Effects of intravenous infusions of vasopressin and angiotensin I1 on central and peripheral noradrenergic function in conscious rabbits

K. P. PATEL,' C. A. WHITEIS, D. D. LUND, AND P. G. SCHMID Veterans Adminisrrution Medical Center, C~rdiovu~scular Center, Iowu Citj , IA, L1.S.A. 52240

and Depcirtrnent of lnternul Medicine, Lrnivrrsiiy oj Iowa, Iowu City, IA, U .S.A . 52242

Received August 12, 1986

PA.TEL, K . P., WHITEIS, 6 . A . , LUND, 1). D.. and SCHMID, P. ti. 1987. Effects of intravenous infusioils of vasopressin and angiotensin IT on central and peripheral noradrenergic function in coilscious rabbits. Can. J . Physiol. P h m a c o l . 65: 765-772.

Vasopressin (AVP) and angiotensin 11 (AII) are proposed to exert part of their cardiovascular effects via different actions within the central nervous system. These peptides are also known to alter central noradrenergic function. In the present study we determined the effects of these peptides administered intravenously on norepinephrine (NE) turnover in discrete brain regions thought to be involved in the regulation of circulation, and sinlultaneously, in various peripheral tissues. An index of NE turnovcr was determined by measuring the decline in tissue NE concentration 75 ~ n i n after administration of a-methyl tyrosine (240 mg.kg-' smin ' , i.p.). During NE synthesis blockade, five separate groups of rabbits were infused intravenously ( 1 h) with either saline, AVP (4 and 16 m~.k~- - ' . rn in - - ' ) , A11 (0.1 pg*kg-'.min '), or phenylephrine (PE) (5 pg-kg .rnlin--'). The low dose of AVP produced an increased index of NE turnover in the median presptic area and the paraventricular nucleus, and concomitant- ly, a decreased index of NE turnover in kidney and skeletal muscle. In contrast, A11 produced an increased index of NE turnovcr in the locus ceruleus and the intestine. Neither the infusion of vehicle nor the infusion of phenylephrine, which increased arterial pressure comparable to AVP and AII, produced detectable changes in indices of central and peripheral norepinephrine turnover. A higher dose of AVP produced a different pattern of changes in NE turnover than the low dose. These results demonstrate that intravenous infusion of the low dose of AVP produced changes in noradrenergic function in specif c central areas known to be involved in autonomic outf ow. Concomitailt with the central changes, there were selective changes in peripheral noradrenergic function. A11 produced changes in both central and peripheral noradrenergic function that differed from the effects of AVP. These desperate actions might account for the previously observed differences in effects of AVP and A11 on baroreflex control of the circulation.

PATEL, K. P.. WHITEIS, C. A., LUND. D. D.. et SCHM~D, P. G. 1987. Effects of intravenous infusions of vasopressin and angiotensin 11 on central and peripheral noradrenergic function in conscious rabbits. Can. J. Physiol. P h m a c o l . 65 : 765-772.

On croit que la vasopressine (AVP) et l'angiotensine I1 (AZI) excrcent une partie de leurs effets cardiovasculaires via diverses actions dans le systkme nenleux central. On sait aussi que ces peptides alterent la fonction noradrknergique centrale. Dans la presente etude. nous avons determine les effets de l'administration intraveineuse de ces peptides sur Ie renouvellement de la norepinephrine (NE) dans des regions ckrebrales que l'on croit Gtrc impliqukes dans la rkgulation de la circulation. ainsi que dans divers tissus pkriphkriques. On a determine un indice de renouvellement de NE en evaluant la diminution de la concentration de NE tissulaire 75 min aprks l'administration d'cr-methyltyrosine (240 mgakg-'.min ', i.p.). Durant le blocage de la synthkse de NE, on a perfuse intravcineusement cinq g roups de lapins (pendant 1 h) avec une solut?on saline, de I'AVP (4 et 16 rn~ekg-'emin-'), de I'AII (O,1 pg-kg-'.min-') ou de la phknylephrine (PE) (5 pg-kg 'omin-'). A faible dose d'AVP, l'indice de renouvellement de NE etait a c d dans la region preoptique mediane et le noyau paraventriculaire et, sirnultankment, I'indice de renouvellement etait reduit dans le rein et dans le muscle squelettlque. A l'oppost5; I'AII provoqua une augmentation de I'indice dans le locus ceruleus et dans I'intestin. Ni la perfusion d'un vehicule ni la celle de norepinephrine, qui auginentkrent la pression artkrielle de maniere comparable a I'AVP et a I'AII, ne provoquercnt de variations dktectables des indices du renouvellement central et periphkrique de noripinkphrine. La dose plus ClevCe d7AVP provoqua un profil de variations du renouvellement diffkrent de celul de la dose faible. Ces resultats denlontrent que une perfusion intraveineuse de la dose faihle d' AVP provoqua des variations de la fonction noradrknergique dans des regions centrales spkifiques connues pour 6tre impliqukes dam la production autonome. SimultanCment aux variations centrales, il y eut des variations selectives de la fonction noradknergique pkriphkrique. L,'AII provoqua des variations, tant dans la fonction noradrenergique centrale que pkripherique, qui dlffkrkrent des effets de 1'AVP. Geci peut expliquer les diffirences, observkes anterieurement, entre 1'AVP et I'AII en ce qui a trait au contriile barorkflexe de la circulation.

[Traduit par la revue]

Introduction Recent studies (Guo and Abboud 1984; Guo et al. 1986;

Imazumi and Thames 1986; Schmid et al. 1985; Sharabi et a1. 1985; Undesser et al. 1985) have demonstrated facilitation by vasopressin (AVP) and attenuation by angiotensin I1 (AII) of baroreflex-mediated bradycardia and sympathoinhibition. These actions of A V P and A11 are proposed to act via the central nervous system (Guo and Abboud 1984; Schmid et al. 1985). A V P (I .v . ) causes bradycardia owing to increased vagal tone (Courtice et al. 1984). This bradycardia has been shown to be of

' ~ u t h o r to whom correspondence should be sent at the first address.

central origin (Courtice et al. 1984). In addition, Undresser st al. (1984) have shown that lesions of area postrema eliminates the facilitation of synrpathoinhibition and bradycardia during intravenous infusion of AVP. Likewise, Ferrario et al. (1972) have demonstrated that lesions of area postrema also abolishes the attenuation of AI17s pressor effect. The fact that lesions of area postrema modify the effects of AVP and A11 suggest that the influence of these peptides is centrally mediated. These results are consistent with a hypothesis that AVP and AII differ in the effects that they produce, yet mediate their action via the central nervous system to nnodulate autonomic outflow.

Various central sites involved in the control of autonomic Printed in Canada : Impri~nt au Canacla

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766 CAN. 9. PHYSIOL. PHARMACOL. VOL. 65, 1987

outflow are innervated by noradrenergic neurons (Swanson and Sawchenko 1983). When either A V P or A11 are given into the lateral cerebral ventricles, they produce increases in noradrener- gic function in many of these brain regions (Sumner and Phillips 1983; Tanaka e t al. 1977; Vcrsteeg and Van Heuvan-Nolsen 1984). In addition, Undesser e t al . (1986) have shown that intracerebroventricular administration of 6-hydroxydopainine abolishes the potentiation of baroreflexes produced by vasopres- sin. However. it is not known if intravenous infusions of AVB and AII can affect central noradrenergic function in conscious rabbits. Fur themore , the regional pattern of peripheral norep- inephrine (NE) turnover produced in response to intravenous infusion of either of these vasoactive peptides is not known.

The present study was undertaken in conscious rabbits t o answer three questions. (i) B o e s intravenous infusion of A V P alter noradrenergic function in various brain areas known to be involved in the control o f cardiovascular function? The results will identify specific central sites that may be involved in the facilitation of baroreflex by AVP. (ii) Boes intravenous infusion of AVP produce a selective change in the peripheral sympathetic outflow in conscious freely moving rabbits'? T h e significance of this finding would be that AVB may b e involved in selectively changing blood flow via altering sympathetic nerve activity. (iii) Does AVB exhibit a different pattern of changes in central and peripheral noradrenergic function c o n ~ p a r e d with AII, another vasoactive peptide? The results will identify differences in cen- tral noradrenergic pathways that may be involved in facilitation and attenuation of baroreflexes by A V P and A l l , respectively.

Methods Preparation ofrcrhhits

Rabbits (New Zealand, 2-2.5 kg) acclimatized to the Baboratoi-y environment ( 12-h light cycle and 20-22°C temperature) were studied in experiments perfomed between 0800 and 1200.

Two days praor the experiment, rabbits anesthetized with sodium - - - - - -

pentobarbital (48-58 mglkg , i . v .) wereimphnteciwfihfemoralarterial and venous cannulae. On the day of the experiment, arterial pressure was recorded for 1 h prior to, and during, the experiment in conscious. freely moving rabbits via Gould P50 pressure transducer on a Gould 2000 Series Recorder. Heart rate was determined using a Gould tacho- graph (Bio-Tac) triggered by the arterial pulsations.

Measurement qfhlE turimwr using the blocknde of NE synthesis

Experimental groups ,4niinals were assigned to five treatment groups: AVP infused (3 and

16 mu-kg ' -min- ') , saline-infused controls, All infused (0.1 pg. kg1.anin-I), and phenylephrine infused (5 pg.kg-'.min-'). Pressor agents or saline vehicle were given by intravenous infusion for 1 h. Control interventions were infusions of saline without detectable effects on arterial pressure or infusions of phenylephrine. which raised arterial pressure comparable to the levels achieved by infusion of AVP and ,411. Fifteen minutes before infusion of saline or drugs, these rabbits received a-methyltyrosine methylester hydrochloride (240 mg/ kg, i.p.; Sigma Chemical, St. Louis, h-20). an inhibitor of tyrosine hydroxylase (Brd ie et al. 1966). A separate group of rabbits in- strumented with the catheters, but not given the synthesis inhibitor, were killed and used as control for endogenous levels of NE.

Tissue sampling In all treatment groups, 75 rnin after a-methyl tyrosine administra-

tion, rabbits were killed with a bolus dose of sodium pentobarbital ( I00 mg, i.v.1. The brain, left kidney, heart, left adrenal gland, a section of the duodenum, and a piece gracilis muscle were quickly removed. The brains were initially frozen on dry ice and then stored at - 7 0 T . Later the brains were sliced in 400-prn sections in a cryostat at 10°C. Using the method developed by Palkovits and Brownstein (1983), specilic

TABLE 1. Coordinates for the microdissection of discrete rcglons

Region Anterior-posterior Lateral Dorsal-ventral

Cerebral cortex

Median prcoptic area

Preoptis area Supraoptic

nucleus ParaventPnculx

nucleaus Anterior

hypothalanlus Posterior

hypothalamus Lmus ceruleus '42

A B

NOTE: Cocprdmates were rtleasured in millitnetre\ from the bregrna according to the Stereofaxk Atlas of A4cBride and Klernm et al. ( 1068).

areas and brain nuclei were identified under a dissecting microscope and punches were taken bilaterally from two adjacent slices using a 16-gauge blunted needle (Table I). The anatomy of the areas was confinned by matching the sites with a stcreotaxic atlas of thc rabbit brain (McBride and K l e m ~ 1968). Subsequently, these central sites were also confirmed to contain norepinephrine by matching the sites with the maps of norepinephrine distribution in central nervous system of rabbits (Blessing et al. 1978).

In peripheral tissues, the heart was dissected to obtaln left ventricle, sinoatrial node, atrioventricular node, and right atrial appendage. Thc kidney was dissected to remove a section oi the renal cortex. A piece of the duodenum was obtained by taking the first 2 cm of the small intestine starting at the level of the stomach. A large piece sf gracilis muscle was taken from the limb contralateral to the arterial catheter.

Ixtr~tinn~flvEJrorn~ti~~u~.~ - - - - - - - -

Samples of brain were sonicated in 250 p L 09'0.05 I % r p E c ~ o T i ~ ~ ~ K ~ containing an internal standard, dihydroxybenzylarnine (DHBA , con- centration in perchloric acid was 1.0 ng150 pL). Fifty microlitres of the hornogenate was taken for assay of protein by the method of Lowry et al. (1951); the remaining 200 pL were centrifuged (15 min, 12 000 k 8 , 4°C). The supernatant was then assayed using high performance liquid chromatography (HPLC) in combination with an electrocheani- cal detector (Pate1 and Wine 1984; Schinid et al. 1982a).

Peripheral tissue samples were weighed and homogenized in a solu- tion prepahed by diluting cold perchloric acid (34.5 mL of 70%). sodium EDTA (0.5 g), sodium metabisulfite ( I .O g). and internal standard (DHBA) in 1 L of triple distilled water. The final concentra- tion of DHBA in the homogenizing solution was 20 ng/mL. Samples in homogenizing solution were centrifuged at 4'C for 30 rnin at 13 000 x g ; supernatants were frozen at -70°C. NE was assayed following alumina extraction by an MPLC separation and electrochemical detec- tion (Patel and Kline 1984; Schmid et al. 1982n). Previous studies showed that the extraction ratio for NE and DHBA in the variou5 tissuc homogenates was constant (Pate1 and Kline 1984).

Measurement of plasma osmc~la l i~ m d AVP ,4t the end of an experiment, blood (2.0 mL,) was collected from the

arterial cannula of each rabbit into heparinized centrifuge tubes placed on ice. Osrnolality was measured by freezing point depression. A l -mL sample of plasma was subjected to an extraction procedure for AVP. Briefly, plasma proteins were precipitated by the addition of' 4 ~laL of cold acetone to 1-8nL plasma samples and the precipitate was removed by centrifugation at 3000 X g for 30 rnin at 4°C. The acetone was extracted with 10 nlL cold petroleum ether. the phases were separated by centrifugation. and the ether phase was discarded. The lower

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PATEI, ET AL.

TABLE 2. Initlal blood pressure and heart rate - -

Control AVP (4 m u ) A W (1 6 m u ) A11 PE

Mean blood pressure (mmHg) 8 0 5 3 8 5 2 1 86 +4 8 4 5 2 8 4 5 2

Heart rate (bcatslmin) 2582 11 2772 12 281211 260513 268210

Now: Values represent rnean -C SE (n = 5 - 8 ) . There were no significant differences among the groups.

aqueous phase containing the vasopressin was taken to dryness in a 37°C water bath under a stream of room air. The residue was redis- solved in a I-mL solution of 0.03% acetic acid and 0.15 M NaCl (Ac-saline) and stored at -20°C. AVP was measured by radiolmmu- noassay (RIA), which has been described in detail previously (Matsu- guchi et al. 198 1). Posterior pituitary reference standard was prepared as described in the United States Pharmacopoeia, except chlorobutanol (0.5% by weight) was added as a preservative. 'This material was stored at 4°C at a concentration of 2.1 U/mL and used as a standard for the RIA.

Data unaly.sis Differences in the index of NE turnover were obtained by comparing

the values for tissue concentration of NE 75 min after a-methyl tyro- sine. A lower concentration of NE remaining 75 min after inhibition of tyrosine hydroxylase would i~nply a higher turnover of NE in brain and peripheral tissue (Bhagat 1967 Brodie ct al. 1966; Kline et al. 1986; Pate1 and Kline 1984; Sharman 1981; Sole et al. 1980; Sumners and Phillips 1983; Versteeg and Van Heuvan-Nolsen 1984; Wijnen et al. 1978).

In an earlier study, tissue NE was determined at different time intervals after inhibition of tyrosine hydroxylase (Pate1 et al. 1981). A plot of NE concentration as a percent of the initial value on a log scale versus time after synthesis blockade gave a straight line. Linear regres- sion analysis gave correlation coefficients ranging from 0.85 to 0.98. The majority of over 50 linear relationships from three species of rats had correlation coefficient greater than 0.90 for cortex, hypothalamus, brain stem, skeletal muscle, kidney, and intestine (Pate1 et al. 1981). Since it was demonstrated that there was a single exponential rela- tionship for the disappearance rate of NE in various tissues after inhibition of tyrosine hydroxylase, the present study has utilized a single time point determination (75 min) to infer the turnover of NE. This approach has been widely accepted in a number of previous studies (Bhagat 1967; Brodle et al. 1966; Kline et al. 1986; Pate1 and Kline 1984; Shannan 1981; Sole et al. 1980; Sumners and Phillips 1983; Versteeg and Van Heuvan-Nolsen 1984; Wijnen et al. 1978). Validity of this approach is also supported by the general agreement between results obtained using this and other separate methods to determine turnover of NE (Brodic et al. 1966; Sharnlan 1981). In addition, a second method to assess turnover of NE (Schmid et al. 1982u) showed corresponding results in 12 of the 14 tissues examined in response to high dose of AVP in four rabbits, further demonstrating the validity of the method used to assess index of NE turnover (K. P. Patel, C. A. Whiteis, D. D. Lund, and P. G. Schmid, unpublished results) .

For peripheral tissues, endogenous concentrations of NE 75 min after a-methyl tyrosine were expressed as a percent of the rnean initial tissue concentrations of NE obtained from zero time controls to accomnlodate the data on a single scale.

Statistical comparisons between these 75-rmin values for control groups and the experimental groups were done by an analysis of variance followed by a least squares means test (Winer 197 1). All data are reported as mean + SEM. A p < 0.05 was considered statistically significant.

Results Arterial pressure and hear? rate

Initial mean arterial pressure and heart rate prior to interven-

Change l 5 t In Blood ,n

PE A V P 16mkl) A V P [dm")

8 "

Pressure (' P irnrnHg) 5

- -4 All

0 Change In Heart 25

Rate iBeats/rn!n) -50

-75

control All P E

AVP (4rnU) A V P ( 1 6rnU)

FIG. 1. Effects of intravenaus infusions of vasopressin, angiotensin 11, phenylephrine, and saline on (a) mean arterial pressure and (b) heart rate. (a) Values represent mean (I+-SE) change in pressure (mmHg) from preinfusion values in each respective group. Preinfusion values were not significantly different among groups. AII-, AVP- (4 and 16 mu), and phenylephrine-infused groups had significantly (p < 0.05) increased arterial pressure compared with saline-infused controls. ( b ) Values represent mean (LSE) change in heart rate (beatsimin) from preinfusion values in each respective group. Preinfusion values were not significantly different among groups. AVP- (4 and 16 m u ) and phenylephtine-infused animals had significantly (p < 0.05) lower heart rates compared with saline-infused controls. N = 5-81group.

tions are shown in Table 2. Arterial pressure increased significantly in a dose-related fashion in response to AVP infu- sions; the increases were initially 9 2 1.3 mmHg in the low-dose AVP-infused group and 12 1.5 mmI-Ig in the high-dose AVP-infused group (Fig. 1 a). A11 and phenylephrine infusions caused significant pressor responses that were comparable to the high-dose AVP and were initially 14 0.6 and 1 3 -f 0.6 m H g , respectively. These arterial pressure responses were sustained over the period of infusions. Vehicle infusion had no detectable effect on arterial pressure.

AVP produced a dose-dependent decrease in heart rite, which recovered partially during the I-h infusion (Fig. 1 b). A11 and vehicle did not affect heart rate. Phenylephrine produced a decrease in heart rate between that of AVP and AII, which recovered by the end of the infusion.

Plasma o s n ~ o l a l i ~ and AVP AVP tended to lower plasina osmolality, although not

significantly (Table 3). AII, phenylephrine, and vehicle infu- sions had no effects on plasma osmolality.

Low and high doses of AVP produced plasma levels of 1 93 + 27 and 763 + 178 ~ U / ~ I P L , respectively, compared with 18.9 + 6.1 pU/mL in the vehicle-infused controls ( I pU -- 2.5 pg of AVP). There were no significant differences in the plasma levels of AVP in animals infused with either AII or phenylepherine compared with the vehicle-infused controls (Table 3).

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CAN. J . PHYSIOL. PHARIMACOE. VOL. 65 , 1987

TABLE 3. Plasma vasopressin and osmolality

Control AVP AVP (saline) (4 m u ) (16 m u ) AII PE

Vasopressin ( P U / ~ L ) 18.9-f-6.1 193.4?27.4* 763.42 178.3* 14.$-+ 1.4 14.023.6

Osmolality (mosrnol/kg water) 29353 286 + 5 28519 289 k 5 29424

NO IE. Values represent rnean & SE (n = 6 - 10). *Significantly different from the control (saline) group.

AVP ; IGnilJ)

control PE

83 AVP (4mU) All

AVP ( IBmU)

60

5 0 [NEJ Left

af ter Alpha- 7 5 rnin 4 0

after Alpha- Methyltyros~ne 30

I P S / ~ J S protein)

2 0

10

0

FIG. 2. Effect of intravenous infusions of AVP, AII, phcnylephrine. and vehicle on the index of NE turnover in the cerebral cortex, the median preoptic area, the preoptic area, and the supraoptic nucleus in conscious rabbits. NE concentration (pgipg protein) 75 rnin after a-methyl tyrosine is expressed as mean & SE. N = 6- lQ/group. *, p < 0.05 compared with vehicle-infused controls.

control PE

AVP (4rnU) All

Locus Ceruleus Ap Area A , Area

FIG. 4. Effect of intravenous infusions of AVP, AII, phenylephrinc, and vehicle on the index of NE turnover in the locus ceruleus (A6 area). dorsal-medial medulla (A2 area), and ventral-lateral medulla (Al area) in conscious rabbits. NE concentration (pg/pg protein) 75 min after a-methyl tyrosine is expressed as mean -t SE. N = 6- l0igroup. *, p < 0.05 compared with vehicle-infused contrr~ls.

TABLE 4. Initial endogenous concentrations of norepinephrine in peripheral tissues

AVP I 1BmU)

EM1 Left t 75 rnin 40

after Alpha- Methyltyrosine ( p g / p g protein)

20

10

0 Paraventricuiar Anter~or Posterlor

Nucleus Hypothalamus Hypothalamus

FIG. 3. Effect of intravenous infusions of AVP, AHI, phenylephrine, and vehicle on the index of NE turnovcr in the paraventricular nucleus, the anterior hypothalamic area, and thc posterior hypothalamic area in conscious rabbits. NE concentration (pgipg protein) 75 min after a-methyl tyrosine is expressed as mean k SE. N = 6- lO/group. *, p < 0.05 cornpared wlth vehicle-infused controls.

Tissue .VE concentration qfier inhibition uftyrosiize hydroxylase as an index of NE turnover

Brain After inhibition of NE synthesis, the infusion of a low dose of

AVP produced significantly greater decreases in tissue concen- trations of NE in the median preoptic area and paraventricular nucleus compared wlth saline-infused controls (Figs. 2 and 3).

Tissue [Norepinephrine]

Kidney Intestine Muscle Adrenal Left ventricle Right atrial

appendage Sinoaortic node Atrioventricular

node

N o ~ E : Values represent mean + SEM (n = 6).

Unlike the low dose of AVP, the high dose of AVP produced a significantly greater decrease in the concentration of NE compared with vehicle in the locus cemleus (A6 region) but not the paraven- tricular nucleus. Like the low dose, however, the high dose of AVP produced a greater decrease in NE concentration cor~~pared with vehicle in the median preoptic nucleus (Figs. 3 and 4). There was alsoa tendency toward agreaterdecrease inNEconcentration in the A2 area of animals infused with the higher dose of AVP compared with vehicle after synthesis blockade ( p < 0.06).

A11 compared witia vehicle produced a significantly greater decrease in NE concentration in the locus ceruleus (Fig. 4). Thcre were no significant differences between groups infused with

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ET AL. 765,

Control n=Q

PE n=B

AVP ( 4 r n ~ ) El A N n=7 n=8

0 AVP ( 16rnU) n = 8 *

[NCJ 80 As Percent Of 0-Time 60 Controls

40

20

0 Kidney Intestine Muscle Adrenal

FIG. 5. Effect of intravenous infusions of AVB, AII. phenylephrine, and vehicle on the index of NE turnover in kidney, skeletal muscle, intestine. and adrenal gland in conscious rabbits. NE concentration (ngjg) 75 nlin after a-methyl tyrosine is expressed as a percent (-+ SE) of 0-time controls. N = 6- 10;group. *, p < 0.05 compared with vehicle-infused controls.

[NE] 80 As Percent Of 0-Time Controls

40

20

0 RAAP SAN AVN i Ventr~cle

FIG. 6. Effect of intravenous infusions of AVP, AII, phenylcphrine, and vehicle on the index of NE turnover in the right atrial appendage (RAAP), the region of the sinoatrial node (SAN), the region of the atrioventricular node (AVN), and the left ventricle (LV) in conscious rabbits. Format as in Fig. 5. N =. 6- l Oigroup. * , p < 0.05 compared with vehicle-infused controls.

phenylephrine and vehicle with regard to NE concentrations in any central site after synthesis blockade.

Peripheral tissues Initial endogenous concentrations of NE for the peripheral

tissues are shown in Table 4. After blockade of NE synthesis, the rabbits infused with the low dose of AVP had significantly higher concentrations of NE in the kidney and skeletal muscle compared with controls infused with vehicle (Fig. 5). In con- trast, the high dose of AVP did not produce any detectable changes in NE concentration in skeletal muscle and kidneys compared with vehicle-infused rabbits.

After blockade of NE synthesis, the rabbits infused with A11 had significantly lower concentrations of NE in the intestine (duodenum) compared with vehicle-infused controls (Fig. 4). Phenylephrine and vehicle were associated with comsponding NE concentrations in all peripheral tissues after NE synthesis blockade (Figs. 5 and 6).

Discussion Results of this study demonstrate three major points. First,

intravenous infusions of AVP were capable of producing significant changes in an index of NE turnover in the median preoptic nucleus, paraventricular nucleus, and locus ceruleus, regions known to be involved in cardiovascular regulation. Second, concomitant with the central changes, low doses of AVP produced decreases in an index of NE turnover in kidney and skeletal muscle but not heart and intestine of conscious freely moving rabbits. Third, the central and peripheral norad- renergic responses to AVP differ from responses to AII, another vasoactive peptide.

Plasnsa levels of AVP range from I pU/mL during conditions of nonnal blood volume and osmolality to in excess of 200 pU/mL during severe hemorrhage, nausea, and vomiting (Liard 1984). AVP infusion rates higher than 4 mU.kg-'.min-' pro- duce plasma levels of vasopressin beyond the basal physiologic- al range (Liard 1 984). However, studies demonstrating central- ly mediated action of AVP on baroreflexes have used infusion rates between 2 and 50 m U - k g ' .min-' (Guo et al. 1986; Schmid et al. 1985; Undesser et al. 1985, 1986). In the present study, AVP was infused at 4 and 16 mLJ.kg-'.min-' to rcpresent low and middle doses of AVP previously observed to effect baroreflexes via its central action. The infusion rate employed in the present study (4 mU.kgl-minu') also corresponds to a rate employed for 1 h by Schwartz et al. (1985) (10 ng-kgl.min-') in their studies to examine the hemodynamic effects of AVP. Although the levels of plasma AVP employed in studies of neural control of circulation are higher than those associated with basal physiological responses (Liard 1984), recent evi- dence (Hasser et al. 1985) that endogenous release of AVP during hemorage and hypertonic saline influence neural control of circulation and sympathetic outflow, suggest that these ac- tions of AVP [nay have physiological implications under certain conditions.

To assess noradrenergic function in various central sites as well as peripheral regions, we employed inhibition of NE synthesis to determine an index of NE turnover. Turnover of NE as measured by inhibition of NE synthesis has been shown to vary in direct relation with changes in neural activity (Bhagat 1967; Sharman 198 1). Our earlier data have demonstrated that multiple deter~l~inations of tissue NE concentration over time conform to an exponential decline in tissues NE concentration after blockade of NE synthesis (Pate1 et al. 198 1). Thus, a single determination of NE concentration in tissues after administra- tion of a-methyl tyrosine provides an accepted index of norad- renergic function (Bhagat 1967; Brodie et al. 1966; Kline et al. 1986; Patel and Kline 1984; Shannan 198 1 ; Sole et al. 1980; Sumners and Phillips 1983; Versteeg and Van Heuvan-Nolsen 1984; Wijnen et al. 1978).

In contrast with our expectation, we did not observe a reflex decrease in turnover of norepinephrine in the peripheral tissues in response to phenylephrine. This may be due to the following reasons. ( i ) The pressure increase (not very large, maximum of 13 mmHg) was produced to match the increase produced by AVP infusion. Previous studies have reported a 10-15% de- crease in nerve activity for pressure increases between 10 and 15 mmHg (Guo et al. 1986; Imazumi et al. 1984; Schmid ct al. 1985; Sharabi et al. 1985). The standard errors of our measure- ments in turnover are in the 5- 10% range. (ii) The response in the sympathetic nerve activity to increases in arterial pressure are transient, i.e., even though the arterial pressure is elevated the decrease in sympathetic nerve activity recovers to nomsal. This may be a function of baroreceptor resetting demonstrated in

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770 CAN. 3. PHYSIOL. PHARMACOL. VOL. 65 , 1987

conscious rabbits by Undesser et al. (1984). (iii) The measure- ment of turnover of norepinephrine is an integral value (total, sum) of integrated neural activity over the entire hour and a small acute change (smaller than 10%) may not be discrimin- ated.

When all these factors are considered together it is evident that an acute decrease in sympathetic nerve activity of 10% or less in magnitude may not be discerned. Nevertheless, AVP did produce significant decreases in turnover of norepinephrine in the kidney and skeletal muscle. Since phenylephrine was used as a control for the increase in arterial pressure, the only conclu- sion we have made in this regard is that a change in pressure per se is not responsible for the decrease in turnover of norep- inephrine observed in animals infused AVP.

&@ects AVB on central nurtrdrenergic-$dnction Numerous investigators (Courtice et al. 1984; Cowley et al.

1974: Liard et all. 1981; Schmid et al. 1985; Undesser et al. 1985, 1986) have suggested that AVP interacts at central site(s) to modify autonomic outflow. Liard et al. (1981) reported that AVP infused in the vertebral artery, at concentrations that do not exert a peripheral effect, produced a decrease in cardiac output, peripheral vascular resistance, and heart rate. AVP given in- travenously has been shown to produce bradycardia, as well as to inhibit peripheral sympathetic activity in rabbits (Guo et al. 1982; h a z u m i and Thames 1986; Sharabi et al. 1985; Undesser et al. 1985). These effects are mediated in part via the central nervous system (Guo et al. 1986; Undesser et al. 1986). Lesions of the area postrema abolished AVP mediated bradycardia and reflex sympathoinhibition (Undesser et al. 1985). Destruction of central catecholaminergic neurons by intracerebroventricularly (ICV) administration of 6-hydroxydopamine eliminates the potentiation of baroreflex gain by vasopressin (Undesser et al. 1986). In the present study, intravenous infusion of AVP pro- duced an increased index of NE turnover in the median preoptic nucleus, paraventricular nucleus, and locus ceruleus of con- scious rabbits. Since these central sites are known to influence autonomic outflow (Saper et al. 1976; Swanson and Sawchenka 19831, it is worth speculating that AVP may produce its effects on peripheral sympathetic neural function in part via changes in noradrenergic function in these central sites.

A major goal of this study was to investigate whether AVP given i.v. might affect central noradrenergic function and if so. how the pattern of central responses to i.v. AVP might contrast with previously reported central responses to AVP given into the cerebral ventricle. Tanaka et al. (1977) showed that AVP administered ICV produced increased turnover of NE in the preoptic area, the locus ceruleus, and the nucleus tractus solitar- ius in rats. Versteeg and Van Heuvan-Nolsen ( 1984) were able to inhibit the effects of ICV AVP with antisera to the hormone. Thus, it appears that AVP given either by intravenous or ICV administration can affect NE turnover in the midbrain and fore- brain, although the pattern of effects associated with the two routes of administration are slightly different. It is not clear whether the different species (rabbits vs. rats) or the different routes of administration (i.v. versus ICV) account for the differ- ing patterns of central noradrenergic responses to AVP in these separate studies.

In the earlier studies of Tanaka et al. (1977) and Versteeg and Van Heuvan-Wolsen (B984), there were no data on arterial pressure responses to ICV administration of AVP. Thus, it was not clear whether the changes in central NE turnover were secondary to pressor responses or more directly related to a central action of AVP. The present study establishes that in-

creases in arterial pressure per se during i.v. administration of AVP cannot account for the central changes in NE turnover since no such changes were observed in central noradrenergic activity with phenylephrine infusion. which produced a compa- rable change in blood pressure. However, the possibility of a permissive role for elevated arterial pressure in the central actions of AVP on noradrenergic function cannot be ruled out.

EfSect of AVP on p~r ipherd nurcidret~crgic fuactdon In the present study, the low dose of AVP decreased the index

of NE turnover in hindlimb skeletal muscle and kidney, consis- tent with decreased lumbar sympathetic nerve activity (Guo et al. 1982; Guo et al. 1986; Sharabi et al. 1985) and renal syns- pathetic nerve activity (Undesser et al. 1985) observed in re- sponse to short (6 min) intravenous AVP infusions. However, the high dose of AVP did not produce the expected decreases in turnover of NE in the present study. The plasma levels of AVP were cornparable between these studies. Therefore, results with the low dose of AVP appear to be consistent in the two studies, whereas results with the high doses of AVP do not. There are two possible explanations for this inconsistency. First, turnover of NE was determined over a l-h infusion of AVP to conscious rabbits whereas the efferent sympathetic nerve activity was recorded over a 2- to 6-min infusion of AVP to anesthetized rabbits (Guo et al. 1982; Guo et all. 1986; Sharabi et al. 1985). It is conceivable that the high levels of plasma AVP for 1 h affected central sites that simillar levels for 6 min may not have affected. This speculation is inferred by the observation of increased noradrenergic activity in the locus cemleus, a sym- pathoexcitatory area. in the present study during infusion of the high dose of AVP but not the low dose. Second, the higher dose of AVP infused for 1 h may have produced a facilitation on NE turnover at the nerve terminal that would offset inhibition of sympathetic nerve activity (Schmid et al. 1982f4). Future studies with infusions of AVP for 1 h and conconlitant nseasurement of sympathetic nerve activity and NE turnover would resolve these questions.

Kline et al. (1986) showed that chronic replacement of AVP in Brattelboro rats by intramuscular injection of AVP tannate for 7 days caused significant decreases in turnover of NE in heart, intestine, skeletal muscle, and kidney. Intravascular volume expansion or other chronic influences of AVP, apart from strict- ly a facilitation of sympathoinhibition, might account for these latter observations. It is unlikeBy that volume expansion could have played a role in the actions of AVP in the present study because infusion periods were limited ( 1 h) and fluid adl-ninistra- tion of 1.9 mL/h was similar in all the groups.

Possible sites of acabion-fir AVP The effect s f AVP on peripheral sympathetic neural function

may be produced by its actions at any one or all of the following sites, based on the current literature: (i) directly on the baroreceptors; (ii) directly or indirectly on the central nervous system (possibly involving the noradrenergic pathways); (iii) directly on the sympathetic ganglia; (iv) directly on the nerve terminal.

First, an effect of AVP on the baroreceptors is controversial ad the present time. Guo et al. (1986) have reported that AVP- mediated sympathoinhibition is partly through the action of AVP at the arterial baroreceptors. Hn contrast, Courtice et al. (1984) report that intravenous AVP infusion has no effect on impulse flow in the baroreceptor fibers frc?a-n the carotid sinus. The present study does not rule out either of thest: possibilities.

The second contention that AVP influences peripheral sym-

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PATEL ET AL

pathetic neural function via the central nervous system is possi- ble, since in previous studies, lesioning the area postrema abol- ished AVP-mediated bradycardia and sympathoinhibition (Undesser et al. 1985). It is of interest to note in the present study that there were changes in noradrenergic activity in central areas known to influence autonomic outflow, i.e., paraventricu- lar nucleus and the median preoptic nucleus (Saper et al. 1976; Swanson and Sawchenko 1983). The medial part of the paraven- tricular nucleus has been shown to contain projections to the interrnediolateral cell column of the spinal cord, indicating a direct link between the paraventricular nucleus and sympathetic preganglionic elements (Saper et al. 1976; Swanson and Saw- chenko 1983). Ure speculate that increases in norepinephrine turnover in the paraventricular nucleus with the low dose of AVP rnight be linked to decreases in noradrenergic activity in the kidney and skeletal muscle.

Similarily, the median preoptic nucleus has been shown to project to the dorsal motor nucleus of the vagus in the medulla (Saper et al. 1976). Courtice et al. (1 984) have shown that the cardioinhibition by AVP is due to increased vagal tone, confirmed by electrophysiological recording from the vagus. Consistent with this, in the present study, there appears to be a dose-response relationship between graded increases in norad- renergic function in the median preoptic nucleus and progres- sive bradycardia during vasopressin infusions (Fig.7). We speculate that the changes in noradrenergic activity in the me- dian preoptic nucleus may be related to the bradycardia pro- duced by AVP. In separate studies in our laboratory, injection of lidocaine into the median preoptic nucleus has significantly suppressed the reflex bradycardia produced by intravenous administration of AVP but not that produced by phenylyephrine (Pate1 and Schmid 1986).

The third contention that AVP has a direct effect at the level of sympathetic ganglia is possible. Wall (1984) have shown that there is decreased transmission of impulses at the rabbit superior cervical ganglia in the presence of AVP. Imiuzunli and Tharnes (1986) have also reported that AVP may be exerting its effects at the level of the sympathetic ganglia to inhibit renal sympathetic nerve activity. However, if the effect of AVP was via the sympathetic ganglia, one would expect a dose-related change in the index of NE turnover in all peripheral tissues examined. Contrary to such expectation, we observed no dose-dependent changes but observed changes in specific tissues in response to the lower dose of AVP.

The fourth contention that AVP exerts its peripheral effect directly at the level of the organ has been tested (Courtice et al. 1984; Scott et al. 1982). Courtice et al. (1 984) have reported that AVP did not have a direct effect on the heart rate, after com- bined blockade of P-adrenergic and muscarinic receptors. In addition. there is no direct effect of AVP on the myocardial cell auton~aticity in viiro (Scott et a1 . 1982). These results together indicate that AVB does not have its kradycardic response direct- ly at the level of the heart. The effect of AVP directly at the peripheral noradrenergic terminal remains to be examined. However, if such an action of AVP was present, one would expect that the changes observed would bc ubiquitous in the peripheral tissues and not selective as were observed in the present study.

Conirc~sekng e fec t of AII corrtparucd wlith AVP on cent/-ul anti gerkphvrai nc~rc~dretzergic function

Both AVP and A11 have been demonstrated to produce effects via the central nervous system (Ferrario et al. 1972; Guo et al. 1986; Undesser et al. 1985; Undesser et al. 1986). They nlay

Change in 1 heart rate

(beats/rnin) 1

60 50 40 30 20 10 0

(Increase turnover)

Concentration of NE remaining 75 min after Q-methyl tyrasine In median preoptic area (pg/pg protein)

FIG. 7. Relationship between the index of NE turnover in the median preoptic area and the change in heart rate in three groups of animals: AVP (4 and 16 mu) and vehicle-infused controls.

both act at the area postrenla initially and then via direct or multisynaptic pathways, influence the autonomic outflow (Fer- rario et al. 1972; Undesser et al. 1985). An ICV administration of AVP or A11 both produce increases in noradrenergic activity in various brain sites; however, the pattern of sites that are activated are different (Sumners and Phillips 1983; Tanaka et al. 1977). Parallel to the disparate affects of ICV AVP and AII, intravenbus AVP produced an increased noradrenergic activity in the paraventricular nucleus, median preoptic area, and the locus cemleus, whereas A11 produced increased noradrenergic activity only in the locus ceruleus.

Intravenous infusion of AVP produces sympathoinhibition (measured as decreased lumbar nerve activity) whereas a com- parable pressor dose of A11 does not (Schmid et al. 1985). These results indicate that AVP inhibits sympathetic activity but A11 does not. In the present study, the low dose of AVB produced decreased index of NE tuinover in the skeletal musc.le. which was not observed in animals infused with AII. Furthemlore, A11 produced an increased index of NE turnover in the intestine, whereas the low dose of AVP showed no difference in the intestine but produced a decreased noradrenergic activity in the kidney. Taken together, these results suggest that AVP and A11 have different effects on central and peripheral noradrenergic function.

Canelusion This study has identified changes in noradrenergic function in

specific sites (median preoptic area, paraventricular nucleus, and locus ceruleus) in the central nervous system known to influence autonomic outflow in response to intravenous infusion of AVP in conscious rabbits. Also, intravenous infusion of low dose of AVP affected peripheral noradrenergic function selec- tively, in that there was decreased noradrenergic activity in the skeletal muscle and kidney but no change in other peripheral tissues. Finally, compared with AVB, there is a different profile of changes in central and peripheral noradrenergic activity in response to intravenous infusions of AII.

Acknowledgements We thank Alberto Subieta and Carol Butz for technical assis-

tance. These studies were supported by Veterans Administra- tion and National Institutes of Health grants HL 20768, HL 24246, and HL 14388.

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'9'92 CAN. J. PMYSIOL. PHARMACOL. VQL. 65, 1987

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