(Hypertension. 1996;27:360-363.)
© 1996 American Heart Association, Inc.
Articles |
Parathyroid Hormone–Related Protein Inhibits Endothelin-1 Production
From the Department of Geriatric Medicine, Osaka (Japan) University Medical School.
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Abstract |
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Abstract The effect of human parathyroid hormone–related protein, a powerful vasodilator, on endothelin-1 production in cultured bovine pulmonary arterial endothelial cells was studied. Treatment with parathyroid hormone–related protein(1-34) at concentrations of 10-9 to 10-6 mol/L for 24 hours caused dose-dependent suppression of the secretion of endothelin-1, with maximal suppression at 10-7 mol/L to 74% of the control value. This inhibitory effect was completely abolished by coincubation with 100 ng/mL pertussis toxin, an inhibitor of GTP binding protein. Furthermore, addition of NG-monomethyl-L-arginine, an inhibitor of nitric oxide synthase, at 10-3 mol/L significantly blocked the suppressive effect of parathyroid hormone–related protein(1-34) on endothelin-1 secretion, and further addition of 5x10-3 mol/L L-arginine significantly attenuated the blocking effect of NG-monomethyl-L-arginine. Parathyroid hormone–related protein(1-34) at 10-7 mol/L resulted in an approximately fivefold increase in intracellular cGMP level. Northern blot analysis revealed that parathyroid hormone–related protein(1-34) inhibited both basal and thrombin-induced endothelin-1 gene expression. These findings suggest that the vasodilating property of parathyroid hormone–related protein may be mediated in part through its inhibitory effect on endothelin-1 production, which is probably mediated through nitric oxide and cGMP in endothelial cells. Thus, a feedback regulatory mechanism may exist between parathyroid hormone–related protein and endothelin-1 in the vascular wall.
Key Words: parathyroid hormone • endothelins • nitric oxide • pertussis toxins
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Introduction |
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TopAbstractIntroductionMethods Results Discussion References |
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Parathyroid hormone–related protein, which originally was found to be responsible for producing hypercalcemia associated with malignant disease,1 2 3 has recently been identified in several normal tissues, including endothelial cells4 and vascular smooth muscle cells.5 In addition to its known hypercalcemic and hyperphosphaturic properties, PTHrP has proven to be a potent vasorelaxant and thereby is considered to have important autocrine and/or paracrine actions in the cardiovascular system.6 7 8 Functional expressions of PTHrP-receptor interaction have been shown to be involved in vasorelaxation in a perfused rat kidney preparation, rabbit renal artery and renal arterioles, norepinephrine-precontracted helical strips of rat aorta, and coronary vessels of isolated perfused rat heart.6 7 8 9 10 11 It has been reported that PTHrP results in hypotension and cardiac stimulation,6 but details of the mechanism remain unclear.
On the other hand, ET-1, a novel endothelium-derived vasoconstrictor peptide,12 recently has been found to stimulate PTHrP expression in cultured rat aortic smooth muscle cells.5 However, endothelin release is inhibited by coculture of endothelial cells with vascular smooth muscle cells,13 which suggests that some inhibitory factors may be produced in the coculture system. It has been reported14 15 16 that the release of endothelin from porcine aorta can be inhibited by endothelium-derived NO. Simeoni et al17 recently described the involvement of NO in the vasodilatory response to PTHrP in the kidney. These interesting findings suggest that PTHrP could be an inhibitory factor against ET-1 and that a relationship might exist between PTHrP and ET-1. The present study investigated whether PTHrP can modulate ET-1 production in endothelial cells.
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Methods |
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Materials
Human PTHrP(1-34) was purchased from Peptide Institute Inc. Pertussis toxin (IAP) was obtained from Funakoshi Co. L-NMMA, L-arginine, D-arginine, thrombin, and BSA were from Sigma Chemical Co. DMEM was from Nissui Pharmaceutical Co, and FCS was from Flow Laboratories. All other chemicals used were commercial products of the highest grade available.
Cell Culture
BPAE cells were a gift from Japanese Cancer
Resources Bank
(Tokyo). BPAE cells were grown in DMEM supplemented with 10% FCS in
100-mm culture dishes at 37°C in humidified 5% CO2 air,
with changes of medium every 2 days. Cells were passaged at confluence
by treatment with 0.05% trypsin/0.02% EDTA in 10 mmol/L
phosphate-buffered saline, followed by two washes with DMEM
containing 10% FCS. Cells at the fifth to ninth passage were used for
experiments.
Determination of ET-1
BPAE cells released with trypsin/EDTA
from confluent stock
cultures were seeded in 24-well culture plates at a density of
5x104 cells/well in DMEM with 10% FCS. At confluence
after 3- or 4-day incubation, the cells were washed twice with
serum-free DMEM and then incubated in 1 mL/well of serum-free
DMEM with or without other compounds for 24 hours. After incubation,
the medium of each well was used for ET-1 determination by a sensitive
sandwich-enzyme immunoassay as described previously.18
The cells were then washed twice with 10 mmol/L phosphate-buffered
saline, followed by addition of 0.5 mL of 0.1 mol/L NaOH to dissolve
the cells for the determination of cell protein content by the method
of Lowry et al19 with BSA used as a standard. ET-1 content
was expressed in nanomoles per gram of cell protein.
Measurement of cGMP
For assay of cGMP, confluent monolayers
of cultured BPAE cells
were preincubated in Earle's salt solution (in mmol/L: NaCl 145, KCl
5, CaCl2 1.8, MgCl2 0.8, glucose 5, HEPES 25,
adjusted with NaOH to pH 7.4). After 15 minutes,
10-7 mol/L PTHrP(1-34) was added. The
reaction was stopped at 0, 1, 5, 15, and 30 minutes by quickly freezing
the cells in liquid nitrogen. Frozen cells were homogenized
in ice-cold 6% trichloroacetic acid, and the extracts were assayed
for cGMP by use of a commercially available radioimmunoassay kit
(Amersham). Protein concentration was measured as above. cGMP content
was expressed in nanomoles per gram of cell protein.
Inhibitors of nonspecific phosphodiesterases, such as 3-isobutyl-1-methylxanthine, were not used in this experiment, since suppression of these enzymes could disturb the physiological response of cGMP in endothelial cells.20
Analysis of RNA
Effects of PTHrP on basal and
thrombin-induced ET-1
expression were examined. BPAE cells were grown to confluence in 100-mm
dishes with DMEM containing 10% FCS. After they were washed twice with
serum-free DMEM, the cells were incubated in serum-free DMEM
containing 0.1% BSA with or without other compounds for 12 hours.
Total RNA extraction and Northern blot analysis were performed
as described previously.21 The human endothelin precursor
cDNA was prepared according to the method of Itoh et al22
from the EcoRI site of plasmid pUC18, designated pHET4-3,
which had cDNA inserts of 
1.17 kb. The cDNA probes for human ET-1
and GAPDH were labeled with [32P]deoxycytidine
triphosphate (111 terabecquerel/mmol) by the random-primed labeling
method. Hybridization with a GAPDH cDNA probe was used to monitor
uniform loading of RNA on Northern blots.
Statistics
Results are expressed as mean±SD.
Statistical analysis
was performed by one-way ANOVA and Student's t test. A
value of P<.05 was considered significant.
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Results |
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TopAbstractIntroductionMethods Results Discussion References |
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Treatment of BPAE cells with PTHrP(1-34) at 10-9 to 10-6 mol/L for 24 hours caused dose-dependent suppression of ET-1 secretion (Fig 1
).
PTHrP(1-34) at 10-8
mol/L and higher concentrations significantly inhibited ET-1 secretion.
Maximal suppression (to about 74% of the control level) occurred at
10-7 mol/L PTHrP(1-34). PTHrP(1-34) at
concentrations >10-7 mol/L had no
further inhibitory effect on ET-1 secretion.
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As shown in Fig 2, the inhibitory
effect of PTHrP(1-34) on ET-1 secretion was completely abolished by
coincubation with 100 ng/mL IAP, an inhibitor of the
inhibitory component of GTP binding protein.23
The effect of PTHrP(1-34) at 10-8 mol/L
on ET-1 secretion was also significantly blocked by addition of
10-3 mol/L L-NMMA, a competitive
inhibitor of NO synthesis.24 Further addition
of 5x10-3 mol/L L-arginine,
a precursor of NO in vivo,25 significantly attenuated the
blocking effect of L-NMMA on the suppressive effect of PTHrP(1-34) on
ET-1 secretion, whereas addition of D-arginine showed no
such effect (Fig 2).
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As shown in Fig 3, PTHrP(1-34) resulted in a
significant increase of cGMP level in cultured
endothelial cells. The time course showed that the
intracellular cGMP content was enhanced approximately fivefold within 1
minute after stimulation by 10-7 mol/L
PTHrP(1-34). The increased cGMP level was maintained for about 15
minutes and then decreased.
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We then examined whether PTHrP can modulate ET-1 gene expression. As
shown in Fig 4, Northern blot analysis revealed
that thrombin at 10 U/mL induced a significant increase in ET-1 mRNA
level in BPAE cells. PTHrP(1-34) significantly inhibited both basal and
thrombin-induced increase of ET-1 mRNA level. No significant
changes of GAPDH mRNA levels were observed after treatment with
thrombin or PTHrP(1-34).
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Discussion |
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TopAbstractIntroductionMethods Results Discussion References |
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Both PTHrP and ET-1 have been demonstrated to be vasoactive peptides.6 7 8 12 PTHrP, like PTH, exhibits vasodilatory properties,6 7 8 9 10 11 whereas ET-1 is a potent vasoconstrictor.12 Although their definitive roles in normal cell physiology have not been established, increasing evidence suggests that both PTHrP and ET-1 may have predominantly local actions and play important roles in the regulation of peripheral vascular resistance.
In the present study, we demonstrated that secretion of ET-1 was clearly inhibited by PTHrP(1-34) in a dose-dependent manner in cultured BPAE cells. Because it has been reported that PTHrP binds to seven transmembrane G protein–coupled receptors, which appear to be the same receptors as those for PTH,26 we investigated the effect of IAP on PTHrP(1-34)-suppressed ET-1 secretion. It was shown that the inhibitory effect of PTHrP(1-34) on ET-1 secretion was completely blocked by IAP, suggesting that the effect of PTHrP(1-34) on ET-1 secretion was through a GTP binding protein–coupled receptor pathway.
Vascular endothelial cells can synthesize NO from L-arginine.25 Endothelium-derived NO inhibits contractions evoked by ET-1 in the aorta of normotensive and spontaneously hypertensive rats.27 Other studies14 15 16 indicated that NO inhibits ET-1 secretion. Simeoni et al17 described the role of NO in the vasodilatory response to PTHrP in the kidney, but it is unclear whether the NO-mediated response to PTHrP has any effect on ET-1 secretion. We observed that L-NMMA, a competitive inhibitor of NO synthase that synthesizes NO from L-arginine,24 25 inhibited the effect of PTHrP on ET-1 secretion, and further addition of L-arginine recovered the inhibitory effect of PTHrP on ET-1 secretion, which suggests that the effect of PTHrP may be related to the NO synthesis system. Furthermore, it is well known that NO can activate soluble guanylate cyclases that increase cGMP production. The increase of intracellular cGMP content stimulated by PTHrP provides further evidence that NO may be involved in the effect of PTHrP on ET-1 secretion. PTHrP not only inhibited the basal expression of ET-1 mRNA but also the increase of ET-1 mRNA expression induced by thrombin, which has been reported to be a potent stimulator of ET-1 gene expression,28 suggesting an effect of PTHrP on ET-1 production at the gene level.
Vasoconstrictive agents such as norepinephrine, endothelin, thrombin, and angiotensin II have been reported to increase PTHrP mRNA expression in cultured rat aortic smooth muscle cells.5 It was reported that endothelin release was inhibited by coculture of endothelial cells with cells of the vascular media,13 suggesting that some inhibitory factors appeared in the coculture system. Combined with the results of our study, these interesting findings lead us to hypothesize that negative feedback regulation may exist in the vessel wall between endothelial cells and smooth muscle cells. It is possible that PTHrP is one of the inhibitory factors for ET-1 production in the vascular wall. Endothelial cells synthesize and secrete ET-1 to induce contraction of vascular smooth muscle cells. If the concentration of ET-1 is sufficiently high, such as 10-7 mol/L, ET-1 will stimulate vascular smooth muscle cells to express PTHrP, which will reduce ET-1 secretion due to feedback inhibition, and PTHrP will exert its vasodilatory property. Although the concentrations of ET-1 required to elicit PTHrP expression are in excess of those observed in plasma,29 because of the polar secretion property of ET-1,30 it is conceivable that ET-1 released locally could achieve higher interstitial concentrations that are probably sufficient for stimulating PTHrP expression. Saijonmaa et al31 reported that ET-1 stimulated its own secretion in human endothelial cells. If PTHrP inhibits ET-1 expression stimulated by ET-1 itself, it will be of importance in the regulation of vascular tone. An imbalance between these vasoactive substances may therefore be responsible for alteration of peripheral vascular resistance. Further studies are required to explore the relationship between PTHrP and ET-1 and their possible role in the regulation of the contractile state of vascular smooth muscle.
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Selected Abbreviations and Acronyms |
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Footnotes |
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Reprint requests to Shigeto Morimoto, MD, Department of Geriatric Medicine, Osaka University Medical School, 2-2 Yamadaoka, Suita City, Osaka 565, Japan.
Received May 29, 1995; first decision June 30, 1995; accepted November 14, 1995.
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