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Renal and systemic effects of synthetic atrial natriuretic factor
Pergamon Press
Life Sciences, Vol. 36, pp. 33-44 Printed in the U.S.A.
RENAL AND SYSTEMIC EFFECTS OF SYNTHETIC ATRIAL NATRIURETIC FACTOR Andrea A. Seymour, Edward H. B l a i n e , Elaine K. Mazack, Shaler G. Smith, Inez I . S t a b i l i t o , Anne B. Haley, Mary A. Napier, Margaret A. Whinnery and Ruth F. Nutt Merck I n s t i t u t e f o r Therapeutic Research Merck Sharp and Dohme Research Laboratories Departments of Pharmacology and Medicinal Chemistry West P o i n t , PA 19486 and Department of Analytical Natural Product Chemistry Rahway, NJ 07065 (Received in final form October 22, 1984)
Summary A synthetic peptide corresponding to a sequence of 26 amino acids contained in endogenous rat a t r i a l n a t r i u r e t i c factor (ANF), was infused into one renal artery of anesthetized dogs for a comprehensive in vivo evaluation of the renal and systemic effects of pure ANF. The results proved conclusively that ANF acted d i r e c t l y on the kidney since urine volume and fractional excretion of sodium, potassium, chloride and calcium were elevated in a dose-related manner in the ANF-treated kidney, but were not s i g n i f i c a n t l y affected in the contralateral saline-infused organ. The maximum effects achieved with the synthetic ANF were higher than any reported following intravenous administration of crude extracts of rat a t r i a and were similar to those produced by thiazide diuretics. In four of the five dogs studied, renal vascular resistance f e l l progressively as doses of ANF were increased. Glomerular f i l t r a t i o n rate was not s i g n i f i c a n t l y elevated during ANF infusion, but was correlated with sodium excretion rates. Even though mean a r t e r i a l pressure was progressively reduced, there was no s i g n i f i c a n t change in heart rate and no stimulation of renin secretion. Arterial cyclic GMP concent r a t i o n was higher in the basal state and rose more rapidly than did renal venous levels, indicating that increases in c i r c u l a t i n g concentrations of a r t e r i a l cyclic GMP originated from an extrarenal source. Dose-related elevations in urinary cyclic GMP excretion could be explained by increased cyclic GMP f i l t r a t i o n , by enhanced production in tubular c e l l s , or by renal tubular secretion. Espec i a l l y in the saline-infused kidney, there was a clear dissociation between excretion of cyclic GMP and fractional sodium excretion. We conclude that the synthetic ANF increased electrolyte excretion via a direct renal action which was not solely dependent upon changes in renal vasculature, renin secretion or cyclic GMP levels. In recent years, e x t r a c t s of the a t r i a of rat hearts were found to stimu l a t e f r a c t i o n a l e x c r e t i o n of sodium, potassium and c h l o r i d e in anesthetized rats ( i - 6 ) . Glomerular f i l t r a t i o n rate (GFR) and renal blood f l o w (RBF) were reported to e i t h e r r i s e (4-6) or to remain unchanged (1-2) at n a t r i u r e t i c 0024-3205/85 $3.00 + .00 Copyright (c) 1985 Pergamon Press Ltd.
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doses. In isolated perfused rat kidneys, a t r i a l extracts increased GFR (7) and either enManced (7) or decreased (8) renal vascular resistance (RVR) depending upon pre-existing vascular tone (7). Extracts of monkey or human atria also stimulated sodium and calcium excretion without affecting either GFR or free water clearance (9) in anesthetized monkeys. In addition to a n a t r i u r e t i c action, a t r i a l extracts relaxed isolated rat aortic strips (i0) and reduced blood pressure of anesthetized rats (1,2,3). F i n a l l y , a t r i a l preparations enhanced the a r t e r i a l plasma concentration and urinary excretion of cyclic 3',5'-guanosine monophosphate(GMP) in anesthetized rats (11). Cyclic GMP levels in kidney minces and in primary cultures of renal tubules were also elevated by treatment with a t r i a l extracts (11). The a c t i v e substance e x t r a c t e d from a t r i a is now g e n e r a l l y known as a t r i a l n a t r i u r e t i c f a c t o r and has been characterized as a peptide (12). Recently, several i n v e s t i g a t o r s have i s o l a t e d and sequenced a series of peptides which possess the b i o l o g i c a l a c t i v i t y of ANF (13-16). A compound, corresponding to the 26 residues of the C-terminal of a 73 amino acid form of ANF (17), and cont a i n e d i n the DNA sequence described f o r a l a r g e r precursor p r o t e i n (18), was s y n t h e t i z e d by a combination of c l a s s i c a l s o l u t i o n and s o l i d phase techniques as described by Seidah et a l . (13). The sequence of the s y n t h e t i c peptide is ArgArg-Ser-Ser-C~s-Phe-G~y-G~y-Ar9-~e-As~-Arg-~e-G~y-A~a-G~n-Ser-G~y-Leu-G~y-C~sAsn-Ser-Phe-Arg-Tyr-COOH w i t h a d i s u l f i d e bridge between the cysteines at posit i o n s 5 and 21. This peptide i s i d e n t i c a l to a t r i o p e p t i n I I reported by C u r r i e et a l . (16) w i t h the a d d i t i o n of Arg-Arg on the N-terminus and of Tyr on the C-terminus. Our s y n t h e t i c p r e p a r a t i o n , which was 82% p e p t i d a l in composition, produced n a t r i u r e t i c a c t i v i t y comparable to t h a t of a t r i a l e x t r a c t s in anesthet i z e d rats (17) and relaxed i s o l a t e d vascular smooth muscle preparations (19). In the present study t h i s a c t i v e p o r t i o n of ANF was evaluated i n anesthet i z e d dogs w i t h o u t the p o t e n t i a l complications of unrelated peptides which were probably present i n the a t r i a l e x t r a c t s . The renal e f f e c t s of ANF were maximized and a d i r e c t renal a c t i o n of ANF was v e r i f i e d by i n f u s i o n of several doses of the s y n t h e t i c peptide d i r e c t l y i n t o one renal a r t e r y w h i l e s a l i n e was d e l i v e r e d i n t o the c o n t r a l a t e r a l kidney. The e f f e c t s of the s y n t h e t i c peptide on mean a r t e r i a l blood pressure (MAP), renal blood f l o w (RBF) and renal funct i o n , i n c l u d i n g GFR, free water reabsorption and f r a c t i o n a l e x c r e t i o n of sodium, potassium, c h l o r i d e and calcium were analyzed. In a d d i t i o n , c y c l i c GMP c o n c e n t r a t i o n s were measured i n u r i n e and renal venous plasma samples from both kidneys and i n a r t e r i a l plasma. For the f i r s t time, the renal c o n t r i b u t i o n to the increase in c y c l i c GMP l e v e l s could be evaluated. C y c l i c 3 ' , 5 ' - a d e n o s i n e monophosphate ( c y c l i c AMP) was measured i n selected samples to v e r i f y the s p e c i f i c i t y of the c y c l i c GMP response. F i n a l l y , plasma renin c o n c e n t r a t i o n s were determined and the e f f e c t s of ANF on a r t e r i a l plasma renin c o n c e n t r a t i o n and renin s e c r e t i o n rates were assessed. Methods
Five female dogs (weighing an average of 13.6 + 0.4 kg) were anesthetized with v i n b a r b i t a l , intubated and ventilated with roo~ a i r via a Harvard respirat o r . Catheters inserted into the abdominal aorta and vena cava via the femoral artery and vein, respectively, provided routes for arterial blood sampling and blood pressure measurements (Micron transducer) and for intravenous infusions of creatinine and an isoosmotic solution (6% dextran and 0.9% sodium chloride). Both kidneys were exposed via b i l a t e r a l flank incisions and catheters were placed in each ureter for timed urine collections. Curved needles (20 or 23 gauge) attached to S i l a s t i c tubing were inserted d i r e c t l y into each renal vein for blood sampling and into each renal artery for direct intrarenal infusions.
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Renal Effects of Atrial Natriuretic
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An electromagnetic f l o w probe (Zepeda) was p o s i t i o n e d around each renal a r t e r y f o r continuous renal blood f l o w d e t e r m i n a t i o n s . In order t h a t glomerular filtration rate could be determined by c a l c u l a t i n g the rate of clearance of exogenous c r e a t i n i n e , a bolus of c r e a t i n i n e (50 mg/kg, i v ) was i n j e c t e d and a s u s t a i n i n g i n f u s i o n ( i mg/kg/min, i v ) was begun at l e a s t 45 minutes before the f i r s t experimental p e r i o d . Mean a r t e r i a l pressure, heart rate and l e f t and r i g h t RBF were c o n t i n u o u s l y recorded on a Buxco system and renal vascular resistances were derived e l e c t r o n i c a l l y . Control sampling was not begun u n t i l a s t a b l e b a s e l i n e was assured. Urine was q u a n t i t a t i v e l y c o l l e c t e d during each i 0 min period and a r t e r i a l blood samples were drawn at the midpoint f o r measurements of hematocrit and plasma c r e a t i n i n e and e l e c t r o l y t e c o n c e n t r a t i o n s . At the end of each p e r i o d , blood was obtained from the aorta and from each renal vein (15 ml t o t a l ) f o r d e t e r m i n a t i o n of plasma renin c o n c e n t r a t i o n s and plasma c y c l i c GMP and c y c l i c AMP l e v e l s . F l u i d losses due to sampling were replaced by a continuous intravenous d r i p of 6% dextran at i to 1.5 ml/min. During the f i r s t two 10 minute measurement periods, saline was infused at 0.19 ml/min into both renal arteries. Since the baseline values were stable, the data from the two control periods were averaged and presented as singular control values throughout t h i s report. Solutions of synthetic ANF dissolved in saline were then delivered into one renal artery during the next 8 periods so that doses of 1.2, 2.4, 4.8, 9.6, 19.2, 38.4, 76.8 and 156 pmole/kg/min were infused for 10 minutes each. Once the ANF administration was complete, saline infusion into the treated kidney was resumed and four 20 minute recovery periods were observed. Creatinine concentrations in urine and a r t e r i a l plasma were measured using a standard colorimetric assay and were used to calculate creatinine clearance, an estimate of GFR. Urinary and plasma concentrations of sodium, potassium and chloride were assessed with ion selective electrodes (Technicon) and calcium levels by atomic absorption spectrophotometry. Fractional electrolyte excretions (FE) were expressed as the percentage of the f i l t e r e d electrolyte load (or GFR multiplied by the plasma concentration) which was excreted in the urine. Plasma renin concentration was determined by radioimmunoassay (Clinical Assays Gamma Coat [1251]) of the amount of angiotensin generated at 37°C from 50 ul of plasma in an excess of homologous renin substrate. Renin secretion rates were the product of the renal plasma flow and the arteriovenous d i f f e r ence in plasma renin concentrations. Cyclic GMP concentration in urine and in a r t e r i a l and renal venous plasma was measured by radioimmunoassay (Amersham Cyclic GMP RIA k i t ) . Plasma samples were f i r s t extracted with ethanol; recoveries averaged 89.6 + 0.3% (n = 251). Cyclic GMP excretion was calculated from the urine volume muTti p l i e d by the urinary cyclic GMP concentration. Arterial load and renal venous effluent are the products of the renal plasma flow and the appropriate plasma concentration. Renal extraction of cyclic GMP was determined from the arteriovenous difference in plasma concentrations times the renal plasma flow. C y c l i c AMP c o n c e n t r a t i o n in selected e x t r a c t e d a r t e r i a l plasma samples and i n u r i n e were measured using the Amersham C y c l i c AMP RIA k i t . C a l c u l a t i o n s are as explained above. A l l data are expressed as the mean + the standard e r r o r of the mean (SEM). S i g n i f i c a n t changes from c o n t r o l values Were i d e n t i f i e d by a p p l i c a t i o n of the Dunnett's t - t e s t . The values from the ANF-treated kidney were compared to those of the s a l i n e - i n f u s e d kidney by paired t - t e s t f o r normally d i s t r i b u t e d samples. When samples did not pass the Wilks Shapiro t e s t f o r n o r m a l i t y , the Wilcoxon
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Renal Effects of Atrial Natriuretic Factor
Vol. 36, No. i, 1985
Sign Rank t e s t was applied i n s t e a d . C o e f f i c i e n t s of d e t e r m i n a t i o n ( r 2) were established by performance of a l i n e a r regression using the l e a s t squares method. Results Sodium e x c r e t i o n from the ANF-infused kidney p r o g r e s s i v e l y rose from a c o n t r o l of 24 + 6 MEq/min to a peak of 184 + 50 ~Eq/min d u r i n 9 a d m i n i s t r a t i o n of 19.2 pmole/kg/min ANF then f e l l to 39 + 9 ~Eq/min i n the f i n a l recovery p e r i o d . Following s i m i l a r response p a t t e r n s , u~ine volume and f r a c t i o n a l e x c r e t i o n s of sodium, c h l o r i d e and calcium ( F i g . i and Table I ) were s i g n i f i c a n t l y elevated above the pretreatment values in the ANF-infused kidney; maximum increases were achieved at i n f u s i o n rates of 9.6 to 38.4 pmole/kg/min. In a d d i t i o n , f r a c t i o n a l potassium e x c r e t i o n from the ANF-treated kidney (Table I) was s i g n i f i c a n t l y greater than t h a t of the c o n t r a l a t e r a l kidney at a l l times a f t e r the 4.8 pmole/ kg/min dose. While free water reabsorption (Table I ) i n the ANF-treated kidney was s i g n i f i c a n t l y higher than pretreatment l e v e l s , t h e r e were no s i g n i f i c a n t d i f f e r e n c e s between measurements made in the two kidneys. I n f u s i o n of the synt h e t i c ANF p r o g r e s s i v e l y reduced MAP ( F i g . i ) but had no s i g n i f i c a n t e f f e c t on heart rate which was 128 + 9 beats/min during c o n t r o l and 139 + 7 beats/min d u r i n g i n f u s i o n of the h i g h e s t dose. TABLE I ANFa (pmo l e / k~/mi n) 0 1.2 2.4 4.8 9.6 19.2 38.4 76.8 156 0 0 0 0 a
Urine Volume b (ml/min) ANF Saline .20+.04 .47¥.21 .73¥.38 .9~.38" i.II¥.28 + 1.09#.27" .96¥.26* .9~.23 .69¥.22 .4~.14 .40¥.10 .36¥.09 .30¥.07
.20+.04 ,2~.05 ,20¥.04 .2~.[6 .22¥.08 ° .28¥.08 ° .2~.05 ° .2~.05 ° .23¥.05 .17¥.04 .15¥.03 ° .I~.03 ° .14¥.03 °
FEK (%) ANF Saline 30+3 2~7 2~3 3~5 41¥5 37#6 41¥7 42¥7 37¥6 29¥4 26¥3 32¥4 33¥3
ANF
FEcI (%) Saline
26+3 1.3+ . i 2~3 4.~1.6 2~3 5.~1.9 2~4 ° 8.0¥2.1 + 22¥4 f 9.5¥2.0 + 2~2 ° 9.9#2.0 + i ~ 3 ° 10.1¥2.1 + 21¥4 ° 9.5¥1.9 + 22¥2 ° 8.0¥1.6 + I~3 ° 5.~1.5 16¥2* f 4.2~-I.i 16¥2*o 4.6¥I .4 20¥2 3.6¥1.0
The f i r s t row of data represents the average periods. Each dose of ANF was infused f o r a recovery period was 20 minutes long, b Each column represents the measurements made or s a l i n e as i n d i c a t e d by the subheadings. * p < 0.05, + p < 0.01, compared to pretreatment ° p < 0,05, f p < 0.01, ANF- vs. s a l i n e - t r e a t e d
1.1+.2 1.3¥.3 1.~.3 1.6¥.5 ° 1.8¥.6 f 2.0¥.7 ° I.~.6 f 2.1~.5 ° 2.6¥.6 +o 1.9¥.5 ° 1.3¥.4 ° I.~.2 ° i.i¥.3 °
Free Water Reabsorption (ml/min) ANF Saline .33+.07 .3~.09 .36¥.08 .4~.09 .5~.12 .60¥.13" .6~.13 + .6~.12 + .6~.19 + .4~.07 .46¥.04 .42¥.05 .3~.Ob
.33+.06 .33¥.05 .33¥.05
. 3 ~ 06 .33¥ .36¥ .3~ .41~ .36T .31¥ .30¥ .3~ .31¥
06 06 04 06 06 05 03 05 05
of two 10-minute clearance lO-minute p e r i o d ; each i n the kidney infused w i t h ANF control. kidney
There were no s t a t i s t i c a l l y s i g n i f i c a n t a l t e r a t i o n s in GFR ( F i g . 1) or i n the renal vascular r e s i s t a n c e and RBF determined d u r i n g the f i n a l minute of each treatment period (Table I I ) . When the peak responses ( t h a t i s , the highest RBF and the lowest RVR) were examined in each dog (Table I l l ) , increases i n RBF and decreases in v a s c u l a r resistance were observed i n 4 of the 5 dogs, Even though GFR appeared to r i s e at some p o i n t during ANF treatment i n each dog (Table I l l ) ,
Vol. 36, No. i, 1985
Renal Effects of Atrial Natriuretic
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FIG. 1 Blood pressure and renal responses t o ANF are shown above, described in the t e x t .
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t h e maximum response occurred at a d i f t e r e n t dose in each animal so t h a t no s t a t i s t i c a l l y s i g n i f i c a n t increase was discernable ( F i g . i ) . There was however a c o r r e l a t i o n between the sodium e x c r e t i o n and GFR ( r z = 0.80) measured during ANF infusions i n t o the t r e a t e d kidney.
Renin s e c r e t i o n from the ANF-treated kidney was v a r i a b l e before and during the course of the peptide i n f u s i o n and, despite a s l i g h t drop during some of the lower doses, was not s i g n i f i c a n t l y d i f f e r e n t from the pretreatment c o n t r o l . In only one instance was t h e r e a s i g n i f i c a n t d i f f e r e n c e betwen the rate of r e n i n release from the ANF- and s a l i n e - i n f u s e d kidneys; however, due to the i n h e r e n t v a r i a b i l i t y of t h i s measurement, small changes may be d i f f i c u l t t o detect under the present experimental c o n d i t i o n s . There were poor c o r r e l a t i o n s between renin s e c r e t i o n and e i t h e r sodium e x c r e t i o n or GFR ( r 2 = 0.12 and 0.16, respectively). A r t e r i a l plasma c y c l i c GMP c o n c e n t r a t i o n rose in a d o s e - r e l a t e d manner from 20 + 3 pmole/ml to a maximum of 102 + 13 pmole/ml a f t e r a d m i n i s t r a t i o n of 156 pmole/kg/min of s y n t h e t i c ANF. Renal-venous c o n c e n t r a t i o n s also increased from 8.6 + 1.0 and 9.3 + 1.0 to 37.8 + 6.0 and 37.2 pmole/ml in the ANF- and
38
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Renal Effects of Atrial Natriuretic Factor
TABLE I I Renal hemodynamics and renin levels at the end of each lO-minute treatment period in 5 anesthetized dogsa ANF (pmole/ kg/min ) 0 1.2 2.4 4.8 9.6 19.2 38.4 76.8 156 0 0 0 0
RBF (ml/min) ANF Saline 146+20 141¥16 147¥17 159¥20 15~22 131¥10 122~ii 120¥10 120¥13 129¥14 122TII 12~13 116¥11
145+ 6 137T 8 132¥ 9 128¥ 8 121¥ 8 120¥6 116¥ 8 120¥ 6 126# 8 129¥15 120¥19 i0~21 96T24"
RVR (mm Hg/ml/min) ANF Saline .88+.09 .8~.06 .79¥.06 .7~.07 .6~.06 .76¥.04 .79T.06 .77#.07 .76#.09 .77¥.08 .84¥.07 .9~.iI .93¥.10
.83+.05 .8~.04 .85¥.04 .93¥.08 .8~.05 .82¥.05 .82#.06 .767.05 .70¥.05 .77¥.09 .94¥.19 1.19¥.30 1.47¥.50
Renin Secretion (ng/min) ANF Saline 457+376 469¥427 679¥531 70Y 44 16~i18 129¥ 91 234¥ 68 45~203 658¥276 156¥136 20~107 607¥277 343T174
PRC (ng/ml/hr)
206+ 79 9.1+3.1 291¥164 12.9¥4.5 569¥302 11.8¥5.9 41~244 13.~6.3 260#124 12.1+--4.4 2 0 1 # 79 11.3¥4.3 446T 61f 9.3¥4.0 30~186 9.7¥4.0 664#232 10.4¥4.5 69~274 12.6¥4.0 705¥291 10.9¥4.4 786¥446 13.4¥4.0 407¥123 17.4¥4.0
a Format as explained in Table I * p < 0.05, compared to pretreatment control f p < 0.01, ANF- vs. Saline-treated saline-infused kidneys, r e s p e c t i v e l y . The calculated rates of c y c l i c GMP i n f l u x and e f f l u x were also s i g n i f i c a n t l y elevated during administration of the synthetic peptide (Fig. 2). As a r t e r i a l levels rose, renal extraction of the c y c l i c nucleotide increased for both kidneys. The f i l t e r e d load of c y c l i c GMP and urinary c y c l i c GMP excretion was also enhanced in a dose-related manner in both the ANF-treatedkidney and in the contralateral organ (Fig. 3). Cyclic AMP levels measured in the same samples were not s i g n i f i c a n t l y altered by ANF treatment. A r t e r i a l plasma c y c l i c AMP was 3.2 + 0.6 and 3.6 + 0.7 pmoles/ml at control and 156 pmole/kg/min, r e s p e c t i v e l y . At the same time points, urinary excretion of c y c l i c AMP was 172 + 34 and 161 + 32 pmole/min from the ANF-treated kidney and 173 + 31 and ib4-+ 25 pmole/mTn from the saline-infused kidney. Discussion The present study offered the f i r s t opportunity to examine in anesthetized dogs the effects of a single peptide synthesized from the amino acid sequence contained in the natural ANF isolated from rat a t r i a . Use of the synthetic compound in the present experiment permitted evaluation of an active ANF fragment in the absence of unrelated a t r i a l peptides. Selection of the anesthetized dog model allowed us to d e l i v e r ANF via one renal artery so that the i p s i l a t e r a l kidney received concentrations of the peptide which exceeded those achieved in the systemic c i r c u l a t i o n . Consequently, b i l a t e r a l renal function could be examined at unequal renal plasma ANF concentrations, a condition which could not be attained during intravenous administration.
Vol. 36, No. i, 1985
39
Renal Effects of Atrial Natriuretic Factor
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ANF- Treafed Kidney
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120 140 160
FIG. 2 The changes i n plasma l e v e l s and renal e x t r a c t i o n of c y c l i c GMP are shown in the top and bottom panels, r e s p e c t i v e l y . Despite the estimated lO-fold d i l u t i o n of the peptide as i t entered the systemic c i r c u l a t i o n , synthetic ANF lowered blood pressure at doses which had no effect on renal function in the saline-infused kidney. Interestingly, there was no reflex tachycardia, a phenomenonwhich has been previously reported following intravenous injection of a t r i a l extracts into conscious rats (20). While both our synthetic ANF and a t r i a l extracts have been shown to relax isolated vascular smooth muscle (10,19), other potential mechanisms which may contribute to the depressor response have yet to be f u l l y explored.
During i n t r a r e n a l i n f u s i o n of the s y n t h e t i c ANF, the peak f r a c t i o n a l e l e c t r o l y t e e x c r e t i o n s measured in the present experiment surpassed those prev i o u s l y reported f o l l o w i n g intravenous i n j e c t i o n of a t r i a l e x t r a c t s i n t o rats ( i , 2). This d i f f e r e n c e may r e s u l t from the route of a d m i n i s t r a t i o n , the use of a more pure s y n t h e t i c ANF p r e p a r a t i o n or a d i f f e r e n c e in species responsiveness. The magnitudes of the increases i n f r a c t i o n a l sodium e x c r e t i o n observed in the present study resemble those produced by t h i a z i d e d i u r e t i c s in anesthetized dogs (21). However, u n l i k e t h i a z i d e s , s y n t h e t i c ANF p r o g r e s s i v e l y reduced MAP; t h e r e f o r e , changes in renal hemodynamics secondary to the depressor e f f e c t may have obscured the t r u e c e i l i n g of the n a t r i u r e t i c response to ANF. Confirmation of the maximum s a l u r e s i s which u l t i m a t e l y may be achieved by pure ANF awaits e v a l u a t i o n of the peptide in other experiments i n which blood pressure i s main-
Vol. 36, No. i, 1985
Renal Effects of Atrial Natriuretic Factor
E
1.5-
4]
+p
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co+oo,.+oT .... o
1.0~9
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=
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O 0 Saline+Treoted Kpdney n=5
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vE
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uu
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CL
R
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[
(prnole/kg/min,iro) O Time (min)
[
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,z I
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100 120 140 160
FIG. 3 The changes in the amount of f i l t e r e d top and bottom panels, r e s p e c t i v e l y .
and excreted c y c l i c GMP are shown in the
t a i n e d at i t s c o n t r o l l e v e l , o t h e r compensatory mechanisms are considered and t a c h y p h y l a x i s has been disproved. We may also p o s t u l a t e t h a t the peptide is at l e a s t p a r t i a l l y i n a c t i v a t e d i n the systemic c i r c u l a t i o n since d i l u t i o n alone would not e x p l a i n the lack of e f f e c t on the c o n t r a l a t e r a l kidney at the highest ANF i n f u s i o n , a dose which is at l e a s t 30 times the m i n i m a l l y e f f e c t i v e i p s i l a t e r a l c o n c e n t r a t i o n . Nevertheless, the present data are c o n s i s t e n t with a d i r e c t renal e f f e c t of ANF since the renal responses of the ANF-treated kidney developed at doses which had no apparent e f f e c t s on c o n t r a l a t e r a l renal function. While f r e e water r e a b s o r p t i o n appeared t o r i s e in the ANF-infused kidney, t h e r e was no s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e when compared to the s a l i n e infused organ. Before meaningful conclusions can be reached concerning the e f f e c t s of ANF on the renal handling of water, ANF must be i n v e s t i g a t e d in experiments in which a n t i d i u r e t i c hormone is e i t h e r i n h i b i t e d or maximally stimulated. Previous studies in anesthetized rats (1-3) and monkeys (9) revealed no effect of injections of a t r i a l extracts on GFR. However, infusion of a t r i a l extracts into rats (4,5) and isolated perfused rat kidneys (7) s i g n i f i c a n t l y elevated GFR, expecially at high doses (5). In the anesthetized dogs in the present study, there were no s t a t i s t i c a l l y significant changes in the ANFinfused kidney when compared to i t s own control level by Dunnett's t - t e s t or
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when compared to the GFR of the s a l i n e - i n f u s e d kidney by the paired t - t e s t . Upon examining the GFR measured during ANF i n f u s i o n in the i n d i v i d u a l animals, the f i l t r a t i o n rate appeared to be somewhat enhanced i n each dog, but the peak occurred at a d i f f e r e n t dose i n each animal. While t h e r e seemed to be a reasonable c o r r e l a t i o n (r 2 = 0.80) between GFR and sodium e x c r e t i o n measured during ANF a d m i n i s t r a t i o n , the r i s e in f r a c t i o n a l sodium e x c r e t i o n (sodium clearance/GFR) suggested t h a t the n a t r i u r e s i s produced by ANF could not be e n t i r e l y a t t r i b u t e d to the increase in GFR. When a l l f i v e dogs were included in the a n a l y s i s of the measurements of RBF and RVR made during the l a s t minute of each I0 minute i n f u s i o n , no s t a t i s t i c a l l y s i g n i f i c a n t changes were i d e n t i f i e d (Table I I ) . However, when the maximum responses to each dose of ANF were examined (Table I l l ) , ANF was found to a t y p i c a l l y lower RBF in one dog in which c o n t r o l RBF was e l e v a t e d . A n a l y s i s of the responses in the other 4 dogs revealed a s i g n i f i c a n t increase in RBF at one dose (19.6 pmole/kg/min) and f a l l s i n RVR at a l l doses of 4.8 pmole/kg/min and h i g h e r . These f i n d i n g s are c o n s i s t e n t w i t h the observations in i s o l a t e d perfused kidneys i n which changes in renal r e s i s t a n c e depended upon the c o n t r o l RVR values. That i s , when i n i t i a l vascular tone was enhanced i n i s o l a t e d perfused kidneys by pretreatment with a v a s o c o n s t r i c t o r (7) and i n 4 of the dogs in the present study, ANF produced v a s o d i l a t i o n . However, when baseline RVR was low, treatment w i t h higher doses of ANF apparently c o n s t r i c t e d the renal vessels. I n t e r e s t i n g l y , i n the one dog i n which ANF increased RVR from an u n u s u a l l y low c o n t r o l l e v e l , a c o n s t r i c t o r dose of ANF (19.2 pmole/kg/min) elevated sodium e x c r e t i o n from a baseline of 36 to 248 uEq/min. While the RVR response was unique to t h a t animal, i t s n a t r i u r e t i c response was not u n l i k e t h a t produced i n the other 4 dogs. S i m i l a r l y , sodium e x c r e t i o n was also reported to r i s e in the i s o l a t e d perfused r a t kidneys i n which vascular r e s i s tance increased ( 7 ) , but was not measured i n the i s o l a t e d kidneys in which RVR f e l l (7, 8). A d d i t i o n a l studies designed to assess i n t r a r e n a l hemodynamics are needed to e l u c i d a t e the s u b t l e , but p o t e n t i a l l y i m p o r t a n t , r e l a t i o n s h i p s between the n a t r i u r e t i c and renal vascular e f f e c t s of ANF. While ANF e x t r a c t s enhanced a r t e r i a l plasma l e v e l s and u r i n a r y e x c r e t i o n of c y c l i c GMP i n anesthetized rats ( i i ) , the source of the c y c l i c GMP generat i o n was not apparent from those e a r l i e r data. In the present i n v e s t i g a t i o n , i t was possible to obtain a more complete p r o f i l e of changes in c y c l i c GMP produced by s y n t h e t i c ANF by measuring f o r the f i r s t time the c y c l i c n u c l e o t i d e c o n c e n t r a t i o n s in the a r t e r i a l plasma, in the renal venous plasma and in the u r i n e from each kidney. In agreement w i t h an e a r l i e r report of the e f f e c t s of i n j e c t i o n of a t r i a l e x t r a c t i n t o rats ( i i ) , s y n t h e t i c ANF i n f u s i o n increased a r t e r i a l c y c l i c GMP c o n c e n t r a t i o n s in a d o s e - r e l a t e d manner. In the present experiment, we were able to demonstrate t h a t at no time before or d u r i n g i n f u sion of the peptide did renal venous e f f l u e n t exceed a r t e r i a l c y c l i c GMP levels. In a d d i t i o n , c y c l i c GMP e f f l u x rates from the ANF-treated kidney were not d i f f e r e n t from the l e v e l s measured i n the blood i s s u i n g from the s a l i n e infused organ. Together, these data suggest t h a t the enhanced l e v e l s of c i r c u l a t i n g c y c l i c GMP r e s u l t i n g from ANF a d m i n i s t r a t i o n o r i g i n a t e d from an e x t r a r e n a l source. The r i s e s in u r i n a r y e x c r e t i o n of c y c l i c GMP during i n f u s i o n of the synt h e t i c ANF could p o t e n t i a l l y r e s u l t from e i t h e r t u b u l a r production of c y c l i c GMP, from renal clearance ( e . g . f i l t r a t i o n and/or s e c r e t i o n ) of the c y c l i c n u c l e o t i d e from the a r t e r i a l plasma or from a combination of the two. The f i r s t e x p l a n a t i o n would be c o n s i s t e n t w i t h the reported s t i m u l a t i o n of c y c l i c GMP i n primary c u l t u r e s of renal t u b u l a r c e l l s ( I I ) . In a d d i t i o n , c y c l i c GMP e x c r e t i o n s before beginning ANF treatment in the present study were 199 + 20 and 193 + 19% of the f i l t e r e d load of the c y c l i c n u c l e o t i d e in the experTmental
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Renal Effects of Atrial Natriuretic Factor
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and saline-infused kidneys, r e s p e c t i v e l y ; there were no s i g n i f i c a n t changes in these r a t i o s during the infusion of the synthetic ANF. These data suggest that e i t h e r c y c l i c GMP was secreted into the tubular f l u i d from the a r t e r i a l plasma or that the c e l l s of the tubules, which contain enzymes involved in c y c l i c GMP metabolism (22,23), produced c y c l i c GMP. F i n a l l y , the enhanced levels of urinary c y c l i c GMP excretion were not l i k e l y to be p r i m a r i l y responsible for the renal e f f e c t s of ANF since c y c l i c GMP excretion from the saline-infused kidney rose to levels s i g n i f i c a n t l y greater than the pretreatment baseline values without development of s i g n i f i c a n t d i u r e s i s or s a l u r e s i s . In view of the poor correlations between renin secretion and either sodium excretion or GFR, the changes in renal function during ANF infusion did not appear to be related to alterations in renin release. In 3 of the 5 dogs control levels of renin secretion were less than IOU ng/min, therefore a reduction in the release rate would have been d i f f i c u l t to detect. There was however, a tendency for renin secretion to f a i l at doses of ANF at which the concurrent depressor response did not exceed 20 mm Hg. At ANF infusions in excess of 38.4 pmole/kg/min, renin secretion was near or above control levels while MAP was reduced by at least 30 mm Hg. Althoughthese data are preliminary due to t h e i r v a r i a b i l i t y , these f a l l s in blood pressure may be expected to e l i c i t some increase in renin release under other circumstances (24). An actual i n h i b i t i o n of basal or stimulated renin secretion by ANF remains to be demonstrated in a study directed toward that purpose. In summary, the synthetic peptide corresponding to a 26 amino acid sequence of ANF was infused into one renal artery of anesthetized dogs and stimulated diuresis and saluresis in a manner which could not be solely explained by changes in RBF or GFR. The higher doses of the peptide also signif i c a n t l y lowered MAP without affecting heart rate or stimulating renin secret i o n . Control a r t e r i a l plasma concentrations of cyclic GMP were greater than the renal venous levels. During ANF infusion, a r t e r i a l cyclic GMP rose more rapidly than did renal venous e f f l u x , indicating an extrarenal source of circulating cyclic GMP. In addition, cyclic GMP excretion was double the rate of f i l t r a t i o n but less than the a r t e r i a l delivery rate. Therefore, cyclic GMP was added to the tubular f l u i d by active secretion from the plasma, by nephrogenous production or a combination of the two processes. These data supply the f i r s t insight into the effects of a pure synthetic ANF peptide on renal function, blood pressure and plasma levels of renin and cyclic GMP in the anesthetized dog.
Acknowledgments We wish to thank Shirley S a t i r i t z for performing the calcium assays. We are also appreciative of the excellent secretarial support of Maryanne Olkowski and Carol Van Arsdale.
References 1. 2. 3. 4. 5. 6.
A.J. DE BOLD, H.Bo BORENSTEIN, A.T. VERESS and H. SONNENBERG, L i f e Sci, 28, 89-94 (1981). R. KEELER, Can. J. Physiol. Pharmacol. 60, I078-IU82 (1982). D.M. POLLACK and R.O. BANKS, C l i n . S c i . ~ 5 , 47-55 (1983). D. BEASLEY, R.L. MALVIN and N. PALIS, Fed__Proc. 43, 502 (1984). J.P. BRIGGS, B. STEIPE, G. SCHUBERT and J. SCHNERMANN, Pflugers Arch. 395, 271 (1982). H.B. BORENSTEIN, W.A. CUPPLES, H. SONNENBERG and V.T. VERESS, J. Physiol. 334, 133 (1983).
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7. 8. 9. i0. ii. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
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M.J.F. CAMARGO, H.D. KLEINERT, S.A. ATLAS, J.E. SEALEY, J.H. LARAGH AND T. MAACK, Am. J. Physiol. 246, F447 (1984). T. OSHIMA, M.G. CURRIE, D.M. GELLER and P. NEEDLEMAN, Circ. Res. 54, 612 (1984). M.N. NEMEH, and J.P. GILMORE, Circ. Res. 53, 420 (1983). R.C. DETH, K. WONG, S. FUKOZAWA, R. ROCCO, J.L. SMART, C.J. LYNCH and R. AWAD, Fed. Proc. 41, 983 (1982). M. CANTIN, R. GARClA, G. THIBAULT, P. HAMET and J. GENEST, J. Mol. Cell. Cardiol. 15 Supp. I , 54 (1983). A.J. DE BOLD, Proc. Soc. Exp. Biol. Med. 170, 133-138 (1982). N.G. SEIDAH, C. LAZURE, M. CHRETIEN, G. THIBAULT, R. GARClA, M. CANTIN, J. GENEST, R.F. NUTT, S.F. BRADY, T.A. LYLE, W.J. PALEVADA, C.D. COLTON, T.M. CICCARONE and D.F. VEBER, Proc. Natl. Acad. Sci. 8__II, 2640 (1984). M.A. NAPIER, R.S. DEWEY, G. ALBERS-SCHONBERG, C.D. BENNETT, J.A. RODKEY, E.A. MARSH, M. WHINNERY, A.A. SEYMOURand E.H. BLAINE, Biochem. Biophys. Res. Commun. 120, 981 (1984). T.G. FLYNN, M.L. DE BOLD and A.J. DE BOLD, Biochem. Biophys. Res. Commun. 117, 859-865 (1983). M.G. CURRIE, D.M. GELLER, B.R. COLE, N.R. SIEGEL, K.F. FOK, S.P. ADAMS, S.R. EUBANKS, G.R. GALLUPPI and P. NEEDLEMAN, Science 223, 67-69 (1984). G. THIBAULT, R. GARCIA, M. CANTIN, J. GENEST, C. LAZURE, N.G. SEIDAH and M. CHRETIEN, FEBS Letters 167, 352-356 (1984). R.A. ZIVIN, d.H. CONDRA, R.A.F. DIXON, N.G. SEIDAH, M, CHRETIEN, M. NEMER, M. CHAMBERLANDand J. DROUIN, Proc. Natl. Acad. Sci, in press. R.J. WINQUIST, E.P. FAISON and R.F. NUTT, Eur. J. Pharamcol. 102, 169 (1984). B.L. PEGRAM, N.C. TRIPPODO, F.E. COLE and A.A. MACPHEE, Fed. Proc. 43, 453 (1984). J.B. PUSCHETT, J. Clin. Pharmacol. 21, 564-574 (1981). A.A. WARGO, L.M. SLOTKOFF, D.A. JOSE, J.C. PELAYO and G.Mo EISNER, Nephron 32, 165-169 (1982). A.N.K. YUSUFI, C. DANCONA, J . - L . NGUYEN and J . - J . HELWIG, Renal Physiol. 6, 80-86 (1983). J.O. DAVIS and R.H. FREEMAN, Physiol. Rev. 56, 1-56 (1976).
Life Sciences, Vol. 36, pp. 33-44 Printed in the U.S.A.
RENAL AND SYSTEMIC EFFECTS OF SYNTHETIC ATRIAL NATRIURETIC FACTOR Andrea A. Seymour, Edward H. B l a i n e , Elaine K. Mazack, Shaler G. Smith, Inez I . S t a b i l i t o , Anne B. Haley, Mary A. Napier, Margaret A. Whinnery and Ruth F. Nutt Merck I n s t i t u t e f o r Therapeutic Research Merck Sharp and Dohme Research Laboratories Departments of Pharmacology and Medicinal Chemistry West P o i n t , PA 19486 and Department of Analytical Natural Product Chemistry Rahway, NJ 07065 (Received in final form October 22, 1984)
Summary A synthetic peptide corresponding to a sequence of 26 amino acids contained in endogenous rat a t r i a l n a t r i u r e t i c factor (ANF), was infused into one renal artery of anesthetized dogs for a comprehensive in vivo evaluation of the renal and systemic effects of pure ANF. The results proved conclusively that ANF acted d i r e c t l y on the kidney since urine volume and fractional excretion of sodium, potassium, chloride and calcium were elevated in a dose-related manner in the ANF-treated kidney, but were not s i g n i f i c a n t l y affected in the contralateral saline-infused organ. The maximum effects achieved with the synthetic ANF were higher than any reported following intravenous administration of crude extracts of rat a t r i a and were similar to those produced by thiazide diuretics. In four of the five dogs studied, renal vascular resistance f e l l progressively as doses of ANF were increased. Glomerular f i l t r a t i o n rate was not s i g n i f i c a n t l y elevated during ANF infusion, but was correlated with sodium excretion rates. Even though mean a r t e r i a l pressure was progressively reduced, there was no s i g n i f i c a n t change in heart rate and no stimulation of renin secretion. Arterial cyclic GMP concent r a t i o n was higher in the basal state and rose more rapidly than did renal venous levels, indicating that increases in c i r c u l a t i n g concentrations of a r t e r i a l cyclic GMP originated from an extrarenal source. Dose-related elevations in urinary cyclic GMP excretion could be explained by increased cyclic GMP f i l t r a t i o n , by enhanced production in tubular c e l l s , or by renal tubular secretion. Espec i a l l y in the saline-infused kidney, there was a clear dissociation between excretion of cyclic GMP and fractional sodium excretion. We conclude that the synthetic ANF increased electrolyte excretion via a direct renal action which was not solely dependent upon changes in renal vasculature, renin secretion or cyclic GMP levels. In recent years, e x t r a c t s of the a t r i a of rat hearts were found to stimu l a t e f r a c t i o n a l e x c r e t i o n of sodium, potassium and c h l o r i d e in anesthetized rats ( i - 6 ) . Glomerular f i l t r a t i o n rate (GFR) and renal blood f l o w (RBF) were reported to e i t h e r r i s e (4-6) or to remain unchanged (1-2) at n a t r i u r e t i c 0024-3205/85 $3.00 + .00 Copyright (c) 1985 Pergamon Press Ltd.
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doses. In isolated perfused rat kidneys, a t r i a l extracts increased GFR (7) and either enManced (7) or decreased (8) renal vascular resistance (RVR) depending upon pre-existing vascular tone (7). Extracts of monkey or human atria also stimulated sodium and calcium excretion without affecting either GFR or free water clearance (9) in anesthetized monkeys. In addition to a n a t r i u r e t i c action, a t r i a l extracts relaxed isolated rat aortic strips (i0) and reduced blood pressure of anesthetized rats (1,2,3). F i n a l l y , a t r i a l preparations enhanced the a r t e r i a l plasma concentration and urinary excretion of cyclic 3',5'-guanosine monophosphate(GMP) in anesthetized rats (11). Cyclic GMP levels in kidney minces and in primary cultures of renal tubules were also elevated by treatment with a t r i a l extracts (11). The a c t i v e substance e x t r a c t e d from a t r i a is now g e n e r a l l y known as a t r i a l n a t r i u r e t i c f a c t o r and has been characterized as a peptide (12). Recently, several i n v e s t i g a t o r s have i s o l a t e d and sequenced a series of peptides which possess the b i o l o g i c a l a c t i v i t y of ANF (13-16). A compound, corresponding to the 26 residues of the C-terminal of a 73 amino acid form of ANF (17), and cont a i n e d i n the DNA sequence described f o r a l a r g e r precursor p r o t e i n (18), was s y n t h e t i z e d by a combination of c l a s s i c a l s o l u t i o n and s o l i d phase techniques as described by Seidah et a l . (13). The sequence of the s y n t h e t i c peptide is ArgArg-Ser-Ser-C~s-Phe-G~y-G~y-Ar9-~e-As~-Arg-~e-G~y-A~a-G~n-Ser-G~y-Leu-G~y-C~sAsn-Ser-Phe-Arg-Tyr-COOH w i t h a d i s u l f i d e bridge between the cysteines at posit i o n s 5 and 21. This peptide i s i d e n t i c a l to a t r i o p e p t i n I I reported by C u r r i e et a l . (16) w i t h the a d d i t i o n of Arg-Arg on the N-terminus and of Tyr on the C-terminus. Our s y n t h e t i c p r e p a r a t i o n , which was 82% p e p t i d a l in composition, produced n a t r i u r e t i c a c t i v i t y comparable to t h a t of a t r i a l e x t r a c t s in anesthet i z e d rats (17) and relaxed i s o l a t e d vascular smooth muscle preparations (19). In the present study t h i s a c t i v e p o r t i o n of ANF was evaluated i n anesthet i z e d dogs w i t h o u t the p o t e n t i a l complications of unrelated peptides which were probably present i n the a t r i a l e x t r a c t s . The renal e f f e c t s of ANF were maximized and a d i r e c t renal a c t i o n of ANF was v e r i f i e d by i n f u s i o n of several doses of the s y n t h e t i c peptide d i r e c t l y i n t o one renal a r t e r y w h i l e s a l i n e was d e l i v e r e d i n t o the c o n t r a l a t e r a l kidney. The e f f e c t s of the s y n t h e t i c peptide on mean a r t e r i a l blood pressure (MAP), renal blood f l o w (RBF) and renal funct i o n , i n c l u d i n g GFR, free water reabsorption and f r a c t i o n a l e x c r e t i o n of sodium, potassium, c h l o r i d e and calcium were analyzed. In a d d i t i o n , c y c l i c GMP c o n c e n t r a t i o n s were measured i n u r i n e and renal venous plasma samples from both kidneys and i n a r t e r i a l plasma. For the f i r s t time, the renal c o n t r i b u t i o n to the increase in c y c l i c GMP l e v e l s could be evaluated. C y c l i c 3 ' , 5 ' - a d e n o s i n e monophosphate ( c y c l i c AMP) was measured i n selected samples to v e r i f y the s p e c i f i c i t y of the c y c l i c GMP response. F i n a l l y , plasma renin c o n c e n t r a t i o n s were determined and the e f f e c t s of ANF on a r t e r i a l plasma renin c o n c e n t r a t i o n and renin s e c r e t i o n rates were assessed. Methods
Five female dogs (weighing an average of 13.6 + 0.4 kg) were anesthetized with v i n b a r b i t a l , intubated and ventilated with roo~ a i r via a Harvard respirat o r . Catheters inserted into the abdominal aorta and vena cava via the femoral artery and vein, respectively, provided routes for arterial blood sampling and blood pressure measurements (Micron transducer) and for intravenous infusions of creatinine and an isoosmotic solution (6% dextran and 0.9% sodium chloride). Both kidneys were exposed via b i l a t e r a l flank incisions and catheters were placed in each ureter for timed urine collections. Curved needles (20 or 23 gauge) attached to S i l a s t i c tubing were inserted d i r e c t l y into each renal vein for blood sampling and into each renal artery for direct intrarenal infusions.
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Factor
35
An electromagnetic f l o w probe (Zepeda) was p o s i t i o n e d around each renal a r t e r y f o r continuous renal blood f l o w d e t e r m i n a t i o n s . In order t h a t glomerular filtration rate could be determined by c a l c u l a t i n g the rate of clearance of exogenous c r e a t i n i n e , a bolus of c r e a t i n i n e (50 mg/kg, i v ) was i n j e c t e d and a s u s t a i n i n g i n f u s i o n ( i mg/kg/min, i v ) was begun at l e a s t 45 minutes before the f i r s t experimental p e r i o d . Mean a r t e r i a l pressure, heart rate and l e f t and r i g h t RBF were c o n t i n u o u s l y recorded on a Buxco system and renal vascular resistances were derived e l e c t r o n i c a l l y . Control sampling was not begun u n t i l a s t a b l e b a s e l i n e was assured. Urine was q u a n t i t a t i v e l y c o l l e c t e d during each i 0 min period and a r t e r i a l blood samples were drawn at the midpoint f o r measurements of hematocrit and plasma c r e a t i n i n e and e l e c t r o l y t e c o n c e n t r a t i o n s . At the end of each p e r i o d , blood was obtained from the aorta and from each renal vein (15 ml t o t a l ) f o r d e t e r m i n a t i o n of plasma renin c o n c e n t r a t i o n s and plasma c y c l i c GMP and c y c l i c AMP l e v e l s . F l u i d losses due to sampling were replaced by a continuous intravenous d r i p of 6% dextran at i to 1.5 ml/min. During the f i r s t two 10 minute measurement periods, saline was infused at 0.19 ml/min into both renal arteries. Since the baseline values were stable, the data from the two control periods were averaged and presented as singular control values throughout t h i s report. Solutions of synthetic ANF dissolved in saline were then delivered into one renal artery during the next 8 periods so that doses of 1.2, 2.4, 4.8, 9.6, 19.2, 38.4, 76.8 and 156 pmole/kg/min were infused for 10 minutes each. Once the ANF administration was complete, saline infusion into the treated kidney was resumed and four 20 minute recovery periods were observed. Creatinine concentrations in urine and a r t e r i a l plasma were measured using a standard colorimetric assay and were used to calculate creatinine clearance, an estimate of GFR. Urinary and plasma concentrations of sodium, potassium and chloride were assessed with ion selective electrodes (Technicon) and calcium levels by atomic absorption spectrophotometry. Fractional electrolyte excretions (FE) were expressed as the percentage of the f i l t e r e d electrolyte load (or GFR multiplied by the plasma concentration) which was excreted in the urine. Plasma renin concentration was determined by radioimmunoassay (Clinical Assays Gamma Coat [1251]) of the amount of angiotensin generated at 37°C from 50 ul of plasma in an excess of homologous renin substrate. Renin secretion rates were the product of the renal plasma flow and the arteriovenous d i f f e r ence in plasma renin concentrations. Cyclic GMP concentration in urine and in a r t e r i a l and renal venous plasma was measured by radioimmunoassay (Amersham Cyclic GMP RIA k i t ) . Plasma samples were f i r s t extracted with ethanol; recoveries averaged 89.6 + 0.3% (n = 251). Cyclic GMP excretion was calculated from the urine volume muTti p l i e d by the urinary cyclic GMP concentration. Arterial load and renal venous effluent are the products of the renal plasma flow and the appropriate plasma concentration. Renal extraction of cyclic GMP was determined from the arteriovenous difference in plasma concentrations times the renal plasma flow. C y c l i c AMP c o n c e n t r a t i o n in selected e x t r a c t e d a r t e r i a l plasma samples and i n u r i n e were measured using the Amersham C y c l i c AMP RIA k i t . C a l c u l a t i o n s are as explained above. A l l data are expressed as the mean + the standard e r r o r of the mean (SEM). S i g n i f i c a n t changes from c o n t r o l values Were i d e n t i f i e d by a p p l i c a t i o n of the Dunnett's t - t e s t . The values from the ANF-treated kidney were compared to those of the s a l i n e - i n f u s e d kidney by paired t - t e s t f o r normally d i s t r i b u t e d samples. When samples did not pass the Wilks Shapiro t e s t f o r n o r m a l i t y , the Wilcoxon
36
Renal Effects of Atrial Natriuretic Factor
Vol. 36, No. i, 1985
Sign Rank t e s t was applied i n s t e a d . C o e f f i c i e n t s of d e t e r m i n a t i o n ( r 2) were established by performance of a l i n e a r regression using the l e a s t squares method. Results Sodium e x c r e t i o n from the ANF-infused kidney p r o g r e s s i v e l y rose from a c o n t r o l of 24 + 6 MEq/min to a peak of 184 + 50 ~Eq/min d u r i n 9 a d m i n i s t r a t i o n of 19.2 pmole/kg/min ANF then f e l l to 39 + 9 ~Eq/min i n the f i n a l recovery p e r i o d . Following s i m i l a r response p a t t e r n s , u~ine volume and f r a c t i o n a l e x c r e t i o n s of sodium, c h l o r i d e and calcium ( F i g . i and Table I ) were s i g n i f i c a n t l y elevated above the pretreatment values in the ANF-infused kidney; maximum increases were achieved at i n f u s i o n rates of 9.6 to 38.4 pmole/kg/min. In a d d i t i o n , f r a c t i o n a l potassium e x c r e t i o n from the ANF-treated kidney (Table I) was s i g n i f i c a n t l y greater than t h a t of the c o n t r a l a t e r a l kidney at a l l times a f t e r the 4.8 pmole/ kg/min dose. While free water reabsorption (Table I ) i n the ANF-treated kidney was s i g n i f i c a n t l y higher than pretreatment l e v e l s , t h e r e were no s i g n i f i c a n t d i f f e r e n c e s between measurements made in the two kidneys. I n f u s i o n of the synt h e t i c ANF p r o g r e s s i v e l y reduced MAP ( F i g . i ) but had no s i g n i f i c a n t e f f e c t on heart rate which was 128 + 9 beats/min during c o n t r o l and 139 + 7 beats/min d u r i n g i n f u s i o n of the h i g h e s t dose. TABLE I ANFa (pmo l e / k~/mi n) 0 1.2 2.4 4.8 9.6 19.2 38.4 76.8 156 0 0 0 0 a
Urine Volume b (ml/min) ANF Saline .20+.04 .47¥.21 .73¥.38 .9~.38" i.II¥.28 + 1.09#.27" .96¥.26* .9~.23 .69¥.22 .4~.14 .40¥.10 .36¥.09 .30¥.07
.20+.04 ,2~.05 ,20¥.04 .2~.[6 .22¥.08 ° .28¥.08 ° .2~.05 ° .2~.05 ° .23¥.05 .17¥.04 .15¥.03 ° .I~.03 ° .14¥.03 °
FEK (%) ANF Saline 30+3 2~7 2~3 3~5 41¥5 37#6 41¥7 42¥7 37¥6 29¥4 26¥3 32¥4 33¥3
ANF
FEcI (%) Saline
26+3 1.3+ . i 2~3 4.~1.6 2~3 5.~1.9 2~4 ° 8.0¥2.1 + 22¥4 f 9.5¥2.0 + 2~2 ° 9.9#2.0 + i ~ 3 ° 10.1¥2.1 + 21¥4 ° 9.5¥1.9 + 22¥2 ° 8.0¥1.6 + I~3 ° 5.~1.5 16¥2* f 4.2~-I.i 16¥2*o 4.6¥I .4 20¥2 3.6¥1.0
The f i r s t row of data represents the average periods. Each dose of ANF was infused f o r a recovery period was 20 minutes long, b Each column represents the measurements made or s a l i n e as i n d i c a t e d by the subheadings. * p < 0.05, + p < 0.01, compared to pretreatment ° p < 0,05, f p < 0.01, ANF- vs. s a l i n e - t r e a t e d
1.1+.2 1.3¥.3 1.~.3 1.6¥.5 ° 1.8¥.6 f 2.0¥.7 ° I.~.6 f 2.1~.5 ° 2.6¥.6 +o 1.9¥.5 ° 1.3¥.4 ° I.~.2 ° i.i¥.3 °
Free Water Reabsorption (ml/min) ANF Saline .33+.07 .3~.09 .36¥.08 .4~.09 .5~.12 .60¥.13" .6~.13 + .6~.12 + .6~.19 + .4~.07 .46¥.04 .42¥.05 .3~.Ob
.33+.06 .33¥.05 .33¥.05
. 3 ~ 06 .33¥ .36¥ .3~ .41~ .36T .31¥ .30¥ .3~ .31¥
06 06 04 06 06 05 03 05 05
of two 10-minute clearance lO-minute p e r i o d ; each i n the kidney infused w i t h ANF control. kidney
There were no s t a t i s t i c a l l y s i g n i f i c a n t a l t e r a t i o n s in GFR ( F i g . 1) or i n the renal vascular r e s i s t a n c e and RBF determined d u r i n g the f i n a l minute of each treatment period (Table I I ) . When the peak responses ( t h a t i s , the highest RBF and the lowest RVR) were examined in each dog (Table I l l ) , increases i n RBF and decreases in v a s c u l a r resistance were observed i n 4 of the 5 dogs, Even though GFR appeared to r i s e at some p o i n t during ANF treatment i n each dog (Table I l l ) ,
Vol. 36, No. i, 1985
Renal Effects of Atrial Natriuretic
Factor
37
~p< 05,÷p< Ol Comparedto Time:O
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FIG. 1 Blood pressure and renal responses t o ANF are shown above, described in the t e x t .
A b b r e v i a t i o n s are
t h e maximum response occurred at a d i f t e r e n t dose in each animal so t h a t no s t a t i s t i c a l l y s i g n i f i c a n t increase was discernable ( F i g . i ) . There was however a c o r r e l a t i o n between the sodium e x c r e t i o n and GFR ( r z = 0.80) measured during ANF infusions i n t o the t r e a t e d kidney.
Renin s e c r e t i o n from the ANF-treated kidney was v a r i a b l e before and during the course of the peptide i n f u s i o n and, despite a s l i g h t drop during some of the lower doses, was not s i g n i f i c a n t l y d i f f e r e n t from the pretreatment c o n t r o l . In only one instance was t h e r e a s i g n i f i c a n t d i f f e r e n c e betwen the rate of r e n i n release from the ANF- and s a l i n e - i n f u s e d kidneys; however, due to the i n h e r e n t v a r i a b i l i t y of t h i s measurement, small changes may be d i f f i c u l t t o detect under the present experimental c o n d i t i o n s . There were poor c o r r e l a t i o n s between renin s e c r e t i o n and e i t h e r sodium e x c r e t i o n or GFR ( r 2 = 0.12 and 0.16, respectively). A r t e r i a l plasma c y c l i c GMP c o n c e n t r a t i o n rose in a d o s e - r e l a t e d manner from 20 + 3 pmole/ml to a maximum of 102 + 13 pmole/ml a f t e r a d m i n i s t r a t i o n of 156 pmole/kg/min of s y n t h e t i c ANF. Renal-venous c o n c e n t r a t i o n s also increased from 8.6 + 1.0 and 9.3 + 1.0 to 37.8 + 6.0 and 37.2 pmole/ml in the ANF- and
38
Vol. 36, No. i, 1985
Renal Effects of Atrial Natriuretic Factor
TABLE I I Renal hemodynamics and renin levels at the end of each lO-minute treatment period in 5 anesthetized dogsa ANF (pmole/ kg/min ) 0 1.2 2.4 4.8 9.6 19.2 38.4 76.8 156 0 0 0 0
RBF (ml/min) ANF Saline 146+20 141¥16 147¥17 159¥20 15~22 131¥10 122~ii 120¥10 120¥13 129¥14 122TII 12~13 116¥11
145+ 6 137T 8 132¥ 9 128¥ 8 121¥ 8 120¥6 116¥ 8 120¥ 6 126# 8 129¥15 120¥19 i0~21 96T24"
RVR (mm Hg/ml/min) ANF Saline .88+.09 .8~.06 .79¥.06 .7~.07 .6~.06 .76¥.04 .79T.06 .77#.07 .76#.09 .77¥.08 .84¥.07 .9~.iI .93¥.10
.83+.05 .8~.04 .85¥.04 .93¥.08 .8~.05 .82¥.05 .82#.06 .767.05 .70¥.05 .77¥.09 .94¥.19 1.19¥.30 1.47¥.50
Renin Secretion (ng/min) ANF Saline 457+376 469¥427 679¥531 70Y 44 16~i18 129¥ 91 234¥ 68 45~203 658¥276 156¥136 20~107 607¥277 343T174
PRC (ng/ml/hr)
206+ 79 9.1+3.1 291¥164 12.9¥4.5 569¥302 11.8¥5.9 41~244 13.~6.3 260#124 12.1+--4.4 2 0 1 # 79 11.3¥4.3 446T 61f 9.3¥4.0 30~186 9.7¥4.0 664#232 10.4¥4.5 69~274 12.6¥4.0 705¥291 10.9¥4.4 786¥446 13.4¥4.0 407¥123 17.4¥4.0
a Format as explained in Table I * p < 0.05, compared to pretreatment control f p < 0.01, ANF- vs. Saline-treated saline-infused kidneys, r e s p e c t i v e l y . The calculated rates of c y c l i c GMP i n f l u x and e f f l u x were also s i g n i f i c a n t l y elevated during administration of the synthetic peptide (Fig. 2). As a r t e r i a l levels rose, renal extraction of the c y c l i c nucleotide increased for both kidneys. The f i l t e r e d load of c y c l i c GMP and urinary c y c l i c GMP excretion was also enhanced in a dose-related manner in both the ANF-treatedkidney and in the contralateral organ (Fig. 3). Cyclic AMP levels measured in the same samples were not s i g n i f i c a n t l y altered by ANF treatment. A r t e r i a l plasma c y c l i c AMP was 3.2 + 0.6 and 3.6 + 0.7 pmoles/ml at control and 156 pmole/kg/min, r e s p e c t i v e l y . At the same time points, urinary excretion of c y c l i c AMP was 172 + 34 and 161 + 32 pmole/min from the ANF-treated kidney and 173 + 31 and ib4-+ 25 pmole/mTn from the saline-infused kidney. Discussion The present study offered the f i r s t opportunity to examine in anesthetized dogs the effects of a single peptide synthesized from the amino acid sequence contained in the natural ANF isolated from rat a t r i a . Use of the synthetic compound in the present experiment permitted evaluation of an active ANF fragment in the absence of unrelated a t r i a l peptides. Selection of the anesthetized dog model allowed us to d e l i v e r ANF via one renal artery so that the i p s i l a t e r a l kidney received concentrations of the peptide which exceeded those achieved in the systemic c i r c u l a t i o n . Consequently, b i l a t e r a l renal function could be examined at unequal renal plasma ANF concentrations, a condition which could not be attained during intravenous administration.
Vol. 36, No. i, 1985
39
Renal Effects of Atrial Natriuretic Factor
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40
Renal Effects of Atrial Natriuretic Factor
Vol, 36, No, i, 1985
ANF- Treafed Kidney
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FIG. 2 The changes i n plasma l e v e l s and renal e x t r a c t i o n of c y c l i c GMP are shown in the top and bottom panels, r e s p e c t i v e l y . Despite the estimated lO-fold d i l u t i o n of the peptide as i t entered the systemic c i r c u l a t i o n , synthetic ANF lowered blood pressure at doses which had no effect on renal function in the saline-infused kidney. Interestingly, there was no reflex tachycardia, a phenomenonwhich has been previously reported following intravenous injection of a t r i a l extracts into conscious rats (20). While both our synthetic ANF and a t r i a l extracts have been shown to relax isolated vascular smooth muscle (10,19), other potential mechanisms which may contribute to the depressor response have yet to be f u l l y explored.
During i n t r a r e n a l i n f u s i o n of the s y n t h e t i c ANF, the peak f r a c t i o n a l e l e c t r o l y t e e x c r e t i o n s measured in the present experiment surpassed those prev i o u s l y reported f o l l o w i n g intravenous i n j e c t i o n of a t r i a l e x t r a c t s i n t o rats ( i , 2). This d i f f e r e n c e may r e s u l t from the route of a d m i n i s t r a t i o n , the use of a more pure s y n t h e t i c ANF p r e p a r a t i o n or a d i f f e r e n c e in species responsiveness. The magnitudes of the increases i n f r a c t i o n a l sodium e x c r e t i o n observed in the present study resemble those produced by t h i a z i d e d i u r e t i c s in anesthetized dogs (21). However, u n l i k e t h i a z i d e s , s y n t h e t i c ANF p r o g r e s s i v e l y reduced MAP; t h e r e f o r e , changes in renal hemodynamics secondary to the depressor e f f e c t may have obscured the t r u e c e i l i n g of the n a t r i u r e t i c response to ANF. Confirmation of the maximum s a l u r e s i s which u l t i m a t e l y may be achieved by pure ANF awaits e v a l u a t i o n of the peptide in other experiments i n which blood pressure i s main-
Vol. 36, No. i, 1985
Renal Effects of Atrial Natriuretic Factor
E
1.5-
4]
+p
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co+oo,.+oT .... o
1.0~9
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(prnole/kg/min,iro) O Time (min)
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FIG. 3 The changes in the amount of f i l t e r e d top and bottom panels, r e s p e c t i v e l y .
and excreted c y c l i c GMP are shown in the
t a i n e d at i t s c o n t r o l l e v e l , o t h e r compensatory mechanisms are considered and t a c h y p h y l a x i s has been disproved. We may also p o s t u l a t e t h a t the peptide is at l e a s t p a r t i a l l y i n a c t i v a t e d i n the systemic c i r c u l a t i o n since d i l u t i o n alone would not e x p l a i n the lack of e f f e c t on the c o n t r a l a t e r a l kidney at the highest ANF i n f u s i o n , a dose which is at l e a s t 30 times the m i n i m a l l y e f f e c t i v e i p s i l a t e r a l c o n c e n t r a t i o n . Nevertheless, the present data are c o n s i s t e n t with a d i r e c t renal e f f e c t of ANF since the renal responses of the ANF-treated kidney developed at doses which had no apparent e f f e c t s on c o n t r a l a t e r a l renal function. While f r e e water r e a b s o r p t i o n appeared t o r i s e in the ANF-infused kidney, t h e r e was no s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e when compared to the s a l i n e infused organ. Before meaningful conclusions can be reached concerning the e f f e c t s of ANF on the renal handling of water, ANF must be i n v e s t i g a t e d in experiments in which a n t i d i u r e t i c hormone is e i t h e r i n h i b i t e d or maximally stimulated. Previous studies in anesthetized rats (1-3) and monkeys (9) revealed no effect of injections of a t r i a l extracts on GFR. However, infusion of a t r i a l extracts into rats (4,5) and isolated perfused rat kidneys (7) s i g n i f i c a n t l y elevated GFR, expecially at high doses (5). In the anesthetized dogs in the present study, there were no s t a t i s t i c a l l y significant changes in the ANFinfused kidney when compared to i t s own control level by Dunnett's t - t e s t or
42
Renal Effects of Atrial Natriuretic
Factor
Vol.
36, No. i, 1985
when compared to the GFR of the s a l i n e - i n f u s e d kidney by the paired t - t e s t . Upon examining the GFR measured during ANF i n f u s i o n in the i n d i v i d u a l animals, the f i l t r a t i o n rate appeared to be somewhat enhanced i n each dog, but the peak occurred at a d i f f e r e n t dose i n each animal. While t h e r e seemed to be a reasonable c o r r e l a t i o n (r 2 = 0.80) between GFR and sodium e x c r e t i o n measured during ANF a d m i n i s t r a t i o n , the r i s e in f r a c t i o n a l sodium e x c r e t i o n (sodium clearance/GFR) suggested t h a t the n a t r i u r e s i s produced by ANF could not be e n t i r e l y a t t r i b u t e d to the increase in GFR. When a l l f i v e dogs were included in the a n a l y s i s of the measurements of RBF and RVR made during the l a s t minute of each I0 minute i n f u s i o n , no s t a t i s t i c a l l y s i g n i f i c a n t changes were i d e n t i f i e d (Table I I ) . However, when the maximum responses to each dose of ANF were examined (Table I l l ) , ANF was found to a t y p i c a l l y lower RBF in one dog in which c o n t r o l RBF was e l e v a t e d . A n a l y s i s of the responses in the other 4 dogs revealed a s i g n i f i c a n t increase in RBF at one dose (19.6 pmole/kg/min) and f a l l s i n RVR at a l l doses of 4.8 pmole/kg/min and h i g h e r . These f i n d i n g s are c o n s i s t e n t w i t h the observations in i s o l a t e d perfused kidneys i n which changes in renal r e s i s t a n c e depended upon the c o n t r o l RVR values. That i s , when i n i t i a l vascular tone was enhanced i n i s o l a t e d perfused kidneys by pretreatment with a v a s o c o n s t r i c t o r (7) and i n 4 of the dogs in the present study, ANF produced v a s o d i l a t i o n . However, when baseline RVR was low, treatment w i t h higher doses of ANF apparently c o n s t r i c t e d the renal vessels. I n t e r e s t i n g l y , i n the one dog i n which ANF increased RVR from an u n u s u a l l y low c o n t r o l l e v e l , a c o n s t r i c t o r dose of ANF (19.2 pmole/kg/min) elevated sodium e x c r e t i o n from a baseline of 36 to 248 uEq/min. While the RVR response was unique to t h a t animal, i t s n a t r i u r e t i c response was not u n l i k e t h a t produced i n the other 4 dogs. S i m i l a r l y , sodium e x c r e t i o n was also reported to r i s e in the i s o l a t e d perfused r a t kidneys i n which vascular r e s i s tance increased ( 7 ) , but was not measured i n the i s o l a t e d kidneys in which RVR f e l l (7, 8). A d d i t i o n a l studies designed to assess i n t r a r e n a l hemodynamics are needed to e l u c i d a t e the s u b t l e , but p o t e n t i a l l y i m p o r t a n t , r e l a t i o n s h i p s between the n a t r i u r e t i c and renal vascular e f f e c t s of ANF. While ANF e x t r a c t s enhanced a r t e r i a l plasma l e v e l s and u r i n a r y e x c r e t i o n of c y c l i c GMP i n anesthetized rats ( i i ) , the source of the c y c l i c GMP generat i o n was not apparent from those e a r l i e r data. In the present i n v e s t i g a t i o n , i t was possible to obtain a more complete p r o f i l e of changes in c y c l i c GMP produced by s y n t h e t i c ANF by measuring f o r the f i r s t time the c y c l i c n u c l e o t i d e c o n c e n t r a t i o n s in the a r t e r i a l plasma, in the renal venous plasma and in the u r i n e from each kidney. In agreement w i t h an e a r l i e r report of the e f f e c t s of i n j e c t i o n of a t r i a l e x t r a c t i n t o rats ( i i ) , s y n t h e t i c ANF i n f u s i o n increased a r t e r i a l c y c l i c GMP c o n c e n t r a t i o n s in a d o s e - r e l a t e d manner. In the present experiment, we were able to demonstrate t h a t at no time before or d u r i n g i n f u sion of the peptide did renal venous e f f l u e n t exceed a r t e r i a l c y c l i c GMP levels. In a d d i t i o n , c y c l i c GMP e f f l u x rates from the ANF-treated kidney were not d i f f e r e n t from the l e v e l s measured i n the blood i s s u i n g from the s a l i n e infused organ. Together, these data suggest t h a t the enhanced l e v e l s of c i r c u l a t i n g c y c l i c GMP r e s u l t i n g from ANF a d m i n i s t r a t i o n o r i g i n a t e d from an e x t r a r e n a l source. The r i s e s in u r i n a r y e x c r e t i o n of c y c l i c GMP during i n f u s i o n of the synt h e t i c ANF could p o t e n t i a l l y r e s u l t from e i t h e r t u b u l a r production of c y c l i c GMP, from renal clearance ( e . g . f i l t r a t i o n and/or s e c r e t i o n ) of the c y c l i c n u c l e o t i d e from the a r t e r i a l plasma or from a combination of the two. The f i r s t e x p l a n a t i o n would be c o n s i s t e n t w i t h the reported s t i m u l a t i o n of c y c l i c GMP i n primary c u l t u r e s of renal t u b u l a r c e l l s ( I I ) . In a d d i t i o n , c y c l i c GMP e x c r e t i o n s before beginning ANF treatment in the present study were 199 + 20 and 193 + 19% of the f i l t e r e d load of the c y c l i c n u c l e o t i d e in the experTmental
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and saline-infused kidneys, r e s p e c t i v e l y ; there were no s i g n i f i c a n t changes in these r a t i o s during the infusion of the synthetic ANF. These data suggest that e i t h e r c y c l i c GMP was secreted into the tubular f l u i d from the a r t e r i a l plasma or that the c e l l s of the tubules, which contain enzymes involved in c y c l i c GMP metabolism (22,23), produced c y c l i c GMP. F i n a l l y , the enhanced levels of urinary c y c l i c GMP excretion were not l i k e l y to be p r i m a r i l y responsible for the renal e f f e c t s of ANF since c y c l i c GMP excretion from the saline-infused kidney rose to levels s i g n i f i c a n t l y greater than the pretreatment baseline values without development of s i g n i f i c a n t d i u r e s i s or s a l u r e s i s . In view of the poor correlations between renin secretion and either sodium excretion or GFR, the changes in renal function during ANF infusion did not appear to be related to alterations in renin release. In 3 of the 5 dogs control levels of renin secretion were less than IOU ng/min, therefore a reduction in the release rate would have been d i f f i c u l t to detect. There was however, a tendency for renin secretion to f a i l at doses of ANF at which the concurrent depressor response did not exceed 20 mm Hg. At ANF infusions in excess of 38.4 pmole/kg/min, renin secretion was near or above control levels while MAP was reduced by at least 30 mm Hg. Althoughthese data are preliminary due to t h e i r v a r i a b i l i t y , these f a l l s in blood pressure may be expected to e l i c i t some increase in renin release under other circumstances (24). An actual i n h i b i t i o n of basal or stimulated renin secretion by ANF remains to be demonstrated in a study directed toward that purpose. In summary, the synthetic peptide corresponding to a 26 amino acid sequence of ANF was infused into one renal artery of anesthetized dogs and stimulated diuresis and saluresis in a manner which could not be solely explained by changes in RBF or GFR. The higher doses of the peptide also signif i c a n t l y lowered MAP without affecting heart rate or stimulating renin secret i o n . Control a r t e r i a l plasma concentrations of cyclic GMP were greater than the renal venous levels. During ANF infusion, a r t e r i a l cyclic GMP rose more rapidly than did renal venous e f f l u x , indicating an extrarenal source of circulating cyclic GMP. In addition, cyclic GMP excretion was double the rate of f i l t r a t i o n but less than the a r t e r i a l delivery rate. Therefore, cyclic GMP was added to the tubular f l u i d by active secretion from the plasma, by nephrogenous production or a combination of the two processes. These data supply the f i r s t insight into the effects of a pure synthetic ANF peptide on renal function, blood pressure and plasma levels of renin and cyclic GMP in the anesthetized dog.
Acknowledgments We wish to thank Shirley S a t i r i t z for performing the calcium assays. We are also appreciative of the excellent secretarial support of Maryanne Olkowski and Carol Van Arsdale.
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