Increased vasopressinergic activity following DOCA administration in the rat

Brain Research Bulktin,

0361~9230/86$3.00 + .OO

Vol. 16, pp. 93-98. 1986. 0 Ankho International Inc. Printed in the U.S.A.

Increased Vasopressinergic Activity Following DOCA Administrationin the Rat YIU-FAI CHEN,’ MARSHALL D. LINDHEIMER AND SUZANNE OPARIL Cardiovascular

Research and Training Center, University of Alabama at Birmingham Department o~~edi~ine~ 3irmingham, AL 35294 and Department of Obstetrics, Gynecology and Medicine, The Pritzker Schooi of Medicine University of Chicago, Chicago, IL 60637 Received 24 June 1985 CHEN, Y.-F., N. D. LINDHEINER AND S. OPARIL. Increased vasopressinergic activityfollowing DOCA administra1986.-The dynamics of hypothalamic arginine vasopressin (AVP) release was examined during the development of hypertension in rats with d~xyco~cos~~~ (DOCA)-NaCI induced hypertension. Experiments were performed in four groups of uninephrectomixed Sprague-Dawley rats 3,7,14 and 21 days after treatment with: (I) DOCA and 1% saline (DOCA-NaCl); (II) DOCA, regular chow, and tap water (DOCA-R-HIO); (III) regular chow and 1% saline (N&l); and (IV) regular chow and tap water (H,O). Systolic blood pressure (BP) was signifmantly elevated in DOCA-NaCl and DOCA-R-&O (183k-5 and 1554 mm Hg, respectively) but not in an additional contro1 group which received DOCA and a low NaCl diet (119zt2 mm Hg). Plasma AVP and h~~c AVP niease were increased in all DOCA treated groups at each time point studied, while plasma osmolality was similar in each group. These studies demonstrate increases in the hypothalamic release of AVPin DOCA-NaCl hypertensive animals, but suggest that they are due to the mineralocorticoid and are independent of blood pressure and NaCl intake.

tion in the rut. BRAIN RES BULL 16( 1) 93-98,

DOCA-NaCl hy~~ension

Hy~t~~amic

vasopressin release

Vasopressin

Sodium

level in relation to the development of hypertension has not been systematically evaluated in the DOCA-NaCl model. In addition, the cont~bution of DOCA per se, without added sodium, to the elevation in plasma AVP has not been examined thoroughly. In the current study the activities of central and peripheral vasopressinergic systems were assessed as indices of hy~th~~ic-hy~physe~ function in the developmental phase of DOCA-NaCl hypertension. AVF released from the hypothalamus, which does not contribute directly to the circulating pool, was measured in order to assess the activity of central vasopressinergic systems which are thought to modulate the function of neuronal pathways involved in blood pressure regulation. Circulating AVP, which is mainly a secretory product of the posterior pituitary, was monitored because it is known to alter blood pressure via a direct pressor effect and by enhancing pressor responses to catecholamines [2]. The release of AVP from isolated superfused hypothalamic tissue and plasma AVP levels were measured in material derived from rats treated for 3, 7, 14 and 21 days with DOCA-NaCl, and from control rats treated with DOCA and ordinary rat chow (DOCA-R-H,O), NaCl alone, or tap H,O alone and from an additional control group which received DOCA and low NaCl chow (DOCA-low NaCl) for 21 days. We observed that the spontaneous release of AVP from isolated hypothalami and plasma AVP levels were increased in

SEVERAL lines of evidence have implicated arginine vasopressin (AVP) in the pathogenesis of deoxycorticosterone acetate (DOCAbNaCl hypertension. Elevated plasma concentrations and urinary excretion of AVP have been reported in DOCA-NaCl hypertensive rats [8, 9, 17, 201. Administration of AVP has been shown to accelerate the appearance of hy~~ension in DOCA-NaCl treated rats 1141. Intravenous injection of specific anti-AVP antisera [In or antagonists of the pressor action of AVP [8] produces an acute reduction in blood pressure (BP) in DOCA-NaCl treated rats. Further, homozygous Brattleboro rats with diabetes insipidus fail to develop hy~~ension when treated with DOCA-NaCl unless they are also given exogenous AVP [3,8]. Nevertheless, controversy persists concerning the role of AVP in the maintenance of DOCA-NaCl hypertension. Bumier ef al. f6] observed no elevation in plasma AVP levels following 4 weeks of DOCA-NaCl treatment in the rat. Further, a number of investigators have noted little or no change in blood pressure following administration of an antagonist of the vascular action of AVP in DOCA-NaCl hypertensive rats [IS-201. The mechanism(s) by which plasma AVP levels in DOCA-NaCl hypertension are elevated are not understood. Whether increased secretion, decreased degradation and/or clearance from the circulation or a combination of these and other mechanisms is responsible is unknown. Further, the time course of appearance of elevations in circulating AVP

‘Requests for reprints should be addressed to Yiu-Fai Chen, Ph.D., C~iov~~ul~ Birmingham, 1012 Zeigler Building, University Station, Birmingham, AL 35294.

93

Research and Training Center, University of Alabama at

CHEN. DOCA treated rats at all time points measured. The observation that both hypothalamic AVP release and plasma AVP levels are increased in DOCA treated animals independent of NaCl intake and hypertension suggests that DOCA administration stimulates the release of AVP from the central nervous system by mechanisms independent of concomitant NaCl intake and blood pressure. The findings of enhanced hypothalamic AVP release and elevated circulating AVP levels in DOCA-low NaCl animals, which maintained normal blood pressures through the course of the experiment. do not provide direct support for an independent (of NaCl) role for either centrally released or circulating AVP in the pathogenesis of hypertension. METHOD

Male Sprague-Dawley rats (Charles River Breeding Laboratories, Wilmington, MA) housed in a constant temperature (24rl”C) and humidity (60*5%) facility with 12 hour dark/light cycles were fed a standard pellet diet (Charles River RMH 1000 formula containing 0.38% (w/w) sodium and 0.73% potassium). All animals underwent a left nephrectomy at 4 weeks of age and 2 weeks later were divided into 4 experimental groups as follows: (1) DOCA-NaCl-implanted SC with silastic strips (silicone rubber, Dow Corning Company, Midland, MI) containing 100 mg/kg of deoxycorticosterone acetate (DOCA) (Sigma Chemical Company, St. Louis, MO) and drank one percent saline ad lib; (2) DOCA-R-H,O-implanted SC with DOCA and drank tap water ad lib; (3) NaCl-sham operated and drank one percent saline ad lib; and (4) H,O-sham operated and drank tap water ad lib. A fifth group (DOCA-low NaCl) was subjected to unilateral nephrectomy and DOCA implantation as described, was fed a low salt diet containing 0.05% sodium (Purina Test Diets, Richmond, IN) and drank distilled water ad lib. Body weight and BP were measured twice weekly. Systolic BP was measured in conscious, prewarmed restrained rats by the tail-cuff method, using an electrophygmomanometer and physiograph recorder (Narco Biosysterns, Houston, TX).

Studies were performed on animals from groups 1-4 at three days, one, two, and three weeks after DOCA implantation or sham operation. Group 5 was studied at three weeks only. Awake rats were rapidly killed by decapitation, care being taken to avoid exciting the animals and squeezing their thoraces, since both of these insults are known to stimulate AVP [16] release. Trunk blood was collected in chilled, heparinized tubes for determination of plasma AVP concentration. Blood was immediately centrifuged at 4°C. Plasma osmolality was determined by freezing-point depression using an Advanced Osmometer (Advanced Instruments, Incorporated, Needham Heights, MA) and the remaining samples were stored at -20°C until used. The whole hypothalamus was quickly separated from the rest of the brain with tine scissors, cutting from the optic chiasma rostrally, from the mammillary bodies caudally, and laterally at the level of the hypothalamic sulci. The dorsal extent of the cut was to a depth of about 2.5-3.0 mm. These fragments, weighing approximately 35 mg, were removed gently and immediately placed in ice cold Krebs-Ringer-

._.

OOCA*l%

LINDHEIMER

ANI) OPAKII

NaCl

A -A OOCA+H20

190

180 !

.-.

1% NaCl

o---o

Hz0

n--¤OOCA

+ Low

N&l

al

?i z L a %

180-

150-

l% 140,0 0

z v)

130 i 120 i

Days After DOCA implantation 1. Changes in systolic BP in uninephrectomired rats during treatment with DOCA-1% NaCI. DOCA-R-H,O. DOCA-Low NaCI. 1% NaCl alone or Hz0 alone for 21 da&. Results represent meansi_SEM. The number of rats per subgroup for each determination is shown in parentheses. Asterisks above the bars designate differences from the H,O. NaCl and DOCA-LSW NaCl groups. *“=p
phosphate solution (NaCl, 120.3 mM; KCl, 4.8 mM; CaCI,, 2.6 mM; MgSOl, 1.2 mM; 295 mOsm; pH 7.4) containing glucose (0.18%), bovine serum albumin (0.1%) and pargyline (3.5x10 .’ M) at pH 7.4. This buffer was also used as the superfusion medium. The procedure for superfusion has been described previously [ 151. Briefly, hypothalamic tissue from each rat was cut into 12-16 pieces and placed in a superfusion chamber which had been constructed from the barrel of a 3 ml plastic syringe, then positioned in a constant temperature bath at 37°C. A volume of 500 ~1 was maintained in each chamber. The superfusion medium was delivered to each chamber from the bottom at a constant rate of 500 @l/10 min by a miniature peristaltic pump (Rainin Instruments, Wobum, MA). Tissue was allowed to equilibrate for 60 min to wash out the nonspecific release of AVP from damaged nerve endings and cell bodies prior to the collection of samples. Effluent (superfusate) was collected continuously for 100 min from tubing which exited from the top of the chamber. Collected superfusates were immediately snap frozen at 20 min intervals in dry ice-ethanol solution and stored at -20°C for AVP measurement. To assess the viability of the superfused tissue, hypothalami from 5 H,O control animals were subjected to chemical stimulation with a pulse of 60 mM K* injected directly into the chamber. Collected superfusate samples were immediately snap frozen at 2 min intervals (X 7) following the K+ bolus and processed as described below.

INCREASED

VASOPRESSIN

IN DOCA-TREATED

95

RAT TABLE

1

EFFECTS OF DGCA AND NaCl ON THE PLASMA OSMOLALITV, ARGININE VASGPRESSIN DURING THE DEVELOPMENT OF DGCA-N&l HYPERTENSION 3 Days

14 Days

7 Days

21 Days

A. Plasma Osmolality (mOsm/k@ 293.6 YZ1.4 (8) 1. H,O

295.0 + 1.7 (7)

296.8 it 1.2 (9)

296.6 Z!Z1.1 (16)

292.4 ir 1.0 (9) 294.5 2 1.4 (9)

293.8 f 1.5 (8) 2%.6 ” 1.4 (10)

2%.3 it 0.9 (8) 294.7 -c 1.6 (9)

294.7 ir 1.3 (18) 297.1 rt l.O(l8)

294.0 r 3.0 (10)

293.7 * 1.0 (10)

296.3 t 1.1 (8)

294.8 +: 1.5 (18)

-

-

-

296.6 ” 0.6 (14)

2. 1% NaCl 3. DOCA1% NaCl 4. DOCA-RH,G 5. DOCALow NaCl 3. Plasma a&nine 1. H,O 2. 1% NaCl 3. DOCA1% NaCI 4. DOCA-RHZ0 5. DOCAlow NaCl

vasopressin @g/ml) 3.0 2 1.0 (8) 2.4 t 0.7 (10) 8.8 z?: 1.1 (9) 15.6 ” 3.7 (lO)*ij

1.5 2 0.3 (7) 2.6 2 0.5 (8) 7.7 + 1.0 (lo)?

2.3 f 0.3 (9) 2.8 + 0.5 (8) 5.1 k 0.6 (9)t

1.4 ?Z0.2 (16) 1.3 it: 0.4 (18) 4.4 rt 0.3 (18)t

17.3 f 3.3 (lo)**

4.1 2t 0.9 (8)

10.2 ~?r1.8 (18)*$

-

-

-

4.8 rt 1.1 (13)t

Values are means f SEM; number of animab is &en in parentheses. *=p
Radioimm~~oa~~ay

Procedures

AVP in plasma samples and hypothahunic superfusates was quantified by radioimmunoassay (RIA). Details of our vasopressin assay have been published previously [il. Briefly, acetone extracts of plasma or unextracted superfusate were assayed at a fmai dilution of 1:3,000,000 with a highly specific antiserum (10169) that does not crossreact with oxytocin, vasotocin, or ~otensin II. The sensitivity of the assay is 0.15 pg/assay tube, with a 50% displacement at 2 pg/assay tube. The chloramine T iodinated tracer has a specific activity of > 1,000 pCi/pg and <5% nonspecific binding.

mkt K’ Pulse

60

14 -

Statistical Analysis Results were expressed as means+SEM. A one-way or two-way anaiysis of variance (ANOVA) was used for both within- and between-group comparisons. Between-group comparisons and comparisons over time were performed using Duncan’s New Multiple Range Test. Results with p
Figure 1 summarizes mean systolic BP’s for the five experimental groups over the course of the study. The BP’s of Hz0 control group, the NaCi group and the DOCA-low NaCi group remained unchanged throughout the three week experimental period. In contrast, blood pressure rose significantly in DOCA-NaCi treated rats (137*3 mm Hg, ~~0.01, ANOVA) as early as seven days after the beginning of treatment, compared to pretreatment levels (123+3 mm Hg). BP continued to rise progressively during the following weeks, to a level of 183+5 mm Hg at the end of three weeks. In rats

ot

1

20

I

I

I

40 60 SO Minutes of Superfurion

I

f

100

120

FIG. 2. Effect of a pulse of 60 mM potassium (ftnal concentration in the chamber) on AVP release from superfused hypothaiamic tissue in virm. Tissue was taken from uninephrectomized HZ0 control rat. Bars represent meansrtSEM. “n” is the number of ob’servations.

receiving DOCA and a standard diet (DOCA-R-H,O), a moderate increase in BP was seen at 14 days (14222 mm Hg vs. pretreatment 12422 mm Hg, pKO.01, ANOVA). BP in the DOCA-R-H,0 group reached 155&3 mm Hg at 21 days of treatment. Body weight did not differ among the five groups during the three week experimentai period. PIasma osmohrhty was not significantly altered by DOCA-NaCi, DOCA or NaCl treatment at any time point (Table IA). No differences

CHEN, LINDHElMER

DOCA/H20

(n

AND OPARi I

= 18)

6-

0’

1

20

_--L_

40

.I___i_

60

80

100

Minutes of Superfusion FIG. 3. Basal secretion rates of AVP from superfused hypothalamic tissue. Effluent samples were taken during the first 20 minutes of superfusion. Results are means%SEM. Numbers of animals are shown in the columns. a=p
FIG. 4. Spontaneous AVP release from superfused unsfimulateu hypothalamic tissue during a 100 minute superfusion period. Bars “n” is the number of observations. represent means?SEM. a=p
among groups were observed when data were analyzed by two-way analysis of variance. The response of hypothalamic AVP release to bolus injection of 60 mM K’ is illustrated in Fig. 2. A 2.5 fold increase in AVP release from stable baseline levels was observed during the first 10 min post K+ injection, confirming the viability of the superfused tissue. Figure 3 summarizes spontaneous release rates of AVP during the first 20 min of superfusion of isolated hypothalamic tissue derived from the five groups of rats. At 21 days of treatment, AVP release rates in the three DOCA treated groups were significantly elevated above both H,O and NaCl controls. The DOCA-R-H,0 treated rats had the highest release rate (12.0-t 1.3 pg/min/Hypo, n= 18), significantly greater than the DOCA-NaCl group (6.910.6 pg/min/Hypo. n=18, ~~0.01). AVP release in the DOCA-low NaCl group (9.6? 1.O pg/min/Hypo, n= 10) was intermediate between the DOCA-NaCl and DOCA-R-H,0 groups and not significantly different from either. AVP release rate was the same in the four groups studied early (3 days) in the course of treatment, then tended to decrease over time in the two control groups. At 21 days of treatment, AVP release rate was significantly (p
Plasma AVP levels are summarized in Table IS. Plasma AVP concentration increased in the DOCA-NaCl group, reaching significantly elevated levels @<0.05, ANOVA compared to H,O control and NaCl treated rats) one week after the initiation of DOCA-NaCl treatment. In the DOCA-R-H,0 group, plasma AVP levels increased more rapidly and to a greater extent than in DOCA-NaCl rats. Animals treated with DOCA and NaCl restriction also showed significant elevations in plasma AVP levels compared to H,O controls. At 21 days of treatment AVP levels in this group were not significantly different from those in the DOCA-NaCI group. In one percent NaCl treated rats, plasma AVP concentration remained unchanged throughout the study and was not different from that of H,O control group. DISCUSSION

Our results demonstrate that the spontaneous release of AVP from isolated superfused hypothalami was increased in unilaterally nephrectomized Sprague-Dawley rats treated with DOCA. Increases in hypothalamic AVP release and circulating AVP levels appeared in DOCA-treated rats independent of blood pressure and NaCl intake. These increments were observed as early as three days following initiation of DOCA treatment, a time when BP was not significantly increased in either the DOCA-NaCl or DOCA-R-H,0 group. Further, animals subjected to DOCA treatment and severe restriction of NaCl intake showed significant increases in plasma AVP levels and hypothalamic AVP release without concomitant elevations in blood pressure. These findings, coupled with the observations that NaCl treated rats showed no increase in AVP release or circulating AVP levels and that plasma osmolality was similar in each group, suggest that the elevation in plasma AVP observed in the DOCA-NaCl model of hypertension is related to DOCA administration rather than NaCl loading or hypertension per se. Further, these results do not provide direct support for an

INCREASED VASOPRESSIN IN DOCA-TREATED RAT independent (of NaCi) role for either centrally released or circulating AVP in the pathogenesis of hypertension in this model. Our observation of increased spontaneous AVP release from isolated superfused hypothalami of DOCA treated animals is the fmt demonstration that central AVP release is altered by mineralocorticoid treatment. The in vitro superfusion technique employed in our experiments has been shown to be useful in studying the mechanism of secretion of several neurohormones [7], including AVP 112,251.DeVito et al. [ 121demonstrated that spontaneous and K+-stimulated AVP release from the hypothalamic-neurohypophyseal tissue unit of SHR is greater than that from tissue of WistarKyoto (WKY) control rats. In addition, Siadek and Blair [23] demonstrated that acetylchoiine causes a concentration dependent stimu~tion of AVP release from organ cultured hy~th~am~ne~ohy~phys~ explants obtained from SHR and WKY donors, and that the acetyichoiinestimulated AVP release is signi&antly greater in the explants from 5- and &week-old SHRs than in explants from age matched WKYs. These data were interpreted by these authors as suggesting that an abnormal hypothalamicpituitary system may play an active role in the development and maintenance of hypertension in the SHR. Our fmdings of increased hypothalamic release and circulating levels of AVP antedating the development of hypertension in DOCA-NaCi and DOCA-R-H,0 treated rats are consistent with the interpretation that increased central neurosecretory activity precedes the development of hypertension in the DOCA-NaCi model. Whether these alterations contribute to the development of DOCA-NaCi hypertension is a question which merits further study. Spontaneous release of AVP from whole hypothalamus superfused in vitro was used in the current study as an index of the activity of central vasopressinergic systems in developing DOCA-NaC1 hypertension. Previous neuroanatomical studies have demonstrated that the hy~~~~ic AVP synthesizing nuclei, including the paraventricular, supraoptic and suprachiasmatic nuclei, send vasopressinergic projections to a number of central neural areas involved in cardiovascular regulation [5]. The latter include the locus coeruieus, nucleus tractus solitarius, dorsal vagal complex, AV3V region, central gray and inte~edioiaterai column of the spinal cord and the blood vessels of the neurohypophysis. Since these vasopressinergic projections make axosomatic and axodendritic connections with target areas thought to be involved in cardiovascular regulation [241, AVP may modulate blood pressure control via these systems. Direct support for this hypothesis was provided by the recent demons~tion that micr~njection of AVP directly into the locus coeruieus produces dose related increases in blood pressure and heart rate in conscious normotensive rats

97

due to altered central nervous system activity with enhanced AVP release or to chaoges in degr&tion and/or clearance rates in the peripheti circulation, mechanisms which have been shown to regulate hormone levels. C&on et al. [81 have reported that both the urinary excretion and the plasma concentration of AVP are elevated within the fast week of DOCA-NaCi treatment in the rat, giving indirect evidence of an increase in AVP production. However, a previous study carried out in our laboratories showed no sign&ant differences in hypothalamic and pituitary AVP stores between DOCA/NaCi hypertensive rats and their water drinking controls [ 181. This does not support the hypothesis that AVP synthesis is increased in the central nervous system of DOCA/NaCi hypertensive rats. Further study is needed to characterize more precisely the dynamics of AVP production and metabolism in brain tissue of the DOCA/NaCi model. Hypothalamic AVP release rates and plasma AVP levels were elevated in DOCA treated rats maintained on low, “normal” and high NaCi intakes, which had normal, slightly elevated and severely elevated blood pressures, respectively. These fmdings, coupled with the observation that one percent NaCi treated rats showed no change in hypothalamic AVP release or plasma AVP levels, indicate that DUCA per se, rather than sodium or blood pressure elevation, stimulates the release of AVP via a central mechanism. Hypothalamic AVP release and plasma AVP were greater in DOCA-R-H20 treated than DOCA-NaCi treated animals throughout the study. The mechanism by which superimposition of NaCi admi~st~tion on DOCA treatment attenuated hy~th~amic AVP release is unce~n, but may have been related to voiume expansion 113,221. In the current study uninephrectomized rats treated with DOCA and maintained on standard laboratory chow and tap water developed hypertension that was less severe and later in onset than that of DOCA-NaCi treated animals. Our finding of increased blood pressure in the DOCA-R-H20 gr&p contrasts with the data of De Champlain [lOI and Reid [21], who found no alteration in blood pressure in rats treated with DOCA and tap water for six to seven weeks. One explanation for this apparent discrepancy may have been the greater NaCl intake of our rats, which consumed a “standard” diet (Charles River RMH-1000 formula) containing 0.38% (w/w) sodium and 0.73% potassium. There is no description of the sodium content of the diet in the previous reports. Our observation that blood pressure was not elevated in animals treated with DOCA and stringent NaCl restriction is consistent with the demonstration of De Champlain et al. [ll] that complete removal of sodium from the diet of DOCA-NaCl hypertensive rats lowered BP to normotensive levels. Taken together, these data indicate that NaCi is essential for DOCA-treated rats to develop or maintain an increased BP.

[41.

The elevated plasma AVP levels seen in DOCA-NaCi treated rats in the present study confvm and extend previous reports of other investigators, who found increased plasma AVP c~cent~tions during the &ariy [8], established [9, 18, 201and malignant 1171stages of DOCA-NaCi hypertension in the rat. Previous studies did not determine whether the increase in plasma AVP seen in DOCA-NaCi hypertension is

ACKNOWLEDGEMENTS The authors thank Cindy Yarbrough for her excellent technical assistance and &bra Roaden and Vicky Chastain for their help in preparation of the manuscript, This work was supported in part by USPHS Grants HL-22544, HL-25451, HL-07457, HL-00707, and HD5572. and by Grant-in-Aid No. 76-777 of the American Heart Association. -

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AND OPARII~

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24. Sofroniew, M. J. Projections from vasopressin, oxytocin and neurophysin neurons to neural target areas in the rat and human. .I Hisrochetn Cyrwhem 28: 475-478, 1980. 25. Wagner, H., H. Haberle and R. E. Lang. Transmitter mediated arginine vasopressin release from superfused hypothalamus and pituitary gland. .I Endocrinol In\,c,sf 1: 215-220. 1978.