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NDT Advance Access originally published online on June 2, 2007
Nephrology Dialysis Transplantation 2007 22(10):2831-2837; doi:10.1093/ndt/gfm269
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© The Author [2007]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org


Parathyroid hormone stimulates the endothelial nitric oxide synthase through protein kinase A and C pathways

Gloria Rashid1, Jacques Bernheim1,2, Janice Green1 and Sydney Benchetrit1,2

1Laboratory of Renal Physiology, Department of Nephrology and Hypertension, Meir Medical Center, Kfar-Saba and 2Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel

Correspondence and offprint requests to: Jacques Bernheim, MD, Department of Nephrology and Hypertension, Meir Medical Center, Tchernichovsky 59, Kfar-Saba 44281, Israel. Email: gloriar{at}clalit.org.il



   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 Acknowledgements
 References
 
Background. Parathyroid hormone (PTH), the major systemic calcium regulating hormone has been implicated in the development of hypertension and the occurrence of uraemic vascular changes. As nitric oxide synthase (NOS) is involved in the production of nitric oxide, and acute PTH effect is characterized by vasodilation, the effect of PTH on the endothelial NOS (eNOS) system was measured in cultured human umbilical cord vein endothelial cells (HUVEC) and the pathways possibly involved were studied.

Methods. The presence of the PTH receptor-1 (PTHR1) on the HUVEC membrane was examined by RT-PCR, immunocytochemistry and western blot. HUVEC were stimulated with 10–12 to 10–10 mol/l PTH. The eNOS mRNA expression was established by RT-PCR and the eNOS protein levels were determined by western blot. The eNOS activity was measured by the conversion of [14C]arginine to [14C]citrulline.

Results. PTHR1 has been found to be expressed in HUVEC and its expression is depressed by increasing concentrations of PTH. PTH induced a significant increase in eNOS mRNA (10–11 mol/l: 1.87 ± 0.16, P = 0.012; 10–10 mol/l: 1.96 ± 0.28, P = 0.007, fold of control), and protein expression. The eNOS activity was also significantly stimulated (10–11 mol/l: 1139 ± 203; 10–10 mol/l: 1323 ± 216 vs control: 621 ± 154 cpm/150 µg protein, P < 0.01). The addition of calphostin C (PKC inhibitor) or Rp-cAMP (PKA inhibitor) reduced the eNOS mRNA, protein expression and activity of PTH-stimulated HUVEC. The combined treatment of calphostin C and Rp-cAMP abolished the eNOS protein expression and activity.

Conclusion. PTH induces an increased activity of the eNOS system; probably both PKA and PKC pathways are involved in this activation. Such data may explain the vasodilation observed after acute treatment with PTH.

Keywords: endothelial cells; nitric oxide synthase; parathyroid hormone; PKA; PKC



   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 Acknowledgements
 References
 
Parathyroid hormone (PTH) has always been considered to play a critical role in maintaining an adequate calcium–phosphorus homoeostasis, which is related to its well-known impact on bones and kidneys [1]. Previous investigations have demonstrated that PTH can also affect the function of various target organs—brain, heart, lungs, pancreas, adrenal glands, testes and cells—lymphocytes, red blood cells and smooth muscles [2,3].

Amino terminal PTH fragment (1–34) and intact PTH are potent vasodilators, as demonstrated in vivo in perfused organs and in vitro in vascular smooth muscle strips or cells [4,5]. The ability to induce vasodilation also seems to be due to a cAMP-induced blockade of smooth muscle cell L-type calcium channel [6,7]. In addition, vascular smooth muscle cell (VSMC) relaxation is related to an increase in ceramide production at low concentrations of PTH [8]. A possible effect of PTH on endothelial cells has been suggested recently by showing that human PTH and human PTH-related protein (hPTHrP) stimulate the release of NO by bovine pulmonary artery endothelial cells, measuring NO using microsensor technology [9]. The aim of the present study was to determine the effect of PTH on the endothelial NOS (eNOS) system in cultured human umbilical cord vein endothelial cells (HUVEC) and the pathways which may be involved.



   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 Acknowledgements
 References
 
Endothelial cell culture and incubation
Endothelial cell cultures were obtained from umbilical cords as previously described [10]. The Ethics Review Committee approved the study and the parturient gave written informed consent. Only umbilical cords from women who had a normal pregnancy and birth were used. Cultured cells were identified as endothelial by their morphology and the presence of vonWillebrand factor. Confluent cultures of HUVEC used for experiments at passages 2–4, were incubated with different concentrations of PTH (fragment 1–34, 10–12–10–10 mol/l, equivalent to 4.1–410 pg/ml, respectively, Sigma, Missouri, USA) for 24–72 h.

The pathways by which PTH(1–34) may have an effect on HUVEC functions were evaluated on cells pretreated for 30 min with protein kinase C (PKC) inhibitor (calphostin C, 50 nmol/l, Sigma) and/or cAMP antagonist (Rp-cAMP, 10 µmol/l, Sigma). Calphostin C inhibits PKC activity by binding to the regulatory domain of PKC. Rp-cAMP is a diasteromer of cAMP that competitively binds to the regulatory subunit of PKA to prevent cAMP-induced dissociation and activation of the enzyme.

To study the effect of PTH receptor blockade, the antagonist, [Nle 8,18, Tyr34]-PTH(7–34) amide (Bachem, Bubendorf, Switzerland), was added to the medium at a final concentration of 0.5 µmol/1, 15 min prior to the addition of the agonist, PTH(1–34).

Expression of PTH receptor type 1 (PTHR1)
HUVEC cultured on glass cover slips were rinsed three times with PBS containing 0.05% BSA (washing buffer), fixed in 4% paraformaldehyde for 10 min. The endogenous peroxidase activity was blocked with 1% hydrogen peroxide for 10 min, rinsed and incubated with 3% BSA for 2 h. Four micrograms per millilitre of monoclonal mouse IgG1 antibody to human PTHR1 (Acris Antibodies, Hiddenhausen, Germany) was added to the cells for 30 min. Control groups were run with non-immune serum. The streptavidine-biotine-peroxide method was performed using Histotain®-Plus Bulk kit (Zymed Lab, CA, USA). The immunoreactive PTHR1 was visualized with AEC substrate kit (Zymed). Counterstaining was performed with haematoxylin solution.

Reverse transcriptase-polymerase chain reaction (RT-PCR)
Expression of the eNOS gene was performed by semi-quantitative multiplex RT-PCR and real-time PCR techniques. Total RNA was extracted from endothelial cells using the PUREscript RNA isolation kit (Gentra systems Inc., MN, USA), according to the manufacturer's instructions. RNA (1 µg) was then reverse transcribed into single-stranded DNA with 200 units of SUPERSCRIPTTMII RNase Reverse Transcriptase (Invitrogen, CA, USA) and oligo (dT)15 primer (Promega, Madison, WI, USA) at 37°C for 45 min, 42°C for 15 min and 99°C for 5 min.

Conventional RT-PCR
Semi-quantitative multiplex RT-PCR amplification was performed on one-tenth of the cDNA solution with 0.5 units of Taq DNA polymerase (Sigma) at a final volume of 50 µl. The PCR conditions and primer sequences were as follows: for eNOS mRNA amplification: forward primer: 5'-CCCCTGCACTATGGAGTCTG-3', reverse primer: 5'-CGTGAGCCCAAAATGTCTTC-3', generating a 582 bp PCR product; β-actin primers sequence: forward primer: 5'-GAGACCTTCAACACCCCAGC-3', reverse primer: 5'-GCTCATTGCCAATGGTGATG-3', generating a 388 bp PCR product; for PTHR1 mRNA amplification: forward primer: 5'-CCTGTCCGGACTACATTTATG-3', reverse primer: 5'-GCCCACGGTGTAAATCATGC-3', generating a 194 bp PCR product; PCR program for eNOS: 30 cycles of 94°C for 30 s, 60.6°C for 30 s and 72°C for 30 s; PCR program for PTHR1: 94°C for 2 min, 30 cycles of 94°C for 30 s, 56°C for 30 s and 72°C for 30 s. All primers were chosen to be complementary to domains in different exons to avoid false-positives caused by DNA contamination of the RNA preparations. RT PCR products were separated on 1.5% agarose (Sigma).

Real-time RT-PCR
To quantify the amounts of eNOS mRNA expression in endothelial cells, real-time RT-PCR was performed with a Light Cycler instrument (Roche Diagnostics GmbH, Mannheim, Germany) in glass capillary tubes. The Light Cycler Fast Start DNA Master SYBR Green I reaction mix (Roche Diagnostics GmbH) and primers for human eNOS and β-actin (the same as conventional PCR) were added to cDNA dilutions. The thermal profile for SYBER Green PCRs was 95°C for 10 min, followed by 35 cycles of 95°C for 10 s, 58°C for 7 s, 72°C for 18 s and 90°C for 5 s. To prove the specificity of the PCR product, a melting curve analysis was performed by 95°C for 5 s, 70°C for 20 s. A dilution series of a standard sample was run with the unknown samples for eNOS and β actin. The eNOS expression was determined by normalization against β-actin expression.

Western blot analysis
Total protein (50 µg) was subjected to electrophoresis on 7.5% SDS–polyacrylamide gels and transferred to a nitrocellulose membrane. The membrane was blocked with 5% skim milk and incubated with eNOS monoclonal antibody (1:2000; Transduction Labs, Lexington, KI, USA). The second antibody was sheep anti-mouse Ig conjugated with horseradish peroxidase (Jackson ImmunoResearch Labs Inc., Pennsylvania, USA). The bound antibodies were visualized with enhanced chemiluminescent reporter system (ECL). The nitrocellulose membranes were stripped and blocked before being reprobed with PTHR1 monoclonal antibody (1:80, Abcam, Cambridge, UK) or {alpha}-Tubulin monoclonal antibody (1:2000; Sigma). The expressions of eNOS, PTHR1 and {alpha}-Tubulin proteins were detected as single bands at 136, 66 and 50 kDa, respectively.

eNOS activity assay
To measure eNOS activity in cell lysates (150 µg total protein) the conversion of [14C]-arginine to [14C]-citrulline was used as previously described by Shah et al. [11].

Intracellular cAMP measurement
Intracellular cAMP levels were evaluated using the competitive enzyme-immunoassay (EIA) ‘cAMP Biotrak EIA’ System (Amersham Biosciences, Buckinghamshire, UK) according to the manufacturer's instructions (non-acetylation protocol). Briefly, cells were rinsed with PBS and lysed with the appropriate lysis buffers. Cellular extract samples were transferred to an antibody-precoated microtitre plate for intracellular cAMP measurement. The assay was based on competition between unlabelled cAMP and a fixed quantity of peroxidase-labelled cAMP, for a limited number of binding sites on a cAMP-specific antibody. OD was measured with spectrophotometer and cAMP levels were calculated as a function of the percentage of bound substrate in each well, corrected for non-specific binding.

Statistical analysis
The results are expressed as mean ± SE. Two-tailed Student paired t-test was used for data analysis. P values of less than 0.05 were considered significant.



   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 Acknowledgements
 References
 
The expression of PTH receptor type 1 (PTHR1) on HUVEC
Immunocytochemical staining by monoclonal PTHR1 antibody demonstrated that HUVEC express PTHR1 on their membrane surface (Figure 1A) and also expressed PTHR1 mRNA (194 bp) when measured by RT-PCR (Figure 1B). PTHR1 protein expression was confirmed by western blot (Figure 1C) and has been found to be down-regulated in the presence of PTH(1–34) (Figure1C).


Figure 1
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  		Fig. 1. Expression of PTHR1 in HUVEC. (A) Immunocytochemical staining of PTHR1 in HUVEC. Anti-PTHR1: staining with anti-PTHR1 antibody. Red staining on the membrane indicates the presence of PTHR1. Negative control: stained with a non-immune serum. (B) Result of representative RT-PCR for PTHR1. (C) Representative result of western blot for protein expression of PTHR1: HUVEC were incubated with 10–11 and 10–10 mol/l PTH(1–34) for 72 h. Similar results were obtained in six identical experiments.

 
PTH increased eNOS mRNA expression in HUVEC
Figure 2 shows the effect of PTH(1–34) on eNOS mRNA expression. PTH (10–11–10–10 mol/l) increased the eNOS mRNA expression after 24 h of incubation as compared to control (0). The relative fold of eNOS signal as detected in conventional PCR or real-time PCR is plotted against 0 (Table 1). PTH(1–34) significantly increased the expression of eNOS mRNA as compared to the control group.


Figure 2
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  		Fig. 2. Effect of PTH(1–34) on eNOS mRNA expression in HUVEC. HUVEC were incubated for 24 h with increasing concentrations of PTH(1–34) (10–12–10–10 mol/l). Total RNA was extracted and the level of eNOS and β-actin mRNA expression were assessed by semiquantitative PCR. Results of representative RT-PCR; similar results were obtained in six independent experiments.

 

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  		Table 1. Effect of PTH on the mRNA expression of eNOS by HUVEC: densitometric analysis and real-time PCRa

 
To test whether the effect of PTH(1–34) on the eNOS system is mediated through PKC or cAMP systems, endothelial cells were pretreated for 30 min with PKC inhibitor (calphostin C, 50 nmol/l) and/or cAMP antagonist (Rp-cAMP, 10 µmol/l). The addition of 50 nmol/l calphostin C or 10 µmol/l Rp-cAMP significantly reduced, but did not completely prevent, the eNOS mRNA expression (Table 2) while the combined treatment of calphostin C and Rp-cAMP on eNOS mRNA expression had a stronger effect (Table 2). Calphostin C and Rp-cAMP without PTH(1–34) had no significant effect on the basal eNOS mRNA expression as measured by real-time PCR (calphostin C: 1.2 ± 0.09, Rp-cAMP: 1.2 ± 0.08, fold of control, P = NS).


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  		Table 2. Effect of calphostin C (PKC inhibitor), Rp-cAMP (cAMP antagonist) and PTH receptor antagonist on eNOS mRNA expression and activity in the presence of PTHa

 
The competitive PTHR antagonist, [Nle8,18, Tyr34]-PTH(7–34) amide, significantly inhibited the PTH-related increase in eNOS mRNA expression (Table 2).

PTH increased eNOS protein expression in HUVEC
We examined whether the increase in eNOS mRNA expression is associated with an increase in eNOS protein levels. PTH(1–34) (10–11–10–10 mol/l) increased eNOS protein levels after 72 h of incubation (Figure 3A). The {alpha}-tubulin levels were similar between groups (Figure 3A). The addition of 50 nmol/l calphostin C or 10 µmol/l Rp-cAMP reduced the eNOS protein expression (Figure 3B). The combined treatment of calphostin C and Rp-cAMP abolished the eNOS protein expression (Figure 3B).


Figure 3
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  		Fig. 3. Effect of PTH(1–34) on eNOS protein expression in HUVEC. Western blot analysis. (A) HUVEC were incubated with 10–12–10–10 mol/l PTH(1–34) for 72 h. Similar results were obtained in six independent experiments. (B) HUVEC pretreated for 30 min with 50 nmol/l calphostin C (PKC inhibitor), and/or 10 µmol/l Rp-cAMP (cAMP antagonist). Similar results were obtained in three independent experiments.

 
PTH increased eNOS activity in HUVEC
PTH(1–34) induced an increase in eNOS activity after 72 h of incubation as compared to the control (0) (10–12 mol/l: 815.2 ± 227, P = NS; 10–11 mol/l: 1138.7 ± 202.6, P = 0.0004; 10–10 mol/l: 1323.4 ± 215.6, P = 0.014 vs 0: 621.9 ± 154.6 cpm/150 µg protein) (Figure 4). The addition of 50 nmol/l calphostin C or 10 µmol/l Rp-cAMP significantly reduced, but did not completely inhibit, the eNOS activity (Table 2). The combined treatment of calphostin C and Rp-cAMP abolished the eNOS activity (Table2). Calphostin C and Rp-cAMP without PTH(1–34) significantly reduced the basal eNOS activity (calphostin C: 32.7 ± 14.4%, P = 0.002; Rp-cAMP: 55.3 ± 17.6%, P = 0.02; calphostin C+RpcAMP: 30.3 ± 11.3%, P = 0.0008 percent of control) (results are not shown as a graph).


Figure 4
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  		Fig. 4. Effect of PTH(1–34) on eNOS activity in HUVEC. HUVEC were incubated for 72 h with PTH(1–34) (10–12–10–10 mol/l). Results are mean ± SE of six experiments. *P < 0.05 vs 0.

 
The competitive PTHR antagonist, [Nle8,18, Tyr34]-PTH(7–34) amide, prevented the PTH-related increase in eNOS activity (Table 2).

PTH-induced elevation of cAMP production in HUVEC
PTH(1–34) induced significantly the cAMP production in HUVEC after 24 h of incubation as compared to the control (0) (10–11 mol/l: 1.4 ± 0.08, P = 0.009; 10–10 mol/l: 1.9 ± 0.01, P = 0.01 fold of control) (Figure 5).


Figure 5
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  		Fig. 5. Intracellular levels of cAMP in HUVEC after PTH(1–34) treatment. HUVEC were incubated for 24 h with PTH(1–34) (10–12–10–10 mol/l). The cells were lysed for intracellular cAMP assay as described in the ‘Methods’ section. Results are expressed as fold of control (0). Data are expressed as mean ± SE of four independent experiments. *P < 0.01 vs 0; #P = 0.03 vs 10–11.

 


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
Acknowledgements
 References
 
PTH receptors are particularly abundant in kidney and bone, but are also expressed in a wide variety of normal tissues [12–14] including VSMC [13]. The expression of the PTH receptor on endothelial cells was not clear in the literature. In the first publication dealing with this subject Rian et al. [15] reported that endothelial cells did not express PTH receptors. On the other hand, more recently others have shown that endothelial cell lines express PTHR1 using the RT-PCR technique [16,17]. We have demonstrated in this study that endothelial PTHR1 is expressed on the HUVEC surface membrane and have confirmed its expression by measuring mRNA and protein levels. This is the first report showing the presence of the PTH receptor on the cell surface membrane of cultured endothelial cells using immunocytochemistry and western blot. To be noted also, that in using in situ hybridization, immunocytochemistry and immunolocalization with electron microscopy, Amizuka et al. [18] showed the presence of PTHR on renal endothelial cells evaluated in histological sections obtained from mice and rat kidney. These findings suggest that the endothelial cell may be a target of PTH through PTHR1. In fact, we have also demonstrated that PTH(1–34) could decrease the PTHR1 protein expression, an effect which to our knowledge has not been previously reported and may be a control response to the cellular action of PTH. We also found that PTH(1–34) significantly stimulates the HUVEC eNOS system. There is a significant increase in eNOS mRNA expression, which is detected at physiological concentrations of PTH. The expression of eNOS protein and the eNOS activity have been found to be enhanced when HUVEC were stimulated with physiological and pathophysiological concentrations of PTH. These results could be in correlation with previous studies reporting that PTH has a strong acute vasodilator action [19–21] detected in using whole animal experiments, isolated vascular strips or perfused organs [4,5]. In addition competitive PTH receptor antagonist, [Nle 8,18, Tyr34]-PTH(7–34) amide, antagonized the PTH(1–34)-stimulated endothelial eNOS expression and activity, a finding which indicates that PTH stimulates the eNOS system through PTH receptors.

Our in vitro protocol could be considered as mimicking the action of PTH on endothelial cells, using increasing concentrations of human active PTH (fragment 1–34) at physiological and pathophysiological concentrations (from 4 to 410 pg/ml). PTH(1–34), a full biological agonist, activates adenylate cyclase and PKC (reviewed in [22]) and has been used in many experimental systems. There are groups who have used the fragment (1–84) [8,17] in endothelial cell experiments but the fragment (1–34) has also been widely used [9]. Bone or kidney cells show an accumulation of cAMP when treated with PTH(1–84) or (1–34), and, to a lesser extent, PTH(2–34) but not after treatment with PTH fragments missing the first two amino acids (reviewed in [22]). Therefore we focused our experimental studies on fragment 1–34.

In our experimental model the effect of PTH(1–34) was measured after 24 h (mRNA expression) or 72 h (protein expression and activity) of incubation. In preliminary results (not shown) we found that PTH(1–34) had a slight effect on eNOS mRNA expression after 3 and 7 h of incubation but the strongest effect occurred after 24 h of incubation. In contrast to our time course of PTH(1–34), Kalinowski et al. [9] demonstrated that PTH/PTHrP induce an elevation of NO release from bovine pulmonary artery endothelial cells within seconds after addition of PTH/PTHrP. Notably, this group used a highly sensitive porphyrinic microsensor.

The vasodilatory action of PTH is obviously not only related to a stimulation of endothelial NO production. The ability to induce vasodilation also seems to be due to a cAMP-induced blockade of the smooth muscle cell L-type calcium channel [6,7]. In addition, VSMC relaxation is related to an increase in ceramide production at low concentrations of PTH [8]. Therefore, according to our results and supported by other reports [8,9], endothelial cells may play a direct, or indirect, role in PTH-induced vasodilation.

The link between PTH and vascular tone is complex. On one hand, PTH may acutely induce vasodilation, while on the other, it has been shown that chronic infusion of the physiological dose of PTH raises blood pressure [24]. The chronic effect of excess PTH on blood pressure has frequently been discussed in the literature. Hypertension and frequent cardiovascular complications were recently reported in cases of primary and secondary hyperparathyroidism [24]. Endothelial vasodilatory function is impaired in patients with hyperparathyroidism [25]. Vaziri et al. [26] have shown that chronic renal insufficiency is associated with reduced eNOS expression in vivo and reduced NO generation, an effect that is reversed by parathyroidectomy. PTH has also been linked to an increase in the VSMC synthesis of 20-hydroxyeicosatetranoic acid, which is a constrictive agent [27]. In the HUVEC line PTH rapidly increases cAMP, which is followed by an elevation in [3H] thymidine incorporation and endothelin-1 secretion [17]. This may explain why PTH affects blood pressure (long term) and eventually why atherosclerosis is more frequent and severe in the presence of hyperparathyroidism [24].

The PTH receptor is a G-protein coupled receptor that activates two G-proteins and thereby two major signal transduction pathways (reviewed in [22,28,29]). By binding to the PTH receptors, PTH may activate either adenylate cyclase, and subsequently PKA, or the phospholipase C (PLC)/PKC pathway [30,31]. PTH normally activates both pathways of classical target cells like chondrocytes, osteoblasts, osteoclasts and kidney-derived cells, while on non-classical target cells, like smooth muscle cells, it activates the adenylate cyclase pathway (reviewed in [22]). Numerous experimental data suggest that PTH exerts vasorelaxant action via cAMP-dependent inhibition of L-type Ca++ channel currents in VSMC [32,33]. There is not much literature regarding the effect of PTH on endothelial cells. Kalinowski et al. [9] first showed that PTH(1–34) activates endothelial NO production through cAMP/PKA and calmodulin pathways. Throckmorton et al. [8] later reported that PTH-induced PKC translocation through a calcium-phospholipid pathway in an endothelial cell line and Isales et al. [17] showed that both endothelial cAMP and intracellular calcium were increased in the HUVEC cell line. In our present study, PKC or cAMP inhibitors partially depressed the human PTH(1–34)-induced eNOS expression and activity, suggesting that both PKC and PKA pathways are involved in endothelial eNOS expression as well as eNOS activity. The combined treatment of PKC and PKA inhibitors completely abolished the eNOS protein expression and eNOS enzyme activity. These findings show that both pathways are involved in endothelial cell function and the response to PTH can be maintained, at least partially, when one of the two is blocked. Our data confirm the results of previous studies, which have shown that PTH activates PKC [8] and cAMP [17] in the immortalized HUVEC cell line (ECV 304). We have also found that PTH has a dose-dependent stimulating effect on intracellular cAMP activation. Calphostin C and Rp-cAMP decreased significantly the basal eNOS activity, suggesting that PKA and PKC pathways are involved in basal eNOS activity and may partly have an independent action on eNOS activity.

In our study we did not deal with the effect of PTHrP, which is interrelated to PTH and is widely expressed in cardiovascular cells (endothelial and smooth muscle cells) [33]. PTH and PTHrP are highly homologous and share the same receptor (PTHR1); both stimulate adenylate cyclase/PKA and phospholipase C/PKC pathways and induce vasorelaxation [22,32,33]. As PTHrP acts as an arterial vasodilator with potency greater than that of PTH [9,23] and endothelial cells are targets of PTH/PTHrP through PTHR1, it is probable that a similar effect of PTHrP might be seen on the eNOS mRNA system using our experimental model. This important point will have to be studied. It should be noted that Kalinowski et al. [9] have already shown that PTH and PTHrP could induce an elevation in endothelial NO release in the presence of hPTH and hPTHrP (at least at a concentration of 10–12 mol/l) [9]. However, if HUVEC may express PTHrP at very low concentrations (33.6 fmol/106 cells; ~130 fmol/mg protein) [34,35], it must be noted that PTH even at a very high concentration (10–6 mol/l) was not found to affect PTHrP secretion in HUVEC [35]. Therefore the findings reported in this study are most probably not related to any effect of PTH on basal secretion of PTHrP.

In conclusion, the presence of PTH receptors on the membrane of cultured HUVEC was clearly demonstrated by immunocytochemistry, western blot and PCR. PTH(1–34) was shown to affect the endothelial eNOS system at all levels of expression and activity (mRNA, proteins, activity) through PTHR1 and that PKA and PKC pathways are involved. It seems reasonable to suggest that the effect of PTH on vascular tone may be in part related to an increase in the eNOS system production and activity.



   Acknowledgements
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
References
 
This work was supported by a Bircher-Benner grant from Tel-Aviv University and the Dr Yechezkiel and Pearl Klayman, Cathedra of Urology of whom J.B. is incumbent.

Conflict of interest statement. None declared.



   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
 References
 

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Received for publication: 5.10.06
Accepted in revised form: 6. 4.07


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