| Background and ObjectiveRostral ventrolateral medulla (RVLM) is a key integrating center of sympathetic nerve activity and cardiovascular regulation. The tonic excitation of the presympathetic or premotor neurons in the RVLM, which project directly to intermediolateral column and are significant in cardiovascular activities control, is the basis of sympathetic nerve activity. Along with nucleus tractus solitarius (NTS), the RVLM is known as target site of central-acting antihypertensive drugs for its high density of I1-imidazoline receptors (I1Rs) andα2-adrenaline receptors (α2ARs).Moxonidine (MOX), a second generation central-acting drug, lowers blood pressure (BP) and heart rate (HR) by inhibiting sympathetic outflow through acting mainly I1Rs within the RVLM (the ratio of MOX-related affinity of I1R toα2AR is 40~70:1). Recently, some studies indicated that the nonspecific inhibition of endogenous NO production in the central nervous system would significantly attenuate the hypotension and bradicardia induced by clonidine or MOX. In addition, it was suggested that NO derived from iNOS is responsible for the central sympathoinhibition and neuronal nitric oxide synthase (nNOS) generated NO for sympathoexcitation in the RVLM. Furthermore, the"balance"between iNOS and nNOS was very important in maintaining hemodynamic stabilization.Through observing the effects of prior central inhibition of iNOS selectively on sympathoinhibition responses induced by MOX and quantifying the iNOS protein expression in the RVLM and the NTS after chronic MOX ICV infusion, it was determined that whether the cardiovascular inhibition of MOX is mediated by iNOS-NO mechanism in the RVLM.MethodsMale Sprague-Dawley (SD) rats were recruited as the experimental subjects. After differential doses of S-methylisothiourea (SMT), a specific iNOS inhibitor, were ICV injected (1nmol, 10 nmol, 100 nmol) or RVLM microinjected (20 pmol, 200 pmol), the effects of pretreatment of SMT on cardiovascular inhibition of MOX (ICV 20 nmol, RVLM 5 nmol or intravenous 50 g/kg, respectively) were studied. In addition, the effets of chronic ICV MOX (20 nmol/d, 2 weeks) infusion on the protein expression of iNOS in the RVLM and the NTS was determined by using immunohistochemistry and Western Blot techniques. In the current study, three independent experiments were performed: acute ICV injection of SMT, RVLM microinjection of SMT, chronic ICV infusion of MOX.1 Acute ICV injection of SMTThe MAP and HR of rats were continuously monitored. First of all, the artificial cerebrospinal fluid (aCSF, 5 l) or different does of SMT (1 nmol, 10 nmol, 100 nmol, 5 l) were ICV injected, and 30min later, MOX (20 nmol, 5 l) was administrated by the same way. Then, the effect of pretreatment with SMT on the moxonidine-induced cardiovascular inhibitory reponses was determined. Twenty-seven male SD rats were randomly divided into five groups: aCSF + aCSF (n=5),aCSF + MOX (n=5),SMT 100 nmol + MOX (n=6),SMT 10 nmol + MOX (n=6) and SMT 1 nmol + MOX (n=5).2 RVLM microinjection of SMT2.1 The MAP, HR and RSNA of rats were continuously monitored. Unilateral RVLM microinjection of aCSF (100 nl) or differential doses of SMT (20 pmol, 200 pmol, 100 nl) was performed followed by MOX (5 nmol, 100nl) microinjection at the same site 10min later. And the effect of SMT on the MOX-induced cardiovascular inhibitory reponses was determined. Nineteen male SD rats were randomly divided into four groups: aCSF + aCSF (n=4),aCSF + MOX (n=5),SMT 200 pmol + MOX (n=5) and SMT 20 pmol + MOX (n=5).2.2 Bilateal RVLM microinjection of aCSF (100 nl) or SMT (20 pmol or 200 pmol in 100 nl) was carried out followed by intravenous MOX (50 g/kg) administration 10min later. Then, the effect of pretreatment with SMT on the moxonidine-induced cardiovascular inhibitory reponses was determined. Sixteen male SD rats were randomly divided into three groups: aCSF + MOX (n=4), SMT 20 pmol + MOX (n=5), SMT 200 pmol + MOX (n=7).3 Chronic ICV infusion of MOXAfter a metal cannula was implanted into the lateral ventricle of each subject, aCSF (5 l) or MOX (20 nmol, 5 l) was daily ICV infused for 2 weeks. At the end of the experiment, MAP and HR were measured, then the brain was removed and the protein expression of iNOS within the RVLM and the NTS was quantified by immunohistochemistry and Western Blot techniques. Twenty male SD rats were randomly divided into tow groups: aCSF (n=10), MOX 20 nmol/d (n=10). Results1 Acute ICV injectionICV injection of MOX (20 nmol) following aCSF significantly reduced MAP (-22±3 mmHg) and HR (-58±13 bpm) (P<0.05). The hypotensive (-20±2 mmHg, -9±4 mmHg, -10±3 mmHg) and bradicardiac (-42±13 bpm, -18±9 bpm, -11±7 bpm) effects of MOX were does-dependently attenuated after SMT (1, 10, 100 nmol) administration.2 RVLM microinjection2.1 Unilateral RVLM microinjection of MOX (5 nmol) significantly reduced MAP (-25±3 mmHg), HR (-45±11 bpm) and RSNA (-44±7%) (P<0.05). Pretreatment with SMT (20, 200 pmol) in the same site significantly attenuated the decreases in MAP (-14+3 mmHg; -6±3 mmHg), HR (-20±7 bpm; -18±9 bpm) and RSNA (-23±4%; -11±3%) at the does-dependent manner (P<0.05).2.2 Intravenous injection of MOX (50 g/kg) significantly reduced MAP (-32±5 mmHg) and HR (-59±14 bpm) (P<0.05). The hypotensive effect (-15±2 mmHg) of MOX were significantly attenuated after bilateral microinjection of SMT (200 pmol at each side) (P<0.05), but the bradicardia (-54±16 bpm) was not significantly affected (P>0.05). Prior microinjection of SMT 20 pmol did not cause any significant effect on the MOX-induced responses (P>0.05).3 Chronic ICV injectionCompared to simple aCSF infusion, both baseline MAP (aCSF: 114±4 mmHg, MOX: 93±3 mmHg) and HR (aCSF: 406±8 bpm, MOX: 338±7 bpm) were significantly reduced after chronic administration of MOX (20 nmol/d for 2 weeks) (P<0.05). In addition, quantified by using immunohistochemistry and Western Blot techniques, iNOS protein expression within the RVLM was greatly upregulated (P<0.05). In the NTS, iNOS protein expression was not different between the two groups (P>0.05).ConclusionsCentral blockade of the endogenous release of NO derived from iNOS prevented the cardiovascular inhibition of MOX. Chronic ICV infusion of MOX selectively upregulated iNOS protein expression in the RVLM. Therefore, the NO mechanism generated by iNOS in the RVLM played an important role in mediating the cardiovascular inhibition of the centrally acting antihypertensive drug MOX.
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