1092
Antihypertensive Action of
Medullipin I Given by Mouth
E. Eric Muirhead, Bennie Brooks, Lawrence W. Byers, Kenji Toba, and Leonard Share
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Perfusion of normal rat kidneys with 5% human albumin in a balanced salt solution bubbled
with oxygen yielded medullipin I (Med I) in the renal venous effluent The presence of Med I
in the renal venous effluent has been established by thin-layer chromatography, by the type of
vasodepressor effect when injected intravenously as a bolus into the hypertensive rat, by
inhibition of the vasodepressor effect of the renal venous effluent by Tween 20 and SKF 525A
(proadifen, inhibitor of cytochrome P-450), and by removal of the liver from the circulation (a
procedure that inhibits extracted Med I). Med I so derived lowered blood pressure of
spontaneously hypertensive rats when injected into the stomach by an indwelling tube or when
given by mouth. The lowering of blood pressure was attended by no change in cardiac output
and no change in heart rate. Med I given by mouth to the spontaneously hypertensive rat is a
vasodilator that suppresses sympathetic tone, acting in the same way as Med I extracted from
renal papillae and given intravenously. Importantly, the antihypertensive action was demonstrated in the spontaneously hypertensive rat, a model of hypertension considered to mimic
idiopathic or essential hypertension of humans. Med I is a promising therapeutic agent for
hypertension. (Hypertension 1991;17:1092-1096)
M
edullipin I (Med I) is a hormone of the
renal papilla.1 It is present in small
amounts12 in the renal venous effluent
(RVE) of normal rats and in large amounts1-3-4 in the
RVE after unclipping of Goldblatt hypertensive rats.
Med I is transported to the liver where it is activated
into medullipin II (Med II).5-6 Med II causes vasodilation,7 suppresses sympathetic tone,8 causes diuresis-natriuresis,9 and in the rat has a suppressive
effect on the central nervous system.8
Med I can be derived by two approaches: 1) by
extraction from fresh renal papillae and concentration
by chromatography1 and 2) by perfusion of normal
kidneys under elevated blood pressure (BP).2-4 The
vasodepressor agent in the perfusate of the latter
previously was shown by extraction and thin-layer
chromatography to have the same RF on the chromatographic plate as Med I extracted from renal
papillae.4 Moreover, the medullipin of renal perfusate
and extracted medullipin have the same biological
properties and apparently represent the same entity.3
Because the medullipins offer promise as antihypertensive agents, it is of interest to determine if Med I
has an antihypertensive action when given by mouth to
the spontaneously hypertensive rat (SHR) of Okamoto and Aoki. We report herein that this is the case.
From the Departments of Pathology and Physiology, University
of Tennessee, Memphis, and Baptist Memorial Hospital, Memphis, Tenn.
Address for correspondence: E. Eric Muirhead, MD, Department
of Pathology, 899 Madison Avenue, 270UE, Memphis, TN 38146.
Methods
National Institutes of Health guidelines10 for animal research were followed precisely.
Med I was derived by perfusing normal rat kidneys
at 180 mm Hg with 5% human albumin in a balanced
salt solution bubbled with oxygen, as described previously.4 The procedure included treating the donor
rat with captopril (10 mg/kg i.p.) and the BurroughsWellcome compound BW 755C (10 mg/kg i.p.) (kindly furnished by Wellcome Research Laboratories,
Beckenham, Kent, UK) to inhibit the renin-angiotensin, cyclooxygenase, and lipoxygenase systems, as reported by Higgs et al.11 Oxygen was bubbled through
the albumin because of the suggestion that Med I
synthesis involves an oxidative step.12-13 The albumin
solution of the RVE was centrifuged at 4,000 rpm for
10 minutes to remove a minor residue and was assayed
for antihypertensive action by injecting a bolus dose
(0.5 ml) intravenously into SHRs. BP was monitored
for 30 minutes before injection, during vasodepression, and until recovery of BP occurred (see Figure 1).
The RVE so derived and assayed for vasodepressor activity was given to SHRs by two routes: by an
indwelling stomach tube and by mouth. The dose by
stomach tube was 10-15 ml and by mouth was 10 ml.
BP was measured directly from the abdominal
aorta by an indwelling catheter.14 Intravenous injections were made by an indwelling catheter in the
inferior vena cava.14 The assay SHRs were purchased
from Taconic Farms, Inc., Germantown, N.Y., and
Muirhead et al
Downloaded from http://hyper.ahajournals.org/ by guest on July 28, 2017
weighed 300-350 g when used. The rats donating the
kidney for perfusion were Sprague-Dawley rats from
Bio-Lab Corp., St. Paul, Minn., and weighed 400475 g on the day of perfusion.
Cardiac output (CO) was measured by the thermodilution method.1516 SHRs weighing at least 350 g
were placed under pentobarbital anesthesia (30
mg/kg i.p.), and the abdominal aorta was cannulated
as described previously.14 In addition, a precision fine
wire thermocouple (0.125 mm in diameter, Omega
Engineering, Inc., Stamford, Conn.) was implanted
in the aortic arch via the left femoral artery. This was
followed by installation of a microbore plastic tube
(0.375 mm i.d.x0.75 mm o.d.) into the right atrium
via the right external jugular vein. Through a midline
incision, an intragastric catheter was placed in the
stomach. This tube was constructed by fusing with a
soldering iron a 15-mm length of PE-90 tube with a
9-mm length of PE-50 tube. An oval bulb (3x1.5
mm) was formed at the tip of the PE-50 segment.
This bulb was implanted just inside the stomach
through an 18-gauge needle opening and anchored to
the outer wall by a purse-string suture.
The four lines, respectively, were channeled subcutaneously through a 17-gauge tube to the back of
the neck. The jugular and aortic tubes were filled
with saline containing heparin (each 50 units, Upjohn
Co., Kalamazoo, Mich.). Penicillin G (20,000 units
i.m.) was injected after the incisions were closed.
After at least 7 days of recovery, the rats were
placed in a wire cage (7.5x7.5x25 cm) having a slot
for travel of the lines and were allowed to acclimate
for 45 minutes before the experiment.
For hemodynamic measurements, mean BP was
recorded from the abdominal aorta via a Statham P
231D transducer (Oxnard, Calif.) connected to a
Brush 2600 recorder (Gould Instruments, Cleveland,
Ohio). Heart rate (HR) was recorded from the
arterial pulse with a Brush Biotach. CO was measured by the rapid injection of 200 [i\ 0.9% NaCl at
20-24°C into the right atrium and recorded by the
thermocouple in the thoracic aorta and a preamplifier designed by the Biomedical Instrument Division
of the University of Tennessee, Memphis, on a Brush
recorder. Rectal temperature was measured by a
Telethermometer (Yellow Springs Instruments Co.,
Yellow Springs, Ohio).
Each CO determination was based on an average of
at least two consecutive dilution curves with a minimum
of 2 minutes between them. In preliminary experiments
in which CO was measured 10 times at 2-minute
intervals, the coefficient of variation was 5.1%.
TABLE 2.
191±6
P
1093
TABLE 1. Effect on Blood Pressure of 5% Albumin Solution Before
Renal Perfusion in Assay Rats
BP, minutes after injection (mm Hg)
HBP
(mm Hg)
20
10
30
40
60
50
174.5±11 179.5+6 176.5±5 177±7 178±9 179±5 176.5±7
>0.8
>0.7
>0.9
>0 7
>0.7
>0 8
P
Dose, 0 5 ml l.v; n = 10. HBP, hypertensive blood pressure; BP,
blood pressure.
Baseline BP was taken continuously for 1 hour and
was followed by three control HR and CO measurements. The rats were allowed to recover. When
baseline measurements were again steady, a bolus
injection of Med I (10-15 ml RVE at 37°C) was given
by gastric tube over 30 seconds. A second and third
HR and CO measurement was performed at the
nadir of BP and after its recovery to prior hypertensive levels. Six SHRs were used in the hemodynamic
measurements. Altogether, 11 SHRs received Med I
by stomach tube.
The localization of the thermocouple in the aorta
was determined at autopsy.
For Med I given by mouth, SHRs were offered
Med I in albumin solution at 37°C to drink (10 ml in
a glass tube). BP was determined for 30 minutes
before this offer. Those rats drinking the entire 10 ml
had their BP followed for at least 50 minutes (n=8).
There were two sets of controls for the 5% albumin
solution: 1) 0.5 ml plain albumin solution (not perfused through kidneys) was injected intravenously as
a bolus into SHRs, and BP was followed (Table 1)
(n = 10); and 2) plain albumin solution (10 ml) was
injected by stomach tube into SHRs, and their BP
was followed (Table 2) (n=4).
Statistical analyses were by Student's t test. Values
of p< 0.05 were considered significant. Results are
given as mean±SEM.
Results
Vasodepressor Action of Renal Venous Effluent
Containing Medullipin I Derived by Perfusion of
Normal Kidneys
Figure 1 depicts the type of vasodepressor effect of
the RVE obtained from normal kidneys perfused
under high pressure. Of special note is the 2-minute
delay between the intravenous bolus injection and
the beginning decline of BP, a characteristic feature
of extracted Med I.1 BP dropped from 200 to 100
mm Hg in approximately 4 minutes, remaining so
depressed for 8 minutes. Recovery required approx-
Effect on Blood Pressure of 5% Albumin Solution by Stomach Tube Before Renal Perfusion In Assay Rats
BP, minutes after injection (mm Hg)
HBP
(mm Hg)
Medullipin I Is Active by Mouth
10
20
30
40
50
60
70
191+5
>0.9
190±15
>0.6
187.5±15
>0J
186±6
>0 5
189±1
>0 9
190±2
>0.8
191±6
>0.9
Dose, 10 ml; n=4. HBP, hypertensive blood pressure; BP, blood pressure.
80
90
192±3
>0.9
197±1.5
<0.02
1094
Hypertension
Vol 17, No 6, Part 2 June 1991
200
FIGURE 1. Representative recording of vasodepressor
effect of renal venous effluent (RVE) (0.5 ml Lv.)
injected into a spontaneously hypertensive rat. 1, Blood
pressure (BP) 200 mm Hg; 2, 0.5 ml RVE injected Lv.;
3, BP 100 mm Hg; 4, 5, 6, BP 140, 180, 200 mm Hg,
respectively. Response lasted 46 minutes.
S 190
100
10 minutes
to 166±6 mm Hg from 194±5 mm Hg (an approximate
20% drop in BP) and occurred in 46 ±12 minutes. By
63 ±4 minutes, recovery of BP had occurred.
Figure 4 shows the lowering of BP when Med I
was given by mouth. As by stomach tube, the
average drop of BP required approximately 30
minutes but remained depressed for at least 70
minutes.
Figure 5 gives the state of BP at the nadir (maximal
effect) of all eight examples, which occurred at
49 ±10 minutes and amounted to 152.5 mm Hg from
imately 40 minutes. In the present study, 16 perfusates (RVEs) of this type were used.
Downloaded from http://hyper.ahajournals.org/ by guest on July 28, 2017
Antihypertensive Action ofMedullipin I in Renal
Venous Effluent
Figure 2 shows the lowering of BP by Med I when
injected by stomach tube. The average results indicated
that approximately 30 minutes was required for BP to
reach its nadir. Recovery began after 50 minutes.
Figure 3 depicts the state of BP at the nadir (the
maximal effect) of all 11 examples. The nadir amounted
200190-
X
E
170-
Q.
03
160P vs. HBP
150-
•-.05
**<.O5
FIGURE 2. Line graph showing lowering of
blood pressure (BP) in spontaneously hypertensive rats after 10-15 ml renal venous effluent containing medullipin I was given by
stomach tube (n=ll). Continuous effect is
shown Values are for all 11 examples at each
point on the time axis. HBP, hypertensive BP.
M±SEM
HBP
10
20
3 0 4 0 5 0 6 0
7 0 8 0 9 0
Time from HBP (mln)
200190HBP
O)
180-
Rtcovwy**
E
a. 170m
160*Pvs.HBP<.005
**Pv8.Recovery >
M±SEM
150-
HBP
10
20
30
40
50
60
Time from HBP (mln)
70
80
FIGURE 3. Plot showing lowering of blood
pressure (BP) m spontaneously hypertensive
rats when medullipin I in renal venous effluent was given by stomach tube (n=ll).
Maximal effect (at nadir) and recovery are
shown. HBP, hypertensive BP.
Muirhead et al
Medullipin I Is Active by Mouth
1095
190180-
S 170-
1-eoH
ffi
150-
Pvs. HBP
* < .005
**<.O2
MiSEM
140HBP
10
20
3 0 4 0 5 0 6 0
Time from HBP (mln)
70
FIGURE 4. Line graph showing lowering of
blood pressure (BP) in spontaneously hypertensive rats after 10 ml renal venous effluent
containing medullipin I was given by mouth
(n=8). Continuous effect is shown. Values
are for all eight examples at each point on the
time axis. HBP, hypertensive BP.
90
80
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190180-
HBP
ji-H
160150Nadir*
140-
HBP
10
20
*Pvs.HBP<.005
**Pv». Recovery > .6
MiSEM
30
40
50
60
Time from HBP (mln)
185 ±4 mm Hg (an approximate 17.5% drop in BP).
By 76 ±8 minutes, recovery had occurred.
Table 1 demonstrates lack of change in BP when 0.5
ml plain 5% albumin was injected intravenously. Table
2 demonstrates lack of change in BP when 10 ml plain
5% albumin solution was injected by stomach tube.
Hemodynamic Effect of Medullipin I in Renal Venous
Effluent Given by Stomach Tube (n=6)
Table 3 relates the hemodynamic findings during the
antihypertensive action of Med I. The drop of BP from
its hypertensive level to the nadir was significant. ReTABLE 3.
70
80
FIGURE 5. Plot showing maximal effect on
blood pressure (BP) in spontaneously hypertensive rats after 10 ml of renal venous effluent containing medullipin I was given by
mouth. HBP, hypertensive BP.
90
covery of BP was complete. There was no significant
change in HR and CO as the BP lowered to the nadir
nor as recovery of BP occurred. Thus, Med I acted as a
vasodilator when given by stomach tube, and sympathetic tone was suppressed (no tachycardia).
Discussion
Using the principle elucidated by Karlstrom et al,2
namely, that Med I is secreted by an isolated kidney
perfused with whole blood under elevated perfusion
pressure, we derived Med I by perfusing isolated normal rat kidneys with 5% albumin solution bubbled with
Hemodynamic Findings After Medullipin I by Stomach Tube
Parameter
Blood pressure (mm Hg)
Heart rate (beats/min)
Cardiac output (mVlOO g wt)
Control
190±9
383 ±24
19±1.1
Nadir
P
158±8
387 ±23
18.6±2
<0.02
>0.9
>0.9
Recovery
194±11
384±29
18.2±1.6
p vs. control
0.75
>0.9
>0 7
Dose, 10 ml of renal venous effluent; n=6. Mean body wt of the six spontaneously hypertensive rats (SHRs) was
328±2 g. Cardiac output for these rats is similar to that reported by Albrecht et al17 in SHRs with established
hypertension using the dye dilution method.
1096
Hypertension Vol 17, No 6, Part 2 June 1991
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oxygen at 180 mm Hg.4 Oxygen was used because prior
observations suggested an oxidative step in the synthesis of Med I by the kidney.12-13 Med I so derived and
Med I extracted from fresh renal papillae and concentrated by chromatography appear to be the same
agent.3 Med I, therefore, has the characteristics of an
antihypertensive hormone of the kidney and most likely
its renomedullary interstitial cells.18
In these experiments, we showed that Med I lowers
BP of hypertensive rats when given by mouth or by
stomach tube. This makes Med I a promising therapeutic agent for hypertension. It is of special interest
that this per os antihypertensive effect occurred in
the SHR, a rat model considered to resemble the
most common type of human hypertension, idiopathic or essential hypertension.
The per os effect of kidney perfusion-derived Med
I was the same as that previously described for
intravenous Med I extracted from fresh renal papillae. In both cases, the agent obtained is a vasodilator7
that suppresses sympathetic tone.8 Moreover, extracted Med I causes diuresis-natriuresis.9
Med I must be converted to Med II by the liver56
to be active. This conversion appears to involve the
cytochrome P-450-dependent en2yme system of the
liver.612 Conversion of Med I injected intravenously
requires 1-2 minutes (illustrated in Figure 1),
whereas the ultimate drop in BP from Med I given by
mouth required some 30 minutes (Figures 2 and 4).
We consider the latter to result from the need for
intestinal enzymes to digest the albumin to which
Med I is attached before its absorption and conveyance to the liver.
The medullipin system and the renin-angiotensin
system are feedback control systems. The former
either lowers BP or keeps it lowered and is antihypertensive. The latter raises BP or keeps it elevated and is
prohypertensive. These systems have a reciprocal behavior toward renal artery perfusion pressure. A drop
in renal artery pressure stimulates the secretion of
renin and the generation of angiotensin II and tends
to shut off the secretion of Med I and the generation
of Med II.4 An elevation of renal artery perfusion
pressure stimulates the secretion of Med I and the
generation of Med II2-4 while suppressing the secretion of renin and the generation of angiotensin II.
There are indications that an excess of one system
can overpower the other system. An excess of circulating angiotensin II can overcome the antihypertensive action of an effective amount of Med II.19 By the
same token, an excess of Med II can modulate the
effectiveness of angiotensin II.20 Thus, the action of
Med I taken by mouth likely will depend on the
dosage available and the extent to which angiotensin
II is operative in a given hypertensive state. In the
SHR, Med I appears to be an effective antihypertensive agent by mouth.
References
1 Muirhead EE, Byers LW, Desiderio DM, Pitcock JA, Brooks
B, Brown PS, Brosius WL: Derivation of antihypertensive
neutral renomedullary hpid from renal venous effluent. / Lab
Clin Med 1982;99:64-75
2. Kailstrora G, Amman V, Folkow B, Gothberg G: Activation
of the humoral antihypertensive system of the kidney increases
diuresis. Hypertension 1988;ll(suppl II):II-597-II-601
3. Muirhead EE: Renomedullary vasodepressor lipids, in Massry
SG, Glassock RJ (eds): Textbook of Nephology Baltimore,
Williams & Wilkins Co, 1989, pp 191-193
4. Muirhead EE, Brooks B, Byers LW, Brown P, Pitcock JA:
Secretion of medullipin I by isolated kidneys perfused under
elevated pressure. Chn Exp Pharmacol Physiol (in press)
5. Muirhead EE, Byers LW, Brooks B: Conversion of the antihypertensive neutral renomedullary lipid by the lrver. Trans
Assoc Am Physicians 1986;98:269-274
6. Muirhead EE, Brooks B, Byers LW, Pitcock JA, Brown PS:
Conversion of medullipin I by the liver. Trans Assoc Am
Physicians 1988;101226-234
7. Dundore RL, Solus EE, Shaffer RA, Muirhead EE, Brody MJ:
Antihypertensive and hemodynamic effects of antihypertensive
neutral renomedullary hpid and two synthetic lipids, Snyder lipid
and selachyl alcohol. / Cardiovasc Pharmacol (in press)
8. Muirhead EE, Folkow B, Byers LW, Aus G, Friberg P,
Gothberg G, Nilsson H, Thoren P: Cardiovascular effects of
antihypertensive renomedullary lipids (ANRL and ANRL).
Acta Physiol Scand 1983;117:465-467
9. Karlstrom G, Amman V, Bergstrom G, Muirhead EE, Rudenstam J, Gothberg G: Renal and circulatory effects of
medullipin I as studied in the in vitro cross-circulated isolated
kidney and intact Wistar-Kyoto (WKY) r&l.Acta Physiol Scand
1989;137.521-533
10. US Dept of Health and Human Services: Guide for the Care
and Use of Laboratory Animals NTH Guide Supplement for
Grants and Contracts, NIH publication No. 85-23. Washington, DC, US Government Printing Office, 1985, pp 1-83
11. Higgs GA, Moncada S, Vane JR: Eicosanoids in inflammation.
Ann Chn Res 1984;16:287-299
12. Muirhead EE, Byers LW, Capdevila J, Brooks B, Pitcock JA,
Brown P. The renal antihypertensive endocrine function: Its
relation to cytochrome P-450. / Hypertens 1989;7:361-369
13 Muirhead EE, Byers LW, Brooks B, Brown P, Pitcock JA:
Biologic contrasts between medullipin I and vasoactive glyceryl compounds. Am J Med Sti 1989,298.93-103
14. Muirhead EE, Brooks B: Blood Pressure Measurement m Rats.
US Dept of Health, Education, and Welfare publication No.
(NIH) 78-1473 Bethesda, Md, National Institutes of Health,
1977, pp 5-14
15 Osborn JW Jr, Barber BJ, Cowley AW Jr Chronic measurement
of cardiac output in anesthetized rats using miniature implanted
thermocouples. Am J Physiol 1986;251:H1361-H1372
16. Cooper T, Pinakatt T, Richardson AM: The use of the thermal
dilution principle for measurement of cardiac output in the
rat. Med Electron Btot Eng 1963;1 61-65
17. Albrecht I, Vizik M, Krecek J: The hemodynamic of the rat
during ontogenesis, with special reference to the age factor in the
development of hypertension, in Okamoto K (ed): Spontaneous
Hypertension, Its Pathogenesis and Complications. New York,
Acknowledgment
Springer-Verlag New York, Inc, 1972, pp 121-127
18 Muirhead EE, Rightsel WA, Leach BE, Byers LW, Pitcock
JA, Brooks B: Reversal of hypertension by transplants and
hpid extracts of cultured renomedullary interstitial cells. Lab
Invest 1977;35:162-172
19 Muirhead EE, Rightsel WA, Pitcock JA, Inagami T: The
renin-angiotensin system in cultured juxtaglomerular cells, in
Fisher JW (ed). Kidney Hormones London, Academic Press,
Inc, vol 3, 1986, pp 247-272
20. Kumar A, Bing RF, Swales JD, Thurston H: Delayed reversal
of Goldblatt hypetension by angiotensin II infusion in the rat
Am J Physiol 1984;246(//eort Circ Physiol 15) H811-H817
We thank Barbara King for her assistance in
preparing this manuscript.
KEY WORDS • vasodilation • antihypertensive agents •
angiotensin II • medullipin • spontaneously hypertensive rats
Antihypertensive action of medullipin I given by mouth.
E E Muirhead, B Brooks, L W Byers, K Toba and L Share
Hypertension. 1991;17:1092-1096
doi: 10.1161/01.HYP.17.6.1092
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Copyright © 1991 American Heart Association, Inc. All rights reserved.
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