Involvement of Nitric Oxide in Cardiorespiratory Regulation in the Nucleus Tractus Solitarii

Neuropharmacology,Vol. 35, No. 5, pp. 625-631, 1996 CopyrightCl 1996Elsevier Science Ltd. All rights reserved Printed in Great Britain 0028-3908/96$15.00 + 0.00

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Involvement of Nitric Oxide in Cardiorespiratory Regulation in the Nucleus Tractus Solitarii S. VITAGLIANO, L. BERRINO,* M. D’AMICO, S. MAIONE, V. DE NOVELLIS and F. ROSSI Institute of Pharmacologyand Toxicolo@,Faculty of Medicine and Surgery,2nd Universityof Naples, via CostantinopoIi16, 80138NapIes,Italy (Accepted21 November 1995) Summary-The aim of this studywas to investigatewhether nitric oxide (NO) is involvedin cardiorespiratory regulation in the nucleus tractus solitarii (NTS). Unilateral microinjections (50 nl) of the NO-donor,sodium nitroprusside (SNP, 40-100-200 mM), into the NTS of anaesthetized rats elicited dose-dependent apnea (7.3 * 2.3 see; 28.6 f 5.7 see; 35.6 t 6.4 see, respectively; n = 6) and a decrease in arterial blood pressure (8.4 * 3.1 mmHg; 18.2 f 5.8mmHg; 25.8 * 6.7nunHg, respectively; n = 6). Similarly, unilateral microinjections (50 nl) of another NO-donor, 3-morpholinosydnonimine(SIN-1, 20-40-100 mM), also induced apnea (5.1 t 2.4 see; 8.7 f 4.3 see; 26.3 f 6.4 see, respectively;n = 6) and a decreasein arterialblood pressure (6.2 f 2.3 mmHg; 11.123.3 mmHg; 18.3 * 6.1 mmHg, respectively; n = 6). The SNP- and SIN-l-induced apnea and arterial blood pressuredecreasewere significantly(p < 0.01) blocked by a 3 min pretreatmentwith two calcium-channelblockers, diltiazem (0.1 mM) and cobalt (10 mM), while lower doses (diltirtzem0.01 and cobalt 1)were ineffective.Microinjectionsof diltiazem (0.01 mM) and cobalt (1 mM) alone did not induce any change in basal cardiorespiratoryvalues, likediltiazem(0.1mM)andcobalt(10mM).Thesedatasuggestthat NO maybe involvedin NTScardiorespiratory regulationvia calcium-channel activation. Copyright 01996 Elsevier Science Ltd. Keywords-Nucleus tractus solitarii, nitric oxide, calcium-channelblockers.

Nitric oxide (NO) is a diffusible messenger, initially identifiedas being an endothelial-derivedrelaxing factor (EDRF) producing vasorelaxation of the vasculature (Rees et al., 1989).Accumulating evidence suggeststhat NO may play an important role as a neurotransmitterin the central nervous system (CNS) (Garthwaite, 1991; Snyder, 1992; Bredt and Snyder, 1992). NO synthase activity has been found in the adult forebrainas well as in the cerebellum and in other different areas of the CNS (Bredt et al., 1990; Forstermann et al., 1990). There is also evidence that NO plays a role in blood pressure regulation by reducing sympathetic outflow from the CNS. In fact, NO injections into both the ventrolateral medulla and the nucleus tractus solitarii (NTS) induced changes in sympathetic renal nerve activity and arterial blood pressure (Shapoval et al., 1991; Mollace et al., 1992; Togashi et al., 1992). The NTS is located in the dorsal medial part of the medulla oblongata, containing most of the known mammalian neurotransmitters involved in cardiovascular,respiratory and gastrointestinal

*Towhomcorrespondence shouldbe addressed.

regulation(Leone and Gordon, 1989;Kessler et al., 1991; Van Giersbergen et al., 1992). Moreover, it has been found that NO and calcium-channel activation via the NMDA receptor play a role in NTS neuronal firing (Harada et al., 1993;Vitagliano et al., 1994).The aim of this study was to investigatewhether NO plays a role in the NTS control of cardiorespiratory activity and the involvementof calcium-channelactivation. METHODS

All experiments were performed on male Sprague– Dawley rats, weighing 260-280 g and housed at constant temperature (21 f 1°C) and relative humidity (55 * 5%), under a regular lightdark schedule(light 7.00 a.m.–7.00 p.m.). Food and water were freely available. On the day of the experiment the animals were anaesthetized with urethane ethyl-carbamate(1.2 g/kg i.p.). Rectal temperature was monitoredand maintainedbetween 37 and 38°C by an infrared heating lamp. The trachea was cannulated and respiratory activity (tidal volume, Vf) was recorded by a pneumotachograph connected to a poligraph (Hellige, mod. 218087, Germany). To record arterial blood pressure a femoral artery was cannulated and

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diameter outside tip, pulled by a verticle pipette puller (David Kopf Instruments, Tujunga, CA, U.S.A.), was stereotaxicallyplaced into the NTS using the following coordinates from the obex: mediolateral 0.9 mm, anteroposterior 0.5 mm and dorsoventral 0.8 mm. Micropipettes were carried in a stereotaxic micromanipulator and connected by a polyethylene PE 10 tubing to a syringe. All drugs were dissolved in saline with 1% fast green and pH was adjusted to 7.0 by (0.1 N) NaOH and HC1 solutions. SNP was administered at 40-100-200 mM doses in 50 nl; SIN-1 20-40-100 mM in 50 nl; diltiazem 0.01-0.1 mM in 50 nl; and cobalt 1–10mM in 50 nl. Sodium nitroprusside, diltiazem hydrochloride, cobalt chloride and fast green were purchased from Sigma Chemical Co., St Louis, MO, U.S.A.; 3-morpholinosydnoniminehydrochloride (SIN1) was kindly provided by Prof. Massimo Di Rosa, Naples, Italy. Control microinjections were carried out with the same volume of solution used to dissolve the connected to the poligraph. Unilateral microinjections drugs. The saline vehicle elicited no changes in arterial were carried out with the rats held in a stereotaxicframe blood pressure and respiratory activity (Tables 1–5). (David Kopf Instruments, Tujunga, CA, U.S.A.). A Microinjection sites were identified at the end of each cervical incision was made and the neck muscles experiment (Fig. 1). Data were expressed as the mean f dissected for the microinjections into the NTS; the standard error (SE). Statistical analysis was performed atlanto-occipital membrane was then cut to expose the using student’st-test and a P value of less than 0.01 was 4th ventricle at the obex level. A double glass micro- consideredsignificant.All experimentswere approvedby pipette (FHC, Brunswick, ME, U.S.A.) with a 40-50 pm the Animal Care and Use Committee of our Institution.

Table 1. Effects of unilateral microinjectionsof SNP (4O-1OW2OO mM) and SIN-1 (20-40-100 mM) on respiratory activity and arterial blood pressure in the NTS of anesthetized rats

Treatment

Number of animals

Apnea duration (see t SE)

Saline SNP 40 SNP 100 SNP 200 SIN-1 20 SIN-1 40 SIN-1 100

6 6 6 6 6 6 6

7,3 ~ 2.3* 28.6 t 5.7* 35.6 f 6.4* 5.1 ? 2.4* 8.7 f 4.3* 26.3 ~ 6.4*

Maximal decrease of arterial blood pressure (mmHg t SE)

lfl

0

8.4 ? 3.1* 18.2 ? 5.8* 25.8 ? 6.7* 62 ~ 2.3* 11.1 * 3.3* 18.3 t 6.1*

“p <0.01 vs saline. Table 2. Effects of a 3 min pretreatmentwith diltiazem (0.01-0.1 mM) on respiratoryand cardiovasculareffects inducedby SNP (40-100-200 mM) in the NTS of anesthetized rats

Treatment

Number of animals

Saline SNP 40 SNP 100 SNP 200 Diltiazem 0.01 Diltiazem 0.1 SNP 40 + D 0.01 SNP 40 + D 0.1 SNP 100 + D 0.01 SNP 100 + D 0.1 SNP 200 + D 0.01 SNP 200 + D 0.1 “p <0.01 vs saline;

6 6 6 6 6 6 6 6 6 6 6 6 **p <0.01

vs

SNP alone.

Apnea duration (see t SE)

0 7.3 ? 2.3” 28.6 ? 5.7” 35.6 z 6.4*

o 0

7.8 * 2.3*

O**

29.5 * 4.6*

o**

34.7 ? 5.8*

o**

Maximal decrease of arterial blood pressure (mmHg * SE) lfl

8.4 * 3.1* 18.2 * 5.8* 25.8 * 6.7* 2*1 3*1 8.3 f 3.6*

o**

18.3 * 5.4* O** 26.2 ? 6.5*

o**

Involvement of NO in cardiorespiratory regulation

627

2

3’

1

Vt(ml) o

‘r

200ABP

(mmHg) 100-

3’

0t SNP Fig. 2. Effects of two microinjections of SNP (40 mM) into the NTS on respiratory activity and arterial blood pressure.

RESULTS

cobalt were reversible after a recovery time of about 45 min. Lower doses of diltiazem (0.01 mM) and cobalt As summarizedin Table I, unilateral microinjections (1 mM) did not antagonize SNP- and SIN-l-induced of the NO-donors, SNP (40-10&200mM) and SIN-1 apnea and hypotension. Injections of these calcium(20-40-100mM) into the NTSelicited dose-dependent blockers (diltiazem 0.014.1 mM and cobalt 1–10mM) apnea and a decrease in arterial blood pressure. All into the NTS did not elicit any changes in the basal animals recovered from the apnea and hypotension. In breathing rate, tidal volume and arterial blood pressure Fig. 2 it is shown that two successiveand equal doses of (Tables 2-5 and Figs 3-5). SNP (40 mM) elicited the same cardiorespiratoryeffects, excluding NO desensitation or tachyphylaxis.A 3 min pretreatmentwith diltiazem (0.1 mM) or cobalt (10 mM) DISCUSSION in the NTS significantly (p < 0.01) antagonized the The aim of this study was to investigate whether NO cardiorespiratoryeffects inducedby both SNP and SIN-1. (Tables 2-5 and Figs 3-5). The effect of diltiazem and plays a role in NTS cardiorespiratorycontrol and if it acts

Table 3. Effects of a 3 min pretreatment with cobalt (1–10mM) on respiratory and cardiovasculareffects inducedby SNP (40-100-200 mM) in the NTS of anesthetized rats

Treatment Saline

SNP40 SNP100 SNP200 Cobalt 1 Cobalt 10 SNP 40 +

CO 1 SNP40+ co 10 SNP100+ co 1 SNP 100+ Co 10 SNP200+ CO 1 SNP200+ co 10

Number of animals

6 6 6 6 6 6 6 6 6 6 6 6

vs SNP alone. *p <0.01 vs saline; **p<0.01

Apnea duration (see k SE)

0 7.3? 2.3* 28.6 t 5.7* 35.6 ? 6.4* o 0 7.5? 2.4* ()** 27.9 ? 5.4* ()** 35.6 t 6.8* O**

Maximal decrease of arterial blood pressure (mmHg t SE) 111 8.4 ~ 3.1* 18.2 ~ 5.8* 25.8 * 6.7* 1~1 z~l 9.1 * 2.2* ()** 20.4 t 4.5* O** 26.62 6.2* O**

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t

200-1

ABP (mmHg) 100-

5’

3’ Ii!!!!!

liiiiil

~diltiazcm ~ SNP Fig.3. Effectsof a pretreatmentof diltiazem(0.1mM),intotheNTSon apneaandhypotensioninducedby SNP(200mM).

via calcium conductance. Our results,in agreementwith other studies (Harada et al., 1993),are consistentwith the hypothesisthat NO regulatescardiorespiratoryactivityin the NTS. In fact, microinjectionsof the NO-donors(SNP and SIN-1) into the NTS of anesthetized rats elicited apnea and hypotension that were prevented by calciumchannel blockers (diltiazem and cobalt). Harada et al. (1993) speculated that an inhibitorof NO formation,NGmonomethyl-L-arginine (L-NMMA), increased arterial blood pressure and renal sympathetic nerve activity by inhibitingthe neuronaleffect of glutamateon the NMDA subtype of receptor within the NTS. This viewpoint is based on previous findings in the cerebellum showing that NO formation is coupled with NMDA receptor

activation (Garthwaite et al., 1991). Our study could suggestthat the apnea and hypotensionelicitedby the NO derived from SIN-1 and SNP mimics NMDA receptor activation in the NTS. Moreover, there is evidence that NMDA receptors control a respiratory off-switch in the cat NTS and that this may be considered a phasic inhibition(Fouts et al., 1988).Diltiazem and cobalt have no effects per se (Tables 2–5) probably because they would interfere with phasic inhibition of NTS glutamatergic neurons.From this point of view, our data seem, at least in part, in disagreementwith the results of Harada and coworkers (1993) who demonstrated a tonic modulatory role within the NTS for NO. Therefore, our data suggest that calcium channels may be involved in

Table4.Effects of a 3 min pretreatmentwith diltiazem (0.01-0.1 mM) on respiratoryand cardiovasculareffects inducedby SIN-1 (20-40-100 mM) in the NTS of anesthetized rats

Treatment Saline SIN-1 20 SIN-1 40 SIN-1 100 Dihiazem 0.01 Diltiazem 0.1 SIN-1 20 + D 0.01 SIN-1 20 + D 0.1 SIN-1 40 + D 0.01 SIN-1 40 + D 0.1 SIN-1 100 + D 0.01 SIN-1 100 + D 0.1

Number of animals 6 6 6 6 6 6 6 6 6 6 6 6

*p<0.01vs saline; **p<0.01vs SIN-1 alone.

Apnea duration (see t SE)

0

Maximal decrease of arterial blood pressure (mmHg * SE)

lfl

5.1 t 2.4* 8.7 f 4.3* 26.3 Y 6.4*

6.2 ? 2.3* 11.1 t 3.3* 18.3 * 6.1*

5.9 * 1.8*

7.1 * 2.2*

9.9 ~ 3.6*

12.2 t 4.2*

0 0

o** o**

25.7 ? 5.9* ()**

1*1 2*1

O** O**

19.6 ? 6.7*

o*”

Involvement of NO in cardiorespiratoryregulation

I 1–1

I

2-

k!!!!!!

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1Vt (ml) o-

I

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629

(mmHg) 100~

I

0-1

? SIN-1

3’

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~

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diltiazem

1

I

? SIN-1

Fig. 4. Effects of a pretreatment of diltiazem (0.1 mM), into the NTS on apnea and hypotensioninduced by SIN-1 (20 mM).

modulatingphasic NO/NMDA-inducedcardiorespiratory changes, but not the tonic control of these neurovegetative functions. In a previous study we showed that the local application of cobalt chloride (100 mM), a divalent cation commonly accepted as a Ca2+-antagonist(Krmeko and Tachibana, 1986), in the NTS reversibly blocks NMDA-induced apnea and hypotension (Vitagliano et al., 1994). The results of the present study are also consistent with the hypothesis that in the NTS NO, like NMDA receptor stimulation, could activate calciumchannels. In fact, we observed that pretreatment with diltiazem, a typical Ca2+-antagonistdrug (Murata et al., 1992), and cobalt inhibited the apnea and hypotension elicited by SIN-1 and SNP. Moreover,it has been shown

that NO can inducerelease of neurotransmittersacting on presynaptic axons (Halbrugge et al., 1991; Prast and PhiIippu, 1992; Lonart et aZ.,1992), as in the hippocampus to induce long-term potentiation (LTP) (Bohme et al., 1991; Haley et al., 1992; Schuman and Madison, 1991). Therefore, we suggest that NO depolarizes presynaptic axons thus eliciting calcium-channelactivation (Pocock et al., 1993; Wiegand et al., 1990); consequently it may cause a release of several neurotransmitters in the synapse such as L-glutamateor Ach that elicit apnea and hypotensionin the NTS (Criscioneet al., 1983;Morin-Surunet al., 1984;Leone et al., 1989).It follows that the application of diltiazem and cobalt, like other calcium blocking agents (nimodipine, nifedipine

Table 5. Effects of a 3 min pretreatmentwith cobalt (1-10 mM) on respiratory and cardiovasculareffects inducedby SIN-1 (20-40-100 mM) in the NTS of anesthetized rats

Treatment Saline SIN-1 20 SIN-1 40 SIN-1 100 Cobalt 1 Cobalt 10 SIN-1 20 + CO 1 SIN-1 20 + Co 10 SIN-1 40 + CO 1 SIN-1 40 + CO 10 SIN-1 100 + co 1 SIN-1 100 + Co 10

Number of animala 6 6 6 6 6 6 6 6 6 6 6 6

*p <0.01 vs saline; **p <0.01 vs SIN-1 alone.

Apnea duration (see * SE) 0

5.1 f 2.4* 8.7 ~ 4.3* 26.3 * 6.4* 0 0

5.4 ~ 2.3* O“*

8.8 ? 4.1* o** 26.7 z 5.8* O**

Maximal decrease of arteriat bkwd pressure (mmHg f SE) 1*1

6.2 * 2.3* 11.1 f 3.3* 18.3 f 6.1” ~~1 1*1

6.7 * 2.3” o**

12.2 * 3.4* O** 17.9 * 6.7* O**

630

S. Vitagliano et al. Iosrc

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2’

1

3’

Vt (ml)

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2’

(mmHg) 100-

~’

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~ Cobalt ? SIN-I ? SIN-1 Fig. 5. Effects of a pretreatment of cobalt (10 mM), into the NTS on apnea and hypotensioninduced by SIN-1 (100 mM).

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