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Possible participation of spinal nitric oxide in the control of the blood pressure in anesthetized rats
Brain Research 764 Ž1997. 67–74
Research report
Possible participation of spinal nitric oxide in the control of the blood pressure in anesthetized rats Marıa ´ del Carmen Garcıa ´ a
a,b,)
, Stella Maris Celuch b , Edda Adler-Graschinsky
b
Catedra de Farmacologıa, UniÕersidad de Buenos Aires, Buenos Aires, Argentina ´ ´ Facultad de Farmacia y Bioquımica, ´ b (CONICET), Junın Instituto de InÕestigaciones Farmacologicas ´ ´ 956, 58 Piso, 1113 Buenos Aires, Argentina Accepted 25 March 1997
Abstract In pentobarbital-anesthetized rats the intrathecal Ži.t.. injection of the nitric oxide ŽNO. precursor, L-arginine Ž10 and 20 m mol., elicited a decrease in the mean blood pressure ŽMBP. whereas the inhibitor of the NO synthase ŽNOS. N G-nitro-L-arginine methyl ester ŽL-NAME; 0.1–10 m mol. produced a dose-dependent pressor effect. The pressor response to L-NAME was prevented by pretreatment with L-arginine. Neither D-arginine nor D-NAME modified the MBP. The NO donor sodium nitroprusside ŽSNP; 0.125 and 0.25 m mol, i.t.. induced a hypotensive response followed by a pressor effect. The dual response to SNP as well as the hypotensive effect of L-arginine were abolished by the guanylate cyclase inhibitor Methylene blue Ž0.3 m mol, i.t... Nicotinic ganglionic blockade by hexamethonium Ž10 mgrkg, i.v.. reduced the hypotensive effects of both L-arginine and SNP and prevented almost completely the pressor effects of either L-NAME or SNP. The pressor effect of L-NAME was abolished by 2-amino-5-phosphonovaleric acid ŽAPV; 30 nmol, i.t.., a selective antagonist of glutamate receptors of the NMDA subtype. These results suggest that in the spinal cord of pentobarbital-anesthetized rats NO exerts both inhibitory and excitatory effects on the preganglionic sympathetic nerve activity related to the control of the BP. The synthesis of NO appears to be tonically activated through the stimulation of spinal glutamate receptors of the NMDA subtype. q 1997 Elsevier Science B.V. Keywords: Nitric oxide; Spinal cord; Blood pressure; L-Arginine; N G -Nitro-L-arginine methyl ester ŽL-NAME.; Sodium nitroprusside; Methylene blue
1. Introduction There is considerable evidence that nitric oxide ŽNO., synthetized from L-arginine in the presence of NO synthase ŽNOS. w21,30x, is involved in the central regulation of the cardiovascular functions in several species such as the rat w27,28,32x, the cat w17,33x, the dog w29x and the rabbit w16x. In the nucleus tractus solitarius, that is the first relay station in the baroreflex arc, NO elicites decreases in the renal sympathetic nerve activity, blood pressure ŽBP. and heart rate ŽHR. w16,23,39x. In addition, NO induces inhibitory effects on renal sympathetic nerve activity, BP and HR through its action on the rostral ventrolateral medulla w33,39,44x and the paraventricular nucleus of the hypothalamus w18x. These studies suggest that NO could have in the brain an overall inhibitory effect on the sympathetic output.
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Corresponding author. Fax: q54 Ž1 . 963-8593; E-mail: [email protected] 0006-8993r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 0 4 2 1 - 6
Preganglionic sympathetic neurons from several mammals w1,5,7,10,31,40x exhibit NOS immunoreactivity as well as a marker for neuronal NOS, the nicotinamide dinucleotide phosphate diaphorase. Moreover, it has been found that sympathetic preganglionic neurons from spontaneously hypertensive rats possess lower NOS activity than the normotensive controls w38x. Although these studies suggest that spinally synthetized NO could regulate the sympathetic nerve activity, the role of this messenger in the modulation of the BP at the level of the spinal cord is not yet fully understood. Feldman et al. w11x observed that the intrathecal Ži.t.. injection of a specific inhibitor of NOS, N G -nitro-L-arginine methyl ester ŽL-NAME., increases the BP of urethane-anesthetized rats and suggested that this effect could be due to a tonic inhibitory action of spinal NO on the sympathetic nerve activity. On the other hand, Lee et al. w22x reported that in urethane-anesthetized rats, the intrathecal injection of L-NAME causes a dose-dependent decrease in the BP whereas the NO donor, sodium nitroprusside ŽNPS., produces pressor effects. These authors proposed that in the spinal cord NO plays a tonic
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excitatory role on the central control of the BP. Moreover, an excitatory action of NO on renal sympathetic preganglionic neurons has been demonstrated by Hakim et al. w15x in the spinal cord of pentobarbital-anesthetized rabbits. The present study was undertaken to further investigate the role of NO in the central control of the BP at the level of the spinal cord in pentobarbital-anesthetized rats. Our results suggest that NO induces excitatory as well as inhibitory effects on the sympathetic preganglionic activity related to the regulation of the BP.
recording made just before the beginning of the drug injection and the value at a given time. 2.3. Intrathecal injection of drugs Drugs and saline solution were injected i.t. in volumes no greater than 4 m l using a 10 m l Hamilton microsyringe. The rate of injection was 1 m lrmin. For bolus i.v. administration of drugs the volume of injection was 0.1 ml and the cannula was flushed with 0.1 ml saline solution. 2.4. Experimental protocols
2. Materials and methods 2.1. Surgical procedures Procedures for the evaluation of cardiovascular effects of intrathecal Ži.t.. injection of drugs were similar to those previously described w13x. Wistar rats of either sex Ž180– 230 g. were anesthetized with sodium pentobarbital. The animals received an initial dose of sodium pentobarbital Ž40 mgrkg, i.p.., supplemented with 20 mgrkg Ži.p.. before introduction of the i.t. catheter. An additional dose Ž20 mgrkg. was given during the experiment when necessary. The depth of anesthesia Žsurgical plane. was monitored by the absence of the eyelid reflex. Although the animals breathed spontaneously, the trachea was cannulated to avoid respiratory disorders related to the accumulation of secretions at the superior airways. A polyethylene cannula was placed in the right femoral artery for recording of the blood pressure ŽBP.. In some experiments, a femoral vein was cannulated for i.v. injection of drugs. While recording the BP, a cannula was positioned at the level of the T12 –L 1 intervertebral space as described by Dib w9x. Briefly, a cannula Žo.d. 0.65 mm. was inserted into the subarachnoid space at the level of the C 8 –T1 vertebrae and gently pushed downward 4.5 cm. The position of the cannula was verified post mortem by opening ventrally the vertebrae to localize the cannula tip. The rectal temperature was maintained at 37–388C by a heating lamp. 2.2. Blood pressure recording and heart rate calculation The BP was measured from the right femoral artery via a Statham P23 1D transducer and recorded on a Grass 7B polygraph ŽQuincy, MA, USA.. The mean BP ŽMBP. was calculated from the formula: diastolic pressureq 1r3 Ž systolic pressurey diastolic pressure .. The heart rate ŽHR. was calculated from the BP record. The BP and HR were allowed to stabilize for at least 30 min before starting the experiment. Changes in MBP and HR induced by i.t. or i.v. injection of drugs refer to the differences between the
Unique doses of either N G-nitro-L-arginine methyl ester ŽL-NAME. or N G -nitro-D-arginine methyl ester ŽD-NAME. or L-arginine or D-arginine or sodium nitroprusside ŽSNP. were injected i.t. The BP and HR were recorded just before injection Žtime 0. and at the following times after the beginning of the injection: 1, 1.5, 2, 4, 6, 8, 10, 15, 20, 25 and 30 min. In some animals, L-NAME Ž2.5 m mol. was administered 5 min after i.t. injection of either L-arginine Ž5 m mol. or 2-amino-5-phosphonovaleric acid ŽAPV; 30 nmol.. In other experimental groups, the animals received either SNP Ž0.25 m mol. or L-arginine Ž10 m mol. 5 min after pretreatment with Methylene blue Ž0.30 m mol, i.t... To analyze the effects of nicotinic ganglionic blockade on the cardiovascular responses to either L-NAME Ž10 m mol, i.t.. or L-arginine Ž10 m mol, i.t.. or SNP Ž0.25 m mol, i.t.., the latter drugs were injected 5 min after i.v. injection of hexamethonium bromide Ž10 mgrkg.. When the drugs to be administered were L-arginine or SNP, a continous i.v. infusion with phenylephrine Ž130 m grml, rate: 0.2–0.3 m grmin. was started immediately after the bolus i.v. injection of hexamethonium and was maintained up to the end of the experiment. 2.5. Drugs 2-Amino-5-phosphonovaleric acid, L-arginine, Darginine, hexamethonium bromide, N G-nitro-L-arginine methyl ester, N G-nitro-D-arginine methyl ester, phenylephrine HCl and sodium nitroprusside were obtained from Sigma Chemical Co. ŽUSA.. Methylene blue was obtained from Merck Quımica Argentina ŽARGENTINA.. All drugs ´ were daily dissolved in saline solution. Drug doses refer to their respective free bases. 2.6. Statistics All values represent the mean " S.E.M.. Statistical differences were assessed either by Student’s t-test or by one-factor analysis of variance followed by the Newman– Keuls test. P values smaller than 0.05 were regarded as significant.
M.d.C. Garcıa ´ et al.r Brain Research 764 (1997) 67–74
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3. Results 3.1. CardioÕascular responses induced by intrathecal injection of L-arginine and L-NAME The biosynthetic precursor of NO, L-arginine, induced a decrease in the MBP at doses of 10 and 20 m mol Ži.t.. whereas a lower dose of the drug Ž5 m mol, i.t.. was devoid of effect. The hypotensive response to 20 m mol L-arginine was accompanied by a slight but significant bradycardia ŽFig. 1.. On the other hand, the changes in MBP and HR produced by i.t. injection of 10 m mol D-arginine Žy1.1 " 0.6 mmHg and y5 " 6 beatsrmin; n s 6. did not differ from the maximal changes induced by i.t. injection of saline solution Žy2.3 " 0.8 mmHg and 5 " 3 beatsrmin; n s 5.. The time-course of the hypotensive effect of 10 m mol L-arginine Ži.t.. is summarized in Fig. 2. The decrease in the MBP was maximal 6–10 min after the beginning of the injection and persisted for at least 30 min. The decrease in
Fig. 2. Time-course of the decrease in the mean blood pressure Ž D MBP, mmHg. induced by L-arginine Ž10 m mol, i.t... Effects of Methylene blue. L-Arginine was injected in 4 min, starting at time 0, either under control conditions Ž`. or in the presence of Methylene blue Žv .. Methylene blue Ž0.3 m mol, i.t.. was injected 5 min before the beginning of i.t. administration of L-arginine. The hatched horizontal bar represents the effect of i.t. injected saline solution. Shown are mean values"S.E.M. for 5 animals per group. a P - 0.01 vs. saline solution and vs. Methylene blue plus L-arginine Žanalysis of variance followed by Newman–Keuls test.. The resting MBP ŽmmHg.rHR Žbeatsrmin. values were: 106.7"2r390"9 in the saline solution group, 98.7"3.4r384"14 in the L-arginine group and 97.7"4.8r382"13 in the Methylene blue plus L-arginine group.
MBP elicited by L-arginine was entirely prevented by an inhibitor of the soluble guanylate cyclase, Methylene blue Ž0.3 m mol; i.t... The NOS inhibitor, L-NAME Ž0.1–10 m mol, i.t.., induced a dose-dependent pressor effect that was accompanied by bradycardia ŽFig. 3.. On the other hand, i.t. injection of D-NAME Ž10 m mol. did not modify the MBP and HR. The maximal changes induced by the drug on these parameters Žy1.3 " 1.9 mmHg and y6 " 7 beatsrmin, respectively; n s 5. did not differ from those induced by saline solution. The time-course of the pressor response to L-NAME Ž2.5 m mol, i.t.. is summarized in Fig. 4. The increase in MBP reached a maximum 8–10 min after the beginning of the injection and persisted for at least 30 min. The pressor effect of L-NAME ŽFig. 4. was abolished by pretreatment with a dose of L-arginine Ž5 m mol, i.t.., which per se did not modify the resting MBP Žsee Fig. 1..
Fig. 1. Maximal changes in mean blood pressure Ž D MBP, mmHg. and heart rate Ž D HR, beatsrmin. induced by i.t. injection of L-arginine. The drug was injected in volumes not greater than 4 m l. The hatched bar represents the effect of i.t. injected saline solution. Shown are mean values"S.E.M. for 4–5 animals per dose. a P - 0.01 vs. saline solution ŽStudent’s t-test.. The resting MBP ŽmmHg.rHR Žbeatsrmin. values were: 106.7"2.0r390"9 in the saline solution group; 105.0"4.7r386 "5 in the 5 m mol L-arginine group, 98.7"3.4r384"14 in the 10 m mol L-arginine group and 99.3"7.1r380"5 in the 20 m mol L-arginine group.
3.2. Effects of a glutamate receptor antagonist on the pressor response induced by L-NAME Since there is evidence that in the central nervous system NO production is linked to the activation of postsynaptic glutamate receptors of the N-methyl-D-aspartate ŽNMDA. subtype w6,14x, we determined whether 2-amino5-phosphonovaleric acid ŽAPV., a selective antagonist for NMDA receptors, modified the pressor response induced by L-NAME. As shown in Fig. 5, the increase in MBP
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Fig. 4. Time-course of the increase in the mean blood pressure Ž D MBP, mmHg. induced by N G -nitro-L-arginine methyl ester ŽL-NAME.. Effects of L-arginine. L-NAME Ž2.5 m mol, '. and saline solution Ž4 m l, n. were i.t. injected in 4 min, starting at time 0. L-Arginine Ž5 m mol. was i.t. injected 5 min before the beginning of the injection of either L-NAME Žv . or saline solution Ž`.. Shown are mean values "S.E.M. for 5 animals per group. a P - 0.05 vs. the corresponding saline control values; b P - 0.05 vs. the corresponding values in the L-arginine plus L-NAME group Žanalysis of variance followed by Newman–Keuls test.. The resting MBP ŽmmHg.rHR Žbeatsrmin. values were: 106.7"2.0r390"9 in the saline solution group, 98.3"5.4r390"7 in the L-arginine plus saline solution group, 96.7"5.1r382"7 in the L-NAME group and 96.7" 4.5r394"18 in the L-arginine plus L-NAME group. Fig. 3. Maximal changes in mean blood pressure Ž D MBP, mmHg. and heart rate Ž D HR, beatsrmin. induced by i.t. injection of N G -nitro-Larginine methyl ester ŽL-NAME.. The drug was injected in volumes not greater than 4 m l. The hatched bar represents the effect of i.t. injected saline solution. Shown are mean values"S.E.M. for 4–5 animals per dose. a P - 0.01 vs. saline solution ŽStudent’s t-test.. The resting MBP ŽmmHg.rHR Žbeatsrmin. values were: 106.7"2.0r390"9 in the saline solution group; 95.0"3.9r377"17 in the 0.1 m mol L-NAME group, 99.1"5.7r397"12 in the 1 m mol L-NAME group, 96.7"5.1r382"7 in the 2.5 m mol L-NAME group, 102.2"4.9r363"22 in the 5 m mol L-NAME group and 103.3"7.8r396"13 in the 10 m mol L-NAME group.
produced by i.t. injection of L-NAME Ž2.5 m mol. was abolished after pretreatment with APV Ž30 nmol, i.t... The NMDA receptor antagonist induced per se, 5 min after its i.t. injection, a decrease in the resting MBP Žy9.0 " 2.7 mmHg; n s 8. that was significantly different from that induced by the i.t. administration of saline solution Žy1 " 1 mmHg; n s 5; P - 0.05.. 3.3. CardioÕascular responses induced by intrathecal injection of sodium nitroprusside Sodium nitroprusside ŽSNP: 0.125 and 0.25 m mol, i.t.. induced biphasic effects on the MBP ŽFig. 6.. The first phase of the response was a transient decrease in the MBP that lasted less than 4 min and reached its maximum 1–2 min after the beginning of the injection. This hypotensive effect was followed by a long-lasting increase in the MBP
Fig. 5. Maximal changes in MBP Ž D MBP, mmHg. induced by i.t. injected N G -nitro-L-arginine methyl ester ŽL-NAME.. Effects of 2-amino5-phosphonovaleric acid ŽAPV.. Intrathecal injection of either L-NAME Ž2.5 m mol. or saline solution was performed in control animals and in animals pretreated with APV. APV Ž30 nmol. was i.t. injected 5 min before the beginning of the i.t. administration of either L-NAME or saline solution. Shown are mean"S.E.M. for 4–5 animals per group. a P 0.001 vs. the corresponding control value ŽStudent’s t-test.. The resting MBP ŽmmHg.rHR Žbeatsrmin. values in the controls were: 106.7" 2r390"9 for saline solution and 96.7"5.1r382"7 for L-NAME. The resting MBP ŽmmHg.rHR Žbeatsrmin. values in APV-treated animals were: 104.3"3.9r406"9 for saline solution and 95.0"6.6r416"18 for L-NAME.
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71
3.4. Effects of blockade of nicotinic ganglionic transmission on the cardioÕascular responses to intrathecal injection of L-arginine, L-NAME and sodium nitroprusside To rule out the possibility that the cardiovascular responses induced by i.t. injected L-NAME, L-arginine and SNP had resulted from peripheral effects due to leakage of the drugs from the site of injection, their effects on MBP were analyzed in rats pretreated with a ganglionic blocking agent, hexamethonium Ž10 mgrkg, i.v... To preclude the possibility that the decrease in the baseline MBP in the presence of hexamathonium could mask the hypotension that is caused by L-arginine and SNP, for these experimental groups the animals received, in addition to the bolus i.v. injection of hexamathonium, a continous infusion of phenylephrine Ž0.2–0.3 m grmin.. Under the latter conditions the baseline MBP values after hexamethonium did not differ from those found in non-pretreated animals ŽTable 1.. As shown in Table 1, hexamethonium pretreatment reduced by 80% the pressor responses induced by L-NAME Ž10 m mol; i.t.. and SNP Ž0.25 m mol; i.t... Regarding the hypotensive responses, they were abolished in the case of L-arginine and reduced by 50% approximately in the case of SNP.
Fig. 6. Changes in mean blood pressure Ž D MBP, mmHg. induced by i.t. injection of sodium nitroprusside ŽSNP.. Effects of Methylene blue. Sodium nitroprusside Ž =, 0.125 m mol; `, 0.25 m mol. was injected in 1 min, starting at time 0. v, Methylene blue Ž0.3 m mol; i.t.. was injected 5 min before SNP Ž0.25 m mol.. The hatched bar represents the effect of i.t. injected saline solution. Shown are mean values"S.E.M. for 4–5 animals per group. a : P - 0.05 vs. saline solution, b P - 0.05 vs. the corresponding values in the Methylene blue plus SNP group Žanalysis of variance followed by Newman–Keuls test.. The resting MBP ŽmmHg.rHR Žbeatsrmin. values were: 100.3"3.0r395"11 in the saline solution group; 99.2"6.3r407"20 in the 0.125 m mol SNP group and 94.7" 7.9r402"13 0.25 m mol in the SNP group; 98.3"4.4r394"23 in the Methylene blue plus SNP group.
that peaked at 8–10 min after the beginning of the injection and persisted at least for 30 min. The changes in the MBP induced by SNP were dose-dependent. As also shown in Fig. 6, the hypotensive as well as the hypertensive responses induced by 0.25 m mol SNP were prevented by Methylene blue Ž0.3 m mol, i.t... The maximal changes in HR induced by 0.125 m mol SNP Žy5 " 5 beatsrmin; n s 4. and by 0.25 m mol SNP Žy14 " 12 beatsrmin; n s 5. did not differ from the maximal change observed after i.t. injection of 1 m l saline solution Ž4 " 2 beatsrmin, n s 5..
4. Discussion The present study shows that in pentobarbitalanesthetized rats, the i.t. injection of the precursor of NO, L-arginine, induces a hypotensive response whereas the NOS inhibitor, L-NAME, causes a dose-dependent pressor effect. These cardiovascular responses are stereospecific since i.t. injection of the corresponding D-isomers had no effects on the BP. The observation that the pressor re-
Table 1 Effects of hexamethonium on the maximal changes in mean blood pressure Ž D MBP, mmHg. induced by intrathecal injection of N G -nitro-L-arginine methyl ester ŽL-NAME., L-arginine ŽL-Arg. and sodium nitroprusside ŽSNP. L-NAME
Ž10 m mol.
L-Arg
Pretreatment None Ž n s 5. Baseline MBP ŽmmHg. Maximal D MBP ŽmmHg.
103.3 " 7.8 q32.3 " 3.8
Ž10 m mol.
SNP Ž0.25 m mol.
Pretreatment Hex Ž n s 4. 68.7 " 8.6 q7.5 " 3.9
None Ž n s 5. d d
97.1 " 1.0 y13.7 " 1.3
Pretreatment Hex q Phe
a
94.2 " 4.2 q0.8 " 3.7
Ž n s 4. d
None Ž n s 5. 96.7 " 6.6 y33.0 " 1.3 q21.7 " 4.3
Hex q Phe b c
a
88.7 " 6.7 y15.4 " 2.6 q4.6 " 2.7
Ž n s 4. b,d c,d
Hexamethonium ŽHex: 10 mgrkg, i.v.. was administered 5–7 min before the beginning of i.t. injection of either L-NAME or L-Arg or SNP. Baseline MBP was measured immediately before i.t. injection of drugs. Shown are mean values" S.E.M.; n s number of animals. a A continous i.v. infusion of phenylephrine ŽPhe: 0.2–0.3 m grmin. was started inmediately after the bolus i.v. injection of hexamethonium and it was maintained up to the end of the experiment. b D MBP during the first phase of the response to SNP. c D MBP during the second phase of the response to SNP. d P - 0.05 vs. the corresponding value in the non-pretreated group.
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sponse to L-NAME was prevented by a low dose of L-arginine is likely to indicate that its cardiovascular effects are linked to NOS inhibition. Moreover, the finding that the effects of L-NAME and L-arginine on BP were significantly reduced by nicotinic ganglionic blockade indicates that they are related to an action at the level of the central nervous system. Taken together, our results suggest that at the level of the thoracolumbar spinal cord endogenous NO can induce inhibitory effects on the sympathetic outflow. In the present study, the hypotensive response to Larginine was antagonized by pretreatment with Methylene blue, a guanylate cyclase inhibitor w2x. This result suggests that activation of guanylate cyclase and thus cGMP synthesis is involved in the inhibitory effect of spinal NO on the sympathetic outflow, as previously proposed for other physiological responses mediated by NO w17,19,20,37x. Nevertheless, mechanisms other than guanylate cyclase inhibition could be involved in the action of Methylene blue. This is because Methylene blue can inactivate NO via the generation of peroxide anion w24,41x and can inhibit the synthesis of NO via a direct inhibition of NOS w25x. In the central nervous system the activation of NOS and hence NO production is linked to the activation of postsynaptic NMDA receptors by glutamate w6,14x. In the spinal cord, preganglionic sympathetic neurons receive tonic excitatory input from glutamatergic neurons arising at the rostroventrolateral medulla w4,8,26x. The excitatory effect of glutamate involves the activation of NMDA receptors on preganglionic sympathetic neurons w4,26,36x. Therefore, the finding that the NMDA receptor antagonist, APV, abolished the pressor response caused by L-NAME could suggest that sympathetic preganglionic neurons synthetize and release NO in response to the tonic activation of NMDA receptors by glutamate. Nevertheless, NOS has been detected not only in the intermediolateral column, but also in other areas of the spinal cord, such as the dorsal horn and around the central canal w40x. Therefore, the participation of NO synthetized at sites other than preganglionic sympathetic neurons cannot be disregarded from our present results. Though only a hypotensive action of endogenous NO was detected under our experimental conditions, the observation that the NO donor SNP induced an initial hypotensive response followed by a sustained pressor effect suggests that in the rat spinal cord NO can produce inhibitory as well as excitatory effects on the sympathetic outflow. These effects of SNP may be related to the activation of guanylate cyclase by NO, since both phases of the response were abolished by Methylene blue. Moreover, both phases of the response to SNP involved central sites of action because pretreatment with hexamethonium significantly reduced the hypotensive effect and produced an almost complete blockade of the pressor response. The remaining hypotensive response to SNP in the presence of hexamethonium probably reflects a peripheral vasorelaxant
effect of the drug after leakage from the site of injection. The present evidence for a dual hypotensive as well as hypertensive effect of NO in the spinal cord may help to explain some conflicting data in the literature which suggest that spinal NO plays either an excitatory w22x or an inhibitory role in BP regulation w11x. In the central nervous system NO diffuses from its site of formation to neighboring nerve terminals where it induces the release of neurotransmitters w45x. Therefore, as a matter of hypothesis, it could be suggested that the effects of NO on the BP in this study are mediated through the release of inhibitory as well as excitatory neurotransmitters in the spinal cord, which either directly or indirectly modify the preganglionic sympathetic nerve activity. In support of this view, there is highly relevant evidence from Wu and Dun that shows that NO exhibits facilitatory effects on both inhibitory w43x and excitatory w42x inputs to sympathetic preganglionic neurons in slice preparations of neonatal rats. The differences in the present study between the effects of enzyme-generated NO and SNP-generated NO could rely on the fact that enzymatic generation of NO occurs only in NOS-containing cells, whereas the formation of NO from SNP may be less confined inasmuch as it is assumed to occur spontaneously in biological tissues w3x. Moreover, according to Schuman and Madison w34x, NO may exert multiple, perhaps opposite, actions depending on the timing and concentration of its application. Therefore, the differences between the effects of L-arginine and SNP may also be related to the concentrations of NO achieved in the spinal cord after i.t. injection of either compound. In this regard, it is of interest that in the rat spinal cord NO may induce pronociceptive effects at low concentrations and antinociceptive effects at higher concentrations w20x. In regards to the heart rate, the finding that L-arginine and SNP did not induce changes in the heart rate, except for the mild bradycardia produced by the highest dose of L-arginine, could suggest that there was not an important diffussion of the drugs from the site of i.t. injection at the T12 –L 1 level toward the T1 –T3 level, where the preganglionic sympathetic neurons giving innervation to the heart are localized w35x. On the other hand, the marked bradycardia induced by L-NAME might be reflexogenic, as it oppossed the pressor effect of the drug. In conclusion, though only the inhibitory action of endogenous NO was detected in our experimental conditions, this study suggests that in the spinal cord of pentobarbital-anesthetized rats NO exerts both inhibitory and excitatory effects on the preganglionic sympathetic nerve activity related to the control of the BP. The synthesis of NO in the spinal cord appears to be tonically activated through the stimulation of NMDA receptors, probably localized at preganglionic sympathetic neurons. It is of interest to note that since differences in the central control of the cardiovascular functions have been found in con-
M.d.C. Garcıa ´ et al.r Brain Research 764 (1997) 67–74
scious compared to anesthetized animals w12x, the generalization of the present results will demand additional experiments in conscious freely moving rats.
w19x
w20x
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Research report
Possible participation of spinal nitric oxide in the control of the blood pressure in anesthetized rats Marıa ´ del Carmen Garcıa ´ a
a,b,)
, Stella Maris Celuch b , Edda Adler-Graschinsky
b
Catedra de Farmacologıa, UniÕersidad de Buenos Aires, Buenos Aires, Argentina ´ ´ Facultad de Farmacia y Bioquımica, ´ b (CONICET), Junın Instituto de InÕestigaciones Farmacologicas ´ ´ 956, 58 Piso, 1113 Buenos Aires, Argentina Accepted 25 March 1997
Abstract In pentobarbital-anesthetized rats the intrathecal Ži.t.. injection of the nitric oxide ŽNO. precursor, L-arginine Ž10 and 20 m mol., elicited a decrease in the mean blood pressure ŽMBP. whereas the inhibitor of the NO synthase ŽNOS. N G-nitro-L-arginine methyl ester ŽL-NAME; 0.1–10 m mol. produced a dose-dependent pressor effect. The pressor response to L-NAME was prevented by pretreatment with L-arginine. Neither D-arginine nor D-NAME modified the MBP. The NO donor sodium nitroprusside ŽSNP; 0.125 and 0.25 m mol, i.t.. induced a hypotensive response followed by a pressor effect. The dual response to SNP as well as the hypotensive effect of L-arginine were abolished by the guanylate cyclase inhibitor Methylene blue Ž0.3 m mol, i.t... Nicotinic ganglionic blockade by hexamethonium Ž10 mgrkg, i.v.. reduced the hypotensive effects of both L-arginine and SNP and prevented almost completely the pressor effects of either L-NAME or SNP. The pressor effect of L-NAME was abolished by 2-amino-5-phosphonovaleric acid ŽAPV; 30 nmol, i.t.., a selective antagonist of glutamate receptors of the NMDA subtype. These results suggest that in the spinal cord of pentobarbital-anesthetized rats NO exerts both inhibitory and excitatory effects on the preganglionic sympathetic nerve activity related to the control of the BP. The synthesis of NO appears to be tonically activated through the stimulation of spinal glutamate receptors of the NMDA subtype. q 1997 Elsevier Science B.V. Keywords: Nitric oxide; Spinal cord; Blood pressure; L-Arginine; N G -Nitro-L-arginine methyl ester ŽL-NAME.; Sodium nitroprusside; Methylene blue
1. Introduction There is considerable evidence that nitric oxide ŽNO., synthetized from L-arginine in the presence of NO synthase ŽNOS. w21,30x, is involved in the central regulation of the cardiovascular functions in several species such as the rat w27,28,32x, the cat w17,33x, the dog w29x and the rabbit w16x. In the nucleus tractus solitarius, that is the first relay station in the baroreflex arc, NO elicites decreases in the renal sympathetic nerve activity, blood pressure ŽBP. and heart rate ŽHR. w16,23,39x. In addition, NO induces inhibitory effects on renal sympathetic nerve activity, BP and HR through its action on the rostral ventrolateral medulla w33,39,44x and the paraventricular nucleus of the hypothalamus w18x. These studies suggest that NO could have in the brain an overall inhibitory effect on the sympathetic output.
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Corresponding author. Fax: q54 Ž1 . 963-8593; E-mail: [email protected] 0006-8993r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 0 4 2 1 - 6
Preganglionic sympathetic neurons from several mammals w1,5,7,10,31,40x exhibit NOS immunoreactivity as well as a marker for neuronal NOS, the nicotinamide dinucleotide phosphate diaphorase. Moreover, it has been found that sympathetic preganglionic neurons from spontaneously hypertensive rats possess lower NOS activity than the normotensive controls w38x. Although these studies suggest that spinally synthetized NO could regulate the sympathetic nerve activity, the role of this messenger in the modulation of the BP at the level of the spinal cord is not yet fully understood. Feldman et al. w11x observed that the intrathecal Ži.t.. injection of a specific inhibitor of NOS, N G -nitro-L-arginine methyl ester ŽL-NAME., increases the BP of urethane-anesthetized rats and suggested that this effect could be due to a tonic inhibitory action of spinal NO on the sympathetic nerve activity. On the other hand, Lee et al. w22x reported that in urethane-anesthetized rats, the intrathecal injection of L-NAME causes a dose-dependent decrease in the BP whereas the NO donor, sodium nitroprusside ŽNPS., produces pressor effects. These authors proposed that in the spinal cord NO plays a tonic
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excitatory role on the central control of the BP. Moreover, an excitatory action of NO on renal sympathetic preganglionic neurons has been demonstrated by Hakim et al. w15x in the spinal cord of pentobarbital-anesthetized rabbits. The present study was undertaken to further investigate the role of NO in the central control of the BP at the level of the spinal cord in pentobarbital-anesthetized rats. Our results suggest that NO induces excitatory as well as inhibitory effects on the sympathetic preganglionic activity related to the regulation of the BP.
recording made just before the beginning of the drug injection and the value at a given time. 2.3. Intrathecal injection of drugs Drugs and saline solution were injected i.t. in volumes no greater than 4 m l using a 10 m l Hamilton microsyringe. The rate of injection was 1 m lrmin. For bolus i.v. administration of drugs the volume of injection was 0.1 ml and the cannula was flushed with 0.1 ml saline solution. 2.4. Experimental protocols
2. Materials and methods 2.1. Surgical procedures Procedures for the evaluation of cardiovascular effects of intrathecal Ži.t.. injection of drugs were similar to those previously described w13x. Wistar rats of either sex Ž180– 230 g. were anesthetized with sodium pentobarbital. The animals received an initial dose of sodium pentobarbital Ž40 mgrkg, i.p.., supplemented with 20 mgrkg Ži.p.. before introduction of the i.t. catheter. An additional dose Ž20 mgrkg. was given during the experiment when necessary. The depth of anesthesia Žsurgical plane. was monitored by the absence of the eyelid reflex. Although the animals breathed spontaneously, the trachea was cannulated to avoid respiratory disorders related to the accumulation of secretions at the superior airways. A polyethylene cannula was placed in the right femoral artery for recording of the blood pressure ŽBP.. In some experiments, a femoral vein was cannulated for i.v. injection of drugs. While recording the BP, a cannula was positioned at the level of the T12 –L 1 intervertebral space as described by Dib w9x. Briefly, a cannula Žo.d. 0.65 mm. was inserted into the subarachnoid space at the level of the C 8 –T1 vertebrae and gently pushed downward 4.5 cm. The position of the cannula was verified post mortem by opening ventrally the vertebrae to localize the cannula tip. The rectal temperature was maintained at 37–388C by a heating lamp. 2.2. Blood pressure recording and heart rate calculation The BP was measured from the right femoral artery via a Statham P23 1D transducer and recorded on a Grass 7B polygraph ŽQuincy, MA, USA.. The mean BP ŽMBP. was calculated from the formula: diastolic pressureq 1r3 Ž systolic pressurey diastolic pressure .. The heart rate ŽHR. was calculated from the BP record. The BP and HR were allowed to stabilize for at least 30 min before starting the experiment. Changes in MBP and HR induced by i.t. or i.v. injection of drugs refer to the differences between the
Unique doses of either N G-nitro-L-arginine methyl ester ŽL-NAME. or N G -nitro-D-arginine methyl ester ŽD-NAME. or L-arginine or D-arginine or sodium nitroprusside ŽSNP. were injected i.t. The BP and HR were recorded just before injection Žtime 0. and at the following times after the beginning of the injection: 1, 1.5, 2, 4, 6, 8, 10, 15, 20, 25 and 30 min. In some animals, L-NAME Ž2.5 m mol. was administered 5 min after i.t. injection of either L-arginine Ž5 m mol. or 2-amino-5-phosphonovaleric acid ŽAPV; 30 nmol.. In other experimental groups, the animals received either SNP Ž0.25 m mol. or L-arginine Ž10 m mol. 5 min after pretreatment with Methylene blue Ž0.30 m mol, i.t... To analyze the effects of nicotinic ganglionic blockade on the cardiovascular responses to either L-NAME Ž10 m mol, i.t.. or L-arginine Ž10 m mol, i.t.. or SNP Ž0.25 m mol, i.t.., the latter drugs were injected 5 min after i.v. injection of hexamethonium bromide Ž10 mgrkg.. When the drugs to be administered were L-arginine or SNP, a continous i.v. infusion with phenylephrine Ž130 m grml, rate: 0.2–0.3 m grmin. was started immediately after the bolus i.v. injection of hexamethonium and was maintained up to the end of the experiment. 2.5. Drugs 2-Amino-5-phosphonovaleric acid, L-arginine, Darginine, hexamethonium bromide, N G-nitro-L-arginine methyl ester, N G-nitro-D-arginine methyl ester, phenylephrine HCl and sodium nitroprusside were obtained from Sigma Chemical Co. ŽUSA.. Methylene blue was obtained from Merck Quımica Argentina ŽARGENTINA.. All drugs ´ were daily dissolved in saline solution. Drug doses refer to their respective free bases. 2.6. Statistics All values represent the mean " S.E.M.. Statistical differences were assessed either by Student’s t-test or by one-factor analysis of variance followed by the Newman– Keuls test. P values smaller than 0.05 were regarded as significant.
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3. Results 3.1. CardioÕascular responses induced by intrathecal injection of L-arginine and L-NAME The biosynthetic precursor of NO, L-arginine, induced a decrease in the MBP at doses of 10 and 20 m mol Ži.t.. whereas a lower dose of the drug Ž5 m mol, i.t.. was devoid of effect. The hypotensive response to 20 m mol L-arginine was accompanied by a slight but significant bradycardia ŽFig. 1.. On the other hand, the changes in MBP and HR produced by i.t. injection of 10 m mol D-arginine Žy1.1 " 0.6 mmHg and y5 " 6 beatsrmin; n s 6. did not differ from the maximal changes induced by i.t. injection of saline solution Žy2.3 " 0.8 mmHg and 5 " 3 beatsrmin; n s 5.. The time-course of the hypotensive effect of 10 m mol L-arginine Ži.t.. is summarized in Fig. 2. The decrease in the MBP was maximal 6–10 min after the beginning of the injection and persisted for at least 30 min. The decrease in
Fig. 2. Time-course of the decrease in the mean blood pressure Ž D MBP, mmHg. induced by L-arginine Ž10 m mol, i.t... Effects of Methylene blue. L-Arginine was injected in 4 min, starting at time 0, either under control conditions Ž`. or in the presence of Methylene blue Žv .. Methylene blue Ž0.3 m mol, i.t.. was injected 5 min before the beginning of i.t. administration of L-arginine. The hatched horizontal bar represents the effect of i.t. injected saline solution. Shown are mean values"S.E.M. for 5 animals per group. a P - 0.01 vs. saline solution and vs. Methylene blue plus L-arginine Žanalysis of variance followed by Newman–Keuls test.. The resting MBP ŽmmHg.rHR Žbeatsrmin. values were: 106.7"2r390"9 in the saline solution group, 98.7"3.4r384"14 in the L-arginine group and 97.7"4.8r382"13 in the Methylene blue plus L-arginine group.
MBP elicited by L-arginine was entirely prevented by an inhibitor of the soluble guanylate cyclase, Methylene blue Ž0.3 m mol; i.t... The NOS inhibitor, L-NAME Ž0.1–10 m mol, i.t.., induced a dose-dependent pressor effect that was accompanied by bradycardia ŽFig. 3.. On the other hand, i.t. injection of D-NAME Ž10 m mol. did not modify the MBP and HR. The maximal changes induced by the drug on these parameters Žy1.3 " 1.9 mmHg and y6 " 7 beatsrmin, respectively; n s 5. did not differ from those induced by saline solution. The time-course of the pressor response to L-NAME Ž2.5 m mol, i.t.. is summarized in Fig. 4. The increase in MBP reached a maximum 8–10 min after the beginning of the injection and persisted for at least 30 min. The pressor effect of L-NAME ŽFig. 4. was abolished by pretreatment with a dose of L-arginine Ž5 m mol, i.t.., which per se did not modify the resting MBP Žsee Fig. 1..
Fig. 1. Maximal changes in mean blood pressure Ž D MBP, mmHg. and heart rate Ž D HR, beatsrmin. induced by i.t. injection of L-arginine. The drug was injected in volumes not greater than 4 m l. The hatched bar represents the effect of i.t. injected saline solution. Shown are mean values"S.E.M. for 4–5 animals per dose. a P - 0.01 vs. saline solution ŽStudent’s t-test.. The resting MBP ŽmmHg.rHR Žbeatsrmin. values were: 106.7"2.0r390"9 in the saline solution group; 105.0"4.7r386 "5 in the 5 m mol L-arginine group, 98.7"3.4r384"14 in the 10 m mol L-arginine group and 99.3"7.1r380"5 in the 20 m mol L-arginine group.
3.2. Effects of a glutamate receptor antagonist on the pressor response induced by L-NAME Since there is evidence that in the central nervous system NO production is linked to the activation of postsynaptic glutamate receptors of the N-methyl-D-aspartate ŽNMDA. subtype w6,14x, we determined whether 2-amino5-phosphonovaleric acid ŽAPV., a selective antagonist for NMDA receptors, modified the pressor response induced by L-NAME. As shown in Fig. 5, the increase in MBP
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Fig. 4. Time-course of the increase in the mean blood pressure Ž D MBP, mmHg. induced by N G -nitro-L-arginine methyl ester ŽL-NAME.. Effects of L-arginine. L-NAME Ž2.5 m mol, '. and saline solution Ž4 m l, n. were i.t. injected in 4 min, starting at time 0. L-Arginine Ž5 m mol. was i.t. injected 5 min before the beginning of the injection of either L-NAME Žv . or saline solution Ž`.. Shown are mean values "S.E.M. for 5 animals per group. a P - 0.05 vs. the corresponding saline control values; b P - 0.05 vs. the corresponding values in the L-arginine plus L-NAME group Žanalysis of variance followed by Newman–Keuls test.. The resting MBP ŽmmHg.rHR Žbeatsrmin. values were: 106.7"2.0r390"9 in the saline solution group, 98.3"5.4r390"7 in the L-arginine plus saline solution group, 96.7"5.1r382"7 in the L-NAME group and 96.7" 4.5r394"18 in the L-arginine plus L-NAME group. Fig. 3. Maximal changes in mean blood pressure Ž D MBP, mmHg. and heart rate Ž D HR, beatsrmin. induced by i.t. injection of N G -nitro-Larginine methyl ester ŽL-NAME.. The drug was injected in volumes not greater than 4 m l. The hatched bar represents the effect of i.t. injected saline solution. Shown are mean values"S.E.M. for 4–5 animals per dose. a P - 0.01 vs. saline solution ŽStudent’s t-test.. The resting MBP ŽmmHg.rHR Žbeatsrmin. values were: 106.7"2.0r390"9 in the saline solution group; 95.0"3.9r377"17 in the 0.1 m mol L-NAME group, 99.1"5.7r397"12 in the 1 m mol L-NAME group, 96.7"5.1r382"7 in the 2.5 m mol L-NAME group, 102.2"4.9r363"22 in the 5 m mol L-NAME group and 103.3"7.8r396"13 in the 10 m mol L-NAME group.
produced by i.t. injection of L-NAME Ž2.5 m mol. was abolished after pretreatment with APV Ž30 nmol, i.t... The NMDA receptor antagonist induced per se, 5 min after its i.t. injection, a decrease in the resting MBP Žy9.0 " 2.7 mmHg; n s 8. that was significantly different from that induced by the i.t. administration of saline solution Žy1 " 1 mmHg; n s 5; P - 0.05.. 3.3. CardioÕascular responses induced by intrathecal injection of sodium nitroprusside Sodium nitroprusside ŽSNP: 0.125 and 0.25 m mol, i.t.. induced biphasic effects on the MBP ŽFig. 6.. The first phase of the response was a transient decrease in the MBP that lasted less than 4 min and reached its maximum 1–2 min after the beginning of the injection. This hypotensive effect was followed by a long-lasting increase in the MBP
Fig. 5. Maximal changes in MBP Ž D MBP, mmHg. induced by i.t. injected N G -nitro-L-arginine methyl ester ŽL-NAME.. Effects of 2-amino5-phosphonovaleric acid ŽAPV.. Intrathecal injection of either L-NAME Ž2.5 m mol. or saline solution was performed in control animals and in animals pretreated with APV. APV Ž30 nmol. was i.t. injected 5 min before the beginning of the i.t. administration of either L-NAME or saline solution. Shown are mean"S.E.M. for 4–5 animals per group. a P 0.001 vs. the corresponding control value ŽStudent’s t-test.. The resting MBP ŽmmHg.rHR Žbeatsrmin. values in the controls were: 106.7" 2r390"9 for saline solution and 96.7"5.1r382"7 for L-NAME. The resting MBP ŽmmHg.rHR Žbeatsrmin. values in APV-treated animals were: 104.3"3.9r406"9 for saline solution and 95.0"6.6r416"18 for L-NAME.
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3.4. Effects of blockade of nicotinic ganglionic transmission on the cardioÕascular responses to intrathecal injection of L-arginine, L-NAME and sodium nitroprusside To rule out the possibility that the cardiovascular responses induced by i.t. injected L-NAME, L-arginine and SNP had resulted from peripheral effects due to leakage of the drugs from the site of injection, their effects on MBP were analyzed in rats pretreated with a ganglionic blocking agent, hexamethonium Ž10 mgrkg, i.v... To preclude the possibility that the decrease in the baseline MBP in the presence of hexamathonium could mask the hypotension that is caused by L-arginine and SNP, for these experimental groups the animals received, in addition to the bolus i.v. injection of hexamathonium, a continous infusion of phenylephrine Ž0.2–0.3 m grmin.. Under the latter conditions the baseline MBP values after hexamethonium did not differ from those found in non-pretreated animals ŽTable 1.. As shown in Table 1, hexamethonium pretreatment reduced by 80% the pressor responses induced by L-NAME Ž10 m mol; i.t.. and SNP Ž0.25 m mol; i.t... Regarding the hypotensive responses, they were abolished in the case of L-arginine and reduced by 50% approximately in the case of SNP.
Fig. 6. Changes in mean blood pressure Ž D MBP, mmHg. induced by i.t. injection of sodium nitroprusside ŽSNP.. Effects of Methylene blue. Sodium nitroprusside Ž =, 0.125 m mol; `, 0.25 m mol. was injected in 1 min, starting at time 0. v, Methylene blue Ž0.3 m mol; i.t.. was injected 5 min before SNP Ž0.25 m mol.. The hatched bar represents the effect of i.t. injected saline solution. Shown are mean values"S.E.M. for 4–5 animals per group. a : P - 0.05 vs. saline solution, b P - 0.05 vs. the corresponding values in the Methylene blue plus SNP group Žanalysis of variance followed by Newman–Keuls test.. The resting MBP ŽmmHg.rHR Žbeatsrmin. values were: 100.3"3.0r395"11 in the saline solution group; 99.2"6.3r407"20 in the 0.125 m mol SNP group and 94.7" 7.9r402"13 0.25 m mol in the SNP group; 98.3"4.4r394"23 in the Methylene blue plus SNP group.
that peaked at 8–10 min after the beginning of the injection and persisted at least for 30 min. The changes in the MBP induced by SNP were dose-dependent. As also shown in Fig. 6, the hypotensive as well as the hypertensive responses induced by 0.25 m mol SNP were prevented by Methylene blue Ž0.3 m mol, i.t... The maximal changes in HR induced by 0.125 m mol SNP Žy5 " 5 beatsrmin; n s 4. and by 0.25 m mol SNP Žy14 " 12 beatsrmin; n s 5. did not differ from the maximal change observed after i.t. injection of 1 m l saline solution Ž4 " 2 beatsrmin, n s 5..
4. Discussion The present study shows that in pentobarbitalanesthetized rats, the i.t. injection of the precursor of NO, L-arginine, induces a hypotensive response whereas the NOS inhibitor, L-NAME, causes a dose-dependent pressor effect. These cardiovascular responses are stereospecific since i.t. injection of the corresponding D-isomers had no effects on the BP. The observation that the pressor re-
Table 1 Effects of hexamethonium on the maximal changes in mean blood pressure Ž D MBP, mmHg. induced by intrathecal injection of N G -nitro-L-arginine methyl ester ŽL-NAME., L-arginine ŽL-Arg. and sodium nitroprusside ŽSNP. L-NAME
Ž10 m mol.
L-Arg
Pretreatment None Ž n s 5. Baseline MBP ŽmmHg. Maximal D MBP ŽmmHg.
103.3 " 7.8 q32.3 " 3.8
Ž10 m mol.
SNP Ž0.25 m mol.
Pretreatment Hex Ž n s 4. 68.7 " 8.6 q7.5 " 3.9
None Ž n s 5. d d
97.1 " 1.0 y13.7 " 1.3
Pretreatment Hex q Phe
a
94.2 " 4.2 q0.8 " 3.7
Ž n s 4. d
None Ž n s 5. 96.7 " 6.6 y33.0 " 1.3 q21.7 " 4.3
Hex q Phe b c
a
88.7 " 6.7 y15.4 " 2.6 q4.6 " 2.7
Ž n s 4. b,d c,d
Hexamethonium ŽHex: 10 mgrkg, i.v.. was administered 5–7 min before the beginning of i.t. injection of either L-NAME or L-Arg or SNP. Baseline MBP was measured immediately before i.t. injection of drugs. Shown are mean values" S.E.M.; n s number of animals. a A continous i.v. infusion of phenylephrine ŽPhe: 0.2–0.3 m grmin. was started inmediately after the bolus i.v. injection of hexamethonium and it was maintained up to the end of the experiment. b D MBP during the first phase of the response to SNP. c D MBP during the second phase of the response to SNP. d P - 0.05 vs. the corresponding value in the non-pretreated group.
72
M.d.C. Garcıa ´ et al.r Brain Research 764 (1997) 67–74
sponse to L-NAME was prevented by a low dose of L-arginine is likely to indicate that its cardiovascular effects are linked to NOS inhibition. Moreover, the finding that the effects of L-NAME and L-arginine on BP were significantly reduced by nicotinic ganglionic blockade indicates that they are related to an action at the level of the central nervous system. Taken together, our results suggest that at the level of the thoracolumbar spinal cord endogenous NO can induce inhibitory effects on the sympathetic outflow. In the present study, the hypotensive response to Larginine was antagonized by pretreatment with Methylene blue, a guanylate cyclase inhibitor w2x. This result suggests that activation of guanylate cyclase and thus cGMP synthesis is involved in the inhibitory effect of spinal NO on the sympathetic outflow, as previously proposed for other physiological responses mediated by NO w17,19,20,37x. Nevertheless, mechanisms other than guanylate cyclase inhibition could be involved in the action of Methylene blue. This is because Methylene blue can inactivate NO via the generation of peroxide anion w24,41x and can inhibit the synthesis of NO via a direct inhibition of NOS w25x. In the central nervous system the activation of NOS and hence NO production is linked to the activation of postsynaptic NMDA receptors by glutamate w6,14x. In the spinal cord, preganglionic sympathetic neurons receive tonic excitatory input from glutamatergic neurons arising at the rostroventrolateral medulla w4,8,26x. The excitatory effect of glutamate involves the activation of NMDA receptors on preganglionic sympathetic neurons w4,26,36x. Therefore, the finding that the NMDA receptor antagonist, APV, abolished the pressor response caused by L-NAME could suggest that sympathetic preganglionic neurons synthetize and release NO in response to the tonic activation of NMDA receptors by glutamate. Nevertheless, NOS has been detected not only in the intermediolateral column, but also in other areas of the spinal cord, such as the dorsal horn and around the central canal w40x. Therefore, the participation of NO synthetized at sites other than preganglionic sympathetic neurons cannot be disregarded from our present results. Though only a hypotensive action of endogenous NO was detected under our experimental conditions, the observation that the NO donor SNP induced an initial hypotensive response followed by a sustained pressor effect suggests that in the rat spinal cord NO can produce inhibitory as well as excitatory effects on the sympathetic outflow. These effects of SNP may be related to the activation of guanylate cyclase by NO, since both phases of the response were abolished by Methylene blue. Moreover, both phases of the response to SNP involved central sites of action because pretreatment with hexamethonium significantly reduced the hypotensive effect and produced an almost complete blockade of the pressor response. The remaining hypotensive response to SNP in the presence of hexamethonium probably reflects a peripheral vasorelaxant
effect of the drug after leakage from the site of injection. The present evidence for a dual hypotensive as well as hypertensive effect of NO in the spinal cord may help to explain some conflicting data in the literature which suggest that spinal NO plays either an excitatory w22x or an inhibitory role in BP regulation w11x. In the central nervous system NO diffuses from its site of formation to neighboring nerve terminals where it induces the release of neurotransmitters w45x. Therefore, as a matter of hypothesis, it could be suggested that the effects of NO on the BP in this study are mediated through the release of inhibitory as well as excitatory neurotransmitters in the spinal cord, which either directly or indirectly modify the preganglionic sympathetic nerve activity. In support of this view, there is highly relevant evidence from Wu and Dun that shows that NO exhibits facilitatory effects on both inhibitory w43x and excitatory w42x inputs to sympathetic preganglionic neurons in slice preparations of neonatal rats. The differences in the present study between the effects of enzyme-generated NO and SNP-generated NO could rely on the fact that enzymatic generation of NO occurs only in NOS-containing cells, whereas the formation of NO from SNP may be less confined inasmuch as it is assumed to occur spontaneously in biological tissues w3x. Moreover, according to Schuman and Madison w34x, NO may exert multiple, perhaps opposite, actions depending on the timing and concentration of its application. Therefore, the differences between the effects of L-arginine and SNP may also be related to the concentrations of NO achieved in the spinal cord after i.t. injection of either compound. In this regard, it is of interest that in the rat spinal cord NO may induce pronociceptive effects at low concentrations and antinociceptive effects at higher concentrations w20x. In regards to the heart rate, the finding that L-arginine and SNP did not induce changes in the heart rate, except for the mild bradycardia produced by the highest dose of L-arginine, could suggest that there was not an important diffussion of the drugs from the site of i.t. injection at the T12 –L 1 level toward the T1 –T3 level, where the preganglionic sympathetic neurons giving innervation to the heart are localized w35x. On the other hand, the marked bradycardia induced by L-NAME might be reflexogenic, as it oppossed the pressor effect of the drug. In conclusion, though only the inhibitory action of endogenous NO was detected in our experimental conditions, this study suggests that in the spinal cord of pentobarbital-anesthetized rats NO exerts both inhibitory and excitatory effects on the preganglionic sympathetic nerve activity related to the control of the BP. The synthesis of NO in the spinal cord appears to be tonically activated through the stimulation of NMDA receptors, probably localized at preganglionic sympathetic neurons. It is of interest to note that since differences in the central control of the cardiovascular functions have been found in con-
M.d.C. Garcıa ´ et al.r Brain Research 764 (1997) 67–74
scious compared to anesthetized animals w12x, the generalization of the present results will demand additional experiments in conscious freely moving rats.
w19x
w20x
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