![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Vol. 291, Issue 3, 982-987, December 1999
Commissariat à l'Energie Atomique,
 |
Abstract |
|---|
 Top
 Abstract
 Introduction
Experimental Procedures
Results
Discussion
References
|
|---|
The hemoregulatory peptide Acetyl-Ser-Asp-Lys-Pro (AcSDKP) has been reported to accumulate in plasma and urine after the oral administration of angiotensin-converting enzyme (ACE) inhibitors in humans. It is unknown whether such an accumulation also occurs in tissues. We administered captopril (3, 10, or 30 mg/kg) orally for 2 weeks to Wistar rats. In a second experiment, captopril (10 mg/kg) was administered for 9 days and was followed by a 1-h i.v. infusion of either AcSDKP (0.1 or 2 mg/kg) or saline on day 9. Captopril alone dose-dependently increased plasma AcSDKP by a factor of 3 to 5 and urine AcSDKP by a factor of 3. It slightly increased renal and pulmonary AcSDKP concentrations but did not affect AcSDKP concentrations in bone marrow and spleen. The combination of AcSDKP (2 mg/kg) and captopril gave very high AcSDKP concentrations in plasma and urine and increases in AcSDKP concentration by factors of 27 in kidney, 5.5 in lung, and 6.9 in the extracellular fraction of bone marrow. In contrast, no change was observed in the AcSDKP concentration in spleen and in the intracellular fraction of bone marrow. In conclusion, during chronic ACE inhibition in rats, AcSDKP levels slightly increased in organs with high ACE contents. No such increase occurred in hematopoietic organs. AcSDKP had to be combined with captopril to significantly increase its concentration in tissues other than the spleen. The possibility of pharmacologically increasing AcSDKP levels in the extracellular fraction of bone marrow may be of value for protecting hematopoietic cells from the toxicity of cancer chemotherapy.
| |
Introduction |
|---|
|
Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
|
|---|
The
hematopoietic tetrapeptide Acetyl-Ser-Asp-Lys-Pro (AcSDKP), originally
isolated from fetal calf bone marrow (Lenfant et al., 1989
), is an
endogenous hematopoiesis regulatory factor that reversibly prevents the
entry of pluripotent hematopoietic stem cells and normal early
progenitors into the S phase of the cell cycle, in vitro and in vivo,
by keeping them in the G0 phase (Lenfant et al.,
1989; Robinson et al., 1992). AcSDKP directly and reversibly inhibits
the growth of the human CD34+ cell subpopulation
in response to growth factors (Bonnet et al., 1993).
In vivo, AcSDKP increases survival in mice treated with lethal doses of
cytosine arabinoside (Bogden et al., 1991), doxorubicin (Masse et al.,
1998), or sublethal irradiation (Watanabe et al., 1996). In humans,
AcSDKP may be useful for protecting normal human hematopoietic stem
cells against damage due to cytotoxic therapy (Carde et al., 1992).
AcSDKP, whose precursor could be thymosin 
4,
is normally present in human plasma (Pradelles et al., 1990; Liozon et
al., 1993) and in circulating mononuclear cells (Pradelles et al., 1990), and it is ubiquitously distributed in vivo (Pradelles et al.,
1991). It may be involved in the proliferation of other cell types such
as hepatocytes (Lombard et al., 1990), renal fibroblasts (Yoshioka et
al., 1998), and cardiac fibroblasts (Rhaleb et al., 1998).
AcSDKP is cleared from plasma by two mechanisms, angiotensin-converting
enzyme (ACE)-mediated hydrolysis and glomerular filtration (Azizi et
al., 1999). ACE is a zinc metallopeptidase that displays activity
toward a broad range of substrates, at least in vitro (Erdös,
1990) and has two homologous N- and C-terminal active domains (Wei et
al., 1992). AcSDKP is preferentially hydrolyzed by the N-terminal
active site of ACE, whereas angiotensin I is cleaved with the same
efficiency by the two domains (Rieger et al., 1993; Rousseau et al.,
1995). AcSDKP hydrolysis is blocked by ACE inhibitors (ACEIs) in vitro
(Rieger et al., 1993) and in vivo (Azizi et al., 1996). ACEIs given as
a single dose to normal subjects or during long-term treatment in
hypertensive patients result in plasma AcSDKP levels five to six times
higher and urine concentrations 40 times higher than those of control
subjects and/or patients (Azizi et al., 1997, 1999). In patients with
renal failure treated with an ACEI, AcSDKP accumulates to very high concentrations (up to 200 times normal levels) (Azizi et al., 1999). It
is unknown whether the accumulation of AcSDKP in plasma during ACE
inhibition, particularly if renal function is severely impaired, has
any long-term biological effect. Such an accumulation of the peptide
may be responsible for the hematological side effects induced by ACE
inhibitors, especially if renal function is impaired (Hirakata et al.,
1984). It has recently been shown that lisinopril prevents the entry
into the cell cycle of murine hematopoietic stem cells in vivo
following irradiation with 2 gray (Gy) (Rousseau-Plasse et al., 1998).
These effects may be due to an ACE inhibition-induced accumulation of
the peptide in tissues, in which the physiological concentration of the
peptide is 100 to 1000 times higher than that in plasma (Pradelles et
al., 1991). It is unclear whether AcSDKP accumulates in tissues if ACE
is inhibited in plasma and tissue.
The aim of this study was to investigate whether, during chronic ACE
inhibition and during the combined administration of the exogenous
peptide with an ACE inhibitor, AcSDKP accumulates not only in plasma
but also in hematopoietic and nonhematopoietic tissues in rats. The ACE
inhibitor captopril was used in this study because it has a high
affinity for the N-terminal active site of ACE (Wei et al., 1992;
Michaud et al., 1997) and because its pharmacokinetic and
pharmacodynamic properties, and especially its tissue distribution,
have been extensively described (Cohen and Kurz, 1982; Cushman
et al., 1989).
| |
Experimental Procedures |
|---|
|
Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
|
|---|
Animals
All experiments were performed on normotensive 8- to 12-week-old Wistar male rats, weighing 300 to 350 g (Iffa-Credo, France). Rats were maintained under a 12-h light 12-h dark cycle. They were fed with commercial rat chow, and tap water was available ad libitum. Rats were used after a 1-week acclimation period and were housed in groups of three to five animals. Captopril was administered via the drinking water. Water consumption was measured daily and body weight weekly. The volumes of the drug solution were adjusted to the drinking habits of individual animals to ensure that the correct dose was taken. Control rats received deionized water. All studies on animals complied with the Décret sur l'Expérimentation Animale (French regulations concerning animal experimentation, Decree 87-848, 19 October 1987).
Experiments
Three consecutive experiments were performed.
Experiment 1: Pilot Study. This study was carried out to find the doses of captopril required to obtain a dose-response curve for plasma AcSDKP levels and plasma renin concentration (PRC) and to find the duration of treatment required to achieve the maximum effect on these two parameters.
Three groups of five rats each were used. Two groups were treated with captopril (10 or 100 mg/kg daily) for 28 days and were compared with a control group. Blood samples were taken on days 14 and 28.Experiment 2: Dose-Response Curves for Effect of Captopril on Plasma, Urine, and Tissue Levels of AcSDKP. Four groups of 10 rats each were studied. Three groups were actively treated with captopril (3, 10, or 30 mg/kg) administered daily for 14 days and these groups were compared with a control group. Blood, urine, and tissue samples were taken on day 14.
For experiments 1 and 2, blood was taken from the jugular vein, under anesthesia with ketamine-xylazine i.p. (20 and 5 mg/kg b.wt., respectively). Urine samples were taken by direct bladder puncture.Experiment 3: Effects of Combined ACE Inhibition and Exogenous AcSDKP Infusion. Six groups of five rats each were investigated: a control group; two groups treated by a 1-h i.v. infusion of AcSDKP at 0.1 mg/kg for one of the groups and 2 mg/kg for the other; a group treated with 10 mg/kg/day of captopril for 9 days, which was then given a 1-h i.v. infusion of vehicle on day 9; and finally, 2 groups treated with 10 mg/kg/day captopril for 9 days, which then received a 1-h i.v. infusion of AcSDKP at either 0.1 or 2 mg/kg on day 9.
On day 9, after the last administration of captopril, the rats were anesthetized with inactin (10 mg/100 g b.wt.) and the right and left jugular veins were cannulated. A bladder catheter was implanted for the collection of the urine samples. At time zero (T0), an i.v. infusion of 0.1 or 2 mg/kg/h AcSDKP or saline in the left jugular vein was started. In all groups, furosemide (1 mg/kg/h) was infused for 1 h to induce constant diuresis for the collection of total urine samples. Blood samples were collected from the right jugular vein in heparinized tubes with ice-cold syringes to evaluate plasma AcSDKP levels. Plasma AcSDKP levels were measured at T0 and 5, 30, and 60 min (T5, T30, T60) after the start of AcSDKP or vehicle infusion. Two urine samples were collected (T0-T30 and T30-T60). Animals were sacrificed after the removal of blood samples. All samples were stored at
30°C before analysis.
Laboratory Methods
Tissue Sampling.
For experiments 2 and 3, spleen, kidneys,
and lungs were rapidly removed and immediately frozen in liquid
nitrogen. Sections of spleen, kidney, and lung (100 mg) were
homogenized (Polytron; Kinematica GmBH, Littau, Switzerland) in 2 M
acetic acid (pH 2.9). The homogenates were centrifuged at
20,000g for 30 min at 4°C. The supernatants were
diluted in 2 M acetic acid to obtain a 10 mg/ml tissue solution, which
was stored at 30°C until assay.
30°C. Bone marrow cells were washed, resuspended in
saline, and stored at 30°C until assay. For intracellular AcSDKP
determination, bone marrow cells were subjected to sonication for
20 s (Vibra Cell; Bioblock, Illkirch, France) after thawing.
Bone Marrow Protein Concentration.
Bone marrow protein
concentration was determined as described by the Bradford method
(Bradford, 1976).
AcSDKP Determination.
AcSDKP was determined in plasma,
urine, and tissue samples by a competitive enzyme immunoassay
(Pradelles et al., 1990) with a detection limit of 0.2 pmol/ml.
Briefly, plasma, urine, bone marrow cells, and tissue supernatants were
extracted with methanol. The resulting extracts were then centrifuged
at 4500 rpm for 15 min. The supernatants were collected and evaporated
to dryness. The precipitates were suspended in assay buffer and
quantified by enzyme-immunoassay.
|
).
PRC Determination.
PRC was determined by measuring the
production in vitro of angiotensin I in the presence of an excess of
angiotensinogen from plasma from binephrectomized rats (Menard and
Catt, 1972).
Statistical Analysis. The area under the curve was calculated by the trapezoidal method for plasma AcSDKP concentration (0-60 min). The data from each experiment were analyzed by one-way ANOVA. The assumptions of ANOVA (homogeneity of variance and normality) were checked for each variable, and natural logarithmic transformation was applied where appropriate. If the F test was significant (p < .05), paired comparisons were performed with the Scheffe test. Only pairwise comparisons with the control group are reported in experiment 3, for clarity and to avoid multiple testing.
Calculations were done with Statview 5.1 statistical software (Abacus Concepts, Inc., Berkeley, CA). Data are expressed as means ± 1 S.D. in the tables. A p <.05 was considered to be significant.| |
Results |
|---|
|
Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
|
|---|
Experiment 1: Pilot Study. For the groups treated with 10 or 100 mg/kg captopril, plasma AcSDKP levels were three to six times higher than those in the control group on both day 14 and day 28 (Table 1). The effect of the two doses of captopril on plasma AcSDKP levels was similar on days 14 and 28, with the maximal effect of captopril on plasma AcSDKP concentrations having already taken effect on day 14. PRC was higher in the captopril-treated groups than in the control group, and this increase was dose dependent (Table 1). Thus, experiment 2 was designed such that 3, 10, or 30 mg/kg captopril was administered over 14 days.
|
Experiment 2: Dose-Response Curves for Effects of Captopril on Plasma, Urine, and Tissue Levels of AcSDKP. In the control group, plasma and urine AcSDKP concentrations were 1.12 ± 0.29 and 28 ± 24 pmol/ml, respectively. AcSDKP concentrations in tissues were much higher than those in plasma, and the highest concentrations were measured in the spleen (Table 2).
|
Experiment 3: Effects of Combined ACE Inhibition and Exogenous
AcSDKP Infusion.
Plasma AcSDKP levels remained stable in the
control group (Fig. 1). As expected,
plasma AcSDKP concentration was three to five times higher in the group
treated with 10 mg/kg captopril than in the control group. AcSDKP
infusion alone dose-dependently increased plasma AcSDKP levels toward a
plateau (AcSDKP 0.1 mg/kg/h, 104 ± 44 pmol/ml at
T60 versus AcSDKP 2 mg/kg/h, 1608 ± 261 pmol/ml at T60) (Fig. 1). If rats were
treated with 10 mg/kg captopril for 9 days before exogenous AcSDKP
infusion, much higher plasma AcSDKP concentrations at
T60 [AcSDKP 0.1 mg/kg, 328 ± 43 pmol/ml versus AcSDKP 2 mg/kg, 6749 ± 1567 pmol/ml (Fig. 1)]
were achieved than if no prior treatment was given. There was a time
lag before the effect of ACE inhibition could be detected during AcSDKP
infusion because the plasma AcSDKP levels achieved at
T5 were similar to those measured if the
peptide was infused alone. In contrast to the single-infusion groups,
no plateau of AcSDKP concentration was reached if the peptide was
infused into rats in which ACE was inhibited. The combined
administration of 0.1 mg/kg AcSDKP with 10 mg/kg captopril resulted in
lower plasma levels of AcSDKP than did the single infusion of 2 mg/kg
AcSDKP. The pattern for urine AcSDKP levels was similar to that for
plasma levels (Table 3).
|
|
| |
Discussion |
|---|
|
Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
|
|---|
The aim of this study was to determine whether AcSDKP concentrations in potential target hematopoietic organs and nonhematopoietic organs could be changed by chronic administration of an ACE inhibitor, an exogenous AcSDKP infusion, or a combination of an ACE inhibitor with an exogenous AcSDKP infusion. We first determined the dose and duration of captopril treatment required to achieve a dose-response curve and a maximal effect on plasma AcSDKP levels (experiment 1). We then performed experiment 2, with captopril doses of 3 to 30 mg/kg/day for 14 days. Experiment 3 was then designed to increase the plasma concentration of AcSDKP above that of organ by administering AcSDKP at high doses to rats in which ACE was inhibited.
Effects on Plasma and Urine AcSDKP Concentrations.
AcSDKP was
present in rat hematopoietic and nonhematopoietic tissues at
concentrations much higher than those in plasma. The highest
concentrations were measured in hematopoietic tissues. These data are
consistent with previous results obtained in mice (Pradelles et al.,
1991). Consistent with previous studies in vitro (Rieger et al., 1993;
Rousseau et al., 1995), in rats (Junot et al., 1999), and in humans
(Azizi et al., 1996, 1997, 1999), our results confirmed that ACE also
was the main enzyme involved in AcSDKP metabolism in rats because both
plasma AcSDKP and urine AcSDKP levels increased if ACE was inhibited by captopril.
). In
a previous experiment, we investigated the pharmacodynamic effect of
increasing doses of captopril (0.01-10 mg/kg) during the 90 min after
i.v. administration to spontaneously hypertensive rats and found that
the ED50 of captopril for the inhibition of
AcSDKP hydrolysis (0.02 mg/kg) was 100 times lower than that of PRC (2 mg/kg) (Junot et al., 1999). ACE inhibitors displayed various potencies
in inhibiting the degradation of different natural or synthetic
substrates of ACE in vitro, among which captopril inhibits AcSDKP
hydrolysis (i.e., the ACE N domain) more potently than angiotensin I
hydrolysis (Michaud et al., 1997). Moreover, the rise in AcSDKP
concentrations in plasma and urine is a direct consequence of ACE
inhibition, whereas the rise in PRC is due to the interruption of the
angiotensin II negative feedback loop on renin secretion (Ménard
et al., 1991), which is itself the result of angiotensin II deprivation
at the level of juxtaglomerular cells during ACE inhibition.
In experiment 3, no plateau in AcSDKP plasma concentration was reached
if ACE was inhibited at the same time as AcSDKP was infused, confirming
that the half-life of the exogeneously infused peptide was considerably
increased compared with the single infusion of the peptide. If the
tetrapeptide was infused singly, a plateau of AcSDKP concentration in
plasma was achieved between T30 and T60. T30
corresponds to approximately four to five times the known half-life of
the exogenous peptide (roughly 5 min) (Ezan et al., 1994). Finally, our
results also showed that AcSDKP was eliminated via glomerular
filtration in rats. The similarities between AcSDKP metabolism in rats
and humans are not surprising because the rat ACE gene has 80 to 90%
sequence homology with the human ACE gene, therefore sharing similar
substrates (Koike et al., 1994).
Effects on Bone Marrow and Spleen AcSDKP Concentrations. For hematopoietic tissues, 3 to 30 mg/kg/day captopril administered over 14 days caused no significant increase in AcSDKP concentrations in total bone marrow and spleen. Captopril (10 mg/kg) had to be combined with high doses of AcSDKP (2 mg/kg) to achieve significantly higher AcSDKP concentrations in the extracellular fraction of bone marrow compared with the control group, whereas there was no significant accumulation of AcSDKP in either the intracellular fraction of bone marrow or spleen supernatants.
These are two possible reasons for the lack of increase in AcSDKP concentration in total bone marrow and spleen when ACE was inhibited: the amount of ACE in hematopoietic organs is small and captopril may be poorly distributed in these organs (its distribution in these organs is not known). The presence or absence of ACE in bone marrow is much debated. Grillon et al. (1990) showed that [3H]AcSDKP is not degraded by murine, rabbit,
or human bone marrow cells after 24 h of incubation. This was
confirmed by Comte et al. (1998) who detected no ACE activity (with
[3H]AcSDKP as substrate) either in supernatants
or cell fractions of murine bone marrow. In contrast, in long-term
human bone marrow cultures in vitro, AcSDKP has been shown to be
synthesized and released into the medium by macrophages, to be stored
by molecules of the extracellular matrix, and to be partly degraded by
stromal cells, probably by ACE present on the cell surface (Li et al., 1997). We detected no increase in AcSDKP concentration in total bone
marrow during ACE inhibition, suggesting that ACE is probably not
present in large amounts in hematopoietic organs such as bone marrow
and spleen.
Therefore, the absence of increase in AcSDKP concentration in the
intracellular fraction of bone marrow and spleen when AcSDKP (2 mg/kg)
is infused with captopril may be due to the persistence of an
unfavorable concentration gradient between these organs and the plasma compartment.
These results suggest that 1) the equilibrium between the production
and degradation of AcSDKP in hematopoietic organs was not affected by
the ACE inhibitor captopril, and 2) in vivo, the peptide is probably
secreted in plasma from bone marrow and other organs and is degraded
later by ACE present at the surface of endothelial cells or in plasma.
Effects on Kidney and Pulmonary AcSDKP Concentrations. For kidney and lung, mildly but significantly higher AcSDKP concentrations were measured in the captopril-treated rats than in control rats (experiment 2). If the peptide (2 mg/kg/h) was administered alone or in combination with captopril, much higher AcSDKP concentrations were achieved. Despite the correction for potential blood contamination, the very high concentrations of AcSDKP in kidney supernatants during combined administration of AcSDKP and captopril (experiment 3) should be interpreted with caution because potential contamination by AcSDKP from urine, in which it is present in large amounts, is difficult to exclude.
The increase in AcSDKP concentrations in kidney and lung homogenates was probably due to the local inhibition of tissue ACE by captopril 10 mg/kg. The 10-mg dose of captopril has previously been shown to induce a significant and persistent ACE inhibition in these tissues (Cohen and Kurz, 1982; Cushman et al., 1989). Moreover, kidneys and lungs are the
organs known to have the highest ACE contents of the entire body
(Erdös, 1990). They were also the two organs with the lowest
basal AcSDKP concentrations in the absence of ACE inhibition in our experiments.
The effect of long-term AcSDKP accumulation in the kidneys and lungs is
unknown, but it has recently been shown that AcSDKP (10
9-105 M) decreases
the proliferation rate of renal fibroblasts in culture in a
dose-dependent manner. This effect was increased by adding captopril to
the culture medium (Yoshioka et al., 1998).
In conclusion, doses of AcSDKP that result in similar plasma levels of
the peptide to those measured in our experiment and that have been
shown to be effective at protecting hematopoietic stem cells from the
cytotoxic effects of anticancer drugs (Bogden et al., 1991; Aidoudi et
al., 1996; Bogden et al., 1998; Masse et al., 1998) did not cause any
significant increase in AcSDKP in target hematopoietic tissues such as
bone marrow and spleen. Therefore, the pharmacological activity of
AcSDKP does not appear to be mediated by its accumulation in the
tissues of target organs. No binding of
[3H]AcSDKP to purified hematopoietic cells has
ever been detected (Comte et al., 1998), so our results suggest that
the pharmacological effects of AcSDKP are indirect, involving
interference with the peripheral production or degradation of other
cytokines that might reach the bone marrow.
| |
Footnotes |
|---|
Accepted for publication August 9, 1999.
Received for publication March 18, 1999.
1 This work was supported by grants from Assistance Publique des Hôpitaux de Paris, Commissariat à l'Energie Atomique, and by the Institut National de la Sante et de la Recherche Médicale program project PROGRES (Programme de Recherche en Santé).
Send reprint requests to: Dr. Michel Azizi, Centre d'investigation clinique, Hôpital Broussais, 96 rue Didot, 75674 Paris Cedex 14, France. E-mail: michel.azizi{at}brs.ap-hop-paris.fr
| |
Abbreviations |
|---|
AcSDKP, Acetyl-Ser-Asp-Lys-Pro; ACE, angiotensin I-converting enzyme; ACEI, angiotensin I-converting enzyme inhibitor; PRC, plasma renin concentration.
| |
References |
|---|
|
Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
|
|---|
This article has been cited by other articles:
|
|
 |
|
  E. Habouzit, H. Richard, H. Sanchez, N. Koulmann, B. Serrurier, R. Monnet, R. Ventura-Clapier, and X. Bigard Decreased muscle ACE activity enhances functional response to endurance training in rats, without change in muscle oxidative capacity or contractile phenotype J Appl Physiol, July 1, 2009; 107(1): 346 - 353. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Kovacs, J. Toth, J. Tarjan, and A. Koller Correlation of flow mediated dilation with inflammatory markers in patients with impaired cardiac function. Beneficial effects of inhibition of ACE Eur J Heart Fail, August 1, 2006; 8(5): 451 - 459. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
*
- -), AcSDKP 2 mg/kg/h (
- -), captopril + AcSDKP 2 mg/kg/h
(