(Hypertension. 1997;30:1009-1014.)
© 1997 American Heart Association, Inc.
Articles |
A Newly Identified Peptide, Proadrenomedullin N-Terminal 20 Peptide, Induces Hypotensive Action via Pertussis Toxin–Sensitive Mechanisms
From the Fourth Department of Internal Medicine, University of Tokyo School of Medicine, Tokyo 112, Japan.
Correspondence to Toshiro Fujita, MD, Fourth Department of Internal Medicine, University of Tokyo School of Medicine, 3-28-6 Mejirodai Bunkyo-ku, Tokyo 112, Japan. E-mail fujita-dis{at}h.u-tokyo.ac.jp
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Abstract |
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Abstract Proadrenomedullin N-terminal 20 peptide (PAMP) and adrenomedullin (AM) are novel hypotensive peptides. Although they are derived from the same gene product, proadrenomedullin, their hypotensive mechanisms are different; PAMP inhibits the release of norepinephrine from the peripheral sympathetic nerve endings, whereas AM fosters vasodilation by elevating intracellular cAMP, possibly via activation of cholera toxin–sensitive G proteins. In PC12 cells, PAMP inhibited N-type calcium channel via activation of pertussis toxin–sensitive mechanisms. To clarify the relationship between the hypotensive effect of PAMP and pertussis toxin-sensitive mechanisms, we administered pertussis vaccine intraperitoneally into rats for 3 consecutive days. By using mesenteric artery preparation, we showed that PAMP's ability to decrease norepinephrine overflow was significantly attenuated in pertussis toxin-treated rat (-18.5±6.9%; P<.05 versus control rats). In electrically stimulated pithed rat, PAMP (20 and 40 nmol/kg) showed a hypotensive effect (-13±5 and -18±7 mm Hg, respectively; P<.05, P<.01), whereas in pertussis vaccine-treated rat it did not (-2±3 and -8±9 mm Hg, respectively; P=NS). Also, in pithed rat, plasma norepinephrine level was significantly elevated by electrical stimulation in both control (0.323±0.035 ng/mL) and pertussis vaccine- treated groups (0.355±0.079 ng/mL). After injection of PAMP (40 nmol/kg), plasma norepinephrine level significantly decreased in the control group (0.225±0.044 ng/mL; P<.01) but not in the pertussis vaccine-treated group (0.392±0.021 ng/mL; P=NS). Moreover, in conscious rats, intravenous administration of PAMP (40 nmol/kg) did not evoke hypotension after pertussis vaccine treatment, although untreated controls had significantly decreased arterial pressure (-5±2 versus -20±3 mm Hg; P<.01). In contrast to PAMP, the administration of AM (1 nmol/kg) significantly reduced the blood pressure of pertussis vaccine-treated as well as control rats (-20±5 versus -18±7 mm Hg; P=NS). These results demonstrate that the ability of PAMP to inhibit norepinephrine release from peripheral sympathetic nerve endings and to decrease blood pressure is pertussis toxin sensitive. Our findings thus suggest that despite being derived from the same gene, PAMP and AM apparently produce hypotension by activating different signaling pathways.
Key Words: sympathetic nervous sytem • pertussis vaccine • rats • norepinephrine
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Introduction |
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Adrenomedullin was originally isolated from pheochromocytoma cells, but it is also produced in and secreted by endothelial cells.1 2 The hypotensive action of AM, which causes vasodilation, is evident from its ability to elevate intracellular cAMP in vascular smooth muscle cells and to raise the intracellular calcium levels in endothelial cells, thereby increasing their rate of nitric oxide synthesis.3 4 5 The DNA sequence encoding the precursor of AM, proadrenomedullin, has been identified in human as well as rat tissue.6 7 The first paired basic amino acid of this precursor (Lys43-Arg44) is a representative site for proteolytic cleavage, yielding the product now called PAMP.6 7 Like AM, PAMP has a hypotensive effect; however, these two peptides from the same precursor seem to control blood flow through different mechanisms. Our previous studies suggest that PAMP reduces norepinephrine (NE) overflow from peripheral sympathetic nerve endings of mesenteric arteries,8 which in turn decreases blood pressure.9
NE release from neurons is regulated mainly by intracellular calcium level because entry of calcium ions into the presynaptic terminal triggers NE release. At the peripheral sympathetic terminal, voltage-sensitive N-type calcium channels are important in the control of NE release.10 Moreover, neurotransmitters can regulate these voltage-sensitive calcium channels via pertussis toxin-sensitive guanine nucleotide-binding proteins (G proteins).11 Although neither the intracellular signaling device nor the specific receptor of PAMP is known, PAMP previously reduced calcium influx entering the cell via N-type calcium channels, and this effect was sensitive to pertussis toxin in differentiated PC12 cells.12 This finding suggests that the hypotensive effect of PAMP results from inhibition of N-type calcium channel function via pertussis toxin-sensitive G proteins, thereby decreasing NE release.
In the present study, we examined this hypothesis in vivo by investigating the effect of PAMP on NE release and blood pressure in PTX-treated rat. In this system, the vaccine blocks the action of G proteins.13 Our results demonstrate that PAMP inhibits the peripheral sympathetic nervous system by suppressing NE release, due to activation of a pertussis toxin-sensitive mechanism.
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Methods |
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Animal Preparation
A total of 74 male Sprague-Dawley rats (8 weeks old, 250 to 300 g, Charles River Japan) were used in this study. All rats were housed in a quiet room with constant temperature (24±1°C), humidity (60±5%), and light (illumination from 6 AM to 6 PM) and allowed free access to regular chow (Oriental Yeast Co) and tap water. The rats were randomly divided into two groups. One group (n=37) received 300 opacity unit/kg PTX in 0.1 mL of saline intraperitoneally for 3 days. The second group (n=37, controls) received 0.1 mL of saline intraperitoneally for 3 days. Body weights were measured every day. All protocols in this study were approved by the ethics committee of our institute, and all surgical procedures conformed to the guidelines established by the US Department of Health and Human Services and published by National Institutes of Health (NIH publication No. 85-23, revised 1985).
Protocol 1: NE Overflow From Sympathetic Nerve Endings
One day after the last injection of PTX or saline, 10 rats from
each group were tested. Isolated mesenteric arteries were prepared by a
modification of Castellucci's method.8 14 The entire
intestines were discarded, and the mesenteric arteries were quickly
connected to the perfusion apparatus. The preparations were
perfused with a Krebs-Henseleit solution by use of a peristaltic pump
(Minipuls 2, Gilson Medical Electronics SA) at a rate of 2 mL/min.
Constituents of the solution were as follows (mmol/L): NaCl
114.5, KCl 4.6, KH2PO4 1.4, MgSO4
2.4, CaCl2 2.5, NaHCO3 25, glucose 5.6. The
solution was continuously oxygenated with a gas mixture of
95% O2/5% CO2 at 37°C. A 30-minute
equilibration period was allowed before starting each experiment.
A platinum electrode placed around the periarterial plexus
of the mesenteric artery was used to stimulate the postganglionic
sympathetic nerve fibers. A standard electrical stimulus of 8 Hz for 1
minute was given every 15 minutes. The perfusate through the
mesenteric vascular preparation was collected into tubes containing 10
mg EDTA at a final concentration of 2.5 mg/mL for measurement of
NE by high-performance liquid
chromatography.8 14 NE was measurable from
1 to 10 000 pg/mL, and NE concentrations in the effluent of
stimulated arteries were 
100 to 500 pg/mL. Samples were
collected every 2 minutes before and after nerve stimulation. PAMP in a
dose of 10 pmol/mL was applied 5 minutes before electrical
stimulation. Electrical stimulation was given twice without and twice
with PAMP every 15 minutes.
Protocol 2: Plasma NE Level and Blood Pressure Change in
Pithed Rats
A total of 40 rats from control and PTX-treated groups were
used. The rats were anesthetized with ether, and both the left
carotid artery and the jugular vein were cannulated with PE-50
polyethylene tubing tapered at the tip. Both catheters were tunneled
subcutaneously to the back of the neck, filled with heparinized saline
(200 U/mL), and plugged with stainless steel pins. The incisions were
closed with sutures. After the tracheal cannulation, the animals were
pithed by inserting a steel rod (1.6 mm OD), which was covered
with enamel except at its end (5-mm length), through the orbit and the
foramen magnum and down into the spinal column to its sacral end. The
animals were then placed on a ventilator (SN-480-7, Shinano) and
respirated artificially through the tracheal cannula by 1 mL/100 g body
wt of stroke volume (60 strokes/min). Both of the vagal nerves were
cut. Body temperature was maintained at 37°C with a thermostatically
controlled heating table.
A steel rod inserted behind the skull and pushed down between the vertebral column and the skin served as the indifferent electrode. The end of the pithing rod was placed at the level of the 7th to 10th thoracic vertebrae. Electrical stimulation (30 V, 0.5 Hz, 1 millisecond for 30 minutes; delivered from an electrical stimulator, SEN3301; Nihon Kohden) was generated between the pithing rod and the indifferent electrode9 15 after a 1 mg/kg intravenous injection of (+)-tubocurarine.
Seven rats from both groups were used for measuring blood pressure change. MAP and heart rate were recorded with a pressure transducer (model TP-200T, Nihon Kohden) that was connected to a thermal array recorder (model WS-641G, Nihon Kohden). PAMP (20 and 40 nmol/kg) or vehicle (0.1 mL of saline) was injected serially as an intravenous bolus into the jugular vein. Each injection was administered 15 minutes after MAP had returned to basal level. The nadir values of the decreases in MAP were considered to be the responses to each peptide dose.
Twelve rats of each group were used for measuring plasma NE change. A volume of 1 mL of arterial blood was drawn for baseline determinations of NE levels 5 minutes after pithing. Afterward, 2 mL of whole blood collected from one control rat and from one PTX-treated rat was transfused into the pithed rats. Seven minutes after electrical stimulation, a second sample for measuring NE (S1) was drawn, and blood was transfused as described above. Seven minutes after collection of S1, 0.1 mL of saline (n=6 for each group) or PAMP (40 nmol/kg in 0.1 mL of saline) (n=6 for each group) was injected intravenously, and a third sample of arterial blood was drawn 5 minutes after injection (S2). The NE level was determined by the trihydrosyindole method after high-performance liquid chromatography separation as previously reported.16 With this method, NE levels of 1 to 10 000 pg/mL can be accurately measured, a range that includes the plasma NE concentration within 40 to 400 ng/mL.
Protocol 3: Response of Blood Pressure and Heart Rate in
Unanesthetized and Unrestrained Rat
As in protocol 1, 1 day after the last injection of PTX or
saline, seven rats from each group were used. Twenty-four hours before
experiments, the rats were anesthetized with ether, and the
surgical procedures and cannula implants, except for tracheal cannula,
were done as described above. Both catheters were plugged with
stainless steel pins. After the surgical procedure, the rats were
placed in a cage that permitted free movement for at least 24 hours to
acclimate them to the new environment. MAP and heart rate were
recorded with a pressure transducer (model TP-200T, Nihon Kohden)
connected to a thermal array recorder (model WS-641G, Nihon
Kohden). The peptides AM (1 nmol/kg) and PAMP (40
nmol/kg) dissolved in 0.1 mL of saline were injected as an
intravenous bolus into the jugular vein in random order.
Each injection was done 15 minutes after MAP and heart rate returned to
basal level.9
To examine the efficacy of PTX administration, carbachol (1, 3, 10, 30, and 100 mg per rat) dissolved in 0.1 mL of saline was injected as a bolus 1 hour after PAMP or AM were administered, and the decreases in heart rate were recorded. Changes in MAP and heart rate were measured at nadir level.
Drugs
PTX was from Takeda Chemicals Co. PAMP and AM were from Peptide
Institute Inc. Tubocurarine, carbachol, guanethidine, and tetrodotoxin
were from Sigma Chemical Co.
Statistics
All results are given as mean±SEM. Data were analyzed
with Student's t test for comparison between two groups. To
analyze the effect of PAMP on NE overflow and blood pressure,
paired t tests were applied, and for comparisons between the
effects of AM and PAMP treatments or the control and PTX groups,
two-way ANOVA and subsequent Dunnett's test were applied. Two-way
ANOVA and Scheffé's test were applied to analyze plasma
NE change by PAMP in pithed rats. To analyze sensitivity for
carbachol, change of heart rate (%) was calculated and median
effective doses (ED50) were obtained and analyzed
by Student's t test. Statistical significance was defined
at a value of P<.05.
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Results |
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Body Weight Change After Administration of PTX
In control rats not given PTX, body weights did not change from test days 1 to 4, respectively (280±10, 283±15, 282±20, and 286±22 g). In the PTX group, body weight were similar to those of controls days 1 to 4, respectively (282±15, 280±12, 276±15, and 280±20 g).
Protocol 1: NE Overflow From Sympathetic Nerve Ending
To examine the effect of PAMP on peripheral
sympathetic nerve endings, we used isolated mesenteric arteries. No NE
overflow was detectable without electrical stimulation. Previously, the
NE overflow tested by six consecutive electrical stimulations did not
differ.8 Both the NE overflow and the pressor responses
induced by electrical stimulation were completely abolished by the
addition of guanethidine (10-5 mol/L)
and tetrodotoxin (10-7 mol/L) to the
perfusate (NE; not detectable), suggesting that this stimulus
adequately reflects the activity of the sympathetic nerves. Average
tissue weight was 3.86±0.78 g in the control group and 3.43±0.98 g in
the PTX group.
In PTX-treated rats, the NE overflow with PAMP (0.260±0.021
ng/g tissue weight) was greater than in control rats
(0.232±0.024 ng/g tissue weight, P<.05). PAMP
decreased the NE overflow from 0.260±0.021 to 0.214±0.019 ng/g
tissue weight (P<.05) (Fig 1A
) in the PTX group, but this decrease
was significantly smaller (-18.5±6.9%) than in the control group
(-37.8±3.5%, P<.05) (Fig 1B).
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Protocol 2: Change of Blood Pressure and Plasma Level NE in
Pithed Rat
To examine the correlation between the peripheral
sympathoinhibitory effect of PAMP and blood pressure, we
used pithed rats in which autonomic regulation of blood pressure is
destroyed. As we reported, PAMP produced hypotension only after
exogenous activation of the peripheral sympathetic nervous
system by electrical stimulation9 ; thus, in this study, we
used only electrically stimulated pithed rats. In all groups, pithing
had no effect on baseline blood pressure (Table 1), and after electrical stimulation,
blood pressures were elevated to the same extent (Table 1). In
controls, PAMP injection decreased blood pressure in a dose-dependent
manner (-13±5 and -18±7 mm Hg), however, in the PTX group,
PAMP failed to decrease blood pressure (-2±3 and -8±9 mm Hg)
(Table 1, Fig 2).
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After pithing, plasma NE levels among all groups were also alike (Table 2). Additionally, electrical stimulation
significantly and equally increased all NE levels. Saline injection did
not affect the mean plasma NE level of either the control or the PTX
group (Table 2). In contrast, as we reported previously,9
PAMP injection reduced plasma NE levels (S2) significantly in the
control group but not in the PTX group (Table 2).
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Protocol 3: Responses of Blood Pressure and Heart Rate in
Unanesthetized and Unrestrained Rats
Finally, we investigated the ability of PTX to block the
hypotensive effect of PAMP in vivo. Baseline MAP and heart rate were
101±4 mm Hg and 420±32 bpm, respectively, in the PTX group and
105±5 mm Hg and 315±28 bpm, respectively, in the control group.
Although PTX did not change the MAP significantly, heart rates were
significantly higher in the PTX group (P<.01), which is
consistent with a previous report.17
Both in the control and in the PTX groups, intravenous
administration of AM in a dose of 1 nmol/kg decreased MAP
similarly (-20±5 vs -18±7 mm Hg). In contrast,
intravenous administration of PAMP in a dose of 40
nmol/kg decreased MAP in the control group but not in the PTX
group (-5±2 vs -20±3 mm Hg, P<.01) (Fig 3).
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MAP indicates
change of mean arterial pressure; PTX, pertussis
vaccine-treated group.Carbachol-induced bradycardia was significantly suppressed in the PTX group. Furthermore, the ED50 was significantly smaller in the control group than in the PTX group (91±8 vs 7±1 mg/rat, P<.01).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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PAMP and AM are the novel hypotensive peptides derived from one precursor.18 In previous studies with isolated rat mesenteric arteries, it was shown that PAMP inhibits NE overflow8 whereas AM caused direct vasodilation,3 which is thought to be the hypotensive mechanism of PAMP and AM.9
Depression of calcium currents in peripheral neurons
by neurotransmitters is thought to constitute an important regulatory
mechanism of neurotransmitter release. Nerve cells have been shown to
express at least two distinct types of ion channels on their cellular
membranes: one type is the neurotransmitter-operated ion channels, and
the other type is the voltage-operated ion channels. PAMP has been
reported to inhibit carbachol-induced calcium influx, which occurs via
voltage-dependent calcium channels.19 Among
voltage-dependent calcium channels, N-type calcium channel plays major
role in regulating calcium influx at the peripheral
sympathetic nerve.10 PAMP also inhibited
-conotoxin-sensitive calcium influx in NGF-treated PC12
cells,12 and pertussis toxin counteracted this inhibition
by PAMP.12 These outcomes suggest that PAMP inhibits
N-type calcium channel function via a pertussis toxin-sensitive
mechanism. PC12 cells can mimic noradrenergic
sympathetic neurons when treated with NGF20 ; however, no
direct evidence of interaction between pertussis toxin sensitivity and
the hypotensive effect of PAMP has yet been shown in vivo.
Here, both ex vivo and in vivo, we showed in three ways that PAMP may interact with pertussis toxin-sensitive mechanisms. First, PTX inhibited the PAMP-induced NE release in isolated, electrically stimulated mesenteric arteries. Second, in an in vivo study using pithed rats, PAMP injection significantly decreased plasma NE levels of controls but not of their PTX-treated counterparts. Third, blood pressure responses to PAMP were completely blocked by PTX both in pithed and conscious rats. In contrast to PAMP, the hypotensive action of AM was not blocked in the PTX-treated group, which confirms the results of former studies.1 5 AM increases intracellular cAMP in platelets1 and also activates phospholipase C and inositol 1,4,5-trisphosphate, thereby elevating intracellular calcium levels in the endothelium.5 Because these actions were blocked by cholera toxin but not PTX,5 AM activates cholera toxin-sensitive systems but is not effective for pertussis toxin-sensitive systems.
The effects of pertussis toxin are multiple, and pertussis toxin
possesses the capacity to inactivate G proteins such as
Gi, Go, and some forms of
Gp.21 Formerly, three consecutive days of PTX
treatment resulted in complete loss of pertussis toxin
ADP-ribosylatable substrates in adipocytes for more than 15 days in
vivo, which suggests prolonged ADP ribosylation of
1-subunits and inactivation of Gi without
altering the level of s and
Gs.13 Thus, PTX appears to be an effective,
specific, and long-term way of inactivating Gi in living
individuals. Moreover, in the present study, PTX significantly
blunted the ability of carbachol to decrease heart rate, which results
from activation of Gi.22 Similarly, we showed
here that the hypotensive action and the inhibitory effect
on NE overflow by PAMP were blocked by PTX. Considering that N-type
calcium channels have been noted as primary targets for
Go23 24 and Gi25 and
that our results in vivo emphasize the impact of PTX on this system, we
can speculate that intracellular signaling of PAMP decreases blood
pressure via pertussis toxin-sensitive G proteins.
The doses of PAMP and AM used in our study both in vivo and ex vivo are far higher than those present in circulating blood26 27 ; therefore, these agents might be effective only at pharmacological doses. However, because mRNAs for PAMP and AM are found in vascular smooth muscle cells and endothelial cells,2 28 they may be local hormones, and at nerve termini the concentration of PAMP might be higher than in plasma.
Finally, cardiomyocyte injury is induced by an excess of catecholamine,29 and patients' prognoses after myocardial infarction30 or congestive heart failure31 correlate with their plasma catecholamine level. Therefore, PAMP may become a new therapeutic agent to prevent further development of cardiogenic disease both by decreasing blood pressure and by sympathoinhibition.
In summary, our data suggest that PAMP decreases NE release from peripheral sympathetic nerve endings via pertussis toxin-sensitive mechanisms; thus, its signaling pathway differs from that of AM, which is derived from the same precursor peptides.
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Selected Abbreviations and Acronyms |
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Received February 25, 1997; first decision March 8, 1997; accepted March 18, 1997.
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References |
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M. Mahata, S. K. Mahata, R. J. Parmer, and D. T. O'Connor Proadrenomedullin N-Terminal 20 Peptide : Minimal Active Region to Regulate Nicotinic Receptors Hypertension, November 1, 1998; 32(5): 907 - 916. [Abstract] [Full Text] [PDF]
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