Augmented Sympathetic Nerve Activity and PressorResponsiveness in DOC A Hypertensive Rats

KAZUO TAKEDA, M.D., AND RUBEN D. BUNAG, M.A.

SUMMARY Spike potentials in abdominal sympathetic Denes were recorded together with aortic bloodpressure before and during electrical stimulation of the posterior hypothalamus in urethane-anesthetized rats.Both the initial rate of sympathetic nerve firing and the subsequent acceleration produced by hypothalamicstimulation were higher in deoxycorticosterone acetate (DOCA) hypertensive than in normotensive rats.Pressor responsiveness was generally augmented, but responses to hypothalamic stimulation were increasedfar more than those to injected noreplnephrine. Vasodepression produced by blocking autonomlc ganglia phar-macologically with pentolinium was also more pronounced in DOCA hypertensive rats than in normotensivecontrols. These results support the conclusion that a centrally induced sympathetic hyperactivity is importantfor maintaining the blood pressure elevation in rats with established DOCA hypertension.(Hypertension 2: 97-101, 1980)

KEY WORDS • blood pressure • DOCA hypertension • ganglion blockade • hypothalamus •norepinephrine • sympathetic nerves

' W O conflicting but almost equally popularviews now exist on whether sympathetichyperactivity participates in causing de-

oxycorticosterone acetate (DOCA) hypertension.Supporting the belief that sympathetic hyperactivity isessential, the hypotension induced by combiningadrenalectomy with chemical sympathectomy hasbeen shown to result in identical blood pressure levelsin adult normotensive and DOCA hypertensive rats.1

Also, to restore blood pressure levels followingsplanchnicotomy during pentobarbital anesthesia, thesplanchnic nerves had to be stimulated at much higherfrequencies in DOCA hypertensive than in other rats.s

Furthermore, destruction of central catechol-aminergic neurons through intracerebroventricular in-jection of 6-hydroxydopamine prevents not only theelevation in tail-cuff systolic pressure3 but also theattendant increase in plasma norepinephrine* in un-anesthetized DOCA hypertensive rats. On the con-trary, however, others have found DOCA hyperten-sion either unaffected in adult rats8"* or made even

From the Department of Pharmacology, College of HealthSciences and Hospital, University of Kansas Medical Center, Kan-sas City, Kansas.

Supported in part by Research Grant HL-14560 from theNational Heart and Lung Institute. Dr. Kazuo Takeda is a postdoc-toral research fellow from the Second Department of Medicine,Kyoto Prcfectural University of Medicine, Kyoto, Japan.

Address for reprints: Ruben D. Buflag, M.D., Department ofPharmacology, University of Kansas Medical Center, 39th andRainbow Blvd., Kansas City, Kansas 66103.

Received January 22,1979; revision accepted September 17,1979.

worse in neonates79 after chemical sympathectomy.Observations that seem contradictory have also

been made in our laboratory: DOCA hypertensive ratshave an enhanced pressor responsiveness tohypothalamic stimulation,10 yet their pressures fellonly to the same levels as those of normotensive ratswhen the hypothalamus was destroyed.11 In anattempt to find a logical explanation for this, werecorded sympathetic nerve activity directly beforeand during hypothalamic stimulation. To determine ifthe sympathetic overactivity thereby revealed wouldaccount for the elevation in blood pressure, we alsomeasured vasodepression occurring after pentolinium-induced ganglion blockade.

Methods

Female Sprague-Dawley rats, 3 weeks old, werepurchased from Charles River Breeding Laboratories(Wilmington, MA). Soon after arrival, all wereanesthetized with sodium amobarbital (8 mg/100 gi.p.) and their left kidneys removed. Silicon rubbermolds containing DOCA (200 mg/kg) were implantedsubcutaneously in eight rats, according to the methodof Ormsbee and Ryan,is while molds of siliconerubber alone were similarly implanted in 10 others.Thereafter, the rats were given 0.9% sodium chloridesolution instead of water for drinking, and systolicpressures were measured weekly using a tail-cuffmethod validated for use in awake rats.13

Two months later, systolic pressures (mmHg ± SEM) averaged 119 ± 4 in sham-operated, and185 ± 9 in DOCA hypertensive rats (p < 0.001); cor-

97

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98 HYPERTENSION VOL 2, No 1, JANUARY-FEBRUARY 1980

responding averages for body weight (232 ± 7 and236 ± 13 g respectively) or heart rate (371 ± 7 and352 ± 12/min respectively) were not significantlydifferent. All rats were then reanesthetized withamobarbital, and a concentric electrode (NE-100 withchronic connectors, Rhodes Medical Instruments,Woodland Hills, CA) was placed in the posteriorhypothalamus at stereotaxic coordinates antero-posterior 4.6, lateral 1.0, and dorsoventral -2.5.10

Electrodes were fixed to the skull with stainless steelscrews and dental cement.14 Following electrode im-plantation, rats were individually caged in an air-conditioned room, fed a standard laboratory chow adlibitum, and allowed to recover for at least 1 weekbefore experimentation.

During experiments, rats were anesthetized withurethane (0.1 g/100 g i.p.), and catheters were insertedinto a jugular vein for drug injection and into thelower abdominal aorta for blood pressure record-ing.16 The abdominal plexus was exposed, and, withthe aid of a stereoscopic microscope, a bipolarstainless-steel electrode (uninsulated tips 1 mm apart)was placed on the major nerve bundle accompanyingthe superior mesenteric artery immediately below theceliac ganglion (hereafter referred to as the "ab-dominal sympathetic nerve"). Nerves and electrodetips were immersed in mineral oil to reduce tissue-drying. Spike potentials were amplified (Grass PI5AC amplifier) and monitored on a storage os-cilloscope (Tektronix 5111). To reduce noise duringthese recordings, spontaneous respiration wasabolished by paralyzing skeletal muscles withdecamethonium bromide (Syncurine, 0.2 mg/100 gi.v.) and connecting the rats to a respirator ventilatedwith a mixture of 50% oxygen and 50% nitrogen.

Analog signals for aortic pressure and nerve activitywere recorded continuously on magnetic tape. Toquantify nerve activity, original analog signals wereplayed back from tape into an ink-writing recorderand simultaneously fed into an amplitude analyzer (F.Haer and Co., Brunswick, ME) to convert individualspikes into uniform pulses and delete backgroundnoise. Because residual activity remaining afterganglion blockade with pentolinium was the same asthat after crushing the nerve, the low-level control ofthe window discriminator was routinely set to filterbackground noise persisting after pentolinium injec-tion. The number of individual pulses per second werecounted with a rate analyzer (F. Haer and Co.) whoseoutput was recorded separately as a histogram,digitized by a computer interface, and then printed bya programmed calculator (Monroe 1860) for rates un-der 100 spikes/sec (higher rates were countedmanually from the histogram).

Blood pressure was recorded continuously by con-necting the aortic catheter through Tygon tubing to asmall-volume-displacement pressure transducer(Statham P23Gb). A square-wave stimulator (GrassS-48 with constant current unit) was used to deliver50, 100, and 200 /xA currents (pulse duration 1 msec,frequency 100/sec) in 10-second trains to the posteriorhypothalamus. Two drugs were routinely injected

through the jugular vein catheter: pentolinium tartrate(Ansolysen), 0.5 mg (salt)/100 g, and norepinephrinebitartrate (Levophed), 100, 200, and 400 ng(base)/100 g.

At the end of each experiment, a 2 mA directcurrent was passed through the hypothalamic elec-trode for 5 seconds to produce a small lesion andthereby facilitate histologic localization of the elec-trode tip. Then, 10% formalin was perfused into thebrain through a needle inserted into the ascending aor-ta via the left ventricle. Excised brains were stored informalin until sectioning; transverse sections 40 nthick were examined through a stereomicroscope, andelectrode tip locations verified by comparison with theatlas by Pellegrino and Cushman.16 All lesion siteswere immediately lateral to the fornix andmamillothalamic tract; in addition to the posteriorhypothalamic nucleus, other structures within or adja-cent to the terminal lesion included the lateralhypothalamic area, premamillary nuclei, zona incerta,and median forebrain bundle.

Data (averages ± SEM) from both rat groups wereanalyzed using / tests for comparing means of in-dependent samples,17 and differences at a 5% level(p < 0.05) were considered significant.

ResultsInitial baselines for both blood pressure and sym-

pathetic nerve activity were elevated in DOCAhypertensive rats. Like the difference shownpreviously with the tail-cuff method when these ratswere awake, mean aortic pressure (mm Hg ± SEM),which averaged 140 ± 7 in DOCA hypertensive rats,was higher (p < 0.001) during urethane anesthesiathan the corresponding average of 112 ± 2 mm Hg insham-operated controls. Similarly, the frequency ofsympathetic nerve firing (spikes/3 sec ± SEM), whichaveraged 74 ± 9 in DOCA hypertensive rats, wassignificantly higher (p < 0.05) than the correspondingaverage of 52 ± 5 spikes/3 sec in sham-operated ones.By contrast, the averages for heart rate/min ± SEM ofstimulation on sympathetic nerve firing were alwayssham-operated rats were not significantly different.

Accelerated Nerve Firing During Hypothalamic StimulationElectrical stimulation of the posterior hypo-

thalamus increased nerve firing rates almost instantlyin all rats. Accelerated rates persisted throughout the10-second period of stimulation, but their magnitudewas usually greatest during the first 3 seconds and thendiminished spontaneously thereafter (though still stay-ing well above prestimulation levels) despite continuedstimulation. In both rat groups, the amount ofacceleration was proportional to the current strength(within the range tested) applied to the hypothalamus;the acceleration occurring during the first 3 seconds ofstimulation with any current was invariably morepronounced in DOCA hypertensive than in sham-operated rats (table 1).

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SYMPATHETIC HYPERACTIVITY IN DOCA HYPERTENSION/Takeda and Buhag 99

TABLE 1. Increases in Firing Rates of Sympathetic Nerves During Hypothalamic Stimulation in Rats Anesthe-tized with Urethane

Currentstrength

50

100

200

Stimulusduration

(sec)

3

6

9

3

6

9

3

6

9

R a t

Sham-operated(spikes/3 sec)

81 =

48 ±

34 ±

192 =i

142 ±

114 ±

t 12

fa 9

t 7

* 19

t 21

= 16

215 ± 23

182 ± 18

153 ± 18

group*

DOCA hypertensive(spikes/3 sec)

161 =fa 21

120 ± 21

79 -

258 ±

173 ±

t 7

8± 25

3± 22

131 ± 14

295 =•= 28

226 =

187 *

" 25

t 22

p value

< 0.005

< 0.005

< 0.025

< 0.05

D A

n.s.

< 0.05

n.s.

n.s.

*Results are presented as increases in absolute firing rates over base line levels obtained in 10 sham-operatedand eight DOCA hypertensive rats,

n.s. = not significant.

Comparison of Responses to Hypothalamic Stimulation andto Injected Norepinephrine

As in previous studies, the effects of hypothalamicstimulation on sympathetic nerve firing were alwaysaccompanied by measurable increases in aorticpressure, which were also much more prominent in

DOCA hypertensive than in sham-operated rats (fig.1). Aside from the pressor effects of hypothalamicstimulation, similar responses induced by injectinggraded doses of norepinephrine were also compared.Both stimuli consistently evoked measurable increasesin mean aortic pressure, which were larger in DOCA

FIGURE 1. Pressor and neural responses to hypothalamic stimulation in a sham-operated (A) and aDOCA hypertensive (B) rat. Upper panels: Tracings of phasic aortic pressure (mm Hg; note scale changebetween panels). Middle panels: Original nerve spike potentials. Lower panels: Integrated nerve activity(spikes/sec). The arrow marks the start of stimulation for 10 seconds at the current strength {\iA) indicatedby the large number.

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100 HYPERTENSION VOL 2, No 1, JANUARY-FEBRUARY 1980

hypertensive than in sham-operated rats (table 2). Ingeneral, pressor responses to hypothalamic stimula-tion tended to be more strongly enhanced than thoseto injected norepinephrine, but these differences inmagnitude of enhancement were small. For instance,in sham-operated rats, hypothalamic stimulation with100 nA produced pressor responses averaging 15 ± 2,which are almost the same as the average of 14 ± 1elicited by 200 ng/100 g of norepinephrine. By con-trast, in DOCA hypertensive rats, the same stimulusstrengths produced average pressor responses of40 ± 4 and 31 ± 4 respectively. With either stimulus,heart rates were almost invariably reduced, but noneof the differences between rat groups was significant.

Vasodepression Caused by Ganglion Blockade

As ganglioplegia was induced with pentoliniumroutinely at the end of each experiment, the ensuingfall in aortic pressure provided a readily availablemeans of estimating the amount of pre-existing sym-pathoadrenal activity. The mean aortic pressure wasreduced more (p < 0.005) in DOCA hypertensive(—78 ± 6 mm Hg) than in sham-operated ( -53 ± 3mm Hg) rats; consequently, baseline pressures afterganglion blockade were almost equal in both groups(65 ± 5 and 61 ± 2 mm Hg respectively).

Discussion

Much of the evidence now being disputed about therole of sympathetic hyperactivity in DOCA hyperten-sion comes from experiments in which the presence orabsence of sympathetic overactivity was inferred in-directly from blood pressure changes produced byeither immuno- or chemical sympathectomy. Im-munosympathectomy completely prevented develop-ment of hypertension in one study,18 but was found in-effective in another published in the same year."Similarly, contradictory results have been reportedfollowing chemical sympathectomy produced with 6-hydroxydopamine. Whereas two reports claimed thatthis drug was successful in protecting against subse-

TABLB 2. Pressor Responsiveness in Urethane-aneslhetizedRats

Stimulus

Hypothalamic

stimulation

G*A)

Norepinephrine

injection

(ng/100 g)

Strength

50

100

200

100

200

400

RatSham-

operated(mm Hg)

6 ± 1

15 ± 2

19 ± 2

9 ± 1

14 ± 1

23 ± 3

group*DOCA

hypertensive(mm Hg)

26 =

40 *

t 4t 4

46 ± 5

22 =i

31 =i

t 4

t 445 =*= 6

'Average =*= SEM changes from base line levels.All p values comparing averages between sham-operated

and DOCA hypertensive groups are < 0.005.

quent elevation of blood pressure,'118 many otherattempts failed,5"7 possibly because sympathectomieswere incomplete. Adrenergic nerve endings in bloodvessels regenerate more rapidly,19 and are less readilydepleted of their catecholamine content followingchemical or immunosympathectomy.10'21

Nevertheless, even more perplexing results wererecently reported by Provoost and de Jong,8- * whofound that the development of DOCA hypertensionwas potentiated, rather than inhibited, in rats sub-jected to neonatal sympathectomy. Destruction ofsympathetic nerves was presumed to be functionallycomplete since pressor responses could not be elicitedwith either tyramine or electrical stimulation ofvarious brain or spinal centers.8 The antihypertensiveprotection previously obtained by others was at-tributed to hypotensive sensitization by anesthesia,but why this occurred only in DOCA hypertensive ratswas left unexplained.

Additional evidence attesting to the existence ofsympathetic hyperactivity in rats with establishedDOCA hypertension can be obtained from our results.Despite wide variations from rat to rat in the frequen-cy of spontaneous nerve firing, baselines for sym-pathetic nerve activity were significantly faster at theonset in DOCA hypertensive than in normotensiverats. And when the posterior hypothalamus was sub-sequently stimulated with graded current strengths,the magnitude of both the added acceleration in nervefiring (table 1) and the ensuing pressor responses(table 2) were invariably stronger in DOCA hyperten-sive rats than in normotensive ones.

It seems logical to assume that this enhancement ofpressor responsiveness was due mainly to the accom-panying increase in sympathetic nerve discharge, butthis cannot be the sole mechanism involved becausepressor responses to injected norepinephrine were alsoaugmented, thereby implying that vascular sensitivityhad also increased. Exactly how much eachmechanism contributed is difficult to determine. Forinstance, upon hypothalamic stimulation with 50 nA,the increase in sympathetic nerve firing was only abouttwo times larger in DOCA hypertensive rats, but thecorresponding increment in pressor response wasalmost four times larger. Since pressor responsivenessto injected norepinephrine was only doubled, the dis-crepancy could conceivably indicate an enhancedrelease of endogenous norepinephrine. Becausedifferences in the magnitude of both the sympatheticnerve firing and pressor responses became smaller asthe strength of the hypothalamic stimulation inten-sified, it seems likely that the extent of the neural con-tribution to the enhanced pressor responsivenessdiminished as current strengths for stimulation wereincreased. Finally, still another sign of sympatho-adrenal hyperactivity is provided by the greatervasodepression resulting in DOCA hypertensive ratsfollowing pharmacologic blockade of autonomicganglia with pentolinium.

If sympathetic overactivity really accounts for mostof the blood pressure elevation once DOCA hyperten-sion becomes established, then interruption of the

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SYMPATHETIC HYPERACTIVITY IN DOCA HYPERTENSION/Takeda and Buhag 101

efferent nerve pathway at or below the site of over-activity should lower the blood pressure moremarkedly in DOCA hypertensive rats than in others.Of the procedures tested thus far (other than immuno-or chemical sympathectomy), a greater hypotensiveeffect did occur following pithing" or section" of thespinal cord, or after pharmacologic ganglionblockade."'" Aside from pentolinium, two otherganglion-blocking drugs, namely, chlorisondamine24

and mecamylamine,26 have also been tried but inter-pretation of the results is complicated by the fact that,regardless of the site of action, other hypotensivedrugs such as furosemide," hydrochlorothiazide,2* ordiazoxide,27 have all been found consistently moreeffective in DOCA hypertensive rats than in normo-tensive controls. Another drug with selective but weakantihypertensive effects that could be centrallymediated is propranolol.28

Because identical blood pressure levels can beproduced in normotensive and DOCA hypertensiverats by cutting the spinal cord at the C6-C7 level,23 butnot by destroying the posterior hypothalamus,11 sym-pathetic nerve hyperactivity may be occurring at siteson the neuraxis lower than the posterior hypo-thalamus. Thus, the apparent contradictionbetween enhanced pressor responsiveness to hypo-thalamic stimulation10 on the one hand, and thelack of a greater hypotensive effect upon hypo-thalamic destruction11 on the other, would beresolved. In line with this, and based on the selectivedecrease of norepinephrine turnover in the brain stemof DOCA hypertensive rats,2*' w Chalmers*0 proposedthat increased sympathetic nerve activity at theperiphery results from reduced inhibition bymedullary noradrenergic neurons terminating at thevasomotor center.

References

1. De Champlain J, van Ameringen M: Regulation of bloodpressure by sympathetic nerve fibers and adrenal medulla innormotensive and hypertensive rats. Circ Res 31: 617, 1972

2. Iriuchijima J, Mizogami S, Sokabe H: Sympathetic nervous ac-tivity in renal and DOC hypertensive rats. Jpn Heart J 16: 36,1975

3. Haeusler G, Finch L, Thoenen L: Central adrencrgic neuronesand the initiation and development of experimental hyperten-sion. Experientia 28: 1200, 1972

4. Reid J, Zivin J, Kopin I: Central and peripheral adrenergicmechanisms in the development of deoxycorticosterone-salinehypertension in rats. Circ Res 37: 569, 1975

5. Clarke D, Smooklcr H, Barry H: Sympathetic nerve functionand DOCA-NaCl induced hypertension. Life Sci 9: 1097, 1970

6. Finch L. Leach G: The contribution of the sympathetic nervoussystem to the development and maintenance of experimentalhypertension in the rat. Br J Pharmacol 39: 317, 1970

7. Finch L, Cohen M, Horst W: Effects of 6-hydroxydopamine atbirth on the development of hypertension in the rat. Life Sci 13:1403, 1973

8. Provoost A, de Jong W: Influence of neonatal chemical sym-pathectomy on the development of hypertension in spon-taneously hypertensive rats (SHR) and in rats with renal or cor-ticosteroid hypertension. Clin Exp Pharmacol Physiol 3:145, 1976

9. Provoost A, de Jong W: Differential development of renal,DOCA-salt, and spontaneous hypertension in the rat afterneonatal sympathectomy. Clin Exp Hyperten 1: 177, 1978

10. Bufiag R, Eferakeya A, Langdon D: Enhancement ofhypothalamic pressor responses in spontaneously hypertensiverats. Am J Physiol 228: 217, 1975

11. Bufiag R, Eferakeya A: Immediate hypotensive aftereffects ofposterior hypothalamic lesions in awake rats with spontaneous,renal, or DOCA hypertension. Cardiovasc Res 10: 663, 1976

12. Ormsbee H, Ryan C: Production of hypertension with deoxy-corticosterone acetate-impregnated silicone rubber implants. JPharm Sci 62: 255, 1973

13. Bufiag R: Validation in awake rats of a tail-cuff method formeasuring systolic pressure. J Appl Physiol 34: 279, 1973

14. Bufiag R, Riley E, Montello M: Sustained pressor respon-siveness to prolonged hypothalamic stimulation in awake rats.Am J Physiol 231: 1708, 1976

15. Takeda K, Buflag R: Sympathetic hyperactivity duringhypothalamic stimulation in spontaneously hypertensive rats. JClin Invest 62: 642, 1978

16. Pellcgrino L, Cushman A: A Stereotaxic Atlas of the RatBrain. New York, Appleton-Century-Crofts, 1967, p 35

17. Snedccor G, Cochran W: Statistical Methods. Ames, IowaState University Press, 1967

18. Ayitey-Smith E, Varma D: An assessment of the role of thesympathetic nervous system in experimental hypertension usingnormal and immunosympathectomized rats. Br J Pharmacol40: 175, 1970

19. Finch L, Haeusler G, Kuhn H, Thoenen H: Rapid recovery ofvascular adrenergic nerves in the rat after chemical sympathec-tomy with 6-hydroxydopamine. Br J Pharmacol 48: 59, 1973

20. Berkowitz B, Spector S, Tarver J: Resistance of noradrenalinein blood vessels to depletion by 6-hydroxydopamine or im-munosympathectomy. Br J Pharmacol 44: 10, 1972

21. Finch L, Haeusler G, Thoenen H: A comparison of the effectsof chemical sympathectomy by 6-hydroxydopamine in newbornand adult rats. Br J Pharmacol 47: 249, 1973

22. Dusting G, Harris G, Rand M: A specific increase in car-diovascular reactivity related to sodium retention in DOCA-salt-treated rats. Clin Sci Mol Mcd 45: 571, 1973

23. van Ameringen M, De Champlain J, Imbeault S: Participationof central noradrenergic neurons in experimental hypertension.Can J Physiol Pharmacol 55: 1246, 1977

24. Dc Champlain J, Krakoff L, Axelrod J: Relationship betweensodium intake and norepinephrine storage during developmentof experimental hypertension. Circ Res 23: 479, 1968

25. Finch L: The neurogenic component in the experimentalhypertensive rat. Pharmacology 5: 245, 1971

26. Stanton H, White J: Hypotensive actions of drugs on un-anesthetized normotensive and 'metacorticoid' hypertensiverats determined by a direct recording technique. Arch Int Phar-macodyn Ther 154: 351, 1965

27. Wohl A. Hausler L, Roth F: Mechanism of the antihyperten-sive effect of diazoxide: in vitro vascular studies in the hyperten-sive rat. J Pharmacol Exp Ther 162: 109, 1968

28. Bufiag R: Propranolol in DOCA hypertensive rats: develop-ment of hypertension inhibited and pressor responsivenessenhanced. Eur J Pharmacol 43: 323, 1977

29. Nakamura K, Gerold M, Thoenen H: Experimental hyperten-sion of the rat: reciprocal changes of norepinephrine turnover inheart and brainstem. Naunyn Schmicdebergs Arch Pharmacol268: 125, 1971

30. Chalmers J: Brain amines and models of experimentalhypertension. Circ Res 36: 469, 1975

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K Takeda and R D Buñagrats.

Augmented sympathetic nerve activity and pressor responsiveness in DOCA hypertensive

Print ISSN: 0194-911X. Online ISSN: 1524-4563 Copyright © 1980 American Heart Association, Inc. All rights reserved.

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