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Nitric oxide signaling pathway mediates the L-arginine-induced cardiovascular effects in the nucleus tractus solitarii of rats
ELSEVIER
PII SOO24-3205(99)00510-X
Life Sciences, Vol. 65, No. 23, pp. 2439-2451, 1999 Copyright 0 1999 Elsevier Science Inc. F?intedin the USA. All rights reserved CKl24-3205/99&see tht matter
NITRIC OXIDE SIGNALING PATHWAY MEDIATES THE L-ARGININE-INDUCED CARDIOVASCULAR EFFECTS IN THE NUCLEUS TRACTUS SOLITARH OF RATS
Hui-Ching Lin’, Fang-Jung WanlX2, Kwok-Kei Cheng3, Ching-Jiunn
Tsengls4
Graduate Institute of Life Sciences’, Institute of Undersea and Hyperbaric Medicine*, National Defense Medical Center, Taipei; Departments of Surgery3, Medical Education and Research4, Veterans General Hospital-Kaohsiung, Taiwan, R.O.C.
(Received in final form July 27, 1999) Summary
We have previously demonstrated that L-arginine produces profound cardiovascular effects when microinjected into the nucleus tractus solitarii (NTS) of the rat. The present study extended our earlier work and examined further the underlying mechanisms of action of L-arginine in the NTS. Our results showed that intra-NTS microinjection of L-arginine (O.l- 10 nmol) elicited dose-dependent depressor and bradycardic effects that were not significantly evoked by equivalent doses of D-arginine. The effects of L-arginine were blocked by pre-injection of 7nitroindazole (0.02-l nmol), a neuronal nitric oxide synthase inhibitor. Additionally, application of the calmodulin inhibitor W-7 (0.01-0.33 nmol) reduced cardiovascular responses to L-arginine (10 nmol) in a dose-dependent manner. Pre-injections of soluble guanylyl cyclase inhibitors, LY83583 (O.Ol0.33 nmol) and IH-[1,2,4]oxadiazolo[4,3-alquinoxalin-l-one (ODQ, 0.03-l pmol) both suppressed the L-arginine-induced depressor and bradycardic effects. Finally, the cardiovascular effects of L-arginine in the NTS were attenuated by HA1004 (0.1-l nmol), a cGMP-dependent protein kinase inhibitor, but not by the protein kinase C inhibitor H-7 (1 nmol). Taken together, the results indicate that the cardiovascular effects produced by L-arginine in the NTS are inhibited by pharmacological interventions that block nitric oxide production and cGMP-PKG signaling pathway within the nucleus. Key Words: neuronal nitric oxide synthase, calmodulin, soluble goanylyl cyclase, cyclic Gh4P
The nucleus tractus solitarii (NTS), located in the dorsal medial part of the medulla oblongata, is an essential component of the central pathway that mediates the principal homeostatic cardiovascular reflexes (1). In the NTS, glutamate neurotransmission is responsible for the delivery of peripheral baroreceptor information (2), and microinjection of L-glutamate into the NTS elicited a baroreceptor reflex-like response (3). Recently, nitric oxide synthase (NOS) has been found in the cerebellum, forebrain and other regions of the adult rat brain (4). Accumulating evidence suggests that nitric oxide (NO) can function as a neurotransmitter or as a second messenger in the CNS (5). Studies implicated further that NO in the brain stem nuclei is involved Corresponding author: Ching-Jiunn Tseng, M.D., Ph.D., Department of Medical Education and Research, Veterans General Hospital-Kaohsiung, 386 Ta-Chung 1st Road, Kaohsiung, Taiwan, R.O.C. Fax: 01 l-886-7-3468056; E-mail: [email protected]
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in the central cardiovascular regulation by reducing sympathetic outflow from the CNS. Specifically, distribution of NOS in the NTS has been demonstrated by histological studies (6), and local application of the NO precursor L-arginine in the NTS increased neuronal activity (78). Conversely, blockade of NO formation in the NTS resulted in an increase of blood pressure or activation of the sympathetic nervous system (9). In summary, these findings suggest that NO in the NTS plays an important role in the modulation of cardiovascular function. The NO signal transducing system has been extensively studied in recent years. In the CNS, NO can be formed by nNOS via a calciurn/calmodulin-dependent manner. The produced NO subsequently binds to soluble guanylyl cyclase (sGC), leading to the production of cGMP (10). A primary action of cGMP production is the stimulation of cGMP-dependent protein kinase (PKG) which then phosphorylates substrate proteins to exert a variety of actions (1 l).The NO signaling pathway participates in the regulation of neural functions such as neurotransmission, long-term potentiation, neurotoxicity, and seizure activity (12-14). However, cGMP-independent mechanisms are also reported to mediate the action of NO in the brain (15, 16). With regard to central cardiovascular regulation, we have reported that microinjection of L-arginine into the NTS of anesthetized rats produced the falls in blood pressure, heart rate and renal sympathetic nerve activity (17). However, L-arginine has been reported to elicit central cardiovascular actions by a NO-independent pathway (18). Furthermore, precise mechanisms underlying the cardiovascular effect of NO in the NTS remain controversial. Thus, although Lewis et al. (19) have shown that S-nitrosocysteine, a NO donor, produced hypotension and bradycardia when microinjected into the NTS, others proposed that this action is independent of the release of NO (20,21). Based on these findings, this study examined the possible mechanisms of action of Larginine in the NTS. Specifically, the purpose of this study was to determine whether the cardiovascular effects of L-arginine after microinjection in the NTS is mediated by NO production and cGMP-PKG signaling pathway within this area. In the present study, we firstly characterized the effects of L-arginine or D-arginine (an isomer of L-arginine) on blood pressure and heart rate in the rat when microinjected into the NTS. Secondly, we examined the effects of pretreatment with 7-nitroindazole (7-NI, a neuronal NOS inhibitor), N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W-7, a calmodulin inhibitor), LY83583 and lH-[1,2,4]oxadiazolo[4,3-alquinoxalin-l-one (ODQ) (inhibitors of guanylyl cyclase) on the L-arginine-induced cardiovascular responses in the NTS. Thirdly, the effects of pretreatment with N-(guanidinothy)-5isoquinolinesulfonamide hydrochloride (HA1004) (a cGMP-dependent protein kinase inhibitor) and l-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7, a protein kinase C inhibitor) on the cardiovascular responses to L-arginine were studied. Methods General methods Male Sprague-Dawley rats (weight 250-350 g) were obtained from National Animal Center (Taipei, Taiwan). Rats were initially anesthetized with combined urethane (400 mg/kg i.p.) and a-chloralose (40 mg/kg i.p.). 3 hours later, the status of anesthesia was checked every 2 hr by pinching the hind limbs of the rat. If the hind limbs withdraw while pinching, maintaining dose of urethane 100 mgikg (i.v.) was administered. A polyethylene cannula was placed in the femoral vein for drug administration. Blood pressure was measured directly through a cannula placed in the femoral artery and connected to a pressure transducer (Gould P23 ID) and polygraph (Gould RS3800). Heart rate was monitored continuously by a tachograph preamplifier (Gould 13-461565). Tracheostomy was performed to maintain airway patency during the experiment.
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Microinjection study For NTS microinjection, rats were placed in a stereotaxic instrument (Kopf), with head flexed downward at a 45” angle. The dorsal surface of the medulla was exposed by limited craniotomy, and animals were then allowed to be stabilized for at least 1 hr before experiments. Single-barrel glass cannulas were prepared (0.031-inch OD, 0.006-inch ID; Richland Glass Co.) that had external tip diameters of 40 pm. The cannulas were connected to a Hamilton microsyringe by a polyvinyl tubing. The injection site in the NTS for drug administration was decided at first by the responsiveness to microinjection of L-glutamate in this area. The glass cannulas were filled with L-glutamate (100 pmoV60nl) and lowered into the NTS with the coordinates anteroposterior 0.0 mm; mediolateral, 0.5 mm; and vertical, 0.4 mm, with the obex as the reference (22). Microinjections in the NTS were given over 10 seconds by air pressure. The minimal acceptable decreases in blood pressure and heart rate were 30 mmHg and 50 beats/min after microinjection of 0.1 nmol of L-glutamate into the NTS. According to our observations, the responsive area is restricted to the intermediate one-third of the NTS; conversely, the administration of the same dose of L-glutamate in adjacent areas failed to elicit the response (23). In the beginning of each experiment, we also assured that saline administered in the NTS did not elicit any effects on BP or HR due to the volume effect caused by injection. In this study, an animal received only several injections in the NTS and each injection volume was restricted to 60 nl. After finishing injection of an agent in the NTS, the single barrel cannula was lifted and washed by distill water at least 3 times. After that, it was filled with a subsequent drug and then reinserted into the NTS according to the coordinates formerly determined using Lglutamate. To adequately control the influence of the vehicles of various agents on the cardiovascular responses to L-arginine, the vehicles were injected in exactly the same protocol as were the active inhibitors. Thus, rats were injected firstly with L-arginine (10 nmol) into unilateral NTS, and the effects on BP and HR were monitored. Rats were then allowed to rest for at least 30 min until BP and HR returned to stable state. After that, L-arginine was administered 15 min after intra-NTS microinjections of either an inhibitor or its vehicle in the rat. In addition, the effects of vehicles of various inhibitors on BP and HR in the NTS were examined. After completion of experiments, 60 nl of sky blue was injected through the cannula, and animals were perfused with saline followed by a solution of 4% formaldehyde and a 30% sucrose solution. Sections of 40 pm of the brain stem structure were stained with cresyl violet for verifying microinjection sites in the NTS.
NADPH-diaphorase histochemistry To identify the distribution of NOS containing neurons in the NTS, coronal sections of 40 pm of the brain stem were stained for NADPH-diaphorase activity. Sections were then incubated at room temperature for 1-2 hr in a solution containing 50 mM Tris-HCl, 1 mM P-NADPH, 0.2 mM nitro blue tetrazolium and 0.2 % Triton X-100 (PH 8.0). Following several rinses in Tris buffer (pH 7.6), stained sections were mounted on gelatinized slides to dehydrate in alcohol and then coverslipped.
Chemicals and Statistical Analysis 7-NI (dissolved in methanol, 25% for 1 nmol and 5% for the other doses), LY83583 (dissolved in 5% methanol), W-7, HA1004 and H-7 were purchased from RBI (Natick, MA, USA). ODQ (from Sigma, St Louis, MO, USA) was first dissolved in dimehtyl sulphoxide (DMSO), and diluted to the final dose 1 pmol of ODQ present in the solution of 0.1% DMSO. P-NADPH, nitro blue tetrazolium, L-arginine (dissolved in the 0.9% sterile saline) and D-arginine (dissolved in the
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0.9% sterile saline) were purchased from Sigma. We did not observe significant effects on mean BP or HR after the administration of 5 %, 25% methanol or 0.1% DMSO in the NTS. These vehicles also did not influence the effects of L-arginine as described below. For statistical analysis, pair t (for within-group comparisons) or unpaired t tests (for betweengroup comparisons) were applied when significant main effects were noted using a one-way ANOVA. Differences with a p value less than 0.05 were taken as significant. All data are presented as mea&SEM. Results NOS-containing neurons in the NTS NOS-containing neurons were visualized using NADPH diaphorase (NADPH-d) histochemistry. A dark blue reaction product formed from reduced tetrazolium salts was detected in neuronal cell bodies and processes. A cluster of NADPH-d-stained cell bodies and processes occupied the rostral-caudal regions of NTS neurons. At obex level, moderate densities of NADPH-d staining cell bodies and processes were found to locate in the ventrolateral NTS, adjacent to the dorsal motor nucleus of vagus (Figure 1). According to our observation, these NADPH-d-positive sites generally concurred to the injection sites of L-arginine that produced profound cardiovascular responses.
nNOS in the L-arginine-induced cardiovascular responses in the NTS In this study, we firstly determined the cardiovascular effects of L-arginine (0.01-10 nmol) or Darginine (0.01-10 mnol) when microinjected into the NTS. The baseline MBP and HR were 105+3 mmHg and 318f9 bpm, respectively. Unilateral microinjections of L-arginine into the NTS induced dose-related depressor and bradycardic responses (Figure 2A). L-arginine at the highest dose (10 mnol) significantly decreased MBP and HR (-32+6 mmHg and -23f5 bpm, ~~0.05, vs. saline group, total n=18). According to our previous report (17), the cardiovascular responses elicited by 33 run01 L-arginine did not differ from that produced by 10 mnol of Larginine in this study. Thus, 10 nmol of .L-arginine seems to produce the near-maximal cardiovascular response in the NTS. To the contrary, microinjection of equivalent dose range of D-arginine, an isomer of L-arginine, did not evoke cardiovascular changes in the NTS (n=12, NS). We also observed that microinjection of L-arginine (10 nmol) into the NTS repeatedly at 15-min intervals still generate similar magnitude of cardiovascular responses with little tachyphylaxis phenomenon (Figure 2B). Furthermore, we investigated whether pre-injection with 7-N& a nNOS inhibitor, influenced the cardiovascular responses to L-arginine in the NTS. Firstly, microinjection of vehicle (25% methanol) did not alter the depressor and bradycardic effects of L-arginine (AMBP -28k5 mmHg, AHR -23+5 bpm). Pre-injection of 7-NI (20 pmol-1 mnol) produced a dose-related attenuation of the L-arginine-evoked depressor and bradycardic effects (Figure 3, ~0.05). 7-NI itself did not alter the basal MBP and I-IR (AMBP -2&3, AHR 6+3 bpm, n=12, NS). Caz+/calmodulin in the L-arginine-induced cardiovascular responses in the NTS To investigate whether the effect of L-arginine in the NTS links to intracellular Ca’+/calmodulin activity, the effect of the calmodulin inhibitor W-7 on the L-arginine-induced cardiovascular responses was explored. Pretreatment with W-7 (0.1 nmol) significantly reduced the depressor but not the bradycardia effect of L-arginine in the NTS (BP from -32+3 mmHg to -12+_3 mmHg,
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Fig. 1 (A) Representative graphs of injection sites in the brainstem of rats in coronal section 14.08 mm caudal to the bregma according to the atlas of Paxinos and Watson (22). Arrowhead indicates the site of injection in the NTS. (B) The NADPH-diaphorase staining cell bodies and processes in the ventrolateral region of the NTS. Higher magnification of the area indicated by the arrow on right side of the NTS is shown in the inset. Scale bars = 200 urn for (A) and (B), and 20 urn for inset. Abbreviations: DMV; dorsal motor nucleus of vagus, NTS; nucleus tractus solitarii.
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n=9, ~~0.05). Nevertheless, both the depressor and bradycardic effects of L-arginine were significantly attenuated by a higher dose (0.33 nmol) of W-7 (AMBP from -32f4 mmHg to -3f3 mmHg and AHR from -19+5 bpm to -12M bpm, respectively, pcO.05, n=8, Figure 4). The cardiovascular effects of L-arginine in the NTS recovered almost completely 90 min after W-7 administration. microinjection of W-7 (0.1-0.33 nmol) into the NTS alone did not alter basal BP but evoked a bradycardic effect (-15+8 bpm, ~~0.05, n=9).
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influence on basal MBP, while evoking a small bradycardic effect (-18+8 bpm at 0.33 nmol, ~~0.05, n=8) that disappeared 15 min after injection. Recent studies reported that a new compound, 1-H-[1,2,4,]oxadiazolo[4,3-alquinoxalin-l-one (ODQ), is a selective and potent inhibitor of soluble guanylyl cyclase (24,25). Thus, we also examined the effects of pretreatment with ODQ on the L-arginine-elicited cardiovascular responses. Our results revealed that pretreatment with ODQ (0.03-l pmol) in the NTS significantly abolished the depressor and bradycardic effects of L-arginine in a dose-dependent manner (Figure 5B). Thus, 1 pin01 of ODQ significantly inhibited depressor and bradycardic responses elicited by 10 nmol L-arginine (AMBP: from -33+4 to -8E2 mmHg, AHR -30&5 to 9*5 bpm, respectively, ~~0.05, n=6). Full recovery of the action of L-arginine was observed 3 to 4 hr after ODQ (1 pmol) administration. On the other hand, microinjections of vehicles (0.1% DMSO) into NTS did not alter the cardiovascular responses to L-arginine, nor did evoke any change in basal MBP and HR (-2f3 mmHg and -8+3 bpm, NS, respectively). These data altogether demonstrated that LY83583 and ODQ, by way of inhibiting sGC activation, significantly reduced cardiovascular effects elicited by L-arginine in the NTS.
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Fig. 3 (A) Bar graph showing cardiovascular effects of L-arginine (L-Arg) in the NTS and their modification by 7-nitroindazole (7-N& a nNOS inhibitor). Baseline values were 95*4 mmHg for MAP and 292rtlO bpm for HR. *p
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Fig. 5 (A) Bar graph showing the effects of LY83583 (0.01-0.33 nmol) pretreatment on the cardiovascular responses to the microinjection of L-arginine (L&g, 10 nmol) in the NTS. (B) The bar graph and tracings showing the effects of ODQ (0.03-l pmol) pretreatment on the cardiovascular responses to L-Arg (10 nmol). *p
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introduced a new compound, ODQ, which displays selective and potent inhibition of sGC activity in vitro. An in vivo study also revealed that ODQ acts as a selective and possibly competitive inhibitor of the sGC targeted by NO (25). Consistent with the effects of LY83583, pretreatment with ODQ significantly blocked the depressor and bradycardic effects produced by L-arginine in the NTS. Moreover, the results showed that the ability of ODQ in the attenuation of cardiovascular responses to L-arginine is more potent than that of LY83583. Thus, 1 pmol of ODQ markedly attenuated the response induced by L-arginine, whereas LY83583 did not show significant action until a higher dose (330 pmol) was applied. The primary action of elevated cGMP level in the cell is the stimulation of cGMP-dependent protein kinase (PKG), which subsequently phosphorylates substrate proteins to exert its actions. In the brain, the NO-cGMP-mediated PKG activation has been identified (34,35). Nevertheless, NO-cGMP signaling system also actively cross-talks with other signaling substrates and second messengers. For example, Maurice and Haslam (36) reported that NO-stimulated increases in cGMP could potentiate CAMP-mediated platelet inhibition by suppressing cyclic nucleotide phosphodiesterases. In the present study, we characterized the possible role of PKG in the NOmediated central cardiovascular regulation. Specifically, the effect of pre-injection of HA-l 004, a PKG inhibitor (37), on the cardiovascular responses to L-arginine in the NTS was examined. The results revealed that HA- 1004 pretreatment significantly attenuated the depressor and bradycardic effects produced by L-arginine. However, caution must be exercised when interpreting this data, because HA-1004 also affects other signal transducing molecules such as PKA or serves as a calcium antagonist (38). Notably, only low doses (0.1-l nmol) of HA-1004 were used in this study to prevent the non-selective actions elicited by this agent. Since HA-1004 is relatively selective for PKG in the lower dose range, the effects of HA-1004 observed here is more likely due to the inactivation of PKG. This view is supported by the finding that H-7, an inhibitor of PKC and PKA (39,40), did not block the L-arginine-induced cardiovascular responses. In contrast, H-7 has been shown to protect against the endotoxin lipopolysaccharide-induced NOS activation and the subsequent PKC formation (41). Taken together, these data support the view that PKG is involved in the cardiovascular responses evoked by NO production in the NTS. In summary, the results support the role of the NO-cGMP-PKG signaling pathway played in the cardiovascular responses to the microinjection of L-arginine in the NTS. Firstly, the cardiovascular effects produced by L-arginine rely on intracellular Ca2’/calmodulin-dependent nNOS activation. Secondly, cGMP formation and PKG activation is involved in the L-arginineinduced cardiovascular responses. Acknowledgments This work was supported by NSC 87-2314-B-075B-015, DOH87-HR-705 Dr. Ching-Jiunn Tseng. Portions of this work has been presented Neuroscience Meeting, October 25-30, 1997, New Orleans, U.S.A.
and VGHKS 86-67 to at the 27th Annual
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(1984). 38. T. ASANO and H. HIDAKA, J. Pharmacol. Exp. Ther. 231 141-145 (1984). 39. S. KAWAMOTO and H. HIDAKA, Biochem. Biophys. Res. Communi. 125 258-264 (1984). 40. J. QUICK, J.A. WARE and P.E. DRIEDGER, Biochem. Biophys. Res. Common. 187 657663 (1992). 41. T.M. McKENNA, J.M. CLEG6, and T.J. WILLIAMS, Shock 2 84-89 (1994).
PII SOO24-3205(99)00510-X
Life Sciences, Vol. 65, No. 23, pp. 2439-2451, 1999 Copyright 0 1999 Elsevier Science Inc. F?intedin the USA. All rights reserved CKl24-3205/99&see tht matter
NITRIC OXIDE SIGNALING PATHWAY MEDIATES THE L-ARGININE-INDUCED CARDIOVASCULAR EFFECTS IN THE NUCLEUS TRACTUS SOLITARH OF RATS
Hui-Ching Lin’, Fang-Jung WanlX2, Kwok-Kei Cheng3, Ching-Jiunn
Tsengls4
Graduate Institute of Life Sciences’, Institute of Undersea and Hyperbaric Medicine*, National Defense Medical Center, Taipei; Departments of Surgery3, Medical Education and Research4, Veterans General Hospital-Kaohsiung, Taiwan, R.O.C.
(Received in final form July 27, 1999) Summary
We have previously demonstrated that L-arginine produces profound cardiovascular effects when microinjected into the nucleus tractus solitarii (NTS) of the rat. The present study extended our earlier work and examined further the underlying mechanisms of action of L-arginine in the NTS. Our results showed that intra-NTS microinjection of L-arginine (O.l- 10 nmol) elicited dose-dependent depressor and bradycardic effects that were not significantly evoked by equivalent doses of D-arginine. The effects of L-arginine were blocked by pre-injection of 7nitroindazole (0.02-l nmol), a neuronal nitric oxide synthase inhibitor. Additionally, application of the calmodulin inhibitor W-7 (0.01-0.33 nmol) reduced cardiovascular responses to L-arginine (10 nmol) in a dose-dependent manner. Pre-injections of soluble guanylyl cyclase inhibitors, LY83583 (O.Ol0.33 nmol) and IH-[1,2,4]oxadiazolo[4,3-alquinoxalin-l-one (ODQ, 0.03-l pmol) both suppressed the L-arginine-induced depressor and bradycardic effects. Finally, the cardiovascular effects of L-arginine in the NTS were attenuated by HA1004 (0.1-l nmol), a cGMP-dependent protein kinase inhibitor, but not by the protein kinase C inhibitor H-7 (1 nmol). Taken together, the results indicate that the cardiovascular effects produced by L-arginine in the NTS are inhibited by pharmacological interventions that block nitric oxide production and cGMP-PKG signaling pathway within the nucleus. Key Words: neuronal nitric oxide synthase, calmodulin, soluble goanylyl cyclase, cyclic Gh4P
The nucleus tractus solitarii (NTS), located in the dorsal medial part of the medulla oblongata, is an essential component of the central pathway that mediates the principal homeostatic cardiovascular reflexes (1). In the NTS, glutamate neurotransmission is responsible for the delivery of peripheral baroreceptor information (2), and microinjection of L-glutamate into the NTS elicited a baroreceptor reflex-like response (3). Recently, nitric oxide synthase (NOS) has been found in the cerebellum, forebrain and other regions of the adult rat brain (4). Accumulating evidence suggests that nitric oxide (NO) can function as a neurotransmitter or as a second messenger in the CNS (5). Studies implicated further that NO in the brain stem nuclei is involved Corresponding author: Ching-Jiunn Tseng, M.D., Ph.D., Department of Medical Education and Research, Veterans General Hospital-Kaohsiung, 386 Ta-Chung 1st Road, Kaohsiung, Taiwan, R.O.C. Fax: 01 l-886-7-3468056; E-mail: [email protected]
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in the central cardiovascular regulation by reducing sympathetic outflow from the CNS. Specifically, distribution of NOS in the NTS has been demonstrated by histological studies (6), and local application of the NO precursor L-arginine in the NTS increased neuronal activity (78). Conversely, blockade of NO formation in the NTS resulted in an increase of blood pressure or activation of the sympathetic nervous system (9). In summary, these findings suggest that NO in the NTS plays an important role in the modulation of cardiovascular function. The NO signal transducing system has been extensively studied in recent years. In the CNS, NO can be formed by nNOS via a calciurn/calmodulin-dependent manner. The produced NO subsequently binds to soluble guanylyl cyclase (sGC), leading to the production of cGMP (10). A primary action of cGMP production is the stimulation of cGMP-dependent protein kinase (PKG) which then phosphorylates substrate proteins to exert a variety of actions (1 l).The NO signaling pathway participates in the regulation of neural functions such as neurotransmission, long-term potentiation, neurotoxicity, and seizure activity (12-14). However, cGMP-independent mechanisms are also reported to mediate the action of NO in the brain (15, 16). With regard to central cardiovascular regulation, we have reported that microinjection of L-arginine into the NTS of anesthetized rats produced the falls in blood pressure, heart rate and renal sympathetic nerve activity (17). However, L-arginine has been reported to elicit central cardiovascular actions by a NO-independent pathway (18). Furthermore, precise mechanisms underlying the cardiovascular effect of NO in the NTS remain controversial. Thus, although Lewis et al. (19) have shown that S-nitrosocysteine, a NO donor, produced hypotension and bradycardia when microinjected into the NTS, others proposed that this action is independent of the release of NO (20,21). Based on these findings, this study examined the possible mechanisms of action of Larginine in the NTS. Specifically, the purpose of this study was to determine whether the cardiovascular effects of L-arginine after microinjection in the NTS is mediated by NO production and cGMP-PKG signaling pathway within this area. In the present study, we firstly characterized the effects of L-arginine or D-arginine (an isomer of L-arginine) on blood pressure and heart rate in the rat when microinjected into the NTS. Secondly, we examined the effects of pretreatment with 7-nitroindazole (7-NI, a neuronal NOS inhibitor), N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W-7, a calmodulin inhibitor), LY83583 and lH-[1,2,4]oxadiazolo[4,3-alquinoxalin-l-one (ODQ) (inhibitors of guanylyl cyclase) on the L-arginine-induced cardiovascular responses in the NTS. Thirdly, the effects of pretreatment with N-(guanidinothy)-5isoquinolinesulfonamide hydrochloride (HA1004) (a cGMP-dependent protein kinase inhibitor) and l-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7, a protein kinase C inhibitor) on the cardiovascular responses to L-arginine were studied. Methods General methods Male Sprague-Dawley rats (weight 250-350 g) were obtained from National Animal Center (Taipei, Taiwan). Rats were initially anesthetized with combined urethane (400 mg/kg i.p.) and a-chloralose (40 mg/kg i.p.). 3 hours later, the status of anesthesia was checked every 2 hr by pinching the hind limbs of the rat. If the hind limbs withdraw while pinching, maintaining dose of urethane 100 mgikg (i.v.) was administered. A polyethylene cannula was placed in the femoral vein for drug administration. Blood pressure was measured directly through a cannula placed in the femoral artery and connected to a pressure transducer (Gould P23 ID) and polygraph (Gould RS3800). Heart rate was monitored continuously by a tachograph preamplifier (Gould 13-461565). Tracheostomy was performed to maintain airway patency during the experiment.
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Microinjection study For NTS microinjection, rats were placed in a stereotaxic instrument (Kopf), with head flexed downward at a 45” angle. The dorsal surface of the medulla was exposed by limited craniotomy, and animals were then allowed to be stabilized for at least 1 hr before experiments. Single-barrel glass cannulas were prepared (0.031-inch OD, 0.006-inch ID; Richland Glass Co.) that had external tip diameters of 40 pm. The cannulas were connected to a Hamilton microsyringe by a polyvinyl tubing. The injection site in the NTS for drug administration was decided at first by the responsiveness to microinjection of L-glutamate in this area. The glass cannulas were filled with L-glutamate (100 pmoV60nl) and lowered into the NTS with the coordinates anteroposterior 0.0 mm; mediolateral, 0.5 mm; and vertical, 0.4 mm, with the obex as the reference (22). Microinjections in the NTS were given over 10 seconds by air pressure. The minimal acceptable decreases in blood pressure and heart rate were 30 mmHg and 50 beats/min after microinjection of 0.1 nmol of L-glutamate into the NTS. According to our observations, the responsive area is restricted to the intermediate one-third of the NTS; conversely, the administration of the same dose of L-glutamate in adjacent areas failed to elicit the response (23). In the beginning of each experiment, we also assured that saline administered in the NTS did not elicit any effects on BP or HR due to the volume effect caused by injection. In this study, an animal received only several injections in the NTS and each injection volume was restricted to 60 nl. After finishing injection of an agent in the NTS, the single barrel cannula was lifted and washed by distill water at least 3 times. After that, it was filled with a subsequent drug and then reinserted into the NTS according to the coordinates formerly determined using Lglutamate. To adequately control the influence of the vehicles of various agents on the cardiovascular responses to L-arginine, the vehicles were injected in exactly the same protocol as were the active inhibitors. Thus, rats were injected firstly with L-arginine (10 nmol) into unilateral NTS, and the effects on BP and HR were monitored. Rats were then allowed to rest for at least 30 min until BP and HR returned to stable state. After that, L-arginine was administered 15 min after intra-NTS microinjections of either an inhibitor or its vehicle in the rat. In addition, the effects of vehicles of various inhibitors on BP and HR in the NTS were examined. After completion of experiments, 60 nl of sky blue was injected through the cannula, and animals were perfused with saline followed by a solution of 4% formaldehyde and a 30% sucrose solution. Sections of 40 pm of the brain stem structure were stained with cresyl violet for verifying microinjection sites in the NTS.
NADPH-diaphorase histochemistry To identify the distribution of NOS containing neurons in the NTS, coronal sections of 40 pm of the brain stem were stained for NADPH-diaphorase activity. Sections were then incubated at room temperature for 1-2 hr in a solution containing 50 mM Tris-HCl, 1 mM P-NADPH, 0.2 mM nitro blue tetrazolium and 0.2 % Triton X-100 (PH 8.0). Following several rinses in Tris buffer (pH 7.6), stained sections were mounted on gelatinized slides to dehydrate in alcohol and then coverslipped.
Chemicals and Statistical Analysis 7-NI (dissolved in methanol, 25% for 1 nmol and 5% for the other doses), LY83583 (dissolved in 5% methanol), W-7, HA1004 and H-7 were purchased from RBI (Natick, MA, USA). ODQ (from Sigma, St Louis, MO, USA) was first dissolved in dimehtyl sulphoxide (DMSO), and diluted to the final dose 1 pmol of ODQ present in the solution of 0.1% DMSO. P-NADPH, nitro blue tetrazolium, L-arginine (dissolved in the 0.9% sterile saline) and D-arginine (dissolved in the
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0.9% sterile saline) were purchased from Sigma. We did not observe significant effects on mean BP or HR after the administration of 5 %, 25% methanol or 0.1% DMSO in the NTS. These vehicles also did not influence the effects of L-arginine as described below. For statistical analysis, pair t (for within-group comparisons) or unpaired t tests (for betweengroup comparisons) were applied when significant main effects were noted using a one-way ANOVA. Differences with a p value less than 0.05 were taken as significant. All data are presented as mea&SEM. Results NOS-containing neurons in the NTS NOS-containing neurons were visualized using NADPH diaphorase (NADPH-d) histochemistry. A dark blue reaction product formed from reduced tetrazolium salts was detected in neuronal cell bodies and processes. A cluster of NADPH-d-stained cell bodies and processes occupied the rostral-caudal regions of NTS neurons. At obex level, moderate densities of NADPH-d staining cell bodies and processes were found to locate in the ventrolateral NTS, adjacent to the dorsal motor nucleus of vagus (Figure 1). According to our observation, these NADPH-d-positive sites generally concurred to the injection sites of L-arginine that produced profound cardiovascular responses.
nNOS in the L-arginine-induced cardiovascular responses in the NTS In this study, we firstly determined the cardiovascular effects of L-arginine (0.01-10 nmol) or Darginine (0.01-10 mnol) when microinjected into the NTS. The baseline MBP and HR were 105+3 mmHg and 318f9 bpm, respectively. Unilateral microinjections of L-arginine into the NTS induced dose-related depressor and bradycardic responses (Figure 2A). L-arginine at the highest dose (10 mnol) significantly decreased MBP and HR (-32+6 mmHg and -23f5 bpm, ~~0.05, vs. saline group, total n=18). According to our previous report (17), the cardiovascular responses elicited by 33 run01 L-arginine did not differ from that produced by 10 mnol of Larginine in this study. Thus, 10 nmol of .L-arginine seems to produce the near-maximal cardiovascular response in the NTS. To the contrary, microinjection of equivalent dose range of D-arginine, an isomer of L-arginine, did not evoke cardiovascular changes in the NTS (n=12, NS). We also observed that microinjection of L-arginine (10 nmol) into the NTS repeatedly at 15-min intervals still generate similar magnitude of cardiovascular responses with little tachyphylaxis phenomenon (Figure 2B). Furthermore, we investigated whether pre-injection with 7-N& a nNOS inhibitor, influenced the cardiovascular responses to L-arginine in the NTS. Firstly, microinjection of vehicle (25% methanol) did not alter the depressor and bradycardic effects of L-arginine (AMBP -28k5 mmHg, AHR -23+5 bpm). Pre-injection of 7-NI (20 pmol-1 mnol) produced a dose-related attenuation of the L-arginine-evoked depressor and bradycardic effects (Figure 3, ~0.05). 7-NI itself did not alter the basal MBP and I-IR (AMBP -2&3, AHR 6+3 bpm, n=12, NS). Caz+/calmodulin in the L-arginine-induced cardiovascular responses in the NTS To investigate whether the effect of L-arginine in the NTS links to intracellular Ca’+/calmodulin activity, the effect of the calmodulin inhibitor W-7 on the L-arginine-induced cardiovascular responses was explored. Pretreatment with W-7 (0.1 nmol) significantly reduced the depressor but not the bradycardia effect of L-arginine in the NTS (BP from -32+3 mmHg to -12+_3 mmHg,
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Fig. 1 (A) Representative graphs of injection sites in the brainstem of rats in coronal section 14.08 mm caudal to the bregma according to the atlas of Paxinos and Watson (22). Arrowhead indicates the site of injection in the NTS. (B) The NADPH-diaphorase staining cell bodies and processes in the ventrolateral region of the NTS. Higher magnification of the area indicated by the arrow on right side of the NTS is shown in the inset. Scale bars = 200 urn for (A) and (B), and 20 urn for inset. Abbreviations: DMV; dorsal motor nucleus of vagus, NTS; nucleus tractus solitarii.
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n=9, ~~0.05). Nevertheless, both the depressor and bradycardic effects of L-arginine were significantly attenuated by a higher dose (0.33 nmol) of W-7 (AMBP from -32f4 mmHg to -3f3 mmHg and AHR from -19+5 bpm to -12M bpm, respectively, pcO.05, n=8, Figure 4). The cardiovascular effects of L-arginine in the NTS recovered almost completely 90 min after W-7 administration. microinjection of W-7 (0.1-0.33 nmol) into the NTS alone did not alter basal BP but evoked a bradycardic effect (-15+8 bpm, ~~0.05, n=9).
-20 I Q
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influence on basal MBP, while evoking a small bradycardic effect (-18+8 bpm at 0.33 nmol, ~~0.05, n=8) that disappeared 15 min after injection. Recent studies reported that a new compound, 1-H-[1,2,4,]oxadiazolo[4,3-alquinoxalin-l-one (ODQ), is a selective and potent inhibitor of soluble guanylyl cyclase (24,25). Thus, we also examined the effects of pretreatment with ODQ on the L-arginine-elicited cardiovascular responses. Our results revealed that pretreatment with ODQ (0.03-l pmol) in the NTS significantly abolished the depressor and bradycardic effects of L-arginine in a dose-dependent manner (Figure 5B). Thus, 1 pin01 of ODQ significantly inhibited depressor and bradycardic responses elicited by 10 nmol L-arginine (AMBP: from -33+4 to -8E2 mmHg, AHR -30&5 to 9*5 bpm, respectively, ~~0.05, n=6). Full recovery of the action of L-arginine was observed 3 to 4 hr after ODQ (1 pmol) administration. On the other hand, microinjections of vehicles (0.1% DMSO) into NTS did not alter the cardiovascular responses to L-arginine, nor did evoke any change in basal MBP and HR (-2f3 mmHg and -8+3 bpm, NS, respectively). These data altogether demonstrated that LY83583 and ODQ, by way of inhibiting sGC activation, significantly reduced cardiovascular effects elicited by L-arginine in the NTS.
L-Arginine (10 nmol)
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Fig. 3 (A) Bar graph showing cardiovascular effects of L-arginine (L-Arg) in the NTS and their modification by 7-nitroindazole (7-N& a nNOS inhibitor). Baseline values were 95*4 mmHg for MAP and 292rtlO bpm for HR. *p
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L-Arginine (10 nmol) 0
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Fig. 5 (A) Bar graph showing the effects of LY83583 (0.01-0.33 nmol) pretreatment on the cardiovascular responses to the microinjection of L-arginine (L&g, 10 nmol) in the NTS. (B) The bar graph and tracings showing the effects of ODQ (0.03-l pmol) pretreatment on the cardiovascular responses to L-Arg (10 nmol). *p
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introduced a new compound, ODQ, which displays selective and potent inhibition of sGC activity in vitro. An in vivo study also revealed that ODQ acts as a selective and possibly competitive inhibitor of the sGC targeted by NO (25). Consistent with the effects of LY83583, pretreatment with ODQ significantly blocked the depressor and bradycardic effects produced by L-arginine in the NTS. Moreover, the results showed that the ability of ODQ in the attenuation of cardiovascular responses to L-arginine is more potent than that of LY83583. Thus, 1 pmol of ODQ markedly attenuated the response induced by L-arginine, whereas LY83583 did not show significant action until a higher dose (330 pmol) was applied. The primary action of elevated cGMP level in the cell is the stimulation of cGMP-dependent protein kinase (PKG), which subsequently phosphorylates substrate proteins to exert its actions. In the brain, the NO-cGMP-mediated PKG activation has been identified (34,35). Nevertheless, NO-cGMP signaling system also actively cross-talks with other signaling substrates and second messengers. For example, Maurice and Haslam (36) reported that NO-stimulated increases in cGMP could potentiate CAMP-mediated platelet inhibition by suppressing cyclic nucleotide phosphodiesterases. In the present study, we characterized the possible role of PKG in the NOmediated central cardiovascular regulation. Specifically, the effect of pre-injection of HA-l 004, a PKG inhibitor (37), on the cardiovascular responses to L-arginine in the NTS was examined. The results revealed that HA- 1004 pretreatment significantly attenuated the depressor and bradycardic effects produced by L-arginine. However, caution must be exercised when interpreting this data, because HA-1004 also affects other signal transducing molecules such as PKA or serves as a calcium antagonist (38). Notably, only low doses (0.1-l nmol) of HA-1004 were used in this study to prevent the non-selective actions elicited by this agent. Since HA-1004 is relatively selective for PKG in the lower dose range, the effects of HA-1004 observed here is more likely due to the inactivation of PKG. This view is supported by the finding that H-7, an inhibitor of PKC and PKA (39,40), did not block the L-arginine-induced cardiovascular responses. In contrast, H-7 has been shown to protect against the endotoxin lipopolysaccharide-induced NOS activation and the subsequent PKC formation (41). Taken together, these data support the view that PKG is involved in the cardiovascular responses evoked by NO production in the NTS. In summary, the results support the role of the NO-cGMP-PKG signaling pathway played in the cardiovascular responses to the microinjection of L-arginine in the NTS. Firstly, the cardiovascular effects produced by L-arginine rely on intracellular Ca2’/calmodulin-dependent nNOS activation. Secondly, cGMP formation and PKG activation is involved in the L-arginineinduced cardiovascular responses. Acknowledgments This work was supported by NSC 87-2314-B-075B-015, DOH87-HR-705 Dr. Ching-Jiunn Tseng. Portions of this work has been presented Neuroscience Meeting, October 25-30, 1997, New Orleans, U.S.A.
and VGHKS 86-67 to at the 27th Annual
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