(Received for publication, August 8, 1995; and in revised form, October 30, 1995)
From the
Ventricular cardiomyocytes have been identified as target cells for parathyroid hormone (PTH). A structurally related peptide hormone, parathyroid hormone-related peptide (PTH-rP), is expressed in the heart. In the present study, it was investigated whether PTH-rP can mimic or modify effects of PTH on cardiomyocytes. The investigated effect was induction of creatine kinase (CK) activity, which is associated with cardiac hypertrophy.
PTH and PTH-rP have a similar secondary structure within the active domain 28-34, with exception of amino acid 29. At this position the hydrophilic glutamine in the PTH molecule corresponds to hydrophobic alanine in the PTH-rP molecule. Synthetic PTH or PTH-rP peptides covering domain 28-34 and recombinant full-length PTH(1-84) were used. PTH(28-48) (100 nM) induced CK activity within 24 h (123 ± 3%; means ± S.D., n = 4). PTH-rP(7-34) (1 nM to 1 µM) failed to induce CK activity in cardiomyocytes. Given simultaneously, PTH-rP (1 µM) reduced the stimulation of CK activity by PTH(1-84), PTH(1-34), and PTH(28-48) by 94 ± 9, 79 ± 8, and 69 ± 14%, respectively (means ± S.D., n = 4). In contrast, PTH-rP(7-34) was sufficient to stimulate proliferation of chicken chondrocytes. Thus, PTH-rP exerts different effects on cardiomyocytes and classical target cells for PTH.
A synthetic hybrid peptide was synthesized,
[Ala]PTH(28-48), in which alanine replaced
glutamine at position 29, as in the PTH-rP molecule. In contrast to
PTH(28-48), this mutated peptide
[Ala
]PTH(28-48) had no intrinsic activity
but antagonized the effect of PTH(1-84) and PTH(28-48) on
cardiomyocytes. The results demonstrate that on cardiomyocytes the
effect of PTH can be antagonized by PTH-rP. This antagonism seems due
to a hydrophobic replacement at position 29.
Cardiac myocytes have been identified as target cells for
parathyroid hormone (PTH)()(1, 2) . We
found recently that PTH exerts a hypertrophic effect on adult
cardiomyocytes, characterized by an increase in protein synthesis and a
selective induction of cytosolic creatine kinase (CK)(3) .
Parathyroid hormone-related peptide (PTH-rP) is a peptide hormone
structurally related to PTH. Both peptide hormones have a strong
homology in the N-terminal part of the molecule and can bind to the
same receptor(4) . In contrast to PTH, which is synthesized in
the parathyroidea, PTH-rP is expressed in many tissues including the
heart(5, 6) . Direct effects of PTH-rP on myocardial
cells have not been investigated before. The aim of the present study
was to compare the effects of PTH-rP and PTH on cardiomyocytes with
their effects on classical target cells for PTH, i.e. chondrocytes. Recombinant full-length PTH and commercially
available synthetic peptides, either covering the protein kinase
C-activating domain and the N-terminally located adenylate cyclase
activating domain of PTH, or N-truncated peptides, covering exclusively
the protein kinase C-activating domain, were used.
Our study focused
on the midregional part of the PTH molecule, covering amino acids
28-34. This part of the molecule has been identified as the core
of a protein kinase C-activating domain of PTH and PTH-rP (7, 8, 9, 10, 11) . The
secondary structure of the two peptide hormones in this region is very
similar(12) , consisting of a helical motif. In PTH, the
hydrophobic amino acids are placed on one side and the hydrophilic
amino acids on the other side (Fig. 1). In PTH-rP, the exception
to this rule is that the hydrophobic alanine is located at position 29, i.e. in a hydrophilic environment. The role of this amino acid
in the structure-function relationship of the two peptide hormones was
analyzed specifically in this study. For this purpose two mutated
proteins were synthesized: mutant
[Ala]PTH(28-48), in which the hydrophilic
glutamine at position 29 of the PTH molecule is replaced by the
hydrophobic alanine, as in the PTH-rP molecule; and
[Ans
]PTH(28-48), in which glutamine is
replaced conservatively by the hydrophilic asparagine.
Figure 1:
Helix wheels of the 28-48 region
of PTH, PTH-rP, and the PTH mutants
[Asn]PTH(28-48) (N29Q) and
[Ala
]PTH(28-48) (A29Q). Hydrophobic amino
acids are hatched. In the PTH mutant
[Asn
]PTH(28-48), a hydrophilic amino acid
is replaced by another hydrophilic amino acid. In the mutant
[Ala
]PTH(28-48) at position 29, glutamine
is replaced by alanine which is at position 29 in
PTH-rP.
Isolated cardiomyocytes from the ventricular myocardium of the adult rat were used as an experimental model. In this preparation other cells are absent and the cardiomyocytes are mechanically quiescent. The parameter under investigation was the induction of cytosolic CK, a characteristic feature of the hypertrophic response of cardiomyocytes to PTH stimulation(3) .
To compare the effects of PTH-rP on
cardiomyocytes with those of classical target cells, we also used
primary cultures of chicken derived chondrocytes. This cell system has
been used previously to identify the protein-kinase C-dependent domain
of PTH(9) . PTH stimulates the proliferation of chicken
chondrocytes, and incorporation of [H]thymidine
was determined as a measure of DNA synthesis.
Four hours after
plating, cultures were washed twice with culture medium to remove round
and non-attached cells. The remaining cultures consisted of >95%
rod-shaped cells. Following this washing procedure, experimental media
were added in which the cells were incubated at 37 °C for the times
indicated. The experimental media consisted of the basic culture medium
(control) and the following additions, as indicated: PTH peptides
(human PTH(1-84), bovine PTH(1-34), human PTH(28-48),
human [Asn]PTH(28-48), human
[Ala
]PTH(28-48)) and PTH-rP peptides
(human PTH-rP(1-34) and human PTH-rP(7-34)). Ascorbic acid
(100 µM) was added to the culture media as an antioxidant.
Chondrocytes were isolated from sterna of 16-day-old embryonic
chicks as described previously(9) . Chondrocytes were seeded
into microtiter plates with 96 wells (6-mm diameter; 14,000
cells/cm) and a 200-µl volume of medium 199 containing
10% fetal calf serum. After 17 h, the medium was replaced by 200 µl
of serum-free medium 199. After 4 days the cultures were used for
determination of DNA synthesis.
The distribution of the cytosolic isoenzymes of creatine kinase, MM, MB, and BB, was analyzed as described previously(3) . The supernatants were applied to 1-ml DEAE-cellulose columns that had been equilibrated with SAE-buffer (composition in mM: 20 NaCl, 5 magnesium acetate, 0.4 EDTA, and 100 Tris/HCl; pH 7.9). The CK-MM isoenzyme eluted directly with this buffer, the CK-MB isoenzyme with change of NaCl concentration to 40 mM and pH to 6.4, the CK-BB with change of NaCl concentration to 250 mM and pH 6.4.
Figure 2: Stimulation of cytosolic CK activity by 24-h exposure of cardiomyocytes to various concentrations of PTH(1-84) or PTH(1-34). Data are given as mean values ± S.D. of four cultures; different from control: *, p < 0.05. Basal activity of CK was 3.2 ± 0.5 units/mg protein.
Figure 3: Schematic drawing of the PTH and PTH-rP variants used in the experiments. Black indicates parts with high homology on the basis of amino acid composition, gray indicates parts with structural homology, and white indicates parts without homology. AC and PKC indicate the position of adenylate cyclase-activating domain and protein kinase C-activating domain of the peptides. The numbers indicate the number of amino acids.
Figure 4: Stimulation of cytosolic CK activity by 24-h exposure of cardiomyocytes to various concentrations of PTH(28-48) or PTH-rP(7-34). Data are given as mean values ± S.D. of four cultures; different from control: *, p < 0.05. Basal activity of CK was 3.7 ± 0.2 units/mg protein.
Figure 5: Inhibition of cytosolic CK activity by 24-h exposure of cardiomyocytes to 100 nM PTH(28-48) and simultaneously PTH-rP(7-34) in various concentrations. Data are given as mean values ± S.D. of four cultures; different from control: *, p < 0.05. The 100% value of stimulated cardiomyocytes incubated with PTH(28-48) but without PTH-rP(7-34) was 4.6 ± 0.3 units/mg protein, and basal activity of untreated controls was 3.4 ± 0.2 units/mg protein.
Figure 6: Inhibition of cytosolic CK activity by 24-h exposure of cardiomyocytes to PTH(1-34) (100 nM) and simultaneously PTH-rP(1-34) in various concentrations. Data are given as mean values ± S.D. of four cultures; different from control: *, p < 0.05. The 100% value of stimulated cardiomyocytes incubated with PTH(1-34) but without PTH-rP was 4.3 ± 0.7 units/mg protein, and basal activity of untreated controls was 3.2 ± 0.3 units/mg protein.
The question was investigated whether the three cytosolic isoenzymes of creatine kinase, i.e. CK-MM, CK-MB, and CK-BB, were differently induced in cells treated with PTH(28-48). PTH(28-48) (300 nM) increased only the activity of CK dimers containing the B-isoform: The CK-MB activity was increased to 175%, and the CK-BB activity to 118%. CK-MM activity was not significantly enhanced (Fig. 7). Simultaneous addition of PTH-rP(7-34) (1 µM) abolished the induction of CK-MB and CK-BB through 100 nM PTH(28-48).
Figure 7: Distribution of isoenzymes of cytosolic CK in cardiomyocytes. Specific activities of isoenzymes (CK-MM, CK-MB, CK-BB) after 24-h incubations in presence of PTH(28-48) (PTH, 100 nM) or PTH(28-48) with PTH-rP(7-34) (PTH+PTH-rP, 100 nM, 1 µM). Mean values of isoenzyme activities under control conditions were set at 100%; these were 2.88 ± 0.09 units/mg protein for CK-MM, 0.17 ± 0.01 units/mg protein for CK-MB, and 0.49 ± 0.14 units/mg protein for CK-BB. Data are means + S.D.; n = 4. Differences from control: *, p < 0.05.
Figure 8: Thymidine incorporation by 24-h exposure of chicken chondrocytes to various concentrations of PTH-rP(1-34) and PTH-rP(7-34). Data are given as mean values ± S.D. of four cultures; different from control: *, p < 0.05. The basal thymidine incorporation of control chondrocytes was 229 ± 31 cpm.
Figure 9:
Stimulation of CK activity by
[Ans]PTH(28-48) and
[Ala
]PTH(28-48) at various concentrations.
Data are given as mean values ± S.D. of four cultures; different
from control: *, p < 0.05. Basal activity of CK was 3.7
± 0.2 units/mg protein.
Figure 10:
Inhibition of cytosolic CK activity in
24-h cultures of cardiomyocytes cultivated in presence of
PTH(28-48) (100 nM) and simultaneously
[Asn]PTH(28-48) or
[Ala
]PTH(28-48) at the indicated
concentrations. Data are means ± S.D. of four cultures;
different from control: *, p < 0.05. The 100% value of
cardiomyocytes cultivated for 24 h in presence of PTH(28-48) (100
nM) was 4.7 ± 0.3 units/mg protein, and basal activity
of untreated controls was 3.6 ± 0.4 units/mg
protein.
Figure 11:
Inhibition of cytosolic CK activity by
24-h exposure of cardiomyocytes to phorbol myristate acetate (PMA) (100 nM) and simultaneously
[Ala]PTH(28-48) in various concentrations.
Data are means + S.D. of four cultures; different from control: *, p < 0.05. The 100% value of stimulated cardiomyocytes
incubated with phorbol myristate acetate (100 nM) but without
[Ala
]PTH(28-48) was 5.1 ± 0.4
units/mg protein, and basal activity for untreated controls was 3.5
± 0.4 units/mg protein.
It was further investigated whether PTH-rP
peptides and the mutated antagonistic PTH peptide are able to
antagonize CK induction of naturally occurring full-length
PTH(1-84) on cardiomyocytes as well. Induction of CK-activity by
PTH(1-84) was antagonized by either PTH-rP(1-34) or
[Ala]PTH(28-48) to 94 ± 8 and 82
± 5%, respectively (Fig. 12).
Figure 12:
Inhibition of cytosolic CK activity by
24-h exposure of cardiomyocytes to PTH(1-84) (10 nM) and
simultaneously PTH-rP(1-34) or
[Ala]PTH(28-48) in various concentrations.
Data are means ± S.D. of four cultures; different from control:
*, p < 0.05. The 100% value of stimulated cardiomyocytes
incubated with PMA but without PTH-rP(1-34) or
[Ala
]PTH(28-48) was 5.7 ± 0.5
units/mg protein and for untreated controls 3.8 ± 0.4 units/mg
protein.
In the present study the question was investigated whether PTH-rP, a peptide expressed in myocardial tissue, can mimic or modulate the ability of PTH to induce cytosolic CK in ventricular cardiomyocytes. The main finding of the present study is that PTH and PTH-rP have comparable effects on classical target cells, i.e. chondrocytes, but PTH-rP cannot mimic the action of PTH in cardiomyocytes. PTH stimulated cytosolic CK activity of cardiomyocytes by inducing the fetal type B-isoform. In contrast, PTH-rP had no such effect.
The present study has revealed for the first time that PTH-rP may act indirectly on cardiomyocytes, even if lacking a direct effect. PTH-rP(1-34) and PTH-rP(7-34) were found to antagonize the ability of PTH(28-48) and full-length PTH(1-84) to induce cytosolic CK. When at position 29 a hydrophobic residue (alanine) was introduced into the PTH(28-48) molecule, a mutant peptide was obtained that was functionally equivalent to PTH-rP(7-34). It antagonized the effect of full-length PTH(1-84) and PTH(28-48) but lacked intrinsic activity. In case of a hydrophilic replacement at position 29, the activity of PTH(28-48) was not altered. The results show that position 29 is essential for the function of the core domain of PTH, possibly due to the formation of a hydrogen bond. The results also suggest that PTH-rP competes with PTH for the same binding site but lacks intrinsic activity due to this structural differences within the core of the functional domain.
The antagonism between
PTH(28-48) and peptides with hydrophobic replacement at position
29 are not due to adverse effects at the intracellular key step (3) for the induction of CK, i.e. protein kinase C. In
experiments where protein kinase C was stimulated directly by a phorbol
ester, [Ala]PTH(28-48) had no antagonistic
effect. This finding supports the hypothesis that the antagonism
between the peptides is due to a competition at sarcolemmal binding
sites.
It has been reported that PTH and PTH-rP differ also in other effects on adult cardiomyocytes; PTH but not PTH-rP was found to increase intracellular calcium(19) . In the murine osteoblastic cell line MCT3T3-E1 (20) and in human placenta(21) , PTH-rP was also found unable to mimic the action of PTH. These studies did not investigate, however, whether PTH-rP antagonized the effects of PTH on the investigated cell types and did not identify the structural cause for a difference in the action between the two peptide hormones.
In conclusion, the results of this study demonstrate a marked
difference for the effects of PTH and PTH-rP on cardiomyocytes but not
on chondrocytes. The results offer the opportunity to design
heart-specific antagonists of PTH similar to
[Ala]PTH(28-48) used in this study. Our
results further indicate that PTH peptides, covering the 28-34
region of PTH, are full biological agonists in respect to CK induction
on cardiomyocytes. In contrast, PTH-rP, which is expressed in
myocardial tissue itself, seems to function as a paracrine modulator of
the hypertrophic effects of PTH in cardiac muscle.