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(Hypertension. 1998;31:719-720.)
© 1998 American Heart Association, Inc.

Letters to the Editor

Central Nervous System Is Not Involved in Initiation of the Pressor Effect of 7-Nitroindazole in Urethane-Anesthetized Rats

Yuri Zagvazdin; Anton Reiner; ; Ibrahim F. Benter

Department of Anatomy and Neurobiology, University of Tennessee, Memphis, Tenn
Department of Pharmacology, Southern College of Optometry, Memphis, Tenn

To the Editor:

We read with interest the article by Sander et al¹ ; it provided a valuable update on the controversies concerning the role of the inhibition of neuronal nitric oxide synthase (NOS) acting within the central sympathetic nervous system (CNS) in the systemic hypertension caused by NOS inhibition. Some authors have suggested that neuronal NO is involved in the tonic restraint of sympathetic vasoconstrictor outflow from the CNS. Removal of such restraint by inhibition of neuronal NOS should, in principle, lead to sympathetic activation and increased systemic blood pressure. After careful analysis of published studies in this area and their own work, Sander et al concluded that central inhibition of NOS does not contribute to the onset of hypertension after systemic NOS inhibition in conscious animals, but it does contribute to the long-term maintenance of such hypertension in conscious rats. Sander et al noted that support for a neurogenic component in NOS-induced hypertension was, however, evident in studies of anesthetized animals. The authors did not clarify whether this effect was on initiation or maintenance of hypertension or both. Some studies in anesthetized rats that were not noted by Sander et al, however, did not support a neurogenic component in the hypertension induced by L-arginine–derived NOS inhibitors.^{2 3 4} On the other hand, the rapid pressor effect of diphenyleneiodonium, an NOS inhibitor that is chemically distinct from N^G-substituted arginine analogs, does appear to be CNS-dependent in anesthetized rats.⁵ The basis of these conflicting data on the role of a neurogenic component in NOS inhibition–mediated hypertension in anesthetized animals is unclear.

One way to more specifically assess the contribution of centrally acting neurally derived NO to systemic hypertension is to use NOS inhibitors that do not affect the endothelial isoform of NOS. 7-Nitroindazole has been proposed to be a relatively selective inhibitor for the neuronal isoform of NOS,⁶ and we believed it might be especially useful in evaluating the specific role of neuronal NO in regulation of arterial blood pressure. We and others have found that 7-nitroindazole induces a moderate pressor effect in unanesthetized as well as urethane-anesthetized rats that achieves a maximal level after 10 to 15 minutes and is maintained unchanged for a subsequent 45 minutes.^{7 8} Various factors (eg, use of volatile anesthetics or barbiturates that blunt NO-dependent vasoresponsiveness and hypertension)^{9 10} may have obscured the moderate pressor effect of 7-nitroindazole in many other studies (for discussion, see Reference 7). The evident rapid pressor effect of 7-nitroindazole in conscious or urethane-anesthetized rats could be the result of inhibition of neuronal NOS in the CNS, leading to a consequent increase in central sympathetic drive.

To test this hypothesis, 11 Sprague-Dawley rats were anesthetized with urethane (1.6 g/kg IP) and artificially ventilated, with blood gas parameters kept within a normal range. Temperature was kept constant at 37°C. Arterial blood pressure was measured through the femoral artery using a blood pressure analyzer (BPA-100, Micro-Med). 7-Nitroindazole dissolved in peanut oil was administered intraperitoneally in a maximally effective dose (50 mg/kg)⁷ in 5 intact rats and in 5 rats 20 to 25 minutes after the spinal cord was pithed with a metallic rod inserted through the right orbit. Mean arterial blood pressure decreased to 56±6 mm Hg after pithing prior to 7-nitroindazole administration, whereas it was 79±6 mm Hg in the intact rats. In all rats, 7-nitroindazole evoked a prominent pressor effect by 10 minutes after administration. In pithed rats, the mean±SEM of the 7-nitroindazole–induced increase in arterial pressure (15±3 mm Hg) at 10 minutes was not significantly different from the mean of the increase in arterial blood pressure in intact rats (18±3 mm Hg). The pressor effect of 7-nitroindazole was dramatically potentiated (39 mm Hg) in 1 additional pithed rat in which arterial blood pressure was elevated to a normal level by intravenous infusion of epinephrine. A similar potentiating effect of epinephrine on the increase in arterial blood pressure induced by N^G-substituted analogs of L-arginine has been reported in pithed, ganglion-blocked, or sympathectomized rats.^{2 3 4}

Our results therefore suggest that a mechanism other than inhibition of the neuronal NOS in the CNS is responsible for the initial pressor effect of 7-nitroindazole. Thus, the conclusion discussed by Sander et al that a central neurogenic component may not be involved in the initiation of the hypertension after inhibition of NO production is not limited to N^G-substituted analogs of L-arginine and can be extended to other classes of NOS inhibitors. It is also important that this conclusion may be valid not only for conscious but also for anesthetized rats, although more detailed studies of anesthetized rats are needed on this point.

References

1. Sander M, Hansen J, Victor RG. The sympathetic nervous system is involved in the maintenance but not initiation of the hypertension induced by N-nitro-L-arginine methyl ester. Hypertension. 1997;30:64–70.[Abstract/Free Full Text]

2. Pegoraro AA, Carretero OA, Sigmon DH, Beierwaltes WH. Sympathetic modulation of endothelium-derived relaxing factor. Hypertension. 1992;19:643–647.[Abstract/Free Full Text]

3. Chyu KY, Guth PH, Ross G. Effect of N^w-nitro-L-arginine methyl ester on arterial pressure and on vasodilator and vasoconstrictor responses: influence of initial vascular tone. Eur J Pharmacol. 1992;212:159–164.[Medline] [Order article via Infotrieve]

4. Huang M, Leblanc ML, Hester RL. Systemic and regional hemodynamics after nitric oxide synthase inhibition: role of neurogenic mechanism. Am J Physiol. 1994;267:R84–R88.[Abstract/Free Full Text]

5. Wang YX, Pang CCY. Functional integrity of the central and sympathetic nervous system is a prerequisite for pressor and tachycardic effects of diphenyleneiodonium, a novel inhibitor of nitric oxide synthase. J Pharmacol Exp Ther. 1993;265:263–272.[Abstract/Free Full Text]

6. Moore PK, Wallace P, Gaffen Z, Hart SL, Babbedge RC. Characterization of the novel nitric oxide synthase inhibitor 7-nitroindazole and related indazoles: antinociceptive and cardiovascular effects. Br J Pharmacol. 1993;110:219–224.[Medline] [Order article via Infotrieve]

7. Zagvazdin Y, Sancesario G, Wang YX, Share L, Fitzgerald MEC, Reiner A. Evidence from its cardiovascular effects that 7-nitroindazole may inhibit endothelial nitric oxide synthase in vivo. Eur J Pharmacol. 1996;303:61–69.[Medline] [Order article via Infotrieve]

8. Prickaerts J, Steinbusch HWM, Smits JFM, de Vente J. Possible role of nitric oxide-cyclic GMP pathway in object recognition memory: effects of 7-nitroindazole and zaprinast. Eur J Pharmacol.. 1997;337:125–136.[Medline] [Order article via Infotrieve]

9. Nakamura K, Mori K. Nitric oxide and anesthesia. Anesth Analg. 1993;77:877–879.[Free Full Text]

10. Terasako K, Nakamura K, Toda H, Kakuyama M, Hatano Y, Mori K. Barbiturates inhibit endothelium-dependent and -independent relaxations mediated by cyclic GMP. Anesth Analg. 1994;78:823–830.[Abstract/Free Full Text]

Response

Mikael Sander; Jim Hansen; ; Ronald G. Victor

Molecular Cardiology Laboratories, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Tex

In replying to the letter by Zagvazdin et al regarding our recent article,¹ we would like to address two issues: (¹ ) the relative contributions of anesthetized versus conscious animal studies and (² ) the specificity of indazoles versus methyl arginines as inhibitors of NOS I, the "neuronal" isoform.

First, studies in anesthetized rather than conscious animals provided the initial evidence that a neurogenic component contributes to the blood pressure–raising effect of methyl-arginine inhibitors of NOS. To test this hypothesis, we used conscious unrestrained rats^{1 2} to avoid the potentially confounding effects of centrally acting general anesthetic agents (as exemplified by the noted controversy in this literature). Our results suggest that in addition to differing effects of general anesthetics, differences in the time course of NO inhibition also account for some of the seemingly contradictory findings in previous studies.

Second, the new data presented by Zagvazdin et al are based on the use of indazole derivatives. These compounds at first were touted as specific inhibitors of NOS I, whereas methyl arginines such as N-nitro-L-arginine methyl ester (L-NAME) are considered promiscuous inhibitors of all NOS isoforms. When administered systemically, the indazoles were found to inhibit NO production in brain, but they caused little or no increase in blood pressure.³ Subsequently, however, Zagvazdin et al⁴ showed that 7-nitroindazole administered intraperitoneally causes a reproducible increase in blood pressure in both urethane-anesthetized and conscious rats. Furthermore, a greater specificity of indazoles versus methyl arginines for inhibition of NOS I over NOS III has not been substantiated by careful kinetic studies of enzymatic activity in vitro,⁵ and they were shown to cause relaxation of vascular smooth muscle⁶ by mechanisms that do not involve NO. This vasodilatory effect may contribute to the smaller blood pressure–raising effect of 7-nitroindazole relative to the methyl arginines. In designing experiments to test our hypothesis, we interpreted these data to suggest no clear advantages in using nitroindazoles over methyl arginines to test our hypothesis. Indeed, another recent study shows that prolonged oral administration of 7-nitroindazole to conscious rats for 4 weeks resulted in a progressive increase in blood pressure,⁷ which parallels our findings using L-NAME.¹ A recent report suggests that newer indazole constructs may have enhanced specificity for inhibition of NOS I.⁸ However, even if this is the case, their use may not fully elucidate the role of neuronally derived versus endothelially derived NO. NOS I is not purely "neuronal" because it is abundantly expressed in mammalian skeletal muscle,⁹ and NOS III is not purely "endothelial" because it is expressed in neurons, where it appears to contribute importantly to neuronal signaling, such as that involved in long-term potentiation.¹⁰

References

1. Sander M, Hansen J, Victor RG. The sympathetic nervous system is involved in the maintenance but not initiation of the hypertension induced by N-nitro-L-arginine methyl ester. Hypertension.. 1997;30:64–70.

2. Sander M, Hansen PG, Victor RG. Sympathetically mediated hypertension caused by chronic inhibition of nitric oxide. Hypertension.. 1995;26:691–695.[Abstract/Free Full Text]

3. Moore PK, Gaffen WZ, Hart SL, Babbedge RC. Characterization of the novel nitric oxide synthase inhibitor 7-nitroindazole and related indazoles: antinociceptive and cardiovascular effects. Br J Pharmacol.. 1993;110:219–224.

4. Zagvazdin Y, Sancesario G, Wang Y-X, Share L, Fitzgerald MEC, Reiner A. Evidence from its cardiovascular effects that 7-nitroindazole may inhibit endothelial nitric oxide synthase in vivo. Eur J Pharmacol.. 1996;303:61–69.

5. Bland-Ward PA, Moore PK. 7-Nitroindazole derivatives are potent inhibitors of brain, endothelium and inducible isoforms of nitric oxide synthase. Life Sci.. 1995;57:PL131–PL135.[Medline] [Order article via Infotrieve]

6. Medhurst AD, Greenlees C, Parsons AA, Smith SJ. Nitric oxide synthase inhibitors 7- and 6-nitroindazole relax smooth muscle in vitro. Eur J Pharmacol.. 1994;256:R5–R6.[Medline] [Order article via Infotrieve]

7. Ollerstam A, Pittner J, Persson AEG, Thorup C. Increased blood pressure in rats after long-term inhibition of the neuronal isoform of nitric oxide synthase. J Clin Invest.. 1997;99:2212–2218.[Medline] [Order article via Infotrieve]

8. Wu J, Wang Y, Rowan MJ, Anwyl R. Evidence for involvement of the neuronal isoform of nitric oxide synthase during induction of long-term potentiation and long-term depression in the rat dentate gyrus in vitro. Neuroscience.. 1997;78:393–398.[Medline] [Order article via Infotrieve]

9. Chang W-J, Iannaccone ST, Lau KS, Masters BSS, McCabe TJ, McMillan K, Padre RC, Spencer MJ, Tidball JG, Stull JT. Neuronal nitric oxide synthase and dystrophin-deficient muscular dystrophy. Proc Natl Acad Sci USA.. 1996;93:9142–9147.[Abstract/Free Full Text]

10. Son H, Hawkins RD, Martin K, Kiebler M, Huang PL, Fishman MC, Kandel ER. Long-term potentiation is reduced in mice that are doubly mutant in endothelial and neuronal nitric oxide synthase. Cell.. 1996;87:1015–1023.[Medline] [Order article via Infotrieve]

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