Pharmacokinetics receDtor blockade

of ANF and urodilatin during cANF and neutral endopeptidase inhibition

ZAID A. ABASSI, JOHN TATE, SALLY HUNSBERGER, HENRY KLEIN, DANIEL TRACHEWSKY, AND HARRY

R. KEISER

Hypertension-Endocrine Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda 20892; and Center for Drug Evaluation and Research Staff College, Food and Drug Administration, Rockville, Maryland Abassi, Zaid A., John Tate, Sally Hunsberger, Henry Klein, Daniel Trachewsky, and Harry R. Keiser. Pharmacokinetics of ANF and urodilatin during cANF receptor blockade and neutral endopeptidase inhibition. Am. J. Physiol. 263 (Endocrinol. Metab. 26): E870-E876,1992.-Urodilatin is a new member of the family of natriuretic peptides. It is of renal origin. Previous reports indicate that urodilatin is natriuretic in lower doses than atria1 natriuretic factor (ANF)-(99-126) and that it might be more effective than ANF in the treatment of cardiovascular edema. The present study was designed to compare the pharmacokinetics of the hydrolysis and clearance of 1251-labeledurodilatin and 1251-ANF. In control rats, the volume of distribution (V,,), metabolic clearance rate (MCR), and distribution half-life (distribution tli2) of urodilatin in plasma were not significantly different from those of ANF. Infusion of clearance (c)ANF-(4- 23), a specific ligand for receptors that clear ANF in excess amounts (i.e., a bolus injection of 100 pg/kg followed by a continuous infusion of IO pg. kg-l. min-l), increased the amount of intact peptide in the plasma to the same extent for both urodilatin and ANF. In addition, cANF-(4 - 23) decreased the V,, and the MCR and increased the distribution tli2 of both peptides to about the same degree. Prior treatment of rats with SQ-28,603, a specific neutral endopeptidase (NEP; EN 3.4.24.11) inhibitor, was without significant effect on the metabolic clearance of urodilatin, whereas it decreased the clearance of ANF by 65%. Furthermore, an infusion of SQ28,603 suppressed the appearance of the hydrolytic products of ANF in blood but not of urodilatin. Moreover, the inhibitor increased the total amount of ANF recovered in the kidneys to five times the control values, whereas it did not alter the renal uptake of urodilatin. These data demonstrate that urodilatin is removed from the circulation mainly by cANF receptors, whereas circulating ANF-(99- 126) is cleared by both cANF receptors and NEP. This may contribute to the differences in the natriuretic potency of these two peptides, despite similar distribution tlj2, MCR, and V,,. urodilatin; atria1 natriuretic factor; neutral endopeptidase inhibitor; clearance atria1 natriuretic factor receptor ligand; metabolic clearance rate; volume of distribution; half-time; hydrolysis of urodilatin; hydrolysis of atria1 natriuretic factor [ (ANF) - (1 - 28) = ANFhormone of cardiac origin (6), with potent diuretic, natriuretic, and vasodilator properties (10, 22). ANF is involved in blood pressure regulation and in the control of sodium and volume homeostasis (3, 10, 22). ANF acts on specific receptors in kidney, blood vessels, adrenal glands, and brain (16, 17, 28). There are at least two types of ANF-( l-28) receptors with different structural and functional features. A small number of ANF receptors are coupled to particulate guanylate cyclase (16, 20), mediate the biological actions of the hormone via an increase in intracellular guanosine 3’-5’.cyclic monophosphate (cGMP; see Refs. 13, 16), and are termed B-receptors. The overwhelming ATRIAL

NATRIURETIC

FACTOR

(99- 126)] is a peptide

20857

majority of the ANF binding sites are not associated with guanylate cyclase or any other second messenger (39) and are called clearance receptors (C-receptors). The B-receptors have a molecular weight of 130,000 and a very high affinity for ANF-( l-28), whereas the C-receptors have a molecular weight of 66,000 and a very high affinity for both ANF-( l-28) and ANF(4-23) (16, 17, 20). Intravenous infusion of clearance (c) ANF-(4-23) into rats decreases the clearance of ANF- (1 - 28) and causes natriuresis and hypotension due to an increase in plasma ANF immunoreactivity (20). However, cANF-(4-23) has no effect in the isolated perfused rat kidney (19,20). In sum, these findings indicate that the C-receptors are not involved in the biological actions of ANF-( l-28) but serve as a nonenzymatic pathway for ANF clearance. Recently, Almeida et al. (2) and Chiu et al. (5) demonstrated that treatment of rats with cANF caused an increase in the initial plasma concentration of 1251-labeled ANF-( 1 - 28) and prolonged its distribution half-life (t1j2) two- to threefold. Moreover, such treatment decreased the volume of distribution at steady state (V,,) and the total body metabolic clearance rate (MCR) of 1251-ANF-( 1 - 28). These findings indicate that C-receptors play a major role in the clearance of ANF from plasma.A second important pathway for ANF-( l-28) removal from the circulation is via the action of the enzyme neutral endopeptidase (NEP) (23, 32). NEP is found in high concentration in the kidney, primarily in the brush border of the proximal tubule and in the glo merulus (30). NEP is also found in the lung, brain, intestine, and white blood cells (25). Berg et al. (4) and Olins et al. (23) have reported that ANF is rapidly inactivated by homogenates of renal NEP via cleavage at the Cys7Phe8 bond. Bilateral nephrectomy in rats doubled the distribution tl12 of exogenous atriopeptin III (18) and increased the plasma levels of endogenous ANF (15), suggesting that the kidney plays an important role in the inactivation of ANF. Hollister and co-workers (14) have reported a significant contribution of the lungs to ANF clearance, and this coincides with the demonstration of large amounts of NEP activity in the lungs (7). Inhibition of NEP activity by specific inhibitors delays the disappearance of administered ANF from the circulation and potentiates its renal and vascular effects in rats (34). In sum, NEP plays a significant role in the metabolism of ANF in plasma. Recently, another natriuretic factor [ANF-(95 - 126)] was isolated from human urine and named urodilatin (29). It has the same amino acid sequence as ANF(99- 126) plus an NH2-terminal extension of four amino

E870 Downloaded from www.physiology.org/journal/ajpendo at Midwestern Univ Lib (132.174.254.157) on February 12, 2019.

PHARMACOKINETICS

OF URODILATIN

acids. Urodilatin is probably produced within the nephron by different posttranslational processing of the prohormone ANF-( 1 - 126) (8,ll). Urodilatin appears to be the major form of ANF immunoreactivity in urine (8, 29). Unlike ANF-(99126), urodilatin is inert to proteolytic degradation by NEP (9). In experimental animals (8, 12) and humans (27), urodilatin, like ANF(99- 126), is natriuretic, diuretic, and a vasodilator. Initial studies indicated that urodilatin has greater natriuretic potency than ANF-(99126) in normal humans (27) and in experimental models of congestive heart failure (1, 24, 36). The mechanisms underlying this greater natriuretic potency have not been clarified. The present study was designed to evaluate the relative contribution of C-receptors and NEP in the pharmacokinetics and hydrolysis of urodilatin by use of the C-receptor blocker cANF-(4-23) and of the NEP inhibitor SQ-28,603 (33). In addition, we compared the pharmacokinetics and hydrolysis of urodilatin and ANF-(99-126). MATERIALS

AND

METHODS

Experiments were performed on male Munich Wistar rats (Harlan Sprague-Dawley Farms, Indianapolis, IN) weighing 220-330 g. The animals were maintained on standard commercial rat Chow and tap water ad libitum. Experimental protocols. Rats were anesthetized with an intraperitoneal injection of 5-set-butyl-5-ethyl-2-thiobarbituric acid (Inactin; 100 mg/kg; Abbott, North Chicago, IL) and underwent tracheostomy. Polyethylene catheters (PE-50) were inserted into a jugular vein for infusion of the various solutions. The urinary bladder was catheterized via a suprapubic incision. A solution of 0.9% saline was infused intravenously at a rate of 1% body wt/h throughout the experiments. Rats received a single intravenous injection of 1 &i of either 1251-ANF-( l-28) or 1251-labeledurodilatin, and the catheter was flushed rapidly with 0.2 ml saline. In the experimental groups, the animals were treated with either 30 mg/kg iv SQ-28,603 (Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ) for 30 min before the injection of tracer or 100 hg/kg iv cANF(4-23) for 15 min before the administration of the tracer, followed by a continuous intravenous infusion of 10 pg. kg-l min-l. One group received both SQ-28,603 and cANF-(4-23). Arterial blood samples, each 200 ~1, were obtained at 0.5, 1.0, 1.5, 2.0, 2.5, 3, 4, 5, 6, 8, 10, 20, and 30 min after the administration of the labeled hormones. Plasma samples, 100 ~1,were treated with 500 ~110% trichloroacetic acid (TCA) at 0°C and centrifuged. The pellets were resuspended two times in 500 ~1 TCA at 0°C and recentrifuged. Radioactivity in either the combined supernatants (TCA soluble) or the pellet (TCA-ppt) was quantitated via a gamma counter (Beckman Instruments, Irvine, CA). The TCA-ppt 1251 radioactivity was shown by high-performance liquid chromatography (HPLC) to be mainly intact ANF, while the TCAsoluble radioactivity was mainly free 1251or hydrolytic products. This agreed with previous reports in the literature (2, 5). After the injection of the labeled peptides (30 min), the rats were killed and their kidneys, lungs, liver, and heart were rapidly removed to determine the radioactivity in these organs as well as in the collected urine. HPLC. The nature of the radioactivity recovered in plasma was determined by reverse-phase HPLC. Samples of arterial blood, 0.75 ml each, were collected at 0.5, 3, and 10 min after bolus injection of 1 &i of either 1251-urodilatin or 1251-ANF to rats treated with either NEP inhibitor (NEP-I), cANF-(4-23) l

AND

ES71

ANF

a combination of NEP-I and cANF-(4-23), or vehicle. Plasma samples, 0.3 ml, were loaded onto cartridge columns (C,, Amersham, Arlington Heights, IL) that had been preactivated with 2 ml 100% methanol and 2 ml distilled water. The columns were subsequently washed with 5 ml 0.1% trifluoroacetic acid (TFA) and 4 ml 80% acetonitrile in 0.1% TFA. The aqueous washings and the eluate were collected separately, and radioactivity was measured with a gamma counter. The aqueous washings contained free 1251while the eluate contained intact peptide. The distribution of the radioactivity between the aqueous washings and the eluate coincided with the distribution of the radioactivity between the TCA-soluble 1251and TCA-ppt 1251,respectively. These findings confirmed the reliability of the TCA method in the separation of intact peptide from the products of hydrolysis. - The- aqueous washings and the eluate were combined and evaporated via a vacuum centrifuge. The residue was reconstituted in 200 ~10.1% TFA and analyzed by reverse-phase HPLC column [Ultrapore C, (4.6 x 75 mm), Beckman Instruments, Fullerton, CA] with a linear gradient of lo-60% acetonitrile in 0.1% TFA over 20 min at a flow rate of 1 ml/min. Fractions were collected every minute, and radioactivity was quantitated via a gamma counter. The HPLC column was calibrated with 1251 and either 1251-urodilatin or 1251-ANF-(99- 126). The nature of the radioactivity recovered in the kidney was determined by reverse-phase HPLC. The kidneys were dissected and immediately homogenized in 5 vol cold 0.1 M HCl. The homogenates were centrifuged, and the supernatants were used for analysis by HPLC as described above. Phurmacokinetic analysis and statistical methods. The program MKMODEL (version 4.40; Holford, N. Biosoft, Milltown, NJ) was used to estimate the pharmacokinetic parameters for each rat. In several of the rats the data after 8 min were not used to esti mate the parameters because the measurements represented entrapped radioactivity. The pha .rmacokinetic parameters were calculated from the plasma disappearance curve of TCA-ppt 1251radioactivity by using a two-compartment model (38) with parameterization of the model as follows concn(

t,

=-dose v

X

e-Llxt

+

(E (L,

L) z

X2-

L,)

X

e-L&t

1

Here, V is volume of the central compartment. L, is distribution or rapid disposition constant, L, is elimination or slow disposition constant, Ex, is L1 x L, x V/clearance. The rapid distribution t 1/2 phase can be calculated from the distribution tlj2 = log(2)/L,. The L, parameter was shown to be significantly different from 0 (P = O.OOOl),which implies that a twocompartment model is more appropriate than a one-compartment model. For both a one-compartment model and a noncompartment model the volume at steady state is the same as the central compartment volume. Dunnett’s test (31) was used to compare the parameters for each experimental group to control. A Bonferroni corrected t test (37) was used to compare the parameters for ANF with those for urodilatin. All results are expressed as means t SE. Atrial peptides. ANF, urodilatin, and des-[Gln1*Ser1gGly20Leu21Gly22]recombinant (r) ANF were purchased from Peninsula Labs, Belmont, CA. 1251-rANF-(99- 126) and 1251-urodilatin (sp act 2,200 Ci/mmol with ~5% TCA-soluble radioactivity) were purchased from New England Nuclear, Boston, MA. RESULTS

Pharmacokinetics of 1251-AAW(99- 126). In the control group, the amount of intact, i.e., TCA-ppt, peptide in plasma decreased rapidly, reaching a plateau 4-5 min after the injection of the labeled ANF (Fig. 1). Pretreatment of the rats with NEP-I, had no effect on the

Downloaded from www.physiology.org/journal/ajpendo at Midwestern Univ Lib (132.174.254.157) on February 12, 2019.

ES72

PHARMACOKINETICS

OF URODILATIN

100000

Y

I

100 I0

Control

-

NEP-I

-

cANF

-

NEP-I + cANF

I IO

II 30

II 20

Time (min) Fig. 1. Disappearance of trichloroacetic acid-precipitable (TCA-ppt) radioactivity, i.e., intact atria1 natriuretic factor (ANF), from plasma after a bolus injection of 1251-ANF-(99- 126) either alone, with SQ28,603 and cANF-(4--23), or a combination of both agents in rats. See MATERIALS AND METHODS for details. Radioactivity in intact peptide is expressed as counts/min (cpm) in 1 ml plasma. Results are means & SE; n = 5 rats in each group. Infusion of cANF-(4-23) alone or with neutral endopeptidase inhibitor (NEP-I) significantly [P < 0.001 by analysis of variance (ANOVA)] delayed disappearance of 1251-ANF-(99- 126) from plasma compared with control rats.

AND ANF

compared with groups treated with either agent alone. In control rats, V,,, MCR, and distribution tli2 were 13.3 t 0.8 ml/100 g body wt, 8.2 t 1.1 ml*minS1* 100 g body wt-‘, and 33 t 2 s, respectively (Table 1). Although NEP-I was without effect on the V,,, it increased the distribution tl,2 to 43 t 4 s (P = not significant) and decreased the MCR to 36 t 5% of the values obtained in control rats. The presence of cANF-(4-23) decreased the V,, and MCR to 8.4 t 0.5 (P 5 0.05) and 1.0 t 0.1 (P s 0.05), respectively, and increased the distribution tl12 to 80 t 3 s (P zs 0.05). The combination of cANF and NEP-I had little or no further effect on these parameters. In the control rats, the hydrolytic products of ANF in plasma, i.e., TCA-soluble radioactivity, increased in a time-dependent manner (Fig. 2). NEP-I significantly decreased the appearance of hydrolytic products throughout the first 20 min of the experiment. In contrast, cANF significantly enhanced the appearance of hydrolytic products in the first 8 min of the experiment, while slightly decreasing their appearance in the last 20 min. The presence of both cANF and NEP-I exerted an additive effect, resulting in an even greater delay in the appearance of hydrolytic products, especially in the latter part of the experiment. 60

rate of disappearance of the intact peptide in plasma. Infusion of cANF-(4- 23), resulted in a significant increase in the initial plasma level of ANF, and this was sustained throughout the experiment. Co-treatment with NEP-I and cANF-(4-23) resulted in a further significant delay in the disappearance of ANF from plasma Table 1. Effect of NEP-I, cANF-(4-23), or the combination on pharmacokinetics of 1251-ANF-(99-126) and 1251-urodilatin v 899

ml/100 g body wt

Hormone

MCR, ml - min-l 100 g body wt-l l

in rats t%?

min

ANF-(1-28)

Control NEP-I cANF NEP-I

Urodilatin Control NEP-I cANF

+ cANF

1320.8 1520.4 8.4t0.5” 7.9t0.2”

8.2tl.l 2.9t0.4” l.oko.l* 1.0~0.04”

0.55t0.03 0.72kO.06 1.3t0.05” 1.2t0.08*

28t5.8 17k3.2 7.4*0.5* 7.0*1.1*

5.9t1.2 7.8+1.2? 1.8+0.1*$ l.OkO.3”

0.73kO.16 0.43t0.08 0.94+0.06t 1.20k0.15

NEP-I + cANF Values expressed as means t SE; 5 rats were included in each group. V,,, volume of distribution at steady state; MCR, metabolic clearance rate; distribution tl/,, plasma half-life of 1251-atrial natriuretic factor (ANF) or 1251-urodilatin after administration of either peptide to normal rats treated with cANF-(4 - 23), neutral endopeptidase inhibitor (NEP-I), or the combination of both drugs. Parameters were calculated by analysis of a two-compartment model (see MATERIALS AND METHODS). * P < 0.05 vs. control rats. t P c 0.05 vs. 1251-ANF-treated rats.

0

1

0

I

10

-

NEP-I

-

cANF

__(I__

NEP-I

1

I

+ cANF I

20

I

30

.

1

40

Time (min) Fig. 2. Appearance of TCA-soluble radioactivity, i.e., hydrolytic products, in plasma after a bolus injection of 1251-ANF-(99- 126) alone, with either NEP-I or cANF-(4-23), or a combination of both agents. Data are from same experiments shown in Fig. 1. Soluble radioactivity is expressed as percentage of total radioactivity in each plasma sample. Values are means t SE; n = 5 rats in each group. Prior treatment of rats with NEP-I alone or in combination with cANF-(4- 23) significantly (P < 0.007 by ANOVA) suppressed appearance of TCA-soluble radioactivity in plasma.

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PHARMACOKINETICS

q

Urine

OF URODILATIN

NEP-I + cANF4-23

KIDNEY

LUNG

HEART

LIVER

Fig. 3. Percentage of total radioactivity injected that was recovered in urine, kidneys, lungs, heart, and liver from rats infused with 1251-ANF(99-1126) alone, with either NEP-I or cANF-(4-23), or a combination of both agents. * Significant difference (P 5 0.05 by unpaired t test) between control and other experimental groups. t Significant difference between rats pretreated with NEP-I alone and cANF-(4- 23) -treated rats.

The radioactivity recovered in the lung, heart, and liver was not altered by NEP-I, whereas the radioactivity recovered in the kidney increased fivefold over the control values (Fig. 3). Infusion of cANF-(4-23) decreased the

ES73

AND ANF

amount of radioactivity recovered in the kidney, lung, and liver, while it increased the radioactivity in the urine. Pharmacokinetics of 1251-urodiZatin. In all groups, there was a rapid fall in the amount of intact urodilatin, i.e., TCA-ppt radioactivity, in plasma in the first 6-10 min, followed by a plateau throughout the rest of the experiment (Fig. 4). NEP-I was without a significant effect on the amount of intact peptide in the plasma. During the infusion of cANF-(4-23), the amount of intact peptide was significantly higher (P < 0.001) in the first 10 min than in control animals. The combination of NEP-I and cANF-(4-23) resulted in a further delay in the disappearance of intact peptide compared with the effect of either NEP-I or cANF-(4-23) alone. Unlike its effect on the MCR of ANF, NEP-I had no significant effect on the MCR of urodilatin, compared with the control values. The presence of cANF-(4-23) significantly (P 5 0.05) decreased the V,, and increased the distribution tl12 of urodilatin in a similar manner to that observed with ANF. In the first few minutes after the infusion of urodilatin, there was a gradual increase of hydrolytic products in plasma, followed by a more rapid increase in the last 20 min of the experiment (Fig. 5). NEP-I caused a small, but not significant, shift in the curve to the right, whereas cANF- (4 - 23) decreased the appearance of hydrolytic products throughout the experiment. The combination of . -

Control NEP-I cANF NEP-I

+ cANF

100000 n z z Q -0 .-s % ‘5 2 3

I

Control

10000

-

NEP-I

-

cANF

-

NEP-I + cANF

‘; Q a 2

1000

E : E 100 0

I

I

I

10

20

30

Time (min) Fig. 4. Disappearance of TCA-ppt radioactivity, i.e., intact urodilatin, from plasma in rats after a single dose of 1251-urodilatin alone, with either NEP-I or cANF-(4-23), or a combination of both agents. Radioactivity in intact peptide is expressed as cpm in 1 ml plasma. Infusion of cANF-(4-23) alone or with NEP-I significantly (P C 0.001 by ANOVA) delayed disappearance of 1251-urodilatin from plasma compared with control.

10

20

30

40

Time (min) Fig. 5. Appearance of TCA-soluble radioactivity, i.e., hydrolytic products, in plasma after administration of 1251-urodilatin. Data are from same experiments shown in Fig. 4. Soluble radioactivity is expressed as percentage of total radioactivity in each plasma sample. NEP-I treatment did not differ significantly from control group, whereas cANF(4-23) treatment alone or in combination with NEP-I differed significantly (P < 0.0001 by ANOVA) from control.

Downloaded from www.physiology.org/journal/ajpendo at Midwestern Univ Lib (132.174.254.157) on February 12, 2019.

PHARMACOKINETICS

n

Control

q

NEP-I

OF

0 URINE

KIDNEY

LUNG

HEART

LIVER

Fig. 6. Percentage of total radioactivity injected that was recovered in urine, kidneys, lungs, heart, and liver from rats infused with 1251-urodilatin alone, with either NEP-I or cANF-(4-23), or a combination of both agents. * Significant difference (P 5 0.05 by unpaired t test) between control and other experimental groups. t Significant difference between rats pretreated with NEP-I alone and cANF-(4- 23)-treated rats.

NEP-I and cANF-(4-23) caused a further decrease in the appearance of hydrolytic products compared with either inhibitor alone. The nature of the radioactivity recovered in plasma was determined by HPLC 0.5, 3, and 10 min after the administration of 1251-urodilatin. In the plasma from control rats, the bulk of total radioactivity at 0.5 min coeluted with intact urodilatin, with a minor peak corresponding to free iodine. At 3 min, the proportion of radioactivity in the peak of intact peptide decreased significantly. At 10 min, most of the radioactivity co-eluted with free iodine. Treatment with NEP-I had no effect on the HPLC profiles of plasma samples compared with control rats. Infusion of cANF-(4-23) alone or combined with NEP-I increased the total eluted radioactivity (N5fold over the control values); however, the HPLC profiles were similar to those in the other groups of rats. HPLC analysis of the accumulated radioactivity in the kidney of control rats and NEP-I-pretreated rats revealed that intact 1251-urodilatin accounted for >90% of the total recovered radioactivity in the kidneys of both groups. On average, 2% of the total injected radioactivity was excreted into the urine (Fig. 6). This proportion was not affected by NEP-I; however, it was increased significantly with either cANF-(4-23) alone or combined with NEP-I. Five percent and 1.5% of the infused radioactivity were recovered in the kidneys and lungs, respectively. These values were not changed significantly by NEP-I, whereas they decreased significantly with cANF-(4 - 23). DISCUSSION

The present study demonstrates that both ANF and urodilatin are removed rapidly from the circulation, due mainly to the action of cANF receptors. Under normal conditions, there were no major differences between the

URODILATIN

AND ANF

distribution tli2, MCR, and V,, of ANF and urodilatin. Furthermore, the total radioactivity recovered in kidneys, lungs, and urine was similar in control rats injected with equal amounts of either 1251-ANF or 1251-urodilatin. In the presence of NEP-I, the amount of 1251-ANF recovered in the kidneys was increased, and its clearance from plasma was reduced, whereas the inhibitor was without effect on these same parameters for 1251-urodilatin. In agreement with Luft et al. (18) and Murthy et al. (21)) the distribution tl/2 of ANF was found to be very short and was not significantly different from that of urodilatin. The analogy of urodilatin with ANF (29) suggests that both peptides share the same receptors. Valentin et al. (35) reported that urodilatin is as effective as ANF in displacing 1251-ANF bound to renal tissue and in stimulating cGMP generation in target cells. Moreover, Saxenhofer and Paul (26) have recently reported that urodilatin and ANF bind to both ANF receptor subtypes (B-ANF and C-ANF receptors) with similar affinities. Our finding that infusion of excess cANF-(4- 23) caused significant decreases in both the MCR and V,, of administered urodilatin and ANF, and increased their retention in plasma, supports the former reports and indicates that cANF receptors are mainly responsible for the short distribution tl,2 of these peptides. The distribution tl12, MCR, and V,, for ANF in our study are much lower than those reported by Almeida et al. (2) and Chiu et al. (5), i.e., distribution tl12 of 1.33 min, MCR of 50-60 ml= min-l 100 g body wt-‘, and V,, of 97-119 ml/100 g body wt. The main reason for these differences is that different methods of data analysis were used. While they used noncompartmental analysis to calculate the pharmacokinetic parameters, we used two-compartmental analysis. Our preference for the latter model was dictated by the pattern of decay of TCAppt 1251 in plasma, which consisted of both a fast component and a slow component, in all experimental groups. Our data were obtained by fitting the decay of plasma TCA-ppt 1251radioactivity into the equation of a biexponential curve via a computer program to overcome the slight inconsistencies in the last portion of the curve. When Almeida et al. (2) described the disappearance of 1251-ANF from plasma by a biexponential curve, the calculated pharmacokinetic parameters were similar to those obtained in the current study. Despite the differences between the absolute values obtained in this study and those of other studies (2,5), the trend was very similar. In all studies, infusion of cANF-(4-23) decreased significantly the MCR and the V,, and increased the distribution t 1/2 of ANF. Moreover, cANF-(4-23) caused a significant reduction in the appearance of degradation products in plasma, in combination with a marked increase in the plasma levels of 1251-ANF. The current study importantly extends earlier studies (2,5) regarding the involvement of cANF receptors in the metabolism of ANF. Besides studying the effect of cANF-(4-23) on the plasma levels of 1251-ANF, we also measured its influence on the amount of radioactivity recovered in various target organs, as well as in urine. In addition to increasing the decreased plasma levels of 1251-ANF cANF-(4-23) the amount of radioactivity accumulated in the kidneys l

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PHARMACOKINETICS

OF URODILATIN

and in the lungs and increased the amount of radioactivity in urine. These findings support the well-established belief that the kidneys and the lungs play major roles in the clearance of ANF, due to the high concentration of cANF receptors in these organs (13,16,20). The similarity between ANF and urodilatin in this regard (see Figs. 3 and 6) indicates that urodilatin is also removed from the circulation in the same tissues and by similar mechanisms, i.e., binding to cANF receptors. Previous studies have reported that ANF is also inactivated, both in vivo and in vitro, via NEP (EN 3.4.24.11; see Refs. 23, 25, 32, 33). In the present study, pretreatment of the rats with SQ-28,603, a specific inhibitor of NEP, was without effect on the rate of disappearance of 1251-ANF from plasma, despite its inhibitory effect on the rate of appearance of the hydrolytic products of the peptide. Further, NEP-I significantly decreased the MCR of 1251-ANF but did not change significantly its V,, or distribution t 1/2. Similar findings have been reported by Chiu et al. (5), who used the same experimental model. In a study by Sybertz et al. (34), inhibition of NEP potentiated the renal action of ANF and decreased its removal from plasma. Taken together with the current study, one may conclude that NEP contributes significantly to the clearance of ANF from the circulation. In our experiments, prior treatment of the rats with NEP-I increased the radioactivity accumulated in the kidney. This increase indicates that 1251-ANF was protected from degradation rather than from a change in the delivery of the hormone to the kidney. Previous studies showed that NEP-I was without effect on glomerular filtration rate or renal blood flow (33). Furthermore, the present study showed that NEP-I had no effect on the plasma levels of 1251-ANF. The absence of hemodynamic changes in response to the inhibitor, in combination with the lack of effect on 1251-ANF plasma levels, supports this conclusion. In addition, the stimulatory effect of NEP-I on the amount of 1251-ANF accumulated in the kidneys coincided with the large amount of NEP activity in the brush border of the proximal tubule of the rat (25). Despite the fact that ANF and urodilatin have very similar structures, it seems that NEP is not involved in the degradation of urodilatin. Although NEP-I decreased the MCR of 1251-ANF by 65%, it was without significant effect on the MCR of urodilatin or on the rate of its hydrolysis. Furthermore, NEP-I, in contrast to its increase in the recovery of 1251-ANF in the kidney, was without effect on the renal uptake of 1251-urodilatin. In addition, NEP-I significantly suppressed the appearance of hydrolytic products of 1251-ANF in blood but not of 1251-urodilatin. The slight, but not significant, suppressive effect of NEP-I on the appearance of hydrolytic products of 1251-urodilatin suggests that urodilatin is not totally protected from NEP inactivation. These findings provide indirect evidence that urodilatin is not destroyed by NEP or at least is not a good substrate for NEP. Our “in vivo” findings confirm, for the first time, the “in vitro” findings of Gagelmann et al. (9), i.e., that urodilatin is resistant to renal degradation. Interestingly, several studies (1,24,27,36) indicate that urodilatin is natriuretic in lower doses than ANF. Although the exact mechanisms

AND ANF

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responsible for these differences between ANF and urodilatin at the renal level remain unclear, these studies suggest that urodilatin escapes inactivation by NEP in the brush border and that more urodilatin reaches the distal sites in the nephron and induces more sodium excretion. The present study supports this explanation; however, other mechanisms such as differences in the hemodynamic responses or in the interaction with other hormonal systems cannot be excluded. Moreover, the comparable levels of ANF and urodilatin in the kidneys of the control rats suggest that a higher potency, rather than higher concentration, accounts for the more potent diuretic activity of urodilatin compared with ANF. Almeida et al. (2) noted that an infusion of cANF caused a significant increase in the initial appearance of TCA-soluble radioactivity in plasma, most likely as a consequence of the higher plasma levels of 1251-ANF obtained under these conditions. In the present study, cANF induced similar alterations in the rate of 1251-ANF disappearance; however, such changes were not evident with urodilatin. The exact reason for this difference is unknown; however, one may speculate that it is due to slight differences in V,,, MCR, and distribution tl12 between the peptides. Furthermore, Chiu et al. (5) reported that cANF was without effect on the initial appearance of TCA-soluble radioactivity, despite use of a similar experimental model. In any event, it seems that this initial portion of the curve represents a negligible proportion of the total hydrolysis of the labeled peptides. In all of the experimental groups, HPLC analysis of the radioactivity in plasma revealed that the relative proportions of TCA-ppt and of TCA-soluble radioactivity corresponded to the percentages of radioactivity that co-eluted with 1251-ANF/1251-urodilatin and free 1251, respectively. It is likely that the main source of the free 1251 is the hydrolysis of the 1251-ANF/1251-urodilatin-cANF receptor complex by lysosomes and the subsequent efflux of free 1251 and other hydrolytic products from the cell into the circulation (2,5). These hydrolytic products contribute to the radioactivity recovered in the urine. In summary, the present study demonstrates that both ANF and urodilatin are removed from the circulation mainly by cANF receptors. In the case of ANF, an additional pathway contributes to its clearance, and this is the enzyme NEP. Our finding, in vivo, that urodilatin is resistant to this enzymatic degradation supports previous in vitro findings and provides a unique explanation for urodilatin’s greater natriuretic potency. We are grateful to E. R. Squibb for the gift of SQ-28,603 Address for reprint requests: Z. A. Abassi, Hypertension-Endocrine Branch, National Institutes of Health, Bldg. 10, Rm. 8C103,9000 Rockville Pike, Bethesda, MD 20892. Received 14 February 1992; accepted in final form 22 June 1992 REFERENCES 1. Abassi, 2. A., J. Powell, E. Golomb, and H. R. Keiser. Renal and systemic effects of urodilatin in experimental high-output heart failure. Am. J. Physiol. 262 (Renal Fluid Electrolyte Physiol. 31): F615F621, 1992. 2. Almeida, F. A., M. Suzuki, R. M. Scarborough, J. H. Lewicki, and T. Maack. Clearance function of type C receptors of atria1 natriuretic factor in rats. Am. J. Physiol. 256 (Regulatory Integrative Camp. Physiol. 25): R469-R475, 1989.

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