Influences to the heart and vessels from the VLM sympathoexcttatory systems. ... tone through those VLM sympatoexcilatory neurons, which are involved in the.
Neurophysloiogy, Vot 28, No. 213, March-lane, 1996
Nitric Oxide and Sympathoexcitatory Cardiovascular Neurons of the Ventrolateral Medulla in Cats L. N . S h a p o v a l , 1 V. F. S a g a c h , 1 L. S. P o b e g a i l o , I a n d L. B. D o l o m a n I
Neirofiziologiya/Neurophysiology, Vol. 28, No. 2/3, pp. 111-120, March-June, 1996.
Received June 23, 1996. In acute experiments on cats, the effects ol injections of nitric oxide (NO) donor's and an inhibitor o[ Its s y n ~ into the sympathoexcttatory neuronal structures in the ventrotateral medulla (VLM) were studied to examine their effects On the peripheral mechanisms of the cardiovascular control. Unilateral in lections of NO donors, nitroglycerine (1.3-5.2 nmol) or sodium nttroprus~de (1.1-4.6 nmol) tnto the sites of the ~'T'mpathoexcttatocy neurons restdin$ tn the VI2,[ Induced the lowering of the systemic arterial pressure (SAP) In a dose-depended fashion. Two types of the hypotensive responses have been distinguished. In the first type responses, lowering of the SAP level was mainly due to a decrease in the peripheral vascular resistance (PVR), while the heart rate (FIR) and stroke volume (SV) were only slightly reduced. In the second type responses, the drop In SAP level resulted mainly from a decrease in the HR and myocardial contractivity. The~e effects were Induced by the limitation of the descending excltalccy Influences to the heart and vessels from the VLM sympathoexcttatory systems. An increase In the NO concentrations In the neuronal structures located 2.5-4.5 mm caudally to the trapezoid bodies resulted In the first type ~ , while that in the sites Immediately adjacent to the caudal sympatholnhtbltory area (0.5-1.5 mm rostrally to the XIlth cranial nerve tools) was a,~ociated with the second type of reactions. Stimulation of the endogenous NO r e ! e ~ from the neurons after I n ~ f l o n s of L-arglnine induced the same cardiovascular shifts as exogentc NO did, and attenuation of NO synthesis following injections of NO antagonist L-NMMA into the VLM neuronal structures evoked hemodynamic shifts of a reverse direction. Injections of NO donors inhibited the reflex rezponses induced by the activation of the carotid sinus receptors. Our data give further evidence for NO involvement in the inhibitory control of the cardiac activity and vascular tone through those VLM sympatoexcilatory neurons, which are involved in the system of central neurogenic cardiovascular control and the activity of which prevent the development o[ hypertension.
the vascular wall and now is considered one of the basic peripheral vasodilators 12-5 ]. An amino acid L-arginine is the physiological precursor for the formation of NO in vascular endothelial cells [6-8 ]. Release of NO from the vascular endothelial cells was shown to be inhibited with a derivative of this amino acid L-NG-monomethylL-arginine (L-NMMA) [9-11 ]. Unexpectedly an enzyme that transforms L-arginine into NO, NO-synthase, was detected in numerous different parts of the brain 112-15]. Physiological experiments gained evidence for the ability of NO to modulate neuronal activity in different portions of the brain, including the VLM [16-181. Studies on the possibility that NO is involved in the activities of VLM neurons revealed that an increase in NO level in the structures of this area (resulting from either administration of exogenous NO or enhancement of production of endogenous NO imme-
INTRODUCTION The chemosensitive neuronal structures localized in the ventrolateral medulla (VLM) are known to contribute considerably to the optimization of the cardiac activity and vascular tone [I ]. Hence, it is clear that the level of the arterial pressure can be dramatically changed with different physiologically active substanses affecting the sympathoexcitatory neurons of this brain region. This is why nitric oxide (NO) has been a substance of special interest in recent years. It represents a basis for endothelium-derived relaxing factor in
I Bogomolets Institute of Physiology, National Academy of Sciences of Ukraine. Kiev, Ukraine.
86 ~90-2977/96/2802/3-00865| 5,00°|997 Ph~numPublishing Corporation
NO and Sympathoexcitatory Medullary Neurons
diately in this brain site) considerably affects the mechanisms of central cardiovascular control. These effects occur via attenuation of descending influences to the heart and vessels, which are mediated by the sympathoexcitatory mechanisms [18-20]. However, both the phenomenology of the hemodynamic shifts evoked by the changes in NO level in the VLM and the m,~chanisms of these re~nonses still have not been satisfactorily studied. In our study, we examined the effects of the changes in the NO levels using injections of NO donors, its physiological precursor in the organism, or its antagonist in the sympathoexcitatory neuronal structures of the VLM; cardio- and hemodynamic shifts and reflex responses of the circulation system were recorded.
METHODS Experiments were carried out on cats weighing 3.0-3.5 kg; chloralose-urethane anesthesia (50 and 500 mg/kg, respectively) was used. The ventral medullary surface was accessed from the ventral side of the skull after the head of an animal had been fixed in a stereotaxic frame. After a part of the larynx, trachea, esophagus, and overlying muscles had been removed, the basio-occipital bone was carefully trepaned as close to the tympanic bullas as possible, without damaging the nerves leaving the skull through the jugular foramen. A tracheostomic tube was inserted at the low neck level, and the animal was artificially ventilated. An arterial cannula was inserted into the femoral artery, and the systemic arterial pressure (SAP) was recorded using a pressure transducer and a tensoamplifier; recorded signals were displayed on an oscilloscope and recorded using a photorecorder and an ink recorder. The heart rate (HR) was measured from SAP pulse fluctuations. The stroke volume of the heart (SV) was recorded with the use of tetrapolar transthoracal impedance rheoplethysmography. Electrodes of special construction were placed on the thorax of a cat. The cardiac output (CO, I/min) and peripheral vascular resistance (PVR, mN.m-9.sec) were calculated. Descending neurogenic influences from the VLM to the vessels were evaluated based on the changes of the background spike activities in the renal nerve (under conditions of multifiber recording). In physiological experiments, this nerve is usually used as a standard of the vasoconstrictor nerve. Similar influences to the heart were determined by the changes in the spike activity in the inferior cardiac nerve. Both nerves were carefully separated, dissected free, transected, and placed on bipolar silver wire electrodes for recording of multifiber el,;ctrical activity. The exposed nerves were immersed in
87
warm mineral oil to prevent their overdrying. The signals recorded from the ,nerves were amplified with a differential amplifier, displayed on an oscilloscope, and recorded with a photorecorder. Permanent recording of the left ventricular pressure was provided with a pressure transducer; it was inserted with a catheter into the left ventricle of the heart via the left atrium apex. Signals were displayed on an oscilloscope and recorded on paper. The first derivative of the left ventricular pressure (dP/dt) was obtained by electronic differentiation from the pressure signal. The pressor baroreceptor reflex was induced with bilateral occlusion of the common carotid arteries. NO-containing drugs (nitroglycerine, 1.3-5.2 nmol and sodium nitroprusside, 1.1-4.6 nmol), NO precursor L-arginine (1.7-6.8 nmol), and NO antagonist I.,-Ncmonomethyl-L-arginine, L-NMMA (4.9 nmol) were injected with a microsyringe into the medullary sites, extended 2.0-6.0 mm caudally to the trapezoid bodies and 3.0-5.0 mm laterally to the midline, through a cannula (external diameter of 300 ,urn). The volume of injection was 300 hi. Methylene blue (50/zM), which inhibits the effects of released NO, was applied to the examined ventral medullary surface using a cotton pledget. In control experiments, the brain area was injected with 300 nl of 0.9% NaCI solution. After an experiment, the medullary sites from the animals euthanized with excessive anesthesia were removed and fixed in I0% formalin. Then 60-/.~m-thick slices were cut in a cryostat, stained with Cresyl violet, and histologically examined. Statistical analysis was performed using techniques for comparison of the paired data. P < 0.05 values were considered statistically significant.
RESULTS Effects of NO on the SAP Level. Control injections of the 0.9% NaCI solution in the tested brain sites did not alter significantly the SAP level. Unilateral injections of nitroglycerine and sodium nitroprusside, the compounds spontaneously releasing NO and so considered NO donors, into the neuronal structures of the examined portion of the brain were followed with a dose-dependent drop in the SAP. The most pronounced SAP reduction was observed after the drugs had been injected into the brain loci 4.0-6.0 mm caudally to the trapezoid bodies and immediately adjacent to the sympathoinhibitory neuronal pools. Injections of 5.2 nmol nitroglycerine and 4.6 nmol sodium nitroprusside into these medullary sites decreased the SAP level by 22.6 % (P < 0.01) and 35.0% (P < 0.001) on average, respectively (Fig. IA, B). The efficacy of microinjections was reduced with shifting of a chemotrode in the rostra; direction. The
88
L N. $hapoval et al.
Hg
mrt
140
2
I
z///,
1
120
I
"////A I00 . # . . .
,..
. . . . .
L,'.~
.....
~//.,~
. . . .
:A
~//,
/:///
~.;::~
/##~#
2
~//~ / / / / /
/ / / J i
/II/I ///II lli/~
60
v///, 9"///
..../A . . . .
.....
80
/~
2
I///I
/////
/i/i:l //I/: :/i:.¢
.....
Y//~
i/ill
9"//2 ?'//2
40 -
////: ##i#i
"/,-//, ://// f////,
"///,4 /////~ l//l/
20-
oi
1
Fig. I. Changes In the arterial pressure induced by Injections of N O donors nitroglycerine (5.2 nmol, n - 14, A) and sodium nitroprnsslde (4.6 amol, n - 13, B), L--arglnlne, the N O precursor (6.8 nmol, n - 14, C), and L - N M M A , the N O antagonist (4.9 nmol, n - 9, D) In the sympathoexcllatory neuronal structures In the ventrolateral medulla. Injectlons were made 4.0-6.0 m m caudally to the trapezoid bodies and 3.5-4.0 m m laterally to the mldllne. I) Control values, 2) 20 se.c after drug injections.
short, for such kind of responses, latency (about 3 sec) and rather sharp increase in the SAP level, reaching the peak 20 sec after the drug administration, were typical of these hypotensive responses. They lasted 180 sec, on average. Similar hypotensive responses were evoked by activation of NO synthesis and endogenous NO increase after microinjections of an NO precursor L-arginine in the mentioned medullary sites (Fig. 1C). Injections of 6.8 nmol L-arginine resulted in the SAP drop by 29.0% (P < 0.01). Inhibition of NO synthesis by L-NMMA injections into the sympalhoexcitatory neuronal groups of the VLM resulted in the development of hypertensive responses (Fig. 1). After 4.9 nmot of L-NMMA (D) had been injected into these structures, the SAP level increased by 22.3%, on average (P < 0.05). Five minutes after methylene blue had been applied to the ventral surface of the medulla, injections of NO donors into the VLM failed to induce any SAP shifts. Since methylene blue blocks the effects, caused with NO release from the cells, through inhibition of intracellular guanylate cyclase, we can suggest that hypotensive responses following NO injections into the populations of the neurons in this brain area are likely to bc realized via this enzyme activation and consequent elevation in the intracellular level of cGMP. Effects of NO on the Background Impulse Activity
in the Renal Nerve. The decrease in the SAP level developed concurrently with inhibition of the background spike activity in the renal nerve. Unilateral NO injections into the sympathoexcitatory structures of the VLM reduced the frequency of bursts in the postganglionic fibers of the renal nerve (we considered this nerve a standard of the vasoconstrictor nerve) up to their complete disappearance. The intervals between bursts became longer, and the amplitude of spike activity decreased. Inhibition of the activity in the renal nerve following injections of two NO donors, nitroglycerine and sodium nitroprusside, was qualitatively and quantitatively similar to that induced by an NO precursor in the organism, L-arginine. A statistically significant decrease in the frequency of bursts occurred with approximately l-see-long delay and peaked up in 3 sec after the drugs were administered in the synpathoexcitatory neuronal structures in the VLM. After injections of the NO antagonist L-NMMA into these structures of the VLM, the background spike activity in the renal nerve was facilitated (Fig. 2). Effects of NO on the Parameteres of the Cardioand Hemodynamics. The analysis of the hemodynamic shifts following sodium nitroprusside (4,6 nmol) or L-arginine (6.8 nmol) injections into the neuronal structures of the VLM allowed us to distinguish two
NO and Sympathoexcitatory Medullary Neurons
89
$~c -f
%
f SAP
2 HR
3
SV
4 CO
5 PVR
_.L.
7O
0
f
2
3
4
5 see
Fig. 2. Changes in spike activity recorded from the renal nerve after injections of NO donors nitroglycerine (1) and sodium nitroprusside (2), L-arginine, the NO precursor (3), and L-NMMA, the NO antagonist (4) tnto the sympathoexcilatory neuronal structures in the venlrolateral medulla, Doses and the sites for injections are the same as tn Fig. 1.
main types of responses. The vascular component is likely to predominantly determine the first type of hemodynamic responses. In these cases, hypotensive shifts resulted from a PVR drop (Fig. 3A); the cardiac component of this response played no significant role in the SAP level decrease. For example, the SAP drop by 24.0% on average (P < 0.001) was due to the PVR decrease by 23.8% (P < 0.05). There were only slight shifts in the HR and SV; their changes were statistically insignificant. Responses of this type were induced with NO donors or NO precursor injected into the neuronal structures located 2.5- 5.5 mm caudally to the trapezoid bodies. The second type of hypotensive responses was based mainly on suppression of the cardiac activity. Rightside injections of NO in these cases caused no pronounced changes in the myocardial contractile activity, but considerably reduced the HR (Fig. 3B). For example, the SAP level decrease by 35.0% (P < 0.001) mostly resulted from a marked HR decrease (by 26.6% on average; P < 0.01). The SV demonstrated a trend toward increase (by 4.8%; P > 0.05), which was likely to be a compensative shift resulting from an HR decrease. The CO decreased, on average, by 23.2% (P > 0.05) and PVR by 17.2% (P > 0.05).
Fig. 3. Changes in the parameters of the central hemodynamics induced by the leftside (A) and rightside (B) injections of 4.6 nmol sodium nitroprusside Into the sympathoexcitatory neuronal structures of the ventrolateral medulla 3.0-4.5 mm (A) and 4.5-6.0 mm (B) caudally to the trapezoid bodies. 1) Systemic arterial pressure (SAP); 2) heart rate (HR); 3) stroke volume (SV); 4) cardiac output( CO); 5) peripheral vascular resistance (PVR). The values before Injections are taken as
10o %.
Inhibition of the VLM sympathoexcitatory neurons by the elevation of NO level resulted in inhibition of the background spike activity in the left cardiac nerve (Fig. 4A). This effect was characterized by a short latency: a statistically significant drop in the frequency of bursts in this nerve was observed 1 sec after the drug injection into the medullary structures and peaked at 3 sec on average. The total duration of suppression of the spike activity was 360 sec on average. Following the leftside injections of NO donor or its precursor, the systolic pressure in the left heart ventricle (Ply) significantly and in a standard manner decreased (by 31.9% on average; P < 0.01). The rate of increase in the intraventricular pressure (dP/dT,,=) was reduced by 39.5?/0 (P < 0.01). The Veragute's index of contractivity (IV) also decreased by 26.1% (P < 0.05). Thus, all parameters characterizing the contractile myocardial activity were found to be reduced (B). Leftside injections of NO donors mostly inhibited inotropic function, while rightside injections suppressed chronotropic function of the heart. It deserves attention that hypotensive responses, which were attributed to inhibition of the cardiac activity, occurred only after injections of sodium nitroprusside or L-arginine into the rather limited medullary area located 0.5-1.5 mm caudally to the area of the XIIth cranial nerve root, which is immediately adjacent to the caudal medullary sympathoinhibitory area. Elevation of the NO level in more rostral sites of the brain failed to change the
90
L.N. Shapoval et al.
iiii!iiiiiiiiiii
increase. The CO and PVR increased by 38.1% (P < 0.05). Injections of sodium nitroprusside into the sympathoexcitatory neuronal pool of the VLM, performed at the peak of the reflex hypertensive response, were followed by a decrease in the SAP level. The amplitude of response became 120.5% of the initial value, which was 22.0% lower than that before the injection (P < 0.01; Fig. 5). In this situation, the hypotensive response mostly was determined by the relative decrease in the SV (P < 0.05). The CO (P < 0.05) and PVR (P < 0.05) were also significantly reduced. Practically the same results were obtained following injections of L-arginine into the tested structures of the medulla. Thus, injeclions of NO donors into the sympathoexcitatory neuronal structures of the VLM determined considerable suppression of the pressor sinocarotid reflex.
;iiii!ii!!ii
DISCUSSION
A
3
% 100
I
!
|
I
I
0
I
2
3
4
sec
B 1
2
.
.
3
.
.
.
4
.
5
!ii!iiiiiiiiiii
80
, 60
,.
i{i!iiiiiiiiiii! :+:+:~+:
40
iiiiiiiiiii!i
Fig. 4. Changes in the spike activity recorded from the left inferior cardiac nerve (A) and parameters of cardiodynamics (B) induced by the leflside injections of 4.6 nmolsodiumnitroprusside into the neuronal structures in the ventrolateral medulla 4.5-5.5 mm caudally to the Irapezoid bodies (0.5-1.5 mm rostrally to the area of the fOOlof the Xmh cranial nerve). Abscissa in A) Time after injections, sec; ordinate) frequency of spike activity. In B: 1) systolic pressure in the left ventricle, 2) rate of intraventricular pressure increase, 3) Veragum's index of contracfivity, 4) rate of myocardial relaxation, and 5) Index of myocardial relaxation. Values of the initial parameters are taken as 100%; n - 12. One or two asterisks show the levels of significance of the differences as compared with the control (P < 0.05 and P < 0.01, respectively).
cardiac activity. These data show that in the VLM, localizations of the pools of sympathoexcitatory neurons, which are involved in the neurogenic control of vascular tone and cardiac activity, are dissimilar. Effects of NO on the Reflex Responses Caused by t h e Activation of the Carotid Sinus Receptors. To study the involvement of the VLM NO-sensitive neurons in the reflex circulatory responses, we examined the effects of injections of NO donors into the sympathoexcitatory neuronal structures of this brain area on a pressor sinocarotid reflex. This reflex was induced with bilateral occlusion of the common carotid arteries; it was expressed as an increase in the SAP level by 42.5% on average (P < 0.001), together with the pronounced reflex facilitation of the spike activity in the renal nerve. The hypertensive response mostly resulted from the SV increase (by 63.4%; P < 0.01). There was a mild decrease in the HR by 13.5% (P > 0.5); it likely was of a compensative nature due to the pronounced SV
Analysis of our findings shows that the effects of NO are comparable with those produced by injections of a well-known inhibitory central transmitter, gammaaminobutyric acid (GABA), into the sympathoexcitatory neuronal structures of the VLM [21]. GABA and NO were both shown to affect through inhibition of descending excitatory influences upon the heart and vessels due to the depression of the activity of the sympathoexcitatory neurons in this region of the brain [18, 19, 21 ]. The effects of NO donors (sodium nitroprusside and nitroglycerine) and the amino acid L-arginine (which enhances the synthesis and release of the endogenous NO in nervous cells and, therefore, is considered the physiological precursor for NO in the organism) were qualitatively and quantitatively similar. It is known [22, 23 ] that in the peripheral vessels the effect of NO is mediated through stimulation of the soluble guanylate cyclase with the consequent elevation of the cGMP level. It is also known that soluble guanylate cyclase is found in the CNS, although it is distributed nonuniformly in different structures of the brain. Cholinergic and adrenergic transmissions and the effects of excitatory amino acids and peptides were reported to be realized through the elevation of the cGMP level. There is a suggestion [7] that in many cases receptor-mediated activation of NO production from L-arginine also leads to an increase in the soluble guanylate cyclase concentration. It is likely that there is a regular process of L-arginine utilization and NO production in the brain structures. This process plays a considerable role in the maintenance of the optimum level of arterial pressure. L-NMMA-induced inhibition of NO synthesis in the brain structures results in a dramatic elevation of the SAP level and intensification of the spike activity in the peripheral vasoconstrictor nerves [18-20, 29 ]. Based on these considerations, one can propose that NO is an
NO and Sympathoexcitatory
Medullary
Neurons
91
%
~'k
3 SV
60
1 SAP
4 CO
5 PVR ak
40
i!!iii!~i~i~i
20
2 HR
iiiiiiiii iiiii}iiiiiiili .......
100
80
Fig. 5. Changes In the parameters of the central hemodynamlcs Induced by injections of 4.6 nM sodium nltropru~ide into the neuronal structur¢~ of the venlrolateral medulla (VLM) at moment corresl~ndlng to the peak of the p ~ slmxarofld reflex. Left columns are the parameters meama~d at the peak of pressor carotid reflex, fight coloumns are those after Injections of sodium nltropross~de Into the slructures of the VIM (the valuns before occlusion of the carotid arteries are accopted as 100%). Indications are the same as In Fiig~- 3 and 4. The leve I,, of significance of differences, Im compared with the control, are shown above the left columns, while those of differences before and after Injections of sodium nltrvprnsslde are shown above the rlght columns,
important endogenous central nitrovasodilator and, hence, the reduction of NO synthesis results in the development of hypertensions. Examining the hemodynamic effects of NO donor injections into the medullary structures showed that there are at least two distinct mechanisms of the SAP decrease. Obviously, they are associated with the peculiarities in the functional organization of the neurons in this brain area. For example, the changes in HR and myocardial contractivity were observed only after injections of NO donors in a very limited medullary zone located at a distance of 0.5-1.5 mm rostrally to the XIIth cranial nerve roots. According to our data, the neurons that are involved in cardial control are localized here. These cells are likely to compose a relatively compact "cardiac center" including both cardioexcitatory and cardioinhibitory neurons located near the area where the Xllth cranial nerve leaves the medulla. Similar results were obtained earlier 121 I after GABA injections into these neuronal structures of the VLM. The existence of functional asymmetry in the descendl,ag sympathoexcitatory influences to the heart also
deserves attention. To a large extent, such functional asymmetry can be explained by corresponding asymmetry in the anatomical organization. The heart is not a paired organ and, therefore, there are definite "lateral" differences in the innervation of its different portions, as well as asymmetry in exertion of the efferent influences on the heart. Stimulation of the right sympathetic cardiac nerve in cats evokes predominantly changes in the chronotropic function of the heart, whereas stimulation of the left nerve results in modulation of the myocardial contractile activity [24, 25]. The neurons involved in the control of the vascular tone are distributed more rostrally within the limits of the examined portion of the medulla. Injections of the drugs that elevate the NO level into these medullary structures resulted in a decrease in the SAP level predominantly because of the decrease in the resistance of the peripheral vessels. In this case, the changes in the cardiac component of this response were insignificant: the HR and myocardial contractile activity remained practically unchanged. The question of the transmitter nature in the neurons of the VLM that are affected by NO is of
92
L N. Shapoval et aL
definite interest. Recently data were obtained on the inhibitory effects of the vascular NO on the release of catecholamines from the adrenergic nerves [26, 27). Nonadrenergic and noncholinergic nerves were also found, whose electrical stimulation enhances NO synthesis and release. One can suggest that these nerves can mediate vasodilation of the arteries [28 ]. It cannot be ruled out that there are specific sites for receptor binding of NO on the membranes of some neurons of the VLM. In addition, NO can nonspecifically inhibit neurons differing in their transmitter nature. The relative contribution of NO synthesized by and released from the vascular endothelial cells and brain neurons to the control of vascular tone remains to be elucidated. Baroreceptors of the a r c u s a o r t a e and carotid sinus are known to be involved in the reflex control of the arterial pressure. Among the central mechanisms involved in this control, the sympathoexcitatory neuronal structures in the VLM obviously play an important role. We think that the role of NO in regulatory reflex effects on the system of circulation is rather active. T h e following phenomenon supports this statement: a pressor sinocarotid reflex is intensively suppressed after injections of NO donors into the sympathoexcitatory structures in the VLM. The sympathetic activities in the renal nerve are not facilitated, and the SAP level is much less increased after i.v. infusion of an inhibitor of NO-synthase after the spinal cord has been transsect at C t - C 2 levels [29]; these findings give further support to the above statement. The results of testing of baroreflexes in awake rabbits after the blockade of NO synthesis [30] also are in agreement with these data. It cannot be ruled out that there is some synergism between the peripheral and central mechanisms of vasomotor control. In this context, NO produced by the neurons possessing NO-synthase can play a key role in the central control of circulation.
REFERENCES 1. L. N. Shapoval, "The role of structures of the ventrolateral medulla in cardiovascular regulation,* Neurophysiology, 24, 717-735 (1992). 2. R. F. Furchgott and J. V. Zawadski, "The obligatory role of endothelial cells in relaxation of arterial smooth muscle by acetylcholine," Nature, 288, 373-376 (1980). 3. L. J. Ignarro, R. E. Byrns, G. M. Buga, and K. S. Wood, "Endothelium-derived relaxing factor from pulmonary artery and vein ~ s pharmacological and chemical properties identical to tho, e of nitric oxide radical," Circ. Res., 61,866-879 (1987). 4. T. M. Griffith, "Endotheltum-derived relaxing factor and the control of flow in conduit and resistance arteries," in: Flow-Dependent Regulation of Vascular Function, J. A. Bevan, G. Kaley, and G. B. Rubanyi (eds.). Oxford Univ. Pres*. New York (1995), pp. 178-213. 5. S. Moncada, M. W. Radomski, and R. M. J. Palmer, "En-
dothelium-derived relaxing factor, Identification as nitric oxide and role in the control of vascular tone and platelet function,* Biochem. PharmacoL, 37, 2'495-2501 (1988). 6. R. M. J. Palmer, D. S. A~hton, and S. Moncada, "Vascular endothelial cells synthesize nitric oxide from L-arginine," Nature, 333, 664-666 (1988). 7. S. Moncada, R. M. J. Palmer, and E. A. Higgs, "BIo6yntheals of nitric oxide from L-arginine. A pathway for regulation of cell function and communication," BiocherrL PharmacoL, 38, 17091715 (1989). 8. H. H. W. Schmidt, M. M. Klein, F. Niroomand, and E. Bohme, "Is arglnine a physiological precursor of endothellum-dertved nitric oxide?," Ear. J. PharmacoL, 148, 293-295 (1988). 9. S. N. Gardiner, A. M. Campion, T. Bennet, et al., =Regional haemodynamic effects of inhibiting endothelial cell production of nitric oxide ,v~th N-monomethyl-L-arginine in conscious rats," in: Nitric Oxide from L-Arginine, a Bioregulatory Syxtem, S. Moncada and E. A. Higgs (eds.), Excerpta Medic.a, Am, terdam, New York, Oxford (1990), pp. 81-89 10. $. Moncada, R. M. J. Palmer, and E. A. Hlggs, =Nitric oxide: physiology, pathophyslology, and pharmacology," Pharmaco/. Rev., 43, 109-142 (1991). 1 t. D. D. Rees, R. M. J. Palmer, H. F. H ~ n o and S. Moncada, "A ~pectfic inhibitor of nitric oxide formation from L-arginbm attenuates endothellum-depcndent relaxation," Br. J. PharmacoL, 96, 418-424 (1989). 12. D. $. Bredt, P. M. Hwang, and S. N. Snyder, "Localization of nitric synthase indicating a neuronal role for nitric oxide," Nature, 247, 868-869 ( I ~ ) . 13, B. Mayer, M. John, B. Hetnzel, et al., "Brain nitric oxld¢ synthase is a biopterin- and fiavtn-containing multi-funcflonni oxide-reducing," FEBS Left., 288, 187-191 (1991). 14. A. Magbool, T. F. C. Batten, and P. N. McWllllam, "Co.. localization of neurotratmmtttcr lmmunoreacttvifles in putative nitric oxide synthesizing neurons of the cat brain stem," jr. Chem. Neuroanat., 8, 191-206 (1995). 15. W. P. Gal, J. P. M ~ n g e r , Yh. Yu, et al., "Nitric oxide synthesizing neurons in the central subnucleus of the nucleus tractus solitarius provide a major Innervation of the rostral nucleus amblguus in the rabbit," J. Camp. NeuraL, 357. 348-361 (1995). 16. T. Gartwaite, S. L. Charles, and R. Ches*-Wiltianm, "Endothelium-derived relaxing factor release on activation of NMDA receptors suggests role as intercellular messenger In the brain," Nature, 336, 385-388 (1988). 17. R. J. Knowles, M. Palacios, R. M. J. Palmer, and S. Moncada, "Nitric oxide in the brain," in: Nitric Oxide from L-Arginine, a Bioregulatory System, S. Moncada and E. A. Higgs (eds.), Excerpta Medica, Amsterdam, New York, Oxford (1990), pp. 139-147. 18. L N. Shapoval, L. S. Pobegailo, and T. N. Kovalenko, "Nitric oxide and cardiovascular control by ventrolateral medulla in cats," Eur. J. Physiol., 430, R37 (1995). 19. L. N. Shapoval, V. F. Sagach, and L. S. Pobegailo, "Nitric oxide influences ventrolateral medullary mechanisms of vasomotor control in the cal," NeuroscL Lett., 132, 47-50 (1991). 20. J. Zanzinger, J. Czachurski, and H. Seller, "Inhibition of basal and reflex-mediated sympathetic activity in the RVLM by nitric oxide," Am. J. Physiol. ReguL lntegr. Camp. Physiol., 268, R958-R962 (1995). 21. L. N. Shapoval, V. F. Sagach, and L. S. Pobegailo, "Chemosensitive ventrolateral medulla in cat: the fine structure and GABA-induced cardiovascular effects," J. Auton. Nerv. Syxt., 36, 159-172 (1991). 22. L. J. lgnarro, P. A. Bush, G. M. Buga, el al., "Nitric oxide and cyclic GMP formation upon electrical field stimulation cause relaxation of corpus caverncaum smooth muscle," Biochent Biophys. Rex. Commun., 170. 843-850 (1990). 23. L. J. Ignarro and P . . L Kadovitz, "The pharmacological and physiological role of cyclic GMP in vascular smooth relaxation," Annu. Rev. Pharmacol. Toxicol., 2.5, 171-191 (1985).
NO and Sympathoexcitatory Medullary Neurons 24. M. Kollai and K. Kolzuml, "Reciprocal and non-reciprocal action of the vagal and sympathetic nerves innervating lhe hearL ~ J. AutorL Nerv. Syst., I, 33-52 (1979). 25. W. C. Randall, "Sympathetic control of the heart, in: Neuro~wl Regulation o/ the Heart, W. C. Randal (ed.), Oxford Univ. Press, New York (1977), pp. 43-94. 26. B. Tesfamartam and R. A. Cohen, "Inhibition of adrenergic vasoconstriction by endothelial cell shear stress," Circ. Res., 63, 720-725 (1988). . 27. B. Tesfamarlam, R. M. Weisbrod, and R. A. Cohen, "Endothellum inhibits responses of rabbit carotid arlery to adrenergic nerve stimulation," Ant J. PhysioL, 253, H792-H798 (1987).
93 28, N. Toda and T, Okamura, "Possible role of nitric oxide tn transmilting information from vasodilatator nerve to cerebroarterial muscle," Biochem. Biophys. Res. Commun., 170, 308313 (1990). 29. 1. Saguma, H. Togashi, M. Yoshioka, et al., "N-methyI-L-arginine, an inhibitor of L-arginine-clerived synthesis, stimulates renal sympathetic nerve activity in vivo," Cir~ Res., 70, 607-61 t (t992). 30. Z.-Y. Du, G. J. Dusting, and O. L. Woodman, "Baroreceptor reflexes and vascular reactivity during inhibition of nitric oxide synthesis in conscious rabbits," Eur. J. P~.rm~co/., 214, 21-26 (1992).
