Introduction

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The atrial natriuretic peptide hormonal system consists of a 126-a.a. prohormone synthesized within myocytes of the heart and stored in storage granules within the heart for release into the circulation (Vesely, 1992, 1995). This hormonal system contains several peptides from the same 126-a.a. prohormone with blood pressure lowering, natriuretic, diuretic and/or kaliuretic (i.e., potassium-excreting) properties (Martin et al., 1990; Vesely et al., 1994a, 1994b) (fig. 1). Thus, peptides consisting of a.a. 1 to 30 (i.e., LANP), 31 to 67 (vessel dilator), 79 to 98 (kaliuretic peptide) and 99 to 126 (ANF) each have blood pressure-lowering, diuretic, natriuretic and/or kaliuretic properties in both humans (Vesely et al., 1994a, 1994b) and animals (Martin et al., 1990; Vesely et al., 1987b). When released into the circulation, these peptides circulate as a 98-a.a. amino terminus and a 28-a.a. carboxyl terminus (i.e., ANF) of this prohormone (Meleagros et al., 1988; Sundsfjord et al., 1988; Winters et al., 1988a, 1988b). In addition, vessel dilator and LANP circulate as distinct entities after having been proteolytically cleaved from the rest of the amino terminus by proteases (Gower et al., 1994; Vesely et al., 1993, 1994b) (fig. 1).

View larger version (35K): [in this window] [in a new window]   Fig. 1.   Origination of LANP, vessel dilator, kaliuretic peptide and ANF from the ANF prohormone. LANP consisting of amino acids (a.a.) 1 to 30, vessel dilator composed of a.a. 31 to 67 and kaliuretic peptide composed of a.a. 79 to 98 originate from the amino terminus of the ANF prohormone, whereas ANF consisting of a.a. 99 to 126 originates from the carboxyl terminus of this 125-a.a. prohormone. Each of these peptide hormones circulate as distinct entities and each lowers blood pressure via vasodilation of blood vessels.

Vessel dilator, LANP and ANF bind to specific receptors (Vesely et al., 1990a, 1990b, 1992); then, each of these peptides enhances the particulate form of the enzyme guanylate cyclase (EC4.6.1.2) as part of their mechanism(s) of action (Vesely et al., 1987a, 1987b). The enhancement of guanylate cyclase by each of the respective atrial natriuretic peptides increases the intracellular messenger cGMP (Vesely et al., 1987a, 1987b; Waldman et al., 1984), which causes vasodilation itself. Vessel dilator and LANP also inhibit renal Na⁺, K⁺-ATPase as part of their mechanism(s) of action causing a natriuresis (Chiou and Vesely, 1995; Gunning et al., 1992). In contradistinction, ANF does not have any effect on renal Na⁺, K⁺-ATPase (Charlton and Baylis, 1990; Chiou and Vesely, 1995; Gunning et al., 1992; Pollock et al., 1983). Vessel dilator and LANP each enhance prostaglandin E₂ synthesis, which appears to be the mediator of the inhibition of Na⁺, K⁺-ATPase by these peptides (Chiou and Vesely, 1995; Gunning et al., 1992).

Vessel dilator, LANP and ANF are released simultaneously with central hypervolemia (Vesely et al., 1989) and with rapid heart rates of 125 beats/min (Ackerman et al., 1992; Ngo et al., 1989, 1990). These peptides are also released simultaneously in vitro from isolated perfused atria by atrial distension (Dietz et al., 1991). The half-life of ANF in the circulation of humans is well characterized with half-lives of 1.2 to 6.9 min (average, 2.5-3 min) being reported (Nakao et al., 1986; Tan et al., 1993; Yandle et al., 1986). The half-lives of vessel dilator and LANP have never been investigated in humans. The present investigation was designed to determine the half-lives, distribution, MC and TBC of vessel dilator and LANP after their exogenous administration at 100 ng/kg of b.wt./min for 1 hr to human subjects followed by a 3-hr postinfusion evaluation of their circulating concentrations.

	Methods

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Materials. The human forms of LANP, vessel dilator and ANF were synthesized by Peninsula Laboratories (Belmont, CA). Before their use in these studies, samples of these commercially synthesized peptides were subjected to HPLC to determine their purity using a Novapak C18 (5-µm) cartridge column (Waters Chromatography Division, Millipore Corporation, Milford, MA). The flow rate for the HPLC study was 1 ml/min with 0.1% trifluoroacetate solvent in pump A and 60% acetonitrile in 0.1% trifluoroacetate in pump B, with a gradient of 0% to 60% acetonitrile achieved in 40 min. This evaluation verified their purity and authenticity compared with the known HPLC elution profile of these peptides (Winters et al., 1989). After determining that the respective peptides were pure, the peptides were dissolved in 0.9% saline solution in the hospital pharmacy, where pyrogen and sterility testing was performed before dispensing the 100 ng/kg b.wt. concentrations of each peptide into two 10-ml syringes. Each 10-ml syringe infused all of its contents over a 30-min time period. After completing the experiment, each of the syringes and the infusion catheter were examined by the RIAs described below to determine the amount of the respective peptides that may have remained within the syringes or tubing. Approximately 5% of each peptide remained on the walls of the syringes and tubing after completion of the infusion. This was determined after completion of the experiment by flushing the syringes three times with 4 ml of 0.9% saline and then measuring by the respective RIAs the amount of each peptide present in the 0.9% saline flush. The amount measured was then compared with the amount infused to determine the percentage that had remained on the walls of the syringes and tubing.

Experimental subjects. Ten healthy subjects (six men and four women; ages, 20-54 years; average, 33 years) were investigated. These subjects had heart rates ranging from 66 to 80 beats/min, with respiration between 12 and 14/min. Each of the volunteers were normotensive with blood pressures of <125/80 mm Hg. These volunteers were divided into two similar groups based on age, blood pressure, heart rate and gender (equal number of men and women in each group) (table 1). For comparison of half-lives and MC, another group of six subjects (average age, 34 years) with blood pressure of <125/80 mm Hg had ANF infused at the same concentration (100 ng/kg/min) as the more recently discovered atrial natriuretic peptides.

Experimental protocol. After obtaining written informed consent from each participant, an Insyte-w 20-gauge 1.5-in catheter was placed into one forearm of each subject for infusion, and a second Insyte-w 20-gauge 1.5-inch catheter was placed in the opposite forearm for blood sampling. A 60-min base-line period preceded each infusion. Each of these peptides was infused by a constant rate infusion pump over a 60-min period. Blood samples were obtained every 20 min during the infusion and at 30-min intervals during the 1-hr preinfusion and 3-hr postinfusion periods. A rate of 100 ng/kg b.wt./min was chosen for infusion of these atrial natriuretic peptides because the rate of release of the amino-terminal ANF prohormone peptides from the atrium of the heart with physiological stimuli is 138 to 292 ng/kg b.wt./min (Ackerman et al., 1992). All subjects were studied in the morning after an overnight fast, beginning their base-line period at 8:00 a.m. Each volunteer was studied in the seated position and received only one peptide infusion. Molar equivalents of the 100 ng/kg b.wt. dose were 29 and 26 pmol/l/kg b.wt. for LANP and vessel dilator, respectively.

Measurement of LANP, vessel dilator and ANF. Each of the blood samples and the results of flushing the syringes and tubing with 4 ml of 0.9% sodium chloride were collected into chilled 5-ml EDTA tubes to prevent proteolytic breakdown of any peptides that might be present. These samples were transported on ice and immediately centrifuged at 3000 × g for 15 min. After centrifugation, each sample was extracted with 100% ethanol (1:2 dilution), vortexed and allowed to stand at 4°C for 30 min (Vesely et al., 1994b; Winters et al., 1989). We have previously described in detail RIAs to measure LANP, vessel dilator, and ANF (Vesely et al., 1994a; Winters et al., 1989). For each RIA, the extracted plasma was first reconstituted in 100 µl of 0.1 M phosphate buffer, pH 7.4, containing 0.05 M NaCl, 0.1% BSA, 0.1% Triton X-100 and 0.01% NaN₃. To the redissolved sample, 100 µl (0.03 mg) of rabbit immunoglobulin G (second antibody) plus 100 µl of LANP, vessel dilator or ANF antisera was added and incubated for 24 hr. Then, 100 µl of ¹²⁵I-labeled atrial peptides (10,000 cpm) were added, mixed and incubated for 18 hr at 4°C. Precipitation of the antibody-bound tracer was accomplished by adding 100 µl of goat anti-rabbit globulin after the above-described 18-hr period and incubating this mixture for 2 hr at room temperature. Each tube was then centrifuged at 3000 × g for 20 min. The supernatant was aspirated, and the pellet was counted in a -counter. All determinations were performed in triplicate.

Statistical analysis. Determination that individuals in the two different groups were equal with respect to age, weight, height, gender, blood pressure, base-line peptide levels, MC rates, half-lives and AUC by noncompartmental analyses were compared using an unpaired t test (EPISTAT, Round Rock, TX). The plasma concentration-time data of vessel dilator, LANP and ANF were analyzed by the computerized least-squares, nonlinearized regression program JANA (Statistical Consultants, Lexington, KY). Compartmental analysis was performed using PCNONLIN (Statistical Consultants) and AUMC.BAS (Ron Kluza, University of Arkansas, Little Rock, AR). In addition, the MC rate of the vessel dilator, LANP and ANF were determined as follows: This MC is equivalent to a central compartment (i.e., systemic circulation) TBC. To confirm that the infusions of LANP vessel dilator, and ANF were actually infusing the correct amount of the respective peptides, respective concentrations of each infusate were determined by RIA measurement from samples collected throughout the infusion. The plasma half-lives of these peptides were determined by nonlinear regression using PCNONLIN. The pharmacokinetic analysis of these peptides assumes that there is no change in the endogenous production of these peptides during the period of study. MRT was calculated using PCNONLIN and AUMC computer programs, which use deriviates of the AUC and AUMC in an equation to do so.

	Results

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Mean blood pressure, heart rate, age, weight, height and BSA of the human volunteers receiving LANP or vessel dilator are delineated in table 1. Base-line and peak values of the respective peptides and compartmental disposition parameters after the infusion of vessel dilator or LANP are enumerated in table 2. The measured cumulative infused doses of LANP and vessel dilator are also detailed in table 2. The concentration-time profiles of each of these peptides during and after the infusions are illustrated in figure 2.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   The concentration-time curves for LANP (), vessel dilator () and ANF (). LANP and vessel dilator remain elevated above base line significantly (P < .001) longer than ANF when evaluated by the unpaired t test.

The half-life of vessel dilator was 107 ± 62 min (mean ± S.D.) (table 3). The half-life of LANP was 28 ± 11 min. The half-life for LANPs was decreased compared with that of vessel dilator at least partially because of the large volume of distribution of subjects 2 and 4, who received LANP. Comparison of the half-lives of LANP, vessel dilator and ANF of all subjects are illustrated in figure 3. In this figure, half-lives of vessel dilator and LANP are significantly longer than the half-life of ANF.

View larger version (64K): [in this window] [in a new window]   Fig. 3.   Comparison of the half-lives and MC of ANF, LANP and vessel dilator in the circulation of healthy humans. The half-lives of LANP and vessel dilator were significantly (P < .01) longer, whereas their MC values were significantly lower (p < .0) than those of ANF when evaluated by the unpaired t test (EPISTAT) (n = 6 for ANF and vessel dilator, 4 for LANP).

The -distribution phase rate constants that reflect the movement of peptides into the circulation and their binding to tissues were then determined. The -distribution phase rate constant (i.e., rate at which the concentration of a hormone or substance decreases in the circulation) for vessel dilator was 0.047 ± 0.014 min¹ (i.e., these reciprocal minutes are the rate at which its concentration decreases per minute), whereas the -distribution rate constant for LANP was 0.108 ± 0.082 min¹, as outlined in table 2. The S.D. of the -distribution rate constant phase was 0.014 min¹ for vessel dilator, whereas the S.D. for the -phase rate constant of LANP was 0.082 min¹ due to the very large -phase distribution rate constant of subject 4 receiving LANP. If this subject was deleted from the calculation of the mean -phase distribution rate constant of LANP, the average -distribution of this peptide in the other subjects was 0.070 ± 0.03 min¹. Furthermore, if subject 2 as well as subject 4 was removed from the calculation, the average -phase distribution rate constant for the other subjects for LANP was 0.048 ± 0.005 min¹, a value very similar to the -phase distribution rate constant for vessel dilator. The -distribution phase rate constant reflects the distribution of these peptides within tissues. The -distribution phase rate constant for vessel dilator was 0.008 min¹ with a S.D. of 0.004 min¹ (table 2). The -distribution phase rate constant for LANP was 0.027 min¹ with a S.D. of 0.010 min¹ (table 2). The -distribution phase is an observed slowing of the clearance of these peptides as a result of their broader distribution in tissues.

Noncompartmental analysis of the infusion of vessel dilator and LANP revealed that the average time for the respective peptides to be retained by the body (i.e., MRT) was 214 ± 34 min for vessel dilator and 178 ± 6 min for LANP (table 3). The MRT reflects the sustained presence of vessel dilator and LANP above base line. MRT is identical to the turnover for an endogenous substance and reflects when 63.2% of an administered dose has been eliminated. The TBC (in ml/min) normalized to 1.73 m² BSA averaged 130 ± 26 ml/min for vessel dilator and 293 ± 82 ml/min for LANP (table 3). The MC rate also normalized to 1.73 m² BSA was 241 ± 51 ml/min for vessel dilator and 249 ± 62 ml/min for LANP (table 3). The MC was significantly less (P < .01) for vessel dilator and LANP compared with ANF (fig. 3).

Comparison of the MC with the TBC revealed a significant difference for vessel dilator (P < .05), whereas this same evaluation revealed no significant difference for LANP (P = .42). The volume of distribution during the clearance of these peptides (V_dss) in liters/kg was similar for vessel dilator (0.098 ± 0.02) and LANP (0.123 ± 0.039) (table 3). The V_dss is the steady-state distribution volume and it differs from the -distribution volume in that it is independent of the clearance of a respective peptide. Comparisons of AUCs by both compartmental and noncompartmental analysis using the unpaired t test demonstrated no difference between vessel dilator and LANP. The AUC for proANF 31-67 was 64,913 ± 14,660 ng/liter/min (compartmental) and 68,642 ± 7092 ng/liter/min (noncompartmental), whereas the AUC for proANF 1-30 was 70,251 ± 9123 ng/liter/min (compartmental) and 64,885 ± 12,484 ng/liter/min (noncompartmental).

	Discussion

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The present investigation revealed that the distribution volumes for vessel dilator and LANP are both slightly larger than the plasma volume. This is what one would expect if a peptide hormone is not subject to reuptake and recirculation. The present data, thus, suggest that vessel dilator and LANP are not taken up by tissues and then rereleased into the circulation.

The half-lives of vessel dilator and LANP determined in the present investigation were both significantly longer than that determined previously for ANF in humans (Nakao et al., 1986; Tan et al., 1993; Yandle et al., 1986). Their longer half-lives and slower metabolic clearance (ANF MC rate; 582 ± 53 ml/min/1.73 m² measured by us and 740-2,581 ml/min/m² reported by Iervasi et al., 1993) is the probable reason that vessel dilator and LANP circulate in healthy humans at 15- to 24-fold higher concentrations than ANF, a peptide hormone derived from the same prohormone from which vessel dilator and LANP originate (fig. 1).

The longer half-lives of vessel dilator and LANP may account for their significantly (p < .001) prolonged natriuretic and diuretic effects in humans compared with ANF (Vesely et al., 1994b). The MRTs for vessel dilator and LANP to be eliminated from the body were 214 ± 34 and 178 ± 12 min, respectively. These values indicate that there is a considerable persistence of these two peptide hormones after their respective infusions into healthy humans and that they are widely distributed outside their initial space (i.e., circulation). The average postdistribution phase half-life (i.e., when the plasma concentrations fell by 50% in the postdistribution phase) of vessel dilator was 107 min. The total postdistribution phase of (214 min) is nearly identical to the MRT (214 ± 34 min) for vessel dilator and indicates that the V_dB is very close to the volume of distribution of the central compartment (VC) or the plasma volume.

LANP had a much longer TBC than did vessel dilator with no marked difference in V_dB. A t test comparison of MRC with TBC for LANP with both corrected to 1.73 m² BSA resulted in a t value of 0.8546, indicating no significant difference between the two methods for determining clearance. The present exogenously administered pharmacological peptide investigation would appear to have physiological significance in that endogenous LANP, vessel dilator and ANF when increased by pacing the heart of dogs, have a very similar temporal pharmacodynamic effect as the exogenously administered peptides (Ackerman et al., 1992). It is important to note that the administration of the peptides in the present investigation caused significant (p < .001) natriuresis and diuresis in the healthy subjects (Vesely et al., 1994b).

MC and TBC are measures of the disappearance of the respective peptides from the circulation. An error often incorporated into the one-compartment model is the difference in the dose and the actual amount of the peptide hormone (or drug) in the body at the end of the infusion, which is the value of the numerator (Ackerman et al., 1990). This error can be eliminated by actually measuring the concentration of the respective peptides at the end of its infusion, as was done in the present investigation.

In summary, this investigation reveals that vessel dilator and LANP hormones have half-lives of 107 and 28 min respectively. The MC rates of 241 ± 51 and 249 ± 62 ml/min/1.73 m² BSA for vessel dilator and LANP, respectively, were similar and are thus cleared much slower than ANF (582 ± 53 ml/min/1.73 m² BSA). The significantly (P < .001) longer half-lives and slower clearance rates of LANP and vessel dilator compared with ANF explain why these peptides circulate at concentrations 15- to 24-fold higher than ANF in healthy humans.