chem. 270: 14582-14586, 1995[Abstract/Free Full Text].

2.   Balligand, J. L., D. Ungureanu-Longrois, W. W. Simmons, D. Pimental, T. Malinski, M. Kapturczak, Z. Taha, C. J. Lowenstein, A. J. Davidoff, R. A. Kelly, T. W. Smith, and T. Michel. Cytokine-inducible nitric oxide synthase (iNOS) expression in cardiac myocytes. J. Biol. Chem. 269: 27580-27588, 1994[Abstract/Free Full Text].

3.   Belhassen, L., R. A. Kelly, T. W. Smith, and J. L. Balligand. Nitric oxide synthase (NOS3) and contractile responsiveness to adrenergic and cholinergic agonists in the heart: regulation of NOS3 transcription in vivo by cyclic adenosine monophosphate in rat cardiac myocytes. J. Clin. Invest. 97: 1908-1915, 1996[Abstract/Free Full Text].

4.   Brady, A. J. B., J. B. Warren, P. A. Poole-Wilson, T. J. Williams, and S. E. Harding. Nitric oxide attenuates cardiac myocyte contraction. Am. J. Physiol. 265 (Heart Circ. Physiol. 34): H176-H182, 1993[Abstract/Free Full Text].

5.   Bredt, D. S., and S. H. Snyder. Isolation of nitric oxide synthases, a calmodulin-requiring enzyme. Proc. Natl. Acad. Sci. USA 87: 682-685, 1990[Abstract].

6.   Busse, R., and A. Mulsch. Calcium-dependent nitric oxide synthase in endothelial cytosol is mediated my calmodulin. FEBS Lett. 265: 133-136, 1990[Medline].

7.   Finkel, M. S., C. V. Oddis, T. D. Jacob, S. L. Watkins, B. G. Hattler, and R. L. Simmons. Negative inotropic effects of cytokines on the heart mediated by nitric oxide. Science 257: 387-389, 1992[Medline].

8.   Gardiner, S. M., A. M. Compton, T. Bennett, R. M. J. Palmer, and S. Moncada. Regional haemodynamic changes during oral ingestion of NG-monomethyl-L-arginine or NG-nitro-L-arginine methyl ester in conscious Brattleboro rats. Br. J. Pharmacol. 101: 10-12, 1990[Abstract].

9.   Hare, J. M., J. F. Keaney, J. L. Balligand, J. Loscalzo, and T. M. Smith. Role of nitric oxide in parasympathetic modulation of beta -adrenergic myocardial contractility in normal dogs. J. Clin. Invest. 95: 360-366, 1995[Medline].

10.   Hartzell, H. C., P. F. Méry, R. Fischmeister, and G. Sabo. Sympathetic regulation of cardiac calcium current is due exclusively to cAMP-dependent phosphorylation. Nature 351: 573-576, 1991[Medline].

11.   Ignarro, L. J., G. M. Buga, K. S. Wood, R. E. Byrns, and G. Chaudhuri. Endothelial-derived relaxing factor produced and released from artery and vein is nitric oxide. Proc. Natl. Acad. Sci. USA 84: 9265-9269, 1987[Abstract].

12.   Kanai, A. J., H. C. Strauss, G. A. Truskey, A. L. Crews, S. Grunfeld, and T. Malinski. Shear stress induces ATP-independent transient nitric oxide release from vascular endothelial cells, measured directly with a porphyrinic microsensor. Circ. Res. 77: 284-293, 1995[Abstract/Free Full Text].

13.   Kharitonov, V. G., A. R. Sundquist, and V. S. Sharma. Kinetics of nitric oxide autoxidation in aqueous solution. J. Biol. Chem. 269: 5881-5883, 1994[Abstract/Free Full Text].

14.   Levy, M. N., T. Yang, and D. W. Wallick. Assessment of beat-by-beat control of heart rate by the autonomic nervous system: molecular biology techniques are necessary, but not sufficient. J. Cardiovasc. Electrophysiol. 4: 183-193, 1993[Medline].

15.   Malinski, T., and Z. Taha. Nitric oxide release from a single cell measured in situ by a porphyrinic-based microsensor. Nature 358: 676-678, 1992[Medline].

16.   Méry, P.-F., L. Hove-Madsen, J.-M. Chesnais, H. C. Hartzell, and R. Fischmeister. Nitric oxide synthase does not participate in negative inotropic effect of acetylcholine in frog heart. Am. J. Physiol. 270 (Heart Circ. Physiol. 39): H1178-H1188, 1996[Abstract/Free Full Text].

17.   Nathan, C. Nitric oxide as a secretory product of mammalian cells. FASEB J. 6: 3051-3064, 1992[Abstract/Free Full Text].

18.   Oddis, C. V., R. L. Simmons, B. G. Hattler, and M. S. Finkel. cAMP enhances inducible nitric oxide synthase mRNA stability in cardiac myocytes. Am. J. Physiol. 269 (Heart Circ. Physiol. 38): H2044-H2050, 1995[Abstract/Free Full Text].

19.   Osterrieder, W., G. Brum, J. Hescheler, W. Trautwein, V. Flockerzi, and F. Hofmann. Injection of subunits of cyclic AMP-dependent protein kinase into cardiac myocytes modulates Ca2+ current. Nature 298: 576-578, 1982[Medline].

20.   Palmer, R. M. J., A. G. Ferrige, and S. Moncada. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature 327: 524-526, 1987[Medline].

21.   Parent, R., M. Al-Obaidi, and M. Lavallee. Nitric oxide formation contributes to beta -adrenergic dilation of resistance coronary vessels in conscious dogs. Circ. Res. 73: 241-251, 1993[Abstract].

22.   Paulus, W. J., P. J. Vantrimpont, and A. M. Shah. Acute effects of nitric oxide on left ventricular relaxation and diastolic distensibility in humans. Circulation 89: 2070-2078, 1994[Abstract].

23.   Reuter, H. Localization of beta  adrerergic receptors and effects of noradrenaline and cyclic nucleotides on action potentials, ionic currents and tension in mammalian cardiac muscle. J. Physiol. (Lond.) 242: 429-451, 1974[Medline].

24.   Rockman, H. A., R. A. Hamilton, R. A. Jones, C. A. Milano, L. Mao, and R. J. Lefkowitz. Enhanced myocardial relaxation in vivo in transgenic mice overexpressing the beta 2-adrenergic receptor is associated with reduced phospholamban protein. J. Clin. Invest. 97: 1618-1623, 1996[Abstract/Free Full Text].

25.   Schulz, R., E. Nava, and S. Moncada. Induction and potential biological relevance of a Ca2+-independent nitric oxide synthase in the myocardium. Br. J. Pharmacol. 105: 575-580, 1992[Abstract].

26.   Snyder, S. H. Nitric oxide: first in a new class of neurotransmitters? Science 257: 494-496, 1992[Medline].

27.   Wood, J., and J. Garthwaite. Models of the diffusional spread of nitric oxide: implication for neuronal nitric oxide signaling and its pharmacological properties. Neuropharmacology 33: 1235-1244, 1994[Medline].

28.   Yatani, A., and A. M. Brown. Rapid beta -adrenergic modulation of cardiac calcium channel currents by a fast G protein pathway. Science 245: 71-74, 1989[Medline].


AJP Cell Physiol 273(4):C1371-C1377
0363-6143/97 $5.00 Copyright © 1997 the American Physiological Society